Optimization of an approach to detect low-concentration MNV-1 and HAV from soil-rich or non-soil post-washing water containing various PCR inhibitory substances

Plant- and soil-originated polymerase chain reaction (PCR) inhibitors like polyphenols, polysaccharides, and humic acid (HA) can impact the sensitivity of foodborne virus detection at a low concentration in post-washing water (PWW). This study aims to efficiently detect each virus by inoculating 102 PFU/mL of murine norovirus 1 (MNV-1) and hepatitis A virus (HAV) into post-washing water (PWW) containing various polymerase chain reaction (PCR) inhibitors, including humic acid (HA), pectin, starch, and gallic acid. To optimize the detection of these viruses, a paper filtration-coupled ultrafiltration (PFC-UF) method was used in combination with the polyaluminum chloride (PAC) and polyvinylpyrrolidone (PVP) RNA extraction method, pectinase treatment, and various commercial kits. The PFC-UF with the OneStep PCR Inhibitor Removal Kit can effectively remove HA in PWW when the HA content was less than 1.0 mg/L. However, PFC-UF with the PVP RNA extraction, pectinase treatment, and chloroform/butanol separation were not effective to improve viruses detection. PFC-UF with OneStep PCR Inhibitor Removal Kit successfully recovered MNV-1 (17.58 ± 7.28%) and HAV (7.37 ± 4.06%) from all 9 samples with high soil content and PCR inhibitors. This study successfully addressed the challenge of recovering viruses under extreme conditions of high soil content and PCR inhibitors. The findings provide a promising platform for validating the surveillance of foodborne viruses in washing water at farm fields or washing facilities.