应用巢式聚合酶链反应和测序检测肺炎支原体23S rRNA中点突变  

Application of nested PCR and sequencing technique to detect point mutations of the 23S rRNA gene of Mycoplasma pneumoniae

作　　者：辛德莉[1] 糜祖煌[2] 韩旭[3] 秦玲[2] 李靖[1] 刘禧杰[1] 马少杰[1] 侯安存[1] 李贵[1] 

机构地区：[1]首都医科大学附属北京友谊医院儿科,100050 [2]无锡市克隆遗传技术研究所 [3]中国石油天然气集团公司中心医院儿科

出　　处：《中华儿科杂志》2008年第7期522-525,共4页Chinese Journal of Pediatrics

基　　金：国家自然科学基金资助项目（30571973）;北京自然科学基金资助项目（7063075）;北京中医药科技管理局资助项目（JJ2005-5）

摘　　要：目的建立快速检测肺炎支原体（Mycoplasrna pneumoniae，MP）耐药性的实验方法并应用于临床以了解MP耐药现状。方法应用巢式PCR直接检测咽拭子标本MP-23S rRNA基因并对扩增产物进行电泳检测或DNA测序分析；通过MP培养及体外药物敏感性试验测定临床分离株的红霉素最小抑菌浓度，以验证23S rRNA基因检测结果的可靠性。结果200例临床标本经MP-IgM抗体、咽拭子MP种特异性16S rRNA基因巢式PCR扩增和MP分离培养测定证实为MP64例。应用巢式PCR技术扩增MP-23S rRNA基因，并对扩增产物进行测序，将基因序列与基因库中MP标准株基因序列进行比较，与标准株序列相同的共计26例，其余38例存在23S rRNA点突变：35例在2063位点发生了A到G的点突变，1例在2063位点有A到C的点突变，2例在2064位点有A到G的点突变；耐药率为59．4％。MP耐药基因检测方法灵敏度达10^2ccu／ml，MP培养及药物敏感性试验证实23S rRNA基因检测结果可靠。结论建立的MP耐药基因检测方法能直接检测临床标本中的MP耐药基因。59％的受检标本23S rRNA基因发生点突变。Objective To establish a quick method to detect drug resistance of Mycoplasma pneumoniae （MP） and study the condition of drug resistance in MP infection. Methods MP 23S rRNA target gene in throat swab specimens from 200 patients with suspected MP infection was detected by using nested PCR and DNA sequencing. The result of 23S rRNA gene detection was confirmed by MP isolation and drug susceptibility test in vitro for reliability. Results Of the 200 clinical specimens, 64 were proved to be positive for MP through MP-IgM antibody, MP specific 16S rRNA nested PCR and MP isolation . The 23S rRNA gene was amplified and the gene sequence was compared with MP reference strain in Genbank, 26 were identical to the reference strain, 38 had a point mutation in 23S rRNA. Among them, 35 had A to G mutation at position 2063, 1 had A to C mutation at position 2063 and 2 had A to G mutation at position 2064,the percentage of drug resistance was 59.4%. The sensitivity of the gene detection method was 10^2 ccu/ml and it was confirmed to be reliable by MP isolation and drug susceptibility test. Conclusions The gene detection method could detect MP drug resistant gene directly from clinical specimen, which has the advantages of high specificity, high sensitivity and quickness. It is of great significance for diagnosis of MP infection because MP isolation is difficult and time-consuming.

关 键 词：支原体 肺炎 抗药性 RNA 核糖体 23S 聚合酶链反应 

分 类 号：R686[医药卫生—骨科学]