The Study Of The Inspection Technology Of GⅡ Norovirus And Live Vibrio Parahaemolyticus In Food Based On Droplet Digital Polymerase Chain Reaction

Droplet digital polymerase chain reaction(ddPCR),as a third generation PCR technique,is a technique which has higher sensitivity and doesn’t be affected by matrix inhibition compared with real-time polymerase chain reaction(qPCR).It would improve the detection level of food microorganism theoretically when it was applied to the detection of food microorganism.GⅡnorovirus is one of pathogens of acute gastroenteritis in human,and one of its propagation path is eating the food which was polluted by it.It will make the forecast and prevention of the risk of norovirus infection be possible if reverse transcription droplet digital polymerase chain reaction(RT-ddPCR)is applied to the detection of GⅡnorovirus in food.(1)The RT-ddPCR method was established by optimization of annealing temperature,which was 55.7℃,and then the sensitivity and repetitiveness of this method was compared with those of reverse transcription real-time polymerase chain reaction(RT-qPCR).The results showed that the sensitivity of RT-ddPCR is 5.40copies/μL,which is higher than RT-qPCR,and the repetitiveness of RT-ddPCR is good in general.(2)The elution precipitation method was optimized,and the optimal condition of this method was determined that the eluent was beef-glycine eluent and precipitant was PEG6000+PEG8000 and precipitation temperature was 4℃and time was 4h.The best mean recovery rate of norovirus reached to be 53.43%in this condition.(3)The detection limit of these method which the elution precipitation method inoptimal condition was combined RT-ddPCR and RT-qPCR respectively to detect the GⅡnorovirus of different dilution which was artificially added in Romaine lettuce were determined to be 7.86×10~2copies/g and 1.44×10~4copies/g,which proved that the former method has a higher sensitivity than the latter one.Vibrio parahaemolyticus is one of pathogenic microorganism of acute gastroenteritis in human,which lies in sea food and offshore area usually.This study combined PMA and ddPCR to form a new method called PMA-ddPCR,and applied this method to detect live cells of Vibrio parahaemolyticus.(1)4 primers and probe were designed and the tlh-2 was selected because of better specificity and sensitivity and repetitiveness compared with other primers.The Chelex-100method was selected to extract the DNA of Vibrio parahaemolyticus according to extraction result compared with kit method and boiling water bath method.(2)The PMA concentration and exposure time were optimized.The minimal PMA concentration adequate to inhibit the DNA amplification of dead cells for 6×10~2~6×10~5CFU/m L and the maximal PMA concentration without inhibition on the DNA amplification of live cells for 6×10~2CFU/m L were studied,and the 16μg/m L of PMA was determined based on above results.The time of exposure was determined to be 8min according to the result of the experiment of exposing different time after adding PMA.(3)The PMA-ddPCR and PMA-qPCR methods were used to detect the bacteria fluid which include live cells of different rate,The results of two methods were found to be similar to the result of plate counting method when detected the bacteria fluid which included live bacteria of>1%rate and the accuracy of the results was higher when live bacteria rate was>10%.(4)The sensitivity of PMA-ddPCR and PMA-q PCR was compared which were all 2×10~1CFU/m L,and the PMA-ddPCR didn’t own a higher sensitivity than PMA-qPCR because of the difficulty of extracting DNA from Vibrio parahaemolyticus of 2CFU/m L(5)The detection limit of detecting Vibrio parahaemolyticus in artificially contaminated shrimp and scallop samples by PMA-ddPCR and PMA-qPCR were determined to be 1.12×10~1CFU/g,1.12×10~2CFU/g,8.96CFU/g and 8.96×10~1CFU/g respectively.The method of PMA-ddPCR had an advantage over PMA-qPCR in the detection live cells of Vibrio parahaemolyticus in low contaminated food samples.The ddPCR(RT-ddPCR)technique was applied to detect GⅡnorovirus and Vibrio parahaemolyticus in order to develop the new detection method which can meet the need of the rapid clearance and safety monitoring of food.The study provided a new path of high throughput and rapid detection of other pathogens of low content for food safety monitoring system.