Plant Health and Environment Laboratory, Ministry for Primary Industries, P.O. Box 2095, Auckland 1140, New Zealand.
Viruses. 2024 Sep 30;16(10):1550. doi: 10.3390/v16101550.
High-throughput sequencing (HTS) technologies may be a useful tool for testing imported plant germplasm for multiple pathogens present in a sample, offering strain-generic detection not offered by most PCR-based assays. Metatranscriptomics (RNAseq) and tiled amplicon PCR (TA-PCR) were tested as HTS-based techniques to detect viruses present in low titres. Strawberry mottle virus (SMoV), an RNA virus, and strawberry vein banding virus (SVBV), a DNA virus, were selected for comparison of RNAseq and TA-PCR with quantitative PCR assays. RNAseq of plant ribosomal RNA-depleted samples of low viral titre was used to obtain datasets from 3 M to 120 M paired-end (PE) reads. RNAseq demonstrated PCR-like sensitivity, able to detect as few as 10 viral copies/µL when 60 million (M) PE reads were generated. The custom TA-PCR primer panels designed for each virus were successfully used to recover most of the reference genomes for each virus. Single- and multiple-target TA-PCR allowed the detection of viruses in samples with around 10 viral copies/µL with a minimum continuous sequence length recovery of 500 bp. The limit of detection of the HTS-based protocols described here is comparable to that of quantitative PCR assays. This work lays the groundwork for an increased flexibility in HTS detection of plant viruses.
高通量测序 (HTS) 技术可能是一种有用的工具，可用于测试样本中存在的多种病原体的进口植物种质，提供大多数基于 PCR 的检测方法无法提供的菌株通用检测。元转录组学 (RNAseq) 和嵌合扩增 PCR (TA-PCR) 被测试为基于 HTS 的技术，以检测低滴度存在的病毒。草莓斑驳病毒 (SMoV)，一种 RNA 病毒和草莓叶脉带病毒 (SVBV)，一种 DNA 病毒，被选择用于比较 RNAseq 和 TA-PCR 与定量 PCR 检测。使用植物核糖体 RNA 耗尽的低病毒滴度样品的 RNAseq 获得了 3 M 至 120 M 配对末端 (PE) 读数的数据集。RNAseq 显示出与 PCR 相似的灵敏度，当生成 6000 万 (M) PE 读数时，能够检测到低至 10 个病毒拷贝/μL。为每种病毒设计的定制 TA-PCR 引物组成功用于回收每种病毒的大部分参考基因组。单靶和多靶 TA-PCR 允许在大约 10 个病毒拷贝/μL 的样品中检测到病毒，最小连续序列长度恢复为 500 bp。这里描述的基于 HTS 的方案的检测限与定量 PCR 检测方法相当。这项工作为 HTS 检测植物病毒的灵活性增加奠定了基础。
