Study On A Multi-PCR Amplification Kit Of Detection Bacteria Aminoglycoside Resistance Genes And Its Application

Four aminoglycoside resistance genes (aph(3′)-â…¡a,aac(6′)-â… b,ant(3″)-â… a,aac(3)-â…¡a) were choosed as target genes. Refer to corresponding sequences that publishedin Genebank and the principles in design primers of multiplex PCR, four pairs ofspecificity primers were designed. Four resistance genes were amplified and sequenced.The result shows that compared with the related resistance gene sequences that publicizedin GenBank, the homologies were aph(3′)-â…¡a (99.4%),aac(6′)-â… b (99.3%),ant(3″)-â… a(99.2%),aac(3)-â…¡a (95.3%), which proved that the primers were correct and specificity.Strains (E99,107) that contained resistance genes were token as the possive control.The best and available range of each amplification system's parameters were found bycommom PCR method. And multiplex PCR amplification system were researched andoptimized, including try to find out the best concentration of Mg²⁺,dNTPs,Taq enzymeand primers, the best annealing temperature and the best cycle number of times. The resultshows that, we have established a multi-PCR method to detection aminoglycosideresistance genes from bactaria successfully. The best parameters of amplification system asfollows: 10×PCR Buffer 5μl, MgCl₂ (25 mmol/L) 5μl, dNTPs (2.5 mmol/L) 4μl,template 5μl (McFarland No 1.0), Taq enzyme (2.5 U/μl) 0.3μl, forward and reverseprimers each (25 mmol/L): aph(3′)-â…¡a (0.5μl),aac(3)-â…¡a (0.9μl), aac(6′)-â… b (0.8μl),ant(3″)-â… a (1.8μl), and ensure the total volume was 50μl by adding ddH₂O. The reactionwas performed with 5 rain initial denaturation at 94℃.The PCR program comprised 32cycles (50 s at 94℃, 45 s at 54℃, 50 s at 72℃). A final elongation was done in 6 minat 72℃followed the last cycle.The best method to prepare template was also choosed and optimized, and 60%glycerin in the template makes Taq enzyme more stabilization, with which we can maketemplate by centrifuge the culture twice without distilling DNA from bacteria..Specificity,sensibility,repeatability test and conservation test of the PCR kit werecarried out after we choosed the best modality of its combination. The result shows that thePCR kit was specific, it's only the target genes that can be detected, had a very highsensibility (3.6×10⁴ CFU/mL) and repeatability. The kit could be stored at -20℃for 4months. 30 strains were detected by two different methods (by the PCR kit and by K-Bmethod), compared the two methods, the result shows that the consistency rate was 90%,but the PCR kit had advance in sensitive,convenient and cost much less time.Aminoglycoside resistance genes of 543 strains (including 484 strains isolated from24 provinces in china, 59 strains isolated from giant pandas in Sichuan) were detected by the PCR Kit., the result shows that the highest detection frequency of aph(3′)-â…¡a gene was46.1%(Beijing), aac(6′)-â… b gene was 11.6%(Zhejing), ant(3″)-â… a gene was 504%(Shandong), aac(3)-â…¡a gene was 42.8%(Gansu). In the strains isolated from giant pandas,the detection frequencies of aph(3′)-â…¡a gene was the highest, and the next wasant(3″)-â… a gene (5.08%),aac(3)-â…¡a (1.69%), aac(6′)-â… b was not observed.One kind of PCR kit of detection aminoglycoside resistance genes from bacteria wasinvented, which can be used as a useful, sensibility and convenient metod to detectionaminoglycoside resistance genes.