四种腺病毒核酸提取方法的比较

Comparison of Four Methods of Genomic DNA Extraction for Detection of Adenovirus by Real Time PCR

目的通过蛋白酶K法、苯酚法、病毒DNA/RNA提取试剂盒、腺病毒荧光PCR检测试剂盒等四种腺病毒核酸提取方法的比较,为进一步研究腺病毒的分子生物学特性选择合适的方法提供参考。方法以腺病毒感染的A549细胞为样品,分别以蛋白酶K法、苯酚法、病毒DNA/RNA提取试剂盒、腺病毒荧光PCR检测试剂盒等四种方法提取核酸,核酸经紫外分光光度计检测A260/A280的比值后,用荧光定量PCR方法检测核酸中腺病毒拷贝数浓度,并记录四种腺病毒核酸提取方法所需的操作时间。结果蛋白酶K法、苯酚法、病毒DNA/RNA提取试剂盒、腺病毒荧光PCR检测试剂盒等四种方法提取核酸的A260/A280的比值依次为1.85532、1.7377、1.81474和1.43934,核酸中腺病毒拷贝数浓度依次为4.9×105copies/mL、3.94×103copies/mL、2.66×106copies/mL和6.15×106copies/mL,提取核酸所需的时间分别为1.5、15、0.5和0.5h。结论四种腺病毒核酸提取方法中,蛋白酶K法简单快捷、成本低廉,适合一般实验室使用;苯酚法适合用于提取细胞培养上清中病毒的核酸;病毒DNA/RNA试剂盒的优点是操作时间短,得到的病毒核酸纯度较高;腺病毒荧光PCR检测试剂盒对病毒的核酸损耗少,操作步骤少,适用于临床标本的腺病毒核酸检测。

Objective The goal of this study was to compare four methods for extraction of Adenovirus DNA. Methods The genomic DNA of infected A549 cells was extracted using K method, phenol method, Takara viral RNA/DNA extraction kit and Huayin Adenovirus detection kit. And the following parameters between groups were compared： （1） The value of A260/A280 of genomic DNA. （2） The number of Adenoviroses copies. （3） The consuming time. Results The values of A260/A280 of nucleic acid extracted using proteinase K method, phenol method, Takara viral RNA/DNA extraction kit and Huayin Adenovirus real-time PCR detection kit were 1.85532, 1.7377, 1.81474 and 1.43934, respectively; the number of viral copies were 4.9×10^5 copies/mL, 3.94×10^3 copies/ mL, 2.66×10^6 copies/mL and 6.15×10^6 copies/mL, respectively; the consuming time were 1.5 h, 15 h, 0.5 h and 0.5 h, respectively. Conclusion According to viral DNA quality, the time required for completion, labor and PCR analytical sensitivity, the results showed difference in terms of experimental purpose between the four methods： （1） Proteinase K method was costless. （2） The phenol method was fit for extraction of the Adenovirus nucleic acid from supernatant of viral culture. （3）Takara viral RNA/DNA extraction kit method was good to get the highest quality viral DNA. （4） Huayin Adenovirus real-time PCR detection kit method was time-saving and suitable for the detection of viral DNA from clinical specimens.