Establishment and application of visual loop-mediated amplification assay on Colletotrichum gloeosporioides

Abstract: 【Objective】Grape(Vitis vinifera L.)is one of the world's most widely cultivated and highyielding fruit crops. Grape anthracnose(Colletotrichum gloeosporioides) is one of the major fungal diseases causing fruit decay. Grape anthracnose mainly damages the fruit. After fruit is infected, there are not clear symptoms at early stage until the fruit is nearly ripe. Grape anthracnose seriously affects grape yield and quality. Because the long latent period from infection to appearance of disease, it is easy to miss the best time of early diagnosis and control of grape anthracnose. Although, PCR method has been well applied, but it needs certain equipment, which greatly limits the application of this method. It is necessary to develop a rapid, accurate, convenient, sensitive, specific and economical method for the detection of grape anthrax pathogen to enable early diagnosis and prevention of the disease.【Methods】The conserved region of ITS genomic DNA of C. gloeosporioides from field was amplified by a group of specific primer sequences of ITS1/ITS4. The sequence of the fragment was obtained by cloning and sequencing, then ho-mology comparison was carried out and the Primer Explorer V4 software was used to design 4(a set)LAMP specific primer sets for grape anthrax disease in 6 regions of the fragment. Using characteristics of calcein combining with Mn2+without fluorescence signal emitting, with the reaction carrying out, the pyrophosphate ion is producing, which is more easily combined with Mn2+, then the system will release calcein with spontaneous fluorescence signal of free calcein, moreover the fluorescence signal is enhanced in the presence of Mg2 +. Thus calcein and Mn Cl2 mixture can be used to detect results by color indication.With fluorescent chromogenic agent(0.05 mmol·L^-1 calcein and 0.5 mmol·L^-1 Mn Cl2mixture) adding to the reaction system, reaction mixture will turn into bright green from orange in positive cases, and it will maintain orange in negative cases. The visible LAMP detection system was established by setting up different reaction temperatures(60-65) and reaction durations(10-90 min) for optimizing reaction conditions. Five grape anthracnose samples and 32 other bacterial and fungal strains from different areas were used to test the specificity of this visible system. Total DNA extracted from infected fruit by CTAB method, which was used as the template to verify the sensitivity of the detection system at different concentrations(gradient template concentrations created by of a series of 10 fold dilution). LAMP primer(TJ-F3/TJ-B3) was used for quantitative PCR and conventional PCR, and the detection sensitivity of LAMP was compared with quantitative PCR and conventional PCR. 27 field samples from Fujian, Jiangxi, Guangxi,Guizhou, Beijing and other regions were measured. The result was compared with the q-PCR detection,and the accuracy of the visual LAMP detection system was demonstrated.【Results】Using the above-mentioned software, we designed a set of LAMP detection primers(inner primers: TJ-FIP/TJ-BIP; primer:TJ-F3/TJ-B3). Only 5 infected samples in the reaction system was specifically amplified with green fluorescence products, the other 32 samples had no positive amplified products, indicating this set of primers had a very good detection specificity. In the sensitivity test, the bright green fluorescence and electrophoresis strips were observed when the template concentration was 5×10-4ng·μL-1, but were not observed at template concentrations of 5×10-5mg·L-1and 5×10-6mg·L-1, indicating the lowest detection concentration of C. gloeosporioides DNA by visual LAMP was 5×10-4mg·L-1. The lowest template concentration for q-PCR and conventional PCR was 5×10-5mg·L-1and 5×10-2mg·L-1, respectively. Hence, the detection sensitivity of visual LAMP system was between q-PCR and conventional PCR. The samples from the five areas were detected by the visual LAMP, and the results showed that a disease incidence of 81.5%,which was the same as the results of q-PCR. The test results verified the accuracy of the LAMP detection system, and showed that it could replace q-PCR as a highly efficient routine method for diagnosis and identification of grape anthrax.【Conclusion】This study designed a set of LAMP specific detection primers for grape anthracnose, using the ITS sequence of the grapevine anthrax pathogen as the template. And the novel visual LAMP detection system was established based on the fluorescence characteristics of calcein and manganese chloride mixture. Through the optimization of the reaction temperature and reaction time, the LAMP detection system of grape anthrax disease was improved. The system proved to have a good detection specificity, and its sensitivity ranked between q-PCR and conventional PCR, with a minimum template concentration of 5×10-4ng·μL^-1. Field detection results were good, and therefore it can be used for the detection and identification of grape anthrax.