Design and development of an in-house multiplex RT-PCR assay for simultaneous detection of HIV-1 and HCV in plasma samples
,
Abstract
Background and Objectives: HIV-1 and HCV infections are life threatening problems in patients who receive blood products. Serological methods have proven useful in detecting these infections, but there are setbacks that make it challenging to detect these infectious agents. By the advent of Nucleic Acid Testing (NAT) methods, especially in multiplex format, more precise detection is possible.
Materials and Methods: We have developed a multiplex RT-PCR assay for simultaneous detection of HIV-1 and HCV. Primers were designed for highly conserved region of genome of each virus. Using these primers and standard plasmids, we determined the limit of detection, clinical and analytical specificity and sensitivity of the assay. Monoplex and multiplex RT-PCR were performed.
Results: Analytical sensitivity was considered to be 100 and 200 copies/ml for HIV-1 and HCV, respectively. High concentration of one virus had no significant effect on the detection of the other one with low concentration. By analysis of 40 samples, clinical sensitivity of the assay was determined to be 97.5%. Using different viral and human genome samples, the specificity of the assay was evaluated to be 100%.
Conclusions: The aim of this study was to develop a reliable, rapid and cost effective method to detect HIV-1 and HCV simultaneously. Results showed that this simple and rapid method is perfectly capable of detecting two viruses in clinical samples.
Alter MJ, Margolis HS, Krawczynski K, Judson FN, Mares A, Alexander WJ, et al. The natural history of community-acquired hepatitis C in the United States. The Sentinel Counties Chronic non-A, non-B Hepatitis Study Team. N Engl J Med 1992; 327: 1899-1905.
Sherman KE, Rouster SD, Chung RT, Rajicic N.Hepatitis C Virus prevalence among patients infected with Human Immunodeficiency Virus: a cross-sectional analysis of the US adult AIDS Clinical Trials Group. Clin Infect Dis 2002; 34: 831-837.
Gibellini D, Gardini F, Vitone F, Schiavone P, Furlini G, Re MC. Simultaneous detection of HCV and HIV-1 by SYBR Green real time multiplex RT-PCR technique in plasma samples. Mol Cell Probes 2006; 20: 223-229.
Thimme R, Spangenberg HC, Blum HE. Hepatitis B or hepatitis C and human immunodeficiency virus infection. J Hepatol 2005; 42 Suppl (1): S37-44.
Gibb DM, Goodall RL, Dunn DT, Healy M, Neave P, Cafferkey M, et al. Mother-to-child transmission of hepatitis C virus: evidence for preventable peripartum transmission. Lancet 2000; 356(9233): 904-907.
Michaels SH, Clark R, Kissinger P. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. N Engl J Med 1998;339: 405-406.
Sulkowski MS, Mast EE, Seeff LB, Thomas DL.Hepatitis C virus infection as an opportunistic disease in persons infected with human immunodeficiency virus. Clin Infect Dis 2000 Suppl 1(1): S77-84.
Sulkowski MS, Moore RD, Mehta SH, Chaisson RE, Thomas DL. Hepatitis C and progression of HIV disease. JAMA 2002; 288: 199-206.
Greub G, Ledergerber B, Battegay M, Grob P, Perrin L, Furrer H, et al. Clinical progression, survival, and immune recovery during antiretroviral therapy in patients with HIV-1 and hepatitis C virus coinfection: the Swiss HIV Cohort Study. Lancet. 2000 Nov 25;356(9244): 1800-5.
Monga HK, Rodriguez-Barradas MC, Breaux K, Khattak K, Troisi CL, Velez M, et al. Hepatitis C virus infection-related morbidity and mortality among patients with human immunodeficiency virus infection. Clin Infect Dis 2001; 33: 240-247.
Busch MP, Kleinman SH, Jackson B, Stramer SL, Hewlett I, Preston S. Committee report. Nucleic acid amplification testing of blood donors for transfusion-transmitted infectious diseases: Report of the Interorganizational Task Force on Nucleic Acid Amplification Testing of Blood Donors. Transfusion 2000 ; 40: 143-159.
Pawlotsky JM. Use and interpretation of virological tests for hepatitis C. Hepatology 2002; 36 (5 Suppl 1): S65-73.
Allain JP. Will genome detection replace serology in blood screening for microbial agents? Baillieres Best Pract Res Clin Haematol 2000; 13: 615-629.
Reichelderfer PS, Coombs RW. Virologic parameters as surrogate markers for clinical outcome in HIV-1 disease: verification, variation, and validation. J Acquir Immune Defic Syndr Hum Retrovirol 1995;10 Suppl 2: S19-24.
Stramer SL, Caglioti S, Strong DM. NAT of the United States and Canadian blood supply. Transfusion 2000;40: 1165-1168.
Meng Q, Wong C, Rangachari A, Tamatsukuri S, Sasaki M, Fiss E, et al. Automated multiplex assay system for simultaneous detection of hepatitis B virus DNA, hepatitis C virus RNA, and human immunodeficiency virus type 1 RNA. J Clin Microbiol 2001; 39: 2937-2945.
Mine H, Emura H, Miyamoto M, Tomono T, Minegishi K, Murokawa H, et al. High throughput screening of 16 million serologically negative blood donors for hepatitis B virus, hepatitis C virus and human immunodeficiency virus type-1 by nucleic acid amplification testing with specific and sensitive multiplex reagent in Japan. J Virol Methods 2003; 112(1-2): 145-151.
Adami V, Falasca E, Dorotea L, Malangone W, Astori G, Marini L, et al. Qualitative multiplex RT-PCR for simultaneous detection of hepatitis C virus and human immunodeficiency virus in plasma samples. Clin Microbiol Infect 2004; 102: 1075-1080.
Candotti D, Temple J, Owusu-Ofori S, Allain JP.Multiplex real-time quantitative RT-PCR assay for hepatitis B virus, hepatitis C virus, and human immunodeficiency virus type 1. J Virol Methods 2004;118: 39-47.
Giachetti C, Linnen JM, Kolk DP, Dockter J, Gillotte- Taylor K, Park M, et al. Highly sensitive multiplex assay for detection of human immunodeficiency virus type 1 and hepatitis C virus RNA. J Clin Microbiol 2002; 40:2408-2419.
Shrestha NK, Tuohy MJ, Hall GS, Reischl U, Gordon SM, Procop GW. Detection and differentiation of Mycobacterium tuberculosis and nontuberculous mycobacterial isolates by real-time PCR. J Clin Microbiol 2003; 41: 5121-5126.
Berry N, Herrera C, Cranage M. Detection, quantification, and characterisation of HIV/SIV. Methods Mol Biol.2011; 665: 133-60.
Beuret C. Simultaneous detection of enteric viruses by multiplex real-time RT-PCR. J Virol Methods 2004;115: 1-8.
Goire N, Freeman K, Tapsall JW, Lambert SB, Nissen MD, Sloots TP, et al. Enhancing gonococcal antimicrobial resistance surveillance: a real-time PCR assay for detection of penicillinase-producing Neisseria gonorrhoeae using non-cultured clinical samples. J Clin Microbiol 15; 2010: 2015.
Richards GP, Watson MA, Kingsley DH. A SYBR green, real-time RT-PCR method to detect and quantitate Norwalk virus in stools. J Virol Methods 2004 1; 116:63-70.
Wei X, Youngpairoj AS, Garrido C, Zahonero N, Corral A, de Mendoza C, et al. Minority HIV mutation detection in dried blood spots indicates high specimen integrity and reveals hidden archived drug resistance. J Clin Virol 2011; 50: 148-152.
Weidner J, Cassens U, Gohde W, Sibrowski W, Odaibo G, Olaleye D, et al. An improved PCR method for detection of HIV-1 proviral DNA of a wide range of subtypes and recombinant forms circulating globally. J Virol Methods 2011; 172: 22-26.
Defoort JP, Martin M, Casano B, Prato S, Camilla C, Fert V. Simultaneous detection of multiplex-amplified human immunodeficiency virus type 1 RNA, hepatitis C virus RNA, and hepatitis B virus DNA using a flow cytometer microsphere-based hybridization assay. J Clin Microbiol 2000; 38: 1066-1071.
Vargo J, Smith K, Knott C, Wang S, Fang C, McDonough S, et al. Clinical specificity and sensitivity of a blood screening assay for detection of HIV-1 and HCV RNA. Transfusion. 2002; 42: 876-885.
De Crignis E, Re MC, Cimatti L, Zecchi L, Gibellini D. HIV-1 and HCV detection in dried blood spots by SYBR Green multiplex real-time RT-PCR. J Virol Methods 2010; 165: 51-56.
Tang J, Zhou L, Duan LL, Feng Y, Cao X, Wang Y.Development and evaluation of a new detection tool- visual DNA microarray for simultaneous and specific detection of human immunodeficiency virus type-1 and hepatitis C virus. Mol Biol Rep 2011; 38: 5341-5348.
| Files | ||
| Issue | Vol 4 No 1 (2012) | |
| Section | Articles | |
| Keywords | ||
| Co-infection HCV HIV-1 Multiplex RT-PCR | ||
| Rights and permissions | |
|
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
