Ultra-sensitive FISH is a useful tool for studying chronic HIV-1 infection

Deichmann, Martin; Haas, Rainer

doi:10.1016/s0166-0934(96)02164-7

Cited by 17 publications

(12 citation statements)

References 15 publications

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“…Surprisingly, RainDrop® dPCR analysis of 8E5 Standard 1 with the PDH/HIV LTR- gag duplex assay revealed a ratio of PDH copies to HIV copies of approximately 3.2:1, whereas according to the literature 12, 18, 19 the expected PDH:HIV ratio should have been exactly 2:1. This surprising finding was confirmed by repeating the dPCR analysis of 8E5 Standard 1 with a different dPCR instrument (QX200™) and with a different region of the HIV genome ( pol ) as PCR target.…”

Section: Resultsmentioning

confidence: 90%

“…This experiment utilised the three different 8E5 cell Standards 1, 2 and 3 (passage 2) simultaneously as calibrators in the same run. HIV DNA copies were calculated per million cells using either the published quantity of 1 HIV DNA copy per 8E5 cell 12, 18, 19 for all three different 8E5 sources or alternatively, the quantity determined empirically for the respective 8E5 extracts by dPCR during the present study.…”

Section: Methodsmentioning

confidence: 99%

“…qPCR requires calibration and for this to be reproducible it is essential that the calibrator must be stable when shared between laboratories. A popular choice of calibrator for quantifying HIV DNA by qPCR is 8E5 (ATCC® CRL-8993) 10–17 , a lymphoblastic leukaemia cell line which has been reported by several studies to contain one integrated HIV genome per cell 12, 18, 19 .…”

Section: Introductionmentioning

confidence: 99%

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Instability of 8E5 calibration standard revealed by digital PCR risks inaccurate quantification of HIV DNA in clinical samples by qPCR

Busby¹,

Whale²,

Ferns

et al. 2017

Sci Rep

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AbtractEstablishing a cure for HIV is hindered by the persistence of latently infected cells which constitute the viral reservoir. Real-time qPCR, used for quantification of this reservoir by measuring HIV DNA, requires external calibration; a common choice of calibrator is the 8E5 cell line, which is assumed to be stable and to contain one HIV provirus per cell. In contrast, digital PCR requires no external calibration and potentially provides ‘absolute’ quantification. We compared the performance of qPCR and dPCR in quantifying HIV DNA in 18 patient samples. HIV DNA was detected in 18 by qPCR and in 15 by dPCR, the difference being due to the smaller sample volume analysed by dPCR. There was good quantitative correlation (R2 = 0.86) between the techniques but on average dPCR values were only 60% of qPCR values. Surprisingly, investigation revealed that this discrepancy was due to loss of HIV DNA from the 8E5 cell calibrant. 8E5 extracts from two other sources were also shown to have significantly less than one HIV DNA copy per cell and progressive loss of HIV from 8E5 cells during culture was demonstrated. We therefore suggest that the copy number of HIV in 8E5 extracts be established by dPCR prior to use as calibrator.

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Section: Resultsmentioning

confidence: 90%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Instability of 8E5 calibration standard revealed by digital PCR risks inaccurate quantification of HIV DNA in clinical samples by qPCR

Busby¹,

Whale²,

Ferns

et al. 2017

Sci Rep

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show abstract

“…[24][25][26] The specificity and sensitivity of this technique were confirmed in rhesus monkey PBMCs ( Fig. 1A and B) and in a rhesus monkey herpesvirus-transformed CD4 1 T cell line ( Fig.…”

Section: Fluorescence In Situ Hybridization Detection Of Cells Producmentioning

confidence: 63%

Detection of Viral RNA in CD4^-CD8^-and CD4^-CD8⁺Lymphocytesin Vivoin Rhesus Monkeys Infected with Simian Immunodeficiency Virus of Macaques

Benito

Chatis

Schmitz

et al. 2001

AIDS Research and Human Retroviruses

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A definition of the specific cell types that support HIV replication early in the course of infection will be important for understanding AIDS pathogenesis and designing strategies for preventing infection. Observations have indicated that the population of lymphocytes susceptible to productive infection extends beyond activated CD4(+) T cells. To explore this issue, we have employed laser scanning cytometry technology and the techniques of lymphocyte surface immunophenotyping followed by fluorescent in situ hybridization to detect simian immunodeficiency virus of macaques (SIVmac) RNA in phenotypically defined rhesus monkey lymphocytes. The immunophenotype of productively infected cells in either a rhesus monkey T cell line or in PBMCs infected in vitro with SIVmac was remarkably similar to that observed in productively infected PBMCs obtained from monkeys during primary infection. We observed low levels or no detectable expression of CD4 on cells infected in vitro or on PBMCs of infected monkeys. However, a substantial number of SIVmac-infected PBMCs both in cultured lymphocytes and sampled directly from infected monkeys expressed CD8 but not CD4. These observations are consistent with the possibility that the CD4 molecule may be modulated off the surface of CD4(+)CD8(-) or CD4(+)CD8(+) lymphocytes after infection or that infection occurred via a CD4-independent mechanism. Moreover, there was no preferential expression of CD25 on cells positive for SIVmac RNA, which might have been predicted if replication of the virus was occurring selectively in activated lymphocytes. These results broaden the range of lymphocytes that support productive SIVmac infection to include CD4(-)CD8(-) and CD4(-)CD8(+) subsets, and are consistent with virus replication occurring in nonactivated cells.

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“…The integration sites of HIV-1 were mapped in two model cell lines by using FISH. This application allowed the analysis of the HIV status at the level of a single cell (73). FISH, using digoxigenin-labeled cosmid probes (119 kbp), was applied to the detection of human cytomegalovirus DNA in peripheral blood leukocytes (74).…”

Section: In Situ Hybridizationmentioning

confidence: 99%

Nucleic Acid Analysis

Christopoulos

1999

Anal. Chem.

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The rapid progress in the Human Genome Project has stimulated the development of diverse analytical methods for mutation detection, elucidation of complex biological problems, molecular diagnosis and prognosis of disease, and assessment of treatment. This review will discuss recent advances in nucleic acid analysis as reported in articles that appeared in Chemical Abstracts between October 1, 1996 and October 1, 1998. Major areas of nucleic acid analysis covered in this review include purification methods, hybridization, in situ hybridization, DNA sequencing, amplification techniques, oligonucleotide arrays, miniaturization of nucleic acid analysis, and nucleic acid sensors.

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Ultra-sensitive FISH is a useful tool for studying chronic HIV-1 infection

Cited by 17 publications

References 15 publications

Instability of 8E5 calibration standard revealed by digital PCR risks inaccurate quantification of HIV DNA in clinical samples by qPCR

Instability of 8E5 calibration standard revealed by digital PCR risks inaccurate quantification of HIV DNA in clinical samples by qPCR

Detection of Viral RNA in CD4^-CD8^-and CD4^-CD8⁺Lymphocytesin Vivoin Rhesus Monkeys Infected with Simian Immunodeficiency Virus of Macaques

Nucleic Acid Analysis

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Ultra-sensitive FISH is a useful tool for studying chronic HIV-1 infection

Cited by 17 publications

References 15 publications

Instability of 8E5 calibration standard revealed by digital PCR risks inaccurate quantification of HIV DNA in clinical samples by qPCR

Instability of 8E5 calibration standard revealed by digital PCR risks inaccurate quantification of HIV DNA in clinical samples by qPCR

Detection of Viral RNA in CD4-CD8-and CD4-CD8+Lymphocytesin Vivoin Rhesus Monkeys Infected with Simian Immunodeficiency Virus of Macaques

Nucleic Acid Analysis

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Detection of Viral RNA in CD4^-CD8^-and CD4^-CD8⁺Lymphocytesin Vivoin Rhesus Monkeys Infected with Simian Immunodeficiency Virus of Macaques