| Hepatitis A virus(HAV)is a non-enveloped single-stranded RNA virus,which is one of the important causes of human acute hepatitis.HAV is mainly transmitted through fecal-oral routes(such as contact with HAV-contaminated food,water,and daily life contact),and occasionally through blood transfusion and blood products.People are generally susceptible to HAV.With the improvement of economic and sanitary conditions and the application of hepatitis A vaccine,the incidence of hepatitis A has decreased,but this also led to a decrease in antibody levels and increased susceptibility in unimmunized pop?lations.It is more likely to cause an outbreak of hepatitis A.HAV is relatively stable in the environment,and has strong resistance to dryness,high temperature,high or low pH,light and ?ltraviolet exposure,etc.Most food-borne viruses have very low infectious doses and can survive for a long time on the surface of food and remain infectious.PCR inhibitors in food are high,which affect the detection rate.Therefore,sensitive and specific methods are needed to evaluate food safety.In order to provide a suitable and sensitive method for the detection of HAV in food matrix,different oyster pretreatment methods,four Quantitative Real-time PCR(RT-qPCR),Microdrop chip digital PCR(RT-ddPCR)and three HAV RNA extraction kits were compared and optimized.Part 1 Comparison of different pretreatment methods for oystersThe HAV live attenuated vaccine was added to the oysters as artificially infected sim?lated shellfish samples,and oysters were treated with protease K(method 1),protease K+ PEG precipitation(method 2),protease K+PEG precipitation+chloroform extraction(method 3)and shellfish pretreatment(method 4)according to the national standard GB/T22287-2008.The data were processed by SPSS25.0.First,use methods 1,2,and 3 to perform statistical analysis on the extracted HAV Ct value,recovery amount,and recovery rate.The res?lts show that the difference between several pretreatment methods is statistically significant(Ct value comparison P=0.027<0.05;recovery amount Comparison P=0.002<0.05;Comparison of recovery rate P=0.002<0.05).The Bonferroni method is used to compare the data in pairs,and it is concluded that the Ct value obtained by method 2 is less than that of methods 1 and 3,and the recovery amount and recovery rate are higher than those of methods 1 and 3.The recovery rate of method 2 is 6.7%±0.8%.The RNA extracted by the three methods was tested after 10-fold dilution.The Ct values of the 10-fold diluted specimens were all higher than the Ct values of the undiluted specimens.The recovery ratios of 10-fold diluted solution/stock solution were 0.84,1.03,1.08.The recovery rate of RNA extracted by methods 2 and 3 after 10-fold dilution is higher than that of the undiluted specimen,indicating that the inhibitor in the digestive gland tissue of oyster has a certain inhibitory effect on the PCR reaction and recovery rate,but the inhibitory effect is not obvious.The method 2 with the best recovery effect was selected and compared with the national standard method 4.Firstly,the oysters artificially quantitative mixed with different concentrations of HAV were pre-treated,and the recovered Ct value,virus recovery amount and recovery rate were statistically analyzed.The res?lts show that the greater the amount of virus added,the lower the Ct value,and the higher the virus recovery amount and recovery rate.Method 2 HAV recovery rate was 3.8%?6.4%(P=0.000<0.001),method 4 HAV recovery rate was 1.0%?11.8%(P=0.026<0.05),the difference was statistically significant.In the comparison of the recovery rate of method 2 and method 4 at the same concentration level,the difference was not statistically significant under high-concentration pollution(P=0.063>0.05).Under medium and low-concentration pollution,the virus recovery rate of method 2 was higher than that of method 4.The differences are statistically significant(P<0.001).When HAV is polluted at medium and low concentrations,method 2 can be selected,and the recovery effect is better.Method 2(Protease K+PEG precipitation method)was used to detect 60 commercially available oysters,and no positive res?lts were detected.In summary,method 2,proteinase K+PEG precipitation method is the best for detecting HAV in oysters.When the amount of HAV contamination is low,select method 2 to recover the amount of virus and the recovery rate is higher.In the future,the number of specimens in different seasons can be increased,and more sensitive and specific oyster pretreatment methods and nucleic acid detection methods can be further explored.Part Two:Comparison of Hepatitis A Virus Nucleic Acid Detection and RNA Extraction MethodsThe four methods of HAV detection were compared,which were Quantitative Real-time PCR(RT-qPCR)(Method A,B,C)and Microdrop chip digital PCR(RT-ddPCR)(Method D).Among them,the optimized methods A and B can detect up to 10 copies/?l for plasmid standards.Methods A,B,and C were used to detect the gradient dilution of HAV vaccine strain.The slope,R2 value,amplification efficiency of the standard curve of methods A,B,and C(-3.446?-3.297,0.991?0.998,95.07%?101.051%)are all within the conventional acceptable range(-3.6?-3.0,>0.99,900?110%),and the amplification efficiency of method C is slightly higher.Detect other viral nucleic acids with good specificity.Methods A,B,and C were used to detect 40 artificially contaminated HAV oysters,commercially available oysters and serum specimens,and HAV vaccine specimens.The positive detection rates of methods A,B,and C were 50%,47.5%,and 55%,respectively.After chi-square test,the difference was not statistically significant(P=0.792>0.05).Optimize the detection conditions of D method,and finally determine that the primer probe concentration is lum,lum,0.25um,the annealing temperature is set to 55?,and the detection range is between 4.6copies/?l?7.5×104 copies/?l.In the specificity test,there are 2?3 positive droplets in Norovirus and Coxsackie virus A group,so when the positive droplets are more than 3,it can be judged as a positive result.There is no significant difference in sensitivity between method A and method D in the detection of gradient dilution of HAV live attenuated vaccine.The recovery rates of A and D methods were not significantly different(P=0.879>0.05),respectively,3.16%± 0.08%,and 3.15%± 0.07%,respectively;the recovery rates of low concentration were 2.65%± 0.49%,2.84%± 0.01%(P=0.642>0.05),and the D method was slightly higher than that of method A,but the difference was not statistically significant.The res?lts of the four nucleic acid detection methods(A,B,C,D)were used to detect 26 oysters on the market,none of which were positive.Three nucleic acid extraction kits,Qiagen52906,Omega R6874-01 and Roche 11858882001,were used to extract HAV RNA from artificially quantitative mixed oyster specimens.The extraction efficiency of Roche kit was higher than Qiagen kit and Omega kit,and the Ct values were 27.81±0.26,29.74±0.34,28.47±0.41,respectively.The difference was statistically significant by one-way analysis of variance(P<0.001).Comparison of inhibitor removal effect,the difference in Ct value of HAV virus 10-fold dilution and stock solution of Roche kit,Qiagen kit,Omega kit is 3.21 ±0.13,3.19±0.19,3.13±0.20,The Roche Nucleic Acid Extraction Kit has a slightly better suppression removal effect(The difference is closer to 3.3).The above res?lts show that there is not much difference between methods A and B,and they can be selected according to the actual situation.Method C can be used when detecting food samples such as shellfish with high PCR inhibitor content.In the detection of vaccines and other samples with high RNA purity,the difference between methods A and D is not obvious,and the recovery rate of method D is higher when detecting low-concentration HAV-contaminated shellfish food.The Roche 11858882001 nucleic acid extraction kit performs well in the detection of artificially contaminated oyster specimens.In summary,the proteinase K+PEG precipitation method can be selected for the pretreatment method of oysters,and the virus recovery rate is higher.Roche 11858882001 nucleic acid extraction kit is more advantageous in extracting HAV RNA from shellfish specimens.When detecting HAV RNA in food,methods A,B,and C can be used according to the actual situation,and method D can be used when detecting low-concentration HAV contaminated food. |
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