Establishment and application of a detection method for hepatitis E virus in shellfish based on nanoplate digital PCR
10.3760/cma.j.cn112866-20250827-00184
- VernacularTitle:基于纳米板数字PCR的贝类中戊型肝炎病毒检测方法的建立与应用
- Author:
Qiuyuan WANG
1
;
Ruiting ZHANG
;
Wenjiao YIN
;
Jingyuan CAO
;
Juan SONG
;
Ke XU
Author Information
1. 中国疾病预防控制中心病毒病预防控制所 国家卫生健康委员会医学病毒和病毒病重点实验室，北京 102206
- Publication Type:Journal Article
- Keywords:
Shellfish;
Hepatitis E virus;
Digital PCR;
Real-time fluorescence quantitative RT-PCR
- From:
Chinese Journal of Experimental and Clinical Virology
2025;39(5):631-637
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish a digital reverse transcription polymerase chain reaction（dRT-PCR）detection method for hepatitis E virus（HEV）using nanoplates，and to provide technical reference for HEV monitoring in shellfish by combining virus enrichment pretreatment methods.Methods:The annealing temperature，primer and probe concentrations of HEV dRT-PCR were optimized，and the specificity of the method was evaluated；the sensitivity of this method for detecting HEV in water samples and oyster extracts was compared. The inhibition rate and recovery rate of HEV detection in artificially contaminated oyster samples were calculated，commercially available oyster samples were tested，and compare them with real-time fluorescence quantitative RT-PCR（qRT-PCR）method.Results:The optimized annealing temperature for HEV dRT-PCR was determined to be 60 ℃，and the final concentrations of primers and probes were 0.4 μmol/L，0.4 μmol/L，and 0.2 μmol/L，respectively，indicating good specificity. The sensitivity of both methods for detecting HEV RNA in water samples was higher than that in oyster extracts. The recovery rates of HEV in oyster specimens contaminated with HEV fecal suspension by dRT-PCR and qRT-PCR were 18.76% and 18.36%，respectively，with no statistically significant difference（ P>0.05）；the inhibition rates were 17.26% and 9.58%，respectively，with statistically significant differences（ P<0.05）；55 commercially available oyster samples were tested，and both methods detected HEV RNA positivity in the same sample. Conclusion:The dRT-PCR method established in this study，combined with “proteinase K digestion，PEG/NaCl precipitation，and chloroform/n-butanol extraction” pretreatment，has a good recovery effect on HEV in shellfish food containing a large amount of PCR inhibitors，and can achieve absolute quantification. It has certain application value in monitoring and risk assessment of HEV in shellfish food.
