E-ISSN 2231-170X | ISSN 2231-1696
 

Original Article 


Simultaneous detection of Hepatitis B virus and Hepatitis C virus in human plasma using Taq-man chemistry

Y. Naresh Kumar, S. Rahmathulla, C. Madhavi, V. V. Ramachandra, S. Vishnupriya, Mohammed Aejaz Habeeb, M. N. Khaja.

Abstract
Designing a rapid, reliable and sensitive assay, for detection of hepatitis B virus and Hepatitis C virus variants by real-time PCR, is challenging at best. A recent approach for quantifying the viral load using the sensitive fluorescence principle, was used in this study. A total of 350 samples were collected from outpatient unit, CLRD. Complete Human HBV DNA and HCV sequences were obtained from the National Centre for Biotechnology Information (NCBI); primers and probes were designed and synthesized from core, surface and x region of Hepatitis B and UTR region of HCV. Real-time based detection was done, using standard kit and in-house generated standards and RT-PCR protocols. A standard curve was generated by using the Smart Cycler II software and serial dilution 102 to 108 of cloned viral regions, the calibration curve was linear in a range from 102 to108 cp/ml for both HBV and HCV, with R2 value of 0.999 and 0.995. Out of 100 predetermined HCV negative samples, 02 samples were found positive with in-house developed RT-PCR assay, the positivity of this sample was confirmed by sequencing the amplified product. Low cost of this assay procedure and précised sample volume will permit the assay to be implemented for routine screening of Hepatitis B and C virus mono-infection and co-infection using Real Time PCR , Nucleic acid Chip technology and Fluorescent End Point detection systems. This assay is reproducible showing limited inter and intra assay variability. We demonstrate that the results of our assay correlated well with the standard kit for HBV and HCV virus monitor.

Key words: Hepatitis B virus, Hepatitis C virus, Nucleic acid test, Real-time PCR


 
ARTICLE TOOLS
Abstract
PDF Fulltext
How to cite this articleHow to cite this article
Citation Tools
Related Records
 Articles by Y. Naresh Kumar
Articles by S. Rahmathulla
Articles by C. Madhavi
Articles by V. V. Ramachandra
Articles by S. Vishnupriya
Articles by Mohammed Aejaz Habeeb
Articles by M. N. Khaja
on Google
on Google Scholar

REFERENCES
1. Lauer GM, Walker BD. Hepatitis C virus infection. New England Journal of Medicine 2001; 345(1):41-52. [DOI via Crossref]    [Pubmed]   
2. Shad JA, McHutchison JG. Current and future therapies of hepatitis C. Clinical Liver Disease 2001; 5(2):335-359. [DOI via Crossref]   
3. Kaneko S, Feinstone SM, Miller RH. Rapid and sensitive method for detection of serum hepatitis B virus DNA using the polymerase chain reaction technique. Journal of Clinical Microbiology 1989; 27(9):1930-1933. [Pubmed]    [PMC Free Fulltext]   
4. Kessler H, Preininger HS, Stelzl E, Daghofer E, Santner BI, Marth E, et al. Identification of different states of hepatitis B virus infection with a quantitative PCR assay. Clinical Diagnostic Laboratory Immunology 2000; 7(2):298-300. [DOI via Crossref]   
5. Noborg U, Gusdal A, Pisa EK, Hedrum A, Lindh M. Auto-mated quantitative analysis of hepatitis B virus DNA by using the Cobas Amplicor HBV Monitor Test. Journal of Clinical Microbiology 1999, 37(9):2793-2797. [Pubmed]    [PMC Free Fulltext]   
6. Roth WK, Weber M, Seiftied E. Feasibility and efficacy of routine PCR screening of blood donations for hepatitis C virus, hepatitis B virus, and HIV-1 in a blood bank setting. Lancet 1999; 353(9150):359-363. [DOI via Crossref]   
7. Martell M, Gomez J, Esteban JI, Sauleda S, Quer J, Cabot B, et al. High-throughput real-time reverse transcription- PCR quantitation of hepatitis C virus RNA. Journal of Clinical Microbiology 1999, 37(2):327-332. [Pubmed]    [PMC Free Fulltext]   
8. Rekha T, Chandrakala M, Madhavi C, Mohammed NK, Mohammed AH, Chitoor MH. Prevalent HBV genotypes and subtypes in a South Indian population. Journal of Clinical Virology 2006; 37: 58-64. [DOI via Crossref]    [Pubmed]   
9. Mayumi S, Fuminaka S, Naoky T, et al. Genotype and phylogenetic characterization of hepatitis B virus among multi-ethnic cohort in Hawaii. World J Gastroenterol 2004; 10(15): 2218-2222.
10. Maria EL, Antonello M, Anna PM, Patrizia F, Angelo B. Sequence analysis of hepatitis B virus genome of a new mutant of ayw subtype isolated in Sardinia. Nucleic Acids Research 1991; 19(18): 5078. [DOI via Crossref]   
11. Fuminaka S, Masashi M, Etsuro O, et al. A novel variant genotype C of hepatitis B virus identified in isolates from Australian Aborigines: complete genome sequence and phylogenetic relatedness. Journal of General Virology 2001; 82:883-892. [DOI via Crossref]    [Pubmed]   
12. Fuat K, Yasuhito T, Kei F, et al. A new subtype (subgenotype) Ac (A3) of hepatitis B virus and recombination between genotypes A and E in Cameroon. Journal of General Virology 2005; 86: 2047-2056. [DOI via Crossref]    [Pubmed]   
13. Lieven S, Sija DG, Caroline VG, et al. A new genotype of hepatitis B virus: complete genome and phylogenetic relatedness. Journal of General Virology 2000; 81:67-74. [DOI via Crossref]    [Pubmed]   
14. Fuminaka S, Hiromitsu K, Subrat AA, et al. Epidemiological and sequence differences between two subtypes (Ae and Aa) of hepatitis B virus genotype A. Journal of General Virology 2004; 85:811-820. [DOI via Crossref]    [Pubmed]   
15. Syria L, Vincent T, Francoise B, et al. Expertise of laboratories in viral load quantification, genotyping, and precore mutant determination for hepatitis B virus in a multicenter study. Journal of Clinical Microbiology 2006; 44:3600-3607. [DOI via Crossref]    [Pubmed]    [PMC Free Fulltext]   
16. Behzad BA, Mafi NA, Tabei SZ, Lankarani KB, Torab A, Moaddeb A. Anti-HBc & HBV-DNA detection in blood donors negative for hepatitis B virus surface antigen in reducing risk of transfusion associated HBV infection. Indian J Med Res 2006; 123:37-42.
17. Ching LL, Edward G, Yun FL, et al. Telbivudine versus lamivudine in patients with chronic hepatitis B. N Engl J Med 2007; 357:2576-88. [DOI via Crossref]    [Pubmed]   
18. Pamela MH, Richard DA, Robert W, David HG. Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci 1991; 88:7276-7280. [DOI via Crossref]   
19. Tania MW, Wendell JM, Thomas LP, James JG, Denise W, Betty AOC. Real-time PCR assay for detection and quantification of hepatitis B virus genotypes A to G. Journal of Clinical Microbiology 2006; 44:3325-3333. [DOI via Crossref]    [Pubmed]    [PMC Free Fulltext]   
20. Boom R, Sol CJA, Heijtink R, Wertheim PMEVD, Van JD. Rapid purification of hepatitis B virus DNA from serum. Journal of Clinical Microbiology 1991; 29:1804-1811. [Pubmed]    [PMC Free Fulltext]   
21. Chopra GS, Gupta PK, Anand AC, Varma PP, Nair V, Ramji R. Real time-PCR HBV-DNA analysis: Significance and First Experience in Armed Forces. MJAFI 2005; 61:234-237. [DOI via Crossref]   
22. Aki A, Kazuaki I, Takeshi T, et al. Quantitation of hepatitis B virus genomic DNA by real-time detection PCR. Journal of Clinical Microbiology 1999; 37:2899-2903.
23. Christoph S and William SM. Hepatitis B virus biology. Microbiology and Molecular Biology Reviews 2000; 64:51-68. [DOI via Crossref]   
24. Xuanyong L and Timothy B. Study of the early steps of the hepatitis B virus life cycle. Int J Med Sci 2004; 1:21-33.

How to Cite this Article
Pubmed Style

Y. Naresh Kumar, S. Rahmathulla, C. Madhavi, V. V. Ramachandra, S. Vishnupriya, Mohammed Aejaz Habeeb, M. N. Khaja. Simultaneous detection of Hepatitis B virus and Hepatitis C virus in human plasma using Taq-man chemistry. J Med Allied Sci. 2011; 1(2): 69-73.


Web Style

Y. Naresh Kumar, S. Rahmathulla, C. Madhavi, V. V. Ramachandra, S. Vishnupriya, Mohammed Aejaz Habeeb, M. N. Khaja. Simultaneous detection of Hepatitis B virus and Hepatitis C virus in human plasma using Taq-man chemistry. https://jmas.in/?mno=210240 [Access: January 12, 2023].


AMA (American Medical Association) Style

Y. Naresh Kumar, S. Rahmathulla, C. Madhavi, V. V. Ramachandra, S. Vishnupriya, Mohammed Aejaz Habeeb, M. N. Khaja. Simultaneous detection of Hepatitis B virus and Hepatitis C virus in human plasma using Taq-man chemistry. J Med Allied Sci. 2011; 1(2): 69-73.



Vancouver/ICMJE Style

Y. Naresh Kumar, S. Rahmathulla, C. Madhavi, V. V. Ramachandra, S. Vishnupriya, Mohammed Aejaz Habeeb, M. N. Khaja. Simultaneous detection of Hepatitis B virus and Hepatitis C virus in human plasma using Taq-man chemistry. J Med Allied Sci. (2011), [cited January 12, 2023]; 1(2): 69-73.



Harvard Style

Y. Naresh Kumar, S. Rahmathulla, C. Madhavi, V. V. Ramachandra, S. Vishnupriya, Mohammed Aejaz Habeeb, M. N. Khaja (2011) Simultaneous detection of Hepatitis B virus and Hepatitis C virus in human plasma using Taq-man chemistry. J Med Allied Sci, 1 (2), 69-73.



Turabian Style

Y. Naresh Kumar, S. Rahmathulla, C. Madhavi, V. V. Ramachandra, S. Vishnupriya, Mohammed Aejaz Habeeb, M. N. Khaja. 2011. Simultaneous detection of Hepatitis B virus and Hepatitis C virus in human plasma using Taq-man chemistry. Journal of Medical and Allied Sciences, 1 (2), 69-73.



Chicago Style

Y. Naresh Kumar, S. Rahmathulla, C. Madhavi, V. V. Ramachandra, S. Vishnupriya, Mohammed Aejaz Habeeb, M. N. Khaja. "Simultaneous detection of Hepatitis B virus and Hepatitis C virus in human plasma using Taq-man chemistry." Journal of Medical and Allied Sciences 1 (2011), 69-73.



MLA (The Modern Language Association) Style

Y. Naresh Kumar, S. Rahmathulla, C. Madhavi, V. V. Ramachandra, S. Vishnupriya, Mohammed Aejaz Habeeb, M. N. Khaja. "Simultaneous detection of Hepatitis B virus and Hepatitis C virus in human plasma using Taq-man chemistry." Journal of Medical and Allied Sciences 1.2 (2011), 69-73. Print.



APA (American Psychological Association) Style

Y. Naresh Kumar, S. Rahmathulla, C. Madhavi, V. V. Ramachandra, S. Vishnupriya, Mohammed Aejaz Habeeb, M. N. Khaja (2011) Simultaneous detection of Hepatitis B virus and Hepatitis C virus in human plasma using Taq-man chemistry. Journal of Medical and Allied Sciences, 1 (2), 69-73.