In this study, we integrated the novel RPA amplification technology (Multienzyme Isothermal Rapid Amplification, MIRA) and CRISPR/Cas12 detection technology into a single tube reaction by using crRNAs targeting suboptimal PAMs substrates for sensitive, specific and speedy detecting GBS without the procedure of nucleic acid purification [39]. The MIRA/CRISPR one-pot system can detect GBS pathogen in unextracted cervical secretion samples, the whole detection process can be shortened to less than 30 min without multiple solution transfer, which shows rapidity and simplicity. We used real-time fluorescence technique and end-time fluorescence intensity under UV light to investigate the GBS detection ability of the one-pot method, the lower limit of detection can be reached to 32 copies and the specificity can reach 100 %, which displays satisfactory sensitivity and specificity. In addition, the lateral flow assay (LFA) strip tests were combined with the one-pot system for intuitive and portable GBS detection, further verifying the superiority of the one-pot system. The detection results of 20 clinical samples (including 8 cases of GBS positive) confirmed the ability of the one-pot system to detect real unpurified samples. Furthermore, we established a portable non-instrumental GBS detection device based on the LFA method, which got rid of the professional and site restrictions of GBS detection and provided a certain basis for the GBS home self-test.
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One-pot MIRA-CRISPR 문의 논문