Establishment Of Bovine Mastitis Pathogens SYBR Green-? Real-time PCR Method

Staphylococcus aureus,Escherichia coli and Streptococcus agalactia are the main pathogens which cause bovine mastitis.This study established a rapidly diagnostic method to detect the above three bacteria separately or together by SYBR Green-? real-time PCR.Simplex PCR:Primer design of single bacteria based on the gene nuc of S.aureus,the gene rrnB of E.coli and the gene turf of S.agalactia.The amplicon of each bacteria was cloned and sequenced then the recombinant plasmid was serial diluted as standard templates to establish the above three kinds of pathogens' standard curve,respectively:S.aureus y=-3.415x+14.02,R2=0.996,E.coli y=-3.498x+9.2777,R2=0.999 and S.agalactia y=-3.298x+10.37,R2=0.998.The efficiency were:96.3%,93.1%and 101.0%,respectively.Minimal detectable concentration of samples were 6.08 × 103 copies/?L,7.18×103 copies/?L and 5.30 × 103 copies/?L,respectively.Melting Curve Analysis indicated high specificity without non-purpose amplicon.Repetitive tests showed that within-batch CV%of S.aureus were 1.56%,0.79%and 1.77%;Between-batch CV%were 1.53%,2.26%and 2.57%;E.coli within-batch CV%were 1.56%,1.52%and 2.46%;between-batch CV%were 3.04%,1.45%,3.75%,respectively.S.agalactia within-batch CV%were2.74%,1.89%and 0.98%.Between batches CV%were 3.02%,1.97%and 1.93%,respectively.As a result,the simplex SYBR Green-I fluorescence quantitative PCR of above three bacteria achieved high specificity,sensitivity and reproducibility.Triplex PCR:based on simplex PCR to qualitative analyze three pathogens according to their different TM value.Melting Curve Analysis indicated the TM values of S.aureus,E.coli and S.agalactia were:79.3-80.3,85.1-86.1 and 81.6-82.6.These data confirmed the possibility to detect three bacteria one time.Melting curve of each pathogen had only one own specific peak,without any non-specific amplicon.Minimal detectable concentration of triplex PCR was inferior to that of simplex PCR.Repetitive tests showed that intra-assay CV%of three kinds of pathogens were:0.1%(S.aureus),0.09%(E.coli)and 0.08%(S.agalactiae),inter-assay CV%were 0.30%(S.aureus),0.44%(E.coli)and 0.43%(S.agalactiae)respectively.The results showed that triplex PCR method also had a high specificity,sensitivity and good stability.In conclusion:This study successfully established simplex and multiplex SYBR Green-I real-time PCR methods for rapidly qualitative or quantitative detection of Staphylococcus aureus,Escherichia coli and Streptococcus agalactiae in cow's mastitis.