An accurate, specific, sensitive, high-throughput method based on a microsphere immunoassay for multiplex detection of three viruses and bacterial fruit blotch bacterium in cucurbits

Development of a multiplex RT-PCR assay for simultaneous detection of Cucumber green mottle mosaic virus and Acidovorax citrulli in watermelon

“…In this study, a multiplex RT-PCR method was established to simultaneously detect CGMMV and A. citrulli , which was successfully applied to field-grown samples and stored seeds. The detection methods of CGMMV or A. citrulli were widely reported, including one-step reverse transcription loop-mediated isothermal amplification for CGMMV detection (Li et al, 2013), multiplex RT-PCR assays for simultaneous detection of seven cucurbit-infecting viruses including CGMMV (Kwon et al, 2014), an ELISA-based assay for detection of A. citrulli (Himananto et al, 2011), a microsphere immunoassay (MIA) for simultaneous detection of multiple plant pathogens, including A. citrulli (Charlermroj et al, 2017). However, the detection object of these reported methods cannot give consideration to both CGMMV and A. citrulli , especially PCR-based methods, probable due to the difficulty of acquirement of suitable amplification template for a RNA virus and a bacterium.…”

“…Although several methods have been reported for detecting both plant bacteria and viruses simultaneously, they are all less-than-perfect. Recently, a method based on a MIA has been developed for multiplex detection of three viruses (potyviruses, Watermelon silver mottle virus , Melon yellow spot virus ) and A. citrulli in cucurbits (Charlermroj et al, 2017). However, it required the specific antibodies that were expensive.…”

First record of melon yellow spot virus in pumpkin and its occurrence in cucurbitaceous crops in Thailand

“…Total RNA was extracted from leaves using TRIzol Reagent (Invitrogen, USA) and cDNA was synthesised using RevertAid First Strand synthesis kit (Invitrogen, USA). The PCR amplification was performed by PCR Master Mix 2X (Invitrogen, USA) using nucleocapsid (N) protein gene specific primers and the PCR conditions followed the method of Charlermroj et al (2017). The PCR product was analysed by 1% agarose gel electrophoresis stained by RedSafe (iNtRON, South Korea).…”