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目的建立一种含内参的,针对登革病毒和基孔肯雅病毒核酸检测的3重荧光定量PCR方法。方法针对登革病毒3’端非结构蛋白编码区和基孔肯雅病毒5’端非结构蛋白编码区设计引物与探针,建立一套含有内参的并且能同时检测登革病毒和基孔肯雅病毒的3重实时荧光定量RT-PCR方法。对其最低检测限和特异度进行验证,通过检测血清标本对该方法进行临床应用评估。结果建立的3重荧光定量PCR登革病毒与基孔肯雅病毒绝对定量标准曲线显示,病毒拷贝数的对数值与CT值之间具有良好的线性关系,梯度稀释后的标准品核酸检测均呈现典型的S型曲线。检测日本脑炎病毒、西尼罗病毒、黄热病毒、辛德毕斯病毒各1株,结果均为阴性,特异度为100%。用该方法检测294份登革热患者血清,灵敏度为100%,特异度为89.3%,阳性预测值为92.1%,阴性预测值为100%;测定33份基孔肯雅热病患者血清的灵敏度、特异度及阴、阳性预测值均为100%。结论建立的含有内参的能同时检测登革病毒与基孔肯雅病毒的3重荧光定量PCR方法具有较高的灵敏度和特异度,可用于登革病毒与基孔肯雅病毒感染检测,为早期筛查和鉴别登革病毒和基孔肯雅病毒感染提供依据。
OBJECTIVE: To establish a 3-fold real-time quantitative PCR method for detection of dengue virus and chikungunya virus nucleic acid with internal reference. Methods Primers and probes were designed according to the non-structural protein coding region of dengue virus 3 ’and the non-structural protein coding region of chikungunya virus. A set of primers and probes were designed to detect both dengue virus and chikungunya 3-fold real-time fluorescence quantitative RT-PCR method of the virus. The minimum limit of detection and specificity were verified, and the clinical application of this method was evaluated by detecting serum samples. Results The absolute quantitative standard curves of dengue virus and chikungunya virus by 3-fold fluorescence quantitative PCR showed that there was a good linear relationship between the logarithmic value of virus copy number and CT value. The gradient-diluted standard nucleic acid test showed Typical S-curve. One Japanese strain of encephalitis virus, West Nile virus, Yellow Fever virus and Sindbis virus was detected. The results were negative and the specificity was 100%. The detection of serum of 294 dengue patients by this method showed 100% sensitivity, 89.3% specificity, 92.1% positive predictive value, and 100% negative predictive value. The sensitivity and specificity of 33 serum samples from patients with Chikungunya Degree and negative, positive predictive value are 100%. Conclusion The established 3-FQ-PCR method with simultaneous detection of Dengue virus and Chikungunya virus containing internal reference has high sensitivity and specificity and can be used for the detection of Dengue virus and Chikungunya virus infection. Screening and identification Dengue virus and Chikungunya virus infection provide the basis.