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International Journal of Current Microbiology and Applied Sciences (IJCMAS)
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National Academy of Agricultural Sciences (NAAS)
NAAS Score: *5.38 (2019)
[Effective from January 1, 2019]
For more details click here

ICV 2017: 100.00
Index Copernicus ICI Journals Master List 2017 - IJCMAS--ICV 2017: 100.00
For more details click here

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PRINT ISSN : 2319-7692
Online ISSN : 2319-7706
Issues : 12 per year
Publisher : Excellent Publishers
Email : editorijcmas@gmail.com / submit@ijcmas.com
Editor-in-chief: Dr.M.Prakash
Index Copernicus ICV 2017: 100.00
NAAS RATING 2018: 5.38

Int.J.Curr.Microbiol.App.Sci.2019.8(9): 2282-2295
DOI: https://doi.org/10.20546/ijcmas.2019.809.264


Development of a Loop-Mediated Isothermal Amplification Assay for Detection of Tomato Leaf Curl New Delhi Virus in Ridge Gourd [Luffa acutangula (L) Roxb.]
Priya Naganur1, 2, U. Premchand1, K. S. Shankarappa1*, R. K. Mesta2, C. Manjunatha3 and C. V. Patil1
1Department of Plant Pathology, College of Horticulture, Bengaluru-560 065, University of Horticultural Sciences,  Bagalkot, Karnataka, India
2Department of Plant Pathology, College of Horticulture, Bagalkot - 587101, University of Horticultural Sciences, Bagalkot, Karnataka, India
3ICAR- Indian Agricultural Research Institute, Regional Station, Wellington - 643 231, Tamil Nadu, India
*Corresponding author
Abstract:

Ridge gourd yellow mosaic disease (RgYMD) caused by the strain of Tomato leaf curl New Delhi virus (ToLCNDV) is an emerging disease in India. Early detection of ToLCNDV in ridge gourd has great significance in the management of the disease. An innovative loop-mediated isothermal amplification (LAMP) assay was standardized for rapid detection of ToLCNDV. Assay was carried out using the set of six primers (F3, B3, FIP, BIP, LF and BF) specific to the coat protein (CP) gene of the virus. Reaction time, temperature, primers concentration and DNA dilutions were optimized for detection of ToLCNDV. The reaction was optimized in a single tube at 63 °C for 45 min and terminated by keeping it at 80 °C for 10 min. The sensitivity of LAMP based detection assay was more than 100 times as that of conventional PCR. The LAMP primers specifically targeted to the CP gene of ToLCNDV resulted in typical water fall like bands by the agarose gel electrophoresis. The amplified LAMP products were also identified by visualization with dyes. No amplification was observed in DNA from tissues of healthy plants and water control either by LAMP or PCR assays. The LAMP assay was validated by testing field samples of RgYMD collected from various ridge gourd growing areas in southern India. The results indicated that the LAMP assay standardized will be useful for monitoring and detection of ToLCNDV associated with RgYMD. The assay has many advantages over PCR with respect to cost, accuracy, sensitivity and specificity.


Keywords: Begomovirus; Coat protein gene; LAMP assay; Ridge gourd; Yellow mosaic disease.
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How to cite this article:

Priya Naganur, U. Premchand, K. S. Shankarappa, R. K. Mesta, C. Manjunatha and Patil, C. V. 2019. Development of a Loop-Mediated Isothermal Amplification Assay for Detection of Tomato Leaf Curl New Delhi Virus in Ridge Gourd [Luffa acutangula (L) Roxb.].Int.J.Curr.Microbiol.App.Sci. 8(9): 2282-2295. doi: https://doi.org/10.20546/ijcmas.2019.809.264