Use of Loop–Mediated Isothermal Amplification for the detection of Squash leaf curl Yunnan virus in Cucurbits

Abstract

A Loop–Mediated Isothermal Amplification (LAMP) assay was developed for detection of Squash leaf curl Yunnan virus isolate TH–PK–2016 (SLCuYV–TH–PK–2016) (Genbank accession KX388157), originally isolated from Cucurbita moschata Decne in Nakhon Pathom province of Thailand. Four LAMP primers (B3, F3, BIP and FIP) were designed from the SLCuYV–TH–PK–2016. Replication-associated protein gene (AC1) to amplify a product at 65 ºC in 60 min. LAMP products were analyzed by 1.5% agarose gel electrophoresis or visualized from SYBR^® Green I. Results demonstrated specific detection of SLCuYV–TH–PK–2016 by the developed LAMP assay with a sensitivity of 134 pg/μl of template DNA equalivalent to polymerase chain reaction (PCR). Thus, LAMP provides an alternative assay for rapid, specific and sensitive diagnosis of SLCuYV–TH–PK–2016 in cucurbit plants.