Establishment and methodological validation of a nucleic acid detection method for human parvovirus B19
10.13303/j.cjbt.issn.1004-549x.2024.11.004
- VernacularTitle:人类细小病毒B19核酸检测方法的建立及方法学验证
- Author:
Yue WANG
1
;
Xiaobei ZHENG
1
;
Yajing ZHENG
2
;
Yu SUN
2
;
Qin GONG
1
;
Linlin ZHANG
1
;
Ying ZHAO
1
;
Dandan YANG
1
;
Yi YANG
1
;
Lin LI
1
;
Boran LI
3
;
Jin ZHANG
1
Author Information
1. Sinopharm Wuhan Biopharmaceuticals Co., Ltd, Wuhan 430207, China
2. Hubei Provincial Drug Administration InspeCtion Center (Hubei Provincial Vaccine InspeCtion Center), Wuhan 430064, China
3. Hubei Provincial Drug Administration, Wuhan 430071, China
- Publication Type:Journal Article
- Keywords:
human parvovirus B19;
real-time fluorescence quantitative PCR;
blood products;
plasma;
nucleic acid testing
- From:
Chinese Journal of Blood Transfusion
2024;37(11):1234-1240
- CountryChina
- Language:Chinese
-
Abstract:
[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 2.0×101 to 1.0×108 IU/mL, a double logarithmic regression analysis was performed between the measured values and the theoretical values, and the regression equation R2≥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 (1 269 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.
