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: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 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.

Objective:A comparison of method for the detection of hepatitis E virus (HEV) in oysters was performed to provide a technical reference for the detection of HEV in oysters.Methods:After pre-treatment of oyster digestive gland specimens artificially contaminated with HEV fecal suspensions by the proteinase K digestion with reference to the European Union ISO/TS 15216-2∶2019, HEV RNA was extracted by four nucleic acid extraction method and assayed by Real time RT-PCR to compare the HEV recoveries; artificially contaminated oyster digestive gland specimens were pretreated by proteinase K digestion, proteinase K digestion + PEG precipitation, and proteinase K digestion + PEG precipitation + chloroform extraction, respectively, and HEV RNA was extracted by the optimal nucleic acid extraction method, which was assayed by real time RT-PCR to compare the HEV recoveries and inhibition rates of the three pretreatment method. The optimal HEV assay was applied to commercially available oyster specimens.Results:The HEV recoveries of the four nucleic acid extraction methods were 1.37%, 2.50%, 4.24% and 7.56%, respectively, with statistically significant differences ( F=847.220, P<0.001); The HEV recoveries for each of the three pre-treatment method were 6.02%, 13.65% and 21.17%, respectively, with statistically significant differences ( F=16.800, P<0.001), and the proteinase K digestion + PEG precipitation + chloroform extraction method had the highest recovery; the inhibition rates of the three method were 13.38%, 20.98% and 8.66%, respectively, and the differences were statistically significant ( F=20.205, P<0.001), with the lowest inhibition rate for the proteinase K digestion + PEG precipitation + chloroform extraction method. One HEV RNA positive specimen was detected in 120 commercially available oyster specimens. Conclusions:In the HEV detection of oyster specimens, pre-treatment with proteinase K digestion + PEG precipitation + chloroform extraction can improve the recovery of HEV from oysters and is more suitable for pre-treatment of oyster specimens; different manufacturers′ viral nucleic acid extraction method have different HEV recoveries and should be compared and screened for superiority before carrying out the assay.

Objective:To compare the four nucleic acid detection method of hepatitis A virus.Methods:Using method A, B, and C real-time fluorescent quantitative RT-PCR(RT-qPCR)and method D droplet chip digital PCR(RT-dPCR)to detect the sensitivity of HAV plasmid and gradient dilution HAV vaccine respectively. Specific detection of related viral nucleic acid was performed. Methods A, B, and C were used to detect 40 artificially contaminated HAV oysters, commercially available oysters and serum samples, and HAV vaccine samples, and compare the detection rates. The recovery rates of method A and D on artificially contaminated oysters were compared with low concentration of HAV.Results:Both method A and B could detect HAV plasmids up to 10 copies/μL. In the detection of HAV vaccine with gradient dilution, the slope, R 2 value and amplification efficiency of method A, B, and C were all within the acceptable range (-3.446~-3.297, 0.991-0.998, -95.07%-101.051%). For 40 specimens from different sources, the positive detection rates of method A, B, and C were 50% (20/40), 47.5% (19/40), 55% (22/40), and the difference was not statistically significant ( χ2=0.467, P=0.792). Methods A and D have no significant difference in the detection sensitivity of gradient dilution vaccines. For the detection of artificially contaminated oysters with low concentration of HAV, the recovery rate of method D was higher than that of method A, but the difference was not statistically significant (F=0.294, P=0.642). Conclusions:There is no significant difference between method A, B, and C, which is more convenient and fast. When detecting low concentrations of HAV in food, Methods D had a slight advantage, but the detection cost is slightly higher. The detection method can be selected according to the actual situation.

Objective To compare the performance of three nucleic acid detection methods for hepatitis E virus.Methods The open reading frame (ORF) 2 gene sequence of HEV genotype 4 representative strain was cloned into pUC57 vector.Plasmid DNA was detected by two real-time quantitative method A and B,and the detection limits were compared.Other samples were used for specificity detection.Serum specimens of acute hepatitis E patients were detected by three method,and the results were compared.Results The lowest detection limit of plasmid DNA by A and B method can both reach 35 copies/reaction,with specificity of 100％.The HEV RNA positive rate of serum samples from acute hepatitis E patients by A,B and C method was 47.8％ (43/90),43.3％ (39/90) and 41.1％ (37/90),with the concordance rate of 88.9％ (80/90).There was no statistically significant difference among the three method (x2=0.8414,P=0.6566).Serum specimens with Ct values below 34.6 detected by method A,or below 35.6 detected by method B,the success rate of amplification by method C was 100％.Conclusions Method A has both higher sensitivity and specificity,and method C is a sensitive gene detection method.

Objective:To compare the diagnostic performance of four IgM antibody diagnostic kits for hepatitis E virus (HEV) in an outbreak.Methods:Four reagent kits were used to detect the IgM antibody to HEV in serum samples of the subjects in an outbreak of hepatitis E, and the HEV RNA was detected; statistical analysis was carried out.Results:The detection rates of HEV IgM antibody with MP, Kehua, Wantai and Beier′s reagent kits were 40.00% (108/270), 29.26% (79/270), 28.52% (77/270) and 20.37% (55 /270), respectively, with a consistency rate of 78.52% (212/270). For HEV RNA positive samples, the sensitivity of MP, Kehua and Wantai’s reagents was 66.67% (32/48) and that of Beier’s reagent was 58.33% (28/48). The sensitivity of MP, Kehua and Wantai’s reagents was 69.81% (37/53) and that of Beier’s reagent was 60.38% (32/53) for sera from patients with acute hepatitis E. The detection rates of HEV IgM antibody in serum samples of RNA positive or acute hepatitis E clinical diagnosis cases of Beier-Kehua and Beier-Wantai were significantly different (McNemar’s test: P<0.05), but there was no significant difference between the detection rates of other reagents (McNemar’s test: P>0.05); for serum samples of HEV RNA negative or clinically excluded cases of acute hepatitis E, the detection rates of HEV IgM antibody of Kehua and Wantai’s reagents were the most similar (McNemar’s test: P>0.05), but the detection result of other reagents were significantly different (McNemar’s test: P<0.05). Conclusions:The diagnostic kits of MP, Kehua and Wantai for HEV IgM antibody have high sensitivity and are suitable for the early serological screening of acute hepatitis E. In the outbreak of hepatitis E, comprehensive diagnosis should be made by combining the detection result of HEV IgM antibody and HEV RNA.