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Yersinia pestis detection by loop-mediated isothermal amplification combined with magnetic bead capture of DNA.

ABSTRACT: We developed a loop-mediated isothermal amplification (LAMP) assay for the detection of Y. pestis by targeting the 3a sequence on chromosome. All 11 species of the genus Yersinia were used to evaluate the specificity of LAMP and PCR, demonstrating that the primers had a high level of specificity. The sensitivity of LAMP or PCR was 2.3 or 23CFU for pure culture, whereas 2.3×104 or 2.3×106CFU for simulated spleen and lung samples. For simulated liver samples, the sensitivity of LAMP was 2.3×106CFU, but PCR was negative at the level of 2.3×107CFU. After simulated spleen and lung samples were treated with magnetic beads, the sensitivity of LAMP or PCR was 2.3×103 or 2.3×106CFU, whereas 2.3×105 or 2.3×107CFU for magnetic bead-treated liver samples. These results indicated that some components in the tissues could inhibit LAMP and PCR, and liver tissue samples had a stronger inhibition to LAMP and PCR than spleen and lung tissue samples. LAMP has a higher sensitivity than PCR, and magnetic bead capture of DNAs could remarkably increase the sensitivity of LAMP. LAMP is a simple, rapid and sensitive assay suitable for application in the field or poverty areas.

SUBMITTER: Feng N

PROVIDER: S-EPMC5790586 | biostudies-literature | 2018 Jan - Mar

REPOSITORIES: biostudies-literature

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Yersinia pestis detection by loop-mediated isothermal amplification combined with magnetic bead capture of DNA.

Feng Na N Zhou Yazhou Y Fan Yanxiao Y Bi Yujing Y Yang Ruifu R Zhou Yusen Y Wang Xiaoyi X

Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology] 20170826 1

We developed a loop-mediated isothermal amplification (LAMP) assay for the detection of Y. pestis by targeting the 3a sequence on chromosome. All 11 species of the genus Yersinia were used to evaluate the specificity of LAMP and PCR, demonstrating that the primers had a high level of specificity. The sensitivity of LAMP or PCR was 2.3 or 23CFU for pure culture, whereas 2.3×10<sup>4</sup> or 2.3×10<sup>6</sup>CFU for simulated spleen and lung samples. For simulated liver samples, the sensitivity ...[more]

PMID: 28887007

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Project description:BACKGROUND:Several tests are available for plague confirmation but bacteriological culture with Yersinia pestis strain isolation remains the gold standard according to the World Health Organization. However, this is a time consuming procedure; requiring specific devices and well-qualified staff. In addition, strain isolation is challenging if antibiotics have been administered prior to sampling. Here, we developed a loop-mediated isothermal amplification (LAMP) technique, a rapid, simple, sensitive and specific technique that would be able to detect Y. pestis in human biological samples. METHODS:LAMP primers were designed to target the caf1 gene which is specific to Y. pestis. The detection limit was determined by testing 10-fold serial dilution of Y. pestis DNA. Cross-reactivity was tested using DNA extracts from 14 pathogens and 47 residual samples from patients suffering from non-plague diseases. Specificity and sensitivity of the LAMP caf1 were assessed on DNA extracts of 160 human biological samples. Then, the performance of the LAMP caf1 assay was compared to conventional PCR and bacteriological culture. RESULTS:The detection limit of the developed Y. pestis LAMP assay was 3.79 pg/μl, similar to conventional PCR. The result could be read out within 45 min and as early as 35 minutes in presence of loop primer, using a simple water bath at 63°C. This is superior to culture with respect to time (requires up to 10 days) and simplicity of equipment compared to PCR. Furthermore, no cross-reactivity was found when tested on DNA extracts from other pathogens and human biological samples from patients with non-plague diseases. Compared to the gold standard, LAMP sensitivity and specificity were 97.9% (95% CI: 89.1%-99.9%) and 94.6% (95% CI: 88.6%-97.9%), respectively. CONCLUSION:LAMP detected Y. pestis effectively with high sensitivity and specificity in human plague biological samples. It can potentially be used in the field during outbreaks in resource limited countries such as Madagascar.

Dataset Information

Yersinia pestis detection by loop-mediated isothermal amplification combined with magnetic bead capture of DNA.

Publications

Yersinia pestis detection by loop-mediated isothermal amplification combined with magnetic bead capture of DNA.

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