Establishment And Applicaion Of Multiplex PCR Dignostic Method For Main Pathogens Of Bovine Mastitis

Mastitis is the most common disease in dairy cows.It is an inflammatory reaction when the breast stimulated by physical,chemical and invasion of pathogens.The disease is responsible for large economic losses each year due to lower milk yield and reduced milk quality.In order to make clear the distribution of the main pathogens and serotypes of mastitis in some dairy farms in China,a multiplex PCR rapid diagnostic technique for major pathogens was established.213 clinical and 144 subclinical mastitis milk samples were collected for bacterial isolation.Staphylococcus aureus and Staphylococcus agalactiae were serotyped by multiplex PCR.We developed the method of multiplex PCR for mastitis diagnosis,which targeting at Trueperella pyogenes,Streptococcus parauberis,Streptococcus agalactiae,Staphylococcus aureus and Escherichia coli,and preliminary assembled the diagnostic kit.The experiment results were prganized as follows:1.The main pathogens were identified by the method of 16S rDNA identification.The results showed that the main bacteria isolated from dairy cow mastitis cases were 116 strains of Escherichia coli(36.94%),66 strains Enterococcus faecalis(21.02%),63 strains Streptococcus agalactiae(20.06%),23strains Klebsiella(7.32%),15 strains Staphylococcus aureus(4.78%),14 strains Enterococcus faecium(4.46%),8 strains Shigella(2.55%),3 strains Streptococcus dysgalactiae(0.96%),3 strains Klebsiella pneumoniae(0.96%),1 strain Streptococcus uberis(0.32%),1 strain Pseudomonas aeruginosa(0.32%),1 strain Trueperella pyogenes(0.32%).The result of identification of serotypes indicated that serotype336PS and 5 isolates accounted for 66.67%and 33.33%in the total of pathogenic Staphylococcus aureus respectively,and serotype?a and?isolates accounted for 60.32%and 39.68%in the total of pathogenic Streptococcus agalactiae respectively.2.We designed 5 pairs primers which specific to gene ISR of Trueperella pyogenes,the gene cfb of Streptococcus agalactiae,the gene phoA of Escherichia coli,the gene 23S rRNA of Streptococcus parauberis and the gene nuc of Staphylococcus aureus.Based on the primers,we established a rapid diagnosic method by multiplex PCR.The additive amount of reaction system and the annealing temperature were optimized.The results showed that the specificity from 97.56%to 100%,the sensitivity from 95.65%to 100%.The detection limit of Staphylococcus aureus was 6.25×10~4 cfu/mL,Escherichia coli was 2.95×10~6 cfu/mL,Trueperella pyogenes was 2.95×10~5 cfu/mL,Streptococcus parauberis was 4.3×10~5 cfu/mL,Streptococcus agalactiae was 3.15×10~5 cfu/mL.3.The reagents were assemblied as a detection kit based on the wethod above.The preservation method and shelf life were determined and evaluated.The results indicated that in the state of 4?,expiration date was 2 months.In the state of-20?,expiration date was 4 months.In the state of-80?,expiration date was 6 months.However,repeated freezing and thawing had great influence on the quality of kit.This study has important guiding significance for the rapid detection of major pathogens of cow mastitis in the future,the establishment of reasonable and effective treatment programs,and the improvement of curative effect.