The Loop-mediated Isothermal Amplification For Detecting Three Plant Parasitic Nematodes

The loop-mediated isothermal amplification(LAMP)is an isothermal nucleic acid amplification technique which uses a DNA polymerase with strand displacement activity.The positive amplification products can be observed directly by naked-eyes with the white precipitation of magnesium pyrophosphate and adding of a fluorescent dye.The LAMP assay is rapid and easy for performance,which has been widely applied in rapid diagnosis of the plant nematodes.In order to provide an efficient and convenient technique for rapid detection of plant parasitic nematode Aphelenchoides besseyi,Meloidogyne camelliae and M.mali,the loop-mediated isothermal amplification(LAMP)assays were developed by designing of the specific primers,optimization of the reaction conditions,and verification of the specificity and sensitivity.The white tip disease caused by A.besseyi is transmited by infested seeds.The rapid and accurate diagnosis is critical for disease control.Five specific primers for LAMP reaction were designed by comparing the ITS ribosomal DNA(rDNA)sequences of A.besseyi deposited in GenBank.The LAMP amplification with highest efficiency was obtained by the optimized conditions of 3.0 mmol/L Mg2+,0.8 mmol/L dNTPs,0.2 mol/L betaine and 60 minutes of extension time.The amplification products can be directly observed by the naked eye after SYBR Green I staining.The developed LAMP assay was able to specifically detect A.besseyi from 11 species of plant nematodes.The detection sensitivity is 1/20 000 of single nematode DNA,which was 10 times higher than the conventional PCR assay.The LAMP assay can complete within 1 hour and without the support of professional experimental apparatus,which can be applied in daily field detection of the nematode disease.M.camelliae and M.mali both are non-Chinese Meloidogyne species listed in the Catalogue of Quarantine Pest for Import Plants to the People's Republic of China.Their invasion risk by introducing and transmission with the imported seedlings and nursery stocks into China is extremely high.In this study,five specific primers for LAMP reaction were designed according to the non-conservative sequences of 28S rDNA from M.camelliae.The LAMP amplification with highest efficiency was obtained by the optimized conditions of 1.0 mmol/L dNTPs,5.0 mmol/L Mg2+,without adding betaine and 60 minutes of extension time.The developed LAMP assay was able to specifically detect M.camelliae.The detection sensitivity is 1/200 000 of single nematode DNA,which was 100 times higher than the conventional PCR assay.In the study for M.mali,six specific primers for LAMP reaciton were designed according to the non-conservative sequences of 28S rDNA.The LAMP amplification with highest efficiency was obtained by the optimized conditions of 0.4 mmol/L dNTPs,5.0 mmol/L Mg2+,without adding betaine,and 60 minutes of extension time.The LAMP products were successfully detected by the agarose gel electrophoresis,SYBR Green I staining and lateral flow dipstick(LFD).The developed LAMP assay was able to specifically detect M.mali from 10 species of plant nematodes.The sensitivity assay is 1/20 000 of single nematode DNA,which was 10 times higher than the conventional PCR assay.In summary,three established LAMP assays will provide the valuable technique supports for rapid diagnosis of A.besseyi in field disease,as well as M.camelliae and M.mali in plant quarantine by Chinese Ports.