Loop-mediated Isothermal Amplification Detection Of Five Poplar Disease Pathogens

Poplar is an important fast-growing commercial tree species in China,with remarkable ecological,economic and social benefits.With the continuous expansion of poplar planting area,the occurrence of poplar diseases is becoming more and more serious.Poplar disease is an important obstacle to the healthy development of poplar plantation.It is very important to carry out research on rapid detection and early warning of disease for scientific prevention and control of diseases.This study focused on the establishment of a rapid,sensitive and specific loop-mediated isothermal amplification primers detection system for five important pathogens of poplar.From the aspects of specific primer design of loop-mediated isothermal amplification primers,specific primer screening,optimization of reaction conditions,sensitivity detection and specificity detection,it is proposed to provide technical solutions for rapid field detection of diseases.1.In this study,five kinds of important pathogenic bacteria of poplar that they are L.quercina sub sp.populi,B.dothidea,C.chrysosperma,M.larici-populina and P.populi were used as test strains.In this study,the specific Loop-mediated isothermal amplification primers of five pathogens were designed on-line by using Primer Explorer V5 software,and the primers were screened.The time of the Loop-mediated isothermal amplification primers reaction and the temperature of the reaction were optimized,and the optimal loop-mediated isothermal amplification primers detection system was established.The reaction systems of the five pathogens are: 2 x U-LAMP Mix reagent 10 μL,FIP,BIP,F3,B3 and Bst DNA polymerase 1 μL,DNA template 2 μL,and dd H2 O 25 μL.The optimal reaction conditions for L.quercina sub sp.populi and B.dothidea were determined to be 60℃ water bath for 50 min and 80℃ water bath for 10 min;The optimal reaction conditions for the three pathogens of C.chrysosperma,M.larici-populina and P.populi are 65℃ water bath for 50 min and 80℃water bath for 10 min.The results can be detected by agarose gel electrophoresis and a color indicator.2.The sensitivity test of the Loop-mediated isothermal amplification primers detectionsystem established in this experiment shows that the sensitivity of the five pathogens detected by the Loop-mediated isothermal amplification primers method is very high.The sensitivity of L.quercina sub sp.populi was 8.6×10-11ng/μL.The sensitivity of B.dothidea was 3.7×10-7ng/μL.The sensitivity of C.chrysosperma was 1.55×10-6ng/μL,that of M.larici-populina was1.07×10-6ng/μL,and that of P.populi was 1.67×10-6ng/μL.By testing 5 kinds of test bacteria and 11 reference bacteria,the results showed that the test results were positive except for the test bacteria,and the results of other reference bacteria were negative,which proved the good specificity of the method.In summary,this study established a feasible Loop-mediated isothermal amplification primers detection method for five important pathogens on poplar trees in China.The rapid,high-efficiency,high-sensitivity and specific detection methods provide technical support for detection warning and scientific prevention and control of the five kinds of diseases of poplar.This research has important scientific and practical significance.