원문정보
초록
영어
Norovirus has become the most common cause of human gastroenteritis in developed countries. Detection procedures of foodborne viruses from foods require several steps. The concentration step using polyethylene glycol (PEG) is time-consuming and the detection efficiency of reverse transcription-polymerase chain reaction (RT-PCR) is affected by inhibitors from food components. In this study, a rapid detection method based on buoyant density centrifugation was
developed to replace the time-consuming chloroform-polyethylene glycol-Tris·Tween method. Feline calicivirus that belongs to the family Caliciviridae was used as a surrogate model for norovirus. After artificial inoculation of feline calcivirus (FCV) to oyster and lettuce, 830 μL of homogenized sample suspension was layered on the top of 670 μL 20% percoll and
centrifuged. Then RNA extraction step was proceeded with the supernatant. By varying several physical conditions, the detection limits were lowered to 2.4×102 PFU per 1 g in oyster and 2.4×100 PFU per 1 g in lettuce. The protocol obtained in this study could be used to develop new detection method for norovirus in foods.
목차
Introduction
Materials and Methods
Oyster and lettuce
Virus and cell
Plaque assay
Detection of FCV using chloroform-polyethylene glycol-Tris · Tween (CPT) method
Detection of FCV based on BDC method
Improvement of detection efficiency using PEGprecipitation
Improvement of detection efficiency by diluting reversetranscription-polymerase chain reaction (RT-PCR)mixture
RNA extraction
RT-PCR assay
Results and Discussion
Quantification of FCV and RT-PCR assay
Comparison of CPT and BDC method to detect FCV inoyster and lettuce
Improvement of detection efficiency using PEGprecipitation
Improvement of detection efficiency by diluting RTPCRmixture
Assessment of the developed protocol
References