CAJ | 학술논문

目的：建立一种多重 PCR 方法用于莫西沙星耐药的艰难梭菌鉴定和初步基因分型。方法根据艰难梭菌 slpA 可变区间核苷酸序列的差异设计5种 slpA 基因型（gr、hr、fr、gc08和078）的特异性 PCR 引物，同时加入检测艰难梭菌管家基因磷酸甘油醛异构酶基因 tpi 的种特异性引物，构建多重PCR 方法；利用9种肠道常见的正常或致病菌验证多重 PCR 方法的特异性，利用46株分属于11个slpA 基因型的艰难梭菌参考菌株来验证方法的检测和分型能力；利用建立的多重 PCR 方法检测39株莫西沙星耐药的临床菌株，以 slpA 测序分型法为参照方法，评估该方法的临床实用性。结果多重PCR 检测9种肠道常见的正常或致病菌 tpi 和5种 slpA 基因型均为阴性；46株艰难梭菌参考菌株 tpi均为阳性，36株分属于5种靶 slpA 基因型（gr、hr、fr、gc08和078）的菌株被正确分型，10株分属于其他6种基因型的参考菌株均无法分型。39株莫西沙星耐药的艰难梭菌临床菌株 tpi 均为阳性，32株检出具体基因型，其中22株为 slpA 基因型 gc08，6株为 hr，2株为 fr，2株为078，与 slpA 测序分型结果一致；7株多重 PCR 无法分型，slpA 测序分型结果显示其基因型均不包含在多重 PCR 分型范围内。结论成功建立一种简单、快捷、临床实验室适用的艰难梭菌检测，且能够分辨出5种 slpA 基因型的多重PCR 方法；莫西沙星耐药的艰难梭菌主要为 slpA 基因型 gc08。
목적：건립일충다중 PCR 방법용우막서사성내약적간난사균감정화초보기인분형。방법근거간난사균 slpA 가변구간핵감산서렬적차이설계5충 slpA 기인형（gr、hr、fr、gc08화078）적특이성 PCR 인물，동시가입검측간난사균관가기인린산감유철이구매기인 tpi 적충특이성인물，구건다중PCR 방법；이용9충장도상견적정상혹치병균험증다중 PCR 방법적특이성，이용46주분속우11개slpA 기인형적간난사균삼고균주래험증방법적검측화분형능력；이용건립적다중 PCR 방법검측39주막서사성내약적림상균주，이 slpA 측서분형법위삼조방법，평고해방법적림상실용성。결과다중PCR 검측9충장도상견적정상혹치병균 tpi 화5충 slpA 기인형균위음성；46주간난사균삼고균주 tpi균위양성，36주분속우5충파 slpA 기인형（gr、hr、fr、gc08화078）적균주피정학분형，10주분속우기타6충기인형적삼고균주균무법분형。39주막서사성내약적간난사균림상균주 tpi 균위양성，32주검출구체기인형，기중22주위 slpA 기인형 gc08，6주위 hr，2주위 fr，2주위078，여 slpA 측서분형결과일치；7주다중 PCR 무법분형，slpA 측서분형결과현시기기인형균불포함재다중 PCR 분형범위내。결론성공건립일충간단、쾌첩、림상실험실괄용적간난사균검측，차능구분변출5충 slpA 기인형적다중PCR 방법；막서사성내약적간난사균주요위 slpA 기인형 gc08。
Objective To develop a multiplex polymerase chain reaction (PCR )method for detecting and genotyping moxifloxacin-resistant Clostridium difficile (C.difficile)isolates.Methods Specific PCR primers of slpA genotypes gr,hr,fr,gc08 and 078 were designed according to the differences of slpA nucleotide sequences in different C.difficile genotypes,and the house-keeping gene tpi specific PCR primers were also added for the construction of multiplex PCR method.Nine common intestinal normal and pathogenic strains were used to verify the specificity of slpA multiplex PCR for the detection of C.difficile.Forty-six C.difficile reference strains,belonging to 11 slpA genotypes,were used to verify the ability of the multiplex PCR method for dectecting and genotyping.Thirty-nine moxifloxacin-resistant clinical isolates were genotyped by the multiplex PCR,and its clinical value was evaluated by comparing with slpA sequence typing (slpA ST)method.Results All the 9 intestinal normal and pathogenic strains were negative when detected by the multiplex PCR.And tpi of 46 C. difficile reference strains were positive,and 36 strains belonging to slpA genotypes gr,hr,fr,gc08 and 078 were genotyped correctly.Other 10 strains which belonged to other 6 genotypes were non-typeable. Among 39 moxifloxacin-resistant clinical isolates,all were positive of tpi,and 32 isolates were typed correctly by the multiplex PCR method,including 22 slpA genotypes gc08,6 genotypes hr,2 genotypes fr,and 2 genotypes 078,which were consistent with slpA ST.However,7 isolates could not be typed by multiplex PCR,which were identified as other genotypes not included in the multiplex PCR by slpA ST. Conclusions A convenient and rapid multiplex PCR method for the detection of C.difficile is established successfully,which can distinguish among five slpA genotypes.slpA genotype gc08 is the common genotype of moxifloxacin-resistant clinical isolates.