Establishment And Application Of LAMP Molecular Detection Techniques For Two Rust Fungi Of Tropical Crops

Plant rust disease is one of the most important diseases which do harm to agricultural production,with high incidence,many hosts and serious economic loss.Among them,the two kinds of pathogens,Puccinia melanocephal and Hemileia vastatrix,are relatively common and serious hazards.The former can cause sugarcane brown rust,and the latter causes coffee leaf rust.At present,there are still no effective measures to control them.Therefore,the development of these two rust detection technologies has a high application value.Due to the superiorities such as high specity,sensitivity and simple processes,the loop-mediated isothermal amplification method(LAMP)has been used frequently in a lot of areas including pathogens rapid detection.In order to establish the LAMP technology of Puccinia melanocephal and Hemileia vastatrix,the study carried out the following researches in five aspects.Firstly,through the analysis of the ITS sequences of Puccinia melanocephal and Hemileia vastatrix,the specific regions of the two pathogens were obtained.Primer explorer 4.0 software(http://primerexplorer.jp/elamp4.0.0)was used to design primers for specific regions.The best primers BRSC4 and HVSC2 were further obtained by primer set screening.Secondly,25 ?L of LAMP reaction system and reaction conditions were optimized based on each of the best primer sets.The most superior Puccinia melanocephal system of LAMP is consisted of 0.2 ?M outer primers F3 and B3?1.2?M inner primers FIP and BIP?1 mM dNTPs?6 mM MgSO4?2.5 ?L 10×buffer?1 U Bst-DNA polymerase?1 ?L the extracted template DNA.The most superior Hemileia vastatrix system of LAMP is consisted of 0.2 ?M outer primers F3 and B3?1.6?M inner primers FIP and BIP?1.4 mM dNTPs?4 mM MgSO4?2.5 ?L 10×buffer?1 U Bst-DNA polymerase?1 ?L the extracted template DNA.The reaction was optimized at 63 ? for 60 min,85 °C for 8 min.Thirdly,using the ITS plasmid DNA of Puccinia melanocephal and Hemileia vastatrix as the templates,we carried out the specific analysis of the samples of other pathogenic fungi on coffee and sugarcane plant,and the DNA of pathogenic bacteria from different genera.Then,the results showed that the ladder-like bands could only be amplified from the ITS plasmid DNA template of Puccinia melanocephal and Hemileia vastatrix,but no bands can be amplified from other non-target template DNAs.Fourthly,the sensitivity of the established LAMP amplification to conventional PCR and nested PCR amplification was compared using the ITS recombinant plasmid DNA dilutions of Puccinia melanocephal and Hemileia vastatrix,respectively.The results showed that the minimum detection limit of LAMP technology for Puccinia melanocephal was 10-3 ng/?L,that of conventional PCR method was 10-1 ng/?L,and that of nested PCR method was 10-3 ng/?L;The minimum detection limit of LAMP technology for Hemileia vastatrix is 10-5 ng/?L,the minimum detection limit for conventional PCR is 10-1 ng/?L,and the minimum detection limit for nested PCR is 10-4 ng/pL.Finally,the established LAMP and conventional PCR and nested PCR were used to detect Puccinia melanocephal and Hemileia vastatrix.The results showed that the frequency of detection by LAMP technique was 80%for Puccinia melanocephal and the detection frequency was 76%by nested PCR and 52%by conventional PCR.For Hemileia vastatrix,the detection frequency of LAMP technology was 97.8%,the detection frequency of nested PCR technology was 95.6%,and the average detection frequency of conventional PCR technology was 89.0%.