Highly Sensitive Detection of Low Concentration Hepatitis A Virus in Small Berries Using Polyethylene Glycol Precipitation enrichment and Pre-Amplification Reverse Trandcription-Quantitative Polymerase Chain Reaction

On the basis of polyethylene glycol (PEG)precipitation enrichment and improved pre-amplified reverse transcription-quantitative polymerase chain reaction (RT-qPCR),a method was established for highly sensitive detection of low-dose contamination of hepatitis A virus (HAV)in small berries. The treatment time of HAV in strawberry enriched by PEG precipitation was optimized,and the pre-amplification steps were set up to accelerate the lysis of sample nucleic acid and enrichment of templates. By using this method,the recovery was 15.51%,the lowest detection sensitivity could be up to 4.49 CCID50/ mL of HAV attenuated vaccine titer,and the sensitivity of HAV plasmid was as low as 3.5 copies/mu L. Compared with ISO/ TS15216-2:2019 standard method,this method had higher detection efficiency and was suitable for detection of trace contaminated HAV in small berries. The positive rate of HAV in 78 batches of commercially available small berry samples was 5.13%,which was higher than that of ISO/TS 15216-2:2019 standard (1.28%). This method showed good applicability and application prospect for detection of low-dose HAV virus contamination in small berries.