Development And Application Of Loop-Mediated Isothermal Amplification For Detection Of Aeromonas Veronii

Aeromonas veronii belongs to Aeromonas which is a common patiogenic bacteria. It arouse septicemia of many kinds of organs in aquatic animal.It also effects human and mammal by by the digestive tract,respiratory tract and leading to dirrhea,bactermia,meningitis and endocarditis. In a word,the bacteria need to attracted more attention.LAMP means Loop-mediated Isothermal Amplification which is a new nucleic acid amplification method developde by Eiken Chemical Co,Ltd in2000.lt is characterized by the use of4different primers specifically designed to recognize6distinct regions on the target gene and the reaction process proceeds at a constant temperature in1hour by a DNA polymerase with strand displacement activity.It provides high amplification efficiency,with DNAbeing amplified109～1010timesThe aerolysin (Aer) gene is a target gene in this study. Useing authoritative soft to design4primers (F3,B3,FIP,BIP) in LAMP and2PCR primers in PCR.The objective is to establish the reaction systerm of LAMP and PCRThe reaction conditions were optimized including Mg2+concentration, dNTP concentration, incuding temperature etc in LAMP.LAMP was carried out in a total25μL reaction containing1.6μM FIP and BIP,0.1μM F3and B3,2.4mM dNTP Mix,5M Betaine,3.0mM Mg2+,1μL Bst DNA polymerase (8U),2.5μL10X Lamp buffer,4.5μL ddH2O,1μL DNA. The systerm reacts incubing in63℃for40min, then terminate the reaction in80℃for10min. The result present in2.5%agarose gel.We choose20μL total reaction containing10μL2×Taq PCR Master Mix,7μL ddH2O,1μL each prime(10μM)r,1μL DNA.Amplification conditions for PCR assay were:cool start,5min at,35cycles of30s at95℃,30s at63℃,1min at72℃and final extension of5min72℃. PCR products were electrophoresed in a2%agarose gel. The size of the fragment of the PCR product is good agreement with the predicted size (240bp)Choosing9bacterial strains was detected by LAMP and PCR respectively,it shows that two methods have same perfect specificity.In sensitivety test,it shows that LAMP and PCR could amplicate DNA templete which minimum limit is respectivly1.8fg/μL and1.8×102fg/μ.It prove that LAMP is more sensitive than PCR.In the animal test, we injected the fish in LD50of fish-Aeromonas Veronii which concentration of bacterial is6×10CFU/mL.Then,we extract the bloody DNA as a template of LAMP and PCR. The result is that LAMP is more earlier than PCR to detect the bacterial.the result shows that LAMP and PCR reaction can capture the bacteria in4h and6h respectively.To sum up, LAMP is possessing better specificity and sensitivety, so as to provide a new way to warn and dignose the Aeromonas veronii effection.