| Root-knot nematodes(RKNs)are one of the most important plant pathogens ith wide host range,resulting serious losses in worldwide agriculture and economically crop.Rice is an important grain crop in China.Meloidogyne graminicola is considered as a major prevalent plant-parasitic nematodes(PPNs)attacking rice,can cause serious harm to a variety of crops.Rapid identification and quantification of M.graminicola in soil is very important to early diagnose and take measures to reduce the impact of PPN diseases and ensure food security.In this study,the loop-mediated isothermal amplification(LAMP)and real-time quantitative PCR assay rapid detection technique were established,species-specific primers of M.graminicola were designed based on the SCAR marker genomic sequence.The methods have been applied in quantitative detection of root-knot nematodes in field samples.The main results are shown as follows.1.In this research,the LAMP species-specific primers of M.graminicola were designed based on the SCAR ribosomal DNA gene,includes a forward primer(Mg-FIP),a reverse internal primer(Mg-BIP),a forward external primer(Mg-F3),a reverse external primer(Mg-B3)and a probe(Mg-HP-FITC).The conventional PCR primers were designed same as the LAMP outer primers,Mg-F3 and Mg-B3.The specificity,sensitivity of LAMP detection of M.graminicola were studied by gel electrophoresis,SYBR Green I dye visualization and LFD.The primers were highly specific and sensitive,and only the samples with M.graminicola DNA showed positive results.The primers were highly specific and sensitive,and only the samples with M.graminicola DNA showed positive results.The quantification results of M.graminicola isolated from naturally infested soil showed that the sensitivity of LAMP was 22 M.graminicola in 100 g soil and higher than conventional PCR,211 M.graminicola in 100 g soil.LAMP was 10-2 second-stage juvenile(J2)in 0.5 g soil and higher than conventional PCR,single J2 in 0.5 g soil.2.The qPCR primers,Mg-F and Mg-B were also designed according to M.graminicola SCAR marker sequence.The standard curve generated by plotting log number of M.graminicola in 0.5 g soil against cycle number was y=-3.146 x+29.708,The qPCR amplification efficiency(E)values and correlation coefficients(R2)were 1.079 and 0.970.No amplification was observed with blank soil.so the nematode enumeration obtained using qPCR was positively correlated with the standard curve.A soil from a rice field which M.graminicola had not been detected by microscope examination was used as a reference soil.Different numbers of M.graminicol were added in 0.5 g reference soil and the DNA was extracted.In addition,the correlation coefficient between the number of M.graminicola from the Baermann funnel method extraction and microscopic counting and standard curve predicted was 0.477,P=0.0160(P<0.05),it indicated they had a significant correlation relationship.3.In this research,the comprehensive comparison analysis of LAMP and qPCR of PCR-based methods was conducted in this research and and compared with conventional PCR,two rapid detection methods were analyzed and compared LAMP method and qPCR more rapid and sensitive reaction and reduces the time and easier detection compared with traditional morphological method and conventional PCR.Moreover,the operation of the LAMP assay did not required sophisticated instruments and tedious operation processes,the results of LAMP could be detected by color changes viewed by the unaided eye.This method removed dependence on special gel-imaging equipment and the expensive fluorescent reagent,detection by only requiring a thermostat water bath or ordinary equipment with a steady heat source.The rapid detection method of qPCR can not only effectively determine the presence of M.graminicola in the field soil,but also carry out quantitative detection,providing more favorable technical support for early warning and effective prevention and control of M.graminicola in rice. |
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