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Simple PCR assays improve the sensitivity of HIV-1 subtype B drug resistance testing and allow linking of resistance mutations.

Johnson JA, Li JF, Wei X, Lipscomb J, Bennett D, Brant A, Cong ME, Spira T, Shafer RW, Heneine W - PLoS ONE (2007)

Bottom Line: We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants.Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing.Combined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations.

View Article: PubMed Central - PubMed

Affiliation: Laboratory Branch, Division of HIV/ADS Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America. JJohnson1@cdc.gov

ABSTRACT

Background: The success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. Currently, there is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, however, requires simple and practical methods for routine testing.

Methodology: We developed highly-sensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We specifically used early wildtype virus samples from the pre-antiretroviral drug era to measure background reactivity and were able to define highly-specific screening cut-offs that are up to 67-fold more sensitive than conventional genotyping. We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing.

Significance: Combined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations.

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Related in: MedlinePlus

Principle of the real-time PCR assay.A. HIV-1 template generated from RT-PCR of viral RNA is subjected to both total copy and mutation-specific real-time reactions. B. The difference in the total copy and mutation-specific reactions (ΔCT) is used to differentiate mutant and wildtype specimens. In this example, the experimental cutoff is a ΔCT of 10.5 cycles. A mutation-specific CT within 10.5 cycles of the total copy reaction CT would indicate the presence of mutant virus.
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pone-0000638-g001: Principle of the real-time PCR assay.A. HIV-1 template generated from RT-PCR of viral RNA is subjected to both total copy and mutation-specific real-time reactions. B. The difference in the total copy and mutation-specific reactions (ΔCT) is used to differentiate mutant and wildtype specimens. In this example, the experimental cutoff is a ΔCT of 10.5 cycles. A mutation-specific CT within 10.5 cycles of the total copy reaction CT would indicate the presence of mutant virus.

Mentions: Real-time PCR-based mutation-specific assays were developed for the protease L90M and the reverse transcriptase M41L, K65R, K70R, K103N, Y181C, M184V, and both T215Y and F resistance-associated mutations in HIV-1 subtype B. Mutation testing was performed in 96-well plates using 2 µL of 1∶20 dilutions of the RT-PCR products, except that samples with viral loads below 5000 copies/mL were not diluted. The principle of the real-time PCR assay is to compare the differential amplifications of a mutation-specific PCR and a PCR that amplifies all viruses in the sample (total virus copy reaction) (Figure 1). The HIV-1 total copy primers, ComFWD and ComREV, span n.t. 258–420 in RT and were used with the common probes, com1P and 2P (Figure 1A, see Table 1). The same common reaction was used for all resistance mutation tests to reduce labor and costs. For multiple mutation screening, several resistance mutation-specific reactions can be performed simultaneously. The cycle number at which the fluorescence emission exceeds the background fluorescence threshold is the threshold cycle (CT) and is the unit of measure for comparing the differences in amplification signals (ΔCT) between the total copy and mutation-specific reactions (Figure 1B). All samples were tested in duplicate with the means of the total copy and mutation-specific CTs used for the determination of the ΔCT.


Simple PCR assays improve the sensitivity of HIV-1 subtype B drug resistance testing and allow linking of resistance mutations.

Johnson JA, Li JF, Wei X, Lipscomb J, Bennett D, Brant A, Cong ME, Spira T, Shafer RW, Heneine W - PLoS ONE (2007)

Principle of the real-time PCR assay.A. HIV-1 template generated from RT-PCR of viral RNA is subjected to both total copy and mutation-specific real-time reactions. B. The difference in the total copy and mutation-specific reactions (ΔCT) is used to differentiate mutant and wildtype specimens. In this example, the experimental cutoff is a ΔCT of 10.5 cycles. A mutation-specific CT within 10.5 cycles of the total copy reaction CT would indicate the presence of mutant virus.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1919426&req=5

pone-0000638-g001: Principle of the real-time PCR assay.A. HIV-1 template generated from RT-PCR of viral RNA is subjected to both total copy and mutation-specific real-time reactions. B. The difference in the total copy and mutation-specific reactions (ΔCT) is used to differentiate mutant and wildtype specimens. In this example, the experimental cutoff is a ΔCT of 10.5 cycles. A mutation-specific CT within 10.5 cycles of the total copy reaction CT would indicate the presence of mutant virus.
Mentions: Real-time PCR-based mutation-specific assays were developed for the protease L90M and the reverse transcriptase M41L, K65R, K70R, K103N, Y181C, M184V, and both T215Y and F resistance-associated mutations in HIV-1 subtype B. Mutation testing was performed in 96-well plates using 2 µL of 1∶20 dilutions of the RT-PCR products, except that samples with viral loads below 5000 copies/mL were not diluted. The principle of the real-time PCR assay is to compare the differential amplifications of a mutation-specific PCR and a PCR that amplifies all viruses in the sample (total virus copy reaction) (Figure 1). The HIV-1 total copy primers, ComFWD and ComREV, span n.t. 258–420 in RT and were used with the common probes, com1P and 2P (Figure 1A, see Table 1). The same common reaction was used for all resistance mutation tests to reduce labor and costs. For multiple mutation screening, several resistance mutation-specific reactions can be performed simultaneously. The cycle number at which the fluorescence emission exceeds the background fluorescence threshold is the threshold cycle (CT) and is the unit of measure for comparing the differences in amplification signals (ΔCT) between the total copy and mutation-specific reactions (Figure 1B). All samples were tested in duplicate with the means of the total copy and mutation-specific CTs used for the determination of the ΔCT.

Bottom Line: We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants.Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing.Combined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations.

View Article: PubMed Central - PubMed

Affiliation: Laboratory Branch, Division of HIV/ADS Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America. JJohnson1@cdc.gov

ABSTRACT

Background: The success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. Currently, there is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, however, requires simple and practical methods for routine testing.

Methodology: We developed highly-sensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We specifically used early wildtype virus samples from the pre-antiretroviral drug era to measure background reactivity and were able to define highly-specific screening cut-offs that are up to 67-fold more sensitive than conventional genotyping. We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing.

Significance: Combined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations.

Show MeSH
Related in: MedlinePlus