Establishment of loop-mediated isothermal amplification(LAMP)for visual rapid detection of Diaporthe phoenicicola of fruit spot disease on Phyllanthus emblica
[Objective]This study is to establish a rapid,convenient,and sensitive loop-mediated iso-thermal amplification(LAMP)method for visual detection of Diaporthe phoenicicola that causes fruit spot disease on Phyllanthus emblica,and to provide theoretical basis for the risk prediction and disease control.[Method]The specific amplification primers for LAMP were designed with the translation exten-sion factor gene EF-1α of D.phoenicicola as the target sequence.With D.phoenicicola DNA as the tem-plate,the LAMP detection reaction system was optimized by adjusting the reaction temperature,reaction time,concentration of dNTPs,concentration of Mg2+and concentration ratio of inner and outer primers.The specificity and sensitivity of the system were tested,and practical detection was conducted on the di-seased fruits of P.emblica.[Result]Primer set 4(EF4-F3/EF4-B3 和 EF4-FIP/EF4-BI)was the best primer pair for LAMP detection of fruit spot disease of P.emblica.The optimal reaction conditions of LAMP test were dNTPs concentration of 1.0 mmol/L,Mg2+concentration of 4 mmol/L,inner to outer primer concentration ratio of 6∶1,reaction temperature of 63 ℃,and reaction time of 50 min.The opti-mized LAMP detection method could specifically detect D.phoenicicola,with a sensitivity of 0.01 ng/μL,and an accuracy rate of 100％in practical application.[Conclusion]In this study,LAMP visual rapid detec-tion method for D.phoenicicola on P.emblica fruit was established for the first time,with a high specifici-ty,high sensitivity,and great simplity in its operation,which can be used for rapid detection in the field.
万方数据
维普
