One-step real-time multiplex reverse transcription-polymerase chain reaction assay with melt curve analysis for detection of potato leafroll virus, potato virus S, potato virus X, and potato virus Y

Abstract

Background Certification of seed potato as free of viruses is essential for stable potato production. Among more than 30 virus species infecting potato, potato leafroll virus (PLRV), potato virus S (PVS), potato virus X (PVX), and potato virus Y (PVY) predominate worldwide and should be the targets of a high-throughput detection protocol for seed potato quarantine. Results We developed an assay based on one-step real-time multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with melt curve analysis for the four viruses and one internal control, potato elongation factor 1 alpha gene (EF1α). Virus-specific primers were derived from conserved regions among randomly selected representatives considering viral genomic diversity. Our assay simultaneously detected representative Japanese isolates of PLRV, O lineage of PVS, PVX, and NTN strain of PVY. The variability of melting temperature (Tm) values for each virus was confirmed using Japanese isolates, and virus species could be identified by the values of 87.6 for PLRV, 85.9 for PVX, 82.2 (Ordinary lineage) to 83.1 (Andean lineage) for PVS, and 79.4 (NA-N strain) to 80.5 (O strain and NTN strain) for PVY on average. The reliability of calculation was validated by comparing the calculated Tm values and measured Tm values and the values had a strong linear correlation (correlation of determination: R2 = 0.9875). Based on the calculated Tm values, representative non-Japanese isolates could also be identified by our assay. For removing false positives, two criteria were set for the evaluation of result; successful amplification was considered as 30.0 ≥ threshold cycle value, and the virus-specific peak higher than the EF1α-specific peak was considered as positive. According to these criteria, our assay could detect PLRV and PVS from 100-fold dilution of potato leaf homogenate and PVX and PVY from 1000-fold in a model assay. Conclusion This new high-throughput detection protocol using one-step real-time mRT-PCR was sensitive enough to detect viruses in a 100-fold dilution of singly-virus contaminated homogenate in a model assay. This protocol can detect the four viruses in one assay and yield faster results for a vast number of samples, and greatly save the labor for seed potato quarantine and field surveys.

Simultaneous detection and identification of Peste des petits ruminants Virus Lineages II and IV by MCA-Based real-time quantitative RT-PCR assay within single reaction

“…Thus, the fluorescence signal of the SYBR Green I was different and could be used to specifically identify PCR products by Melting curve analysis (MCA). MCA in conjunction with real-time PCR, including dsDNA dye SYBR Green I and TaqMan probe-based types, has been widely used in previous studies including and single-nucleotide polymorphism(SNP)analysis [24][25][26]. The advantage of probe-based MCA is its ability for multiplex detection using a probe with specific melting temperatures.…”

Preparation of the luciferase-labeled antibody for improving the detection sensitivity of viral antigen

“…With the ecological and environmental changes, novel viral infectious diseases emerge continuously; viral diseases are becoming a more significant threat to health [ 1 – 3 ]. During the early stages of virus infection, rapid detection and accurate identification of the pathogen can determine the source of infection and infection route, thereby enabling effective prevention and control of further spread [ 4 , 5 ].…”