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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.


Comparison of the results between the MALDI-TOF MS and direct sequencing.Results of MALDI-TOF MS and direct sequencing for the PCR products obtained from clinical specimens are demonstrated. a) i) A peak at molecular mass 6565.3 indicated a A (M184) to G (184V) mutation in codon 184 of the RT region. ii) A combined result of peaks at molecular mass 6736.5 (left) and 6423.3 (middle) indicated a AA (K103) to AC (N103) mutation in codon 103 of the RT region. The codon is indicated by a box in the sequencing chromatogram. b) The mixed genotypes at codon 184 and 190 detected by MALDI-TOF MS as shown in the table were concordant to the sequencing chromatogram. The specific site of mixed genotype is indicated by a box.
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pone.0153641.g004: Comparison of the results between the MALDI-TOF MS and direct sequencing.Results of MALDI-TOF MS and direct sequencing for the PCR products obtained from clinical specimens are demonstrated. a) i) A peak at molecular mass 6565.3 indicated a A (M184) to G (184V) mutation in codon 184 of the RT region. ii) A combined result of peaks at molecular mass 6736.5 (left) and 6423.3 (middle) indicated a AA (K103) to AC (N103) mutation in codon 103 of the RT region. The codon is indicated by a box in the sequencing chromatogram. b) The mixed genotypes at codon 184 and 190 detected by MALDI-TOF MS as shown in the table were concordant to the sequencing chromatogram. The specific site of mixed genotype is indicated by a box.

Mentions: To validate the MALDI-TOF MS result, the same PCR products of the 159 specimens used for MALDI-TOF MS analysis were sequenced by conventional DNA sequencing. 154 out of 159 specimens were sequenced successfully and the sequences were submitted to Stanford University HIV drug resistance database for generating the antiretroviral drug resistance profile of each specimen. The drug resistance profiles generated from the sequences of drug resistant specimens were concordant to the MALDI-TOF MS results except patient 12_46, after excluded the failed detections. Indeed, the virus of this patient had a 184V mutation in the pol gene fragment when the sequencing chromatogram was carefully analyzed (Fig 4B). A comparison of the MALDI-TOF MS results and the drug resistance profiles of drug resistant specimens from the database is shown in Table 3. Representative results demonstrating the concordance between the chromatograms of DNA sequencing and the MALDI-TOF MS results at two different drug resistance mutation sites (M184V and K103N) are shown in Fig 4A.


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)

Comparison of the results between the MALDI-TOF MS and direct sequencing.Results of MALDI-TOF MS and direct sequencing for the PCR products obtained from clinical specimens are demonstrated. a) i) A peak at molecular mass 6565.3 indicated a A (M184) to G (184V) mutation in codon 184 of the RT region. ii) A combined result of peaks at molecular mass 6736.5 (left) and 6423.3 (middle) indicated a AA (K103) to AC (N103) mutation in codon 103 of the RT region. The codon is indicated by a box in the sequencing chromatogram. b) The mixed genotypes at codon 184 and 190 detected by MALDI-TOF MS as shown in the table were concordant to the sequencing chromatogram. The specific site of mixed genotype is indicated by a box.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153641.g004: Comparison of the results between the MALDI-TOF MS and direct sequencing.Results of MALDI-TOF MS and direct sequencing for the PCR products obtained from clinical specimens are demonstrated. a) i) A peak at molecular mass 6565.3 indicated a A (M184) to G (184V) mutation in codon 184 of the RT region. ii) A combined result of peaks at molecular mass 6736.5 (left) and 6423.3 (middle) indicated a AA (K103) to AC (N103) mutation in codon 103 of the RT region. The codon is indicated by a box in the sequencing chromatogram. b) The mixed genotypes at codon 184 and 190 detected by MALDI-TOF MS as shown in the table were concordant to the sequencing chromatogram. The specific site of mixed genotype is indicated by a box.
Mentions: To validate the MALDI-TOF MS result, the same PCR products of the 159 specimens used for MALDI-TOF MS analysis were sequenced by conventional DNA sequencing. 154 out of 159 specimens were sequenced successfully and the sequences were submitted to Stanford University HIV drug resistance database for generating the antiretroviral drug resistance profile of each specimen. The drug resistance profiles generated from the sequences of drug resistant specimens were concordant to the MALDI-TOF MS results except patient 12_46, after excluded the failed detections. Indeed, the virus of this patient had a 184V mutation in the pol gene fragment when the sequencing chromatogram was carefully analyzed (Fig 4B). A comparison of the MALDI-TOF MS results and the drug resistance profiles of drug resistant specimens from the database is shown in Table 3. Representative results demonstrating the concordance between the chromatograms of DNA sequencing and the MALDI-TOF MS results at two different drug resistance mutation sites (M184V and K103N) are shown in Fig 4A.

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.