新型冠状病毒肺炎康复者恢复期血浆中多重病原体检测方法的验证

Verification of detection method for multiple pathogens in plasma of convalescent patients with Coronavirus Disease 2019

目的对新型冠状病毒肺炎(Coronavirus Disease 2019,COVID-19)康复者恢复期血浆中多重病原体检测方法进行系统性验证。方法根据血浆样本实际情况,并结合试剂盒要求,分别对COVID-19康复者恢复期血浆中多重病原体分子生物学检测方法进行专属性、重复性、中间精密度及检测限验证,并对50份COVID-19康复者恢复期血浆样本进行方法适用性确认。结果干扰试验和交叉试验结果均显示阳性样本和阴性样本检测均未受影响;同一检测人员或不同检测人员在不同时间相同检测条件下重复检测阳性对照4种病原体熔解温度(melting temperature,Tm)值的RSD分别为0.07%、0.14%、0.07%、0.14%和0.06%、0.23%、0.23%、0.20%,阴性对照内部质控(internal control,IC)和扩增质控(amplification quality control,AC)1和2 Tm值的RSD分别为0.07%、0.01%、0.07%、0.14%和0.11%、0.10%、0.15%、0.22%,重复性和中间精密度RSD均分别小于15%和20%,符合要求;确定了22种病原体的最低检测限;50份COVID-19康复者恢复期血浆样本均未检出病原体。结论COVID-19康复者恢复期血浆中病原体检测方法专属性强、稳定可靠、重复性好,适用于COVID-19康复者恢复期血浆中病原体检测。

Objective To systematically verify the detection method for multiple pathogens in plasma of convalescent patients(CPs)with Coronavirus Disease 2019(COVID-19).Methods According to the actual situation of plasma samples and the requirements of kit,the molecular biological detection method for multiple pathogens in plasma of CPs with COVID-19 was systematically verified for specificity,reproducibility,intermediate precision and limit of detection(LOD),and confirmed for applicability by detecting 50 plasma samples of CPs with COVID-19.Results The results of interference test and cross test showed that the detection of positive samples and negative samples were not affected;The RSDs of melting temperature values(Tm)of the positive control four pathogens by the same or different test personnels at different time under the same test conditions were 0.07%,0.14%,0.07%,0.14%and 0.06%,0.23%,0.23%,0.20%,and those of internal control(IC)and amplification control(AC)1 and 2 were 0.07%,0.01%,0.07%,0.14%and 0.11%,0.10%,0.15%,0.22%,respectively.Meanwhile,the RSDs of reproducibility and intermediate precision were less than 15%and20%respectively,which met the requirements;The minimum LOD of 22 pathogens were determined;No pathogen was detected in 50 plasma samples of CPs with COVID-19.Conclusion The method for detecting pathogens in plasma of CPs with COVID-19 was specific,stable,reliable and reproducible,which was suitable for the detection of pathogens in plasma of CPs with COVID-19.