Development of a quantitative real-time polymerase chain reaction assay specific for Anaplasma phagocytophila

According to the gltA gene sequences specific for Anaplasma phagocytophila,a pair of primers and one TaqMan-MGB probe were designed. And a quantitative real-time polymerase chain reaction (RtPCR) was developed with primers, probe, and template (gltA gene DNA fragment of A. phagocytophila). The standard curve was established with thegltA gene DNA in DNA sequence detection system (ABI 7900HT) and the relationship between the values of threshold cycle (C_ t) and the DNA copy number was found to be linear (r=0.996). The sensitivity of RtPCR was about 100 times higher than that of the nested PCR used to detect homologous DNA, accompanied by high species-specificity and good repeatability. This RtPCR was used to detect ticks and splenic samples of wild mice in forest and the positive and negative results were corresponded with that detected by nested PCR specific for the pathogen; however, it was more sensitive and reliable than nested PCR. These results suggest that the quantitative real-time PCR is highly specific and sensitive for detection of A. phagocytophila. It is very useful for detection of tiny DNA of A. phagocytophila in samples of animals infected with A. phagocytophila.