人类细小病毒B19核酸检测方法的建立及方法学验证

Establishment and methodological validation of a nucleic acid detection method for human parvovirus B19

目的建立人类细小病毒B19实时荧光定量PCR核酸检测方法,并对该方法进行系统的方法学验证。方法针对B19病毒3种基因型的高度保守区设计特异性引物和探针,建立B19定量扩增标准曲线。对该方法的准确度、精密度(重复性和中间精密度)、线性范围、定量限、检测限、特异性及防交叉污染,基因分型及抗干扰能力进行验证。结果当B19病毒定量参考品范围为2.0×10^(1)~1.0×10^(8)IU/mL时,实测值与理论值进行双对数回归分析,回归方程R^(2)≥0.98,线性范围相关性良好。定量限为20 IU/mL,检出率100%。检出限为10 IU/mL,检出率95.23%。针对B19病毒3种基因型标本均能有效检出。针对甲型肝炎病毒、乙型肝炎病毒、丙型肝炎病毒、人类免疫缺陷病毒、人巨细胞病毒、戊型肝炎病毒、梅毒螺旋体7种非B19病原体血浆均为非反应性,种属特异性良好。同时在其他7种同时病原体存在时,弱阳性浓度为E3 IU/mL的阳性标本能够稳定检出,B19核酸检测方法不受干扰。当血红蛋白浓度为431 mg/dL,甘油三酯(1269浊度),非结合性胆红素浓度为20 mg/dL时,本方法针对以上3种常见血浆干扰物质均为非反应性。同时在3种常见血浆干扰物质存在时,弱阳性浓度为E3 IU/mL的阳性标本能够稳定检出,B19核酸检测方法不受干扰。S1(E5 IU/mL)、S2(E4 IU/mL)2个浓度水平标准物质检测值与靶值的偏差均≤±0.5 log值。阳性标本1(浓度水平E5 IU/mL)和阳性标本2(浓度水平E4 IU/mL)日内日间精密度确认和连续5d精密度确认的CV值均≤5%。结论本方法特异性强,灵敏度高,线性范围广,稳定可靠,准确度高,可用于原料血浆中人类细小病毒B19核酸检测。

Objective To establish a real-time fluorescence quantitative PCR nucleic acid detection method of human parvovirus B19 and validate the method systematically.Methods Specific primers and probes for the highly conserved regions of the three genotypes of B19 virus were designed,and B19 quantitative amplification standard curves were established.The accuracy,precision(repeatability and intermediate precision),linear range,quantification limit,detection limit,specificity,anti cross contamination,genotyping and anti-interference ability of this method were verified.Results When the quantitative reference range for B19 virus was 2.0×10^(1) to 1.0×10^(8) IU/mL,a double logarithmic regression analysis was performed between the measured values and the theoretical values,and the regression equation R^(2)≥0.98 showed good linear correlation.The quantification limit was 20 IU/mL,with a detection rate of 100%.The detection limit was 10 IU/mL,and the detection rate is 95.23%.Three genotypes of B19 virus samples can be effectively detected.The plasma of seven non B19 pathogens,including hepatitis A virus,hepatitis B virus,hepatitis C virus,human immuno-deficiency virus,human cytomegalovirus,hepatitis E virus and Treponema pallidum,was non reactive and has good species specificity.Simultaneously,in the presence of seven other concurrent pathogens,positive samples with a weak positive concentration of E3 IU/mL could be stably detected,and the B19 nucleic acid testing method was not interfered with.When the hemoglobin concentration was 431 mg/dL,triglycerides(1269 turbidity)and unconjugated bilirubin concentration was 20 mg/dL,this method was non reactive for all three common plasma interfering substances.In the presence of three common plasma interfering substances,positive samples with a weak positive concentration of E3 IU/mL could be stably detected,and the B19 nucleic acid testing method was not interfered with.The deviation between the detection values of standard substances at two concentration levels of S1(E5 IU/mL)and S2(E4 IU/mL)and the target values were≤±0.5 log value.The CV values of positive sample 1(concentration level E5 IU/mL)and positive sample 2(concentration level E4 IU/mL)for daily precision confirmation and continuous 5-day intra-day precision confirmation were both≤5%.Conclusion This method has strong specificity,high sensitivity,wide linear range,stability,reliability and high accuracy,and can be used for the detection of human parvovirus B19 nucleic acid in plasma.