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 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.

Fig 1. Visualization of LAMP products.

(A) stained with SYBR Green I and observed under natural light (Tube 1: Y. pestis, Tube 2: negative control); (B) with agarose gel electrophoresis (Lane 1: Y. pestis, Lane 2: negative control, Lane 3: DNA ladder marker 100 bp).

Fig 2. Optimization of LAMP caf1.

Optimal reaction was found by varying LAMP reaction parameters. Amplification was verified by naked eye with color change from orange to green (positive test) and without color change remaining orange (negative test) and by agarose gel electrophoresis. Optimization results of (A) the reaction time: 30 min (Tubes and Lanes 1–4), 45 min (Tubes and Lanes 5–8) and 60 min (Tubes and Lanes 9–12) (B) the reaction temperature: 57°C (Tubes and Lanes 1–4), 59°C (Tubes and Lanes 5–8), 61°C (Tubes and Lanes 9–12), 63°C (Tubes and Lanes 13–16), 65°C (Tubes and Lanes 17–20), 67°C (Tubes and Lanes 21–24) and 69°C (Tubes and Lanes 25–28) (C) the betaine concentration: 0 M (Tubes 1–4) and 0.95 M (Tubes 5–8).

Fig 3. PCR amplifying the target sequence in 7 Y. pestis strains with the outer primers (F3 and B3).

Lanes 1 to 7: Y. pestis DNA; Lane 8: no DNA template (sterile distilled water); Lane 9: DNA ladder marker 100 bp.

Fig 4. Detection limit of LAMP and PCR caf1.

Ten-fold serial dilutions of Y. pestis DNA extract were tested. (A) Visualization of color change by the naked eye. (B) Confirmation of results by agarose gel electrophoresis. Tubes and Lanes 1, 2, 3, 4, 5, 6, and 7: Y. pestis DNA extracts undiluted stock, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, and no DNA template (sterile distilled water) respectively, Lane 8: DNA ladder marker 100 bp. (C) Detection limit of conventional PCR caf1. Lanes 1, 2, 3, 4, 5, 6, and 7: 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, Y. pestis DNA extracts undiluted stock and no DNA template (sterile distilled water) respectively, Lane 8: DNA ladder marker 100 bp.

Fig 5. Cross-reactions of LAMP caf1.

LAMP reaction was assessed for DNA amplification of 14 pathogens: (A) Eye visualization of LAMP reaction after SYBR Green I addition, (B) visualization after gel electrophoresis migration of the LAMP products. Tubes and Lanes 1: Y. enterocolitica, 2: extraction control, 3: Y. pseudotuberculosis, 4: E. cloacae, 5: E. coli, 6: S. sonnei, 7: P. mirabilis, 8: S. odorifera, 9: S. marcescens, 10: P. aeruginosa, 11: S. aureus, 12: M. tuberculosis (1), 13: M. tuberculosis (2), 14: M. tuberculosis (3), 15: M. tuberculosis (4), 16: P. vivax, 17: P. falciparum, 18: T. solium,19: Y. pestis, 20: no DNA template (sterile distilled water) and Lane 21: DNA ladder marker 100 bp.