Improved Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) for the Rapid and Sensitive Detection of Yam mosaic virus

Abstract

Yam (Dioscorea spp.) productivity is constrained significantly by the lack of a formal seed system. Vegetative propagation, through tuber setts as 'seed' yams, encourages the recycling of virus-infected planting materials, contributing to high virus incidence and yield losses. Efforts are ongoing to increase the production of high-quality seed yams in a formal seed system to reduce virus-induced yield losses and enhance the crop's productivity and food security. Specific and sensitive diagnostic tests are imperative to prevent the multiplication of virus-infected materials contributing to a sustainable seed yam certification system. During routine indexing of yam accessions, discrepancies were observed between the results obtained from the reverse transcription loop-mediated isothermal amplification (RT-LAMP) test and those from reverse transcription polymerase chain reaction (RT-PCR); RT-LAMP failed to detect Yam mosaic virus (YMV) in some samples that tested positive by RT-PCR. This prompted the design of a new set of LAMP primers, YMV1-OPT primers. These primers detected as little as 0.1 fg/µL of purified RNA obtained from a YMV-infected plant, a sensitivity equivalent to that obtained with RT-PCR. RT-LAMP using YMV1-OPT primers is recommended for all future virus-indexing of seed yams for YMV, offering a rapid, sensitive, and cost-effective approach.

Keywords: RT-LAMP; West Africa; Yam mosaic virus; isothermal amplification; seed systems; virus diagnostics; yam.

Figure 1

The detection of YMV in Dioscorea rotundata samples. (A)-The detection of YMV and actin by the reverse transcription polymerase chain reaction (RT-PCR) [22,25], M-100 bp Gene ruler DNA ladder size 100–3000 (ThermoFisher Scientific), Well 1-Gh1, 2-Gh2, 3-Gh3, 4-Gh4, 5-Gh5, 6-Nig1, + = YMV-positive control and NTC = non-template control; (B)-The detection of YMV by reverse transcription loop-mediated isothermal amplification (RT-LAMP) using primers by Nkere et al., 2018; NTC-Non-template control; +ve = YMV-positive control.

Figure 2

Yam plant (Nig1) showing mild mottle symptoms.

Figure 3

A representative figure showing a partial alignment of YMV coat protein sequences, highlighting the YMV1-OPT LAMP primers designed in this study. YMV1_OPT-F3 = Forward outer primer; YMV1_OPT-B3 = Reverse outer primer; YMV1_OPT-F1C + YMV1_OPT-F2 = Forward inner primer; YMV1_OPT-B1C + YMV1_OPT-B2 = Reverse inner primer; YMV1_OPT-LF = Forward loop primer; YMV1_OPT-LB = Reverse loop primer.

Figure 4

YMV detection by RT-LAMP using YMV1-OPT primers designed in this study. Template RNAs are the same tested by RT-PCR and RT-LAMP by Nkere et al. (2018); +ve control = YMV-positive control; NTC = Non-template control.

Figure 5

Sensitivity of the improved RT-LAMP and comparison with RT-PCR. (A)-YMV amplification from purified total RNA extracts by RT-LAMP using YMV1-OPT primers; Stock- Stock RNA from YMV-positive plant (100 ng/µL). The LAMP assay was conducted using the GenieIII machine (OptiGene), which allows only eight reactions in a run. (B)-YMV amplification from crude RNA extracts by RT-LAMP using YMV1-OPT primers. (C)-YMV amplification by RT-PCR from purified total RNA; M-1 kb DNA ladder, size 0.5 kb−10 kb (New England Biolabs); S-Stock RNA (100 ng/µL); - = YMV-negative control.

Figure 6

A phylogenetic tree based on the partial nucleotide sequence of the coat protein region of YMV. (▼) YMV CP sequences from D. rotundata obtained in this study; (▼) YMV CP sequences from D. alata obtained in this study; (●) YMV CP sequences from D. rotundata downloaded from NCBI; (●) the sequence of Pepper veinal mottle virus partial CP gene used as an outgroup. The tree was generated using the neighbour-joining method in MEGA-X with 1000 bootstrap replications. Branches < 70% were collapsed. The scale bar represents the number of nucleotide substitutions per site.