Improvement in the sensitivity of viroid detection by adapting the reverse transcription step in one-step RT-qPCR assays

Leichtfried, Thomas; Reisenzein, Helga; Steinkellner, Siegrid; Gottsberger, Richard

doi:10.1016/j.jviromet.2021.114123

Journal of Virological Methods

2021

DOI: 10.1016/j.jviromet.2021.114123

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Improvement in the sensitivity of viroid detection by adapting the reverse transcription step in one-step RT-qPCR assays

Thomas Leichtfried

Helga Reisenzein

Siegrid Steinkellner

et al.

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Cited by 2 publications

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“…Meanwhile, a weak hybridisation signal through 2D-PAGE and northern blot has been reported by Vadamalai (2005), showing that the CCCVd-like RNA in the oil palm samples was present at low concentration. Although the viroid concentration in oil palm is low, this weakness can potentially be solved by using a real-time PCR as reported by previous studies on other viroids (Botermans et al, 2020;Leichtfried et al, 2021;Seigner et al, 2020;Tsushima and Fuji, 2022).…”

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Comparison of Real-Time Pcr, Conventional PCR and Rt-Lamp for the Detection of Coconut Cadang-Cadang Viroid Variant in Oil Palm

ROSLAN¹

2022

JOPR

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Real-time quantitative polymerase chain reaction (real-time PCR) was designed for the detection of oil palmCoconut Cadang-cadang Viroid (CCCVd) 246 nt variant. The primers and probe specifically designed for the real-time PCR were optimised with 300 nM of probe against a primer concentration of 400 nM for the detection of oil palm CCCVd variants. Oil palm CCCVd 246 nt variant was successfully detected using realtime PCR in leaf samples collected from 14 symptomatic oil palm from various regions. The sensitivity of real-time PCR was compared with reverse transcription loop-mediated isothermal amplification (RT-LAMP) and conventional PCR. The latter two techniques were reported earlier to be able to detect CCCVd variants in oil palm. Conventional PCR analysis using CCCVd full-length primers detected the presence of CCCVd 246 nt variant in 10 while RT-LAMP detected only seven positive samples from the total 14 field samples.The real-time PCR was highly sensitive and reliable compared to the two detection techniques. The primers designed for the real-time PCR were specific as only the oil palm CCCVd variant plasmid was detected with high fluorescence and lowest quantification cycle (Cq value) compared to the other tested viroids (ASSVd, CTiVd, ELVd, HLVd and PLMVd). The present study has proven the reliability of the technique, thus, highly recommending the next step of developing the high-throughput diagnostic procedure for the eventual use in epidemiological monitoring programmes.

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confidence: 99%

Comparison of Real-Time Pcr, Conventional PCR and Rt-Lamp for the Detection of Coconut Cadang-Cadang Viroid Variant in Oil Palm

ROSLAN¹

2022

JOPR

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Characterization of Tomato Brown Rugose Fruit Virus (ToBRFV) Detected in Czech Republic

et al. 2023

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Tomato is the most consumed vegetable in the world. The tomato brown rugose fruit virus (ToBRFV) is an important destructive virus that damages tomatoes and peppers with significant economic impact. The detection and characterization of this important viral pathogen were evaluated at the molecular and morphological level. The viral isolate was purified and inoculated on tomato and pepper plants. Small RNAs were sequenced in both plants and the profiles were compared. The complete genome of the isolate was obtained, and microRNA (miRNA) profiles were unveiled by small RNA sequencing. Symptoms caused by the isolate were also described and the morphology of the isolate was observed by transmission electron microscopy. Our results contribute to further understanding of the role of miRNAs in ToBRFV pathogenesis, which may be crucial for understanding disease symptom development in tomatoes and peppers.

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Improvement in the sensitivity of viroid detection by adapting the reverse transcription step in one-step RT-qPCR assays

Cited by 2 publications

References 16 publications

Comparison of Real-Time Pcr, Conventional PCR and Rt-Lamp for the Detection of Coconut Cadang-Cadang Viroid Variant in Oil Palm

Comparison of Real-Time Pcr, Conventional PCR and Rt-Lamp for the Detection of Coconut Cadang-Cadang Viroid Variant in Oil Palm

Characterization of Tomato Brown Rugose Fruit Virus (ToBRFV) Detected in Czech Republic

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