Establishment Of Real-time RPA Method For Detection Of Common Pathogenic Bacteria In Aquatic Products

China is a large aquatic product country,with aquatic product output accounting for more than one third of the world's total output.It is also the world's largest seafood consumer market.FAO predicts that in 2020,China's per capita consumption of aquatic products will reach 36 kilograms.However,due to the aquaculture,transportation,storage and other links,aquatic product are easily contaminated by pathogenic microorganisms resulting in food safety problems.It has been received widely attention.At present,the detection methods and standards of pathogenic bacteria in aquatic products have shortcomings such as long time and complicated operation.They are difficult to meet the requirements of rapid detection of food safety.Therefore,the rapid and accurate detection of pathogenic bacteria in aquatic products has important practical significance.This experiment aimed five kinds of common and harmful aquatic food pathogenic bacteria: Aeromonas hydrophila,Salmonella,Escherichia coli O157:H7,Vibrio parahaemolyticus and Listeria monocytogenes to establish real-time RPA.It can be used for rapid detection of clinical samples,possessing the advantages of simple operation,high sensitivity,effective detection of aquatic food common pathogens.This reserch includes the following:1.Establishment of Real-time RPA detection method for common pathogenic bacteria in aquatic products: the conserved sequence of five aquatic food pathogens was determined,respectively targeting the Aeromonas hydrophila hly gene(accession number: CP006870),Salmonella invA gene(accession number: U43238),Escherichia coli O157:H7 Z0372 gene(accession number:AE005174),Vibrio parahaemolyticus tlh gene(accession number:MH047288.1)and Listeria monocytogenes hlyA gene(accession number: HE999704).Probe and primer were designed for combination screening and the reaction conditions were optimized.The optimization reaction were 39?,30 min and 10 uM.Under the optimized conditions,the minimum detection amount of Aeromonas hydrophila,Salmonella,Vibrio parahaemolyticus and Listeria monocytogenes was 100 copies/?L,and Escherichia coli O157:H7 was 10 copies/?L.The sensitivity was more than 10 times higher than that of conventional PCR method.They have no cross reaction with other pathogens with strong specificity.The intra-group and inter-group variation coefficients of the five bacteria were all less than 10%,with good reproducibility.2.Evaluation of the effect of real-time RPA technique on clinical samples and artificially spiked samples: 61 samples were detected by using traditional detection methods,real-time RPA and PCR.The positive detection rates of Listeria monocytogenes,pathogenic Escherichia coli O157:H7,Salmonella and Vibrio parahaemolyticus were 0%.Compared with traditional methods,the sensitivity and specificity of the real-time RPA method for the detection of Aeromonas hydrophila were 73.7%(14/19)and 100%(42/42).Compared with the traditional detection method,the sensitivity and specificity of PCR were 57.89%(11/19)and 97.61%(41/42).The results showed that the sensitivity and specificity of the real-time RPA were higher than that of PCR.The detection sensitivity of the artificially spiked samples for each bacterium was4 CFU/25 g Aeromonas hydrophila;42 CFU/25 g Listeria monocytogenes;20 CFU/25 g Vibrio parahaemolyticus;5 CFU/25 g Salmonella;15 CFU/25 g Escherichia coli O157:H7.And the minimum detection limit of real-time RPA is 10-100 times lower than that of PCR method.In this study,real-time RPA detection method was established for five common pathogenic bacteria.Compared with traditional methods and PCR detection methods,it has advantages such as short reaction time,no need for expensive equipment and reducing the influence of matrix related inhibitors.Thus it is suitable for rapid detection of pathogenic bacteria in the field.Real-time RPA detection methods for five kinds of pathogens were established,and the evaluation results of clinical samples and artificially spiked samples showed that the real-time RPA detection technology was highly sensitive and specific,which was suitable for the detection of pathogenic bacteria in grass-roots or port aquatic products,and provided a strong guarantee for food safety.