Development And Application Of The Detection Kit Of Sulfonamides-resistance Genes In Bacteria By Multi-PCR Technology

In order to establish the special, sensitive and rapid detection technology ofsulfonamides-resistance genes in bacteria, and provide the powerful technical method forthe clinic medication supervising, a rapid method is developed. Three pairs of specificprimers are designed according to the correlative Sul1, Sul2 and Sul3 gene sequencesdownload from the GenBank. A detection kit based on multi-polymerase chain reactiontechnique was developed and primarily applied.The positive isolates FJ504 were obtained by single PCR method. The homologies ofnucleotide sequence were compared between FJ504 strain and other strains publicized inGenBank. The homologies were above 98%. The multi-PCR was established on the basisof simple PCR technique with the positive control FJ504 and the negative controlATCC25922 strain. The reaction conditions were optimized, including the concentration ofMg²⁺, dNTPs, Taq polymerase and primers, the annealing temperature and the cycle times.Other ingredient was fixed when adjusted one ingredient. Optimized system had a totalvolume of 50μL containing 10×PCR buffer 5μL, MgCl₂ (25 mmol/L)5μL, dNTPs(2.5 mmol/L) 4μL, primer of sul1, sul2 and sul3 (25μmol/L) were 1.4μL, 0.4μL and1.2μL respectively, Taq DNA polymerase(2.5 U/μL) 0.35μL, DNA template 5μL.The reaction was performed with 5 min initial denaturation at 94℃.The PCR programcomprised 32 cycles (50 s at 94℃,45 s at 55℃,50 s at 72℃).A final elongation of 6min at 72℃followed the last cycle.By using the PCR kit, specific DNA fragment from positive strain could be amplified,whereas no corresponding fragment was found from the standard staphylacoccus aureusATCC29213 strain, the standard Escherichia coli ATCC25926 strain, the Infectionsbronchitis virus and the rabbit plague virus as control samples. It was shown that the PCRkit had a good specificity and repeatability. The result indicated that a minimum of 2×10⁴cfu/mL could be detected by using the PCR kit. The kit containing primer but no TaqDNA polymerase could be stored for 4 months at 4℃.or for 5 months at -20℃.A clinical detection has been carried on 50 strains, the consistency rate of the result inmulti-PCR with that in the drug-sensitive test was 90%, but the multi-PCR kit detected the potential resistance of some strains.The other clinical detection has been carried on above seven hundreds strainsincluding above 730 strains collected from 24 provinces in our country using the PCR Kit.The results showed that the resistance rates of the isolates from swine and chicken tosulfonamides were 79.2% and 77.1% respectively, while that of the isolates from yak andwild animals were only 36.4% and 20.8% respectively. The detection rates ofsulfonamides-resistant genes in E.coil strains, Salmonella strains and Staphylacoccusaureus strains were 83.2%, 68.0% and 73.8% respectively. The detection rate ofsulfonamides-resistant genes in E.coil strains was 70.9%, which was notably higher thanthat of 70-80's.It was shown that the sulfonamides-resistance genes in isolates fromanimals.can be detected by this kit, and the positive rate of drug-resistant strain increasedgradually.In conclusion, the PCR kit developed in this study had the advantage of accuracy,sensitive and rapidity, providing a new useful tool for the detection ofsulfonamides-resistance genes in bacteria.