Study On Real-time Fluorescent Duplex SRCA Method For Detection Of Salmonella And Shigella In Food

The disease caused by foodbome pathogenic bacteria is one of the focuses of food safety and public health in china.Salmonella and Shigella are two common food-borne pathogens,both of which belong to Gram-negative bacteria of the enterobacteriaceae.Salmonella and Shigella and very similar in morphological and biochemical characteristics.They are often found in dairy products,vegetable salads,poultry and other foods,causing food poisoning.Salmonella can cause gastroenteritis,typhoid and paratyphoid.Shigella can cause dysentery.Therefore,the establishment of a rapid and simple method for simultaneous detection of Salmonella and Shigella in food has important practical significance for timely detection of pathogenic bacteria to avoid food poisoning.In this study,Salmonella and Shigella were used as the target bacteria.After combination of SRCA technology and fluorescent technology,a real-time fluorescent saltatory rolling circle amplification method(RF-SRCA)was established for the first time for simultaneous detection of Salmonella and Shigella in food.The genome sequences of Salmonella and Shigella were compared,and the Salmonella(GenBank No.ALFE01000410.1)and Shigella(GenBank No.LC111517.1)sequences with good homology and high conservatism were selected for RF-SRCA primer design.After primer screening and optimization,the primers with high specificing were chosen.By optimizing the reaction system,a single RF-SRCA reaction system for Salmonella and Shigella was established,respectively.The duplex RF-SRCA methods based on single RF-SRCA reaction system and reaction conditions.The RF-SRCA reaction was carried out in a final volume of 20?L containing dNTPs(0.63 mM),Mg2+(3.00 mM),2?L 10 × ThermoPol Reaction Buffer,0.25 ?M of each Salmonella forward and reverse primer concentration,0.50 ?M of each Shigella forward and reverse primer concentration,1 ?Lof each Salmonella and Shigella DNA template,8 U of Bst DNA polymerase,EvaGreen(0.8 ?L),adding sterile deionized water to make up the system to 20?L.The duplex RF-SRCA amplification was conducted as fllows:45 cycles of 62? for 1 min.The amplification products were analyzed by the melting curve.The melting was conducted as fllows:95? for 15 s,70?for 1 min,heating to 95? at a rate of 0.1?/s.The fluorescence signal was collect from 70? to generate a melting curve.Salmonella and Shigella in food were detected and identified according to the difference of melting temperature between the two products.The duplex RF-SRCA final system was used for specificity and sensitivity tests.The specificity of the duplex RF-SRCA assay was evaluated using 44 known bacterial strains,including 14 strains of Salmonella,7 strains of Shigella,23 strains of non-target bacteria,and sterile deionized water was used as a negative control.The test results showed that the RF-SRCA product of Salmonella and Shigella have a relatively stable Tm values.The Tm value of 14 strains of Salmonella falls between 88.89?89.84?.The Tm value of 7 strains of Shigella falls between 84.24?85.34?.The negative control and non-target bacteria presented negative results.The sensitivity tests of single RF-SRCA and duplex RF-SRCA were performed,and compared with the duplex real-time fluorescent PCR(RT-PCR)method.The results showed that the sensitivity of single RF-SRCA for detecting Salmonella and Shigella was 2 fg/?L.The sensitivity of duplex RF-SRCA to detect Salmonella and Shigella simultaneously was 20 fg/?L,and the sensitivity of duplex RT-PCR was 2 pg/?L.Over all,the detection sensitivity of duplex RF-SRCA was 10 times lower than that of single RF-SRCA,and the sensitivity was 100 times higher than duplex RT-PCR.In the artificial contamination test,the skimmed milk was contaminated with Salmonella and Shigella.The DNA template extracted from the bacterial solution of each dilution was tested for single RF-SRCA,duplex RF-SRCA and duplex RT-PCR,respectively.The results showed that the detection limit of single RF-SRCA for Salmonella and Shigella was 100 CFU/mL.The detection limit of duplex RF-SRCA for simultaneous detection of Salmonella and Shigella was 101 CFU/mL,and the detection limit of duplex RT-PCR was 103 CFU/mL.The results showed that the detection limit of duplex RF-SRCA was 10 times higher than that of single RF-SRCA,but it was 100 times lower than that of duplex RT-PCR.In actual sample testing,the duplex RF-SRCA method was compared with the duplex RF-PCR method and the national standard method.Using 62 samples,the positive detection rate of Salmonella by duplex RF-SRCA method,duplex RF-PCR method and national standard method,respectively.The positive detection rate of Salmonella were 9.7%,8.0%and 6.5%,respectively.the positive detection rate of Shigella by duplex RF-SRCA method,duplex RF-PCR method and national standard method,respectively.The positive detection rate of Shigella were 6.4%,4.8%and 3.2%respectively.In summary,a duplex RF-SRCA detection method was established in this study to achieve rapid,sensitive and efficient detection and identification of Salmonella and Shigella in food at the same time.Compared with the duplex RF-PCR method,this method had higher sensitivity and lower detection limit.The positive detection rate was higher than the duplex RF-PCR method and the national standard method.The duplex RF-SRCA method was to operate and the results could be directly determined.The duplex RF-SRCA has great significance for the rapid detection of Salmonella and Shigella in food and provided a new method and new ideas for food safety.