Study On The RPA Assay For Detection Of Salmonella And Legionella Pneumophila

Worldwide,especially in some parts of China,the air pollution is serious,which provides a good breeding ground for the bacteria in the water and the atmosphere,and poses a great threat to human health,Legionella is often detected in cooling towers,and also in air conditioning systems and water systems of public places,while the Salmonella could contaminate the natural water sources such as rivers and lakes by animal waste or carcesses.The symptoms of legionnaires’ disease are not obvious,and easy to be misdiagnosed or missed diagnosis.Because of its transmission mode,it’s easy to spread and has a high mortality rate.In recent years,the incidence of legionnaires’disease increased quite significantly in the world,and there are more and more reports of Legionella cases in provinces and regions of China.Timely and accurate diagnosis becomes the important precondition for treatment and prevention of legionnaires’ disease.RPA-LFD,the rapid detection method of Legionella pneumophila established by this study,provides more possibilities for this.Nowadays,due to the resistance of drugs and the adaptation to environments of food-borne microorganism,the case of food poisoning caused by pathogenic bacteria is still a common occurrence,to which health and safety organization at home and abroad paid long-term attention.In the cases of food poisoning around the world,the proportion of Salmonella is absolutely more than other strains,so Salmonella is a key target of food safety testing.But once people eat the food contaminated by Salmonella,they are easily infected.Therefore,it is of great significance to explore and develop the technology of rapid detection of Salmonella.In this study,primers of PCR,RPA and qPCR targeting mip gene(GenBank Accession No.AE017354 of Legionnella pneumophila and fimY gene(GenBank Accession No.JQ665438.1)of Salmonella spp.were designed to construct the standard plasmid and develop the rapid detection methods.Several groups of RPA primers were screened by parallel experiments.After the primers determined,the RPA reaction system was optimized,including the prediction of reaction time,the optimization of reaction temperature and the optimization of Mg2+ concentration.A total of 25 μL visual RPA reaction system was optimized as the buffer(dNTPs added)of 12.5 μL,LEG SIGMA FP primer of 1 μL,LEG SIGMA RP primer of 1 μL,template DNA of 1 μL,RPA Enzymes of 5 μL,280 mM MgAC of 1.25 μL,SDW of 3.25 μL.The best reaction temperature and time were 37 ℃ and 20 min,respectively.Through the specific experimental comparison,the two groups of RPA primers and the system were proved to have a strong specificity which were no cross reaction with other pathogenic microorganisms.The sensitivity of the two groups were also performed well.The detection limits of Legionella and Salmonella were 1.6×102 and 1.29 × 102 CFU/mL,respectively.Results were in accordance with qPCR assay.Samples were collected from different areas of Hangzhou,such as natural water,public water,air conditioning and other water samples.We used RPA-LFD,qPCR and conventional culture methods(Legionella pneumophila reference national standard HG/T 4323-2012;Salmonella reference national standard GB 47894-2016)to detect Salmonella and Legionella pneumophila.As a result,Salmonella and Legionella pneumophila were not detected in the samples.In this study,combined recombinase polymerase amplification(RPA)and lateral flow immunoassay techniques(LFD),a rapid RPA-LFD method for detection of two pathogenic bacteria of Legionella pneumophila and Salmonella was established.On the basis of RPA technology with high specificity,high sensitivity,convenience and low requirement for equipment technology,we use lateral flow immunoassay techniques(LFD)combined with the direct observed test strip,make the results of the RPA test quickly and intuitively show out.It provides a new,rapid,simple and efficient method for the detection of pathogenic bacteria,especially in the remote and poor areas.