Abstract:
Objective To establish a loop-mediated isothermal amplification (LAMP) technology to provide a sensitive, specific and simple detection assay for Orientia tsutsugamushi.
Methods Primers were designed with online software Primer Explorer V4 based on groEL gene of Orientia tsutsugamushi. The reaction conditions were optimized. The standard Orientia tsutsugamushi strains was used to evaluate the sensitivity and specificity. A total of 42 blood samples of scrub typhus cases were evaluated using LAMP, conventional PCR and real time fluorescence quantitative PCR.
Results Rapid detection of Orientia tsutsugamushi by colorimetric LAMP assay was completed within 80 min at temperature 61 ℃−65 ℃. The detection limit for LAMP was 10 copies/μL, which was two order of magnitude higher than conventional PCR and one order of magnitude higher than real time fluorescence quantitative PCR. The detection results for 8 pathogens of common natural foci diseases were all negative with a specificity of 100%. A total of 42 blood samples of scrub typhus cases were detected by LAMP, conventional PCR and real time fluorescence quantitative PCR, with the same positive rates of 21.4% (9/42).
Conclusion The visual LAMP assay to detect Orientia tsutsugamushi is a rapid, simple method with high sensitivity and specificity. The method can be suggested to use in primary medical and health institutions.
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