Development of Colorimetric Loop-Mediated Isothermal Amplification (LAMP) Technique for Rapid, Sensitive and Convenient Detection of Chrysanthemum Chlorotic Mottle Viroid (CChMVd) in Chrysanthemum
Salit Supakitthanakorn, Karnjana Vichittragoontavorn, Kaewalin Kunasakdakul, Nilita Mukjang, Chatsuda Phuakjaiphaeo and On-Uma Ruangwong* Author for corresponding; e-mail address: on-uma.r@cmu.ac.th
Volume: Vol.49 No.5 (September 2022)
Research Article
DOI: https://doi.org/10.12982/CMJS.2022.079
Received: 11 May 2022, Revised: 5 August 2022, Accepted: 19 August 2022, Published: -
Citation: Supakitthanakorn S., Vichittragoontavorn K., Kunasakdakul K., Mukjang N., Phuakjaiphaeo C. and Ruangwong O., Development of Colorimetric Loop-Mediated Isothermal Amplification (LAMP) Technique for Rapid, Sensitive and Convenient Detection of Chrysanthemum Chlorotic Mottle Viroid (CChMVd) in Chrysanthemum, Chiang Mai Journal of Science, 2022; 49(5): 1296-1306. DOI 10.12982/CMJS.2022.079.
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.