不同液化处理及核酸共提取方法对痰样本中病毒核酸的提取效果比较  

Effects of different homogenization and nucleic acid co-extraction methods on viral RNA and DNA extrac- tion from sputum

作　　者：何慧[1] 陈懿[1] 潘平[1] 潘富根[2] 余道军[1] 

机构地区：[1]浙江中医药大学附属杭州第一医院检验科,杭州市第一人民医院检验科,杭州310006 [2]浙江大学医学院附属第四医院检验科,浙江义乌322000

出　　处：《国际流行病学传染病学杂志》2016年第2期81-85,共5页International Journal of Epidemiology and Infectious Disease

基　　金：浙江省医药卫生科技计划项目（2015ZDA025）;杭州市科技发展计划（20130733Q04）;杭州市卫生科技计划重大项目（2012ZD001）

摘　　要：目的比较不同痰液液化方法及不同核酸提取方法共提取痰液中病毒RNA和DNA的效果，并比较其优劣。方法将痰液样本分为二硫苏糖醇（DTT）、氢氧化钠（NaOH）液化痰液组，以生理盐水为对照，分别使用TIANampVirusDNA／RNAKit（方法1）、KBW酶法（方法2）、KBW通用法（方法3）和单一核酸提取试剂盒（方法4）提取核酸，微量核酸分析仪检测核酸纯度，多重定量PCR方法检测核酸的浓度。结果核酸纯度测定结果：生理盐水组中方法2和方法3，DTT处理组中方法1和方法2，NaOH处理组中方法3所提DNA纯度略大于1．8；各组中采用方法l所提RNA纯度均明显超过2，方法4及NaOH处理组中方法3所提R-A纯度均低于1．8，其余纯度均在正常范围内。PCR浓度测定结果：腺病毒（DNA）Ct值：生理盐水组〈NaOH处理组（q=35．09，P〈0．05），生理盐水组和DTT处理组间差异无统计学意义（q=2．54，P〉0．05）；提取方法1〈方法3（q=12．68，P〈0．05），方法1、方法2与方法4之间差异无统计学意义（q=0．84、1．69、2．53，P〉0．05）；呼吸道合胞病毒（RNA）Ct值：生理盐水组〈DIT组〈NaOH组（F=152．76，P〈0．01）；提取方法l〈方法2〈方法3（q=6．98、3．75，P〈0．05），方法2与方法4之间差异无统计学意义（q=1．28，P〉0．05）。结论痰液液化前处理会影响病毒RNA和DNA的共提取，TIANampVirusDNA／RNAKit和KBW酶法对痰样本中病毒核酸共提取效果较好。Objective To evaluate the effects of different homogenization and nucleic acid co-extraction methods on viral RNA and DNA co-extraction from sputum. Methods Sputum was liquefied by 0.1% Dithiothreitol （DTT） （group B） and 4% NaOH （group C） respectively, comparing with saline solutions （group A）. TIANamp Virus DNA/RNA Kit （method I ）, KBW enzyme kit （method 2）, KBW univeral kit （method 3） and TAKARA extraction kit （method 4） were used to extract nucleic acid from each group. The purity of nucleic acid extracted by different methods was evaluated by nano ultramicro nucleic acid protein tester, while the concentration of nucleic acid was evaluated by multiple quantitative polymerase chain reactions （mqPCR）. Results The nucleic acid purity： the ratio of DNA purity was slightly higher than 1.8 by method 2 and 3 in group A, method 1 and 2 in group B, and method 3 in group C; the ratio of RNA purity was higer than 2 by method 1 in each group, and was lower than 1.8 by method 4 in each group and method 3 in group C; and the rest results were within the normal range. The concentration of nucleic acid by mqPCR： the adenoviruse （DNA） Ct value： group A〈group C （q=35.09,P〈0.05）, and there was no statistical difference between group A and B （q=2.54,P〉0.05）; method 1 〈method 3 （q=12.68,P〈0.05）, and there were no statistical differences between method 1,2 and 4 （q=0.84,1.69,2.53,P〉0.05）; the respiratory syncytial viruse （RNA） Ct value： group A〈group B〈group C （F=152.76, P〈0.01）, while method 1 〈method 2〈method 3 （q---6.98,3.75 ,P〈0.05）, and there were no statistical differences between method 2 and 4（q=1.28, P〉0.05 ）. Conclusions The sputum homogenization methods have an adverse impact on nucleic acid extration. The co-extraction effects of by TIANamp Virus DNA/RNA Kit and KBW enzyme kit are better than the other methods.

关 键 词：痰 痰液液化 核酸共提取 多重定量PCR 腺病毒 呼吸道合胞病毒 

分 类 号：R440[医药卫生—诊断学]