Objective:In order to make up for the missed detection of the genus Rocahepevirus (HEV-C) infection in the current clinical test method and to quickly differentiate the infection from the hepatitis A virus (HAV) infection, a triple real-time fluorescence quantitative RT-PCR(qRT-PCR)detection method was established to simultaneously detect HAV, genus Paslahepevirus (HEV-A), and HEV-C. Methods:Three pairs of amplification primers and three TaqMan probes were selected to optimize the reaction system and amplification temperature to construct a triple qRT-PCR method ; standard curves were established by plasmid and the sensitivity of the method was evaluated; specificity was evaluated by other viruses; stability was evaluated by nucleic acid of HAV, HEV-A and HEV-C; samples from suspected hepatitis A or E cases were detected and compared with HAV (or HEV) IgM antibody and nested RT-PCR (nRT-PCR) detection result.Results:The correlation coefficient R2 of three standard curves of triple qRT-PCR were all greater than 0.99, and the slopes were close to -3.3. The minimum detection limits of HAV, HEV-A and HEV-C plasmids were 8 copies/μl, 5 copies/μl and 8 copies/μl, respectively. There was no cross reaction with hepatitis B virus and hepatitis C virus, etc. The coefficients of variation of intra-and inter-batch tests were less than 5%. The positive rates of HAV IgM antibody test, HAV nRT-PCR and triple qRT-PCR were 95.1% (58/61), 83.6% (51/61) and 83.6% (51/61) for serum from suspected hepatitis A cases, respectively. The coincidence rate of HAV IgM antibody test and HAV nRT-PCR (or triple qRT-PCR) was 85.2% (52/61), the difference was statistically significant ( χ2=4.00, P=0.039). The positive rates of HEV IgM antibody test, HEV nRT-PCR and triple qRT-PCR were 89.7%(61/68), 69.1%(47/68) and 72.1%(49/68) for serum from suspected hepatitis E cases, respectively. The coincidence rate of HEV IgM antibody test and triple qRT-PCR was 79.4% (54/68), difference was statistically significant ( χ2=8.64, P=0.002). The positive rates of HEV nRT-PCR and triple qRT-PCR for stool samples from suspected hepatitis E cases were 80.0% (20/25) and 76.0% (19/25), respectively; the coincidence rate was 96% (24/25), with no significant difference ( χ2=0, P>0.05). Conclusions:This study established a triple qRT-PCR detection method that can simultaneously and rapidly detect hepatitis A virus, genus Paslahepevirus or genus Rocahepevirus. It has high sensitivity, specificity and stability, and is suitable for rapid detection of specimens from patients recently infected with hepatitis A virus or hepatitis E virus.

Objective:To compare enrichment and nucleic acid detection method for hepatitis E virus (HEV) in simulated cola samples.Methods:Cola samples experimentally contaminated with HEV were enriched using positively charged filter membrane-direct lysis (Method 1), tangential flow ultrafiltration membrane-direct lysis (Method 2), and Method 3 and 4 (with the addition of a PCR inhibitor removal step on the basis of Method 1 and 2, respectively), and were assayed by real-time fluorescence quantitative RT-PCR(RT-qPCR), and the recoveries and inhibition rates were compared. Digital RT-PCR(RT-dPCR) and RT-qPCR were applied to detect the recovery of HEV in different medium and low concentrations of experimentally contaminated cola samples; and the inhibition rate and sensitivity of HEV RNA detection in different matrices.Methods 3 was selected for virus enrichment of 8 commercially available cola specimens, RT-qPCR and RT-dPCR for HEV RNA detection.Results:The HEV recoveries of method 3 and 4 (10.44% and 10.16%) were higher than those of method 1 and 2 (4.89% and 0.32%), and the differences were statistically significant ( P<0.05). The inhibition rates of method 3 and 4 were smaller than the inhibition rates of method 1 and 2. The recoveries of HEV in medium concentration artificially contaminated cola samples by RT-qPCR and RT-dPCR were 17.04% and 16.28%, respectively, and for low concentration artificially contaminated cola samples were 6.91% and 4.65%, respectively, and the differences in recoveries between the two assays at the same concentration were not statistically significant ( P=0.260, P=0.107 ); Cola matrix inhibits the detection of both RT-qPCR and RT-dPCR assays. Eight commercially available cola specimens were negative for HEV. Conclusions:Detection of HEV in cola beverages can be done by positively charged filter membrane-direct lysis + inhibitor removal (method 3) or tangential flow ultrafiltration membrane-direct lysis + inhibitor removal (method 4) enrichment, followed by RT-dPCR or RT-qPCR, with a high recovery of virus detection.

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.

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.

Objective:In order to make up for the missed detection of the genus Rocahepevirus (HEV-C) infection in the current clinical test method and to quickly differentiate the infection from the hepatitis A virus (HAV) infection, a triple real-time fluorescence quantitative RT-PCR(qRT-PCR)detection method was established to simultaneously detect HAV, genus Paslahepevirus (HEV-A), and HEV-C. Methods:Three pairs of amplification primers and three TaqMan probes were selected to optimize the reaction system and amplification temperature to construct a triple qRT-PCR method ; standard curves were established by plasmid and the sensitivity of the method was evaluated; specificity was evaluated by other viruses; stability was evaluated by nucleic acid of HAV, HEV-A and HEV-C; samples from suspected hepatitis A or E cases were detected and compared with HAV (or HEV) IgM antibody and nested RT-PCR (nRT-PCR) detection result.Results:The correlation coefficient R2 of three standard curves of triple qRT-PCR were all greater than 0.99, and the slopes were close to -3.3. The minimum detection limits of HAV, HEV-A and HEV-C plasmids were 8 copies/μl, 5 copies/μl and 8 copies/μl, respectively. There was no cross reaction with hepatitis B virus and hepatitis C virus, etc. The coefficients of variation of intra-and inter-batch tests were less than 5%. The positive rates of HAV IgM antibody test, HAV nRT-PCR and triple qRT-PCR were 95.1% (58/61), 83.6% (51/61) and 83.6% (51/61) for serum from suspected hepatitis A cases, respectively. The coincidence rate of HAV IgM antibody test and HAV nRT-PCR (or triple qRT-PCR) was 85.2% (52/61), the difference was statistically significant ( χ2=4.00, P=0.039). The positive rates of HEV IgM antibody test, HEV nRT-PCR and triple qRT-PCR were 89.7%(61/68), 69.1%(47/68) and 72.1%(49/68) for serum from suspected hepatitis E cases, respectively. The coincidence rate of HEV IgM antibody test and triple qRT-PCR was 79.4% (54/68), difference was statistically significant ( χ2=8.64, P=0.002). The positive rates of HEV nRT-PCR and triple qRT-PCR for stool samples from suspected hepatitis E cases were 80.0% (20/25) and 76.0% (19/25), respectively; the coincidence rate was 96% (24/25), with no significant difference ( χ2=0, P>0.05). Conclusions:This study established a triple qRT-PCR detection method that can simultaneously and rapidly detect hepatitis A virus, genus Paslahepevirus or genus Rocahepevirus. It has high sensitivity, specificity and stability, and is suitable for rapid detection of specimens from patients recently infected with hepatitis A virus or hepatitis E virus.

Objective:To compare enrichment and nucleic acid detection method for hepatitis E virus (HEV) in simulated cola samples.Methods:Cola samples experimentally contaminated with HEV were enriched using positively charged filter membrane-direct lysis (Method 1), tangential flow ultrafiltration membrane-direct lysis (Method 2), and Method 3 and 4 (with the addition of a PCR inhibitor removal step on the basis of Method 1 and 2, respectively), and were assayed by real-time fluorescence quantitative RT-PCR(RT-qPCR), and the recoveries and inhibition rates were compared. Digital RT-PCR(RT-dPCR) and RT-qPCR were applied to detect the recovery of HEV in different medium and low concentrations of experimentally contaminated cola samples; and the inhibition rate and sensitivity of HEV RNA detection in different matrices.Methods 3 was selected for virus enrichment of 8 commercially available cola specimens, RT-qPCR and RT-dPCR for HEV RNA detection.Results:The HEV recoveries of method 3 and 4 (10.44% and 10.16%) were higher than those of method 1 and 2 (4.89% and 0.32%), and the differences were statistically significant ( P<0.05). The inhibition rates of method 3 and 4 were smaller than the inhibition rates of method 1 and 2. The recoveries of HEV in medium concentration artificially contaminated cola samples by RT-qPCR and RT-dPCR were 17.04% and 16.28%, respectively, and for low concentration artificially contaminated cola samples were 6.91% and 4.65%, respectively, and the differences in recoveries between the two assays at the same concentration were not statistically significant ( P=0.260, P=0.107 ); Cola matrix inhibits the detection of both RT-qPCR and RT-dPCR assays. Eight commercially available cola specimens were negative for HEV. Conclusions:Detection of HEV in cola beverages can be done by positively charged filter membrane-direct lysis + inhibitor removal (method 3) or tangential flow ultrafiltration membrane-direct lysis + inhibitor removal (method 4) enrichment, followed by RT-dPCR or RT-qPCR, with a high recovery of virus detection.

Objective:To compare the detection method of hepatitis E virus (HEV) in simulated water samples, and to provide a reference for the detection of HEV in water.Methods:HEV fecal suspension was added to tap water or distilled water simulated water samples, and pretreatment was carried out by electropositive filter-organic eluent elution method (Method 1) to compare the extraction effect of the three nucleic acid extraction kits, A, B, and C. The simulated water samples were pre-treated by Method 1, 2 (electropositive filter-direct lysis method), 3 (tangential-flow ultrafiltration membrane-organic eluent elution method), and 4 (tangential-flow ultrafiltration membrane-direct lysis method) for pretreatment, A kit for nucleic acid extraction, Real time RT-PCR method for detection and comparison of the recovery rate; comparison of the recovery rate of different concentrations of HEV in simulated water samples; comparing the inhibitory effects of inhibitors in tap water samples on real time RT-PCR; and detection of HEV in different batches of tap water specimens.Results:Kit A nucleic acid extraction was better; the recoveries of method 1, 2, 3 and 4 were 7.31%, 39.88%, 6.85% and 64.88%, respectively, which showed a statistically significant difference in the recoveries ( F=114.069, P<0.001). The recoveries of method 4 with the addition of high, medium and low concentrations of HEV were 65.26%, 42.76% and 32.79%, respectively. The inhibition of all four pre-treatment method was less than 75%, which meets the requirements of ISO (15216-2∶2019). Twenty tap water specimens were tested for HEV and the result were negative. Conclusions:This study showed that the two membranes better recovered in combination with direct lysis, respectively; Methods 4 had a higher recovery in the detection of HEV in small volumes of distilled or tap water, but it was limited by the volume of water samples, turbidity, and so on. Suitable method can be selected for different water quality and laboratory conditions.

Objective:To optimize and compare method for hepatitis E virus (HEV) nucleic acid detection from pig liver, and provide technical references for HEV detection in animal viscera specimens.Methods:Three methods (PBS homogenization treatment, proteinase K treatment, chloroform extraction method) were used to pretreat and extract viral nucleic acid form pig liver, which was artificially contaminated with HEV fecal suspensions, and HEV RT-qPCR was used to compare the HEV recovery rate and inhibition rate. The optimized HEV method was applied to commercially available pig liver specimens, and HEV genotyping was performed on positive specimens.Results:The HEV recovery rate of PBS homogenization treatment, proteinase K treatment and chloroform extraction method was 9.88%, 0.19% and 17.28%, respectively. The recovery rate of proteinase K treatment was less than 1%, and it was discarded; t-test was performed to compare recovery rates of the other two methods, which showed statistically significant differences ( t=26.801, P<0.001), the chloroform extraction method had a higher recovery rate. The inhibition rates of the three methods were all less than 75%, within the range of the ISO/TS 15216-2∶2019 standard. Among 192 commercially available pig liver specimens, 17 specimens were detected positive for HEV RNA, with a nucleic acid positive rate of 8.85%; five specimens were successfully genotyped for HEV, all of which were genotype 4. Conclusions:The virus recovery effect was good when chloroform extraction method was used for pig liver pretreatment; moreover, this method could detect HEV RNA from commercially available pig livers, which indicate that it can be used for virus detection in food.

Objective:The two detection method for the detection of hepatitis A virus (HAV) in strawberries were optimized and compared to select the best detection method for the detection of hepatitis A virus in strawberries.Methods:Different concentrations of HAV were inoculated on the surface of known negative frozen strawberry specimens, the concentration of beef extract powder in alkaline elution-PEG concentration method was optimized, the optimal nucleic acid extraction kit was selected, the optimal lysis buffer volume in direct lysis method was optimized, and the recovered viral load was detected by Real-time fluorescence quantitative RT-PCR. SPSS26.0 was used to statistically analyze the data, and the optimized two method were used for the detection of actual specimens.Results:The concentration of beef extract powder by the optimized alkaline elution-PEG concentration method was selected at 3%, and the viral nucleic acid extraction kit was selected as kit B. Six ml of lysis buffer was selected for the optimized direct lysis method. The recovery rates of HAV virus by alkaline elution-PEG concentration and direct lysis were compared with the HAV addition levels, and the HAV virus recovery rates of the two method were 21.50±1.06% and 5.82±0.01%, respectively, and the result showed that the differences were statistically significant. A total of 60 strawberry specimens from four regions were tested at the same time, and the result were all negative.Conclusions:The optimized alkaline elution-PEG concentration method has higher sensitivity and is more suitable for the detection of HAV in strawberry specimens.

Objective:To establish a digital droplet RT-PCR(dRT-PCR) method for Hepatitis A virus (HAV), and compare it with Real time RT-PCR(RT-qPCR) method, and select the best method for detecting hepatitis A virus in strawberry.Methods:Extract HAV vaccine RNA, optimize the reaction conditions of dRT-PCR and evaluate its specificity; Alkaline elution -PEG concentration method was used to extract nucleic acid from strawberry samples. At the same time, dRT-PCR and RT-qPCR method were used to detect the sensitivity and inhibition rate of HAV vaccine RNA in pure water and strawberry matrix, and the recovery rate of HAV in artificially contaminated strawberry was compared, which was applied to the detection of commercially available samples.Results:The optimal annealing temperature for dRT-PCR reaction was 60 ℃, and the optimal concentrations of primers and probes were 0.4 μmol/L、0.4 μmol/L and 0.2 μmol/L, with good specificity. There is no significant difference in sensitivity between the two method in detecting HAV vaccine RNA in pure water and strawberry matrix. The inhibition rate of dRT-PCR is low. The recovery rates of dRT-PCR and RT-qRCR in the detection of strawberry samples contaminated with HAV at higher concentrations were 12.90±0.006% and 30.12±0.02%, respectively. The recovery rates of lower concentrations of HAV contaminated strawberry samples were 18.27±0.07% and 10.85±0.03%, respectively, and the difference was statistically significant ( P<0.05). When strawberry samples on the market were tested, the result of both method were negative. Conclusions:The sensitivity of dRT-PCR method established in this study is not significantly different from that of RT-qPCR in detecting HAV RNA in different substrates, but dRT-PCR has good tolerance to PCR reaction inhibitors and high recovery rate when detecting low concentration HAV. Both detection method can be used for quantitative detection of hepatitis A virus in strawberry, and can be selected according to the actual situation.