Application of LAMP and TaqMan qPCR for the rapid diagnosis of Anaplasma Capra (an emerging tick-borne zoonotic pathogen) and comparison with Nested-PCR

Anaplasma capra was first detected in goats from China in 2012, and in the process, its presence has been demonstrated in humans, domestic animals, wild animals, and ticks in three continents (Asia, Europe, and Africa). Although there is limited information, it is thought that the agent may cause clinical symptoms in humans and animals. This study aimed to develop two molecular diagnostic assays [Taqman real-time PCR (qPCR) and Loop-Mediated Isothermal Amplification (LAMP)] that can be used in the diagnosis of A. capra, including known genotypes, by targeting the groEL gene, and to compare the limit of detection (LoD) and the effectiveness in field samples with nested-PCR. The detection limits of groEL-nested-PCR, qPCR, and LAMP methods were also investigated. PCR, qPCR, and both colorimetric and conventional LAMP assays were able to identify 8.25 × 104, 8.25 × 103, and 8.25 × 102 copies of A. capra DNA/µL in the sample, respectively. According to this finding, qPCR and LAMP had detection limits that were 10 and 100 times greater than PCR’s, respectively. As a result of 150 field samples (sheep, goat, cattle, buffalo, dog, and cat) analyses, two (1.33%) positives in nested-PCR, four (2.66%) positives in qPCR, and five (3.33%) positives in LAMP were detected. The level of agreement between assays was evaluated using Cohen’s kappa test, and consistency was observed between the methods. In conclusion, this study has demonstrated that LAMP and qPCR, which are being used for the first time to diagnose A.capra by targeting the groEL gene, are more sensitive than nested-PCR. These tests, which have a high level of sensitivity, will be beneficial in understanding the pathogen’s epidemiology.