Visual single cell detection of dual-pathogens based on multiplex super PCR (MS-PCR) and asymmetric tailing HCR (AT-HCR)

To prevent the propagation of pathogens and minimize the damage to humans, an ultrasensitive and colorimetric dual detection method for pathogens was developed and validated based on the Multiplex Super Polymerase Chain Reaction (MS-PCR) and Asymmetric Tailing Hybridization Chain Reaction (AT-HCR). In the 5-min MS-PCR, dual pathogens were recognized and amplified simultaneously, generating plenty of dsDNA products with protruding ssDNA toeholds. Since a blocker of oxyethyleneglycol bridge was exquisitely inserted into two-set forward primers, which hindered the polymerase extension. Then, two-set ssDNA toeholds initiated two-set AT-HCR in 20-min and 60-min visual analysis depending on G-quadruplex DNAzyme was induced by Terminal deoxynucleotidyl Transferase (TdT) at the tails of AT-HCR at 3′-OH. During the process, the first magnetic separation was conducted after MS-PCR based on our home-made XP beads, to remove primer-dimers and other impurities in 30-min. The second magnetic separation was performed before TdT catalysis based on dynabeads, to eliminate unreacted AT-HCR hairpins in 15-min. To challenge the practical application capability of this strategy, the detection of analyte in fat-free milk was also tested and demonstrated similar detection capability to visualize single cell. Therefore, the proposed sensor exhibited a high selectivity and sensitivity and is thus applicable for visual single cell detection of dual-pathogens of Salmonella spp. and Staphylococcus aureus simultaneously in 130-min. Generally, it provides a visual, ultrasensitive, rapid and universal detective method for dual-target analysis.