Dataset Information

Amino Acid Prevalence of HIV-1 pol Mutations by Direct Polymerase Chain Reaction and Single Genome Sequencing.

ABSTRACT: The presence of many highly unusual HIV-1 mutations at a minority variant threshold by next-generation sequence (NGS) may indicate that a high proportion of variants at or just above the threshold represent PCR errors. The validity of this hypothesis depends on the concept that highly unusual mutations detected by population-based sequencing are also highly unusual within a person's virus population. Highly unusual mutations were defined as mutations with a prevalence <0.01% in group M HIV-1 direct PCR population-based Sanger sequences in the Stanford HIV Drug Resistance Database. Single genome Sanger sequences [single genome sequences (SGSs)] were analyzed because they are not subject to PCR error. Permutation analyses compared the proportion of highly unusual mutations in SGSs with the empirical frequencies of these mutations in repeated random selections of population-based sequences. We created a database of 11,258 pol SGSs in 963 plasma samples from 345 persons with active virus replication and analyzed the subset of samples containing 10 or more SGSs. Highly unusual mutations occurred more commonly in samples undergoing SGS compared with population-based sequencing in protease (3.9% vs. 0.8%; p?

SUBMITTER: Tzou PL

PROVIDER: S-EPMC6806363 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Amino Acid Prevalence of HIV-1 <i>pol</i> Mutations by Direct Polymerase Chain Reaction and Single Genome Sequencing.

Tzou Philip L PL Rhee Soo-Yon SY Shafer Robert W RW

AIDS research and human retroviruses 20190826 10

The presence of many highly unusual HIV-1 mutations at a minority variant threshold by next-generation sequence (NGS) may indicate that a high proportion of variants at or just above the threshold represent PCR errors. The validity of this hypothesis depends on the concept that highly unusual mutations detected by population-based sequencing are also highly unusual within a person's virus population. Highly unusual mutations were defined as mutations with a prevalence <0.01% in group M HIV-1 dir ...[more]

PMID: 31317771

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Project description:Background and aimDifferent polymerase chain reaction (PCR) techniques have and are still being used for the direct detection of Brucella DNA in serum samples of different animal species and humans without being validated or properly validated, resulting in discrepancies. Thus, this study aimed to evaluate the diagnostic performance of the TaqMan Real-Time-PCR (RT-PCR) targeting the bcsp31 gene versus conventional PCR for the accurate diagnosis of brucellosis at the genus level in cattle sera.Materials and methodsOne hundred and eighty-four serum samples were collected from bacteriologically positive and negative cows with ages ranging from 1 to 5 years old at some infected private farms in the Nile Delta under quarantine measures as well as brucellosis free farms. These samples were classified into four groups after serological diagnosis and investigated by TaqMan RT-PCR and conventional PCR targeting the IS711 gene for Brucella DNA detection. The diagnostic performance characteristics of both PCR techniques were estimated considering the bacteriological results as a gold standard.ResultsTaqMan RT-PCR revealed superiority over conventional PCR; it was able to detect Brucella DNA in 95% (67/70) and 89% (25/28) of the cattle sera samples belonging to Group 1 (serologically and bacteriologically positive) and Group 2 (serologically negative but bacteriologically positive), respectively. On evaluating the diagnostic performance, TaqMan RT-PCR showed superior diagnostic sensitivity (93.9%), diagnostic specificity (88.4%), performance index (182.3), almost perfect kappa agreement (0.825±0.042), strong positive correlation (r=0.826), high accuracy based on the receiver operating characteristic (ROC) curve, and area under the ROC curve (0.911) at p<0.05 and CI of 95%.ConclusionA cattle serum sample is not the metric of choice for targeting Brucella genomic DNA by conventional PCR. The time-saving and rapid TaqMan RT-PCR method revealed a better diagnostic performance in the detection of Brucella DNA in cattle sera. Such performance offered by TaqMan RT-PCR may be considered a step toward the possibility of using such technology in the direct differentiation between Brucella-infected and -vaccinated cattle immunized by smooth vaccines from cattle sera using primers specific for such vaccines.

Dataset Information

Amino Acid Prevalence of HIV-1 pol Mutations by Direct Polymerase Chain Reaction and Single Genome Sequencing.

Publications

Amino Acid Prevalence of HIV-1 <i>pol</i> Mutations by Direct Polymerase Chain Reaction and Single Genome Sequencing.

Similar Datasets

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