Development And Application Of A QPCR Method For The IS900 And F57 Genes Of Mycobacterium Paratuberculosis In Cattle

Cattle paratuberculosis is a chronic digestive system disease of ruminants caused by Mycobacterium paratuberculosis(MAP),which has caused huge economic losses to the cattle industry in China.In order to establish a fast,simple,specific,and sensitive detection method for Mycobacterium paratuberculosis,this experiment designed specific primers and probes within their highly conserved regions based on the sequences of the IS900 and F57 genes of Mycobacterium paratuberculosis published on Gen Bank.By optimizing the primers,probe concentration,and annealing temperature,PCR detection methods for the IS900 and F57 genes of Mycobacterium paratuberculosis were established,respectively Double gene fluorescence quantitative PCR detection method of Mycobacterium paratuberculosis,and the quantitative PCR method established in this experiment was used to conduct molecular epidemiology investigation of Mycobacterium paratuberculosis in cattle farms in some regions of the country.The results showed that the two established detection methods for Mycobacterium paratuberculosis could only detect the IS900 and F57 nucleic acids of Mycobacterium paratuberculosis from various pathogens of bovine diarrhea diseases,indicating that the method established in this experiment has good specificity.The target band amplified by MAP F57 is approximately 222 bp,and the minimum detectable copy number is 1×10~4copies/μL.The target band amplified by MAP IS900 is approximately 328 bp,and the minimum detectable copy number is 1×10~3copies/μL.In the dual fluorescence quantitative PCR detection method,the minimum nucleic acid detection amount of F57 can reach 5copies/μL.The minimum nucleic acid detection amount of IS900 can reach 2.5 copies/μL.Both have high sensitivity.Both detection methods have repeatedly amplified the target fragment with the same size as the target band.The intra batch and inter batch variation coefficients of the dual fluorescence quantitative PCR detection method are both less than1%,indicating that the method established in this experiment has good repeatability.The dual gene fluorescence quantitative PCR detection method established in this experiment was applied to detect 363 nucleic acid samples from cattle farms in some regions of China.The results showed that a total of 57 positive samples were detected;Among them,52 were F57 positive,14 were IS900 positive,and 9 were double gene positive samples.The positive rates of cattle farms in Inner Mongolia,Ningxia,Xinjiang,Shanxi,Henan,and Heilongjiang were 13.90%,5.55%,26.82%,21.42%,10.34%,and68.18%,respectively.This study provides different detection methods for rapid detection of Mycobacterium paratuberculosis,with simple operation,strong specificity,high sensitivity,and good stability,providing technical support for epidemiological investigation and rapid diagnosis of Mycobacterium paratuberculosis.