Dataset Information

Quantitative detection of human Malawi polyomavirus in nasopharyngeal aspirates, sera, and feces in Beijing, China, using real-time TaqMan-based PCR.

ABSTRACT: Human Malawi polyomavirus (MWPyV) was discovered in 2012, but its prevalence and clinical characteristics are largely unknown.We used real-time TaqMan-based PCR to detect MWPyV in the feces (n = 174) of children with diarrhea, nasopharyngeal aspirates (n = 887) from children with respiratory infections, and sera (n = 200) from healthy adults, and analyzed its clinical characteristics statistically. All the MWPyV-positive specimens were also screened for other common respiratory viruses.Sixteen specimens were positive for MWPyV, including 13 (1.47%) respiratory samples and three (1.7%) fecal samples. The samples were all co-infected with other respiratory viruses, most commonly with influenza viruses (69.2%) and human coronaviruses (30.7%). The MWPyV-positive children were diagnosed with bronchopneumonia or viral diarrhea. They ranged in age from 12 days to 9 years, and the most frequent symptoms were cough and fever.Real-time PCR is an effective tool for the detection of MWPyV in different types of samples. MWPyV infection mainly occurs in young children, and fecal-oral transmission is a possible route of its transmission.

SUBMITTER: Ma FL

PROVIDER: S-EPMC5557062 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Quantitative detection of human Malawi polyomavirus in nasopharyngeal aspirates, sera, and feces in Beijing, China, using real-time TaqMan-based PCR.

Ma Fen-Lian FL Li Dan-di DD Wei Tian-Li TL Li Jin-Song JS Zheng Li-Shu LS

Virology journal 20170814 1

<h4>Background</h4>Human Malawi polyomavirus (MWPyV) was discovered in 2012, but its prevalence and clinical characteristics are largely unknown.<h4>Methods</h4>We used real-time TaqMan-based PCR to detect MWPyV in the feces (n = 174) of children with diarrhea, nasopharyngeal aspirates (n = 887) from children with respiratory infections, and sera (n = 200) from healthy adults, and analyzed its clinical characteristics statistically. All the MWPyV-positive specimens were also screened for other c ...[more]

PMID: 28806976

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Project description:BackgroundDNA genotyping with mutation-specific TaqMan(R) probes (Applied Biosystems) is broadly used in detection of single-nucleotide polymorphisms but is less so for somatic mutations because of its limited selectivity for low-level mutations. We recently described coamplification at lower denaturation temperature-PCR (COLD-PCR), a method that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences during the PCR. We demonstrate that combining COLD-PCR with TaqMan technology provides TaqMan genotyping with the selectivity needed to detect low-level somatic mutations.MethodsMinor-groove binder-based or common TaqMan probes were designed to contain a nucleotide that matches the desired mutation approximately in the middle of the probe. The critical denaturation temperature (T(c)) of each amplicon was then experimentally determined. COLD-PCR/TaqMan genotyping was performed in 2 steps: denaturation at the T(c), followed by annealing and extension at a single temperature (fast COLD-PCR). The threshold cycle was used to identify mutations on the basis of serial dilutions of mutant DNA into wild-type DNA and to identify TP53 (tumor protein p53) and EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] mutations in tumors.ResultsCOLD-PCR/TaqMan genotyping identified G>A mutations within TP53 exon 8 (codon 273 mutation hot spot) and C>T mutations within the EGFR gene (drug-resistance mutation T790M) with a selectivity improvement of 15- to 30-fold over regular PCR/TaqMan genotyping. A second round of COLD-PCR/TaqMan genotyping improved the selectivity by another 15- to 30-fold and enabled detection of 1 mutant in 2000 wild-type alleles. Use of COLD-PCR/TaqMan genotyping allowed quantitative identification of low-level TP53 and T790 mutations in colon tumor samples and in non-small-cell lung cancer cell lines treated with kinase inhibitors.ConclusionsThe major improvement in selectivity provided by COLD-PCR enables the popular TaqMan genotyping method to become a powerful tool for detecting low-level mutations in clinical samples.

Dataset Information

Quantitative detection of human Malawi polyomavirus in nasopharyngeal aspirates, sera, and feces in Beijing, China, using real-time TaqMan-based PCR.

Publications

Quantitative detection of human Malawi polyomavirus in nasopharyngeal aspirates, sera, and feces in Beijing, China, using real-time TaqMan-based PCR.

Similar Datasets

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