Stem-loop-primer assisted isothermal amplification enabling high-specific and ultrasensitive nucleic acid detection.

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.