摘要
目的研究磁珠法、离心柱法及一步法对新型冠状病毒(2019-nCoV)核酸提取效率的差异。方法2021年3月5日,在南京大学医学院附属泰康仙林鼓楼医院核酸采样室采集本科室员工咽拭子标本10份,将阳性质控品混入其中,作为模拟阳性标本。用磁珠法、离心柱法及一步法分别提取模拟阳性样本及其稀释样本RNA、人源性标本及其稀释样本RNA,用微量紫外分光光度计测量核酸的纯度(A_(260)/A_(280)比值)及浓度,并进行荧光定量PCR扩增实验,比较其循环阈值(CT)和提取效率。用3种方法提取模拟弱阳性标本,比较其扩增后的CT值差异。服从正态分布的计量资料以xˉ±s表示,组间比较采用t检验,多组间比较采用单因素方差分析。结果磁珠法、离心柱法、一步法提取的2019-nCoV核酸均能扩增出阳性结果。磁珠法与一步法提取的RNA扩增CT值差异无统计学意义(t=‒0.995,P=0.376)。离心柱法提取的RNA较另外2种方法提取的RNA扩增后CT值小,离心柱法、磁珠法、一步法提取的模拟阳性样本扩增后ORF1ab基因的CT值分别为29.28±0.06、30.82±0.14、29.79±0.01,差异有统计学意义(F=11.196,P=0.041);E基因的CT值分别为28.52±0.40、27.33±0.78、27.38±0.13,差异有统计学意义(F=3.407,P=0.169);N基因的CT值分别为28.61±1.02、27.24±0.20、27.25±0.47,差异有统计学意义(F=2.880,P=0.020);离心柱法、磁珠法、一步法提取的人源性标本扩增后人源基因CT值分别为19.68±0.36、20.14±0.06和20.58±0.49,差异有统计学意义(F=4.904,P=0.048)。人源性标本稀释2倍即对扩增人源基因CT值有影响,未稀释标本较稀释2倍标本CT值较小,相差2.95±0.22,差异有统计学意义(t=‒3.025,P=0.039)。一步法、磁珠法、离心柱法提取时间分别为(15.00±1.50)、(20.00±1.50)和(40.00±5.5)min,差异有统计学意义(F=688,P=0.027)。结论磁珠法、离心柱法及一步法均可用于2019-nCoV核酸提取,离心柱法提取效率�
Objective To study the difference in the extraction efficiency of the novel coronavirus(2019-nCoV)nucleic acid by using magnetic beads method,centrifugal column method and one-step method.Methods On March 5,2021,10 throat swabs were collected from the staff working in the nucleic acid sampling room in Department of Clinical Laboratory,Affiliated Taikang Xianlin Drum Tower Hospital,Medical School of Nanjing University.The positive quality control samples were mixed into the swabs and used as mock positive samples.The RNA was extracted from simulated positive samples and their diluted samples by using magnetic beads method,centrifugation column method and one-step method.The purity(A_(260)/A_(280) ratio)and concentration of the nucleic acid obtained were measured by micro-uv photometry,and fluorescence quantitative PCR was performed to compare the CT value and extraction efficiency.The three methods were used to extract the simulated weak positive specimens and to compare the difference of CT values after amplification.The measurement data that followed normal distribution were expressed by xˉ±s,the t test was used for comparing in the same group,and single factor analysis of variance was used for comparing among multiple groups.A P value smaller than 0.05 indicated a significant difference.Results 2019-nCoV nucleic acid extracted by magnetic bead method,centrifugal column method and one-step method could amplify positive results.There was no significant difference between the CT value of RNA amplification extracted by magnetic bead method and one-step method(t=‒0.995,P=0.376).The CT values of orf1ab gene amplified by centrifugal column method,magnetic bead method and one-step method were 29.28±0.06,30.82±0.14 and 29.79±0.01 respectively(F=11.196,P=0.041).The CT values of E gene were 28.52±0.40,27.33±0.78 and 27.38±0.13 respectively(F=3.407,P=0.169).The CT values of N gene were 28.61±1.02,27.24±0.20 and 27.25±0.47,respectively(F=2.880,P=0.020).The CT values of human genes extracted by centrifugal colum
作者
周慧聪
王子铭
胡悦
张洁
朱艳婷
黄梅
严孝岭
虞伟
Zhou Huicong;Wang Ziming;Hu Yue;Zhang Jie;Zhu Yanting;Huang Mei;Yan Xiaoling;Yu Wei(Department of Clinical Laboratory,Affiliated Taikang Xianlin Drum Tower Hospital,Medical School of Nanjing University,Nanjing 210046,China)
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2022年第4期393-398,共6页
Chinese Journal of Laboratory Medicine
基金
南京大学医学院附属泰康仙林鼓楼医院院管课题(TKKYZD20211609)。
