| Haemophilus parasuis(Hps), also known as Glasser’s disease, is caused byHaemophilus. Haemophilus is a common bacterium that exists in respiratory. Thisbacterium doesn’t show any clinical symptoms under the normal circumstances, but itcan cause many kinds of diseases under specific conditions. This bacterium is abundantin our environment, on a global basis. In the recent years, as the poultry industry grows,this disease is becoming an epidemic that affects the global pig-breeding industry. Itoften becomes the origin bacteria, and when combined with other bacteria, causes largescale economic loss for the industry. Therefore, establishing diagnostic method that’srapid and accurate is imperative.At the present time, the main diagnostic methods for Hps are: bacterial isolatecultivation, immunological(antibody monitor and measurement) as well as PCR.However, bacterial isolation method is extremely complicated, time-consuming, andnon-flexible. Immunological method has its own limitation in its flexibility. PCR is moreflexible, sensible and accurate, but it requires expensive equipment and complicatedprocedure, and therefore is hard to be applied extensively.LAMP is a new nucleic acid duplication technology developed by Japanese scientistNotomi. This technology can reproduce rapidly in a stable temperature condition. It hasbeen adopted vastly due to its high efficiency, short cycle, low cost, and simpleoperation.This experiment was carried out using LAMP technology to diagnose Hps usingHps’s OMP P2as a target. Based on LAPM principle, utilizing Primer Explorer3.0for6specific sequences of OMP P2gene,4specific primers were designed. Under theinfluence of DNA and BstDNA polymerase, one can expand DNA module withconsistent temperature, and optimize the expanding conditions such as F3:FIP, B3:BIPinducer, MgSO4, dNTP and Bst’s condensity, reaction time and reaction temperature, andcan confirm LAMP’s best reaction system for25μL: FIP and BIP each of1.6mmol/L, F3and B3each of0.2mmol/L; Magnesium ions concentration level is:3.0mmol/L; dNTPconcentration level is:3.0mmol/L;8U’s BstNDA Polymerase quantity is:0.8μL,2.5μL10×BstDNA Polymerase Reaction buffer,2μLDNA module, and use Sterile DDWsupplement system. LAMP’s basic reaction process is: first put the reaction mixture in 62℃water and allow it to react for40min, then put it in80℃water for10min to stop thereaction. After the reaction, observe with bare eyes and see if there is creamy coloredMagnesium pyrophosphate sedimentation to determine if LAMP has taken place.Fluorescent dyes or1%Agarose gel electrophoresis can also be used.Using PCR method as a comparison, LAMP’s sensitivity was measured. Resultsshow that PCR’s lowest DNA testing quality was20pg/μL; and that of LAMP was0.2pg/μL. LAMP’s level of sensitivity was100higher than that of PCR’s. In the meantime,LAMP method was used for testing the following diseases: Actinobacilluspleuropneumoniae actinobacillus, Escherichia coli, Salmonella, Staphylococcus aureus,Streptococcus agalactiae, Porcine Parvovirus, Porcine Pseudorabies, TransmissibleGastroenteritis of Swine, Swine Mycoplasma pneumonia. Results showed that these9diseases’ LAMP reactions were all negative, further approving that LAMP method was abetter method.In summary, this experiment has established the LAMP system that is the rapid,sensitive, and high efficient diagnostic method for Hps. This method is easy to operate,has a low demand on operator’s technical skills, and it doesn’t require expensiveequipment. It has good potential for fundamental lab applications. |
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