Novel application of Locked Nucleic Acid chemistry for a Taqman assay for measuring diverse human immunodeficiency virus type 1 subtypes

Li, Peilin; Ruel, Theodore; Fujimoto, Katsuya; Hatano, Hiroyu; Yukl, Steven A.; Eller, Leigh Anne; Liegler, Teri; Kamya, Moses R.; Gassasira, Anne; Dorsey, Grant; Rosenthal, Philip; Havlir, Diane V.; Wong, Joseph K.

doi:10.1016/j.jviromet.2010.09.011

Cited by 16 publications

(19 citation statements)

References 41 publications

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“…25 Assay 2 targeted a conserved region of the HIV-1 gag gene and made use of locked nucleic acid technology (LNA, Exiqon, Woburn, MA) for probe construction. 22 Ten microliter PCR reactions consisted of primers and probe in 1 · QuantiTect probe master mix (Qiagen, Valencia, CA). Probes were labeled with 6-FAM at the 5¢-end and Black Hole quencher 1 at the 3¢-end (Eurofins Genomics, Huntsville, AL).…”

Section: Hiv-1 Assaysmentioning

confidence: 99%

“…Plasmid controls for the two HIV-1 proviral assays, 22,25 the two viral RNA real-time PCR assays, 21,24 and assays for the b-globin and b-actin reference genes were constructed by amplifying gene fragments using the appropriate primers and AmpliTaq Gold DNA polymerase. For DNA assays, the PCR fragments were generated by amplifying regions of the HIV-1 genome using the 8E5 cell line DNA as a template (American Type Culture Collection, Manassa, VA).…”

Section: Construction Of Plasmid Controls For Real-time Pcr Assaysmentioning

confidence: 99%

“…20 Assays for determining HIV-1 viral load have targeted conserved regions of the long terminal repeat (LTR) domain and the gag and pol (integrase) genes of HIV-1. [21][22][23][24] Two laboratory-developed probe-based qRT-PCR assays that detect overlapping regions of the HIV-1 LTR 21,24 were used in the present study to measure HIV-1 RNA levels in infected biopsy tissues and supernatant fluids. Assays to detect HIV-1 proviral load have targeted the LTR as well as the gag and pol (integrase) regions of the HIV-1 genome.…”

Section: Introductionmentioning

confidence: 99%

“…Assays to detect HIV-1 proviral load have targeted the LTR as well as the gag and pol (integrase) regions of the HIV-1 genome. 22,23,25 An assay targeting the LTR/gag boundary and another assay targeting the HIV-1 gag gene were chosen to monitor HIV-1 provirus accumulation. 22,25 Integration of HIV-1 into the host genome is essential for viral transmission as replication occurs mainly from integrated DNA.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

The Molecular Characterization of Intestinal Explant HIV Infection Using Polymerase Chain Reaction-Based Techniques

Janocko

Althouse

Brand

et al. 2015

AIDS Research and Human Retroviruses

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Section: Hiv-1 Assaysmentioning

confidence: 99%

Section: Construction Of Plasmid Controls For Real-time Pcr Assaysmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

The Molecular Characterization of Intestinal Explant HIV Infection Using Polymerase Chain Reaction-Based Techniques

Janocko

Althouse

Brand

et al. 2015

AIDS Research and Human Retroviruses

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“…Dipstick and chip technologies are still in development (2,10,11). Therefore, recent "in-house" or generic real-time RT-PCR assays have shown diverse advantages and are relatively inexpensive (the real-time technology is widely used in HIV and other viral infection detections) (12)(13)(14).…”

mentioning

confidence: 99%

Single Real-Time Reverse Transcription-PCR Assay for Detection and Quantification of Genetically Diverse HIV-1, SIVcpz, and SIVgor Strains

et al. 2013

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Although antiretroviral treatment availability has improved, the virological monitoring of patients remains largely uneven across regions. In addition, viral quantification tests are suffering from human immunodeficiency virus type 1 (HIV-1) genetic diversity, fueled by the emergence of new recombinants and of lentiviruses from nonhuman primates. We developed a real-time reverse transcription-PCR (RT-PCR) assay that is relatively inexpensive and able to detect and quantify all circulating forms of HIV-1 and its simian immunodeficiency virus (SIV) precursors, SIVcpz and SIVgor. Primers and a probe were designed to detect all variants of the HIV-1/SIVcpz/SIVgor lineage. HIV-1 M plasma (n ‫؍‬ 190; 1.68 to 7.78 log 10 copies/ml) representing eight subtypes, nine circulating recombinant forms, and 21 unique recombinant forms were tested. The mean PCR efficiency was 99%, with low coefficients of intra-and interassay variation (<5%) and a limit of quantification of <2.50 log 10 copies/ml, with a 200-l plasma volume. On the studied range, the specificity and the analytical sensitivity were 100 and 97.4%, respectively. The viral loads were highly correlated (r ‫؍‬ 0.95, P < 0.0001) with the reference method (generic HIV assay; Biocentric) and had no systematic difference, irrespective of genotype. Furthermore, 22 HIV-1 O plasmas were screened and were better quantified compared to the gold-standard RealTime HIV-1 assay (Abbott), including four samples that were only quantified by our assay. Finally, we could quantify SIVcpzPtt and SIVcpzPts from chimpanzee plasma (n ‫؍‬ 5) and amplify SIVcpz and SIVgor from feces. Thus, the newly developed real-time RT-PCR assay detects and quantifies strains from the HIV-1/SIVcpz/SIVgor lineage, including a wide diversity of group M strains and HIV-1 O. It can therefore be useful in geographical areas of high HIV diversity and at risk for the emergence of new HIV variants.

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Fluorescent Nucleic Acid Analogues in Research and Clinical Diagnostics

Astakhova

2015

RNA Technologies

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Novel application of Locked Nucleic Acid chemistry for a Taqman assay for measuring diverse human immunodeficiency virus type 1 subtypes

Cited by 16 publications

References 41 publications

The Molecular Characterization of Intestinal Explant HIV Infection Using Polymerase Chain Reaction-Based Techniques

The Molecular Characterization of Intestinal Explant HIV Infection Using Polymerase Chain Reaction-Based Techniques

Single Real-Time Reverse Transcription-PCR Assay for Detection and Quantification of Genetically Diverse HIV-1, SIVcpz, and SIVgor Strains

Fluorescent Nucleic Acid Analogues in Research and Clinical Diagnostics

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