SENSITIVE AND SPECIFIC DETECTION OF FOODBORNE PATHOGENS IN PORK BY A LOOPMEDIATED ISOTHERMAL AMPLIFICATION METHODOLOGY

ZHIYONG XIONG*, JUNPING ZHANG**, MIN KANG**

* School of Chemical Engineering and Materials Science, Beijing Institute of Technology Zhuhai, Zhuhai 519088, China - **ZhuHai Maternal and CHILD Health's Hospital, Zhuhai 519001, China

A loop-mediated isothermal amplification (LAMP) method was used in this experiment for rapid detection of the food-borne L. monocytogenes and Salmonella by targeting gene hemolysin gene hly and invA gene with the official design software. The optimal reaction system of L. monocytogenes was found to be composed of 1.6μM (each) of the primers FIP and BIP, 0.2μM (each) of the primers F3 and B3,0.8μM (each) of the primers LP and LB,1M Betaine, 4 mM MgSO4, 10×Thermopol reaction buffer, 4 μL DNA template and 1μ of Bst DNA polymerase. And the reaction mixture of Salmonella consisted of 4 μL of dNTPs(10mM stock solution), 0.5 μL of MgSO4(150mM of stock solution), 2.5 μL of 10xThermopol Reaction Buffer, 6 μL of Betiane, each of 40 pmol/μL inner primers Fip/Bip 2.5 μL ,each of 10 pmol/μL outer primers F3/B3 1.25 μL, 2 μL of template DNA, 1 μL Bst DNA polymerase (8000U/mL of stock solution),1.5 μL ddH2O, total 25 μL reaction system. The detection sensitivity and detection limit of LAMP for L. monocytogenes were 1.43×101 cfu/mL and 2.85×102 cfu/g, lower than the sensitivity and detection limits of the established PCR, 3.57×102 cfu/mL and 7.13×103 cfu/g respectively. For Salmonella, the LAMP method detection limit is 105 cfu/mL, while the PCR method is 107 cfu / mL.