Establishment And Application Of Triple PCR Detection Method For Pathogenic Bacteria Staphylococcus Aureus, Streptococcus Agalactiae And Pseudomonas Aeruginosa In Cows With Recessive Mastitis

Test 1: Establishment of multiple PCR detection methods for three pathogenic bacteria of cow subclinical mastitis.Specific primers were designed based on the S.aureus NUC thermotolerant gene,S.agalactiae 16 S rRNA gene,and P.aeruginosa ETA gene sequence.The optimal detection system of multiplex PCR was established by optimizing the annealing temperature,template ratio and primer ratio of PCR reaction,and the sensitivity and specificity tests were carried out simultaneously.The results showed that the detection method could simultaneously amplify a specific fragment of S.aureus 153 bp,S.agalactiae 270 bp and P.aeruginosa 375 bp and a universal fragment of bacterial 16 S rRNA 1500 bp,while for the control group,Bacillus,Escherichia coli,faecal bacterium,Citrobacter,Acinetobacter,and Staphylococcus aureus can only amplify a universal fragment of 1500 bp;the sequence of the amplified gene is homologous to the sequence of the three bacteria on Genbank.99%,with strong specificity.The minimum sensitivity of the system to S.aureus,S.agalactiae,and P.aeruginosa for 24 h enrichment culture was 4 x 104 cfu/mL,8 x 104 cfu/mL,and 1.5 x 106 cfu/mL,respectively.The corresponding DNA concentrations were 5.97 x 10-2 ng/μL,6.68 x 10-2 ng/μL,and 5 x 10-2 ng/μL,respectively.Test 2: Comparison of two detection methods for S.aureus,S.agalactiae and P.aeruginosa in milkIn order to compare the differences between S.aureus,S.agalactiae and P.aeruginosa in the detection of multiplex PCR and traditional bacterial isolation and identification methods,100 milk samples were sent to Sichuan dairy farms(5 of which were normal milk samples.35 samples of milk for subclinical mastitis and 60 samples of clinical mastitis)were tested.The results showed that milk samples of S.aureus,S.agalactiae and P.aeruginosa were detected in 60 clinical mastitis milk samples,and 14,14 and 8 samples were obtained by bacterial isolation and identification,respectively.The methods were 14 parts,13 parts and 12 parts,respectively.The detection coincidence rates of the two methods were 100%,96.6% and 93.3%,respectively.In 35 milk samples of subclinical mastitis,the milk samples of S.aureus,S.agalactiae and P.aeruginosa were detected,and 10,20 and 1 copies were respectively determined by bacterial isolation and identification,and the multiplex PCR was 6 respectively.The fractions of 19 parts,0 parts,and the detected anastomoses were 88.5%,97.1%,and 97.1%,respectively.In 5 normal milk samples,no bacteria were detected in either method.The total number of milk samples of S.aureus,S.agalactiae and P.aeruginosa was 24,34 and 9 respectively.The detection rate was 24%,34% and 9%.The multiplex PCR assays were 20,32,and 12,respectively;the detection rates were 20%,32%,and 12%.The detection coincidence rates of S.aureus,S.agalactiae and P.aeruginosa in all samples were 96%,98%,and 97%,respectively.Trial 3: Effects of three pathogen infections of S.aureus,S.agalactiae and P.aeruginosa on milk composition of cows with subclinical mastitisIn order to investigate the effects of S.aureus,S.agalactiae and P.aeruginosa infection on the milk components of dairy cows with subclinical mastitis(such as fat,protein,carbohydrate,inorganic salts,non-fat solids,etc.),according to the results of milk sample identification,select In the subclinical mastitis,only 4 samples of S.aureus-positive milk samples were recorded as group A,and only 14 samples of S.agalactiae-positive milk samples were recorded as group B,and S.aureus+S.agalactiae-positive was detected.5 samples of milk samples were recorded as group C,and 5 samples of normal milk samples(control group)were recorded as group D.Only one of the milk samples positive for S.aureus+S.agalactiae+P.aeruginosa was recorded as group E(but not included in the statistical analysis),and the milk component was measured.RESULTS: In the A-D group,the levels of fat,protein,carbohydrate,non-fat solids,and inorganic salts in the control group(D group)were significantly higher than those in the other groups(groups A,B,and C)(P ≤ 0.05),wherein the content of fat,protein,carbohydrates,and non-fat solids in milk is extremely different(P ≤ 0.01).The single-infected group A(S.aureus)and group B(S.agalactiae)in the protein,carbohydrate,non-fat solids,inorganic salts and other indicators were higher than the mixed infection group C(S.aureus + S.agalactiae),but no significant difference(P>0.05).The fat content of group C was significantly lower than that of group A and B(P ≤ 0.01).Among them,the content of fat,protein,carbohydrate,non-fat solids and other indicators in group E were lower than the average of groups A,B,C,and D.In summary,the milk quality of S.aureus,S.agalactiae and P.aeruginosa infection caused by subclinical mastitis decreased,and the contents of fat,protein,carbohydrate,inorganic salt and non-fat solids in milk decreased significantly.In conclusion: 1.According to S.aureus NUC heat-resistant gene,S.agalactiae 16 S rRNA gene and P.aeruginosa ETA gene sequence,three pairs of specific primers were designed to establish multiplex PCR detection method.2.Two methods were used to detect S.aureus,S.agalactiae and P.aeruginosa pathogens by bacteriological detection and multiplex PCR.The coincidence rate of the test results was 96%,98% and 97%.Comparing the two methods,the coincidence rate is higher and the difference is not significant.3.The milk quality of cows with subclinical mastitis caused by S.aureus,S.agalactiae and P.aeruginosa is reduced,and the contents of fat,protein,carbohydrate,inorganic salts and non-fat solids in the milk are significantly decreased.