Procedure for rapid concentration and detection of enteric viruses from berries and vegetables.
Several hepatitis A virus (HAV) and norovirus (NV) outbreaks due to consumption of berries and vegetables have been reported during recent years. To facilitate the detection of enteric viruses that may be present on different fresh and frozen products, we developed a rapid and sensitive detection method for HAV, NV, and rotavirus (RV). Initial experiments focused on optimizing the composition of the elution buffer, improving the viral concentration method, and evaluating the performance of various extraction kits. Viruses were extracted from the food surface by a direct elution method in a glycine-Tris (pH 9.5) buffer containing 1% beef extract and concentrated by ultrafiltration. Occasionally, PCR inhibitors were present in the processed berry samples, which gave relatively poor detection limits. However, this problem was overcome by adding a pectinase treatment in the protocol, which markedly improved the sensitivity of the method. After optimization, this concentration method was applied in combination with real-time reverse transcription-PCR (RT-PCR) using specific primers in various types of berries and vegetables. The average detection limits were 1 50% tissue culture infective dose (TCID(50)), 54 RT-PCR units, and 0.02 TCID(50) per 15 g of food for HAV, NV, and RV, respectively. Based on our results, it is concluded that this procedure is suitable to detect and quantify enteric viruses within 6 h and can be applied for surveillance of enteric viruses in fresh and frozen products.
Butot
S
S
Quality and Safety Assurance Department, Nestlé Research Center, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland.
Putallaz
T
T
Sánchez
G
G
D002460:Cell Line; D005506:Food Contamination; D031985:Fragaria / Q000821:virology; D005638:Fruit / Q000821:virology*; D017957:Hepatitis A Virus, Human / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D018545:Lactuca / Q000821:virology; D029322:Norovirus / Q000235:genetics / Q000302:isolation & purification*; D019697:Onions / Q000821:virology; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012401:Rotavirus / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014675:Vegetables / Q000821:virology*
D012367:RNA, Viral