Department of Biomedical Engineering, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA.
Department of Biomedical Engineering, University of Connecticut, 260 Glenbrook Road, Storrs, CT 06029, USA.
Analyst. 2023 Feb 13;148(4):926-931. doi: 10.1039/d2an01863b.
We developed a single-tube one-step gel-based reverse transcription-recombinase polymerase amplification (RT-RPA)/polymerase chain reaction (PCR) (termed "SOG RT-RPA/PCR") to detect the human immunodeficiency virus (HIV). To improve the assay sensitivity, the RNA template is pre-amplified by RT-RPA prior to PCR. To simplify the detection process and shorten the assay time, we embedded PCR reagents into agarose gel, constructing it to physically separate the reagents from the RT-RPA reaction solution in a single tube. Due to the thermodynamic properties of agarose, the RT-RPA reaction first occurs independently on top of the PCR gel at a low temperature (, 39 °C) during the SOG RT-RPA/PCR assay. Then, the RPA amplicons directly serve as the template for the second PCR amplification reaction, which begins when the PCR agarose dissolves due to the elevated reaction temperature, eliminating the need for multiple manual operations and amplicon transfer. With our SOG RT-RPA/PCR assay, we could detect 6.3 copies of HIV RNA per test, which is a 10-fold higher sensitivity than that of standalone real-time RT-PCR and RT-RPA. In addition, due to the high amplification efficiency of RPA, the SOG RT-RPA/PCR assay shows stronger fluorescence detection signals and a shorter detection time compared to the standalone real-time RT-PCR assay. Furthermore, we detected HIV viral RNA in clinical plasma samples and validated the superior performance of our assay. Thus, the SOG RT-RPA/PCR assay offers a powerful method for simple, rapid, and highly sensitive nucleic acid-based molecular detection of infectious diseases.
我们开发了一种单管一步凝胶基逆转录-重组酶聚合酶扩增（RT-RPA）/聚合酶链反应（PCR）（称为“SOG RT-RPA/PCR”），用于检测人类免疫缺陷病毒（HIV）。为了提高检测的灵敏度，在 PCR 之前，通过 RT-RPA 对 RNA 模板进行预扩增。为了简化检测过程并缩短检测时间，我们将 PCR 试剂嵌入琼脂糖凝胶中，在单管中构建，将试剂与 RT-RPA 反应溶液物理分离。由于琼脂糖的热力学性质，在 SOG RT-RPA/PCR 检测中，RT-RPA 反应首先在低温（，39°C）下在 PCR 凝胶的顶部独立发生。然后，RPA 扩增子直接作为第二个 PCR 扩增反应的模板，当由于升高的反应温度而使 PCR 琼脂糖溶解时，该反应开始，从而无需进行多次手动操作和扩增子转移。通过我们的 SOG RT-RPA/PCR 检测，我们可以检测到每个测试 6.3 个 HIV RNA 拷贝，这比独立的实时 RT-PCR 和 RT-RPA 高 10 倍。此外，由于 RPA 的高扩增效率，与独立的实时 RT-PCR 检测相比，SOG RT-RPA/PCR 检测显示出更强的荧光检测信号和更短的检测时间。此外，我们还在临床血浆样本中检测到 HIV 病毒 RNA，并验证了我们检测方法的优越性能。因此，SOG RT-RPA/PCR 检测为传染病的简单、快速和高灵敏度核酸分子检测提供了一种强大的方法。
