摘要
目的 评价基于核酸自动提取仪的实时荧光PCR（FQ-PCR）法在人巨细胞病毒（HCMV）核酸定量检测中的性能。方法 取高浓度值的HCMV标准品进行梯度稀释,评价灵敏度;取低值、中值、高值标准品评价精密度;取高浓度的HCMV抗凝血浆梯度稀释验证线性范围;使用HCMV DNA阴性抗凝血浆稀释标准品至最低灵敏度浓度500 copy/ml,验证最低检出限;取高浓度血浆样本和生理盐水,进行交叉污染验证试验。分别用核酸自动提取仪与手工法提取50份血浆标本的HCMV核酸,评价两方法的线性关系。结果 灵敏度方面,呈良好的线性关系（r=0. 998）。精密度方面,3个样本的重复性实验CV值均〈5%。线性范围验证显示在5. 0×10^2copy/ml-5. 0×10^7copy/ml时呈现良好的线性关系（r=0. 991,P 〈0. 05）。实验不存在交叉污染情况。2种核酸提取方法具有良好的相关性。结论 基于安普利Anadas9850全自动核酸提取仪的FQ-PCR法在HCMV核酸定量检测中性能良好,能够较好地满足临床检测需求。
Objective To evaluate the performance of real - time fluorescence PCR （ FQ - PCR） method based on nucleic acid automatic extractor in quantitative detection of human cytomegalovirus （HCMV） nucleic acid. Methods HCMV standard plasma at high concentration was serially diluted to access sensitivity; standard plasma at low concentration, medium concentra- tion and high concentration were used to evaluate precision; anticoagulant plasma at high concentration were serially diluted to access linear range; standard plasma were diluted by HCMV DNA negative anticoagulant plasma to the minimum sensitivity of 500 Copies/m1 to verify the minimum detection limit; plasma samples at high concentration and physiological saline were used to verify cross - contamination. The HCMV nucleic acid of 50 plasma samples were extracted by automatic nucleic acid extraction and manual method respectively, and the linear relationship of the two methods was evaluated. Results In terms of sensitivity, a good linear relationship was observed （ r = 0. 998 8 ）. In terms of precision, the CV values of the repeated experiments of the three samples were less than 5%. Linear range validation showed a good linear relationship within 5.0 × 10^2 Copies/ml - 5.0 × 10^7 Copies/ml （r = 0.991, P 〈 0.05）. In cross contamination, there was no cross contamination in the experiment. The two nucleic acid extraction methods had good correlation. Conclusion The FQ - PCR method based on the AMPLLY anadas 9850 automatic nucleic acid extractor is good in the quantitative detection of HCMV nucleic acid, which can meet the clinical testing requirements well.
作者
刘林娜
章婷婷
余坚
陈向南
陈占国
LIU Lin-na;ZHANG Ting-ting;YU Jian;CHEN Xiang-nan;CHEN Zhan-guo(Department of Laboratory Medicine,the Second Affiliated Hospital of Wenzhou Medical University,Wenzhou,Zhejiang 325000,China)
出处
《中国卫生检验杂志》
CAS
2018年第22期2712-2714,2717,共4页
Chinese Journal of Health Laboratory Technology
基金
浙江省医药卫生科技计划项目(2017KY112)
温州市公益性科技计划项目(Y20170201)
关键词
核酸自动提取仪
巨细胞病毒
荧光定量PCR
性能评价
Automatic nucleic acid extractor
Cytomegalovirus
Real time PCR
Performance evaluation
