Multiplex qPCR Assay for HIV-1 Proviral DNA Detection and Subtype Characterization: Exploiting Quenching of Multiple Fluorophores with a Single Quencher Operating in Trans

Contact Quenching

Authors

  • Daisy Patel Wobble Base Bioresearch Private limited, Pune, INDIA.
  • Krishna H. Goyani Wobble Base Bioresearch Private limited, Pune, INDIA.
  • Isha Sharma Parul University, Waghodia, Vadodara, Gujarat, INDIA.
  • Shalin Vaniawala New Civil Hospital, Jay Prakash Narayan Marg, Surat, INDIA.
  • Pratap N. Mukhopadhyaya Wobble Base Bioresearch Private limited, Pune, INDIA. https://orcid.org/0009-0002-9460-8684

DOI:

https://doi.org/10.55544/jrasb.4.2.1

Keywords:

HIV-1 proviral DNA, multiplex qPCR, subtype characterization, quenching in trans, fluorescent probes

Abstract

Accurate detection and quantification of HIV-1 proviral DNA are critical for effective patient monitoring and therapeutic decision-making. In this study, we developed a multiplexed quantitative PCR (qPCR) assay designed to detect HIV-1 proviral DNA, determine viral subtype, specifically identifying the predominant subtype C and validate assay performance using an internal control. Gene-specific primers were engineered by appending an 8-base biotag followed by a common 18-base sequence at the 5′ end, enabling the simultaneous amplification of multiple target sequences. Fluorescent probes labeled with FAM, SUN/VIC, and Cy5 were employed for detection, and a novel strategy involving quenching of labeled probes in trans was implemented to enhance assay flexibility and cost-effectiveness compared to conventional cis-quenched probes. The assay was initially optimized using synthetic linear double-stranded DNA templates representing the HIV-1 gag region, while externally added human chromosomal DNA served as a control for PCR inhibition. Validation was performed on a panel of 11 clinical samples previously analyzed for drug resistance mutations. Results indicated robust amplification of HIV-1 proviral DNA, accurate subtype determination, and reliable internal control performance, with profiles closely matching those obtained by gold standard sequencing-based assays. One sample exhibited PCR inhibition, underscoring the need for internal control monitoring. Overall, the multiplexed qPCR assay provides a sensitive, specific, and efficient tool for comprehensive HIV-1 reservoir quantification and molecular epidemiological studies, potentially informing improved clinical management and personalized treatment strategies. Furthermore, this novel methodology significantly reduces reagent costs and processing time while maintaining high sensitivity, making it ideal for routine clinical and research applications.

Downloads

Download data is not yet available.

Author Biographies

Krishna H. Goyani, Wobble Base Bioresearch Private limited, Pune, INDIA.

Team Lead

Isha Sharma, Parul University, Waghodia, Vadodara, Gujarat, INDIA.

Intern

Shalin Vaniawala, New Civil Hospital, Jay Prakash Narayan Marg, Surat, INDIA.

Collaborator

References

Candotti, D., Jung, M., Huraux, J.-M., Kerouedan, D., MʼPele, P., Gentilini, M., Rosenheim, M., & Agut, H. (1991). Genetic variability affects the detection of HIV by polymerase chain reaction. AIDS, 5(8), 1003–1008. https://doi.org/10.1097/00002030-199108000-00012

Genesca, J., Shih, J. W.-K., Yuan-Bu Wang, R., & Alter, H. J. (1990). Clinical Correlation and Genetic Polymorphism of the Human Immunodeficiency Virus Proviral DNA Obtained after Polymerase Chain Reaction Amplification. Journal of Infectious Diseases, 162(5), 1025–1030. https://doi.org/10.1093/infdis/162.5.1025

Jackson, J. B., Wolinsky, S. M., Hollinger, F. B., Otto, P., Bremer, J. W., Drew, J., & Lin, H. J. (1993). Establishment of a quality assurance program for human immunodeficiency virus type 1 DNA polymerase chain reaction assays by the AIDS Clinical Trials Group. ACTG PCR Working Group, and the ACTG PCR Virology Laboratories. Journal of Clinical Microbiology, 31(12), 3123–3128. https://doi.org/10.1128/jcm.31.12.3123-3128.1993

Menzo, S., Bagnarelli, P., Clementi, M., Varaldo, P. E., Giacca, M., & Manzin, A. (1992). Absolute quantitation of viremia in human immunodeficiency virus infection by competitive reverse transcription and polymerase chain reaction. Journal of Clinical Microbiology, 30(7), 1752–1757. https://doi.org/10.1128/jcm.30.7.1752-1757.1992

Balakrishnan, P., Vidya, S., Ramratnam, B., Thyagarajan, S. P., Solomon, S., Mayer, K. H., Newstein, M., Kantor, R., Katzenstein, D., & Kumarasamy, N. (2005). HIV Type 1 Genotypic Variation in an Antiretroviral Treatment-Naive Population in Southern India. AIDS Research and Human Retroviruses, 21(4), 301–305. https://doi.org/10.1089/aid.2005.21.301

Hirsch, M. S., Günthard, H. F., Kuritzkes, D. R., Clotet, B., Yeni, P. G., Richman, D. D., Jacobsen, D. M., Mellors, J. W., Brun‐Vézinet, F., Schapiro, J. M., Hammer, S. M., Johnson, V. A., & Pillay, D. (2008). Antiretroviral Drug Resistance Testing in Adult HIV‐1 Infection: 2008 Recommendations of an International AIDS Society–USA Panel. Clinical Infectious Diseases, 47(2), 266–285. https://doi.org/10.1086/589297

Sahni, A. K., Prasad, V. V. S. P., & Seth, P. (2002). Genomic diversity of human immunodeficiency virus type-1 in India. International Journal of STD & AIDS, 13(2), 115–118. https://doi.org/10.1258/0956462021924749

Seatla, K. K., Ramaabya, D., Jarvis, J. N., Kasvosve, I., Gaolatlhe, T., Gaseitsiwe, S., Diphoko, T., Avalos, A., Rowley, C. F., Mosepele, M., Mine, M., & Moyo, S. (2018). Four-class drug-resistant HIV-1 subtype C in a treatment-experienced individual on dolutegravir-based antiretroviral therapy in Botswana. AIDS, 32(13), 1899–1902. https://doi.org/10.1097/qad.0000000000001920

Shankarappa, R., Kingsley, L., Chatterjee, R., Roy, P., Gupta, P., Ding, M., Neogi, D., Harrison, L., Learn, G. H., Mullins, J. I., & Ghosh, A. (2001). Human immunodeficiency virus type 1 env sequences from Calcutta in eastern India: identification of features that distinguish subtype C sequences in India from other subtype C sequences. Journal of Virology, 75(21), 10479–10487. https://doi.org/10.1128/jvi.75.21.10479-10487.2001

Brookmeyer, R., Bollinger, R., Quinn, T., Rodrigues, J., Shepherd, M., & Mehendale, S. (1995). The AIDS epidemic in India: a new method for estimating current human immunodeficiency virus (HIV) incidence rates. American Journal of Epidemiology, 142(7), 709–713. https://doi.org/10.1093/oxfordjournals.aje.a117700

Lucas, G. M., Ogburn, E. L., Laeyendecker, O., Srikrishnan, A. K., Mehta, S. H., Agrawal, A., Iqbal, S., Solomon, S., Celentano, D. D., Mcfall, A. M., Solomon, S. S., & Kumar, M. S. (2015). High HIV burden among people who inject drugs in 15 Indian cities. AIDS, 29(5), 619–628. https://doi.org/10.1097/qad.0000000000000592

Shri, N., Patel, P., Jha, R. P., Singh, M., Dhamnetiya, D., & Bhattacharyya, K. (2023). Long-term trends of HIV/AIDS incidence in India: an application of joinpoint and age-period-cohort analyses: a gendered perspective. Frontiers in Public Health, 11. https://doi.org/10.3389/fpubh.2023.1093310

Solomon, S. S., Mehta, S. H., Mcfall, A. M., Celentano, D. D., Balakrishnan, P., Laeyendecker, O., Solomon, S., Nandagopal, P., Anand, S., Ogburn, E. L., Srikrishnan, A. K., Vasudevan, C. K., & Lucas, G. M. (2015). High HIV prevalence and incidence among MSM across 12 cities in India. AIDS, 29(6), 723–731. https://doi.org/10.1097/qad.0000000000000602

Sood, S., Shefner-Rogers, C. L., & Sengupta, M. (2006). The Impact of a Mass Media Campaign on HIV/AIDS Knowledge and Behavior Change in North India: Results from a Longitudinal Study. Asian Journal of Communication, 16(3), 231–250. https://doi.org/10.1080/01292980600857740

Vickerman, P., Demers, E., Verma, S., Boily, M.-C., Pickles, M., Alary, M., Lowndes, C. M., Moses, S., Foss, A. M., & Deering, K. (2010). To what extent is the HIV epidemic in southern India driven by commercial sex? A modelling analysis. AIDS, 24(16), 2563–2572. https://doi.org/10.1097/qad.0b013e32833e8663

Jangam, S. R., Mcfall, S. M., Kelso, D. M., & Yamada, D. H. (2009). Rapid, Point-of-Care Extraction of Human Immunodeficiency Virus Type 1 Proviral DNA from Whole Blood for Detection by Real-Time PCR. Journal of Clinical Microbiology, 47(8), 2363–2368. https://doi.org/10.1128/jcm.r00092-09

Steuler, H., Wildemann, B., & Storch-Hagenlocher, B. (1992). Distinct Populations of Human Immunodeficiency Virus Type 1 in Blood and Cerebrospinal Fluid. AIDS Research and Human Retroviruses, 8(1), 53–59. https://doi.org/10.1089/aid.1992.8.53

Cassol, S., Spadoro, J., Fauvel, M., Major, C., Lapointe, N., Gomez, P., Read, S., Butcher, A., Kinard, S., & Sy, T. (1994). Rapid screening for early detection of mother-to-child transmission of human immunodeficiency virus type 1. Journal of Clinical Microbiology, 32(11), 2641–2645. https://doi.org/10.1128/jcm.32.11.2641-2645.1994

Nyambi, P. N., Larouzé, B., Dazza, M.-C., Glowaczower, E., Van Der Groen, G., Louwagie, J., Ville, Y., Delaporte, E., Peeters, M., Kerouedan, D., & Bedjabaga, I. (1996). Mother-to-Child Transmission of Human T-Cell Lymphotropic Virus Types I and II (HTLV-I/II) in Gabon: A Prospective Follow-up of 4 Years. Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology, 12(2), 187–192. https://doi.org/10.1097/00042560-199606010-00013

Siddappa, N. B., Dice, B., Jayasuryan, N., Mahadevan, A., Shankar, S. K., Shankarappa, R., Hu, F., Satish, B., Sreekanthan, K., Satish, K. S., Dash, P. K., Ranga, U., Venu, K., Satishchandra, P., Keefe, R., Chatterjee, R., & Ravi, V. (2004). Identification of subtype C human immunodeficiency virus type 1 by subtype-specific PCR and its use in the characterization of viruses circulating in the southern parts of India. Journal of Clinical Microbiology, 42(6), 2742–2751. https://doi.org/10.1128/jcm.42.6.2742-2751.2004

Acharya A, Vaniawala S, Shah P, Misra RN, Wani M, Mukhopadhyaya PN. Development, validation and clinical evaluation of a low cost in-house HIV-1 drug resistance genotyping assay for Indian patients. PLoS One. 2014 Aug 26;9(8):e105790. doi: 10.1371/journal.pone.0105790. PMID: 25157501; PMCID: PMC4144911.

Downloads

Published

2025-04-05

How to Cite

Patel, D., Goyani, K. H., Sharma, I., Vaniawala, S., & Mukhopadhyaya, P. N. (2025). Multiplex qPCR Assay for HIV-1 Proviral DNA Detection and Subtype Characterization: Exploiting Quenching of Multiple Fluorophores with a Single Quencher Operating in Trans: Contact Quenching. Journal for Research in Applied Sciences and Biotechnology, 4(2), 1–10. https://doi.org/10.55544/jrasb.4.2.1

Issue

Vol. 4 No. 2 (2025): April Issue

Section

Articles

License

Copyright (c) 2025 Daisy Patel, Krishna H. Goyani, Isha Sharma, Shalin Vaniawala, Pratap N. Mukhopadhyaya

Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.