Dataset Information

Evaluation of AMPLICOR Neisseria gonorrhoeae PCR using cppB nested PCR and 16S rRNA PCR.

ABSTRACT: Certain strains of Neisseria subflava and Neisseria cinerea are known to produce false-positive results with the AMPLICOR Neisseria gonorrhoeae PCR (Roche Diagnostic Systems, Branchburg, N.J.). The analytical sensitivity and analytical specificity of three PCR tests were assessed with 3 geographically diverse N. gonorrhoeae strains and 30 non-N. gonorrhoeae Neisseria spp. The sensitivities of the in-house nested cppB gene and the 16S rRNA PCR methods were greater than that of the AMPLICOR N. gonorrhoeae PCR with purified DNA from all 3 N. gonorrhoeae strains. Six of 14 clinical strains of N. subflava (1 from a vaginal swab, 5 from respiratory sites) produced false-positive AMPLICOR N. gonorrhoeae PCR results and were negative by the two other PCR methods. When applied to 207 clinical specimens selected from a population with a high prevalence ( approximately 9%) of infection, the results for 15 of 96 (15.6%) AMPLICOR-positive specimens and 14 of 17 (82.3%) AMPLICOR-equivocal specimens were not confirmed by the more sensitive nested cppB PCR method. Only 2 of 94 (2.1%) of AMPLICOR N. gonorrhoeae PCR-negative specimens from the same population tested positive by the nested cppB method. These results suggest that for this population the AMPLICOR N. gonorrhoeae PCR test is suitable as a screening test only and all positive results should be confirmed by a PCR method that is more specific and at least as sensitive. This study also illustrates that caution should be used when introducing commercially available nucleic acid amplification-based diagnostic tests into the regimens of tests used for populations not previously tested with these products.

SUBMITTER: Farrell DJ

PROVIDER: S-EPMC84316 | biostudies-literature | 1999 Feb

REPOSITORIES: biostudies-literature

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Evaluation of AMPLICOR Neisseria gonorrhoeae PCR using cppB nested PCR and 16S rRNA PCR.

Farrell D J DJ

Journal of clinical microbiology 19990201 2

Certain strains of Neisseria subflava and Neisseria cinerea are known to produce false-positive results with the AMPLICOR Neisseria gonorrhoeae PCR (Roche Diagnostic Systems, Branchburg, N.J.). The analytical sensitivity and analytical specificity of three PCR tests were assessed with 3 geographically diverse N. gonorrhoeae strains and 30 non-N. gonorrhoeae Neisseria spp. The sensitivities of the in-house nested cppB gene and the 16S rRNA PCR methods were greater than that of the AMPLICOR N. gon ...[more]

PMID: 9889224

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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

Evaluation of AMPLICOR Neisseria gonorrhoeae PCR using cppB nested PCR and 16S rRNA PCR.

Publications

Evaluation of AMPLICOR Neisseria gonorrhoeae PCR using cppB nested PCR and 16S rRNA PCR.

Similar Datasets

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