一种快速核酸提取试剂在流感病毒检测中的应用评价

目的 探讨一种快速核酸提取试剂在流感病毒检测中的适用性及有效性。方法 选取流感病毒H3亚型、乙型Yamagata系细胞株和甲型H1N1型鸡胚株各1株，对其倍比稀释液及2015年北京市流感病原学监测阳性的67份咽拭子样本，同时采用快速提取法及离心柱提取法提取病毒核酸，经实时荧光PCR法进行定性、定量检测，检测结果采用SPSS 19.0软件进行统计分析。结果 快速提取法提取细胞培养液、鸡胚培养液中流感病毒核酸的最低检测限高于离心柱法10倍，咽拭子样本则高100倍；快速提取法提取核酸后检测到的病毒载量均低于离心柱法，其中对含有病毒量较高的毒株检测到的病毒载量低于离心柱法10倍，而对病毒量较少的咽拭子样本，则低100倍；快速提取法稳定性优于离心柱法；提取后的核酸-80℃保存10 d后冻融复测，快速提取法核酸损失量大于离心柱法。67份离心柱提取法检测阳性咽拭子样本使用快速提取法提取核酸后检测，两种方法阳性符合率总计为92.54%，其中10～102拷贝/ml组，阳性符合率为76.92%，102～103拷贝/ml组，阳性符合率为92.59%，103拷贝/ml组，阳性符合率为100%。结论 快速提取法具有稳定性高、易于操作、不易污染的优势，适用于细胞培养液、鸡胚培养液等病毒含量较高的大量样本中流感病毒核酸提取，适于检测样本量大、人手不足、设备欠缺的基层实验室使用；但-80℃冻融后核酸损失量较大，不宜反复冻融后使用；传统的离心柱法对于病毒含量较少的咽拭子样本及核酸需被反复冻融使用时具有优势。

Application of a rapid nucleic acid extraction kit in detection of influenza virus nucleic acid

Objective To evaluate the applicability and effectiveness of a rapid nucleic acid extraction kit in the detection of influenza virus. Methods Ten-fold dilutions from influenza virus (one H3 strain, one BY cell strain and one pdm09 H1N1 embryo strain) and 67 throat swabs positive for influenza virus collected in Beijing in 2015 were selected for the study. The viral nucleic acids were extracted by the novel rapid extraction method and spin column extraction method simultaneously. Real-time fluorescent PCR were used for qualitative and quantitative detections, and the results were analyzed with software SPSS 19.0. Results For the influenza virus cell strain and embryo strain, the detection limitation of the rapid extraction kit for influenza virus nucleic acid was 10 times higher than that of the spin column extraction method and 100 times higher than that of swab detection. The virus load detected by rapid extraction was lower than that by spin column extraction (10 times lower for samples with more viral particles and 100 times lower for swabs with less viral particles). For the stability, the rapid extraction kit was better than the spin column extraction method. The nucleic acid loss caused by rapid extraction due to re-freezing and re-thawing at -80℃ for 10 days was greater than the spin column extraction. Based on the quantitative detection results of spin column extraction, 67 swabs were divided into different viral load groups. By real time RT-PCR for qualitative detection, the total positive consistent rate for rapid extraction and spin column extraction was 92.54%. For the 10-102 copies/ml group, the positive consistent rates were 76.92%, 92.59% for 102-103 copies/ml group, 100% for103 copies/ml group. Conclusion The novel rapid nucleic acid extraction kit has some advantages such as high stability, easy to operate, hard to be polluted, and is appropriate for the nucleic acid extraction from the strains such as cell culture fluid, chick embryo culture fluid which contain more viral particles. Though the novel rapid nucleic acid extraction was more suitable for grass root laboratories which deal with large number of samples, lack staff and equipment, it is not appropriate to use nucleic acid after re-freezing and re-thawing due to the loss of nucleic acid after freezing at -80℃.The traditional spin column extraction method has advantages for the nucleic acid extraction from swabs which contain less viral particles and is more appropriate for the nucleic acid to be reused after re-freezing and re-thawing.