Development of the colorimetric loop-mediated isothermal amplification technique for rapid and sensitive detection of chrysanthemum stunt viroid in chrysanthemum

Abstract

Chrysanthemum stunt viroid (CSVd) is a serious pathogen infecting chrysanthemum worldwide. To improve and enhance the detection procedure, a colorimetric loop-mediated isothermal amplification (LAMP) technique was developed. Six LAMP primers were newly designed and tested to determine the optimal conditions using a recombinant plasmid of CSVd as a DNA template. The optimal conditions for colorimetric LAMP were incubation at 65°C for 45 min. Under these conditions, a ladder-like pattern of LAMP products was detected along with a change of color from pink to yellow in the positive reactions. Limits of the detection (LOD) of colorimetric LAMP were up to 1 fg ∙ μl–1 of plasmid DNA concentration which was 104 times greater than that of polymerase chain reaction (PCR). The developed colorimetric LAMP was not cross reacted to other viruses and viroids. From detection of actual samples and chrysanthemum plantlets which were obtained from meristem tip culture, the colorimetric LAMP showed effective potential in detecting CSVd. Therefore, the colorimetric LAMP can be used as a main technique to detect CSVd and ensure CSVd-free chrysanthemum plantlet production due to its accuracy, rapidness and sensitivity.

Keywords

• chrysanthemum stunt viroid
• lamp
• molecular detection
• ornamental plant
• pospiviroid