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

Abstract

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.

A Novel, Precise and High-Throughput Technology for Viroid Detection in Cannabis (MFDetectTM)

“…The RT-qPCR assay has been reported to detect different viroids in different plant species [ 36 , 37 , 38 , 39 , 40 ]. Although the efficiency of this assay is high for detection of viroids [ 41 ], the specificity of primers and probes makes it complex and not cost-effective for large-scale detection [ 42 , 43 ]. The earliest reports confirming the detection of HLVd in cannabis were also based on RT-qPCR [ 9 , 10 ].…”

A novel, precise and high-throughput technology for viroid detection in cannabis (MFDetect™)

“…The RT-PCR assay has been reported to detect different viroids in different plant species (Nakahara et al, 1999, Luigi and Faggioli, 2011, Luigi and Faggioli, 2013, Malandraki et al, 2015, Hagemann et al, 2021. Although the efficiency of this assay is up to the mark for the detection of viroids (Roslan et al, 2023), the specificity of primers and probes makes it complex and not cost-effective for large-scale detection (Gardner et al, 2003, Nagy et al, 2017. This study describes the MFDetect TM system that relies on a unique set of primers and one-step RT-LAMP plus RT-PCR for detecting HLVd in infected plants.…”