<br>用于高灵敏度 HIV 分子检测的单管一步式凝胶 RT-RPA/PCR,Analyst

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用于高灵敏度 HIV 分子检测的单管一步式凝胶 RT-RPA/PCR
Analyst ( IF 3.6 ) Pub Date : 2023-01-06 , DOI: 10.1039/d2an01863b
Naoki Uno ₁ , Ziyue Li _{1,

2} , Changchun Liu ₁

Affiliation

我们开发了一种单管一步式凝胶逆转录重组酶聚合酶扩增（RT-RPA）/聚合酶链式反应（PCR）（称为“SOG RT-RPA/PCR”）来检测人类免疫缺陷病毒（HIV）。为了提高检测灵敏度，在 PCR 之前通过 RT-RPA 预扩增 RNA 模板。为了简化检测过程并缩短测定时间，我们将PCR试剂嵌入琼脂糖凝胶中，构建琼脂糖凝胶以在单管中将试剂与RT-RPA反应溶液物理分离。由于琼脂糖的热力学特性，在SOG RT-RPA/PCR测定期间，RT-RPA反应首先在低温（例如39℃）下在PCR凝胶顶部独立发生。然后，RPA 扩增子直接作为第二次 PCR 扩增反应的模板，当 PCR 琼脂糖因反应温度升高而溶解时，第二次 PCR 扩增反应开始，无需多次手动操作和扩增子转移。通过我们的 SOG RT-RPA/PCR 测定，我们每次测试可以检测到 6.3 个 HIV RNA 拷贝，这比独立的实时 RT-PCR 和 RT-RPA 的灵敏度高 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，我们在临床血浆样本中检测到了 HIV 病毒 RNA，并验证了我们的检测方法的卓越性能。因此，SOG RT-RPA/PCR 检测为简单、快速、高灵敏度的基于核酸的传染病分子检测提供了一种强大的方法。

"点击查看英文标题和摘要"

Single-tube one-step gel-based RT-RPA/PCR for highly sensitive molecular detection of HIV

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 (e.g., 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.

更新日期：2023-01-06

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