多重荧光定量PCR测定登革热Ⅰ~Ⅳ型方法的建立与初步评价
评价。 方法 针对登革热Ⅰ～Ⅳ型使用自建的引物探针对反应条件及温度进行调整，且对阳性样本进行不同浓度的稀释，检测自建多重荧光定量PCR在登革热Ⅰ～Ⅳ型测定中的重复性、灵敏性及特异性。 结果 6份登革热阳性样本通过自建的检测方法来进行检测，全部能够显示明显的S曲线扩增，且与原有确证方法相比，不同型别的结果一致。其他10份病毒阳性样本及阴性样本全部没有显示扩增曲线，提示自建的检测方法特异性良好。分别检测登革热Ⅰ～Ⅳ型，变异系数（CV%）值水平范围：0.51～5.51，提示重复性较好。使用自建的多重荧光定量PCR对登革热不同型别进行检测的平均用时与ELISA法相比无显著差异（t=1.561，P>0.05）。通过自建的多重荧光定量PCR测定通过仪器来完成核酸的自动提取，在仪器当中加样、上机之后不需要另外的操作，充分降低了发生污染的风险。 结论 本次研究设计建立的登革热Ⅰ～Ⅳ型多重荧光定量PCR检测方法，在重复性、灵敏性及特异性方面均较为理想;且相比ELISA法在加样后无需另外手动操作，更加简易方便。
[关键词] 登革热;荧光定量PCR;血清型;病毒载量
[中图分类号] R512.8 [文献标识码] A [文章编号] 1673-9701（2019）34-0008-06
Establishment and preliminary evaluation of multiplex quantitative PCR for determination of dengue fever type Ⅰ-Ⅳ
FEI Yunxia1 ZHANG Xiangbo2 HE Tao2 ZHANG Yuting1 GAO Yidan3 GAO Ling3 WANG Jie3
CHEN Gongying3
1.Zhejiang University of Traditional Chinese Medicine， Hangzhou 310053， China; 2.Hangzhou Normal University， Hangzhou 310053， China; 3.Department of Infectious Diseases and Liver Diseases， Affiliated Hospital of Hangzhou Normal University， Hangzhou 310053， China
[Abstract] Objective To establish and evaluate the method of multiplex quantitative PCR for the determination of dengue fever type Ⅰ-Ⅳ. Methods For the dengue type Ⅰ-Ⅳ， the self-built primer probe was used to adjust the reaction conditions and temperature， and the positive samples were diluted at different concentrations. The repeatability， sensitivity and specificity of self-built multiplex quantitative PCR in the determination of dengue type Ⅰ-Ⅳ. Results Six dengue-positive samples were tested by self-built detection methods， all of which showed significant S-curve amplification， and the results of the different types were consistent compared with the original confirmation method. The other 10 virus-positive samples and negative samples did not show amplification curves， suggesting that the self-built detection method was specific. The dengue type Ⅰ-Ⅳ type was detected separately， and the coefficient of variation（CV%） value range was 0.51-5.51， indicating good repeatability. The mean time to test different types of dengue using self-built multiplex quantitative PCR was not significantly different from ELISA（t=1.561， P>0.05）. Through the self-built multiplex quantitative PCR assay， the automatic extraction of nucleic acid was completed by the instrument， and no additional operation was required after the sample was loaded in the instrument and the machine was taken on， which completely reduced the risk of contamination. Conclusion The dengue type Ⅰ～Ⅳ multiplex quantitative PCR method established in this study is ideal in terms of repeatability， sensitivity and specificity. Compared with ELISA， no additional manual operation is required after the sample is added. It is easy and convenient.