快速诊断早期棘阿米巴角膜炎qPCR方法的建立及应用

Establishment and use of a real-time PCR technique to rapidly diagnosis early Acanthamoeba keratitis

目的建立检测棘阿米巴18srDNA的TaqMan探针实时荧光定量PCR(Real-time PCR or qPCR)方法,为无创早期诊断棘阿米巴病提供基因诊断标准方法。方法提取分离自土壤和水中的5株棘阿米巴DNA,测序后与GenBank中搜索到的棘阿米巴67条序列进行比对,在物种保守区域设计Real-time引物和TaqMan探针,通过T克隆载体制作标准品并测序,建立方法的标准曲线,测定最低检测浓度,同时用该方法检测其他病原微生物及临床疑似棘阿米巴角膜炎病人眼分泌物,实验数据通过JMP5.0.1和测评软件进行棘阿米巴角膜炎F检验和诊断测试评估。结果qPCR和培养法检测180例疑似棘阿米巴角膜炎病人眼分泌物,阳性率分别为(5±1)%和(2.9±1.2)%,差异有统计学意义(F=13,P<0.01)。以qPCR法测定的标准品CT值为纵坐标(Y),以标本浓度的对数为横坐标(X)建立标准曲线:Y=-3.24X+40.74,R>0.99。用建立的qPCR方法测定棘阿米巴18srDNA最低浓度为10拷贝/UL。用qPCR检测其他病原微生物,结果均为阴性。通过诊断测试评估软件比较两种方法检测临床疑似病人眼分泌物结果,95%置信区间内qPCR方法的灵敏度为100%,传统实验室培养法为62.5%。结论棘阿米巴18srDNA Real-time PCR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的筛查。

Objective The aim of this study was to establish a new technique for the sensitive and noninvasive early diagnosis of Acanthamoeba keratitis （AK）. Methods DNA was extracted from 5 strains of Acanthamoeba isolated from soil and water samples. PCR products of the 18s rDNA gene in Acanthamoeba were sequenced. These sequences were compared to 67 sets of sequences of the 18s rDNA gene of Acanthamoeba in GenBank. Real-time PCR （qPCR） primers and TaqMan probes were designed in accordance with sequences of the 18s rDNA gene of Acanthamoeba. A T cloning vector was used to produce a standard sample that was then sequenced. A standard curve was created by subjecting different diluted concentrations of the standard sample to qPCR. The minimum detectable sample concentration was determined. Conventional laboratory cultures and qPCR were used to test for other pathogenic microorganisms besides Acanthamoeba and to test for Acanthamoeba in eye secretions from patients suspected of having AK. Data were subjected to an F-test in JMPS. 0.1 and diagnostic assessment software. Results In total, 180 samples were tested using cultures and qPCR. Acanthamoeba was detected by cultures at a rate of （5±1）% and by qPCR at a rate of （2.9±1.2）%. The difference in the rate of detection was statistically significantly （F= 13 in the F-test, P〈0.01）. The CT value for qPCR of the standard sample served as the vertical axis （Y）, and the log of the sample concentration served as the horizontal axis （X） to create a standard curve with Y= -3.24, X＋40.74, R〉0.99. The detection limit for detection of Acantharnoeba 18S rDNA by qPCR was 10 copies/UL. The proposed qPCR technique did not detect other pathogenic microorganisms. Diag nostic assessment software was used to compare cultures and qPCR of eye secretions from patients suspected of having AK. qPCR had greater sensitivity （100 %） than cultures （62.5 %） in the 95 % confidence intervals. Conclusion Realtime PCR with a TaqMan probe is a noninvasive, sensitive, specific, highly efficient, economical, and effective way to test for AK and screen patients with suspected AK.