Abstract
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. 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. 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. 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.
Highlights
Plague is a zoonotic disease caused by a gram-negative bacterium, Yersinia pestis, which has been responsible for three major historical pandemics leading to millions of deaths
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 and simplicity of equipment compared to PCR
We developed a rapid, simple and sensitive/specific loop-mediated isothermal amplification (LAMP) method assay for the detection of the caf1 gene sequence that is specific to Y. pestis and to evaluate its performance on biological samples from plague suspected patients from Madagascar
Summary
Plague is a zoonotic disease caused by a gram-negative bacterium, Yersinia pestis, which has been responsible for three major historical pandemics leading to millions of deaths. Plague is endemic in some American, Asian and African countries including Madagascar which reports the vast majority of human plague cases across the globe (85.93% of global cases in 2015) [1]. The World Health Organization (WHO) has classified plague as a re-emerging infectious disease. 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. This is a time consuming procedure; requiring specific devices and well-qualified staff. Strain isolation is challenging if antibiotics have been administered prior to sampling. 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
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