Simultaneous detection of nine antibiotic resistance-related genes in Streptococcus agalactiae using multiplex PCR and reverse line blot hybridization assay
- VernacularTitle:多重PCR/反向线点杂交检测B族溶血性链球菌耐药相关基因的研究
- Author:
Xian-Yu ZENG
;
Hui WANG
;
Wei-Zhen WANG
;
Yi-Qun DUAN
;
Fan-Rong KONG
;
L GWENDOLYN
;
- Publication Type:Journal Article
- Keywords:
Streptococcus agalactiae;
Antibiotic resistant gene detection;
Multiplex PCR;
Two-hybrid system trchniques
- From:
Chinese Journal of Laboratory Medicine
2001;0(04):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop a multiplex PCR-based reverse line blot(mPCR/RLB) hybridization assay to detect,simultaneously,seven genes encoding AR-erm(A/TR),erm(B),mef(A/ E),tet(M),tet(O),aphA-3,aad-6 and two AR-related genes,int-Tn and mreA in group B streptococcus.Methods Nine pairs of specific primers and Oligonucleotide probes targeting erm(A/TR), erm(B),mef(A/E),tet(M),tet(O),aphA-3,aad-6 int-Tn and mreA respectively were modified according to former studies or designed in this study.The primers and probes were labeled with biotin and amino,respectively.The nine genes were amplified simultaneously in the same tube.PCR product hybridized with the probes labeled in the BiodyneC nylon membrane to detect the nine genes.To detect the sensitivity and specificity of the method developed,PCR with single pair of primer targeting each gene were tested in 318 isolates tested and the results were compared with the one abtained by RLB.Results The nine resistance-related genes could be successfully detected by mPCR/RLB assay developed in this study.Based on sequencing,21 of 22 isolates with mef had mef(E)and eight of 353 with int-Tn had an atypical sequence.Except for the above 29 genes,all the others corresponded well with the results obtained by single pair primer PCR.Conclusion The mPCR/RLB assay developed in this study is simple,rapid and suitable for surveillance of antibiotic resistance in GBS.
