人类细小病毒B19的3种基因型核酸检测系统的建立及验证

Establishment and verification of a nucleic acid detection system for three genotypes of human parvovirus B19

摘要

目的: 建立人类细小病毒 B19 实时荧光定量聚合酶链式反应检测方法,并对该方法进行系统性的方法学验证。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,构建重组载体制备参考品质粒,建立 B19 定量扩增标准曲线。对建立的 B19 核酸检测系统的线性范围、准确度、精密度 (重复性和中间精密度) 、检测限、基因分型以及特异性抗干扰能力进行验证。结果: 当 B19 病毒定量参考品范围为 1×10⁴∼1×10⁸ IU⋅mL^-1时,实测值与理论值进行双对数回归分析,回归方程 R²⩾0.9800,线性关系良好。B19 阳性样本1和阳性样本2浓度水平标准物质检测值与靶值的偏差均 ⩽±0.5lg 值。B19 阳性标本1和阳性标本2批内、批间精密度确认和连续重复检测精密度确认的 CV 值均 ⩽5% 。NS1 和 VP1 基因的检出限分别为 57.8 和 5.78 IU⋅mL^-1,检出率 ⩾95% 。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、梅毒螺旋体等 7 种非 B19 病原体血浆均为非反应性,种属特异性良好。在其他7种病原体同时存在时,B19 阳性标本能够稳定检出,B19 核酸检测方法不受干扰。本方法针对常见血浆干扰物质,如血红蛋白、三酰甘油及结合性胆红素均为非反应性,同时在以上 3 种常见血浆干扰物质存在时,B19 阳性标本能够稳定检出,B19 核酸检测方法不受干扰。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。

Abstract

Objective: To establish a real-time fluorescence quantitative polymerase chain reaction (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, recombinant vectors were constructed to prepare reference-grade plasmids, and B19 quantitative amplification standard curves were established.The linear range, accuracy, precision (repeatability and intermediate precision), detection limit, specificity, genotyping and anti-interference ability of this method were verified. Results: When the quantitative reference range for B19 virus was 1×10⁴∼1×10⁸ IU⋅mL^-1, a double logarithmic regression analysis was performed between the measured values and the theoretical values, and the regression equation R²⩾0.9800 showed good linear correlation.The deviation between the detection values of B19 standard substances at two concentration levels of sample 1 and sample 2 and the target values was ⩽±0.5lg value.The CV values of B19 positive sample 1 and sample 2 for intra-batch, inter-batch precision confirmation and continuous repeated detection precision confirmation were both ⩽5%.The detection limits of NS1 and VP1 genes were 57.8 and 5.78 IU⋅mL^-1, respectively, with a detection rate ⩾95%.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 had good species specificity.At the same time, in the presence of seven other concurrent pathogens, the positive samples were stably detected, and the B19 nucleic acid testing method was not interfered with.This method was non-reactive to common plasma interfering substances, including hemoglobin, triglycerides and conjugated bilirubin.Meanwhile, in the presence of the above three common plasma interfering substances, B19-positive specimens were stably detected, and the B19 nucleic acid detection method was not affected. Conclusion: This method has strong specificity, high sensitivity, wide linear range, good stability and reliability, and high accuracy, and can be used for the detection of human parvovirus B19 nucleic acid in plasma.

关键词

人类细小病毒B19 / 实时荧光定量聚合酶链式反应检测方法 / 血液制品 / 血浆 / 核酸检测

Key words

human parvovirus B19 / real-time fluorescence quantitative polymerase chain reaction / blood products / plasma / nucleic acid testing

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基金资助

湖北省药品监督检验研究院2022年度重点实验室开放课题(2022HBKFX002)