人类细小病毒B19核酸检测方法的建立及方法学验证

Establishment and validation of nucleic acid amplification method for the detection of human parvovirus B19 in plasma

目的:建立人类细小病毒B19实时荧光定量PCR(Q-PCR)检测方法,并对该方法进行方法学验证。方法:针对B19病毒三种基因型的高度保守区设计特异性引物和探针,建立B19 Q-PCR标准曲线。并分别从检测限和定量限、型别覆盖能力、特异性、定量范围及定量线性、精密度、回收率、不同机型、以及不同混样模式进行方法学验证。结果:当B19病毒定量参考品范围为2.0×10^(1)~1.0×10^(8) IU·mL^(-1)时,R^(2)>0.99,扩增效率>97.2%,线性范围相关性良好。定量下限为20 IU·mL^(-1),检测下限为10 IU·mL^(-1),针对B19病毒3种基因型样本均能检出,具有相同的定量下限和检测下限。特异性结果显示,本方法检测甲型肝炎病毒、乙型肝炎病毒、丙型肝炎病毒、人类免疫缺陷病毒1型、人巨细胞病毒、戊型肝炎病毒、单纯疱疹病毒1型、梅毒螺旋体等核酸阳性血浆,均无非特异反应,且在上述病原体存在的情况下,B19病毒的定量下限和检测下限均能稳定检出,不受干扰。回收率结果显示,本方法回收率在50%-150%之间,RSD<30%。此外,本方法的精密性变异系数均小于3%,在ABI 7500和伯乐CFX-96机型上,相关技术指标均能达标。且采用48混合96混检测模式检测终浓度10^(4) IU·mL^(-1)和20 IU·mL^(-1)的样本,定量要求符合标准。结论:本方法线性范围广、灵敏度高、特异性和精密度好,回收率高,且可用于不同品牌的荧光定量PCR仪,适用范围广,检测模式灵活,可用于人血浆B19病毒核酸筛查,本研究将为血液制品质量标准提高提供技术储备。

Objective:The study aims to establish a real-time fluorescence quantitative PCR(Q-PCR)method for the detection of parvovirus B19 in human plasma and to verify the method.Methods:Specific primers and probes were designed for the highly conserved regions of three genotypes of B19 virus,and Q-PCR standard curves of B19 virus was constructed.The method was verified from various aspects,including the limit of detection,limit of quantification,genotypes coverage ability,specificity,quantification range and quantification linearity,precision,recovery rate,and different models of pooled samples.Results:This study demonstrated that the method had outstanding linear relationship.When the quantitative reference range of B19 virus was 2.0×10^(1)-1.0×10^(8) IU·mL^(-1),R^(2) was higher than 0.99 and amplification efficiency was higher than 97.2%.The lower limit of quantification was 20 IU·mL^(-1),the lower limit of detection was 10 IU·mL^(-1),and the three genotypes of B19 virus could be completely detected in the range.Specificity results showed that this method was nonspecific for the detection of nucleic acid positive plasma,containing pathogens as hepatitis A virus,hepatitis B virus,hepatitis C virus,human immunodeficiency virus type 1,human cytomegalovirus,hepatitis E virus,herpes simplex virus type 1(HSV-1),treponemapallidum.Even in the presence of these pathogens,the lower limit of quantification and the lower limit of detection of the method could be completely detected without interference.The recovery results showed that the recovery rate of this method was 50%-150%with RSD<30%.Moreover,the precision variation coefficient of this method was less than 3%,and the relevant technical indexes met the standards in ABI 7500 and Bole CFX-96.Finally,48 and 96 plasma pooled models were used to detect the B19 virus load(10^(4) IU·mL^(-1) and 20 IU·mL^(-1)),and the quantitative requirements met the standard.Conclusion:This method has a wide linearity range,high sensitivity and specificity,reliable precision,and high recovery rate.It can be used in two different brands of fluorescence quantitative PCR instruments,with a wide range of application and flexible detection mode,and can be used for nucleic acid screening of B19 virus in human plasma.This study will provide technical reserve for improving the quality and safety of blood product.