LAMP结合示差脉冲伏安法快速检测肺炎克雷伯菌的方法建立及临床应用

Rapid detection of Klebsiella pneumoniae based on loop-mediated isothermal amplification with differential pulse voltammetry

目的利用环介导恒温扩增(LAMP)技术结合示差脉冲伏安(DPV)法,建立快速检测肺炎克雷伯菌的方法,并将其应用于小儿腹泻病原菌分析。方法通过基因比对与引物设计,建立针对肺炎克雷伯菌的LAMP检测方法。采用LAMP技术对肺炎克雷伯菌和其他8种干扰菌以及对不同浓度肺炎克雷伯菌进行扩增,结合DPV法评价其特异性及灵敏度。将200份腹泻患儿的粪便标本同时采用LAMP方法与培养法进行检验,对比2种方法检测肺炎克雷伯菌的阳性率。统计近2年该院门诊及住院小儿腹泻病例,初步分析其常见腹泻致病菌种类及比例。结果设计出针对肺炎克雷伯菌的LAMP检测方法,该法只针对肺炎克雷伯菌有扩增,对其他干扰菌不扩增,显示出良好的特异性,LAMP法最低检测限为10CFU/mL,显示出较高的灵敏度。200份腹泻患儿粪便标本,采用LAMP法与培养鉴定法检测的肺炎克雷伯菌阳性率差异无统计学意义(P>0.05)。近2年内该院小儿腹泻粪便标本中共检出致病菌709株,主要病原菌分别为志贺菌(34.23%)、大肠杆菌(15.40%)、铜绿假单胞菌(12.96%)、弗劳地枸橼酸杆菌(7.82%)、沙门菌(6.36%)、白色念珠菌(6.11%)、肺炎克雷伯菌(4.40%)、产气荚膜梭菌(3.67%)、变形杆菌(3.42%)、其他菌(5.63%)。结论设计出了针对肺炎克雷伯菌的LAMP检测方法,该法具有良好的特异性和较高的灵敏度,能够用于肺炎克雷伯菌的快速检验。

Objective To construct method based on loop-mediated isothermal amplification （LAMP）and differential pulse vol-tammetry （DPV）for the rapid detection of Klebsiellapneumoniae in children with diarrhea.Methods By gene alignment and prim-er design,LAMP method for the detection of Klebsiella pneumoniae was constructed.Klebsiella pneumoniae,other 8 kinds of in-terference bacteria and different concentration of Klebsiella pneumoniae were amplified by using LAMP technology.Specificity and sensitivity were evaluated by using DPV method.LAMP and culture identification were performed to analyze 200 stool samples of children with diarrhea,and the positive rates of the two methods were compared.Infantile diarrhea cases of outpatient and inpatient in recent two years were analyzed to identify common diarrhea bacteria species and their proportions.Results LAMP method for detection of Klebsiella pneumoniae was successfully constructed,which was specific and sensitive.Positive rates of culture identifi-cation and LAMP for the detection of 200 stool samples were without significant difference.A total of 709 strains of pathogenic bacteria were detected in stool samples of children with diarrhea treated in this hospital in recent two years,including Shigella （34.23%）,Escherichia coli （15.40%）,Pseudomonas aeruginosa （12.96%）,Citrobacter freundii （7.82%）,Salmonella （6.36%）,Candida albicans （6.11%）,Klebsiella pneumoniae （4.40%）,Clostridium perfringens （3.67%）,Bacillus proteus （3.42%）and other strains （5.63%）.Conclusion LAMP detection method of Klebsiella pneumoniae was successfully construc-ted,which was with fine specificity and sensitivity,and could be used for rapid detection of Klebsiella pneumoniae.