原料血浆中HBV/HCV/HIV-1核酸检测方法的建立及验证  

Establishment and verification of nucleic acid testing methodfor HBV/HCV/HIV-1 in source plasma

作　　者：邓琨 张林林 喻剑虹 杨玲 陈鄂湘 童剑啸 程郁菁 刘莹 彭干 郑宵蓓 李璞 赵传波 彭丹 张金 王月 DENG Kun;ZHANG Lin-lin;YU Jian-hong;YANG Ling;CHEN E-xiang;TONG Jian-xiao;CHENG Yu-jing;LIU Ying;PENG Gan;ZHENG Xiao-bei;LI Pu;ZHAO Chuan-bo;PENG Dan;ZHANG Jin;WANG Yue(Sinopharm Wuhan Plasma-derived Biotherapies Co.,Ltd.,Wuhan 430207,China)

机构地区：[1]国药集团武汉血液制品有限公司,武汉430207

出　　处：《中国新药杂志》2022年第21期2128-2135,共8页Chinese Journal of New Drugs

基　　金：国家重点科技专项资助项目(2020YFC0841800)。

摘　　要：目的:建立原料血浆中乙型肝炎病毒(HBV)/丙型肝炎病毒(HCV)/人类免疫缺陷病毒(HIV-1)核酸检测方法,并进行系统性验证。方法:分别从灵敏度、精密度、线性、特异性及抗干扰因素等方面,对核酸检测系统进行系统性验证,并筛查1 140 601份原料血浆。结果:分析灵敏度验证盘中HBV(3 IU·mL^(-1))样本、HCV(20 IU·mL^(-1))样本及HIV-1(40 IU·mL^(-1))样本各20份进行单人份检测,样本检出率为100%,高于95%。重复性:实验员A重复检测HBV(3 IU·mL^(-1))样本、HCV(20 IU·mL^(-1))样本及HIV-1(40 IU·mL^(-1))样本各20次,CT值CV分别为2.08%,1.82%,4.70%,均≤5%,实验员B重复检测样本各20次,各项目CT值CV分别为1.33%,2.08%,4.70%,均≤5%;中间精密度:2名实验员各项目CT值CV分别为2.80%,2.26%,3.80%,均≤10%。线性验证:HBV/HCV/HIV-1企业工作参考品10~5, 10~4, 10~3, 10~2浓度水平扩增线性梯度明显,重复性好。特异性验证:将系统性能验证盘作为供试品进行检测,检测结果应与系统性能验证盘预期结果一致。干扰因素验证:当样本中分别含0.19%~1.52%的枸橼酸钠、48~431 mg·dL的溶血血红蛋白及141~1 269福尔马肼浊度的乳糜均对检测无影响。1 140 601份原料血浆中有175份反应性样本,NAT反应性样本:HIV-1样本3份,HCV样本11份,HBV样本132份;ELISA反应性样本:HIV样本0份,HCV样本15份,HBV样本14份。男性样本(100份)高于女性样本(75份)。血型占比O型(37.71%)>A型(29.71%)>B型(21.14%)>AB型(11.43%)。45~55岁年龄段占比最高达64.57%,18~30岁年龄段占比最少仅为4.57%。结论:该方法稳定准确、精密度高、特异性强、抗干扰能力强,适用于原料血浆中HBV/HCV/HIV-1核酸检测。核酸检测与血清学检测有机结合可有效降低输血感染风险,必要的献血浆者追踪随访和更进一步的实验室确证分析有助于血清学检测和核酸检测不一致结果的解释和分析。Objective: To establish and verify a nucleic acid testing(NAT) method for HBV/HCV/HIV-1 in source plasma. Methods: The NAT detection method was systematically verified from the aspects of sensitivity, precision, linearity, specificity, and anti-interference factors, and 1 140 601 source plasma samples were screened. Results: Sensitivity: 20 individual samples of HBV(3 IU·mL^(-1)), HCV(20 IU·mL^(-1)) and HIV-1(40 IU·mL^(-1)) were analyzed in the analytical sensitivity verification plate. The detection rate was 100%, which was higher than 95%. Precision: Repeatability: For HBV(3 IU·mL^(-1)), HCV(20 IU·mL^(-1)) and HIV-1(40 IU·mL^(-1)), the tests were repeated for 20 times by experimenter A. CV of CT value was 2.08%, 1.82%, and 4.70%, respectively, which were all ≤5%. The tests were repeated for 20 times by experimenter B. CV of CT value was 1.33%, 2.08%, and 4.70%, respectively, which were all ≤5%. Intermediate precision: CV of CT value of the two experimenters were 2.80%, 2.26% and 3.80%, all ≤10%. Linear verification: The amplification linear gradient of HBV/HCV/HIV-1 enterprise work reference, which was concentration level of 10~5, 10~4, 10~3, 10~2, was obvious and of good repeatability. Specificity verification: The system performance verification plate was tested as the test sample, and the results was consistent with the expected results of the system performance verification plate. Interference factor verification: There was no interference when the sample contained 0.19%~1.52% sodium citrate, 48~431 mg·dLhemolytic hemoglobin and 141~1 269 formalin turbidity. There were 175 reactive samples in 1 140 601 source plasma. NAT reactive samples: HIV-1 3, HCV 11, HBV 132;ELISA reactive samples: HIV 0, HCV 15, HBV 14;male samples(100) was higher than female samples(75). Blood type: type O(37.71%) > A(29.71%) > B(21.14%) > AB(11.43%). The age group of 45~55 accounted for the highest proportion of 64.57% and the age group of 18~30 the lowest proportion of 4.57%. Conclusion: The developed method is stable

关 键 词：核酸检测 原料血浆 人类免疫缺陷病毒 丙型肝炎病毒 乙型肝炎病毒 

分 类 号：R969[医药卫生—药理学]