用多重PCR方法进行志贺菌超广谱β-内酰胺酶基因分型

Multiplex PCR assay for dissemination and diversity of extended-spectrum β-lactamase genes in Shigella isolates

目的建立了一种志贺菌超广谱β-内酰胺酶（extended—spectrum β—lactamases，ESBLs）基因型分类的多重PCR方法，鉴别杭州市1998-2007年产ESBLs志贺菌散发菌株基因型。方法用纸片扩散药敏法对1998-2007年杭州分离出的195株志贸菌进行产ESBLs筛选，利用多重PCR鉴定CTX—M、TEM、SHV、OXA-1等4个基因型别，并用8次单个PCR验证多重PCR结果。结果在195株志贺菌中共筛选到17株产ESBLs菌株，阳性率为8．72％，与2005年全国监测点结果（阳性率为6．10％）比较，差异无统计学意义（X^2=1，464，P=0.226）；多重PCR结果显示ESBLs基因型分别为：CTX-M型17株（CTX—M-9组亚型11株，CTX—M-1组亚型6株），TEM型2株，OXA-1型10株，无SHV型；多重PCR结果与8次单个PCR结果一致率为94．12％，其中1株OXA-1型结果不一致。结论建立了一种志贺菌ESBLs基因型分类的多重PCR方法，杭州市志贺菌中产ESBLs的比例与全国监测点水平相似，但存在上升的可能。

Objective To develop a rapid and simple multiplex polymerase chain reaction （PCR） method which discriminates extended-spectrum β-lactamases （ESBLs）genes in sporadic Shigella isolates from 1998 to 2007 in Hangzhou city, China. Methods After ESBLs screening according to the Clinical and Laboratory Standards Institute （CLSI） method, CTX-M,TEM, SHV and OXA-1 encoding genes were detected by using a multiplex PCR method, and the results were verified by 8 single gene PCR amplification. Results Seventeen isolates harbored ESBLs genes among 195 Shigella isolates（ 8.72% ）. Genes encoding CTX-M （17 strains ） , TEM （ 2 strains）, OXA-1 （ 10 strains） and SHV （ 0 strains） were discriminated with multiplex PCR analysis,which coincided with eight single gene PCR analysis at 94. 12%. Conclusion Multiplex PCR should be a suitable tool for initial rapid screening and discriminating ESBLs genes in Shigella isolates. With similar trend of national surveillance data, the proportion of sporadic Shigella isolates harhouring ESBLs genes might probably be on increase.