| Bovine mastitis is one of the common diseases of dairy cow, reduction in milk production and poor quality often lead to some public health problems, so the disease for dairy farmers and dairy processing industry is a heavy economic burden, the measures for the prevention and control of the industry sustainable development is particularly important. This study selected a cattle farm in Nanning regularly collected milk samples of mastitis were monitored to investigate the incidence situation. At the same time the main pathogenic bacteria be isolated and drug sensitivity test be done; selection of the genus Staphylococcus aureus NUC gene, Streptococcus conserved sequence extension factor Tu gene, Escherichia coli B gene conserved sequence, establish a rapid detection (visual lamp method); specific studies are as follows:Test one:Nanning Dairy Mastitis dynamic monitoring2015 from March to October in Nanning, Guangxi,50 individual milk sample and three bulk tank milk sample was taken each month randomly,respectively adopt somatic cell count test, CMT and recessive mastitis cows test original milk samples for testing. The results show that the CMT method and somatic cell count in the basic consistent, although the CMT method is the classical method, but only the results of estimate and error, and strip is not very good reaction results, so cattle in the usual mastitis prevention and control supervision is measured on the best use of somatic cell count.Test two:the main mastitis pathogen identification and drug sensitivity testIn oder to understanding major mastitis pathogens Staphylococcus bacteria staphylococci, streptococci and Escherichia coli resistance,228 milk samples were randomly collected for somatic cell count testing and screened 75 copies of the isolation, identification and drug sensitivity test of bacteria detection, of which 167 suspicious strains, and the students of identification to determine the 28 strains of Staphylococcus aureus,16.8%, Streptococcus agalactiae 10 strains, stop Streptococcus lactis 4 strains, Streptococcus uberis 2 strains, accounted for 9.6%, Escherichia coli 35 strains, accounted for 21.0%. Separation of Staphylococcus aureus, Streptococcus, Escherichia coli resistance rate were 25.9% and 35.3%、38.3%, Escherichia coli isolated strains to penicillin has been 100% resistance, to erythromycin, amikacin resistance is higher, to ampicillin and enrofloxacin also appeared in the resistance;Staphylococcus aureus to ampicillin and has 100% resistance, enrofloxacin, erythromycin also had higher resistance, and of acetyl roxithromycin, doxycycline has been part of the resistance; Streptococcus of amikacin has 100% resistance. Among them, three kinds of bacteria had high sensitivity to gentamicin and cefazolin.Test three:establishment and application of visual LAMP detection methodAiming at the conserved sequence of the nuc gene of Staphylococcus aureus, Streptococcus, the elongation factor Tu gene and the B gene of Escherichia coli, the gene of Escherichia coli was designed by using online software ExplorerV4 Primer. Is internal and external primers concentration ratio has been optimized, so as to establish consists of three kinds of main pathogenic bacteria in dairy cattle mastitis visual lamp detection method, and the 66 clinical milk samples were detected. The results show that the reaction system of 25 u L lamp visualization method with a liquid volume is basically the same, reaction conditions are basically the same, only inner primers concentration slightly has the difference, so detection can prior to the reaction solution configuration at the same time. The results are simple and clear, no need to have a high professional quality. In this study, three strains of pathogenic bacteria and other test standards were amplified, and the results were consistent with the traditional method and the PCR method, and the specificity was good. DNA detection limit of LAMP to Streptococcus, Staphylococcus aureus and Escherichia coli were 56 fg/L,46.5 fg/L,8.00fg/L. The DNA templates obtained from artificial pollution samples were obtained by boiling method, microwave method and reagent box method, and the microwave method was the best choice in terms of time and cost. In the visualization through the distribution of liquid added in advance calcein and containing chelators Mn2+ MnCl2 solution, in order to achieve the visual observation of the effect, also can avoid like other fluorescent dyes reaction after the addition and aerosol generating, which in a certain extent can avoid the occurrence of false positive results. |
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