Establishment Of Loop-mediated Isothermal Amplification (LAMP) Methods For Detection Of Listeria Monocytogenes And Yersinia Enterocolitica

Listeria monocytogenes （LM） and Yersinia enterocolitica （YE） both belong tofoodborne zoonotic and psychrotrophic bacteria. LM can be alive and reproductivefrom0～40oC. LM infects human through LM-contaminated food and causes asevere foodborne disease with33%of lethal rate. YE shows productive at2～40oCwhile it can be alive and slow growth at0oC. LM and YE perform high pollutiveability in food processing and cold-storage, especially in process of the meat coldchain production. At present, LM and YE are both hazard wide pathogen which areharmful to the public health and safety not only in developing countries but also indeveloped countries. So it is significant to establish a rapid detection method fordetecting pathogenic LM and YE.In this study, new nucleic acid amplification methods named LAMP （loop-mediatedisothermal amplification） showing the characteristics of simplity, rapidity, idealsensitivity and high specificity were established to target hly gene of LM and outLgene of YE for detecting the pathogenic LM and YE, respectively or simultaneously.Two inner-primers （BIP and FIP） and outer-primers （B3and F3） focused on6regionsof the targrt gene were designed using primers software to amplify the target gene.Some parameters in the LAMP reaction such as Mg2+, betaine, dNTPs, ratio of innerprimers to outer primers, reaction temperature and time were optimized usingdifferent concentrations. The optimal reaction system of the LAMP detecting thegenomic DNA （gDNA） of LM was composed of6mM Mg²⁺,1.8mM dNTPs,1Mbetaine,0.2μM outer-primers,1.0μM inter-primers,1μL Bst DNA polymerase （8U/μL）,2.5μL10×thermpol reaction buffer, and2μL gDNA in25μL of reactionmixture, and kept at65oC for60min. Here the detection limits of LM were77.5 fg/μL of LM gDNA and77CFU/mL of LM bacteria numbers. The optimized reactionsystem of YE included2.5μL10×thermpol reaction buffer,1μL Bst DNA polymera-se （8U/μL）,5mM Mg²⁺,1.8mM dNTPs,1M betaine,0.2μM outer-primers,0.8μMinter-primers,2μL gDNA in25μL of reaction mixture, and incubated at63oC for60min. The LAMP method on detection of YE succeeded to be developed with thedetection limit of48.5fg/μL gDNA and80CFU/mL bacteria numbers in mocksamples. The primers of LM and YE were put in one reaction system to detect LM orYE, simultaneously. The reaction parameters were used following the list of6mMMg2+,1.8mM dNTPs,1M betaine,0.2μM outer-primers （LM）,1.0μM inter-primers（LM）,0.2μM outer-primers （YE）,1.0μM inter-primers （YE）,1μL Bst DNA polym-erase （8U/μL）,2.5μL10×thermpol reaction buffer,2μL gDNA with appropriatevolume distilled water in the total25μL of reaction mixture, and incubating65oC for60min. The detection limits of gDNA and bacteria numbers in mock samples wererespectively82.4fg/μL and84CFU/mL to detect LM, and56.7fg/μL and86CFU/mL to detect YE. As for the mixed LM and YE （1:1），the detection limits were69.4fg/mL to gDNA and94CFU/mL to bacteria numbers. This study will bebenefitial for establishing the detection kit of LM and YE.