JP2013055947A - Use of both rd9 and is6110 as nucleic acid targets for diagnosis of tuberculosis, and provision of multiplex-compliant is6110 and rd9 targets - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide uses of both RD9 and IS6110 as nucleic acid targets for specific and high-sensitive detection of mycobacteria belonging to Mycobacterium tuberculosis complex (MtbC), and/or for specific and high-sensitive discrimination of M. tuberculosis and M. canetti on one hand, from the other strain of the MtbC on the other hand.SOLUTION: The invention relates to the use for in vitro diagnosis of tuberculosis; relating to the use of IS6110 targeting amplified primer pair or hybridization probe, and RD9 targeting amplified primer pair or hybridization probe. The MtbC detection and the strain discrimination can be performed by a single amplification process (multiplex PCR). The means by the invention is an advantageous means for diagnosis of tuberculosis.

Description

  The present invention relates to tuberculosis, particularly tuberculosis diagnosis. The means of the invention use both RD9 and IS6110 as nucleic acid targets for the diagnosis of tuberculosis. The invention in particular allows discrimination between mycobacterial species. This allows for an accurate diagnosis, thereby providing an advantageous means for selecting an appropriate treatment.

[Summary of Invention]
The present invention relates to tuberculosis, particularly tuberculosis diagnosis. The means of the invention use both RD9 and IS6110 as nucleic acid targets for the diagnosis of tuberculosis. The present invention therefore provides a particularly suitable means for the detection of mycobacteria belonging to the Mycobacterium tuberculosis complex (MtbC). The means of the present invention further comprises one M.M. M. tuberculosis and M. tuberculosis. Allows discrimination of canetti from other strains of the other MtbC (eg, M. bovis).

  According to an advantageous feature, the present invention provides multiplex-compliant RD9 and IS6110 targets, which are particularly suitable for performing multiplex PCR, so that the MtbC detection and the species identification are simple. This can be achieved with a single amplification process. According to the multiplex PCR embodiment, the means of the invention advantageously allows the amplification of two different nucleic acid targets in the multiplex, namely the RD9 target and the IS6110 target. By means of the present invention, the detection of mycobacteria belonging to MtbC and the other M.b. Tuberculosis and M.M. Only one single amplification step is required for canetti identification. The means of the invention also have the advantage of fast and reliable performance: the results are available on the same day as the patient sample is taken. They are also specific, sensitive and reliable.

  The World Health Organization estimates that one third of the world's population is infected with Mycobacterium tuberculosis, and 5-10% of people infected with Mycobacterium tuberculosis (but not infected with HIV) He often evaluates that he will become ill or infectious in his life. In 2003, it estimated that 1,750,000 deaths were caused by tuberculosis. HIV and Mycobacterium tuberculosis further accelerate each other's progression and form a lethal combination.

  Strains that are naturally resistant to antituberculosis drugs were recorded in every country studied. For example, M.M. Bobis is naturally resistant to pyrazidamide. In addition, Mycobacterium tuberculosis strains that have acquired resistance to major anti-tuberculosis agents have emerged primarily due to inconsistent, incomplete or irregular adaptations to medicine. A particularly dangerous form of drug-resistant tuberculosis is multidrug-resistant tuberculosis, which is defined as a disease caused by Mycobacterium tuberculosis that is resistant to at least two of the most potent anti-tuberculosis agents, isoniazid and rifampicin. . The proportion of multidrug-resistant tuberculosis is high in some countries, particularly the former Soviet Union, threatening tuberculosis prevention efforts.

  Bacteria that cause tuberculosis in mammals (humans and / or animals) are mycobacteria classified into what is known as the Mycobacterium tuberculosis group (MtbC). The Mycobacterium tuberculosis group includes in particular the following mycobacteria: including Mycobacterium africanum, Mycobacterium bovis (attenuated M. bovis BCG-Calmet-Guerin-) ), Mycobacterium canetti, Mycobacterium caprae, Mycobacterium microti, Mycobacterium pinnipedii, Mycobacterium tuberculosis.

  Mycobacteria classified into the Mycobacterium tuberculosis group are characterized by having 99.9% homology at the nucleotide level and having the same 16S rRNA sequence.

  Despite the very high conservation of their housekeeping genes, the Mycobacterium of Mycobacterium tuberculosis varies widely with respect to host tropism, phenotype and pathogenicity. Some are exclusively human pathogens (M. tuberculosis, M. africanum, M. canetti), some are exclusively rodent pathogens (M. microchi), while others have a broad host range (M. Bovis, M. Kaprae). M.M. Bovis and M.M. Caprae can cause disease in a wide range of domestic or wild animals such as cattle or goats as well as humans. M.M. Microchis has been reported to infect small rodents such as voles, and more recently to humans (Niemann et al., 2000 Emerg. Infect. Dis. 6: 539-542; Kubica et al. , 2003, J. Clin. Microbiol. 41 3070-3077.). M.M. Pinnipedi appears to be the natural host to seal, but this organism is also a guinea pig, rabbit, human, Brazilian tapir (Tapilus terrestris) and possibly a bovine pathogen ( Cousin et al., 2003, Int. J. Syst. Evol. Microbiol 53, 1305-1314).

Fourteen Regions of Difference ranging from 2 to 12.7 kb in size have been identified, Although not in Bovis BCG, It is present in the tuberculosis H37Rv genome. They are referred to as RD1 to RD14 (see Gordon et al. 1999, Mol. Microbiol. 32, 643-655; Behr et al. 1999, Science 284, 1520-1523). The RD nomenclature used in this application is that of Brosch et al. (Brosch et al. 2000, Molecular Genetics of Mycobacteria, eds. Hatfull, GF, & Jacobs, WR, Jr. Am. Microbiol., Washington, DC), pp. 19-36). Ten of the 14 BCG ^- H37Rv ⁺ RD regions are highly conserved among M. tuberculosis strains with respect to other members of the M. tuberculosis group. (RD1, RD2, RD4, RD7, RD8, RD9, RD10, RD12, RD13, RD14; Brosch et al. 2002, PNAS USA, 99 (6), 3684-3629).

Compared to other members of the Mycobacterium tuberculosis group, Six regions have been described that are not present in the tuberculosis H37Rv genome:
5 H37Rv related deletions (referred to as RvD1 to RvD5) and -M. A region known as tuberculosis specific deletion 1 (TbD1) (Gordon et al. 1999, Mol. Microbiol. 32, 643-655; Brosch et al. 1999, Infect. Immun. 67, 5768-5774).

  TbD1 is particularly notable for almost all M.P. It was first identified as a 2,153 bp region missing from the mmpL6 gene of the tuberculosis strain (Brosch et al. 2002, PNAS USA 99: 3684-3690). Based on the presence or absence of the TbD1 region, M.M. Tuberculosis strains can be divided into “ancestral” and “modern” types, respectively. The strains of Beijing, Haarlem and Africa that cause major epidemics are modern (Kremer et al. 1999, J. Clin. Microbiol. 37, 2607-2618; Brosch et al. 2002, PNAS USA, 99 (6 ), 3684-3690).

  The clinical signs and symptoms of tuberculosis are not specific enough to constitute a diagnosis of tuberculosis on its own and on its own.

  Diagnosis is therefore carried out through mycobacterial analysis of the biological sample, and means the detection of the presence or absence of mycobacteria belonging to MtbC.

  Various biological and molecular mycobacterial characteristics were exploited to identify MtbC isolates.

  A series of classical tests based on growth, phenotype and biochemical properties have traditionally been used to distinguish members of MtbC (Haas et al. 1997, J. Clin. Microbiol. 35, 663). 666; Niemann et al. 2000, J. Clin. Microbiol 38, 3231-3234). However, these tests can be slow, cumbersome, inaccurate, irreproducible and time consuming. Furthermore, they do not give obvious results in every case and therefore prevent their use as a means for the diagnosis of tuberculosis.

Current detection means for tuberculosis diagnosis include in particular:
-Microscopy (direct observation and / or cell staining),
-Cell culture,
Gen-Probe® kit.

Mycobacterium is a non-motile polymorphic gonococcus. M.M. Bovis colonies are smooth and recessive (ie small colonies), while Tuberculus colonies are coarse and dominant (ie, large colonies). Direct observation of bacterial colonies, however, is not very specific. Nor is it very sensitive. This is because the sample contains a relatively high concentration of mycobacterial cells (> 10 ⁴ / mL).

  Organisms belonging to the genus Mycobacterium have a unique cell envelope that contains mycolic acid and has a high lipid content. Because the cells are hydrophobic and tend to come together, they do not pass normal dyes (eg Gram stain). They are known as “acid-fast bacteria” because of their lipid-rich cell envelope, and are relatively impermeable to various basic dyes, unless the dye is combined with phenol. Once stained, the cells are said to be “acidic” because they are resistant to decolorization by acidified organic solvents. Currently, two types of acid-fast staining are performed in clinical mycobacterial research. One type is carbole fuchsin (Ciel-Neursen [ZN] or Kinyouun method, others are fluorescent dyes (auramin or auramine-rhodamine). Characteristically, they are acid and alcohol dependent Retain fuchsin or auramine staining after continuous or combined treatment, sensitivity is not optimal, despite the superior acid microscopic specificity for mycobacterium species. Is affected by a number of factors, such as disease prevalence and severity, sample type, quality of sample collection, number of mycobacteria present in the sample, method of treatment (directly or enriched), Influenced by centrifuge method, staining technique, and test quality, at least 100 (in low-income countries) ) And preferably 300 (in industrialized countries), it is recommended that negative results be reported only after testing microscopic immersion fields (or equivalent fluorescent field fields). Therefore, when microscopic observations are performed correctly, it can be time consuming and tedious, and false negatives due to fatigue can also contribute to reduced sensitivity.

  Mycobacterium is a slow-growing bacterium with a normal generation time of 12-18 hours. Colonies usually become visible only after an incubation period of 3 to 10 weeks. Samples containing mycobacterial cells at low concentrations further require several subcultures. Mycobacterium culture in a specific medium is a technique that enables identification of a specific mycobacterium species contained in a biological sample. However, it takes time, especially in patients who are at the beginning of the infection process.

  Nucleic acid hybridization tests have been developed to detect mycobacteria in biological samples. The first test utilized direct probe hybridization. However, the concentration of mycobacterial cells in a sample taken from a patient is usually too low to give a positive hybridization signal. Therefore, a test utilizing PCR amplification was developed. Gen-Probe (registered trademark) kit commercialized as "Amplified Mycobacterium tuberculosis direct test" kit, or MTD kit (Gen-Probe Inc., San Diego, Calif., California, California) USA) utilized amplification of MtbC specific rRNA (transcription-mediated amplification) followed by amplicon detection (hybridization protection assay) by the Gen-Probe HPA method. A positive MTD test indicates that the sample contains MtbC mycobacteria. However, due to inhibition problems and due to insufficient sensitivity, a negative MTD test cannot rule out the possibility that an MtbC organism is isolated from a sample. The MTD test also does not distinguish between various mycobacterial species; Bovis and M.M. Does not distinguish from tuberculosis.

  Those people who received BCG vaccination Due to the fact that Bovis is positive (eg, their urine sample is M. Bovis positive), the diagnosis of tuberculosis becomes even more difficult. Therefore, although not a tuberculosis carrier, May be Bovis positive. Under such circumstances, the biological sample is M.I. Not Bovis, especially M.I. The detection of whether it contains tuberculosic MtbC mycobacteria is of primary importance.

  Therefore, it can be applied to samples collected from mammals, especially from humans. Tuberculosis was obtained from other major MtbC strains, especially M. There is a need for diagnostic means that can be distinguished from Bovis. Rapid and reliable MtbC + diagnostic tests, and preferably biological samples are A rapid and reliable diagnosis of whether there is a suspicion of containing Mycobacterium belonging to MtbC that is not Bovis (MtbC + Mb−), most preferably the biological sample is Suspected of containing tuberculosic cells (Mt +) or it is There is a need for a quick and reliable diagnosis of whether tuberculosis cells are not included (Mt−). The present invention provides such tests and means. The means of the present invention are particularly suitable for amplification, particularly suitable for PCR amplification and as an advantageous feature suitable for multiplex PCR. According to the multiplex PCR embodiment of the present invention, two different nucleic acid targets can be amplified in multiplex, i.e., in a single amplification step: one target is properly selected within IS6110, and others The target is appropriately selected within RD9.

  IS6110 and RD9 are known to those skilled in the art. IS6110 sequences are in the name of INSTITUT PASTUR, EP 0 490 951 B1 (US 5,597,911; US 5,807,672; US 5,837,455) and EP 0 461 045 B1 ( US 5,776,693). M.M. The IS6110 sequence of Tuberculus H37Rv (IS6110 reference sequence) is available under accession number X17348 M29899. The RD9 sequence is described, for example, in WO 00/55632 in the name of Pasteur Institute (EP 1 161 562 A1; and the corresponding US patent application). M.M. The RD9 sequence of Tuberculos H37Rv (IS6110 reference sequence) includes cobL, Rv2073c, Rv2074 and Rv2075c sequences. M.M. The RD9 sequence of tuberculosis H37Rv It corresponds to the sequence from position 250,271 to position 254,176 of the tuberculosis H37Rv genome, which is available as access number BX842578 (segment 7/13 of the M. tuberculosis H37Rv genome).

  To the best of Applicants' knowledge, the prior art, however, does not suggest combining detection of IS6110 with detection of RD9; the prior art does not disclose suitable means for detection of IS6110 and RD9 by multiplex PCR; and The prior art does not disclose IS6110 and RD9 subsequences (eg, SEQ ID NO: 2 and SEQ ID NO: 8 according to the present invention) that are selected as multiplex-compliant targets according to the present invention.

  The means of the invention are particularly suitable for providing very detailed and accurate mycobacterial information in a single amplification process. The present invention thereby provides a fast, reliable, specific and sensitive means for the diagnosis of tuberculosis. Diagnosis can thus be fast and accurate, which is a direct benefit for the patient. The means of the present invention can also be used as a means of assessing whether a particular medical treatment may or may have any effect on an infection. They are M.M. To distinguish tuberculosis from other major MtbC strains, Tuberculosis In order to distinguish from Bovis, the means of the present invention further allow for sufficient consideration of resistance to anti-tuberculosis drugs and thereby avoid the application of inappropriate treatment. They further avoid the consequent occurrence of resistant Bacillus.

FIG. The subsequence of RD9 of tuberculosis H37RV (1464 nucleotide sequence Rv2075c; SEQ ID NO: 19) and SEQ ID NO: 7 according to the present invention (positions 351-600 within Rv2075c) are shown in bold. The RD9 target sequence of SEQ ID NO: 8 of the present invention (positions 390-561 within Rv2075c) is shown in bold and underlined. FIG. The IS6110 sequence (1360 nucleotides; SEQ ID NO: 20) of tuberculosis H37RV is shown. The subsequence of SEQ ID NO: 1 (positions 361-600), selected in IS6110 according to the invention, is represented in bold. The IS6110 target sequence of SEQ ID NO: 2 (positions 372-572) according to the present invention is shown in bold and underlined.

Detailed Description of the Invention

In this application, each of Mycobacterium africanum, Mycobacterium bovis, Mycobacterium canetti, Mycobacterium kaprae, Mycobacterium microchi, Mycobacterium pinnipedi, and Mycobacterium tuberculosis are It is understood that they constitute different species in terms of scientific names. M.M. Bovis Bovis and M.M. Attenuated M. bovis derived Includes Bovis BCG (eg, M. Bovis Pasteur, or M. Bovis Tokyo). Each of the species is known to those skilled in the art as shown by extensive literature in the art (eg, Kremer et al. 1999, J. Clin. Microbiol. 37, 2607-2618; Goh et al. 2001, J. Clin. Microbiol. 39, 3705-3708; Van Solingen et al. 1998, J. Clin. Microbio. 36, 1840-1845). Illustrative strains specifically include the following:
-ATCC 27294, ATCC 25177, ATCC 51910 (M. tuberculosis);
-ATCC 25420, ATCC 35711 (M. Africannam (subtype I));
-ATCC 19422, ATCC 35782, ATCC 11152 (M. Microti);
-ATCC 19210, ATCC 35725, ATCC 35726, ATCC 35730 (M. Bovis);
-ATCC 27290, ATCC 35736, ATCC 35737 (M. Bovis BCG);
-M. Caprae's CIP1057776 strain (available from the Institut Pasteur Collection, or ATCC number BAA 824 from ATCC).
ATCC is the American Type Culture Collection (10801 University Boulevard Manassas, Virginia 20110-2209). Pasteur Institute Collection or CIP ("Collection de l'Institut Pasteur") is Collection de l'Institut Pasteur, Pasteur Institute (25-28 ru du Ductor Roux, F-75724 Paris Cedex 15, France) is there.

  The present invention results from the proper selection and use of two mycobacterial nucleic acids, namely RD9 and IS6110. IS6110 and RD9 are known to those skilled in the art. IS6110 is an IS-like factor, which is The tuberculosis reference strain H37Rv consists of 1360 nucleotides. M.M. The IS6110 sequence of tuberculosis H37Rv is available as access number X17348 M29899. RD9 is a different region, which is The tuberculosis reference strain H37Rv consists of 1464 nucleotides. M.M. The RD9 sequence of tuberculosis H37Rv It corresponds to position 250, 271-254, 176 of the genome sequence of tuberculosis H37Rv, which is available under access number BX842578 (segment 7/13 of M. tuberculosis H37Rv genome).

The use of RD9 and IS6110 according to the present invention can Tuberculosis and M.M. Canetti's from the other MtbC on the other side, especially M.I. Allows identification from Bovis. The use of both RD9 and IS6110 enables the following:
-Detection of MtbC mycobacteria,
-One M.M. Tuberculosis and M.M. Identification of Canetti from the other MtbC and other strains.

The present invention provides for the specific and sensitive detection of Mycobacterium of Mycobacterium tuberculosis group (MtbC) and / or other of said MtbC of Mycobacterium tuberculosis and Mycobacterium canetti. It relates to the use of both RD9 and IS6110 as nucleic acid targets for specific and sensitive discrimination from strains. The present invention relates to the use of RD9 and IS6110 as nucleic acid targets for in vitro diagnosis of tuberculosis. The present invention therefore provides for the specific and sensitive detection of Mycobacterium of the Mycobacterium tuberculosis group (MtbC) and / or for the Mycobacterium tuberculosis and Mycobacterium canetti of the other said MtbC. For both specific and sensitive discrimination from other strains of
At least one IS6110 targeted amplification primer pair or hybridization probe, and at least one RD9 targeted amplification primer pair or hybridization probe.
Said use is suitable for in vitro diagnosis of tuberculosis. Preferably, the at least one IS6110 targeted amplification primer pair or hybridization probe is an IS6110 specific amplification primer pair or hybridization probe, and the at least one RD9 targeted amplification primer pair or hybridization probe is RD9. Specific amplification primer pairs or hybridization probes. A specific primer pair or probe refers herein to a primer pair or probe that does not cross-react with any mycobacterial nucleic acid other than its target (IS6110 or RD9), and preferably does not cross-react with any human nucleic acid. means.

The means of the invention are particularly suitable for amplification and especially PCR amplification. As an advantageous feature, the means of the invention are particularly suitable for multiplex PCR. The present invention therefore relates to the use of RD9 and IS6110 as nucleic acid targets, wherein said IS6110 and RD9 are targeted in multiplex PCR. According to the multiplex PCR embodiment of the present invention, two different nucleic acid targets are targeted in amplification in a multiplex, ie amplification in a single amplification step: one target is suitably within the IS6110 nucleic acid. Selected and other targets are appropriately selected within the RD9 nucleic acid. Specific RD9 and IS6110 nucleic acid targets selected according to the present invention allow:
-Detection of MtbC mycobacteria,
-One M.M. Tuberculosis and M.M. Canetti's distinction from other strains of the other MtbC, and-the design of primers that can be used in multiplex, ie specific and sensitive even when used in a single multiplex amplification step.

According to the present invention, when the presence (+) or absence (-) of RD9 and IS6110 is evaluated in a biological sample, the RD9-IS6110 + reaction is It means that it contains the Mycobacterium of Mycobacterium tuberculosis group that is not tuberculosis (ie MtbC + Mt−). Any other RD9IS6110 reaction, except RD9-IS6110- Tuberculosis or M.P. It is meant to include the Mycobacterium of the Mycobacterium tuberculosis group that is canetti (ie MtbC + and (Mt + or Canetti +)).

The present invention further provides M.I. Provide a detailed diagnosis within the scope of the tuberculosis species:
-RD9 + IS6110-reaction is based on a biological sample of ancestral M.P. -RD9 + IS6110 + reaction means that the biological sample is a modern M. A tuberculosis strain, or the very rare M. Means including either Canetti.
The present invention therefore provides ancestral M.P. Tuberculosis strains were identified as modern Distinguish from tuberculosis strains and avoid the problem of false negative results that often occurred when IS6110 was used as the only marker in the prior art.

  An ancestral strain is herein referred to as M. pneumoniae. Tuberculosis TbD1 + strain, while The tuberculosis strain is M. pneumoniae. It is defined as the tuberculosis TbD1- strain. An ancestral M. containing TbD1 region. Tuberculosis strain no. The sequences of genes mmpS6 and mmpL6 from 74 have been deposited in the EMBL database as access number AJ426486 (TbD1, 2,153 bp, AJ426486 extends from position 340 to position 2492). Brosch et al. 2002, PNAS USA 99: 3684-3689, and the TbD1 primer can be used to assess whether TbD1 is present in a biological sample; in the supporting material of Brosch et al., Supra. See Table 1 (TBD1intS.F and TBD1intS.R primers (CGT TCA ACC CCA AAC AGG TA-SEQ ID NO: 15 and AAT CGA ACT CGT GGA ACA CC-SEQ ID NO: 16, respectively) and TBD1fla f and TBD1fla1-R primers (CTA CCT CAT CTT CCG GTC CA-SEQ ID NO: 17- and CAT AGA TCC, respectively) CGG ACA TGG TG-SEQ ID NO: 18-) is described).

  Modern M.M. Examples of tuberculosis strains are in particular M. cerevisiae. Tuberculosis isolate H37Rv, and Tuberculosis isolate H37Ra. M.M. The complete genome of the tuberculosis isolate H37Rv has been sequenced (Cole et al. 1998, Nature, 393, 537-544). The sequence is also available under ID MTBH37RV (access number AL123456). TbD1 (M. tuberculosis specific deletion 1) is an M. tuberculosis strain comprising representative strains from major infectious diseases (eg, Haarlem, Beijing and Africa clusters). It is not present in about 87% of tuberculosis strains (Kremer et al. 1999, J. Clin. Microbiol. 37, 2607-2618; Brosch et al. 2002, PNAS USA, 99 (6), 3684-3687).

  M.M. Canetti has a rare prevalence among human patients with tuberculosis-like symptoms. Therefore, the RD9 + IS6110 + reaction most frequently refers to MtbC + and Mt + (ie, Mycobacterium in the Mycobacterium tuberculosis group that is M. tuberculosis).

Nevertheless, M.M. The presence of tuberculosis (ie Mt +) and M. If one wishes to identify the presence of Canetti (ie Canetti +), one skilled in the art can utilize appropriate means. In fact, M.M. Canetti A very rare and smooth variant of tuberculosis, usually isolated from patients from or linked to Africa. It contains the same 16S rRNA sequence as M.P. M. tuberculosis, which is shared with other members of the complex and includes all RD and RvD, and TbD1 regions. Canetti strains differ in many respects, including polymorphisms of specific housekeeping genes, IS1081 copy number, colony morphology, and cell wall lipid content, particularly in the following ways:
i. M.M. Canetti has been shown to retain 26 unique spacer sequences in the DR region that are not present in any other member of the Mycobacterium tuberculosis group (Van Embden et al. 2000, J. Bacteriol. 182, 2393). 2401);
ii. M.M. Canetti is described in Brosch et al. 2002, PNAS USA, 99 (6), 3684-3689, characterized by the absence of a specific RD ^can region, which partially overlaps with RD12 and often RD12-can It is called deletion. M.M. In connection with the tuberculosis H37Rv genome, The RD12-can deletion sequence of Canetti isolate 14000059 has been deposited in the EMBL database with accession number AJ583833;
iii. M.M. Canetti is characterized by the presence of a single copy of the IS1081 insert (M. Bovis IS1081 is available as access number X61270);
iv. M.M. Canetti cells form smooth colonies, while M. Tuberculus cells form coarse colonies.

  Therefore, the skilled person From Canetti In any situation where tuberculosis is to be identified, any one or more of the i to iv features can be used.

Summarized as in Table 3 below.

  The pattern shown in Table 3 above is typical, that is, it is found in the majority, if not all, of the strains belonging to the indicated species.

  The invention further provides a nucleic acid target selected within the RD9 and IS6110 nucleic acids. These selected RD9 and IS6110 nucleic acid targets have the very high level of specificity required for diagnostic applications: their detection is confused with microorganisms or any other nucleic acid from mammals Without fail, indicating that RD9 and IS6110 nucleic acids are present in the sample being evaluated. These selected targets can advantageously be used in multiplex (ie, the same amplification process), while allowing for the design of primers that are very sensitive and highly specific. Such a primer does not result in cross-hybridization, so it retains the required specificity and maintains the same sensitivity as 1 Mycobacterium CFU per sample (see example below).

Selected RD9 and IS6110 nucleic acid targets of the present invention have the following sequences:
The sequence of SEQ ID NO: 2 of the IS6110 target, or a sequence that is completely complementary to the full length of said SEQ ID NO: 2,
-The sequence of SEQ ID NO: 8 of the RD9 target or a sequence that is completely complementary to the full length of said SEQ ID NO: 8.
The IS6110 target of SEQ ID NO: 2 has M.P. positions 372 to 572 (including the start and end positions). Fragment of tuberculosis H37Rv IS6110 (accession number X17348 M29899):

The RD9 target of SEQ ID NO: 8 is It is a fragment of tuberculosis H37Rv RD9. M.M. The RD9 sequence of the tuberculosis H37Rv genome extends from position 250,271 to position 254,176 of the H37Rv sequence available as access number BX842578. M.M. The Rv2075c sequence contained within the RD9 sequence of tuberculosis H37Rv ranges from position 252,713 to position 254,176 of the H37Rv sequence available under accession number BX842578. The RD9 target of SEQ ID NO: 8 extends from position 390 to 561 (including the start and end positions) of the Rv2075c sequence, ie the following sequence:

The present invention thus detects Mycobacterium of the Mycobacterium tuberculosis group (MtbC) and / or distinguishes one Mycobacterium tuberculosis and Mycobacterium canetti from the other MtbC other strains. A set of two polynucleotides suitable for use as a reference template sequence in the design of primers that can be used in a multiplex for a single amplification step,
Wherein one of the two reference template polynucleotides is a fragment of RD9 and the other is a fragment of IS6110;
Wherein the IS6110 fragment is present in the sequence of SEQ ID NO: 2 or in a sequence that is completely complementary to the full length of SEQ ID NO: 2,
And wherein the RD9 fragment is present in the sequence of SEQ ID NO: 8, or in a sequence that is completely complementary to the full length of SEQ ID NO: 8.

  Said IS6110 fragment is conveniently A fragment spanning positions 372 to 572 of the IS6110 sequence of the tuberculosis strain H37Rv (including the start and end positions) (the M. tuberculosis H37Rv IS6110 sequence is available as access number X17348 M29899). Said RD9 fragment is conveniently A fragment spanning positions 390 to 561 of the RD9 sequence of the tuberculosis strain H37Rv (including the start and end positions) (the sequence of segment 7/13 of the M. tuberculosis H37Rv genome is available as accession number BX842578; Corresponding to positions 252, 713-254, 176 of this array).

  Primers selected from such reference template sequences are still specific and so as to result in amplification of the selected IS6110 target of SEQ ID NO: 2 and amplification of the selected RD9 target of SEQ ID NO: 8 It can be used in multiplex in a single amplification step while providing sensitive detection and discrimination.

  The invention also relates to each of the individual said reference template sequences, i.e. to detect Mycobacterium of Mycobacterium tuberculosis (MtbC) and / or to detect one Mycobacterium tuberculosis and Mycobacterium canetti on the other A polynucleotide suitable for use as a reference template sequence in primer design that can be used in multiplex in a single amplification step to distinguish from other strains of said MtbC, wherein the reference template polynucleotide is Selected from said set of two polynucleotides.

  The present invention therefore provides specific and sensitive amplification primers and probes that maintain sensitivity and properties even when used in multiplex, and thus can achieve the MtbC detection and the species discrimination in a single amplification step. I will provide a. These primers and probes are advantageously designed with reference to the IS6110 and RD9 targets.

  Amplification methods, particularly polymerase chain reaction (PCR) and PCR-based methods are known in the art (Molecular Cloning: A Laboratory Manual, Maniatis, Fritsch, and Sambrook, CSHL Press, Molecular Biologist; PCR Primer: A Laboratory Manual, Diffenbach and Dveksler, CSHL Press; The Polymerase Chain Reaction, Mullis, Ferre, and Birs, Ne bis; fication, Ferre, Birkhauser Boston Press.).

  The present invention particularly relates to multiplex PCR in biological samples, and more particularly at least duplex PCR (two targets, ie IS6110 and RD9), most preferably at least triplex PCR (two targets, ie IS6110 and RD9, and one internal Describe nucleic acids (oligonucleotides and polynucleotides) suitable for the implementation of (standard).

  Multiplex PCR is herein understood as any PCR reaction aimed at amplification of more than one target. For example, multiplex PCR includes duplex PCR (2 targets), triplex PCR (3 targets) and higher multiplex PCR.

  Of course, the nucleic acids according to the present invention are also suitable for other protocols including simplex protocols, multiplex protocols, end-point protocols, qualitative protocols, quantitative protocols, combinations thereof and the like. Nucleic acid is here understood as any nucleic acid: it can be synthetic or non-synthetic, recombinant or natural, linear or circular. This includes DNA and RNA. Nucleic acids can be either single stranded or double stranded, or even triple stranded. It can be derived from a variety of biological sources, such as from microorganisms (such as bacteria) or higher organisms, such as mammalian cells (such as human cells or non-human mammalian cells). Nucleic acids can also include total DNA, total RNA, genomic DNA, mitochondrial DNA, plasmid DNA, BAC DNA, and mixtures thereof. Furthermore, nucleic acids can be considered in various states of purity. A sample is understood here as either a natural or non-natural sample. Preferably, the sample is derived from a biological source. The sample may also be derived from cell culture supernatant. In a preferred embodiment, the sample is or is derived from bronchial aspirate, bronchial lavage, sputum, lung tissue, cerebrospinal fluid, blood, urine. The sample may also arise from a preliminary process. For example, the sample may be available from purification and / or extraction methods. In particular, the sample may result from a separation and / or purification and / or extraction process performed on a biological sample. The sample can also be a control sample. Control samples include samples as qualitative controls, positive controls, negative controls, quantitative controls and calibration controls. The control can be internal as well as external. Any sample according to the present invention may exist several times. For example, the sample can be provided in 2x, 3x, 4x, multiples, which is advantageous in the case of quantitative experiments.

  Those skilled in the art are familiar with the concept of target templates. The target template can be any nucleic acid whose presence is assessed by the method. The target template is probably but not necessarily a nucleic acid (PCR amplicon) that is amplified by the method.

  Polynucleotide is here understood as any polymer of nucleotides, where nucleotides can be ribonucleotides, deoxyribonucleotides, dideoxyribonucleotides, denatured nucleotides and the like. The nucleotide is preferably single stranded, but can also be double stranded. The length of the polynucleotide can vary and is preferably less than 500 nucleotides (nt), preferably in the range of 50-400 nt, more preferably 100-300 nt, even more preferably 150-250 nt. For example, the selected IS6110 target of SEQ ID NO: 2 is 201 nt, and the selected RD9 target of SEQ ID NO: 8 is 172 nt.

  Oligonucleotide is herein understood as any short polymer of nucleotides, where nucleotides can be ribonucleotides, deoxyribonucleotides, dideoxyribonucleotides, denatured nucleotides, and the like. The oligonucleotide is preferably single stranded. The length of the oligonucleotide can vary and is usually less than 150 nucleotides (nt), preferably in the range of 10-100 nt, more preferably 15-60 nt, even more preferably 18-50 nt. The oligonucleotide can be chemically modified, for example, radioactivity, fluorescence, biotin labeling, tagging or marking by dig labeling. The oligonucleotides according to the invention can be either forward (sense) or reverse (antisense). In addition, although a preferred function may be mentioned in connection with some oligonucleotides according to the invention, several functions may be envisaged for a given oligonucleotide and according to the invention It must be emphasized that it is clear that it may be used in different ways. For example, oligonucleotides can be used as either primers or probes. Also, when an oligonucleotide is described as useful as a probe, one skilled in the art will understand that the complementary sequence of this oligonucleotide is equally useful as a probe targeting the same amplicon. Furthermore, it is clear that any primer suitable for multiplex analysis can also be used in a simplex protocol within the meaning of the present invention. The oligonucleotides according to the invention include in particular PCR primers and probes. Unless otherwise specified, nucleic acid sequences are shown in the 5 'to 3' direction. The oligonucleotide can be present in a desired solvent and a desired concentration of solution / suspension in many forms, eg, in a dry state. Those skilled in the art will know which solvents, concentrations, and storage conditions are appropriate for the oligonucleotides of the present invention. In particular, those skilled in the art will know how to make the oligonucleotide as a stock solution. The oligonucleotides according to the invention can also assume various degrees of purity, as can be determined by the person skilled in the art, for example by HPLC chromatography.

  In this application, when a product, such as a nucleic acid, polynucleotide, oligonucleotide, primer, probe, amplicon, reference template sequence, etc., contains a defined sequence, the scope of such expression is the product Should be understood to include the situation in the defined sequence.

  The concept of primer or PCR primer is known to those skilled in the art. For example, it includes an oligonucleotide that can anneal to the target template under appropriate stringency conditions and allows for polymerase chain extension. Typical primer lengths are 10-30 nt, preferably 10-18 nt, most preferably 15-18 nt, such as 15, 16, 17 or 18 nt. Suitable stringency conditions include in particular high stringency conditions. Illustrative conditions of high stringency are the temperature and ionic conditions used during nucleic acid hybridization and / or washing. “Very stringent conditions” as well as factors affecting the rate of hybridization are known to those skilled in the art and are described, for example, in Maniatis et al. 1982, Molecular Cloning, Cold Spring Harbor Laboratory. In general, very stringent conditions are selected about 5 to 20 ° C. below the thermal melting point (Tm) of the oligonucleotide at a defined ionic strength and pH. Tm is the temperature (under defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a perfectly matched probe. For DNA-DNA hybrids, Tm can be estimated with the equation Tm = 69.3 + 0.41 × (CG)% −650 / L (Meinkoth and Wahl, Anal. Biochem. 1984, 138: 267-284). Where L is the probe strength in nucleotides. “Very stringent conditions” also applies to the cleaning method. Other illustrative conditions of strictness are shown in the examples described below.

  The concept of a probe is also known to those skilled in the art. For example, it includes an oligonucleotide that can anneal to a target template under desirable hybridization conditions. The typical length of the probe is 15-60 nt, preferably 15-40 nt, more preferably 15-30 nt, most preferably 15-28 nt, such as 16-27 nt. Preferably, the probe is fluorescently labeled. However, it will be apparent to those skilled in the art that in certain circumstances, primers may be used as probes and vice versa. Furthermore, it is emphasized that the products according to the invention, in particular the oligonucleotides, are not particularly limited to the intended use mentioned here, but rather should be interpreted broadly regardless of the indicated purpose. For example, a claim to a product (oligonucleotide) for a particular use should be construed to mean a product (oligonucleotide) that is actually suitable for the specified use. Thus, oligonucleotides suitable for use as primers in a multiplex protocol are also clearly suitable for simplex protocols within the meaning of the present invention. Various formats (types) of probes are known in the art, including Taqman ™ probes (hydrolysis probes), molecular beacons (beacon probes or molecular beacon probes) and Scorpion ™ probes. Nucleic acid probes may include intercalating agents, such as one or more intercalating dyes, within their nucleotide sequence.

In a preferred embodiment, the probes according to the invention can all be synthesized and used in a molecular beacon format. The structure of molecular beacons is as follows. A short nucleotide sequence unrelated to the target sequence (the so-called beacon arm) is therefore covalently bound to both ends of the probe. The short unrelated arm is thus bound to the probe 5 'and labeled with a fluorescent component (ie, fluorescent dye or fluorescent marker). Another but still unrelated arm binds to the 3 ′ end of the probe and is labeled with a fluorescence quenching moiety. For this reason, molecular beacons have fluorophores and quenchers at opposite ends. The 5 ′ short arms are completely complementary to those of 3 ′, and they can anneal together, and therefore can assume a hairpin structure if they do not hybridize to the target in solution. In this hairpin higher order structure, the quencher and the fluorescent dye are sufficiently close to each other to enable efficient quenching of the fluorophore. However, when the probe encounters the target molecule, the values of beacon arm Tm and probe Tm are optimally selected (theoretically: probe Tm> beacon arm Tm> primer Tm, where Tm is the target Annealing is preferred with respect to the hairpin conformation. Fluorescence can be emitted when the fluorophore and quencher are moved away from each other and the fluorophore is illuminated with appropriate light excitation. As PCR progresses, amplification products accumulate, and the amount of fluorescence in any given cycle depends on the amount of amplification product present at that time. (See, eg, Sanjay Tyagi and Fred Russell Kramer, Nature Biotechnology 1996, volume 14, pages 303-308; Nature Biotechnology 1998, volume 16, pages 49-53). Of course, it is also possible to connect a fluorophore to the 3 ′ end while attaching a quencher to the 5 ′ end. Schematically, the probe can have the following formula (molecular beacon format):
5 'fluorophore-(arm 1)-probe (arm 2)-quencher 3'
5 'quencher-(arm 1)-probe (arm 2)-fluorophore 3'
Here, arm 1 and arm 2 are all short, allowing hairpin structure formation under appropriate stringency conditions, for example in the range of 3-10 nucleotides, preferably 5, 6, 7 nucleotides. Nucleotide sequences can be made, ie, Arm 1 and Arm 2 are fully complementary to anneal under the desired stringency conditions (standard PCR stringency conditions are, for example, 55-65 ° C. Including annealing temperature and 4 to 8 mM Mg concentration). However, arm 1 and arm 2 are independent of the target sequence of the probe, that is, the hairpin conformation due to annealing between arm 1 and arm 2 is essentially unproliferated in the probe. It is the only possible secondary structure. Those skilled in the art will know how to select such an arm for a given probe. For example, possible beacon formats include:
CGTGCG- (probe sequence) -CGCACG
ATGCGGG- (probe sequence) -CCCGCAT.

  A fluorophore is herein understood as any fluorescent marker / dye known in the art. Examples of such suitable fluorescent markers are Fam, Hex, Tet, Joe, Rox, Tamra, Max, Edans, Cy dyes (eg Cy5), fluorescein, coumarin, eosin, rhodamine, Bodipy, Alexa, Cascade Blue (Cascade Blue), Yakima Yellow, Lucifer Yellow and Texas Red (all of which are trademarks). A quencher is herein understood as any quencher known in the art. Examples of such quenchers include Dabcyl, Dark Quencher, Eclipse Dark Quencher, Elquencher, Tamra, BHQ and QSY (all of which are trademarks) Including. Those skilled in the art will know which dye / quencher combinations are suitable for designing probes. In a preferred embodiment according to the present invention, the spectral characteristics of the probes can be selected so as not to interfere with each other. In particular, when probes are used in multiplex, each single probe can have its own fluorophore that is spectrally significantly different from each other, ie, the absorption / emission spectra are essentially non-overlapping. It is. This advantageously allows low noise multiplex detection for all single probes and ensures that the individual signals do not interfere with each other in the detection. Examples of dyes that can be used together in a multiplex include Fam and Tamra, Fam and Tamra and Texas Red. In accordance with the present invention, all provided oligonucleotides and polynucleotides can be either maintained separately, partially mixed or fully mixed. The oligonucleotides and polynucleotides can be provided in a dry form or dissolved in a suitable solvent, as will be appreciated by those skilled in the art. Suitable solvents include TE, PCR grade water and the like.

  The present invention relates to a primer pair designed to result in amplification of the selected IS6110 target of SEQ ID NO: 2 and a primer pair designed to result in amplification of the selected RD9 target of SEQ ID NO: 8 About.

  The present invention thus relates to a set of primers comprising at least two pairs of primers. The at least two pairs of primers can be used for specific and sensitive detection of Mycobacterium of Mycobacterium tuberculosis (MtbC) and / or for one Mycobacterium tuberculosis and Mycobacterium canetti to the other MtbC. It is suitable for specific and sensitive discrimination from other strains. The at least two pairs of primers have sequences that can be used in a multiplex and single amplification process while still providing specific and sensitive detection and discrimination. The at least two pairs of primers are an IS6110 primer pair consisting of an IS6110 forward primer and an IS6110 reverse primer, and an RD9 primer pair consisting of an RD9 forward primer and an RD9 reverse primer. The RD9 forward primer is a 5 'terminal fragment of 10-18 nucleotides of the sequence of SEQ ID NO: 8, and the 5' terminal fragment starts at the very first 5 'nucleotide of SEQ ID NO: 8. The RD9 reverse primer is a 10 to 18 nucleotide 5 ′ terminal fragment of a sequence that is completely complementary to the SEQ ID NO: 8 over the entire length of the SEQ ID NO: 8, and the 5 ′ terminal fragment is a sequence of the complementary sequence Start at the very first 5 'nucleotide. The IS6110 forward primer is a 5 'terminal fragment of 10 to 18 nucleotides of the sequence of SEQ ID NO: 2, and the 5' terminal fragment starts at the very first 5 'nucleotide of SEQ ID NO: 2. The IS6110 reverse primer is a 10 to 18 nucleotide 5 ′ terminal fragment of a sequence that is completely complementary to the SEQ ID NO: 2 over the entire length of the SEQ ID NO: 2, and the 5 ′ terminal fragment is of the complementary sequence Start at the very first 5 'nucleotide. Said respective primers are preferably 10 to 18 nucleotides, 11 to 18 nucleotides, 12 to 18 nucleotides, 13 to 18 nucleotides, 14 to 18 nucleotides, 15 to 18 nucleotides, 16 to 18 nucleotides, 17 to 18 nucleotides, for example It is a 17 nucleotide fragment. Said RD9 forward primer may conveniently comprise the sequence of SEQ ID NO: 11 (RD389 primer). Said RD9 reverse primer may conveniently comprise the sequence of SEQ ID NO: 12 (RD561 primer). Said IS6110 forward primer may conveniently comprise the sequence of SEQ ID NO: 5 (IS368 primer). Said IS6110 reverse primer may conveniently comprise the sequence of SEQ ID NO: 6 (IS569 primer). Preferably, the IS6110 forward and reverse primers are (independently of) 10-18 nucleotides, such as 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotides. Most preferably, the IS6110 forward and reverse primers are 14-18 nucleotides (independently of each other), such as 15-18, 15-17, such as 15, 16 or 17 nucleotides. Preferably, the RD9 forward and reverse primers are 10-18 nucleotides (independently of each other), eg, 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotides. Most preferably, the RD9 forward and reverse primers are 14-18 nucleotides (independent of each other), such as 15-18, 15-17, such as 15, 16 or 17 nucleotides. The present invention also relates to the respective primer pairs and the respective primers as individual entities. The present invention therefore comprises a primer pair selected from said set of at least two primer pairs (ie RD9 primer pair or IS6110 primer) and such primer pairs (ie RD9 forward primer, RD9 reverse primer, IS6110 forward). Primer, IS6110 reverse primer).

M.M. Primer pair designed to amplify the selected IS6110 target of SEQ ID NO: 2 as a primer for amplification of a biological sample containing MtbC mycobacterium different from the tuberculosis reference strain H37Rv, and the selected of SEQ ID NO: 8 Using primer pairs designed to allow amplification of RD9 targets would result in the generation of IS6110 and / or RD9 amplicons, the sequences of which are specific MtbC present in the sample. Depending on the nucleic acid sequence of the Mycobacterium strain, it may be slightly different from the reference sequence of SEQ ID NO: 2 and / or SEQ ID NO: 8, respectively.
As an example:
-Modern M.I. If the present invention is carried out on a biological sample containing MtbC mycobacteria that is tuberculosis, both RD9 and IS6110 amplicons will be obtained.
-Ancestral M. If the present invention is carried out on a biological sample containing MtbC mycobacteria that is tuberculosis, an RD9 amplicon will be obtained, but an IS6110 amplicon will not be obtained.
-M. Not tuberculosis, and When the present invention is practiced with a biological sample containing non-canetti MtbC mycobacteria, an IS6110 amplicon is obtained, but an RD9 amplicon is not obtained.
MtbC mycobacteria contained in the biological sample are As soon as it is not a tuberculosis H37Rv, the resulting amplicon may have variant sequences compared to the IS6110 and RD9 reference sequences of SEQ ID NO: 2 and SEQ ID NO: 8. Such variant sequences will each have approximately the same size as the reference sequence of SEQ ID NO: 2 or SEQ ID NO: 8. For example, an IS6110 amplicon obtainable by practice of the present invention is believed to have a size ranging from 190 to 210 nucleotides, preferably 195 to 205 nucleotides, most preferably 199 to 203 nucleotides, and RD9 amplicons obtainable by practice of the invention have a size ranging from 160 to 185 nucleotides, preferably from 165 to 180 nucleotides, most preferably from 170 to 174 nucleotides. Such variant sequences will show a high degree of identity with said reference sequence of SEQ ID NO: 2 or SEQ ID NO: 8, respectively. For example, the IS6110 amplicon obtainable by practice of the present invention has at least 95% sequence identity with the reference sequence of SEQ ID NO: 2, and the RD9 amplicon obtainable by practice of the present invention is referenced in SEQ ID NO: 8 It will have at least 95% sequence identity with the sequence. Preferably, the minimum sequence identity will be 96%, 97%, 98% or 99%. The percent identity is calculated over the entire length of the reference sequence of each SEQ ID NO: 2 or SEQ ID NO: 8. Such variant sequences with a high degree of identity are instead hybridized under very stringent conditions to the IS6110 reference sequence of SEQ ID NO: 2 or to the RD9 reference sequence of SEQ ID NO: 8. It may be characterized by the fact that it does. “Very stringent conditions” as well as factors affecting the rate of hybridization are known to those skilled in the art and are described, for example, in Maniatis et al. 1982, Molecular Cloning, Cold Spring Harbor Laboratory. Illustrative “very stringent conditions” include, for example, hybridization of 6 × SSC or 6 × SSPE in aqueous solution at 68 ° C. or in the presence of 50% formamide at 42 ° C. and 0.1 × SSC for 2-60 minutes at room temperature. And washing conditions of the variant sequence hybridized with the reference sequence in an aqueous solution containing 0.2 SDS, followed by a temperature of about 12-20 ° C. below the calculated Tm of the detected hybrid for 2-60 minutes Incubation in a solution containing 0.1 × SSC. Other illustrative very strict conditions are shown in the examples below.

Therefore, the present invention also provides MtbC mycobacteria, and especially M. It relates to such an IS6110 and / or RD9 amplicon obtainable by the practice of the present invention in a biological sample comprising MtbC mycobacteria different from the tuberculosis reference strain H37Rv. The invention relates in particular to:
Any polynucleotide having a size ranging from 190 to 210 nucleotides, preferably from 195 to 205 nucleotides, most preferably from 199 to 203 nucleotides,
O At least 95% of the sequence is identical to the reference sequence of SEQ ID NO: 2 or the sequence completely complementary to SEQ ID NO: 2 over the entire length of SEQ ID NO: 2 (preferably 96%, 97%, 98% or 99% minimal sequence identity), and / or ○ completely complementary to SEQ ID NO: 2 over the full length of SEQ ID NO: 2 under the very stringent conditions A polynucleotide that hybridizes to the sequence;
And any polynucleotide having a size ranging from -160 to 185 nucleotides, preferably from 165 to 180 nucleotides, most preferably from 170 to 174 nucleotides,
O At least 95% of the sequence is identical to the reference sequence of SEQ ID NO: 8 or the sequence completely complementary to SEQ ID NO: 8 over the entire length of SEQ ID NO: 8 (preferably 96%, 97%, 98% or 99% minimum sequence identity), and / or o completely complementary to the reference sequence of SEQ ID NO: 8 or over the entire length of SEQ ID NO: 8 under very stringent conditions A polynucleotide that hybridizes to a sequence.

The present invention relates to a probe suitable for detection of an amplicon that can be obtained using an IS6110 primer pair and an RD9 primer pair as primers in an amplification reaction performed on a biological sample. Conveniently, the probe is:
The sequence of SEQ ID NO: 8, or a sequence that is completely complementary to SEQ ID NO: 8 over the entire length of SEQ ID NO: 8, or the sequence of SEQ ID NO: 8 or a fragment of at least 15 nucleotides of the complementary sequence, or Or a sequence that is completely complementary to SEQ ID NO: 2 over the entire length of SEQ ID NO: 2, or a sequence of at least 15 nucleotides of the sequence of SEQ ID NO: 2 or the complementary sequence.
Preferably, the fragment of at least 15 nucleotides of SEQ ID NO: 2 or SEQ ID NO: 8 is less than 30 nucleotides, most preferably 15-28 nucleotides, eg 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 nucleotides, most preferably 20-25 nucleotides, for example 23 nucleotides.
The invention relates inter alia to probes that target the following amplicons:
The sequence of SEQ ID NO: 8, or a sequence that is completely complementary to SEQ ID NO: 8 over the entire length of SEQ ID NO: 8, or the sequence of SEQ ID NO: 8 or a fragment of at least 15 nucleotides and less than 29 nucleotides of the complementary sequence, or -The sequence of SEQ ID NO: 2, or a sequence that is completely complementary to SEQ ID NO: 2 over the entire length of SEQ ID NO: 2, or a sequence of said SEQ ID NO: 2 or a fragment of at least 15 nucleotides and less than 29 nucleotides of said complementary sequence.
An advantageous probe suitable for detection of the RD9 amplicon is a fragment of a sequence that is complementary to SEQ ID NO: 8, wherein said fragment comprises the sequence of SEQ ID NO: 9 (RD441 probe). Preferably, the probe targeting RD9 is less than 30 nucleotides, most preferably 15-28 nucleotides, eg 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 nucleotides, most preferably 20-25 nucleotides, for example 23 nucleotides. An advantageous probe suitable for detection of the IS6110 amplicon is characterized as a fragment of SEQ ID NO: 2 comprising the sequence of SEQ ID NO: 3 (IS503 probe). Preferably, the probe targeting IS6110 is less than 30 nucleotides, most preferably 15-28 nucleotides, eg 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 nucleotides, most preferably 20-25 nucleotides, for example 23 nucleotides. The probes of the present invention may include one or more intercalating agents, such as one or more intercalating dyes, in their sequence. The probe of the present invention comprises at least one beacon arm, preferably two beacon arms, one at each of its ends (eg, CGTGCCG- (probe sequence) -CGCACG; or ATGCGGG- (probe sequence) -CCGCCAT). It's okay. Advantageous beaconing probes include in particular the probe of SEQ ID NO: 10 (which is the probe of SEQ ID NO: 9 in the beacon format) and the probe of SEQ ID NO: 4 (which is the probe of SEQ ID NO: 3 in the beacon format). Including. The probe of the present invention may comprise a fluorophore at its 5 ′ end and / or a quencher at its 3 ′ end, for example Tamra or Fam at the 5 ′ end and / or Dabcyl at the 3 ′ end.

  The present invention also relates to a PCR set comprising at least one primer pair of the present invention and at least one probe of the present invention. In particular, a PCR set comprising at least one RD9 primer pair of the present invention and at least one RD9 probe of the present invention, as well as at least one IS6110 primer pair of the present invention and at least one IS6110 probe of the present invention. It relates to the PCR set.

A suitable internal standard (IC) is also provided that is particularly suitable for the practice of the present invention. The present invention is suitable for use as an internal standard (IC) in the specific and sensitive detection of Mycobacterium of Mycobacterium tuberculosis (MtbC) and / or one of Mycobacterium tuberculosis and Mycobacterium It relates to oligonucleotides and polynucleotides suitable for specific and sensitive discrimination from Canetti, on the other hand from other strains of said MtbC. The IC oligonucleotides or polynucleotides of the present invention conveniently include the following:
a. Two primer binding site sequences, wherein one of the two primer binding site sequences located 5 ′ relative to the other of the two primer binding site sequences is a primer pair of the present invention (IS6110 primer pair or Is identical to one member of the RD9 primer pair) and the other of the two primer binding site sequences is completely complementary to the other member of the same primer pair of the invention; and b. A sequence that is not related to Mycobacterium and is located between the two primer binding site sequences.
Preferably, the sequence not related to the Mycobacterium is also not related to any human nucleic acid, most preferably not related to any mammalian nucleic acid.
An illustrative IC may be 92 bases:
-17 bases located in the 5 'terminal region and 17 bases located in the 3' terminal region; these are identical and, respectively, completely complementary to the IS6110 primer (in SEQ ID NO: 5 and 6, respectively) IS368 and IS569);
-The remaining 58 bases (random sequence) unrelated to mycobacterium.
For example, the kit of the invention may comprise an IC oligonucleotide or polynucleotide amplified by IS6110 primer pair, such as the IC oligonucleotide of SEQ ID NO: 13.

  The present invention further provides IC probes that are particularly suitable for the detection of IC oligonucleotides or polynucleotides of the present invention (by annealing to said control sequence). An illustrative IC probe is the probe of SEQ ID NO: 14 (SIM ATTO 590).

The present invention is also suitable for specific and sensitive detection of Mycobacterium of Mycobacterium tuberculosis group (MtbC) and / or one of said MtbC of Mycobacterium tuberculosis and Mycobacterium canetti. The invention relates to kits suitable for specific and sensitive discrimination from other strains, as well as kits for in vitro diagnosis of tuberculosis. The kit of the present invention includes the following:
-At least one set of two pairs of primers according to the invention, and / or-at least one primer pair of the invention, and / or-at least one primer of the invention, and / or-at least one set of two according to the invention. One reference template oligonucleotide, and / or at least one reference template polynucleotide according to the invention, and / or at least one probe of any one of the invention.
The kit according to the invention may in particular comprise a PCR set according to the invention (ie at least one primer pair according to the invention and at least one probe according to the invention). In the kit according to the invention, the nucleic acids (primers, probes) can be maintained separately, partially mixed or completely mixed. The nucleic acid can be provided in a dry form or dissolved in a suitable solvent, as will be appreciated by those skilled in the art. Suitable solvents include TE, PCR grade water and the like. Such reagents are known to those skilled in the art and are water, such as nuclease free water, DNAse free water, PCR grade water; salts such as magnesium, potassium; buffers such as Tris; enzymes such as Taq , Vent, Pfu (all of which are trademarks), activatable polymerases, polymerases such as reverse transcriptase; nucleotides such as deoxynucleotides, dideoxynucleotides, dNTPs, dATP, dTTP, dCTP, dGTP, dUTP Other agents such as DTT and / or RNase inhibitors; and polynucleotides such as polyT, polydT and other oligonucleotides (eg primers). The kit according to the invention may comprise a PCR control. Such controls are known in the art and include qualitative controls, positive controls, negative controls, internal standards (IC), quantitative controls, internal quantitative controls and calibration ranges. The internal standard for the PCR process may be a template that is not related to the target template in the PCR process. Such controls may also include control primers and / or control probes. For example, in the case of MtbC mycobacterial detection, as an internal standard, from among genes whose presence is excluded from human-derived samples (eg, derived from plant genes) and whose size and GC content are equal to those of the target sequence It is possible to use a selected polynucleotide. The kit of the present invention may comprise the IC oligonucleotide or polynucleotide of the present invention and / or the IC probe of the present invention. The kit according to the invention may comprise means for extracting and / or purifying nucleic acids from biological samples, for example bronchial aspirate, bronchial lavage fluid, sputum, cerebrospinal fluid, urine, blood, serum, plasma. Such means are well known to those skilled in the art. The kit of the present invention may include a lysis buffer for nucleic acid extraction, for example, a lysis buffer containing an anionic resin that binds to divalent ions. Lysis is preferably performed in the presence of a surfactant and an anionic resin that binds to divalent ions such as Chelex X-100 beads. As an advantageous feature, the anionic resin is also a chaotropic agent. Examples of suitable lysis buffers are Tris 10 mM (Sigma, ref: T-6791), EDTA 1 mM (Sigma, ref: E-1644), pH 8.3 buffer, 8% Chelex X-100 resin ( Bio-Rad, ref: 142-1253), 0.5% NP-40 (Sigma, ref: Igepal I-3021), 0.5% Tween 20 (VWR, ref: 28829296). The kit according to the invention may comprise instructions for its use. The instructions may conveniently be a leaflet, a card, etc. The instructions may also exist in two forms: detailed information gathering thorough information about the kit and its use, possibly including literature data; basic information necessary for its use Collected, for example, quick guide form or notes in the form of cards. In a preferred embodiment, the kit is a diagnostic kit, in particular an in vitro diagnostic kit, ie a tuberculosis diagnostic kit.

The present invention also provides for detecting Mycobacterium of Mycobacterium tuberculosis (MtbC) in a biological sample and / or other of said MtbC of one Mycobacterium tuberculosis and Mycobacterium canetti. It relates to a method for discrimination from strains. The method of the invention comprises the detection of two target nucleic acid sequences,
Wherein one of the two target sequences is IS6110 or a fragment thereof, and the other target sequence is RD9 or a fragment thereof,
Here, the detection of the presence of the RD9 target and the presence or absence of the IS6110 target is described in M.M. Tuberculosis or M.P. It represents that the mycobacterium belonging to Cantetti MtbC is present in the biological sample,
Here, detection of the absence of the RD9 target and the presence of the IS6110 target can It represents that mycobacteria belonging to MtbC that are not tuberculosis are present in the biological sample,
Here, detection of the absence of the RD9 target and the absence of the IS6110 target indicates that mycobacteria belonging to MtbC are not present in the biological sample.

  Detection of the presence of the RD9 target and the presence or absence of the IS6110 target is notably described in M.M. It represents that mycobacteria belonging to MtbC which is not Bovis are present in the biological sample.

  Conveniently, particularly for performing multiplex PCR of the method, the RD9 target sequence is present in the sequence of SEQ ID NO: 8, or a sequence that is completely complementary to SEQ ID NO: 8 over the entire length of SEQ ID NO: 8, And the IS6110 target sequence is present in the sequence of SEQ ID NO: 2 or a sequence that is completely complementary to SEQ ID NO: 2 over the entire length of SEQ ID NO: 2.

  Detection of the presence of the RD9 target and the absence of the IS6110 target is described in M.M. Canetti or ancestor type It represents that mycobacteria belonging to MtbC, which is one of the tuberculosis strains, are present in the biological sample. Detection of the presence of the RD9 target and the presence of the IS6110 target is described in M.M. Canetti or modern M.I. It represents that mycobacteria belonging to MtbC, which is one of the tuberculosis strains, are present in the biological sample. Detection of the absence of the RD9 target and the presence of the IS6110 target can Bovis, M.M. Africannam, M.M. Kaprae, M.M. Microchi or M.I. It represents that mycobacteria belonging to MtbC, which is pinnipedi, are present in the biological sample. According to an advantageous feature of the invention, the method can be performed by PCR. According to a very advantageous feature of the invention, the method is particularly suitable for multiplex PCR using at least one primer pair of the invention (at least one IS6110 and / or at least one RD9 primer pair) as amplification primers. Can be executed. The methods of the invention may include the use of at least one probe of the invention as a hybridization probe and / or a detection probe.

The present invention is an in vitro method for the diagnosis of tuberculosis, comprising:
The presence or absence of MtbC mycobacteria in the biological sample, and / or
M. of biological sample. Tuberculosis or M.P. The presence or absence of a Canetti strain and / or
M. of biological sample. The presence or absence of MtbC mycobacteria that are not tuberculosis
It relates to a method characterized in that it is determined by the execution of said method of MtbC detection and / or MtbC species identification in said biological sample.

  The present invention further provides a method for extracting nucleic acid from a biological sample for MtbC analysis. Lysis is preferably performed in the presence of a surfactant and an anionic resin that binds to divalent ions such as Chelex X-100 beads. As an advantageous feature, the anionic resin is also a chaotropic agent. Examples of suitable lysis buffers are Tris 10 mM (Sigma, ref: T-6791), EDTA 1 mM (Sigma, ref: E-1644), pH 8.3 buffer, 8% Chelex X-100 resin ( Bio-Rad, ref: 142-1253), 0.5% NP-40 (Sigma, ref: Igepal I-3021), 0.5% Tween 20 (VWR, ref: 28829296).

  The invention is illustrated in the following examples, which are given for illustrative purposes only.

[Example 1 (in vitro bacterial sample-specificity and sensitivity)]
1. Materials and methods:
1.1. Nucleic acid extraction:
Bacterial cells were collected from cultures of 100 different strains (54 bacterial strains other than Mycobacterium; 26 Mycobacterium strains not belonging to MtbC; 5 strains of M. bovis, including 4 M. bovis BCG; 23 6 strains other than M. tuberculosis or M. bovis but belonging to MtbC-M. pinnipedi, M. caprae, 3 strains M. africanum, M. microti-; Canetti).

  Lysis was performed in the presence of surfactant and Chelex X-100 beads (ie, an anionic resin that binds to divalent ions). As an advantageous feature, the anionic resin is a chaotropic agent.

  Composition of lysis buffer: Tris 10 mM (Sigma, ref: T-6791), EDTA 1 mM (Sigma, ref: E-1644), pH 8.3 buffer solution 8% Chelex X-100 resin (Bio-Rad , Ref: 142-1253), 0.5% NP-40 (Sigma, ref: Igepal I-3021), 0.5% Tween 20 (VWR, ref: 28829296).

Method:
-200 [mu] l biological sample-400 [mu] l lysis buffer-Add 10 [mu] l liquid internal standard (IC)-Vortex for 30 seconds-Incubate for 10 minutes at 95 [deg.] C-Centrifuge for 5 minutes at 8000 rpm-PCR with 10 [mu] l supernatant.

  The negative control was performed by replacing 200 μl of biological sample with 200 μl of phosphate buffer (pH 6.8, 25 mM).

IC is added during the extraction stage. It is in liquid form. 10 μl of IC is added to 200 μl of biological sample. IC solution comprises in 10μl of IC 9.6 10 ⁴ copies, therefore, 1.6 10 ³ copies after PCR test per extraction is obtained (1/60 dilution).

1.2. Primers and probes:
1.2.1. IS6110 system IS6110 EMBL data =
Location MTIS 6110 1360 bp DNA linear BCT 07-JUL-2002
Definition Mycobacterium tuberculosis IS6110 IS-like element Access number X17348 M29899
Version X173348.1 GI: 48695
Keyword Insertion element IS6110; transposon source Mycobacterium tuberculosis H37Rv.

Subsequence selected in IS6110 =

The underline in SEQ ID NO: 1 represents the primer and probe sequences used in the IS6110 system:
-372 to 387: IS368 forward primer;
-506 to 528: IS503 probe;
556 to 572: IS569 reverse primer complementary sequence.

IS6110 amplified target sequence in single stranded format =

IS6110 probe:
IS503 probe = CGACCACATCAACCGGGAGCCA (SEQ ID NO: 3)
Molecular Beacon (R) Arm = ATGCGGG and CCGCAT
Reporter dyes and quenchers = Tamra® and Dabcyl®
IS503 in beacon (registered trademark) format: IS503MB probe = 5′-Tamra-ATGCGGCGACCACCATCAACCGGGAGCCCACCCGCAT-Dabcyl-3 ′ (SEQ ID NO: 4).

IS6110 forward primer:
IS368 primer = 5′-CAGCACGCTATATACCC-3 ′ (SEQ ID NO: 5).

IS6110 reverse primer:
IS569 primer = 5′-GCTGATGTGCTCTCTTGA-3 ′ (SEQ ID NO: 6).

1.2.2. RD9 system:
Position BX842578 1464 bp DNA linear BCT 10-JUN-2004
Definition Mycobacterium tuberculosis H37Rv complete genome; segment 7/13.
Access number BX842578 REGION: Complementary (252713 ... 254176)
Position 250, 271 to position 254, 176: Sequence of RD9 Position 252 of BX842578, position 254, 176: Sequence of Rv2075c (sub-sequence of RD9).

Subsequence selected within RD9 (subsequence selected within Rv2075c included in RD9):

The underline in SEQ ID NO: 7 means the primer and probe sequences used in the RD9 system:
-390 to 406: RD389 forward primer;
-441 to 464: complementary sequence of the RD441 probe;
545 to 561: complementary sequence of the RD561 reverse primer.

RD9 amplified target sequence in single stranded format =

RD9 probe:
RD441 probe = GCTATACGAGGACTCCACGCTGGG (SEQ ID NO: 9)
Molecular Beacon (R) Arm = CTGTGCG and CGCCG
Reporter dyes and quenchers = Fam and Dabcyl®
RD441: RD441MB probe under beacon (registered trademark) = 5′-Fam-CGTGCGGCTTATAGAGGACTACTACCGCTGGCGCACG-Dabcyl-3 ′ (SEQ ID NO: 10).

RD9 forward primer:
RD389 = 5'-TAAGCGCCTGCGGATGTG-3 '(SEQ ID NO: 11).

RD9 reverse primer:
RD 561 = 5'-GGCGTTGAGCTGGAAAG-3 '(SEQ ID NO: 12).

1.3. Internal standard (IC):
The internal standard (IC) exists in a single stranded sequence. The IC is selected to include a sequence that is unrelated to the amplified target sequence, which is unsuitable for one of the primer pairs (IS6110 primer pair, or RD9 primer pair) used in the PCR reaction. Adjacent to the target sequence 5 'and 3' of the sequence.

  The IC comprises a sequence in its 5 ′ end region and another sequence in its 3 ′ end region, wherein the sequence contained in said 3 ′ hybridizes with one member of the IS6110 primer pair, and The sequence contained 5 'hybridizes to the complementary sequence of the other members of the same IS6110 primer pair. Preferably, the sequence contained in said 3 ′ is completely complementary to one member of the IS6110 primer pair, and the sequence contained in said 5 ′ has the same sequence as the other members of the same IS6110 primer pair . Alternatively, the RD9 primer pair can be used in place of the IS6110 primer pair: the purpose is performed in the PCR process, but between the sequence contained in the 5 ′ and the sequence contained in the 3 ′, the mycobacteria Having an IC amplified by one of a primer pair comprising a sequence unrelated to um, preferably a sequence unrelated to bacterial and mammalian nucleic acids. The IC of this example contains a sequence identical to one member of the IS6110 primer pair in its 5 'end region and a sequence completely complementary to the other member of the same IS6110 primer pair in its 3' end region.

The IC in this example is present at 92 bases:
17 bases located in the 5 ′ end region and 17 bases located in the 3 ′ end region; these are identical and are completely complementary to the S6110 primer, respectively (IS368 and IS569 of SEQ ID NOs: 5 and 6, respectively) ),
-The remaining 58 bases (random sequence) unrelated to mycobacterium.
The IC of this example has the following sequence.

The underline in SEQ ID NO: 13 indicates the following (5 ′ to 3 ′ direction):
-IS386 (IS6110 forward primer of SEQ ID NO: 5)
-IC probe-The target sequence of IS569 (ie the complementary sequence of the IS6110 reverse primer of SEQ ID NO: 6).

  IC probe = SIM ATTO 590 = GACGTGGCAGCCATGAGATGGG (SEQ ID NO: 14), a beacon arm is added to the 5 ′ and 3 ′ ends.

1.4. Illustrative experimental conditions for triplex PCR:
reagent:
Hot Start Taq Polymerase Qiagen (5 U / μl, ref203205) (including PCR buffer)
dNTP: Promega ref U151x (4 × 25 mM)
_{MgCl 2: Sigma, ref M-} 2670
PVP10: Sigma, ref: PVP-10
Glycerol: VWR, ref: 24388295.

Thermal cycler iQ1 (Bio-Rad).

Composition of 1 × PCR mix 1 × PCR mix (0.2 μM RD9 probe (SEQ ID NO: 10) in beacon format, 0.2 μM IC probe (SEQ ID NO: 14) in beacon format, 0.4 μM IS6110 probe in beacon format ( SEQ ID NO: 4), 0.5 μM of each of the four primers, 5% glycerol, 0.3% PVP10, 2 U Taq polymerase, 1 mM dNTP and a final concentration of 5 mM MgCl ₂ ). Add 10 μl of DNA to this mix.

Thermal cycle-1st cycle: 15 minutes at 95 ° C,
Repeated 50 times:
Second cycle: 30 seconds at 95 ° C. Third cycle: 30 seconds at 59 ° C. Fourth cycle: 30 seconds at 72 ° C.—Maintain at 20 ° C. until the instrument is opened.

2. result:
2.1. Primer and probe specificity when used in multiplex:
Selected IS6110 and RD9 primers and probes are MtbC specific: they do not show significant homology with nucleic acids other than MtbC mycobacteria. Advantageously, the selected IS6110 and RD9 primers and probes are specific even when used in a triplex.

These “triplex-grade” specificities can be illustrated in bacterial lysates. The presence of DNA was confirmed by effective amplification of 16S DNA. A positive IC reaction confirms the validity of the tuberculosis PCR analysis. The triplex results were as follows:

Detailed results are shown in Tables 5-1 to 5-4 below.

2.2. PCR sensitivity:
Triplex PCR was performed using previously measured M. p. This was done in duplicate with a tuberculosis H37Rv lysate. The results are as follows (Table 6).

PCR detection tolerance limits:
-For IS6110, 12 CFU / ml (equivalent to 0.12 copies per PCR)
-For RD9, 120 CFU / ml (equivalent to 1.2 copies per PCR).

  The selected IS61110 and RD9 primers are conveniently Has tuberculosis (+ very rare M. canetti) specificity and retains this specificity even in multiplex use. They also allow very sensitive detection (almost as low as 1 copy per sample).

[Example 2 (Sample collected from human-Comparative example)]:
Multiplex PCR was performed as described in Example 1 above, but with samples taken from human patients (Percy Hospital (France)):
A sample of cerebrospinal fluid (CDF),
A sample of bronchial lavage fluid,
-A sample of bronchial aspirate,
-A sample of saliva.

  Prior to analysis, the breathable sample is subjected to digestion-decontamination pretreatment. In practice, most samples subjected to mycobacterial culture are contaminated with various organisms that can rapidly grow from mycobacteria. For this reason, mycobacteria are optimally recovered from clinical material using methods that release mucin and mycobacteria trapped by cells to reduce or eliminate contaminating bacteria. The most widely used digestion-decontamination method is the N-acetyl-L-cysteine (NALC) -NaOH method. Samples were decontaminated with 1% (final concentration) sodium hydroxide-N-acetylcysteine according to standard procedures and centrifuged at 3,000 × g for 20 minutes. Concentrated (Metchock, B. G., F. S. Nolte, and R. J. Wallace, Jr. 1999. Mycobacterium, p. 399-437. In P. R. Murray, E. J. Baron, M. M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology, 7th ed., American Society for Microbi. olology, Washington, D.C.). The pellet is resuspended in phosphate buffer (0.067M, pH 6.8). The sample is now ready for analysis by microscopic examination, culture or molecular biology. Cerebrospinal fluid (CDF) samples are analyzed directly.

The comparative analysis was performed as follows:
-By bacterial culture and microscopy, or
-Perform by Gen-Probe MTD test.

  Bacterial cultures are described in Kent and Kubica (“Public health mycobacteriology: a guide for the level III laboratory,” 1985, U.S. Dpt. F Public Health and Human Health and Human Health. (“Mycobacterium”, 1991, pages 304-339; described in In A. Barlows et al. (Ed.), Manual of Clinical Microbiology, American Society for Microbiology. Microbiology. Microbiology. US). I went there. In particular, Lowenstein-Jensen (LJ) culture was performed as described by Petran et al. (1971, Health Lab. Sci. 8: 225-230). The LJ medium contains mineral salt, potato starch, glycerin, malachite green (sterile) and egg (albumin). Culture is performed in a screw cap tube. Three drops of the centrifuged pellet are resuspended in phosphate buffer and placed on top of the gelose slope. Seed 2-6 tubes (depending on sample volume) and incubate at 37 ° C. Cap the tube loosely (to allow evaporation of any excess liquid) and incubate in a tilted position. Samples are observed once a week for at least 8 weeks. Weekly observations allow detection of contamination with commensal bacteria or the presence of fast growing mycobacteria. When colonies occur, a Ziehl-Neelsen stained smear is performed to confirm the presence of acid alcohol resistant bacteria before stating that the culture is positive for mycobacteria.

Microscopic observation tests are described in Kent and Kubica (as described in "Public health mycobacteriology: a guide for the level III laboratory", 1985, US Dpt. Of Public Health and Human Health. And by fuchsin staining. The recommended interpretation for reporting smear results is shown in Table 7.

  Gen-Probe MTD testing was performed according to manufacturer's recommendations. This is performed with the Gen-Probe (R) kit commercialized as the "Amplified (TM) Mycobacterium tuberculosis direction test" kit (Gen-Probe Inc., San Diego, California 92121, USA). The Gen-Probe MTD test utilizes the amplification and detection of MtbC specific rRNA. Briefly, a biological sample is sonicated, subjected to heat denaturation treatment, followed by amplification by Gen-Probe TMA method (transcription-mediated amplification), and amplicon detection by Gen-Probe HPA method (hybridization protection assay). To serve. A positive MTD test means that the sample contains MtbC mycobacteria (due to inhibition and inadequate sensitivity issues, a negative MTD test, however, may isolate MtbC organisms in the sample. Is not excluded).

The results are as follows.

In Table 8,
^{* Indicates} M.M. Tuberculosis or very rare M. pneumoniae. Canetti,
ND = not detected,
np = do not test
MtbC other than Mt or Mc belongs to MtbC. Tuberculosis (Mt) or M.P. Mycobacterium strains that are not any of Canetti (Mc),
ba = bronchial aspirate,
bw = bronchial lavage fluid,
Spiked = artificially infected sample,
CSF = cerebrospinal fluid,
Respectively.

  The results obtained with the primers and probes of the invention performed in multiplex PCR correlate with those obtained with conventional culture methods and Gen-Probe kits. The present invention is much quicker than conventional culture methods. The present invention is also more accurate than the Gen-Probe MTD test: the MTD test only gives a MtbC positive / MtbC negative response; The conclusion of tuberculosis is that the patient has symptoms like tuberculosis and is MTD positive. It is only a hypothesis based on the fact that it is likely that the tuberculosis strain is infected. In the present invention, the strain is M. Be tuberculosis (or very unusual and easily identifiable M. canetti) and it is not any other strain of MtbC, and in particular it Ensure that it is not Bovis (ie, a strain that may be present in the sample for BCG inoculation). The present invention further provides ancestral M.P. Tuberculosis (RD9 + IS6110-reaction) and modern M.P. Allows discrimination between tuberculosis (RD9 + IS6110 + reaction).

  The contents of all cited documents or publications (both patent documents or scientific publications) are incorporated herein by reference.

Claims (43)

Specific and sensitive detection of Mycobacterium of Mycobacterium tuberculosis group (MtbC) and / or specificity from one of the other MtbC strains of Mycobacterium tuberculosis and Mycobacterium canetti Use the following for efficient and sensitive identification:
At least one IS6110 targeted amplification primer pair or hybridization probe, and at least one RD9 targeted amplification primer pair or hybridization probe. The following uses for in vitro diagnosis of tuberculosis:
At least one IS6110 targeted amplification primer pair or hybridization probe, and at least one RD9 targeted amplification primer pair or hybridization probe.   Use according to claim 1 or 2, wherein the IS6110 and RD9 are targeted in multiplex PCR. Specific and sensitive detection of Mycobacterium of Mycobacterium tuberculosis (MtbC) and / or specific Mycobacterium tuberculosis and Mycobacterium canetti from the other MtbC other strains A set of two pairs of primers suitable for highly sensitive discrimination,
Wherein the two pairs of primers have sequences that can be used in a multiplex and single amplification process while still providing specific and sensitive detection and discrimination;
Here, the two pairs of primers are IS6110 primer pair consisting of IS6110 forward primer and IS6110 reverse primer, and RD9 primer pair consisting of RD9 forward primer and RD9 reverse primer,
Wherein the RD9 forward primer is a 10 'to 18 nucleotide 5' terminal fragment of the sequence of SEQ ID NO: 8, the 5 'terminal fragment starts at the very first 5' nucleotide of SEQ ID NO: 8,
Wherein the RD9 reverse primer is a 10 to 18 nucleotide 5 ′ terminal fragment of the sequence that is completely complementary to the SEQ ID NO: 8 over the entire length of the SEQ ID NO: 8, and the 5 ′ terminal fragment is the complementary Starting from the very first 5 ′ nucleotide of the target sequence,
Wherein the IS6110 forward primer is a 10 to 18 nucleotide 5 ′ terminal fragment of the sequence of SEQ ID NO: 2, the 5 ′ terminal fragment starts at the very first 5 ′ nucleotide of SEQ ID NO: 2,
Here, the IS6110 reverse primer is a 10 to 18 nucleotide 5 ′ terminal fragment of the sequence that is completely complementary to the SEQ ID NO: 2 over the entire length of the SEQ ID NO: 2, and the 5 ′ terminal fragment is the complementary Starting from the very first 5 ′ nucleotide of the target sequence,
A set of two pairs of primers.   The set of two pairs of primers according to claim 4, wherein the RD9 forward primer comprises the sequence of SEQ ID NO: 11.   The set of two pairs of primers according to claim 4, wherein the RD9 reverse primer comprises the sequence of SEQ ID NO: 12.   The set of two pairs of primers according to claim 4, wherein the IS6110 forward primer comprises the sequence of SEQ ID NO: 5.   The set of two pairs of primers according to claim 4, wherein the IS6110 reverse primer comprises the sequence of SEQ ID NO: 5.   A primer pair selected from the set of two pairs of primer pairs according to any one of claims 4 to 8.   A primer selected from the primer pair according to claim 9.   The primer according to claim 10, which is an RD9 primer.   The primer according to claim 10, which is an IS6110 primer.   The primer according to claim 12, which is an IS6110 reverse primer. Single amplification to detect Mycobacterium of Mycobacterium tuberculosis (MtbC) and / or to distinguish one Mycobacterium tuberculosis and Mycobacterium canetti from the other MtbC other strains A set of two polynucleotides suitable for use as a reference template sequence in the design of primers that can be used in a multiplex of steps,
Wherein one of the two reference template polynucleotides is a fragment of RD9 and the other is a fragment of IS6110;
Wherein the RD9 fragment is present in the sequence of SEQ ID NO: 8 or in a sequence that is completely complementary to the full length of SEQ ID NO: 8,
And wherein the IS6110 fragment is present in the sequence of SEQ ID NO: 2 or in a sequence that is completely complementary to the full length of SEQ ID NO: 2,
A set of two polynucleotides. Single amplification to detect Mycobacterium of Mycobacterium tuberculosis (MtbC) and / or to distinguish one Mycobacterium tuberculosis and Mycobacterium canetti from the other MtbC other strains A polynucleotide suitable for use as a reference template sequence in the design of a primer that can be used in a multiplex of steps, comprising:
Wherein the reference template polynucleotide is selected from the set of claim 14.
Polynucleotide. An IS6110 amplicon obtained by using the set of two primer pairs according to any one of claims 4 to 8 as an amplification primer for a biological sample,
a. Having a size of 199 to 203 nucleotides;
b. Has a minimum of 98% sequence identity with the reference sequence of SEQ ID NO: 2 or with a sequence that is completely complementary to SEQ ID NO: 2 over the entire length of SEQ ID NO: 2,
An amplicon characterized by that. An RD9 amplicon obtained by using the set of two primer pairs according to any one of claims 4 to 8 as an amplification primer for a biological sample,
a. Having a size of 170 to 174 nucleotides;
b. Has a minimum of 98% sequence identity with the reference sequence of SEQ ID NO: 8, or with a sequence that is completely complementary to SEQ ID NO: 8 over the entire length of SEQ ID NO: 8;
An amplicon characterized by that. A probe suitable for detecting an amplicon obtained by using the set of two primer pairs according to any one of claims 4 to 8 as a primer in an amplification reaction performed on a biological sample, The probe is:
The sequence of SEQ ID NO: 8, or a sequence that is completely complementary to SEQ ID NO: 8 over the entire length of SEQ ID NO: 8, or the sequence of SEQ ID NO: 8 or a fragment of at least 15 nucleotides and less than 29 nucleotides of the complementary sequence;
Or-the sequence of SEQ ID NO: 2, or a sequence that is completely complementary to SEQ ID NO: 2 over the entire length of SEQ ID NO: 2, or a fragment of at least 15 nucleotides and less than 29 nucleotides of the sequence of SEQ ID NO: 2 or the complementary sequence A probe characterized by being.   The probe of claim 18 further comprising at least one beacon arm.   19. A probe according to claim 18 suitable for the detection of an RD9 amplicon, wherein it is a fragment of a sequence complementary to SEQ ID NO: 8, wherein said fragment comprises the sequence of SEQ ID NO: 9. Amplicon.   19. A probe according to claim 18 suitable for the detection of an IS6110 amplicon, characterized in that it is a fragment of SEQ ID NO: 2 comprising the sequence of SEQ ID NO: 3.   21. Probe according to claim 19 or 20, characterized in that it has the sequence SEQ ID NO: 10.   The probe according to claim 19 or 21, which has the sequence of SEQ ID NO: 4.   24. A probe according to any one of claims 18 to 23, further comprising a fluorophore at the 5 'end and / or a quencher at the 3' end.   25. A PCR set comprising at least one probe according to any one of claims 18 to 24 and at least one primer pair according to claim 9. Suitable for use as an internal standard (IC) in the specific and sensitive detection of Mycobacterium of Mycobacterium tuberculosis (MtbC) and / or one of Mycobacterium tuberculosis and Mycobacterium canetti, the other Oligonucleotides or polynucleotides suitable for specific and sensitive discrimination from other strains of MtbC, wherein the IC oligonucleotide or polynucleotide comprises: nucleotide:
a. 10. Two primer binding site sequences; wherein one of the two primer binding site sequences located 5 'relative to the other of the two primer binding site sequences is one member of a primer pair according to claim 9 And the other of the two primer binding site sequences is completely complementary to the other members of the same primer pair of claim 9; and b. A sequence that is not related to Mycobacterium and is located between the two primer binding site sequences.   27. An oligonucleotide suitable for use as an IC according to claim 26, characterized in that it has the sequence of SEQ ID NO: 13.   28. A probe particularly adapted for the detection of an IC oligonucleotide according to claim 27, characterized in that it has the sequence SEQ ID NO: 14. For the specific and sensitive detection of Mycobacterium of Mycobacterium tuberculosis (MtbC) and / or from one of the other MtbC strains of Mycobacterium tuberculosis and Mycobacterium canetti A kit for specific and sensitive identification comprising the following:
-At least one set of two pairs of primers according to any one of claims 4 to 8,
At least one primer pair according to claim 9,
At least one primer according to any one of claims 10 to 13,
At least one set of two reference template polynucleotides according to claim 14,
At least one reference template polynucleotide according to claim 15,
-Probe according to any one of claims 18 to 24, at least one. A kit for in vitro diagnosis of tuberculosis comprising at least one of the following elements:
-At least one set of two pairs of primers according to any one of claims 4 to 8,
At least one primer pair according to claim 9,
At least one primer according to any one of claims 10 to 13,
At least one set of two reference template polynucleotides according to claim 14,
At least one reference template polynucleotide according to claim 15,
-Probe according to any one of claims 18 to 24, at least one.   31. A kit according to claim 29 or 30, further comprising an IC oligonucleotide or polynucleotide according to claim 25 or 26 and / or an IC probe according to claim 27.   32. The kit according to any one of claims 29 to 31, further comprising a lysis buffer for nucleic acid extraction.   The kit of claim 32, wherein the lysis buffer comprises an anionic resin that binds to divalent ions. For detecting Mycobacterium of Mycobacterium tuberculosis (MtbC) in a biological sample and / or for distinguishing one Mycobacterium tuberculosis and Mycobacterium canetti from other strains of the other MtbC A method for comprising the detection of two target nucleic acid sequences:
Wherein one of the two target sequences is IS6110 or a fragment thereof, and the other target sequence is RD9 or a fragment thereof,
Here, the detection of the presence of the RD9 target and the presence or absence of the IS6110 target is described in M.M. Tuberculosis or M.P. It represents that mycobacteria belonging to MtbC which is one of canetti is present in the biological sample,
Here, detection of the absence of the RD9 target and the presence of the IS6110 target can It represents that mycobacteria belonging to MtbC that are not tuberculosis are present in the biological sample,
Here, detection of the absence of the RD9 target and the absence of the IS6110 target indicates that mycobacteria belonging to MtbC are not present in the biological sample. 35. The method of claim 34, comprising:
The RD9 target sequence is present in the sequence of SEQ ID NO: 8, or a sequence that is completely complementary to SEQ ID NO: 8 over the entire length of SEQ ID NO: 8, and the IS6110 target sequence is the sequence of SEQ ID NO: 2, or SEQ ID NO: Exists in a sequence that is completely complementary to SEQ ID NO: 2 over the entire length of 2.   36. The method of claim 34 or 35, wherein the detection of the presence of the RD9 target and the absence of the IS6110 target is a M.P. Canetti or ancestor type A method characterized by expressing that mycobacteria belonging to MtbC which is one of tuberculosis strains are present in the biological sample.   36. The method of claim 34 or 35, wherein the detection of the presence of the RD9 target and the presence of the IS6110 target is a M.P. Canetti or modern M.I. A method characterized by expressing that mycobacteria belonging to MtbC which is one of tuberculosis strains are present in the biological sample.   36. The method of claim 34 or 35, wherein the detection of the absence of the RD9 target and the presence of the IS6110 target is a M.P. Bovis, M.M. Africannam, M.M. Kaprae, M.M. Microchi or M.I. A method characterized in that mycobacteria belonging to MtbC, which is pinnipedi, are present in the biological sample.   39. Method according to any one of claims 34 to 38, characterized in that it is carried out by PCR.   40. The method of claim 39, wherein the PCR is performed in multiplex.   41. The method according to claim 39 or 40, wherein the PCR is performed using the two pairs of primers according to any one of claims 4 to 8 as amplification primers.   The method according to claim 39 or 41, wherein the PCR is carried out using at least one probe according to any one of claims 18 to 24 as a detection probe. An in vitro method for the diagnosis of tuberculosis,
The presence or absence of MtbC mycobacteria in the biological sample and / or M. Tuberculosis or M.P. The presence or absence of a Canetti strain and / or M. The presence or absence of MtbC mycobacteria that are not tuberculosis
43. A method as determined by performing the method of any one of claims 34 to 42 on the biological sample.

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