一种高效检测致病性钩端螺旋体PCR改良方法及效果评价  

Evaluation of an improved PCR assay in detection of pathogenic Leptospira

作　　者：王蓉[1] 马敏 王忠发 刘吉洛 WANG Rong;MA Min;WANG Zhong-fa;LIU Ji-luo(Department of Viral Research,Ningbo Municipal Center for Disease Control and Prevention,Ningbo,Zhejiang 315010,China;Dinghai Center for Disease Control and Prevention,Zhoushan,Zhejiang 316000,China;Department of Naval Medicine,Naval Military Medical University,Shanghai 200433,China)

机构地区：[1]宁波市疾病预防控制中心病毒研究所,浙江宁波315010 [2]定海区疾病预防控制中心,浙江舟山316000 [3]海军军医大学海军医学系,上海200433

出　　处：《中国媒介生物学及控制杂志》2023年第1期77-81,共5页Chinese Journal of Vector Biology and Control

基　　金：上海市公共卫生体系建设三年行动计划(2020-2022年)重点学科建设(GWV-10.1-XK17)。

摘　　要：目的比较评价1种致病性钩端螺旋体(钩体)的聚合酶链式反应(PCR)改良方法与3种现行标准方法的检测效果,用于病人早期诊断与宿主动物监测。方法对《全国病媒生物病原学监测方案(试行)》推荐致病性钩体检测方法(方法3)增加一步反转录反应过程,不改变引物和探针,形成改良方法(方法4)。分别采用方法4、卫生行业标准推荐方法(方法1)、检验检疫行业标准推荐方法(方法2)和方法3,4种PCR方法平行检测不同稀释度的钩体阳性标本、病人早期血清及鼠肾标本,评价其检测效果。结果方法4的灵敏度分别是方法1、2、3的10000、10000及1000倍。仅有方法4能在病人早期血清中检出钩体核酸。4种方法对于100份鼠肾标本中的钩体检出率分别是2.00%、4.00%、8.00%和40.00%。方法4与方法1、2、3的阳性检出率差异有统计学意义(均P<0.001),方法1、2、3之间的阳性检出率差异无统计学意义(P>0.05)。结论获得的改良方法对钩体病人早期血清和鼠肾标本的核酸检测效果,均明显优于其他3种现行标准检测方法。Objective To compare and evaluate the detection effect between an improved polymerase chain reaction(PCR)method and three current standard methods for pathogenic Leptospira,and to provide an improved method in early diagnosis of patients and host animal monitoring.Methods A step of reverse transcription process was added to the recommended pathogenic Leptospira detection method(method 3)in National Vector Etiology Surveillance Program(Trial),without changing the primers and probes,to form an improved method(method 4).Four PCR methods,i.e.,Method 4,the recommended method of Industry Standards for Health Care(method 1),the recommended method of Industry Standards for Inspection and Quarantine(method 2),and method 3,were used to detect Leptospira positive samples with series dilutions,early serum samples of patients,and rodent kidney specimens in parallel,and to evaluate the detection effect.Results The sensitivity of method 4 was 10000 times that of methods 1 and 2,and 1000 times that of method 3.Leptospira nucleic acids in early serum of patients were only detected by method 4.The detection rates of Leptospira in 100 rodent kidney specimens were 2.00%,4.00%,8.00%,and 40.00%by the four methods,respectively.There was a significant difference in the positive detection rate between method 4 and methods 1,2,and 3(all P<0.001)and no significant difference between methods 1,2,and 3(P>0.05).Conclusion The improved method has a superior detection effect to the other three current standard methods in the early diagnosis of patients with leptospirosis and the rodent kidney.

关 键 词：钩端螺旋体 聚合酶链式反应 16S rRNA 

分 类 号：R377.5[医药卫生—病原生物学] R183[医药卫生—基础医学]