Development Of The Detection Kit Of Tetracycline-resistance Genes In Bacterial Isolates From Animals By Multi-PCR Method

The positive control was obtained from the samples reserved in our laboratory by PCR method.The negative control is the DNA extract from E.coli ATCC25922.A multi-PCR detection method was established.In order to fred out the best reaction parameter we optimized Mg²⁺,dNTPs,Taq enzyme and primers concentration,the best annealing temperature and the best cycle number of times.The final reaction system was shown as follows:10×PCR Buffer 5μL,MgCl₂ (25 mmol/L) 3.0μL,dNTPs(2.5 mmol/L) 4μL,DNA template 5μL,Taq enzyme(2.5 U/μL) 0.35μL,forward and reverse primers each(25 mmol/L):tetA(1.0μL),tetC(0.3μL),tetM(0.4μL),and adding ddH₂O to make sure the total volume was 50μL.The reaction was performed with 5 rain initial denaturation at 94℃.The PCR program comprised 35 cycles(60 s at 94℃,60 s at 56℃,60 s at 72℃).A final elongation was done in 10 rain at 72℃followed the last cycle.We've Choosen 100 strains bacterial randomly test their resistance by K-B method and multi-PCR kit.The result shows that the drug-resistance rate of tetracycline was 92%,the Doxycycline was 84%,and Minocycline was 75%.The detection frequency of tetA,tetC and tetM was 46%,38%,33%respectively.The consistency rate of the result in multi-PCR kit with that in the drug-sensitive test was 89%.Besides,we compared the detection result of single PCR and multi-PCR kit.The uniformity of the two methods has reached 100%.Specificity,sensibility,repeatability test and conservation test of the PCR kit were carried out. Result shows that the PCR kit was specific;only the target genes that can be detected.What's more,it had a very high sensibility(4.0×10⁴ cfu/mL) and repeatability(100%).The kit can be stored at -20℃for 6 months.The strains isolated from 24 provinces in our country were detected by using the PCR Kit. Result shows that the tetracycline-resistance genes in different bacterial,the bacterial isolates from different animals and E.coli isolated from different years has great distinction,and the positive rate of drug-resistant strain increased gradually.In conclusion,we have invented a new rapidity,accuracy and low cost PCR kit,which can act as a useful tool in the detection of tetracycline-resistance genes in bacterial isolates from animals.