| Listeria monocytogenes, belonging to Listeria genus, is a kind of food-borne pathogenic bacteria which can cause human and animal diseases. It can be found in the environment widely. Ingestion of foods contaminated with Listeria monocytogenes leads to people and animals sepsis, abortion and meningitis and the mortality is as high as 30% ~ 70%. The detection, identification, and prevention technology of Listeria monocytogenes has attracted great attention. The traditional detection method has been unable to meet the modern requirements of rapid detection.Loop-mediated isothermal amplification (LAMP) is a new microbiological method. Because it can provide a sensitive, specific, simple and cost-effective test in detecting microbes and viruses, the loop-mediated isothermal amplification method (LAMP) has been used frequently in a lot of areas including the rapid detection of pathogens. But the method is easily to cause false positive which leads to its limitation. To avoid the false positive that aroused by complementary multiple primers under the same condition, the study is to select and improve the LAMP primers combined with the structure of primers and to improve the detection precision of LAMP. And it is called Stemloop-Primer LAMP Method (SLP-LAMP).Stemloop-primers were devised by targeting hlyA gene through primer explorer software (https://primerexplorer.jp/lamp4.0.0/index.html) to develop a LAMP amplification-based detection of total Listeria monocytogenes. The reaction conditions were optimized including temperature, time and Mg2+concentration etc. The SLP-LAMP mixture was made in 25μL of reaction mixture containing a 1.0μmol/L of each inner primer (FIP and BIP), a 0.2μmol/L of each outer primer (F3 and B3), 0.4 mmol/L of each deoxynucleoside triphosphate, 4 mmol/L MgCl2, 10×Bst DNA polymerase reaction buffer, 8U/μL of the Bst DNA polymerase 0.6μL, 1.5μL isolated DNA templates and sterilized double-distilled water. The mixture was incubated at 63℃for 50 min and then heated to 80℃for 5 min to terminate the reaction. Detection results were determined by visible white precipitate or agarose gel electrophoresis.The specificity of this method were studied with 3 Listeria monocytogenes strains and 19 non-Listeria monocytogenes strains. The results indicated Listeria monocytogenes was positive and other strains were negative. The effects of 6 methods of extracting DNA from artificially polluted milk powder were compared and the results indicated that no strict requirements for the method of extracting DNA were not needed.SLP-LAMP technology rapidly detecting Listeria monocytogenes was explored and the sensitivity, artificial contamination detection limit were determined in this study. Also, the results of the method were compared with the ordinary PCR detection method. The results showed that the detection sensitivity and detection limit of the SLP-LAMP method for purely cultured Listeria monocytogenes were 2.45×10~1 CFU/mL and 7.32×10~1 CFU/mL respectively. It only lasted about 2 hours from samples processing to report results and DNA was extracted by DNA extraction kit. In the control, the results showed that the detection sensitivity and detection limit of the PCR method for purely cultured Listeria monocytogenes were 2.45×10~3 CFU/mL and 7.32×10~3 CFU/mL respectively and it took 4 hours.The results indicated that sensitivity, specificity, simplity and cost-effectivity of Listeria monocytogenes detection by SLP-LAMP were superior to those of other methods. And the effectiveness of this method to avoid the false positive that aroused by complementary multiple primers was determined. Given that SLP-LAMP is cheaper, more convenient, and faster, it will be readily accepted by many quarantine institutes requiring microbiological detection methods. |
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