Development and evaluation of hepatitis B virus RNA detection method based on microfluidic chip-based digital PCR
10.3760/cma.j.cn114452-20240730-00418
- VernacularTitle:基于微流控芯片式数字PCR乙型肝炎病毒RNA检测方法的构建与评价
- Author:
Qunfang HUANG
1
;
Rubing XIE
;
Yanping LAN
;
Zhen XUN
;
Can LIU
;
Qishui OU
Author Information
1. 福建医科大学附属第一医院检验科 福建省检验医学重点实验室 福建医科大学基因诊断研究中心 福建省临床免疫学检验临床医学研究中心，福州 350005
- Publication Type:Journal Article
- Keywords:
Polymerase chain reaction;
Microfluidic chip-based digital PCR;
Hepatitis B virus;
RNA
- From:
Chinese Journal of Laboratory Medicine
2025;48(1):103-109
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish a microfluidic chip-based digital PCR (cdPCR) method for detecting hepatitis B virus (HBV) RNA and evaluate its application in patients with chronic HBV infection.Methods:A total of 135 patients with chronic HBV infection were recruited from the First Affiliated Hospital of Fujian Medical University and stratified into two groups based on HBV DNA levels: HBV DNA>100 IU/ml ( n=85) and HBV DNA≤100 IU/ml ( n=50). Additionally, healthy individuals and subjects infected with other viruses ( n=15) served as controls. Primers and probes targeting the HBV pre-C/C region were designed to optimize the microfluidic cdPCR method, and its linear range, limit of detection (LOD), specificity, and precision were assessed. Serum HBV RNA levels were measured using the self-developed method and two commercial kits. Pearson correlation was applied to evaluate the relationships between HBV RNA and other HBV markers. Results:The optimized microfluidic cdPCR method featured a denaturation time of 10 seconds, an annealing/extension temperature of 62 ℃, and primer and probe concentrations of 0.3 μmol/L and 0.2 μmol/L, respectively. The method demonstrated a linear range of 103-10? copies/ml and an LOD of 102 copies/ml. The intra-assay coefficient of variation ( CV) for plasmid standards at 10? and 10? copies/ml were 1.06% and 0.82%, respectively, while the inter-assay CVs were 0.75% and 0.44%. Specificity analysis confirmed the absence of positive signals in sera from healthy controls and subjects infected with other pathogens. In the HBV DNA>100 IU/ml group, the detection rate of the self-developed cdPCR method was 81.18% (69/85), significantly higher than the 64.71% (55/85) achieved by commercial kit B ( P<0.016 7). However, in the HBV DNA≤100 IU/ml group, no significant differences were observed among the three methods ( P>0.05). HBV RNA levels were positively correlated with HBV DNA ( r=0.67), hepatitis B surface antigen ( r=0.53), and hepatitis B e antigen ( r=0.44) (all P<0.001). Conclusion:A microfluidic cdPCR assay for the quantitative detection of HBV RNA has been successfully developed. This assay demonstrates high sensitivity, specificity, and robust detection capability, even for low HBV DNA-concentration samples.
