Dataset Information

Diagnosis of Genus Helicobacter through a hemi-nested PCR assay of 16S rRNA.

ABSTRACT: The present study aimed to establish a genus-specific PCR-based assay to detect helicobacters using 16S rRNA gene as the target template. We designed the hemi-nested primers based on sequences of 16S rRNA gene of 34 types of Helicobacter species. The inclusivity, sensitivity, and specificity of the PCR assay using these primers were examined in three different models, comprising feces simulated samples, BLAB/c mice infection model and clinic patients samples. The detection sensitivity of Helicobacter pylori, Helicobacter hepaticus and Helicobacter bilis strains from feces simulated samples was all 102 CFU/ml. We successfully detected H. hepaticus and H. bilis in the liver, cecum and feces of experimentally infected mice. H. pylori was successfully detected in the feces samples from 3 patients infected with H. pylori while not in the feces samples from 3 healthy human. However, the C97/C05-C97/C98 PCR assay detected H. pylori in the 2 positive samples. Due to the PCR assay's excellent inclusivity, high sensitivity and specificity it may be used to detect the presence of Helicobacters.

SUBMITTER: Qin H

PROVIDER: S-EPMC4881236 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Diagnosis of Genus Helicobacter through a hemi-nested PCR assay of 16S rRNA.

Qin Heping H Tang Guodu G Yi Ping P Pan Xinyi X Huang Huali H Chang Renjie R Shi Zhe Z Ashraf Muhammad Aqeel MA

Saudi pharmaceutical journal : SPJ : the official publication of the Saudi Pharmaceutical Society 20160426 3

The present study aimed to establish a genus-specific PCR-based assay to detect helicobacters using 16S rRNA gene as the target template. We designed the hemi-nested primers based on sequences of 16S rRNA gene of 34 types of Helicobacter species. The inclusivity, sensitivity, and specificity of the PCR assay using these primers were examined in three different models, comprising feces simulated samples, BLAB/c mice infection model and clinic patients samples. The detection sensitivity of Helicob ...[more]

PMID: 27275113

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Project description:BackgroundSequencing of the PCR-amplified 16S rRNA gene has become a common approach to microbial community investigations in the fields of human health and environmental sciences. This approach, however, is difficult when the amount of DNA is too low to be amplified by standard PCR. Nested PCR can be employed as it can amplify samples with DNA concentration several-fold lower than standard PCR. However, potential biases with nested PCRs that could affect measurement of community structure have received little attention.ResultsIn this study, we used 17 DNAs extracted from vaginal swabs and 12 DNAs extracted from stool samples to study the influence of nested PCR amplification of the 16S rRNA gene on the estimation of microbial community structure using Illumina MiSeq sequencing. Nested and standard PCR methods were compared on alpha- and beta-diversity metrics and relative abundances of bacterial genera. The effects of number of cycles in the first round of PCR (10 vs. 20) and microbial diversity (relatively low in vagina vs. high in stool) were also investigated. Vaginal swab samples showed no significant difference in alpha diversity or community structure between nested PCR and standard PCR (one round of 40 cycles). Stool samples showed significant differences in alpha diversity (except Shannon's index) and relative abundance of 13 genera between nested PCR with 20 cycles in the first round and standard PCR (P<0.01), but not between nested PCR with 10 cycles in the first round and standard PCR. Operational taxonomic units (OTUs) that had low relative abundance (sum of relative abundance <0.167) accounted for most of the distortion (>27% of total OTUs in stool).ConclusionsNested PCR introduced bias in estimated diversity and community structure. The bias was more significant for communities with relatively higher diversity and when more cycles were applied in the first round of PCR. We conclude that nested PCR could be used when standard PCR does not work. However, rare taxa detected by nested PCR should be validated by other technologies.

Dataset Information

Diagnosis of Genus Helicobacter through a hemi-nested PCR assay of 16S rRNA.

Publications

Diagnosis of Genus Helicobacter through a hemi-nested PCR assay of 16S rRNA.

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