Establishment Of Method For Citrus Bacterial Canker Disease (Xanthomonas Axonopodis Pv. Citri) With Loop-mediated Isothermal Amplification

Citrus bacterial canker disease (Xanthomonas axonopodis pv.citri) is a popular quarantine disease in globe world. which caused by Xanthomonas axonopodis pv.citri(Xac). It is a key quarantine objects in the citrus planting areas because of its great damage, wide distribution,quickly spreading speed, obstinacy and difficulty of prevent.After isolation and culture, pathogenicity test, polymerase chain reaction, enzyme linked immunoassay, a new method -detection of Citrus bacterial canker disease with loop-mediated isothermal amplification was established.Loop-mediated isothermal amplification is a new gene amplification technology which created by Notomi on 2000. Starting with the principles of main technology, four primers were designed based on specific gene sequence, and Bst DNA polymerase large fragment was used in the reaction to amplificating target gene fragment under the isothermal conditions.A bacterial antagonistic strain was isolated from suspected samples which collected from HenShan. Results showed that bacteria which isolated from samples was Xanthomonas axonopodis pv.citri.By the way of antigen preparation, antibody preparation, antibody purification, the optimal experimental conditions were selected. The concentration of antigen and antibody were 1:400 (1.5×105 cfu/mL) and 1:800 (3.9μg/mL),5% skim milk as blocking solution acting for two hours and the concentration of enzyme labeled antibody was 2.6μg/mL acting for 60 min, at last adding substrate to gain the best appearing color results.The polymerase chain reaction of Xanthomonas axonopodis pv.citri showed that pre-degeneration for 4 min at 95℃, and degeneration for 30sec at 94℃, then 30sec at 59℃, finally 30sec at 72℃. About 413 bp was amplified by this method. The amplification results showed negative results after X.oryzae pv. Oryza,Pseudomonas solanacearum (E.F.Smith),Erwinia carotovora subsp.carotovora (Jones) Bergey,Clavibacter michiganense (Smith) subsp.sepedonicus were detected by PCR.The positive results was showed after detecting disease spot grinding solution of samples showing CBCD symptoms.The specific primers (F3,B3,FIP,BIP) were designed based on the conserved protein gene of Xanthomonas axonopodis pv.citri.Then a new amplification method-loop-mediated isothermal amplification was set up.Pre-degeneration for 7min at 95℃, and cooled for 8min with ice-bath,90min incubation at 64℃,3min inactivation at 80℃. The results showed that LAMP would amplicated Xanthomonas axonopodis pv.citri with rapidly,high sensitivity and specificity, and its detection limit was 60cfu/mL.