Rapid Detection Of Two Foodborne Pathogens By QPCR And LAMP Technologies Analysis

In this paper,the foodborne pathogens Listeria monocytogenes and Staphylococcus aureus were the main research objects.Bacterial DNA extracted by different methods were applied for rapid detection of L.monocytogenes and S.aureus,by real-time PCR?qPCR?and loop-mediated isothermal amplification?LAMP?methods.The detection sensitivity for each assay was determined.In addition,the aptamers magnetic capture and LAMP?AMC-LAMP?were combined for sensitive detection of L.monocytogenes.The main experimental results were as follows:1.The qPCR was used to detection L.monocytogenes and S.aureus.Results showed that the detection limits were 1.12 CFU/mL for L.monocytogenes and 1.04 CFU/mL for S.aureus,using DNA extracted by Commercialized DNA extraction kit from pure cultures.With DNA extracted from artificially contaminated fish samples,the qPCR detection limits were 1.12×10¹ CFU/mL for L.monocytogenes,and 1.04×10¹CFU/mL for S.aureus.When the two methods were compared,bacterial DNA extraction by the column method was 32%of the kit method,and the sensitivity of subsequent qPCR detection was reduced by 10 times.However,the extraction time was 90 min for kit method,whereas it was11 min for column method.Thus,column method is more suitable for rapid extraction of bacterial from a large number of samples with low sensitivity requirements.2.LAMP assays were developed for specific detection of L.monocytogenes and S.aureus.Results showed that the two pathogens had the best amplification at 60?.Under this circumstance,the detection time was 32:31 min for L.monocytogenes and 30:43 min for S.aureus;Detection limits were 2.0×10¹ CFU/mL for L.monocytogenes and1.3×10¹ CFU/mL for S.aureus,using bacterial DNA extracted by kit method from pure cultures.With DNA extracted from artificially contaminated fish samples,detection limits were 2.0×10² CFU/mL for L.monocytogenes and 1.3×10² CFU/mL for S.aureus.Similarly,the DNA extraction efficiency of the column method was lower,and the sensitivity of subsequent LAMP detection was also reduced by 10 times.When the LAMP reaction solution was premixed and stored at-20?,sensitivity of the assay was not changed within 28 days for detection of S.aureus.3.Combined with bacterial DNA extracted by the column method,we developed a fast and on-site LAMP Premixed Kit of S.aureus.By premixing and dispensing the LAMP reaction solution?reaction buffer,Bst polymerase,primers and ddH₂O?,the storage temperature,shelf life,repeatability,and reliability of the LAMP Premixed Kit were tested.The results showed that the LAMP Premixed Kit had a shelf life of 300 days under storage conditions of-20?or-80?,and the reliability and sensitivity of the LAMP Premixed Kit were good during the shelf life.4.We combined the aptamers magnetic capture with LAMP to establish AMC-LAMP for sensitive detection of L.monocytogenes.Four aptamers with higher binding affinity to L.monocytogenes were selected to construct aptamers-conjugated magnetic particles,for capturing of L.monocytogenes.The capture efficiencies were ranged from 37.66-75.35%.The optimum conditions for LAMP reaction were 1.6?M for FIP/BIP primers,0.4?M for FLP/BLP primers,and 0.2?M for F3/B3 primers.And the reaction temperature was set as 63?.AMC-LAMP had a detection limit of 5 CFU/mL,and the total assay time was approximate 3h.These results indicate the developed AMC-LAMP was a potential useful method for rapid screening and on-site detection of L.monocytogenes in food samples.