Dataset Information

Development of a Multiplex Real-Time PCR Assay for Rapid Detection of Tigecycline Resistance Gene tet(X) Variants from Bacterial, Fecal, and Environmental Samples.

ABSTRACT: We developed a multiplex real-time SYBR green-based PCR assay for rapid detection of tet(X) and its variants, including tet(X1) and tet(X2) and high-level tigecycline resistance genes tet(X3), tet(X4), and tet(X5). We showed that the real-time PCR assay developed had high linearity (R 2 ? 0.996), sensitivity (low detection limit), and specificity (only the target gene could be amplified significantly) and further evaluated it using bacterial, fecal, and environmental samples.

SUBMITTER: Fu Y

PROVIDER: S-EPMC7179268 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Development of a Multiplex Real-Time PCR Assay for Rapid Detection of Tigecycline Resistance Gene tet(X) Variants from Bacterial, Fecal, and Environmental Samples.

Fu Yulin Y Liu Dejun D Song Huangwei H Liu Zhihai Z Jiang Haiyang H Wang Yang Y

Antimicrobial agents and chemotherapy 20200324 4

We developed a multiplex real-time SYBR green-based PCR assay for rapid detection of tet(X) and its variants, including tet(X1) and tet(X2) and high-level tigecycline resistance genes tet(X3), tet(X4), and tet(X5). We showed that the real-time PCR assay developed had high linearity (R2 ≥ 0.996), sensitivity (low detection limit), and specificity (only the target gene could be amplified significantly) and further evaluated it using bacte ...[more]

PMID: 32041710

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Project description:This study developed a new single-tube multiplex real-time PCR method for detecting toxigenic C. difficile directly from fecal samples using tcdA, tcdB, cdtB, and internal gene tpi as targets, which could be performed on kinds of polymerase chain reaction device including point-of-care testing (POCT), with improved detection efficiency. The specificity, sensitivity, and repeatability of each gene was evaluated using 69 C. difficile isolates and 74 fecal samples. Results were compared with established PCR, qPCR, and ELISA methods. Interspecies specificity was 100% based on six common intestinal pathogens (Escherichia coli, Enterococcus Faecium, Enterococcus faecalis, Clostridium perfringens, Bacteroides fragilis, Clostridium botulinum). The lower detection limit (LDL) for tcdA, tcdB, and cdtB with pure C. difficile DNA was 101,100, and 100 copies/μL, respectively, the coefficients of variation among different experimental batches and within each experimental batch were both less than 3%, which shows that this method has strong repeatability. And the LDL of fecal DNA was 5 × 100, 5 × 103, and 5 × 102 colony-forming units (CFU)/g, respectively. In addition, the efficiency for detection of tcdA was compared with established PCR and real-time PCR methods, demonstrating high consistency (98.4%) and similar sensitivity. ELISA was used to confirm inconsistent results, which were identical with our method. The sensitivity and specificity for detecting toxigenic C. difficile in fecal samples were 96.49% and 94.12% compared with the toxigenic culture (TC). This method effectively identified the toxigenic and non-toxigenic strains with high specificity, sensitivity, and repeatability, and could reduce the false positive rate of tcdA, and accurately identify the typical Asian strain RT017, making it potentially contribute to the surveillance of CDI in China.Supplementary informationThe online version contains supplementary material available at 10.1007/s13205-022-03434-6.

Dataset Information

Development of a Multiplex Real-Time PCR Assay for Rapid Detection of Tigecycline Resistance Gene tet(X) Variants from Bacterial, Fecal, and Environmental Samples.

Publications

Development of a Multiplex Real-Time PCR Assay for Rapid Detection of Tigecycline Resistance Gene <i>tet</i>(X) Variants from Bacterial, Fecal, and Environmental Samples.

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