Development and evaluation of loop-mediated isothermal amplification for detection of Yersinia pestis in plague biological samples

Abstract

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

Yersinia pestis antibiotic resistance: a systematic review

“…Another LAMP assay targeting the caf1 gene was developed for detection of Y. pestis in plague biological samples. The sensitivity and specificity of this test were quite reliable compared with the gold standard [31]. In fact, the diagnosis of Y. pestis is generally confirmed by a combination of several detection methods.…”

A Novel Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Yersinia pestis

“…It is extremely difficult to design paired loop primers for Y. pestis LAMP assays when targeting caf1 . In the previously reported Y. pestis LAMP assays, only one assay included two paired loop primers ( Singh et al, 2020 ), while other assays used only one single loop primer in their assay development ( Nunes et al, 2014 ; Randriantseheno et al, 2020 ), which possibly was because the primers failed to generate. Using caf1A , we were able to generate paired loop primers, which gave us a total of six caf1A -specific primers ( Table 1 ) for our LAMP assay development.…”

“…LAMP techniques are increasingly used in molecular diagnostics as a potential alternative to PCR-based methods ( Pal et al, 2018 ; Bodulev and Sakharov, 2020 ; Mukama et al, 2020 ) and already have been successfully applied for detection of bacteria, fungi, parasites, and viruses ( Kuboki et al, 2003 ; Gonçalves et al, 2014 ; Lass et al, 2017 ; Lu et al, 2020 ). Several LAMP assays for Y. pestis detection have been developed as well ( Nunes et al, 2014 ; Feng et al, 2018 ; Randriantseheno et al, 2020 ; Singh et al, 2020 ).…”

“…Nevertheless, not all three plasmids are present in all Y. pestis strains ( Butler, 1972 ; Filippov et al, 1990 ; Meka-Mechenko, 2003 ). Previously developed Y. pestis LAMP assays have mainly used caf1 ( Nunes et al, 2014 ; Randriantseheno et al, 2020 ; Singh et al, 2020 ), a highly specific gene that is situated in the pMT1 plasmid and plays an essential role for Y. pestis virulence by expressing the capsular antigen fraction 1 (Caf1) protein ( Sebbane et al, 2009 ; Al-Jawdah et al, 2019 ). However, paired loop primers seemed difficult to generate when using the caf1 gene.…”

“…However, paired loop primers seemed difficult to generate when using the caf1 gene. There was only one loop primer included in previous assays ( Nunes et al, 2014 ; Randriantseheno et al, 2020 ). In this study, we developed a new LAMP assay for rapid detection of Y. pestis .…”