Rapid detection of squash leaf curl virus by loop-mediated isothermal amplification.
A loop-mediated isothermal amplification (LAMP) assay was employed to develop a simple and efficient system for the detection of squash leaf curl virus (SLCV) in diseased plants of squash (Cucurbita pepo) and melon (Cucumis melo). Completion of LAMP assay required 30-60 min under isothermal conditions at 65 degrees C by employing a set of four primers targeting SLCV. Although the sensitivity of the LAMP assay and the polymerase chain reaction (PCR) assay was comparable at high virus concentrations, the LAMP assay was by a 10-fold dilution factor more sensitive than the PCR assay for the detection of SLCV in diseased plants. No reaction was detected in the tissues of healthy plants by either the LAMP or the PCR. The LAMP products can be visualized by staining directly in the tube with SYBR Safe DNA gel stain dye. The sensitivity of the SYBR Safe DNA gel stain is similar to analysis by gel electrophoresis. Although both the LAMP and the PCR methods were capable of detecting SLCV in infected tissues of squash and melon, the LAMP method would be more useful than the PCR method for detection of SLCV infection in cucurbitaceous plants because it is more rapid, simple, accurate and sensitive.
Kuan
Cheng-Ping
CP
Division of Biotechnology, Taiwan Agricultural Research Institute, No. 189 Chung Cheng Rd, Wufeng, Taichung 41362, Taiwan. pcr123@tari.gov.tw
Wu
Min-Tze
MT
Lu
Yi-Lin
YL
Huang
Hung-Chang
HC
D053839:Begomovirus / Q000235:genetics / Q000302:isolation & purification*; D028463:Cucumis melo / Q000821:virology*; D028464:Cucurbita / Q000821:virology*; D017931:DNA Primers / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010935:Plant Diseases / Q000821:virology*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D013997:Time Factors; D014773:Virology / Q000379:methods*
D017931:DNA Primers