Development of Nested PCR and Duplex Real-Time Fluorescence Quantitative PCR Assay for the Simultaneous Detection of Theileria equi and Babesia caballi

• Published in: Frontiers in veterinary science 2022-05, Vol.9, p.873190-873190
• Main Authors: Lv, Kunying, Zhang, Yiwei, Yang, Yixin, Liu, Zheng, Deng, Liang
• Format: Article
• Language: English
• Subjects: Babesia caballi; duplex real-time fluorescence quantitative PCR; equine piroplasmosis; nested PCR; simultaneous detection; Theileria equi; Veterinary Science
• ISSN: 2297-1769; 2297-1769
• DOI: 10.3389/fvets.2022.873190

Equine piroplasmosis (EP) is a type of blood protozoan disease caused by tick-borne parasites, ( ), ( ) and . While many studies have been conducted on EP diagnosis, diagnostic methods exhibiting high sensitivity and specificity remain lacking. Therefore, nested PCR (nPCR) and duplex real-time fluorescence quantitative PCR (qPCR) that can simultaneously detect both and causing agents were established and compared. The two techniques were used to analyze 36 horse blood samples for EP. This set of samples was also detected by a multinested PCR (mnPCR) targeting the gene of and the gene of . By nPCR, duplex real-time fluorescence qPCR and mnPCR, infections with were detected in 16.67% (6/36), 2.78% (1/36), 19.44% (7/36) of the horses, respectively. The prevalence was 58.33% (21/36) by the nPCR, 33.33% (12/36) by the duplex real-time fluorescence qPCR and 2.78% (1/36) by the mnPCR. The overall prevalence of infection with mixed parasites by nPCR was 5.56% (2/36), by duplex real-time fluorescence qPCR was 2.78% (1/36) and by mnPCR 0% (0/36). Results suggest that nPCR can detect and positive samples with good specificity and sensitivity, although distinguishing between the two parasites requires an electrophoresis with 4% agarose gels. The duplex real-time fluorescence qPCR can readily distinguish between and infection, but with low sensitivity.