两种不同释放剂核酸提取法的新型冠状病毒RT-PCR内标检测结果比较  

Results comparing on the internal control of RT-PCR on SARS-CoV-2 by two different nucleic acid extraction methods of releasant

作　　者：刘婷 欧阳政德 吴文金 林孝德 冯志刚 LIU Ting;OUYANG Zheng-de;WU Wen-jin;LIN Xiao-de;FENG Zhi-gang(Department of Clinical Laboratory,Bao'an Hospital of traditional Chinese medicine(Group),Shenzhen,Guangdong 518133,China)

机构地区：[1]深圳市宝安中医院(集团)检验科,广东深圳518133

出　　处：《中国热带医学》2021年第2期186-190,共5页China Tropical Medicine

摘　　要：目的对新型冠状病毒核酸释放剂一步提取法进行优化,提高内标在荧光定量逆转录PCR中的检出效率,降低因内标未检出带来的复查率,提高核酸检测报告时效性。方法对深圳市宝安中医院PCR实验室的新型冠状病毒核酸检测标本,采用本实验室优化方法用释放剂一步法提取核酸,与试剂盒说明书原方法进行比较,观察两种方法提取核酸后内标扩增曲线及Ct值的不同;比较前后复查率改变。结果优化前,各样本内标扩增曲线在Ct 24~44之间散在分布,部分样本内标未出现扩增曲线。经优化后,各样本内标的扩增曲线集中在Ct 22~34之间,且全部呈现典型"S"型曲线;各样本内标的扩增起始循环Ct值明显小于优化前(t=5.937 0, P<0.01),同时优化前后阳性对照N基因及ORF1a/b基因的Ct值差异无统计学意义(P>0.05);优化前后,阳性质评标本N基因(F=1.032, P=0.970 1)与ORF1a/b基因(F=1.262, P=0.784 8),Ct值差异均无统计学意义(P>0.05)。优化后样本总体复查率较优化前显著降低(F=3.899 0, P=0.025 8)。结论优化后的一步法提取核酸方法简单、便捷、用时短,在不影响新型冠状病毒阳性标本N基因及ORF1a/b基因Ct值的同时,内标检出结果明显优化,降低了复查率,缩短了报告发放时限,提高检测效率。Objective Extraction of nucleic acid of SARS-Co V-2 with releasing agent by one-step method wasoptimized to improve the detection rate of the internal control(IC) in RT-PCR, reduce the re-examination rate caused by thenon-detection of IC, and improve the time efficiency of report.Methods The nucleic acid test samples of severe acuterespiratory syndrome coronavirus 2(SARS-Co V-2) from the PCR laboratory of Bao’an Hospital of Traditional Chinese Medicine, were applied to extract nucleic acid with releasing agent one-step method that optimized by the laboratory, andcompared with the original method in the kit specification:To observe the changes of IC amplification curve and cycle threshold(Ct);to compare the change of the review rate before and after.Results The amplification curves of IC were scattered betweenCt24-44 before the optimization, and there were no amplification curves in several samples when unoptimized. After theoptimization, the amplification curves were concentrated in the range of Ct 22-34,and all showed typical "S" shape;the Ct ofsamples were significantly lower than that before the optimized(t=5.937 0, P<0.01), meanwhile, there were no differencebetween the Ct of N and ORF1 a/b gene in the positive control before and after optimization(P>0.05);the Ct of N(F=1.032, P=0.970 1) and ORF1 a/b(F=1.262, P=0.784 8) gene in the positive quality evaluation samples were in the same case(P>0.05).The overall sample review rate was also significantly reduced(F=3.899 0, P=0.025 8).ConclusionThe optimized one-stepmethod for nucleic acid extraction is simple, convenience, and short test time. Without affecting the Ct of N and ORF1 a/b genein the SARS-Co V-2 positive samples, the results after amplification are obviously better than before, and reduced the rate ofreview, shorted the reporting time, which ease the burden of the huge detection pressure.

关 键 词：新型冠状病毒 核酸提取 逆转录PCR 内标 

分 类 号：R563.1+4[医药卫生—呼吸系统]