WO2009122422A1

WO2009122422A1 - Probes and primers for detection of hepatitis b virus and a method thereof

Info

Publication number: WO2009122422A1
Application number: PCT/IN2008/000384
Authority: WO
Inventors: Jagannath Manjula; Vasamsetty Venkata Ramachandra Rao; Chandra Madhavi; Chandrasekhar Bhaskaran Nair; Pillarisetty Venkata Subbarao
Original assignee: Bigtec Private Limited
Priority date: 2008-03-31
Filing date: 2008-06-19
Publication date: 2009-10-08

Abstract

The present invention gives a detailed description of methods for determining the presence and/ or amount of HBV (Hepatitis B virus) nucleic acids in blood/serum/plasma samples by employing dual labeled probes. The designed dual labeled probe can be used for qualitative and quantitative detection of HBV nucleic acids in an infected sample by employing Real time PCR.

Description

PROBES AND PRIMERS FOR DETECTION OF HEPATITIS B VIRUS AND A

METHOD THEREOF

FIELD OF THE INVENTION

The present invention gives a detailed description of methods for determining the presence and/ or amount of HBV (Hepatitis B virus) nucleic acids in blood/serum/plasma sample by employing dual labeled probes. The designed dual labeled probe can be used for qualitative and quantitative detection of HBV nucleic acids in an infected sample by employing Real time PCR.

BACKGROUND AND PRIOR ART OF THE INVENTION

The related art of interest describes various methods for detecting Hepatitis B Virus, but none discloses the present invention. There is a need for an effective probes and primers for detecting Hepatitis B Virus. The related art will be discussed in the order of perceived relevance to the present invention.

Recent studies have shown that throughout the world number of people infected with hepatitis B virus (HBV) amounts to about 300 million. The HBV causes acute and chronic hepatitis (type B hepatitis), and in severe cases cirrhosis and hepatic carcinoma. Seven genotypes of HBV designated as genotype A to G have been reported by Norder, H., et al, (1993) and Stuyver, L. et. al. (2000).

The currently used methods for the diagnosis of HBV is based on ELISA(Enzyme Linked immune sorbent assay) which are based on the presence of serum markers such as, HbeAg, HbsAg, or anti-HBc IgM, anti-HBe, anti-HBs, or anti-HBc IgGs.

PCR based assays for the direct detection of HBV nucleic acids in the blood/serum or plasma of an infected subject may provide an advantage in determining the exact viral load of an infected patient which will be useful for a physician to know the exact stage of infection. This may further help the physician to provide a proper therapy for the patient. Quantifying the exact viral load can also help in monitoring the progress of anti-viral therapy.

International Patent Application No: PCT/US93/09233, European Patent Application Nos. 0 593 789 Al and 0 860 505 Al have proposed methods for the detection of HBV nucleic acids based on polymerase chain reaction (PCR). Further the U.S. Patent No. 5,736, 316 describes a sandwich hybridization assay for the detection of HBV nucleic acids. Since ELISA based methods cannot give an insight into the exact viral load there is a need to look for a method which can give the quantitative measure of the viral load. None of the above inventions and patents, taken either singly or in combination, is seen to describe the instant invention as claimed. Thus, the probes and the respective primers provided in instant invention will therefore help in detection of Hepatitis B Virus gene.

OBJECTIVES QF THE PRESENT INVENTION

The principle objective of the present invention is to provide a method for the detection of Hepatitis B Virus.

Another objective of the present invention is to develop probes and necessary primers for detecting Hepatitis B Virus.

Yet another objective of the present invention is to provide a PCR reaction mixture for detection of Hepatitis B Virus genes.

Still another objective of the present invention is to provide a kit for detection of

Hepatitis B Virus.

Still another objective of the present invention is to determine the presence and/ or amount of Hepatitis B Virus nucleic acids in blood/serum/plasma samples using dual labeled probe which is labeled with a fluorescent dye and a quencher. The mode of detection is by measuring the increase in fluorescence during PCR.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Fig; 1 Histogram showing specificity of Probe 1 [SEQ ID No. 1] in comparison with standard

Fig: 2 Histogram showing sensitivity of probe 1 [SEQ ID No. 1] in comparison with standard

Fig: 3 Flow chart showing details of sample panel

STATEMENT OF THE INVENTION

Accordingly, the present invention provides probe of SEQ ID No. 1, primers of SEQ ID Nos. 2 and 3, a PCR reaction mixture for detection of Hepatitis B Virus, said mixture comprising nucleic acid amplification reagents, dual labeled probe set forth in SEQ ID No. 1, primers of SEQ ID Nos. 2 and 3, and sample, a method of detecting Hepatitis B Virus, said method comprising steps of: forming a reaction mixture comprising nucleic acid amplification reagents, probe of SEQ ID No. 1 with corresponding primers of SEQ ID Nos. 2 and 3, a test sample; and subjecting the reaction mixture to amplification by PCR to obtain copies of the target sequence for detecting the Hepatitis B Virus, and a kit for detection of Hepatitis B Virus, said kit comprising dual labeled probe of SEQ ID No. 1 primers of SEQ ID Nos.2 and 3 and amplification reagents.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention is in relation to probe of SEQ ID No. 1.

In another embodiment of the present invention said probe is a dual labeled probe.

In yet another embodiment of the present invention said probe is for detection of Hepatitis B Virus.

In still another embodiment of the present invention said probe is conjugated to detectable labels having fluorophore at 5' end and a quencher at 3' end.

In still another embodiment of the present invention said fluorophore is selected from a group comprising fluorescein and fluorescein derivatives FAM, VIC₅ JOE, 5-(2'- aminoethyl)aminonaphthalene-l-sulphonic acid, coumarin and coumarin derivatives, lucifer yellow, texas red, tetramethylrhodamine, 6-Carboxy Fluorescein, tetrachloro-6- carboxyfluoroscein, 5-carboxyrhodamine and cyanine dyes.

In still another embodiment of the present invention said quencher is selected from a group comprising Tetra Methyl Rhodamine, 4'-(4-dimethylaminophenylazo)benzoic acid, 4-dimethylaminophenylazophenyl-4'-maleimide, tetramethylrhodamine, carboxytetramethylrhodamine and BHQ dyes.

In still another embodiment of the present invention, said detection is both qualitative and quantitative in nature.

In still another embodiment of the present invention, said probe does not show any cross reactivity with HIV and HCV containing DNA samples.

In still another embodiment of the present invention, the SEQ ID No. 1 is designated for X-gene region of Hepatitis B virus. Primers of SEQ ID Nos. 2 and 3.

In still another embodiment of the present invention, said primers are sense and anti sense primers respectively.

In still another embodiment of the present invention, said primers are for dual labeled probe of SEQ ID No. 1 for detection of Hepatitis B Virus.

The present invention is in relation to a PCR reaction mixture for detection of Hepatitis B Virus, said mixture comprising nucleic acid amplification reagents, dual labeled probe set forth in SEQ ID No. 1, primers of SEQ ID Nos. 2 and 3, and sample.

In another embodiment of the present invention, said sample is selected from a group comprising blood, serum and plasma.

The present invention is in relation to a method of detecting Hepatitis B Virus, said method comprising steps of: forming a reaction mixture comprising nucleic acid amplification reagents, probe of SEQ ID No. 1 with corresponding primers of SEQ ID Nos. 2 and 3, a test sample; and subjecting the reaction mixture to amplification by PCR to obtain copies of the target sequence for detecting the Hepatitis B Virus.

In another embodiment of the present invention, said probe of SEQ ID No. 1 is dual labeled probe.

In yet another embodiment of the present invention, said primers of SEQ ID Nos. 2 and 3 are sense and anti sense primers respectively.

In still another embodiment of the present invention said PCR is real time PCR.

In still another embodiment of the present invention said sample is selected from a group comprising blood, serum and plasma.

In still another embodiment of the present invention said amplification reagents include magnesium chloride, Taq polymerase and buffer for amplification.

The present invention is in relation to a kit for detection of Hepatitis B Virus, said kit comprising dual labeled probe of SEQ ID No. 1 primers of SEQ ID Nos.2 and 3 and amplification reagents. HBV Sequence retrieval and analysis:

HBV data base was thoroughly searched for identifying most conserved regions specific to HBV genome.

• Overall 980 full length genome sequences of HBV from different origins were collected from online databases (www.ncbi.com > all data bases > Core nucleotide)

• Those sequences that are of non-human origin were excluded from the study i.e. from lab animal models.

• Total 980 full length sequences were further analyzed for retrieving the genotype sets and serotype sets. Details are as follows,

Table: 1

FASTA format was obtained for all the sequences in order to their origin database format and sequence positions.

Each and every set were analyzed for Phylogenetic distances between them i.e. intra genotype variations and also for their cluster differentiation.

After the multiple sequence analysis (Kalign software) those sequences that are found to be having greater distances were excluded from the further analysis steps.

Out of these 83, 83, 10O₅ 69, 25, 37, 4 and 9 belongs to Genotype A....H, which were further analyzed to determine the consensus sequence and inter genotype variations and positions of variations. • The most promising regions with conserved regions were selected for designing the primer and probe sets. o Conserved regions with in Surface, Core, Pre-Core, X and polymerase genes were obtained and analyzed for designing oligo sets.

• Out of these regions X gene region was selected for primer and probe designing.

• Different possible primer sets were designed from the X region as per the primer design rules and conditions i.e. Homo-dimer, hetero-dimer, Stem-Loop structure, 3' stability, GC content, melting temperature.

• Finally a set of primer and probe was designed for the X gene. These primers and probe were further analyzed by using efficient software programs.

The present invention provides methods and composition for determining the presence and/ or amount of HBV (Hepatitis B virus) nucleic acids in blood/serum/plasma samples. The designed dual labeled probe can be used for qualitative and quantitative detection of HBV nucleic acids in an infected sample by employing Real time PCR. The dual labeled probe has a size ranging from 20-22 nucleotides. The designed probe has a fluorophore at the 5'end and a quencher at the 3' end. The fluorophore at the 5' end is FAM (6-Carboxy Fluorescein) and the quencher at the 3' end is TAMRA (Tetra Methyl Rhodamine). The current invention is used for the detection of hepatitis B virus present in blood/serum/plasma samples. The method used for detection is by monitoring the increase in fluorescence during the PCR.

According to the present invention the "dual labeled probe" refers to a short sequence of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), modified RNA or DNA, or analogues of RNA or DNA. The dual labeled probe can specifically hybridise to nucleic acids from all hepatitis B virus (HBV) genotypes. The dual labeled probe according to the present invention is generally between about 20-22 nucleotides in length. The dual labeled probe mentioned here specifically hybridise to the HBV nucleic acid sequence without exhibiting non-specific hybridisation to non-HBV nucleic acids. The dual labeled probe described here can be used in the amplification and/or detection of nucleic acids from each of the reported genotypes of HBV designated A to G.

The dual labeled probe employed here follows the principles of Taqman chemistry. TaqMan probes also called Double-Dye oligonucleotide or dual labeled probes, are the most widely used type of probes. They were developed by Roche [Basel, Switzerland] and ABI [Foster City, USA] from an assay that originally used a radio labeled probe [Holland et al. 1991] and consist of a single -stranded probe sequence that is complementary to one of the strands of the amplicon. A fluorophore is attached to one end of the probe and a quencher to the other end. The fluorophore when excited passes its energy, via FRET (Fluorescence resonance energy transfer), to the quencher. Traditionally the FRET pair has been FAM (6-Carboxy Fluorescein) as the fluorophore and TAMRA (Tetra Methyl Rodamine) as the quencher. TAMRA is excited by the energy from the FAM and fluoresces. As this fluorescence is detected at different wavelength to FAM the background level of FAM is low. The probe binds to the amplicon during each annealing step of the PCR. When the Taq polymerase extends from the primer bound to the amplicon it displaces the 5' end of the probe, which is then degraded by the 5'-3' exonuclease activity of the Taq polymerase. Cleavage continues until the remaining probe melts off the amplicon. This process releases the fluorophore and quencher into solution, specially separating them compared to when they were held together by the probe. This leads to an irreversible increase in fluorescence from the FAM and a decrease in the TAMRA. TaqMan probes can be used for variety of applications like a. Detection of specific gene marker and its quantification b. Detection of mutant genes c. Detection of Biomarkers

AU these applications are broadly a part of diagnosis at molecular level.

The dual labeled probe according to the present invention, therefore, is further provided in combination with two primers that can be used to specifically amplify and detect HBV nucleic acid sequences in a test sample by real time PCR.

The PROBE 1 [SEQ ID No.l] and the corresponding primers [SEQ ID No. 2 and SEQ ID No. 3] have a sequence as described in Table 2. Table: 2

The dual labeled probe according to the present invention can find use in research or clinical applications for the detection and/or quantitation of HB V. nucleic acids which may further help to diagnose HBV infection in an infected individual or to monitor the HBV viral load in an infected individual.

To gain a better understanding of the above invention, an elaborate study was done on an established sample panel. A sample panel consisting of 50 HBV positives with (HBsAg +ve, HBeAg -ve, HBV DNA -ve), (HBsAg +ve, HBeAg -ve, HBV DNA +ve), (HBsAg +ve, HBeAg +ve, HBV DNA +ve), 50 HBV negative samples, 5 HCV and 5 HIV positive samples were established by ELISA. The details of the sample panel are as follows :-

The technology of the instant Application is further elaborated with the help of following examples. However, the examples should not be construed to limit the scope of the invention. The following Examples represent preferred embodiments of the present invention.

Example: 1 Sample Panel

Total no of samples selected for the present panel is 110.

POSITIVE PANEL :( n=50)

HBsAg(+) HBeAg (+) HBV DNA (+) - 25 samples

HBsAg(+) HBeAg (-) HBV DNA (+) - 25 samples

All this 50 samples were negative for HCV and HIV.

NEGATIVE PANEL :( n=50)

HBsAg(-), HCV(-) and HIV(-) 50 samples. To check the cross reactivity with other infectious agents, anti-HCV (n=5) and HIV (n=5) samples were included in the panel. Which were negative for HBsAg. A flow chart for sample panel is provided in Fig: 3

Table. 4; Negative Sample Panel

Example: 2

DNA was isolated from all the 110 serum samples using a Chemagen kit. Real time PCR reactions were carried out for all the 110 DNA samples using the dual labeled probe designated as PROBE 1 and its corresponding primers. The sensitivity of the dual labeled probe in picking up the infected samples was compared with a Sacace HBV DNA standard kit. Same concentrations of Real time-PCR reagents, template and primers were used in each case and also cycling conditions were kept constant for all the reactions. Possibility of cross reactivity with Hepatitis C virus (HCV) and Human immunodeficiency virus (HIV) samples was ruled out by carrying out reactions with DNA which were positive for HIV or HCV infections but negative for HBV. The conditions of the PCR were as follows: Step 1: 95° C for 10 sec Step 2: 95° C for 5 sec Step 3: 60° C for 20 sec Steps 2 & 3 were repeated for 44 cycles Example: 3

Results obtained showed that the dual labeled probe designated as PROBE 1 picked up only the HBV positive samples and did not show any false amplification for the negative samples. The designed dual labeled probe did not show any cross reactivity with HIV or HCV containing DNA samples.

PROBE 1 which was designed for the X region picked up all the 25 predetermined positives (HBeAg +ve, DNA +ve) within 35 cycles (positive sample cut off) thus showing 100% specificity. Out of these 25 positives, 5 (20%) were detected with an increase in cycle number compared to the Sacace standard kit. Maximum increase in cycle number was found to be 2.97 and minimum was 0.13.

Table. 5: SACACE vs PROBE 1 HBeAg Positive

Note:

Total no of samples showing Cycle Threshold (CT) increase with standard = 5. Negative sample number = 0

Percentage of samples with increased CT = 20%.

Max CT difference = 2.97cycles.

Min CT difference = 0.13 cycles.

In case of HBeAg -ve, DNA +ve samples, 24 out of 25 predetermined positives were detected within 35 cycles (96% specificity). Out of these 24 positives, 10 (41%) were detected with an increase in cycle number compared to the Sacace standard kit. Maximum increase in cycle number was found to be 5.95 and minimum was 0.05.

Table: 6 SACACE vs. PROBE 1 HBeAg Negative

Note:

Total no of positive samples showing Cycle Threshold (CT) increase with standard

10.

Negative sample number = 1

Percentage of samples with increased CT = 41%. Max CT difference = 5.95 cycles. Min CT difference = 0.05 cycles.

Table. 7: Standard Kit values with respect to cycles

The specificity of probe 1 [SEQ ID No. 1] is provided in Fig: 1, based on the number of samples detected by PROBE 1 in comparison to the Standard Sacace kit The sensitivity of probe 1 [SEQ ID No.l] is provided in Fig: 2, based on early detection of samples in comparison to Standard Sacace kit.

Example: 3 Conclusion

> None of the negative samples showed false positive with the designed dual labeled probe designated as PROBE 1

> The dual labeled probe did not show Cross reactivity with respect to samples of HCV and HIV

> PROBE 1, which is the dual labeled probe designed for X gene showed good specificity and sensitivity (98%). Out of 50 positive samples 49 were found positive and the undetected one was a low copy number sample. A combination of X gene based primers and the probe showed 100% and 96% specificity with HBeAg +ve and HBeAg -ve samples, respectively.

> Based on the overall evaluation studies, PROBE 1 designed for X gene was considered to be the best for HBV detection References

1) Norder, H., et al., (1993) J. Gen. Virol., 74: 1341-1348

2) Stuyver, L. et. al. , (2000) Journal of General Virology, 81: 67-74

3) International Patent Application No. PCT/US93/09233

4) European Patent Application Nos.O 593 789 Al

5) European Patent Application Nos.O 860 505 Al

6) U. S. Patent No. 5,736, 316

7) US Patent No. 7, 015, 317

8) (WO/2003/093797) Polynucleotides for the detection and quantification of Hepatitis B virus nucleic acids

9) United States Patent7262292 Oligonucleotides and methods for detecting hepatitis B viral nucleic acids

10) European Patent EP1783211

SEQUENCE LISTING <1 IO BIGTEC PRIVATE LIMITED

<120> PROBES AND PRIMERS FOR DETECTION OF HEPATITIS B VIRUS AND A

METHOD THEREOF

<130> PCT0831/RB/MSG

<150> 00786/CHE/2008 <151> 2008-03-31

<160> 3

<170> Patentln version 3.3

<210> 1

<211> 22

<212> DNA

<213> Artificial sequence

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<223> PROBE 1

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<210> 2

<211> 20

<212> DNA

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<223> Primer 2

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<212> DNA

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<223> Primer 3

<400> 3 cattcggtgg gcgttcac 18

Claims

WE CLAIM

1) Probe of SEQ ID No. 1.

2) The probe as claimed in claim 1, wherein said probe is a dual labeled probe.

3) The probe as claimed in claim 1, wherein said probe is for detection of Hepatitis B Virus.

4) The probe as claimed in claim 1, wherein said probe is conjugated to detectable labels having fluorophbre at 5' end and a quencher at 3' end.

5) The probe as claimed in claim 4, wherein said fluorophore is selected from a group comprising fluorescein and fluorescein derivatives FAM, VIC, JOE, 5- (2'-aminoethyl)aminonaphthalene-l-sulphonic acid, coumarin and coumarin derivatives, lucifer yellow, texas red, tetramethylrhodamine, 6-Carboxy Fluorescein, tetrachloro-6-carboxyfluoroscein, 5-carboxyrhodamine and cyanine dyes.

6) The probe as claimed in claim 4, wherein said quencher is selected from a group comprising Tetra Methyl Rhodamine, 4'-(4-dimethylaminophenylazo)benzoic acid, 4-dimethylaminophenylazophenyl-4'-maleimide, tetramethylrhodamine, carboxytetramethylrhodamine and BHQ dyes.

7) The probe as claimed in claim 3, wherein said detection is both qualitative and quantitative in nature.

8) The probe as claimed in claim 1, wherein said probe does not show any cross reactivity with HIV and HCV containing DNA samples.

9) The probe as claimed in claim 1, wherein the SEQ ID No. 1 is designated for X- gene region of Hepatitis B virus.

10) Primers of SEQ ID Nos. 2 and 3.

11) The primers as claimed in claim 10, wherein said primers are sense and anti sense primers respectively.

12) The primers as claimed in claim 10, wherein said primers are for dual labeled probe of SEQ ID No. 1 for detection of Hepatitis B Virus

13) A PCR reaction mixture for detection of Hepatitis B Virus, said mixture comprising nucleic acid amplification reagents, dual labeled probe set forth in SEQ ID No. 1, primers of SEQ ID Nos. 2 and 3, and sample.

14) The PCR reaction mixture as claimed in claim 13, wherein said sample is selected from a group comprising blood, serum and plasma.

15) A method of detecting Hepatitis B Virus, said method comprising steps of: (a) forming a reaction mixture comprising nucleic acid amplification reagents, probe of SEQ ID No. 1 with corresponding primers of SEQ E) Nos. 2 and 3, a test sample; and

(b) subjecting the reaction mixture to amplification by PCR to obtain copies of the target sequence for detecting the Hepatitis B Virus.

16) The method as claimed in claim 15, wherein said probe of SEQ E) No. 1 is dual labeled probe.

17) The method as claimed in claim 15, wherein said primers of SEQ ID Nos. 2 and 3 are sense and anti sense primers respectively.

18) The method as claimed in claim 15, wherein said PCR is real time PCR.

19) The method as claimed in claim 15, wherein said sample is selected from a group comprising blood, serum and plasma.

20) The method as claimed in claim 15, wherein said amplification reagents include magnesium chloride, Taq polymerase and buffer for amplification.

21) A kit for detection of Hepatitis B Virus, said kit comprising dual labeled probe of SEQ ID No. 1 primers of SEQ E) Nos.2 and 3 and amplification reagents.