Establishment of real-time PCR assay to detect Anaplasma phagocytophilum Msp2 gene with TaqMan MGB probe

Objective To establish a highly specific and sensitive real-time PCR assay to detect Anaplasma phagocytophilum. Methods A pair of primers and a Taq Man MGB probe were designed according to the Msp2 outer membrane gene sequence. By using real-time PCR assay, Anaplasma phagocytophilum was detected in ticks samples collected from Hubei and Hebei provinces. Results A linear relationship between threshold cycle(Ct)of the quantitative real-time PCR and the DNA copy number could be demonstrated(r=0.99). The standard curve showed that 35 copies target genes per reaction can be detected by this assay. The lowest detection limit of this assay was 2 CFU per μl. The assay showed high species specificity and good reproducibility. A total of 426 tick samples were detected by this assay, including 253 haemaphysalis hystricis in 49 groups, 7 samples were positive. Conclusion The results suggested that the real-time PCR assay with TaqMan MGB is highly specific and sensitive for the detection of Anaplasma phagocytophilum and may be used in the diagnosis of this infection.