Detection and quantitation of two cucurbit criniviruses in mixed infection by real-time RT-PCR

• Published in: Journal of virological methods Vol. 193; no. 2; pp. 320 - 326
• Main Authors: Abrahamian, Peter E., Seblani, Rewa, Sobh, Hana, Abou-Jawdah, Yusuf
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.11.2013
• Subjects: CCYV; Coinfection - virology; Crinivirus - classification; Crinivirus - genetics; Crinivirus - isolation & purification; crops; Cucurbit chlorotic yellows virus; Cucurbit yellow stunting disorder virus; Cucurbita - virology; Cucurbitaceae; CYSDV; DNA Primers - genetics; genes; greenhouses; heat-shock protein 70; HSP70 Heat-Shock Proteins - genetics; mixed infection; Multiplex detection; Multiplex Polymerase Chain Reaction - methods; Oligonucleotide Probes - genetics; Plant Diseases - virology; quantitative polymerase chain reaction; Real-Time Polymerase Chain Reaction - methods; reverse transcriptase polymerase chain reaction; Reverse Transcriptase Polymerase Chain Reaction - methods; RT-qPCR; screening; Sensitivity and Specificity; viral load; Viral Proteins - genetics; viruses; Cucurbit yellow stunting disorder virus; Cucurbit chlorotic yellows virus; CCYV; RT-qPCR; CYSDV; Multiplex detection
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2013.06.004

•Multiplex RT-PCR was developed to differentiate between CCYV and CYSDV in mixed infection.•RT-qPCR assay was developed for specific detection of CCYV and CYSDV.•RT-qPCR was 100–1000 times more sensitive than RT-PCR.•An internal plant gene was used for normalization in quantitation of both viruses in mixed infection. Cucurbit chlorotic yellows virus (CCYV) and Cucurbit yellow stunting disorder virus (CYSDV) are whitefly-transmitted criniviruses infecting cucurbit crops inducing similar symptoms. Single and multiplex RT-PCR protocols were developed and evaluated on cucurbit samples collected from commercial greenhouses. Primers and probes were designed from the highly conserved heat shock protein 70 homolog (Hsp70h) gene. Conventional RT-PCR and multiplex RT-PCR assays showed high specificity and suitability for routine screening. TaqMan-based quantitative real-time RT-PCR (RT-qPCR) protocols were also developed for the detection and quantitation of both viruses occurring in single or mixed infection. The assays proved to be highly specific with no cross amplification. RT-qPCR assays showed a 100–1000 times improved sensitivity over conventional RT-PCR. Virus titers in mixed infections were compared to singly infected plants by RT-qPCR. CYSDV and CCYV titers decreased in double infected plants. This paper reports highly specific conventional RT-PCR and quantitative real-time PCR assays for detection, quantitation and differentiation between two closely related cucurbit-infecting criniviruses.