JP2007075018A - Method for detecting campylobacter coli - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for specifically and rapidly detecting Campylobacter coli with high sensitivity. <P>SOLUTION: The method for detecting the Campylobacter coli comprises detecting nucleic acid amplification by using an oligonucleotide primer specifically hybridizing with an optional base sequence designed from a genetic sequence of an aspartate kinase of the Campylobacter coli. Furthermore, a kit for detecting the Campylobacter coli is provided. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for detecting Campylobacter coli, and more particularly to a method for diagnosing Campylobacter coli infection using a highly sensitive detection method for genes.

  Campylobacter is an infectious food poisoning bacterium whose virulence factor has not been established, but its main pathology is thought to be tissue invasive. The name “Campylobacter” is the Greek word for “curved Neisseria gonorrhoeae” and is a coiled spiral mold. The size is 1.5-5 × 0.2-0.5 μm Gram-negative bacilli, with one flagella in one or both poles, and moves like a corkscrew. It cannot grow in the atmosphere and grows and survives in a microaerobic environment with an oxygen concentration of 3 to 15%.

  The first isolated Campylobacter was Campylobacter fetus, which was discovered in 1913 from a fetal abortion fetus. Originally treated as Vibrio from its form, Campylobacter jejuni was isolated from animal enteritis in 1948 and Campylobacter coli in 1948, and became independent from Vibrio in 1963 as Campylobacter.

  Campylobacter was confirmed to be pathogenic to humans in 1972 and has a short history. The selective separation medium (skilow agar) developed by Skilow in 1977 facilitated separation from stool and has been widely recognized as a causative bacterium of infectious enteritis. In Japan, Campylobacter jejuni and Campylobacter coli were designated as food poisoning causative bacteria in 1982, and in 2003, they became one of the causative bacteria of infectious gastroenteritis of class 5 infectious diseases, which were fixed points in the infectious disease law.

  Therefore, if Campylobacter food poisoning is suspected, it must be delivered to the nearest health center within 24 hours. However, Campylobacter food poisoning has a relatively long incubation period of 2 to 10 days, during which many foods are consumed or discarded, infection with even a small number of bacteria is established, and is easily killed in normal air For this reason, it is extremely difficult to identify the source of infection.

  In the case of enteritis, meat is important as a source of infection, and in particular, it is isolated from chicken at a high rate, and there are many reports. In addition, it has been confirmed that raw vegetables were cooked without washing the appliances that cooked the contaminated chicken, resulting in secondary contamination of the raw vegetables. On the other hand, it can survive for a relatively long time in river water, and therefore it can occur on a large scale due to well water contamination.

  In inflammatory bowel disease that is common in developed countries, acute abdominal pain and watery diarrhea including fresh blood, mucus, and white blood cells last for a few days, and there are many cases that cause fever as a prodrome. Rarely, severe peripheral neuritis called Guillain-Barre syndrome may occur 1 to 2 weeks after the onset of diarrhea. It usually heals spontaneously, but severe cases require coping therapy and chemotherapy.

  The first-line drug is a macrolide drug such as erythromycin. Cephem drugs are naturally resistant, and new quinolone drugs are becoming more resistant, so care must be taken.

  The standard test method for Campylobacter is the isolation culture method. Food materials are used to identify the source of infection, and stool specimens are used for diagnosis. As with many other food poisoning bacteria tests, there are two separate culture methods: a direct culture method and a culture method with enrichment culture.

  For example, in the case of a separation culture method in which enrichment culture is added, the pretreated food material is cultured in a enrichment medium under microaerobic conditions at 42 ° C. for 1 day, and then inoculated into a selective isolation medium. Incubate at 42 ° C for 2 days. Thereafter, morphological observation and motility of a colony suspected of Campylobacter are examined with a phase contrast microscope or a dark field microscope. As a result, colonies suspected of Campylobacter are purely cultured on a blood agar medium not containing a selective agent, and biochemical properties are examined for identification.

  However, since it takes about 5 days to separate and identify from enrichment, genetic diagnosis techniques such as PCR are being introduced for the purpose of speeding up (Non-patent Document 1). Thus, although the PCR method is recognized as a rapid method in Campylobacter examination, further rapidity and improved sensitivity and a simple gene detection method are required.

  The present inventors can achieve the object of the present invention by using a detection method that is faster, more sensitive, specific, and simpler than the currently known method, separation culture method and PCR method, that is, LAMP method. It was.

D. Linton, et. al. , “Journal of Clinical Microbiology” 35: 2568-2572 (1997).

  An object of the present invention is to detect Campylobacter coli for the inspection of Campylobacter coli infection. Another object of the present invention is to inspect the presence or absence of Campylobacter coli in foods contaminated by Campylobacter coli.

  As a result of intensive studies to solve the above problems, the present inventors have produced oligonucleotide primers that selectively hybridize with the Aspartate Kinase gene sequence of Campylobacter coli, and the Aspartate Kinase gene of Campylobacter coli by the LAMP method. The inventors have found that Campylobacter coli can be detected with high sensitivity by amplifying the sequence, and the present invention has been completed.

That is, the present invention has the following configuration.
(1) Oligo designed from any nucleotide sequence selected from nucleotide sequences Nos. 69 to 287 of the Aspartate Kinase gene of Campylobacter coli represented by SEQ ID NO: 1, or a complementary nucleotide sequence thereto Nucleotide primer.
(2) including at least 15 consecutive bases selected from the base sequence represented by SEQ ID NO: 2 to 9 selected from the base sequence of Aspartate Kinase gene of Campylobacter coli or the base sequence complementary thereto (1) Oligonucleotide primer.
(3) Select a base sequence region of F3c, F2c, F1c from the 3 ′ end side on the target nucleic acid of Aspartate Kinase gene of Campylobacter coli, and select a base sequence region of B3, B2, B1 from the 5 ′ end side, When the complementary base sequence is F3, F2, F1, and B3c, B2c, B1c, it is characterized by comprising at least one base sequence selected from the following (a) to (d) (1 The oligonucleotide primer according to (2).
(A) A base sequence having the F2 region of the target nucleic acid on the 3 ′ end side and the F1c region of the target nucleic acid on the 5 ′ end side.
(B) A base sequence having the F3 region of the target nucleic acid.
(C) A base sequence having the B2 region of the target nucleic acid on the 3 ′ end side and the B1c region of the target nucleic acid on the 5 ′ end side.
(D) A base sequence having a B3 region of the target nucleic acid.
(4) A base sequence specific to the Aspartate Kinase gene of Campylobacter coli can be amplified and comprises a base sequence selected from the following (a) to (b) from the 5 ′ end to the 3 ′ end: The oligonucleotide primer according to (1) to (3).
(A) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 2)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 3) -3'.
(B) 5 '-(base sequence of SEQ ID NO: 6)-(arbitrary base sequence having 0 to 50 bases)-(base sequence complementary to the base sequence of SEQ ID NO: 7) -3'.
(5) A method for detecting Campylobacter coli, comprising amplifying the target nucleic acid region of the Aspartate Kinase gene of Campylobacter coli using the oligonucleotide primers according to (1) to (4).
(6) The method for detecting Campylobacter coli according to (5), wherein the amplification reaction of the target nucleic acid region of the Aspartate Kinase gene of Campylobacter coli is the LAMP method.
(7) The presence or absence of Aspartate Kinase of Campylobacter coli is detected by detecting amplification of the target nucleic acid region of Aspartate Kinase gene of Campylobacter coli using the oligonucleotide primers described in (1) to (4). A method for detecting Campylobacter coli.
(8) In the method for detecting Campylobacter coli, a kit comprising the oligonucleotide primer according to (1) to (4).

  According to the present invention, Campylobacter coli can be detected specifically, sensitively and rapidly. Hereinafter, the present invention will be described in detail.

  A sample for identifying Campylobacter coli by nucleic acid amplification may be a clinical test material such as cultured bacterial cells, feces, sputum, urine, blood, tissue, food material, or wiped sample.

  In order to use clinical specimen materials such as stool, vomit, urine, blood, tissue, or food materials or wiped specimens as LAMP samples, phenol and chloroform are used after degrading tissue cell-derived proteins by proteolytic enzymes. Extracted nucleic acids obtained using commercially available extraction kits (eg, Qiagen DNeasy Tissue Kit, Gentra PureGene Cell and Tissue Kit, etc.) as well as general DNA extraction and purification methods such as those used Is a sample. Alternatively, it includes the case where an unpurified sample processing solution can be used as a specimen for rapid detection.

  In order to amplify such a nucleic acid derived from a living body, a new nucleic acid amplification method, which has recently been developed by Natomi et al. And does not require temperature control, which is indispensable for the PCR method, is called a LAMP (Loop-Mediated Isolation Amplification) method. This can be achieved by a loop-mediated isothermal amplification method (Patent Publication WO 00/28082 pamphlet).

  This LAMP method anneals its 3 'end to the template nucleotide and uses it as a starting point for complementary strand synthesis, and by combining primers that anneal to the loop formed at this time, isothermal complementary strand synthesis reaction It is a nucleic acid amplification method that enables

  In the LAMP method, since the 3 ′ end of the primer always anneals to the region derived from the sample, the check mechanism based on complementary binding of the base sequence functions repeatedly, resulting in high sensitivity and specificity. It enables highly efficient nucleic acid amplification reactions.

  The oligonucleotide primers used for nucleic acid amplification of the LAMP method are a total of 6 regions of the nucleotide sequence of the template nucleic acid, that is, the region of F3c, F2c, F1c from the 3 ′ end side, and the region of B3, B2, B1 from the 5 ′ end side These are at least four types of primers that recognize the base sequences of the primers, and are referred to as inner primer F and B and outer primer F and B, respectively. Further, complementary base sequences of F1c, F2c, and F3c are referred to as F1, F2, and F3, and complementary base sequences of B1, B2, and B3 are referred to as B1c, B2c, and B3c, respectively.

  The inner primer is a nucleic acid synthesis reaction product that recognizes “a specific nucleotide sequence region” on the target base sequence and has a base sequence that gives a synthesis origin at the 3 ′ end, and at the same time, uses this primer as the origin An oligonucleotide having a base sequence complementary to an arbitrary region at the 5 ′ end. Here, a primer including “a base sequence selected from F2” and “a base sequence selected from F1c” is an inner primer F (hereinafter referred to as IPF), and “a base sequence selected from B2” and “B1c are selected. A primer including “a base sequence” is referred to as an inner primer B (hereinafter referred to as IPB). The inner primer is between “a base sequence selected from F2” and “a base sequence selected from F1c” or “a base sequence selected from B2” and “a base sequence selected from B1c”. You may have arbitrary base sequences of 0-50 bases.

  On the other hand, an outer primer is an oligonucleotide having a base sequence that recognizes “a specific nucleotide sequence region existing on the 3 ′ end side of a“ specific nucleotide sequence region ”” on the target base sequence and provides a starting point for synthesis. is there. Here, a primer including “a base sequence selected from F3” is referred to as an outer primer F (hereinafter referred to as OPF), and a primer including “a base sequence selected from B3” is referred to as an outer primer B (hereinafter referred to as OPB).

  Here, F in each primer is a primer display that complementarily binds to the sense strand of the target base sequence and provides a starting point for synthesis, while B is complementary to the antisense strand of the target base sequence. The primer display provides a starting point for synthesis. Here, the length of the oligonucleotide used as a primer is 10 bases or more, preferably 15 bases or more, and may be either chemically synthesized or natural. Each primer may be a single oligonucleotide, It may be a mixture of oligonucleotides.

  In the nucleic acid amplification by the LAMP method, in addition to the inner primer and the outer primer, another primer, that is, a loop primer can be used. The loop primer (Loop Primer) is a primer having a base sequence complementary to the base sequence of the single-stranded part of the loop structure on the 5 'end side of the dumbbell structure. When this primer is used, the starting point of nucleic acid synthesis is increased, and the reaction time can be shortened and the detection sensitivity can be increased (Patent Document WO 02/24902 pamphlet).

  The base sequence of the loop primer may be selected from the base sequence of the target gene or its complementary strand as long as it is complementary to the base sequence of the single-stranded portion of the loop structure on the 5 ′ end side of the dumbbell structure described above, Other base sequences may be used. Further, one or two kinds of loop primers may be used.

  From the nucleotide sequence derived from Campylobacter coli, the present inventors obtained the nucleotide sequence of the LAMP method primer that can rapidly amplify the specific nucleotide sequence of Campylobacter coli from the nucleotide sequence of Aspartate Kinase gene represented by SEQ ID NO: 1. As a result of earnest research on the combination, the following primer set was selected.

IPF: 5'-CCGCCATTGCGGATATTTTCAAGTAGAGCCTTGCATGAGTGC-3 '(SEQ ID NO: 10)
OPF: 5'-TATGGCGAGCTTGCGGTA-3 '(SEQ ID NO: 4)
IPB: 5'-TGGATGGAGTCCCGTCGTTTAGAATAAAACAAAGGTTCGCCCCT-3 '(SEQ ID NO: 11)
OPB: 5'-AGCCCTTTGTTCATCACACA-3 '(SEQ ID NO: 12)
LPF: 5'-AAAATCGCTCATTTATTTATCAAGTCC-3 '(SEQ ID NO: 13)
LPB: 5'-ATGGCCGCAAGATTAAGATACC-3 '(SEQ ID NO: 9)

  The enzyme used in nucleic acid synthesis is not particularly limited as long as it is a template-dependent nucleic acid synthase having strand displacement activity. Examples of such enzymes include Bst DNA polymerase (large fragment), Bca (exo-) DNA polymerase, Klenow fragment of E. coli DNA polymerase I, and preferably Bst DNA polymerase (large fragment).

  Known techniques can be applied to the detection of nucleic acid amplification products by the LAMP method. For example, a labeled oligonucleotide that specifically recognizes the amplified base sequence, a fluorescent intercalator method (Japanese Patent Laid-Open No. 2001-242169), or the reaction solution after completion of the reaction is directly subjected to agarose gel electrophoresis. Can be easily detected. In agarose gel electrophoresis, a large number of bands with different base lengths are detected in a ladder form from the LAMP amplification product.

  Further, in the LAMP method, a large amount of substrate is consumed by nucleic acid synthesis, and pyrophosphate as a by-product reacts with the coexisting magnesium to become magnesium pyrophosphate, and the reaction solution becomes clouded to the extent that can be confirmed with the naked eye. Therefore, it is also possible to confirm the increase in turbidity during the reaction and the turbidity after completion of the reaction using a measuring instrument such as a spectrophotometer (Patent Document WO 01/83817 pamphlet). ).

  Various reagents necessary for detecting nucleic acid amplification using the primer of the present invention can be packaged in advance to form a kit. Specifically, various oligonucleotides necessary as a primer or loop primer of the present invention, four types of dNTPs serving as a substrate for nucleic acid synthesis, a template-dependent nucleic acid synthase having a strand displacement activity, and conditions suitable for enzyme reaction Provided buffers and salts, protective agents for stabilizing enzymes and templates, and reagents necessary for detection of reaction products as necessary are provided as kits.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

Example 1. Confirmation of detection sensitivity The detection sensitivity of the LAMP method and the PCR method were compared.

1. Preparation of Sample and Reagent (1) Preparation of Sample Campylobacter coli 5889 strain was cultured under microaerobic conditions using a blood agar medium. The grown colonies were collected with a sterilized cotton swab, suspended in physiological saline, prepared in McFarland 1 from the absorbance at 620 nm to prepare a 3 × 10 ⁸ cfu / mL bacterial suspension, and then TE A bacterial suspension of 1.2 × 10 ⁷ cfu / mL was prepared by diluting 25 times with a buffer (Nippon Gene). The suspension was diluted stepwise with TE buffer 10 times, and heat-treated at 95 ° C. for 5 minutes was used as a sample containing template DNA.

(2) Primers and methods used in the PCR method The PCR method uses the Aspartate Kinase gene as a target. A PCR method developed by Linton et al., That is, a method in which a reaction was carried out using an upstream primer (SEQ ID NO: 14) and the same downstream primer (SEQ ID NO: 15) was used.

(3) Primers used in the LAMP method As primers, IPF (SEQ ID NO: 10), OPF (SEQ ID NO: 4), IPB (SEQ ID NO: 11), OPB (SEQ ID NO: 12), LPF (SEQ ID NO: 13), LPB (SEQ ID NO: 9) ) Was used.

2. Reaction by nucleic acid amplification method (1) Reaction by PCR method PCR reaction was performed by the method described in Non-Patent Document 1.
<Reaction solution composition and reaction conditions for PCR>
Each reagent was prepared as follows per reaction.
・ 1 mol / L Tris-HCl pH 8.3 1.0 μL
・ 1mol / L KCl 2.5μL
・ 25 mmol / L MgCl ₂ 5.0 μL
・ 2.5 mM dNTPs mixture 4.0 μL
・ DDW (sterilized ultrapure water) 32.05μL
100 pmol / μL PCR-CC18F (SEQ ID NO: 14) 0.14 μL
100 pmol / μL PCR-CC519R (SEQ ID NO: 15) 0.14 μL
・ 5U / μL TaKaRa Taq Polymerase 0.25μL

  To the reaction solution, 5.0 μL of the sample at each dilution stage was added to carry out each PCR reaction with a final reaction volume of 50.0 μL. The temperature cycle conditions for the PCR reaction were heat denaturation at 94 ° C. for 1 minute, annealing at 60 ° C. for 1 minute, and polymerase extension reaction at 72 ° C. for 1 minute for a total of 25 cycles to complete the reaction. The time required was about 2 hours.

(2) Reaction by LAMP method For amplification by LAMP method, each reagent concentration in 25 μL of the final reaction solution was prepared as follows. In addition, the synthesis | combination of the primer requested the operon biotechnology company, and used what was purified by HPLC (high performance liquid chromatography).

Reaction solution composition 20 mM Tris-HCl pH 8.8
10 mM KCl
8 mM MgSO ₄
1.4 mM dNTPs
10 mM (NH ₄ ) ₂ SO ₄
0.8M Betaine (SIGMA)
0.1% Tween20
1.6μM IPF
1.6μM IPB
0.4μM OPF
0.4μM OPB
0.8μM LPF
0.8μM LPB
8U Bst DNA polymerase (NEB)

  For the LAMP reaction, add 5 μL of each concentration of sample solution to 20 μL of the above reagent to make 25 μL of the final reaction solution in a 0.2 mL dedicated tube at 65 ° C. for 60 minutes, real-time turbidity measurement device LA-200 (Eiken Chemical) Was used to detect the reaction in real time. As shown in FIG. 1, it was possible to detect Campylobacter coli diluted to 6 cfu / test within 60 minutes. PCR performed as a comparison was detectable up to a dilution of 600 cfu / test (FIG. 2). The detection sensitivity of the LAMP method was superior to that of the PCR method. In addition, the LAMP method was superior in terms of rapidity as compared with the PCR method requiring an amplification reaction of about 2 hours and further electrophoresis analysis.

Example 2 Primer evaluation (specificity test)
Campylobacter coli 2 strain, Campylobacter jejuni 16 strain and Campylobacter fetus 1 strain shown in Table 1 were cultured under microaerobic conditions using a blood agar medium. The grown colonies of each bacterial cell were collected with a sterilized cotton swab, suspended in physiological saline, and adjusted to a concentration of each bacterial cell of 1.2 × 10 ⁶ cfu / mL by absorbance at 620 nm. A suspension obtained by heating at 95 ° C. for 5 minutes was used as a specimen containing template DNA.

In addition, 37 non-Campylobacter spp. 37 strains shown in Table 2 were cultured under aerobic conditions using a blood agar medium. However, Helicobacter pylori was cultured under microaerobic conditions, and Legionella was cultured on a BCYEα agar medium. Colonies of the grown bacterial cells were collected with a sterilized cotton swab, suspended in physiological saline, and the bacterial cell concentration was measured by absorbance at 620 nm. DNA extraction was carried out from a certain amount of bacterial cells (2 × 10 ⁹ cells) using DNeasy Tissue Kit (manufactured by Qiagen) according to the operation described in the kit specifications. The extracted DNA concentration was measured by absorbance at 260 nm, and DNAs derived from all bacterial species were respectively prepared at 2 mg / mL and used as specimens.

  The nucleic acid amplification method was performed according to Example 1.

  As shown in Table 1, all of the Campylobacter coli 2 strains were positive, and the Campylobacter jejuni 16 strain and the Campylobacter fetus 1 strain were negative. In addition, the 37 non-Campylobacter species 37 strains shown in Table 2 were all negative. Similar results were obtained with the PCR method, and the specificity was the same for both methods.

  According to the present invention, an oligonucleotide primer that selectively hybridizes to the Aspartate Kinase gene sequence of Campylobacter coli is prepared, and the base sequence specific to Campylobacter coli is amplified by the LAMP method, whereby Campylobacter coli is specific. It can be detected with high sensitivity and speed.

The detection sensitivity of the LAMP method by the real-time turbidity method is shown. The horizontal axis represents time (minutes), and the vertical axis represents absorbance (turbidity) at 650 nm.

The electrophoretic diagram which shows the detection sensitivity by PCR method.

Explanation of symbols

Lane 1: Marker (TaKaRa 100bp Ladder Marker)
Lane 2: 6 × 10 ⁴ cfu / test
Lane 3: 6 × 10 ³ cfu / test
Lane 4: 6 × 10 ² cfu / test
Lane 5: 6 × 10 ¹ cfu / test
Lane 6: 6 × 10 ⁰ cfu / test
Lane 7: Negative Control

Claims (8)

  An oligonucleotide primer designed from an arbitrary base sequence selected from the base sequences of Nos. 69 to 287 of the Aspartate Kinase gene of Campylobacter coli represented by SEQ ID NO: 1, or a base sequence complementary thereto.   The oligonucleotide according to claim 1, comprising at least 15 consecutive nucleotides selected from the nucleotide sequence represented by SEQ ID NOs: 2 to 9 selected from the nucleotide sequence of the Aspartate Kinase gene of Campylobacter coli or a complementary nucleotide sequence thereto. Primer. Select the base sequence region of F3c, F2c, F1c from the 3 ′ end side on the target nucleic acid of the Aspartate Kinase gene of Campylobacter coli, and select the base sequence region of B3, B2, B1 from the 5 ′ end side, and each complementary base 3. When the sequence is F3, F2, F1, and B3c, B2c, B1c, it comprises at least one kind of base sequence selected from the following (a) to (d): The described oligonucleotide primer.
(A) A base sequence having the F2 region of the target nucleic acid on the 3 ′ end side and the F1c region of the target nucleic acid on the 5 ′ end side.
(B) A base sequence having the F3 region of the target nucleic acid.
(C) A base sequence having the B2 region of the target nucleic acid on the 3 ′ end side and the B1c region of the target nucleic acid on the 5 ′ end side.
(D) A base sequence having a B3 region of the target nucleic acid. The base sequence specific to the Aspartate Kinase gene of Campylobacter coli can be amplified and consists of a base sequence selected from the following (a) to (b) from the 5 ′ end to the 3 ′ end: The oligonucleotide primer of -3.
(A) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 2)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 3) -3'.
(B) 5 '-(base sequence of SEQ ID NO: 6)-(arbitrary base sequence having 0 to 50 bases)-(base sequence complementary to the base sequence of SEQ ID NO: 7) -3'.   A method for detecting Campylobacter coli, comprising performing an amplification reaction of a target nucleic acid region of an Aspartate Kinase gene of Campylobacter coli using the oligonucleotide primer according to claim 1.   6. The method for detecting Campylobacter coli according to claim 5, wherein the amplification reaction of the target nucleic acid region of the Aspartate Kinase gene of Campylobacter coli is the LAMP method.   Detection of Campylobacter coli characterized in that the presence or absence of Aspartate Kinase of Campylobacter coli is detected by detecting amplification of the target nucleic acid region of Aspartate Kinase gene of Campylobacter coli using the oligonucleotide primer according to claim 1. Method. In the method for detecting Campylobacter coli, the kit comprises the oligonucleotide primer according to claim 1.