Fluorescence Quantitative PCR For Detecting Anaplasma Marginale And Sequence Analysis Of The MSP4 Gene Of Anaplasma Marginale

The objectives of the present study were to examine sequence variation in the major surface protein 4 (MSP4) gene of Anaplasma.marginale isolates from Hunan province. The pMSP4 sequences were amplified from each A. marginale sample and the amplicons were cloned into pGEM-T Easy vector, respectively. The inserts were successfully sequenced, and the length of pMSP4 were 530 bp. Sequence comparison revealed that the similarity in pMSP4 sequences among Hunan isolates that of the A. marginale available in GenBankTM were more than 98.3%. There is no significant variation in pMSP4 sequences within A. marginale, while inter-species difference is obvious. The results indicate that the pMSP4 sequences provide useful genetic markers for molecular identification of A. marginale. The results of this study lay down the foundation for further study on molecular epidemiology and diagnostics of A. marginale.The second part of this study, To develop a SYBR Greenl fluorescent quantitative PCR method for detecting Anaplasma.marginale MSP4 gene and estimate it with lab technology. using A. marginale MSP4 sequences to design pair of primers AmspF4/R4 and constructing standard plasmid containing the pMSP4 gene,the SYBR GREEN quantitative Real Time PCR based on pMSP4 gene has been established, which is precise, quick, sensitive, specific, repeatable and contamination-free. This method can detect 104 copies of standard plasmid DNA perμL. The detection of Eperythrogoonosis, swine cysticercosis,heterakis and Histomoniasis was negative. Compare with traditional PCR method, The method is more specific, repeatable, sensitive and accurate.