Establishment And Application Of LAMP Method For Rapid Detection Of Two Food-borne Pathogens

1. Background and ObjectiveIn recent years, with the accelerated process of economic globalization, food safety has become a worldwide public health attention. Food-borne pathogens are the primary cause of food-borne illness, causing great harm to human health. It is a significant risk of food safety. Recently, the methods for food-borne pathogens are time-consuming and laborious and also are lower sensitivity.Enterobacter Sakazakii is one of the important food-borne pathogens which was parasitic in the intestine of humans and animals. Enterobacter sakazakii can infect children by the digestive tract,mainly through powdered infant formula. Then causing neonatal meningitis, septicemias and necrotizing colitis, and it also causes neurological sequelae or death. In2002, the International Commission for Microbiological Specifications for Foods(ICMSF) ranked the organism as ’Severe hazard for specific populations, and threatening life or led to chronic sequelae’. In2005, State General Administration of Quality Supervision, Inspection and Quarantine promulgate the industry standard of detection Enterobacter sakazakii:SN/T1632.1; Then in2006, they formulate market access requirements made of Enterobacter sakazakii in infant formula milk powder of each batch will be seized. Recently, the methods for isolation and identification of E. sakazakii were conventional culture-and biochemical-based assays and FDA which are time-consuming and laborious and also are lower sensitivity, requiring more than3-7days. So some new molecular biological methods including immunological methods, PCR, Real-time PCR and nucleic acid hybridization has been built.Shigella dysenteriae is a class with strong infectious and serious hazards gram-negative enteric pathogens. Shigellosis is one of the important infectious diseases in developing countries, especially in the world. Recently, the methods for identification of Shigella dysenteriae were conventional methods which are time-consuming and laborious and also are lower sensitivity, requiring more than3-5days. Recently, a number of new molecular biological methods for rapid detection of bacteria have been reported, including immunological methods, ELISA, PCR, Real-time PCR and nucleic acid hybridization. The PCR methods provide powerful tools for rapid, specific, and sensitive detection of food-borne pathogens and are considered reliable alternatives for traditional bacteriological methods. However, owing to the expensive systems required and complicate electrophoretic analysis, this application is still not very common in laboratories.Therefore, the development of cost-effective, simple, sensitive, specific, and rapid detection methods is still needed for the detection of Enterobacter Sakazakii and Shigella dysenteriae. Loop-mediated isothermal amplification (LAMP), a rapid technique for amplifying DNA, has been reported by Notomi et al in the year of2000. The use of four LAMP primers which were designed to recognize six distinct regions on the target gene assures specific amplification. LAMP does not entail the use of expensive equipment.It has high specificity and isothermal amplification characteristics.2. Methods2.1Establishment and application of LAMP method for E. SakazakiiA set of four primers, two outer primers (F3and B3) and two inner primers (FIP and BIP), was designed specifically to recognize the Outer Membrane protein A gene (OmpA) of Enterobacter sakazakii, which were designed by analyzing a conserved region of the ompA gene (GenBank accession number DQ000206) with Primer Explorer version4.0software. The reaction conditions of LAMP were established and optimized. DNA was extracted by E.sakazakii ATCC29544.To determine the detection limit of the LAMP assay, E. sakazakii from ATCC25944was diluted1:10serially sevenfold into a minimum concentration equivalent to that of a single bacterium. And taking100ul bacteria of various dilutions to plate count.DNA templates were then prepared from each dilution by the method described above and aliquots (2μl) were subjected to both LAMP and PCR amplifications. The lower limits of detection (cfu/ml) were reported.3strains of E.sakazakii and12non-E.sakazakii strains were used to determine LAMP specificity. DNA templates were made from fresh overnight bacterial cultures and aliquots were subjected to both LAMP and PCR amplifications.36samples were determined by LAMP and PCR amplifications which comparison with FDA.2.2Establishment and application of LAMP method for Shigella dysenteriaeA set of six primers, two outer primers(F3and B3), two inner primers(FIP and BIP) and two loop primers(LF and LB), was designed specifically to recognize the ipaH gene of Shigella, which were designed by analyzing a conserved region of the ipaH gene (GenBank Accession number:M76445) with Primer Explorer version4.0software. The reaction conditions of LAMP were established and optimized. DNA was extracted by Shigella dysenteriae CMCC48097.Shigella dysenteriae from CMCC48097was diluted1:10serially sevenfold into a minimum concentration equivalent to that of a single bacterium. And taking100μl bacteria of various dilutions to plate count. DNA templates were then prepared from each dilution by the method described above and aliquots (2μl) were subjected to both LAMP and PCR amplifications. The lower limits of detection (cfu/ml) were reported.4strains of Shigella dysenteriae and other non-Shigella dysenteriae strains were used to determine LAMP specificity. And applied to the detection Shigella dysenteriae of artificially contaminated pork samples.The test results compared with the conventional PCR.3. Results3.1The detection results of E.sakazakiiThe most optimal reaction conditions of LAMP of E.sakazakii contained (total volume of25μl)2.5μl10X Bst ThermoPol buffer,0.6mM of dNTP,1μl (8U) Bst DNA polymerase,6mM MgCl2,0.8M Betaine,1.6μmol each of FIP and BIP primers,0.4μmol each of the F3and B3primers, and2μl of DNA sample. The most optimal reaction temperature and time of the LAMP assay for the OmpA gene were58℃and60min, respectively. Product was analyzed in2.0%agarose gel electrophoresis.The LAMP assay detection limit was detecting20cfu/ml of pure E.sakazakii. The traditional PCR assay detection limit was200cfu/ml. LAMP detection sensitivity is10times the sensitivity of conventional PCR.36milk samples were detected by LAMP, two samples were have E. sakazakii, the positive rate was5.6%. The test result was consistent with the FDA method.3.2The detection results of Shigella dysenteriaeThe most optimal reaction conditions of LAMP of Shigella dysenteriae contained (total volume of25μl)2.5μl10×Bst ThermoPol buffer,5mM MgCl2,0.3mM dNTPS,0.6mM Betain,1.6μmol each of FIP and BIP primers,0.4μmol each of the F3and B3primers,1.6μmol each of LF and LB primers, and2μl of DNA sample,and8U of Bst DNA polymerase.The mixture was incubated at63℃for60min. Product was analyzed in2.5%agarose gelelectrophoresis.The LAMP assay detection limit was detecting53cfu/ml of pure S. dysenteriae and68cfu/ml in raw meat. The traditional PCR assay detection limit was530cfu/ml and680cfu/ml respectively. LAMP detection sensitivity is10times the sensitivity of conventional PCR. The whole detection process can be completed within2h.4. ConclusionThis research establishe a fast LAMP detection technique which detect Enterobacter sakazakii in infant formula milk and Shigella dysenteriae in meats. And the sensitivity, specificity and accuracy test have been done. Then achieve preliminary application in the actual sample. LAMP can directly applied to detect pathogens on the milk powder, meat products, dairy products. It can detect food-pathogenic bacteria in real samples,which generally spending20-28hours, shorten than the traditional methods. The LAMP detection limit was101cfu/ml in pure culture, up to10-fold more sensitive than that of PCR.The complete LAMP detection system was markedly faster than PCR and conventional methods. LAMP does not require sophisticated equipment, only a water bath or heat block is required to accomplish the whole reaction process, which is of great value for application in poorly equipped laboratories or in large-scale epidemiological studies conducted in isolated areas. The most practical characteristic of LAMP is the visual detection of amplification through the addition of fluorescent dyes such as SYBR Green I. Based on these advantages, we conclude that LAMP is a useful and practical tool for the early diagnosis of these two food-borne pathogens and evaluation of chemotherapeutic effectiveness in treatment of these disease. The LAMP assay established in this study is cost-effective, easy to perform, and readily adaptable for field diagnosis and disease surveillance in real samples.