Microsphere integrated convective amplification sensor for all-in-one-pot detection of Mycoplasma pneumoniae

Abstract

The integration of isothermal amplification with CRISPR-Cas systems play a crucial role in the point-of-care test of infectious pathogens, owing to their specificity, sensitivity, and portability. However, these systems lack rigorous internal amplification control for monitoring false negatives and rely on carefully designed guide sequences, which barriers to their further promotion and application. Additionally, the complexity of sample purification and the laborious preparation of liquid reagents significantly limit these systems to ready-to-use and direct sample-to-answer detection in on-site diagnosis. Here, we combined ready-to-use lyophilized microsphere, thermal lysis of pathogens, guide-sequence-independent convective amplification, internal amplification control for monitoring negative false and a portable multi-channel instrument to develop a microsphere integrated convective amplification sensor (MICASOR) system for on-site detection. MICASOR integrated the overall workflow of DNA testing into one-pot to achieve all-in-one-pot detection for on-site diagnosis. MICASOR is a rapid, potable, ready-to-use and guide-sequence-independent platform and can monitor false negatives. MICASOR can detect as low as 1 copy/μL of clinical samples of Mycoplasma pneumoniae within 15 minutes. Validated by 30 real clinical samples, MICASOR showed 100 % agreement with gold standard real-time PCR. These results suggest that MICASOR has great potential to promote the control of infectious diseases in the outpatients, communities and poor resource areas.