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Sequenced strains used in this study as a positive control, susceptibility quality control bacteria the ATCC25922 extraction of DNA was used as a negative control, PCR assay for detection of bacteria of animal origin tetracycline drug resistance gene triple. For the of Mg 2 , dNTPs, Taq enzyme, primers such as the amount and annealing temperature cycles cycle parameters to optimize and assembly kit. Optimization Results are as follows: 10 × PCR Buffer 5μL concentration of 25mM/LMgCl 2 3.0μL, 2.5mmol / L of the concentration of each dGTP, dCTP, dATP and dTTP mixture 4μL concentration of 2.5U/ULTaq DNA polymerase 0.35μL, concentration was 25mmol / L three kinds of resistance genes tetA gene, tetC gene and tetM gene specificity downstream primer material (25 mmol / L), respectively 1.0μL, 0.3 μL 0.4 μL. Placing a sterile thin-walled PCR reaction tube, add the prepared template 5μL supply water to a total volume of 50μL 20μL mineral oil cap can. After repeated experiments to optimize the reaction conditions of the kit, the optimal PCR amplification cycle parameters: denaturation at 94 ° C for 5min; then enter the cycle: 94 ° C denaturation 60s, 56 ° C annealing 60s, 72 ° C extension 60s, a total of 35 cycles final extension at 72 ℃ for 10min, electrophoretic observations. Application of the test kit and traditional susceptibility testing methods (K-B method) simultaneous detection of 100 bacterial resistance genotype and phenotype, and compare the two testing process and test results. The results showed that the highest rate of bacterial resistance to tetracycline: 92% (92/100), followed by doxycycline resistance rates: 84% (84/100) of minocycline-resistant The rate is relatively the lowest, as follows: 75% (75/100). 100 to be tested bacteria, the tetA gene detection rate of 46% (46/100), tetC gene detection rate: 38% of (38/100), the tetM gene detection rate: 33% ( 33/100). Two detection results meet the rate of 89%. More conventional resistance gene detection methods: single-plex PCR methods and kits using triplex PCR method, the test results was 100%. On this basis, the kit specificity, sensitivity, reproducibility and shelf life testing of the system. The test results show that: This kit is highly specific, specific amplification purposes only resistance gene. This kit has a high sensitivity (4.0 × 10 4 cfu / mL); stability and performance, showed a good intra-and inter-reproducibility (100%). The kit has a shelf life of six months under -20 ℃. The kit developed resistance testing of a sample of 24 Chinese provinces. The results show that the distribution of animal source bacteria of different species, different sources of animals, different era of the E. coli tetracycline-class drug resistance gene there is a big difference. Different decades from the 1970s to today, the E. coli tet gene detection rate was a clear upward trend. In this study, the first at home and abroad to establish the animal source bacteria tetracycline class drug resistance gene triplex PCR detection kit and preliminary application, both at home and abroad have not been reported. The results of this study provide a fast, economical and accurate method for the detection of animal origin bacterial tetracycline resistance gene.
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