Development of Colorimetric Loop-Mediated Isothermal Amplification (LAMP) Technique for Rapid, Sensitive and Convenient Detection of Chrysanthemum Chlorotic Mottle Viroid (CChMVd) in Chrysanthemum

Abstract

Chrysanthemum chlorotic mottle viroid (CChMVd) is a serious pathogen infecting chrysanthemum worldwide. To improve and enhance the detection procedure, the colorimetric loop-mediated isothermal amplifi cation (LAMP) technique was developed. Six LAMP primers were newly designed and tested the optimal conditions using a recombinant plasmid of CChMVd as a DNA template. The optimal conditions for colorimetric LAMP were incubation at 65°C for 60 min. At these conditions, the ladder-liked pattern LAMP products were detected along with the change of color from pink to yellow in the positive reactions. The sensitivity of colorimetric LAMP was up to 2.18 × 103 copies of recombinant plasmid concentration which was 104-fold greater than that of polymerase chain reaction (PCR). The developed colorimetric LAMP was not cross reacted to other viruse s and viroid. Effi ciency of colorimetric reverse transcription (RT)-LAMP for detecting CChMVd in chrysanthemum plantlets obtained from meristem tip culture was evaluated to verify and ensure CChMVd-free plantlets production. The results showed that 18 chrysanthemum plantlets were free of CChMVd contamination. Therefore, the colorimetric RT-LAMP can be used to detect CChMVd routinely due to its accuracy, rapidness and sensitivity.