Establishment And Evaluation Of Multiplex PCR For Detection Of Main Pathogenic Bacteria Of Endometritis In Tibetan Sheep

Endometrial inflammation is one of the diseases threatening the health of Tibetan sheep,which seriously hinders the healthy development of Tibetan sheep breeding industry.Its cause is a reproductive disorder caused by pathogenic microorganisms infecting the uterus of sheep,resulting in emaciation and weight loss of sheep,affecting the reproductive performance of sheep.Because sheep endometritis does not appear systemic fever,inflammation,pain and other clinical symptoms,especially latent endometritis,it is easy to be ignored by breeders,which is also the main reason for the increased culling rate of Tibetan sheep,causing serious economic losses to the sheep breeding industry.In this study,a total of 600 Tibetan sheep uteri from Qinghai pastoral areas were collected,single PCR was performed on the samples and a multiplex PCR method was established,and 30 positives were randomly selected for pathogen isolation and identification tests.The results obtained are as follows:(1)A single PCR method was established by culturing standard strains and extracting the genome,selecting specific primers,which had good specificity and high sensitivity.Among the 600 samples detected by single PCR,47.33%were infected with GBS,34.83%with E.coli,6.5%with S.aureus,24.67%with mixed infection of E.coli and GBS,and Salmonella and T.pyogenes were not detected.(2)A multiplex PCR method was established by using the cfb gene of GBS,phoA gene of E.coli and nuc gene of S.aureus as target genes and the genome of standard strains as template.The positive samples detected by single PCR were selected and detected by multiplex PCR.As a result,45.50%were infected with GBS,33.50%with E.coli,6.5%with S.aureus,and 23.67%with mixed infections of E.coli and GBS.Compared with single PCR method,the coincidence rate of single infection of GBS detected by multiplex PCR was 95.65%,that of E.coli was 95.12%,that of S.aureus was 100%,and that of mixed infection of E.coli and GBS was 95.95%.The minimum detection concentrations for the three main pathogens were S.aureus 2.4×10~5 CFU/mL,E.coli 1.03×10~5 CFU/mL,and GBS 2.8×10~5 CFU/mL.(3)Through the isolation and identification of 30 samples,GBS,S.aureus,E.coli and other species were isolated and identified,which were basically consistent with the detection results of multiplex PCR.The results show the multiplex PCR method was successfully established and the main pathogens of endometritis in Qinghai Tibetan sheep were GBS,E.coli and S.aureus.