DEVELOPMENT OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) ASSAY FOR RAPID DETECTION OF Colletotrichum gloeosporioides IN MANGO
NANTHAWAN HADTHAMARD
Division of Postharvest Technology, School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi (Bang Khun Thian), Bangkok 10150, Thailand
PIYASAK CHAUMPLUK
Department of Botany, Faculty of Science, Chulalongkorn University 10330, Bangkok, Thailand
MANTANA BUANONG
Division of Postharvest Technology, School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi (Bang Khun Thian), Bangkok 10150, Thailand
PANIDA BOONYARITTHONGCHAI
Division of Postharvest Technology, School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi (Bang Khun Thian), Bangkok 10150, Thailand and Postharvest Technology Innovation Center, Ministry of Higher Education, Science, Research and Innovation, Bangkok 10400, Thailand and Postharvest Technology Innovation Center, Ministry of Higher Education, Science, Research and Innovation, Bangkok 10400, Thailand.
NITHAN TANTITANAWAT
Ruamrudee International School, 6 Ramkhamhaeng 184, Min Buri, Bangkok 10510, Thailand
CHALERMCHAI WONGS-AREE *
Division of Postharvest Technology, School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi (Bang Khun Thian), Bangkok 10150, Thailand and Postharvest Technology Innovation Center, Ministry of Higher Education, Science, Research and Innovation, Bangkok 10400, Thailand
*Author to whom correspondence should be addressed.
Abstract
Anthracnose (Colletotrichum gloeosporioides) is a severe disease from fungi infection causing low productivity of vegetables and fruits. Especially mangoes (Mangifera indica L.), which is a highly economic tropical fruit of Thailand, are exported in the third of the world. Hence, this research aimed to develop the condition of LAMP assay for the detection and identification of Colletotrichum species from mangoes. In the present study, the specificity of a primer set of loop-mediated isothermal amplification (LAMP) targeting glutamine synthase (GLN) and tubulin 2 (TUB2) genes was investigated. The result showed only GLN gene target could successfully detect C. gloeosporioides and presented a high sensitivity at 10 ng/ml, the reaction was performed under a thermal cycler for about 65 °C for 60 minutes. A positive reaction was identified visually from pink color to yellow color and confirmed by 1.5% agarose gel electrophoresis. In conclusion, the LAMP assay for anthracnose in mangoes provided a rapid reaction and no need for complex instruments or systems such as PCR machine. The LAMP assay may therefore be applied in routine diagnosis in the laboratory and for in-field.
Keywords: Anthracnose, Colletotrichum, glutamine synthase, tubulin, LAMP