多种比例混合样本对新型冠状病毒核酸检测结果的影响  

Impact of mixed samples in various proportions on the detection of novel coronavirus nucleic acids

作　　者：张自豪 程菲[1] 刘双[1] 谢江 刘兆宇 于梅[1] 于帅[2] ZHANG Zi-hao;CHENG Fei;LIU Shuang;XIE Jiang;LIU Zhao-yu;YU Mei;YU Shuai(Hainan Hospital of Chinese PLA General Hospital,Sanya,Hainan 572013,China)

机构地区：[1]解放军总医院海南医院检验科,海南三亚572013 [2]解放军总医院海南医院热带医学科,海南三亚572013

出　　处：《中华医院感染学杂志》2021年第19期2886-2890,共5页Chinese Journal of Nosocomiology

基　　金：国家老年疾病临床医学研究中心开放课题基金资助项目(NCRCG-PLAGH-2017018);国家慢性肾病临床医学研究中心开放课题基金资助项目(kfkt202020);解放军总医院科研扶持基金资助项目(2018FC-WJFWZX-1-15)。

摘　　要：目的利用真实的新型冠状病毒(SARS-CoV-2)核酸检测阳性咽拭子样本与不同比例SARS-CoV-2核酸检测阴性样本混合稀释,分析SARS-CoV-2核酸检测结果Ct值的变化,评估多种比例混合样本对新型冠状病毒核酸检测的影响。方法选择解放军总医院海南医院已检测的不同SARS-CoV-2病毒载量的8个咽拭子标本,分别与随机4份等体积阴性咽拭子标本、9份等体积阴性咽拭子样本混合、19份等体积阴性咽拭子样本混合,各取200μl进行实时荧光反转录聚合酶链式反应(RT-PCR)检测。另将8个阳性咽拭子样本分别与1份等量咽拭子阴性标本混合,分别进行RT-PCR。结果总体积200μl的多混样本加样,ORF1ab基因Ct值从22.67~39.54,N基因Ct值从24.21~42.37,其中1例单ORF1ab基因阳性,1例单N基因再阳性。随着混合样本数量的增多,Ct值逐渐增大,高Ct值样本,随着混合样本数量的增多,开始出现阴性值。全部样本在5混下全部检出,10混及20混均漏检3例低Ct值样本。比较不同稀释混合比例同原倍样本检出结果的差异,发现2混、5混对Ct值无显著影响,10混、20混对Ct值影响显著(P<0.05)。8个原始样本O基因Ct值从23.54~38.96,N基因Ct值从24.87~35.26,其中1例单O基因阳性,2例阴性。ORF1ab基因与N基因Ct值无明显差异,且2混合样本阳性比例7/8,高于原倍标本6/8。结论随着各地疫情的逐步解除,国外输入及无症状感染者的跨地区输入,仍然给防控带来巨大压力,有限的检测能力是新型冠状病毒肺炎(COVID-19)大流行的主要瓶颈,需持续较长时间进行大规模筛查,占用大量人力资源、社会资源和经济资源,多样本混合尤其是10人份以下样本混合检测将有效提升检测效率、降低检测成本,提高防控能力。OBJECTIVE To analyze the changes in Ct values of SARS-CoV-2nucleic acid test results by using real novel coronavirus(SARS-CoV-2)nucleic acid test positive pharyngeal swab samples mixed with different ratios of SARS-CoV-2nucleic acid test negative samples at dilution,and to evaluate the impact of mixed samples in multiple proportions on the nucleic acid test of SARS-CoV-2.METHODS Eight pharyngeal swab specimens with different SARS-CoV-2viral loads that had been tested at Hainan Hospital of Chinese PLA General Hospital were selected and randomly mixed with four equal-volume negative swab specimens,nine equal-volume negative samples and 19equal-volume negative swab samples,respectively,and 200μl of each specimen were taken for RT-PCR detection.In addition,8positive samples were mixed respectively with 1equal-volume negative specimen,and RT-PCR was performed separately.RESULTS The Ct values of ORF1ab gene ranged from 22.67to 39.54and N gene Ct values ranged from 24.21to 42.37by adding a total volume of 200μl of the multi-mixed samples.Among them,1 case was positive for single ORF1ab gene and 1case was re-positive for single N gene.With the number of mixed samples increased,the Ct values gradually increased,and the samples with high Ct values began to show negative values as the number of mixed samples increased.All samples were detected at 5mixes,and 3cases of low Ct value samples were missed in both 10and 20mixes.Comparing the differences between the detection results of different dilution mixing ratios and the original samples,it was found that 2-mix and 5-mix had no significant effect on Ct values,while 10-mix and 20-mix had a significant effect on Ct values(P<0.05).The Ct values of the O gene of 8original samples ranged from 23.54-38.96,and the Ct value of the N gene ranged from 24.87-35.26,of which,1case was positive for single O gene and 2cases were negative.There was no significant difference between the Ct value of ORF1ab gene and N gene,and the positive ratio of 2-mixed samples was 7/8,higher than 6/8in th

关 键 词：混合样本 新型冠状病毒 实时荧光反转录聚合酶链式反应 

分 类 号：R378[医药卫生—病原生物学]