Department of Clinical Laboratory, Affiliated Hospital of North Sichuan Medical College, Nanchong, 637000, PR China; Department of Laboratory Medicine, North Sichuan Medical College, Nanchong, 637000, PR China; Translational Medicine Research Center, North Sichuan Medical College, Nanchong, 637000, PR China.
Department of Clinical Laboratory, Affiliated Hospital of North Sichuan Medical College, Nanchong, 637000, PR China.
Biosens Bioelectron. 2021 Jul 15;184:113239. doi: 10.1016/j.bios.2021.113239. Epub 2021 Apr 12.
It is highly desired to perform accurate and rapid nucleic acid detections for disease diagnosis at resource-limited setting, such as small clinics, remote areas and home. However, the challenges in sample handling, expensive equipment and complicated operation make canonical polymerase chain reaction (PCR) impossible to run the point-of-care testing (POCT). Herein we report a novel nucleic acid detection method, named stem-loop-primer assisted isothermal amplification (SPA), which specifically and sensitively amplifies target nucleic acid by using Bst DNA polymerase, a pair of canonical PCR primers and their stem-loop derivatives. The stem-loop-primers are easily designed by adding a stem-loop sequence to the canonical PCR primers at 5'-ends. In contrast to loop-mediated isothermal amplification (LAMP), which is a widespread isothermal amplification technology, our SPA is more specific and convenient to design and run. Further, we have demonstrated that SPA can specifically detect type 16, 18, 52 and 58 Human Papilloma viruses (HPV) in cervical samples, suggesting its specificity and robustness for nucleic acid detection. Moreover, pH indicator based colorimetric SPA was developed, which offered 100% accuracy for HPV16 detection in cervical samples, thereby demonstrating its great potential for POCT nucleic acid testing.
在资源有限的环境下（如小诊所、偏远地区和家庭），人们非常希望能够进行准确、快速的疾病诊断核酸检测。然而，样本处理、昂贵的设备和复杂的操作等挑战使得经典聚合酶链反应（PCR）无法在即时检测（POCT）中进行。在此，我们报告了一种新型核酸检测方法，称为茎环引物辅助等温扩增（SPA），该方法通过使用 Bst DNA 聚合酶、一对经典 PCR 引物及其茎环衍生物特异性且灵敏地扩增靶核酸。茎环引物可通过在 5'端添加茎环序列来轻松设计经典 PCR 引物。与环介导等温扩增（LAMP）相比，SPA 更具特异性和方便设计和运行。此外，我们已经证明 SPA 可以特异性地检测宫颈样本中的 16、18、52 和 58 型人乳头瘤病毒（HPV），表明其在核酸检测方面具有特异性和稳健性。此外，我们还开发了基于 pH 指示剂的比色 SPA，该方法在宫颈样本中 HPV16 的检测中具有 100%的准确率，从而证明了其在 POCT 核酸检测中的巨大潜力。
