Rapid, Simple, and Sensitive Detection of Anaplasma phagocytophilum by Loop-Mediated Isothermal Amplification of the msp2 Gene

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Abstract
Anaplasma phagocytophilum is the causative agent of human granulocytic anaplasmosis, which is prevalent throughout China. In this study, we describe a rapid, simple, and sensitive loop-mediated isothermal amplification (LAMP) assay targeting the msp2 gene of A. phagocytophilum that is ideal for application in rural areas in China. This assay has the potential to detect A. phagocytophilum early in infection as an alternative to existing methods. A total of 42 suspected cases of infection with A. phagocytophilum , 15 serologically confirmed and 27 probable cases, were analyzed by the msp2 LAMP assay. To validate the accuracy of LAMP, previously established nested-PCR and real-time PCR assays were utilized. The sensitivity of LAMP was 25 copies per reaction (approximately 1,250 copies per ml blood) for A. phagocytophilum , and the assay did not detect false positives among 27 members of the order Rickettsiales and 17 common clinical pathogens. To evaluate the clinical applicability of the LAMP assay, a total of 42 clinical samples were examined. A positive LAMP result was obtained for 12 of the confirmed cases and for 14 of 27 suspected cases, while only 1 confirmed case and 3 cases (2 confirmed cases and 1 suspected case) were detected by nested PCR and real-time PCR, respectively. The LAMP assay described in this study demonstrated a high level of sensitivity comparable with that of nested PCR and real-time PCR for the detection of A. phagocytophilum . This LAMP assay is a valuable method for rapid, cost-effective, and simple detection of A. phagocytophilum in the rural areas of China.

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