摘要
目的采用TaqMan探针法建立检测棘阿米巴18srDNA的实时荧光定量PCR（Real—timePCR）方法，为早期诊断棘阿米巴病提供基因诊断标准方法。方法提取实验室培养的4株土壤中分离的和1株水中分离的不同基因型棘阿米巴DNA，测序后在物种保守区域设计Real—time引物和TaqMan探针，用建立的方法检测动物模型兔眼分泌物、大肠杆菌、人巨细胞病毒、卡式肺孢子虫、疑似棘阿米巴角膜炎160例病人眼分泌物。结果与传统实验室培养病原相比，所建立的qPCR检测棘阿米巴角膜炎方法测定大肠杆菌、人巨细胞病毒和卡式肺孢子虫与该研究的实验室分离棘阿米巴物种没有交叉反应，检测结果均为阴性。而160例疑似病人，用培养法检测出感染棘阿米巴患者为5例，用qPCR方法检测出6例，其中5例为培养法测出患者，qPCR方法检测阳性率（3．75％）略高于普通培养法（3．13％），但无统计学意义（P〉O．05）。qPCR方法在95％置信区间中灵敏度100％（47．95％～100％）高于普通培养法83．33％（36．10％～97．24％）。结论所建立测定棘阿米巴18srDNA的Real-timePCR基因检测方法具有无创伤性、高效、特异、经济等特点，适用于疑似棘阿米巴角膜炎病人门诊筛选的有效方法。
The aim of this study is to develop a new molecular based method for the high-sensitivity and early diagnosis of Acanthamoeba keratitis. We adopt the methods as follow： DNA was extracted from 4 strains of Acanthamoeba isolated from soil samples and 1 strain of Acanthamoeba from water sample. PCR product of 18s rDNA gene in Acanthamoeba was se- quenced. Real-time PCR （qPCR） primers and TaqMan probes were designed based on 18s rDNA gene sequence of Acanthamoe- ba. The rabbit eye secretions, Escherichia coli, human cytomegalovirus, Pneurnocystis carinii and 160 suspected patients with eye secretion were examined for Acantharnoeba keratitis by traditional laboratory culture and qPCR methods. Then we got the result that compared with traditional laboratory culture pathogen, the established qPCR detection method for separation and de- termination of Acanthamoeba species had no cross reaction with E. coli, human cytomegalovirus, and Pneumocysfis carinii. All test results were negative. For the 160 suspected cases, 5 positive cases with Acantharnoeba keratitis were identified by the culture method, while 6 positive cases with Acanthamoeba keratitis were detected by qPCR methods. The detection rate of qPCR method （3.75 ~） was slightly higher than that of conventional culture method （3, 13 %）, but not in significant level. However, the qPCR method had higher sensitivity than that of the culture method. The most important is that this method is a noninvasive measurement. So the real-time PCR with TaqMan probe detection method for the determination of Baraki Amy Ba is sensitive, specific, high efficiency, economic and effective method in screening and early diagnosis of suspected Acanthamoe-ba keratitis patient＇ s clinic.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2014年第2期158-162,共5页
Chinese Journal of Zoonoses
基金
浙江省自然科学基金(No.Y2080973)
温州市科技局(No.Y20090012)联合资助~~
