The amplification of specific nucleic acid sequences with high specificity and sensitivity is an essential technique for pathogen detection. Recombinase polymerase amplification (RPA) is a rapid isothermal amplification method. Here, we demonstrate the end-point and real-time detection of Spongospora subterranea f. sp. subterranea (Sss) using RPA and potato mop-top virus (PMTV) using reverse transcription (RT)-RPA. Oligonucleotide primers were designed for amplification that target the internal transcribed spacer 1 (ITS1) region and the coat protein readthrough (CP-RT) domain for the detection of Sss and PMTV, respectively. Our data showed that real-time RPA can detect 100 of Sss sporosori per gram of soil, while real-time RT-RPA could detect in ~1 ng total RNA of the PMTV-infected tuber. For Sss detection, the R² value for real-time RPA and real-time PCR was 98.0% by linear regression analysis in the concentration range of 100–34,000 sporosori per gram of soil. The developed RPA assay here may provide a useful alternative tool for the rapid, simple and reliable detection of Sss and PMTV in diagnostic laboratories and in-field testing.

中文翻译：

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重组酶聚合酶扩增（RPA）用于快速等温检测海绵下孢子f。sp。地下和马铃薯拖布顶病毒

具有高特异性和灵敏度的特定核酸序列的扩增是病原体检测的必不可少的技术。重组酶聚合酶扩增（RPA）是一种快速的等温扩增方法。在这里，我们演示了海绵夜蛾f的端点和实时检测。sp。地下（sss）使用RPA，而马铃薯拖把病毒（PMTV）使用逆转录（RT）-RPA。设计寡核苷酸引物用于扩增，该引物分别靶向内部转录的间隔区1（ITS1）和外壳蛋白通读（CP-RT）域，分别用于检测Sss和PMTV。我们的数据显示，实时RPA可以检测到每克土壤中100株Sss孢子菌，而实时RT-RPA可以检测到被PMTV感染的块茎的约1 ng总RNA。对于Sss检测，通过线性回归分析，在每克土壤100–34,000孢子孢菌的浓度范围内，实时RPA和实时PCR的R ²值为98.0％。这里开发的RPA测定法可为诊断实验室和现场测试中的Sss和PMTV的快速，简单和可靠的检测提供有用的替代工具。