Development Of Real-time Quantitative PCRs For Detection Anaplasma Ovis And A. Bovis

Anaplasma is a tick-borne endoglobular rickettsial pathogen of ruminants, which causedisease are called Anaplasmosis. The known pathogenes include A. marginale, A. ovis, A.centrale, A. bovis, A. phagocytophilum and A. platys. The establishment of efficient, rapid andaccurate diagnostic method, has an important role in the detection and surveillence of thedisease. Real-time quantitative PCR technique has the merits of specific, sensitive,reproducible, accurate and fast, so that it has became the popular technology for research andclinical diagnosis.At present, there is no Real-time quantitative PCR for detecting A. ovis andA. bovis in the world.Cloned and sequenced citrate synthase gene (gltA) for12isolates A. ovis, then,species-specific primers and TaqMan probe were designed based on gltA gene, a real timequantitative PCR assay was developed for A. ovis. No cross-reactions were observed with A.marginale, A. bovis, A. phagocytophilum, Chlamydia psittaci, Mycoplasma mycoides,Borrelia burgdorferi s. l., Babesia sp.(Xinjiang isolate) and Theileria luwenshuni. Analyticsensitivity results revealed that the Real-time PCR assay could detect as few as10copies ofgltA gene. The intra-assay and inter-assay CVs were satisfactory low (0.26%to0.97%,0.42%to1.5%, respectively), indicating high reproducibility of the established real-time PCR.Theperformance of the Real-time PCR was assessed by testing254field samples and comparingwith the results from a conventional PCR, the detection rates (25.6%versus9.06%).Theresults demonstrated that the Real-time PCR was significantly more sensitive than theconventional PCR. Our results indicated that the Real-time PCR is a useful approach fordetecting A. ovis infections.Species-specific primers and TaqMan MGB probe were designed based on16S rRNAgene of A. bovis and similar pathogens, a real time quantitative PCR assay was developed forA. bovis No cross-reactions were observed with A. marginale, A. ovis, A. phagocytophilum,Theileria ovis, Theileria luwenshuni, Theileria sinensis, Babesia sp. Xinjiang isolate, Babesiabovis, Babesia bigemina, Chlamydia psittaci, Mycoplasma mycoides, Borrelia burgdorferi s.l., Toxoplasma gondii. Analytic sensitivity results revealed that the Real-time PCR assaycould detect as few as10copies of16S rRNA gene.The intra-assay and inter-assay CVs weresatisfactory low (0.10%to1.01%,0.61%to1.88%, respectively), indicating highreproducibility of the established real-time PCR. The performance of the Real-time PCR wasassessed by testing196filed samples and comparing with the results from a nested PCR, thedetection rates (62.2%versus56.6%).The results demonstrated that the Real-time PCR wassignificantly more sensitive than the nested PCR. Our results indicated that the Real-timePCR is a useful approach for detecting A. bovis infections.