Establishment Of LAMP Method For Detection To Klebsiella Pneumoniae And Listeria Monocytogenes In Milk And Meat

In recent years,many cases of human and animal morbidity and death caused by Listeria monocytogenes and Klebsiella pneumoniae have attracted attention.Listeria monocytogenes is a pathogenic bacterium of listeriosis,which infects humans and animals with symptoms of gastroenteritis such as fever,vomiting,nausea and diarrhea.In severe cases,it can lead to invasive diseases,such as abortion,meningitis and septicaemia.Klebsiella pneumoniae is highly pathogenic to humans,and is one of the significantconditionity pathogens and iatrogenic infection bacteria.It is highly pathogenic to humans and animals,and can cause pneumonia,uteritis,mastitis and other suppurative inflammation,and even sepsis.The detection limit of Listeria monocytogenes in food is 0 in relevant food safety regulations.In 2000,Japanese scholar Notomi et al.published loop-mediated isothermal amplification(LAMP)technology,which attracted the attention of scholars all over the world.LAMP method has the advantage of high specificity and can complete a large number of amplification in a short time under constant temperature.The amplification was accompanied by a large amount of pyrophosphate precipitation,allowing the results to be visually discernable.In recent years,LAMP has gradually become a research hotspot due to its advantages.Compared with bacteriological detection methods,LAMP has shorter reaction time and more convenient operation.Compared with PCR,the instrument cost involved in LAMP reaction process is lower,and there are no denaturation,annealing and extension stages of PCR reaction.In this study,hydroxyl naphthol blue dye was added into the reaction system.As the reaction progressed,the solution changed from purple to light blue.The color change can distinguish the presence or absence of amplification reaction.In this paper,by screening specific sequences and designing primers,a dual visual LAMP system was established for the detection of these two strains.The main research results are as follows:(1)LAMP detection methods for Klebsiella pneumoniae and Listeria monocytogenes in milk and meat were successfully established,and the final reaction system was:Buffer 2.5 ?L,internal primer 0.5?L,external primer 1?L,dNTPs 3?L,MgSO4 1?L,BST chain replacement enzyme 1.5?L,template 1.5?L and sterile water supplement system 25?L.The established minimum detection limits of Klebsiella pneumoniae and Listeria monocytogenes were 3.326×10 4ng/?L and 2.212×10 4ng/?L respectively.(2)Visual LAMP method was established with hydroxynaphthol blue as indicator.The established visual LAMP method was used to detect the clinical samples.The results showed that the positive reaction solution was light blue and the negative reaction was dark blue.(3)The established visual LAMP method was used to detect 50 muscle samples and 50 milk samples collected clinically.16 tissue samples were positive,and 7 milk samples were positive,with positive rates of 0.32 and 0.14,respectively.Meanwhile,PCR method was established to detect the samples by referring to the literature and published methods.The final established dual visual LAMP method was more than 90%consistent with the published PCR positive results.(4)A strain of bacteria was isolated from muscle tissue.Through bacterial isolation and culture,PCR,LAMP and gene sequencing,the strain was proved to be Listeria monocytogenes.(5)The 16S universal primer was used to amplify the DNA of the isolates and the amplified products were sequenced.The sequencing results were compared with the NCBI public sequences.Through the comparison results,it was found that the isolates were in high agreement with Listeria monocytogenes(the entry number is KY05330.1),which proved that the isolates were Listeria monocytogenes.