聚合酶链反应-限制性片段长度多态性方法快速检测儿童胃黏膜中幽门螺杆菌对克拉霉素的耐药性  

Rapid detection of clarithromycin resistant Helicobacter pylori from children＇s gastric biopsy specimens by ploymerase chain reaction-restriction fragment length polymophism

作　　者：颜伟慧[1] 陈洁[1] 余金丹[1] 李中跃[1] 黄晓磊[1] 章许平[1] 

机构地区：[1]浙江大学医学院附属儿童医院消化科,杭州310003

出　　处：《中华儿科杂志》2009年第11期848-851,共4页Chinese Journal of Pediatrics

摘　　要：目的探索聚合酶链反应-限制性片段长度多态性（polymerasechainreaction．restrictionfragmentlengthpolymophism，PCR—RFLP）分析用于快速检测儿童胃黏膜中幽门螺杆菌（helicobacterpylori，Hp）对克拉霉素耐药性的可行性。方法所有胃黏膜标本均来源于2006年9月至2007年2月本院进行胃镜检查的患儿，从快速尿素酶试验阳性者中随机抽取39例标本。以分离培养-琼脂稀释法测定Hp对克拉霉素的最低抑菌浓度（MIC值），同时PCR—RFLP快速法检测23SrRNA基因突变情况，分别计算耐药率。以琼脂稀释法为金标准，评估PCR—RFLP法的敏感性、特异性及阳性预测值、阴性预测值。结果琼脂稀释法共检测出克拉霉素耐药Hp13例，敏感Hp26例，耐药率为33．3％。PCR—RFLP快速法检测出克拉霉素耐药Hp14例，敏感Hp25例，耐药率为35．9％。以琼脂稀释法为金标准，PCR—RFLP法的敏感性和特异性均为92．3％，阳性预测值和阴性预测值分别为85．7％、96．0％。两种方法比较，差异无统计学意义（X2=0．06，P〉0．05）。Hp对克拉霉素的耐药率较本院2004年报道（16／108株）明显增高，差异有统计学意义（X2=6．20，P〈0．05）。结论我院就诊儿童感染Hp对克拉霉素的耐药率呈上升趋势。采用PCR．RFLP分析检测儿童胃黏膜中Hp对克拉霉素的耐药性，操作方便快速、结果敏感可靠，有望为Hp根除治疗选择抗生素提供依据。Objective Infection with clarithromycin-resistant Helicobacter pylori （Hp） is often predictive of treatment failure. Susceptibility testing for Hp could guide therapy of Hp infections. However, agar dilution approved by the Clinical and Laboratory Standards Institute （CLSI） to test for antimicrobial susceptibility of Hp is time consuming （results are often not available in a week or more）. So a more expeditious method is necessary. The purpose of this study was to evaluate polymerase chain reactionrestriction fragment length polymorphism （PCR-RFLP） test performed directly on gastric biopsy specimen from children to detect 23S rRNA mutations （A2143G and A2144G） indicating clarithromycin resistance. Methods All biopsy specimens were derived from patients presenting with upper gastrointestinal symptoms, submitted to endoscopy in the Affiliated Children＇s Hospital, Zhejiang University School of Medicine from September 2006 to February 2007. No patients had undergone eradication therapy. Thirty-nine samples ramdonly selected from positive specimens by rapid urease test, were homogenized in 500 μl brucella broth with 30% glycerol. The 200 μl homogenized fluid was used to purify genomic DNA with the kit according to the instructions provided by manufacturer, and the rest was used to isolate Hp strains by culturing. All the Hp isolates were tested for clarithromycin susceptibility with the agar dilution and classified as resistant if the minimum inhibitory concentrations （MIC） exceeded 1 μg/ml. Simultaneously, PCR-RFLP analysis was performed in order to identify 23S rRNA mutations （A2143G and A2144G）. Finally, the two methods were compared by statistics. The agar dilution was used as a standard to dertermine the sensitivity and specificity of the PCR-RFLP assay. Results Of the 39 samples, agar dilution and PCR-RFLP method respectively detected 13 （33.3%） and 14 （35.9%） clarithromycin-resistant gastric specimens. The sensitivity and specificity of PCR-RFLP for the detection of Hp i

关 键 词：儿童 螺杆菌 幽门 克拉霉素 耐药性 

分 类 号：R686[医药卫生—骨科学]