Division of Agro-Environmental Research, Hokkaido Agricultural Research Center, National Agriculture and Food Research Organization (NARO), Hitsujigaoka 1, Toyohira, Sapporo, Hokkaido, 062-8555, Japan.
Graduate School of Agriculture, Hokkaido University, Kita 8, Nishi 5, Kita-ku, Sapporo, Hokkaido, 060-0808, Japan.
Virol J. 2021 Jun 29;18(1):131. doi: 10.1186/s12985-021-01591-3.
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.
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: R = 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.
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.
种薯的病毒认证对于稳定的马铃薯生产至关重要。在感染马铃薯的 30 多种病毒中，马铃薯卷叶病毒（PLRV）、马铃薯 Y 病毒（PVY）、马铃薯 X 病毒（PVX）和马铃薯 S 病毒（PVS）占主导地位，应该成为种薯检疫高通量检测方案的目标。
我们开发了一种基于一步实时多重逆转录聚合酶链反应（mRT-PCR）和熔解曲线分析的检测方法，可同时检测这四种病毒和一个内参基因（马铃薯伸长因子 1α基因，EF1α）。病毒特异性引物是从随机选择的代表种中衍生而来的，考虑到病毒基因组的多样性。我们的检测方法同时检测了代表日本的 PLRV、O 谱系的 PVS、PVX 和 NTN 株 PVY 的分离株。使用日本分离株验证了每个病毒的熔解温度（Tm）值的可变性，并且可以通过 87.6°C 的 PLRV、85.9°C 的 PVX、82.2°C（普通谱系）到 83.1°C（安第斯谱系）的 PVS 和 79.4°C（NA-N 株）到 80.5°C（O 株和 NTN 株）的平均 Tm 值来识别病毒种。通过比较计算的 Tm 值和测量的 Tm 值以及值的强线性相关性（决定系数：R=0.9875）验证了计算可靠性。根据计算的 Tm 值，我们的检测方法也可以鉴定代表非日本的分离株。为了消除假阳性，对结果评估设定了两个标准；成功扩增被认为是 30.0≥阈值循环值，并且病毒特异性峰高于 EF1α 特异性峰被认为是阳性。根据这些标准，我们的检测方法可以在模型检测中从 100 倍稀释的马铃薯叶匀浆中检测到 PLRV 和 PVS，从 1000 倍稀释的 PVX 和 PVY 中检测到。
这种使用一步实时 mRT-PCR 的高通量检测方案在模型检测中足以灵敏地检测到 100 倍稀释的单一病毒污染匀浆中的病毒。该方案可在一次检测中检测到这四种病毒，并为大量样本提供更快的结果，大大节省种薯检疫和田间调查的劳动力。
