title: Rapid detection method for hepatitis A virus from lettuce by a combination of filtration and integrated cell culture-real-time reverse transcription PCR.
abstract: We have developed a rapid and simple method for filtration using a positively charged membrane to concentrate hepatitis A virus (HAV) from lettuce and an integrated cell culture-real-time reverse transcription PCR (ICC-real-time RT-PCR) to detect infectious HAV. The most  suitable buffer for HAV concentration by filtration was 100 mM Tris-HCl, 50 mM glycine (pH 9.5). Filtration using the NanoCeram matrix was compared with polyethylene glycol precipitation for viral concentration from lettuce inoculated with 6 log RNA copies of HAV. The recovery rate of filtration  was statistically higher than that of polyethylene glycol precipitation (47.3 versus 24.9%, respectively). The sensitivity of ICC-real-time RT-PCR for detection of infectious HAV was determined by inoculation of FRhK-4 cells with HAV (4 log to 0 log RNA copies). ICC-real-time RT-PCR  detected infectious HAV on average 5 days earlier than cytopathic effects at all inoculation levels. HAV recovered from lettuce (approximately 3 log RNA copies) was also analyzed with ICC-real-time RT-PCR. Infectious HAV was detected within 2 days postinfection by ICC-real-time  RT-PCR, whereas cytopathic effects were not observed until 7 days postinfection. Coupled with a virus concentration and purification system using a positively charged membrane, ICC-real-time RT-PCR has the potential to become a novel and rapid method for the detection of infectious HAV  in vegetables.
authors: Department of Public Health, College of Veterinary Medicine, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea.
authors: Ji-Yeon
authors: JY
authors: Hyeon
authors: Jung-Whan
authors: JW
authors: Chon
authors: Chankyu
authors: C
authors: Park
authors: Joong-Bok
authors: JB
authors: Lee
authors: In-Soo
authors: IS
authors: Choi
authors: Moo-Sang
authors: MS
authors: Kim
authors: Kun-Ho
authors: KH
authors: Seo
mesh_terms: D018929:Cell Culture Techniques; D015169:Colony Count, Microbial; D003257:Consumer Product Safety; D005374:Filtration / Q000379:methods*; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D030041:Hepatitis A virus / Q000302:isolation & purification*; D006801:Humans; D018545:Lactuca / Q000821:virology*; D012367:RNA, Viral / Q000032:analysis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014774:Virulence
chemical_list: D012367:RNA, Viral