Xu R, Falardeau J, Avis T J, Tambong J T
Agriculture and Agri-Food Canada, Ottawa, ON, Canada.
Department of Chemistry, Carleton University, Ottawa, ON, Canada.
Lett Appl Microbiol. 2016 Feb;62(2):153-9. doi: 10.1111/lam.12522.
The aim of this study was to develop and validate a HybProbes-based real-time PCR assay targeting the trpB gene for specific identification of Streptomyces scabies and Streptomyces europaeiscabiei. Four primer pairs and a fluorescent probe were designed and evaluated for specificity in identifying S. scabies and Streptomyces europaeiscabiei, the potato common scab pathogens. The specificity of the HybProbes-based real-time PCR assay was evaluated using 46 bacterial strains, 23 Streptomyces strains and 23 non-Streptomyces bacterial species. Specific and strong fluorescence signals were detected from all nine strains of S. scabies and Streptomyces europaeiscabiei. No fluorescence signal was detected from 14 strains of other Streptomyces species and all non-Streptomyces strains. The identification was corroborated by the melting curve analysis that was performed immediately after the amplification step. Eight of the nine S. scabies and S. europaeiscabiei strains exhibited a unique melting peak, at Tm of 69·1°C while one strain, Warba-6, had a melt peak at Tm of 65·4°C. This difference in Tm peaks could be attributed to a guanine to cytosine mutation in strain Warba-6 at the region spanning the donor HybProbe. The reported HybProbes assay provides a more specific tool for accurate identification of S. scabies and S. europaeiscabiei strains.
This study reports a novel assay based on HybProbes chemistry for rapid and accurate identification of the potato common scab pathogens. Since the HybProbes chemistry requires two probes for positive identification, the assay is considered to be more specific than conventional PCR or TaqMan real-time PCR. The developed assay would be a useful tool with great potential in early diagnosis and detection of common scab pathogens of potatoes in infected plants or for surveillance of potatoes grown in soil environment.
本研究的目的是开发并验证一种基于杂交探针的实时荧光定量PCR检测方法，该方法靶向trpB基因，用于特异性鉴定疮痂链霉菌和欧洲疮痂链霉菌。设计了四对引物和一个荧光探针，并评估了它们在鉴定疮痂链霉菌和欧洲疮痂链霉菌（马铃薯普通疮痂病的病原菌）方面的特异性。使用46株细菌菌株（23株链霉菌菌株和23种非链霉菌细菌物种）对基于杂交探针的实时荧光定量PCR检测方法的特异性进行了评估。在所有9株疮痂链霉菌和欧洲疮痂链霉菌中均检测到特异性强的荧光信号。在14株其他链霉菌菌株和所有非链霉菌菌株中均未检测到荧光信号。扩增步骤后立即进行的熔解曲线分析证实了该鉴定结果。9株疮痂链霉菌和欧洲疮痂链霉菌菌株中的8株呈现出独特的熔解峰，熔解温度为69.1°C，而其中一株Warba - 6菌株的熔解峰熔解温度为65.4°C。Tm峰的这种差异可能归因于Warba - 6菌株在跨越供体杂交探针的区域发生了鸟嘌呤到胞嘧啶的突变。所报道的杂交探针检测方法为准确鉴定疮痂链霉菌和欧洲疮痂链霉菌菌株提供了一种更特异的工具。
本研究报道了一种基于杂交探针化学的新型检测方法，用于快速准确地鉴定马铃薯普通疮痂病的病原菌。由于杂交探针化学方法需要两个探针进行阳性鉴定，因此该检测方法被认为比传统PCR或TaqMan实时荧光定量PCR更特异。所开发的检测方法将是一种有用的工具，在早期诊断和检测受感染植物中马铃薯普通疮痂病的病原菌或监测土壤环境中种植的马铃薯方面具有巨大潜力。
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