ORIGINAL ARTICLE
Development of the colorimetric loop-mediated isothermal amplification technique for rapid and sensitive detection of chrysanthemum stunt viroid in chrysanthemum
1
Division of Plant Pathology, Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University, Chiang Mai, Thailand
2
Plant Protection Center, Royal Project Foundation, Chiang Mai, Thailand
A - Research concept and design; B - Collection and/or assembly of data; C - Data analysis and interpretation; D - Writing the article; E - Critical revision of the article; F - Final approval of article
Submission date: 2022-05-03
Acceptance date: 2022-07-25
Online publication date: 2022-08-25
Corresponding author
On-Uma Ruangwong
Division of Plant Pathology, Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University, Chiang Mai, Thailand
Division of Plant Pathology, Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University, Chiang Mai, Thailand
Journal of Plant Protection Research 2022;62(3):272-280
HIGHLIGHTS
- Colorimetric LAMP could detect CSVd within 45 min by incubation at 65 degree celsius.
- The results of detection were easily distinguished by visualization of color change from pink to yellow in a positive reaction.
- Limits of the detection of colorimetric LAMP were up to 1 fg/ul of CSVd plasmid DNA.
- Colorimetric LAMP was specific to only CSVd.
- Evaluation of colorimetric LAMP was conducted to detect CSVd from chrysanthemum.
KEYWORDS
TOPICS
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.
FUNDING
This research work was supported by Royal Project
Foundation (Grant No. 3060-A149) and Thailand
Graduate Institute of Science and Technology (TGIST)
(Grant No. SCA-C0-2562-9705-TH).
RESPONSIBLE EDITOR
Julia Minicka
CONFLICT OF INTEREST
The authors have declared that no conflict of interests exist.
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| eISSN: | 1899-007X |
| ISSN: | 1427-4345 |
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