Optimization And Preliminary Application Of LAMP Detection System For Salmonella And BVD Virus In Calf Diarrhea

Diarrhea is a common disease in the four seasons,which seriously affects the growth and development of calves.Microbial infection,the body of the calves and the internal digestive organs are not fully developed,poor self protection,climate change,as well as the diet is not clean or malnutrition,are the factors of diarrhea.When the epidemic diarrhea occurs in calves,the cause is mainly caused by intestinal pathogens,including bacteria,viruses,parasites,followed by nutritional factors and environmental factors,the most common pathogens infection are bacterial and viral infections.When the diarrhea caused by bacterial and viral infections,just rely on the stool state is difficult to distinguish,but also need to combine with the epidemiological characteristics,clinical symptoms of the initial diagnosis,separated by molecular biology method to diagnose the final pathogen.1.Establishment of Salmonella LAMP Detection System for Calf Diarrhea In order to establish the optimal system for LAMP detection of calf diarrhea,four specific LAMP primiers were designed accoeding to the conserved fragment of Salmonella inv A(GI:164523712),and the LAMP detection system of Salmonella was established.In order to optimize the system,the temperature,Mg2+concentration,d NTPs concentration,Bst polymerase concentration,the ratio of internal and external primer concentration were repeatedly explored,and finally the best reaction temperature of LAMP detection system was 64 ℃,Mg2+concentration DNTPs concentration and Bst polymerase concentration were 8 m M,1.2 m M,8U,the concentration of the outer primers F3 and B3 was 0.2 μM,and the optimum primer concentration ratio was 1: 6,that is,the concentration of the primer FIP and BIP was 1.2 μM.The specificity of the system was strong.2% gel electrophoresis revealed that only the Salmonella showed a good LAMP characteristic ladder band,and there were no characteristic bands in Escherichia coli,Haemophilus influenzae and Mycobacterium tuberculosis.Compared with PCR,the optimized system shows high sensitivity and the detection limit is 1-2 orders of magnitude higher than PCR(10-100 times).2.Establishment of BVDv LAMP Detection System for Calf DiarrheaIn order to verify the LAMP is suitable for different types of nucleic acid template,this study has designed four specific primers on the 5’UTR gene(Gen Bank accession number: AY278459.1)of the bovine viral diarrhea virus(BVDv),the LAMP detection system was also optimized for the Mg2+ concentration,reaction temperature,Bst polymerase concentration,d NTPs concentration and primer concentration ratio.Finally,the optimal reaction temperature of BVDv LAMP detection system was 64℃,Mg2+ concentration,d NTPs concentration,Bst polymerase concentration were 6m M,1.6m M,8U,and the optimal primer concentration ratio was1:6,outer primer F3 and B3 concentration were 0.5μM,FIP and BIP primers were 3μM,AMV reverse transcriptase was 1μL,RNA enzyme inhibitor was0.5μL.Compared with rotavirus,coronavirus also showed a high degree of specificity,the sensitivity of the optimized system was 1 orders of magnitude higher than that of PCR.3.Preliminary application of LAMP detection system for calf diarrheaOn the basis of the previous studies,the first two experiments established the LAMP detection system of Salmonella and BVD virus,which showed the specificity and sensitivity of LAMP,the purpose of this study is to find a molecular biology method that can be applied to rapid detection of simple,convenient calves common diarrhea pathogens.Therefore,we select E.coli,Mycobacterium tuberculosis,rotavirus,coronavirus,Cryptosporidium and tapeworm(including BVD virus and Salmonella),the establishment of these pathogens of the classic LAMP detection system for on-site testing to verify the effectiveness of LAMP in clinical application effect.Compared with the classical LAMP system and and the optimized LAMP system of Salmonella and BVD virus,it can be found that the sensitivity of the optimized system is improved,which indicates that the LAMP detection system can be optimized to achieve good detection effect.As a result of the field test can not be electrophoresis,for LAMP product detection using SYBR GREEN I colorimetric method,the color is more sensitive,and the use of the experimental process invented a new method of dye addition,is good to avoid the occurrence of false positives.Finally,the LAMP is compared with the results of laboratory PCR.It shows that the LAMP method has high accuracy and sensitivity,and the method is simple,does not require the use of expensive instruments,the whole reaction is only about 1 hour,which can be applied to rapid detection of clinical sites,promotion of the farms and the primary laboratory,has a broad application prospect.