Detection Of Three Food-home Virus By RT-PCR-reverse Blot Hybridization

Food-borne virus is one of the major causes of food-borne diseases. There are about 30% of the population in the developed countries have suffered from food-borne diseases each year. In addition, diarrhea and related virus infect more than 2.7 billion patients and lead 240 million children under the age of five to die in developing countries every year. Rotavirus (RV), Norovirus (NV) and Hapatitis A virus (HAV) are well known to be the most common food-borne viruses in the world, which have been spread from person to person by excreta. Epidemiological investigation indicates that the infectant water and food are the most important vehicle in the transmission of RV,NV and HAV.Electron microscopy, ELISA and RT-PCR are the mainly methods to detect RNA virus including RV, NV and HAV. However, electron microscopy is very expensive and has required the higher environment and operators. The sensitivity of ELISA is less than RT-PCR. Therefore, RT-PCR has become the "gold standard" of RV, NV and HAV detection. The aim of this study was to develop a RT-PCR-Reverse Blot Hybridization method for detecting RV, NV and HAV which Not only can effectively improve the detection sensitivity, reduce false positive risk, but also without any instrument, inexpensive, testing results visible. (1) This study used QIAamp Viral RNA Mini Kit for Viral RNA extraction.Oligonucleotide primers targeting Human rotavirus inner capsid protein VP6 gene, GII Norovirus RNA-dependent RNA polymerase conserved RdRp gene and Hepatitis A virus partial gene for VP3-VP1 junction polyprotein were used to amplify viral RNA. Primer concentration, annealing temperature and PCR circles were optimized. The sensitivity of the RT-PCR was 279.3pg (RV),93.7pg (NV),117.3pg (HAV).(2) The amplicon of the three virus were cloned into vecter pGEM-T to for the recombinant plasmid pGEM-RV, pGEM-NV and pGEM-HAV, which was further amplified in an PCR process in reverse blot hybridization.(3) Membranes, deliquation of alkaline phosphatase, addition of RT-PCR amplification, UV cross-link time, enzymatic reaction and washing condition after reverse blot hybridization and enzymatic reaction were optimized. The effects of the time and temperature of reverse blot hybridization on the probe were also examined and discussed to develop a RT-PCR-Reverse Blot Hybridization method for detecting RV, NV HAV. The method of the RT-PCR-Reverse Blot Hybridization has lowest detectable limit of 27.93pg(RV),9.37pg(NV) and 1.173pg(HAV) which was lower than the RT-PCR-Gel Electrophoresis 10～100 times.(4) Detect the 7 Clinical samples by research method to test the availability of the RT-PCR-Reverse Blot Hybridization and compare with RT-PCR-Gel Electrophoresis result. Positive result in 6 of 7 samples was found by RT-PCR-Reverse Blot Hybridization which is in accord with RT-PCR-Gel Electrophoresis. Reverse Spots Hybrid Method is more sensitive than Gel Electrophoresis.