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A DNA Probing System for the Detection of Cucurbit Chlorotic Yellows Virus
Flow chart of bead-based DNA probing system. (A) The complementary anti-tag sequence coupled to a bead set, (B) Amplification products of biotin at 5’ end side, (C) Hybridization process with streptavdin added, and (D) Read by the LiquiChip system.
Flow chart of bead-based DNA probing system. (A) The complementary anti-tag sequence coupled to a bead set, (B) Amplification products of biotin at 5’ end side, (C) Hybridization process with streptavdin added, and (D) Read by the LiquiChip system.

Author:Cheng-Ping Kuan*, Ching-Shan Tseng, Wen-Shiue Chang, Hsin-Der Shih, Ying-Huey Cheng, and Ting-Ching Deng

Abstract:

      In this paper, a PCR assay coupled with microspheres to rapidly screen field samples for Cucurbit chlorotic yellows virus (CCYV) survey on melons was developed. The RT-PCR was designed to have the same binding sites for the forward and reverse primers of the CCYV viral coat protein (CP) gene as the target amplicon, but it had a unique internal sequence used for the probe site. The amplification of the CCYV viral RNA and the detection by LiquiChip were monitored with two different fluorescent probes in a multiplex format, one specific for the CCYV CP gene and the other for the plant chloroplast source gene, ndhB. The detection sensitivity of multiplex RT-PCR by LiquiChip was 100 times higher than only detection by RT-PCR for CCYV. The hybridized beads were processed and analyzed on LiquidChip instrument, which detected the presence of CCYV RNA and quantity of each PCR product from melon samples. Therefore, it may have great advantage to the certification program of melons.

Key words:Cucurbit chlorotic yellows virus (CCYV), Nucleic acid probe, Detection

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Journal of
Taiwan Agricultural Research
Vol. 72, No.3 (2023) ISSN: 2790-086X Vol. 72, No.3 (2023)
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