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Rapid and Simultaneous Detection of Major Drug Resistance Mutations in Reverse Transcriptase Gene for HIV-1 CRF01_AE, CRF07_BC and Subtype B in China Using Sequenom MassARRAY® System.

Cheung KW, Peng Q, He L, Cai K, Jiang Q, Zhou B, To SW, Yam WC, Liu L, Chen Z, Wang H - PLoS ONE (2016)

Bottom Line: The development of a rapid, high-throughput and cost-effective HIV-1 drug resistance (HIV-DR) testing system is a challenge for areas consisting different HIV-1 strains.Importantly, the MALDI-TOF MS results were concordant to the drug resistance profiles of patients obtained from conventional sequencing analysis after excluded the failed detections.In conclusion, this study evaluated the use of Sequenom MassARRAY® system for high-throughput detection of HIV-DR mutations towards the commonly used reverse transcriptase inhibitors in China.

View Article: PubMed Central - PubMed

Affiliation: AIDS Institute and Department of Microbiology, Research Centre for Infection and Immunity, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong SAR, People's Republic of China.

ABSTRACT
The development of a rapid, high-throughput and cost-effective HIV-1 drug resistance (HIV-DR) testing system is a challenge for areas consisting different HIV-1 strains. In this study, we established a broadly reactive multiplex assay that could simultaneously detect major drug resistance mutations at 8 loci, which are associated with resistance to commonly used nucleoside reverse transcriptase inhibitors (NRTIs) and Non-nucleoside reverse transcriptase inhibitors (NNRTIs), in specimens of HIV-1 CRF01_AE, CRF07_BC and subtype B, the three major circulating strains in China, using the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) provided by Sequenom MassARRAY® system. To establish the assay, pol gene fragments were prepared from the plasma viral RNA of 159 patients by nested PCR and the presence of wild type and mutant alleles at the 8 loci were analyzed by MALDI-TOF MS. In terms of loci, the detection rate of the alleles was greater than 97% for M41L, K65R, M184V and G190A, 91.2% for K101E/Q/P, 91.2% for T215F/Y, 89.9% for K103N/S and 80.5% for L210W. In terms of individuals, 80% of the alleles were detected in 95.4% CRF01_AE patients, 100% CRF07_BC patients and 83.3% subtype B patients. Importantly, the MALDI-TOF MS results were concordant to the drug resistance profiles of patients obtained from conventional sequencing analysis after excluded the failed detections. Using plasmid templates, the assay was estimated to be sensitive to detect drug resistant variants at level about 20% of the circulating viral population. The capability of this assay to detect mixed viral populations was further verified by two different patient specimens. In conclusion, this study evaluated the use of Sequenom MassARRAY® system for high-throughput detection of HIV-DR mutations towards the commonly used reverse transcriptase inhibitors in China.

No MeSH data available.


Evaluation of the sensitivity of the MALDI-TOF MS for detection of mixed genotypes.Results of MALDI-TOF MS and direct sequencing for the PCR products obtained from different mixtures of the wild type plasmid (M184) and the mutant plasmid (184V) are demonstrated. The ratio of the plasmids in the mixtures are as indicated. The 184V mutant could be detected when the ratio of mutant plasmid to wild type plasmid was as low as 2 to 8 in both MALDI-TOF MS and direct sequencing. The site of mutation is indicated by a box in the sequencing chromatogram.
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pone.0153641.g003: Evaluation of the sensitivity of the MALDI-TOF MS for detection of mixed genotypes.Results of MALDI-TOF MS and direct sequencing for the PCR products obtained from different mixtures of the wild type plasmid (M184) and the mutant plasmid (184V) are demonstrated. The ratio of the plasmids in the mixtures are as indicated. The 184V mutant could be detected when the ratio of mutant plasmid to wild type plasmid was as low as 2 to 8 in both MALDI-TOF MS and direct sequencing. The site of mutation is indicated by a box in the sequencing chromatogram.

Mentions: Since drug resistant HIV-1 may exist in very low frequency, it is necessary to understand the sensitivity of this MALDI-TOF MS based assay for determining the presence of low abundance mutant virus in mixed viral populations. To evaluate the sensitivity of this assay, a total of 100ng plasmid mixture was first prepared by mixing plasmids carrying wild type (WT) genotype (M184 in the RT region) and plasmids carrying mutant genotype (184V in the RT region) in ratios as indicated in Fig 3. The mixtures were then subjected to nested PCR and analyzed by the MALDI-TOF MS. Result in Fig 3 demonstrated the 184V mutant could be detected when mutant to wild type ratio was as low as 2 to 8 (20%). This result was close to the clinical and other conventional genotypic assays, which detected drug resistant variants at level about 20% of the circulating viral population [6,19].


Rapid and Simultaneous Detection of Major Drug Resistance Mutations in Reverse Transcriptase Gene for HIV-1 CRF01_AE, CRF07_BC and Subtype B in China Using Sequenom MassARRAY® System.

Cheung KW, Peng Q, He L, Cai K, Jiang Q, Zhou B, To SW, Yam WC, Liu L, Chen Z, Wang H - PLoS ONE (2016)

Evaluation of the sensitivity of the MALDI-TOF MS for detection of mixed genotypes.Results of MALDI-TOF MS and direct sequencing for the PCR products obtained from different mixtures of the wild type plasmid (M184) and the mutant plasmid (184V) are demonstrated. The ratio of the plasmids in the mixtures are as indicated. The 184V mutant could be detected when the ratio of mutant plasmid to wild type plasmid was as low as 2 to 8 in both MALDI-TOF MS and direct sequencing. The site of mutation is indicated by a box in the sequencing chromatogram.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836728&req=5

pone.0153641.g003: Evaluation of the sensitivity of the MALDI-TOF MS for detection of mixed genotypes.Results of MALDI-TOF MS and direct sequencing for the PCR products obtained from different mixtures of the wild type plasmid (M184) and the mutant plasmid (184V) are demonstrated. The ratio of the plasmids in the mixtures are as indicated. The 184V mutant could be detected when the ratio of mutant plasmid to wild type plasmid was as low as 2 to 8 in both MALDI-TOF MS and direct sequencing. The site of mutation is indicated by a box in the sequencing chromatogram.
Mentions: Since drug resistant HIV-1 may exist in very low frequency, it is necessary to understand the sensitivity of this MALDI-TOF MS based assay for determining the presence of low abundance mutant virus in mixed viral populations. To evaluate the sensitivity of this assay, a total of 100ng plasmid mixture was first prepared by mixing plasmids carrying wild type (WT) genotype (M184 in the RT region) and plasmids carrying mutant genotype (184V in the RT region) in ratios as indicated in Fig 3. The mixtures were then subjected to nested PCR and analyzed by the MALDI-TOF MS. Result in Fig 3 demonstrated the 184V mutant could be detected when mutant to wild type ratio was as low as 2 to 8 (20%). This result was close to the clinical and other conventional genotypic assays, which detected drug resistant variants at level about 20% of the circulating viral population [6,19].

Bottom Line: The development of a rapid, high-throughput and cost-effective HIV-1 drug resistance (HIV-DR) testing system is a challenge for areas consisting different HIV-1 strains.Importantly, the MALDI-TOF MS results were concordant to the drug resistance profiles of patients obtained from conventional sequencing analysis after excluded the failed detections.In conclusion, this study evaluated the use of Sequenom MassARRAY® system for high-throughput detection of HIV-DR mutations towards the commonly used reverse transcriptase inhibitors in China.

View Article: PubMed Central - PubMed

Affiliation: AIDS Institute and Department of Microbiology, Research Centre for Infection and Immunity, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong SAR, People's Republic of China.

ABSTRACT
The development of a rapid, high-throughput and cost-effective HIV-1 drug resistance (HIV-DR) testing system is a challenge for areas consisting different HIV-1 strains. In this study, we established a broadly reactive multiplex assay that could simultaneously detect major drug resistance mutations at 8 loci, which are associated with resistance to commonly used nucleoside reverse transcriptase inhibitors (NRTIs) and Non-nucleoside reverse transcriptase inhibitors (NNRTIs), in specimens of HIV-1 CRF01_AE, CRF07_BC and subtype B, the three major circulating strains in China, using the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) provided by Sequenom MassARRAY® system. To establish the assay, pol gene fragments were prepared from the plasma viral RNA of 159 patients by nested PCR and the presence of wild type and mutant alleles at the 8 loci were analyzed by MALDI-TOF MS. In terms of loci, the detection rate of the alleles was greater than 97% for M41L, K65R, M184V and G190A, 91.2% for K101E/Q/P, 91.2% for T215F/Y, 89.9% for K103N/S and 80.5% for L210W. In terms of individuals, 80% of the alleles were detected in 95.4% CRF01_AE patients, 100% CRF07_BC patients and 83.3% subtype B patients. Importantly, the MALDI-TOF MS results were concordant to the drug resistance profiles of patients obtained from conventional sequencing analysis after excluded the failed detections. Using plasmid templates, the assay was estimated to be sensitive to detect drug resistant variants at level about 20% of the circulating viral population. The capability of this assay to detect mixed viral populations was further verified by two different patient specimens. In conclusion, this study evaluated the use of Sequenom MassARRAY® system for high-throughput detection of HIV-DR mutations towards the commonly used reverse transcriptase inhibitors in China.

No MeSH data available.