An Improved Tat/Rev Induced Limiting Dilution Assay With Enhanced Sensitivity and Breadth of Detection
Tat/Rev Induced Limiting Dilution Assay (TILDA) is instrumental in estimating the size of latent reservoirs of HIV-1. Here, we report an optimized TILDA containing a broader detection range compared to the reported methods and high sensitivity. Giving priority to sequence conservation, we positioned...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2021-08-01
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| Series: | Frontiers in Immunology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2021.715644/full |
| _version_ | 1818643649979744256 |
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| author | Kavita Mehta Yuvrajsinh Gohil Swarnima Mishra Anish D’silva Afzal Amanullah Deepak Selvam Neelam Pargain Narendra Nala G. N. Sanjeeva Udaykumar Ranga |
| author_facet | Kavita Mehta Yuvrajsinh Gohil Swarnima Mishra Anish D’silva Afzal Amanullah Deepak Selvam Neelam Pargain Narendra Nala G. N. Sanjeeva Udaykumar Ranga |
| author_sort | Kavita Mehta |
| collection | DOAJ |
| description | Tat/Rev Induced Limiting Dilution Assay (TILDA) is instrumental in estimating the size of latent reservoirs of HIV-1. Here, we report an optimized TILDA containing a broader detection range compared to the reported methods and high sensitivity. Giving priority to sequence conservation, we positioned the two forward primers and the probe in exon-1 of HIV-1. The reverse primers are positioned in highly conserved regions of exon-7. The optimized TILDA detected eight molecular clones belonging to five major genetic subtypes of HIV-1 with a comparable detection sensitivity. Using the optimized assay, we show that only a minor proportion of CD4+ T cells of primary clinical samples can spontaneously generate multiply spliced viral transcripts. A significantly larger proportion of the cells produced viral transcripts following activation. The optimized TILDA is suitable to characterize HIV-1 latent reservoirs and the therapeutic strategies intended to target the reservoir size. |
| first_indexed | 2024-12-17T00:02:19Z |
| format | Article |
| id | doaj.art-dc7b381e7bbe443f9a239ab57fab3e47 |
| institution | Directory Open Access Journal |
| issn | 1664-3224 |
| language | English |
| last_indexed | 2024-12-17T00:02:19Z |
| publishDate | 2021-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj.art-dc7b381e7bbe443f9a239ab57fab3e472022-12-21T22:11:02ZengFrontiers Media S.A.Frontiers in Immunology1664-32242021-08-011210.3389/fimmu.2021.715644715644An Improved Tat/Rev Induced Limiting Dilution Assay With Enhanced Sensitivity and Breadth of DetectionKavita Mehta0Yuvrajsinh Gohil1Swarnima Mishra2Anish D’silva3Afzal Amanullah4Deepak Selvam5Neelam Pargain6Narendra Nala7G. N. Sanjeeva8Udaykumar Ranga9Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, IndiaMolecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, IndiaMolecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, IndiaMolecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, IndiaMolecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, IndiaMolecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, IndiaMolecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, IndiaMolecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, IndiaDepartment of Pediatric Genetics, Indira Gandhi Institute of Child Health, Bengaluru, IndiaMolecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, IndiaTat/Rev Induced Limiting Dilution Assay (TILDA) is instrumental in estimating the size of latent reservoirs of HIV-1. Here, we report an optimized TILDA containing a broader detection range compared to the reported methods and high sensitivity. Giving priority to sequence conservation, we positioned the two forward primers and the probe in exon-1 of HIV-1. The reverse primers are positioned in highly conserved regions of exon-7. The optimized TILDA detected eight molecular clones belonging to five major genetic subtypes of HIV-1 with a comparable detection sensitivity. Using the optimized assay, we show that only a minor proportion of CD4+ T cells of primary clinical samples can spontaneously generate multiply spliced viral transcripts. A significantly larger proportion of the cells produced viral transcripts following activation. The optimized TILDA is suitable to characterize HIV-1 latent reservoirs and the therapeutic strategies intended to target the reservoir size.https://www.frontiersin.org/articles/10.3389/fimmu.2021.715644/fulllatent reservoirHIV latencyHIV-1TILDAtranscriptional silence |
| spellingShingle | Kavita Mehta Yuvrajsinh Gohil Swarnima Mishra Anish D’silva Afzal Amanullah Deepak Selvam Neelam Pargain Narendra Nala G. N. Sanjeeva Udaykumar Ranga An Improved Tat/Rev Induced Limiting Dilution Assay With Enhanced Sensitivity and Breadth of Detection Frontiers in Immunology latent reservoir HIV latency HIV-1 TILDA transcriptional silence |
| title | An Improved Tat/Rev Induced Limiting Dilution Assay With Enhanced Sensitivity and Breadth of Detection |
| title_full | An Improved Tat/Rev Induced Limiting Dilution Assay With Enhanced Sensitivity and Breadth of Detection |
| title_fullStr | An Improved Tat/Rev Induced Limiting Dilution Assay With Enhanced Sensitivity and Breadth of Detection |
| title_full_unstemmed | An Improved Tat/Rev Induced Limiting Dilution Assay With Enhanced Sensitivity and Breadth of Detection |
| title_short | An Improved Tat/Rev Induced Limiting Dilution Assay With Enhanced Sensitivity and Breadth of Detection |
| title_sort | improved tat rev induced limiting dilution assay with enhanced sensitivity and breadth of detection |
| topic | latent reservoir HIV latency HIV-1 TILDA transcriptional silence |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2021.715644/full |
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