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Study on a Loop Mediated Isothermal Amplification (LAMP) for the Rapid Detection of Listeria Monocytogenes in Meat

Author: WangXiaoLi
Tutor: ZhangWei
School: Agricultural University of Hebei
Course: Microbiology
Keywords: loop-mediated isothermal amplification LAMP detection Listeria monocytogenes meat
CLC: R155.5
Type: Master's thesis
Year: 2010
Downloads: 241
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Abstract

Listeria monocytogenes, belonging to Listeria genus, is a kind of food-borne pathogenic bacteria which can cause human and animal diseases. It is widely distributed in the environment,where its primary habitat may be soil, decaying vegetation and foods. Ingestion of foods contaminated with L. monocytogenes can result in listeriosis, a severe infectious disease characterized by meningitis,septicemia and abortion etc. The fatality rate is high(30%~40%). Listeriosis predominantly affects certain risk groups, including pregnant women, newborns, elderly people, and immumocompromised patients. The increasing incidences of L. monocytogenes in food-borne outbreaks draw people,s attention. Traditional method for routine detection of L. monocytogenes is complex and time-consuming. It takes from 5 to 7 days with low sensitivity. This method can not fulfil the need for detecting food-borne pathogens rapidly and sensitive.We used the hlyA sequence of L. monocytogenes(CMCC54001) as target sequences and used Primer Explorer software, version3(http://primerexplorer.jp/lamp3.0.0/index.html), to design LAMP primers. The reaction conditions were optimized including temperature,time and Mg2+ concentration etc. The LAMP mixture was made in 25μL of reaction mixture containing a 1.2μM concentration of each inner primer(FIP and BIP), a 0.2μM concentration of each outer primer(F3 and B3), a 0.6μM concentration of the loop primer(LB), 1.6μM each deoxynucleoside triphosphate, 5μM MgSO4,10×Bst DNA polymerase reaction buffer, 8 U of the Bst DNA polymerase large fragment, 2μL isolated DNA templates and sterilized double-distilled water. The mixture was incubated at 64℃for 20 min and then heated to 80℃for 10 min to terminate the reaction. We can obtain the results of detection when the white precipitate was observed by visual examination.Listeria monocytogenes were cultured for 12 h. After incubation, the number of colonies was counted,and serially diluted tenfold in NaCl(9%). Each dilution was used for LAMP and PCR detection. We extracted DNA using the same chemical reagents method. The detection limit of the LAMP assay for pure bacterial culture was 2.4 CFU/mL. In contrast, the detection on limit of the PCR assay for pure bacterial culture was 240 CFU/mL.The detection limit of artificially contamination was 68 CFU/g with LAMP detection for two hour.In contrast, the detection limit of artificially contaminated was 6800 CFU/g with PCR detection for four hours.Our results showed this LAMP assay had a high specificity for the detection of L. monocytogenes by amplifying a fragment of hlyA gene of four L. monocytogenes strains rather than other five strains of Listeria spp. and thirteen non-Listeria spp. strains.The effect of four methods of extraction DNA from L. monocytogenes in artifical contamination were compared, LAMP, the method of extracting DNA, no strict requirements.The result indicates: LAMP has the potential to replace PCR because of its simplicity, tapidity, specificity and cost-effectiveness. In our study,we had developed a new and rapid molecular biological method for detection of L. monocytogenes in food sample. For the rapid detection of foodborne pathogens build a technology platform.

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