Establishment And Application Of PCR Nucleic Acid Immunochromatographic Strip For Detection Of Four Important Pathogenic Bacteria In Dairy Cow Mastitis

Bovine mastitis is a major disease that affects cows worldwide and is caused by inflammation of the mammary glands.The main cause of dairy cow mastitis comes from the infection of pathogenic microorganisms is including bacteria,fungi,viruses,and mycoplasma.Among them,bacterial infections account for the main parts in terms of detection rate,the distribution trend,with Streptococcus agalactiae,Staphylococcus aureus,Escherichia coli and Streptococcus dysgalactiae as the highest four bacterial pathogens.In order to find an efficient,convenient,sensitive and specific detection method for timely and effective treatment and reducing the loss of animal farmers,in this study,a PCR nucleic acid immunochromatographic test strip for detecting the common pathogens of dairy cow mastitis was developed.Based on the sequence alignment analysis published by GenBank,specific primers were designed for the highly conserved nuc gene of S.aureus,cfb gene of S.agalactiae and the 16-23s rDNA sequences of E.coli and S.dysgalactiae.By modifying FITC of the forward primer and DIG of the reverse primer;we prepare 10nm colloidal gold solution by citric acid reduction method,and optimize 10 μL 0.1 mol/L potassium carbonate solution to adjust the labeled antibody pH and 5 μL fluorescein isothiocyanate antibody conjugated gold particles,blocking solution(10%BSA,0.25%PEG2000,0.05%Tween-20,0.05%Triton-x100),gold standard pad treatment solution(5%sucrose,0.3%Tween-20,1%trehalose,0.25%PEG2000),and the sample pad treatment solution A(0.05%Sodium chloride,0.5%Triton-x100.To prepare the sample pad with pH 8.0 0.01M Tris-Hcl solution,we selected NC membrane(Sartorius CN 140),and spray 1mg/mL FITC antibody on the T line of the NC membrane,and 1mg/mL goat anti-mouse secondary antibody on the C line.Then the PCR nucleic acid immunochromatography test strip was assembled.The pure culture of bacteria was tested,and the results showed that the prepared PCR nucleic acid immunochromatographic test strip had good specificity in detecting S.agalactiae,S.aureus,E.coli and S.dysgalactiae.The performance is consistent with the results of gel electrophoresis.The minimum detection limits of nucleic acid immunochromatography test strips for S.agalactiae(2×10~2 CFU/mL),S.aureus(4×10~2 CFU/mL),and S.dysgalactiae(1.6×10~3 CFU/mL)were 10,10,10 times higher than gel electrophoresis method,respectively.The minimum detection limit of test strips of E.coli(5.7×10~3 CFU/mL)was equivalent to that of gel electrophoresis method.Though comprehensive optimization of test results and experimental materials and with unified standards,the PCR nucleic acid immunochromatographic test strip detection kit was assembled.The kit showed good repeatability between batches and within batches,and the test strip is sealed in an aluminum foil bag.The repeatability test after 6 months of storage at 4℃ still has good stability.In the detection of artificially contaminated fresh milk samples,the test strips showed good specificity;and in the sensitivity test,The minimum detection limits of nucleic acid immunochromatography test strips for S.agalactiae(2×10~3 CFU/mL),S.aureus(4×10~5 CFU/mL),E.coli(5.7×10~5 CFU/mL)and S.dysgalactiae(1.6×10~4 CFU/mL)were 10,10,10,100 times higher than gel electrophoresis method,respectively.The results indicated that the PCR nucleic acid immunochromatographic test strip detection method established in this study could avoid the complex operation of gel electrophoresis after nucleic acid amplification,and incorporating test strip detection technology makes the detection method safer,simpler,faster and more convenient,which could be used as a new method to replace nucleic acid gel electrophoresis,with an important guiding significance for the rapid detection of dairy cow mastitis pathogens and reasonable medication.This technology would become a universal detection technology method and have a good application prospect.