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Developed One-step Reverse Transcription Loop-Mediated Isothermal Amplification (Rt-Lamp) Assay for Detection of Strawberry Latent Ring Spot Virus

Developed One-step Reverse Transcription Loop-Mediated Isothermal Amplification (Rt-Lamp) Assay for Detection of Strawberry Latent Ring Spot Virus

Ahmed A. Kheder

Virus and Phytoplasma Research Department, Plant Pathology Research Institute, AgriculturalResearch Center, Giza, Egypt.

ABSTRACT

Strawberry latent ring spot virus (SLRSV) is an important virus which is responsible for considerable losses in horticulture and vegetable crops production including strawberry plants. SLRSV was detected and characterized using serological (DAS-ELISA), biological and molecular diagnostic techniques. Virus incidence was performed in different locations in five governorates during 2018-2020. Forty-one samples out of 693(5.94%) reacted positively to SLRSV. Specifically, the percentages of infection were 7.3, 6.7, 5.4, 5.16 and 2.63% in El-Qalyubia, El-Beheira, El- Menoufia, El-Sharkia and El-Ismailia governorates respectively. SLRSV was mechanically transmitted from infected strawberry plants onto ten herbaceous plant species, different types of symptoms have been observed associated to virus infection on indicator hosts; Mentha spicata, Chenopodium quinoa, Nicotiana tabacum cv. White Burley and Vicia faba after 15, 18, 20 and 22dpi respectively. Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify ~497bp for coat protein gene in RNA 2, the amplified product was confirmed with direct sequences. Phylogenetic analysis results indicated that SLRSV-Eg isolate under study (acc. no. MT648777.1), showed 65.9 – 99.5% nucleotide similarity with available homologous sequences from other crops. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay which is one of the most promising molecular diagnostic techniques was applied. The amplified products were analyzed using amplification curves and electrophoresis. In this study SLRSV was characterized and detected with DAS-ELISA and molecular diagnostic techniques the obtained results concluded that the virus was varied due to locations, also all tested cultivars were susceptible to virus infection and the sensitivity of the RT-LAMP protocol was tenfold higher than that of conventional RT-PCR. Finally it is recommended to use LAMP assay which is useful for researchers, seed production specialists and was suggested to the Egyptian quarantine system which is interested in determining strawberry virus infections by using a single assay.

 

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Pakistan Journal of Zoology

October

Vol. 53, Iss. 5, Pages 1603-2000

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