The PCR-HCR dual signal amplification strategy for ultrasensitive detection of Escherichia coli O157:H7 in milk

In this study, we established a novel polymerase chain reaction-hybridization chain reaction (PCR-HCR) dual signal amplification assay for sensitive detection of Escherichia coli O157:H7 in milk. In this assay, a blocker forward primer and a biotin reverse primer based on E. coli O157:H7 specific fliCh7 gene were specifically designed for PCR amplification resulting in a single-double stranded DNA with biotin (ss-dsDNA~biotin). These PCR products, ss-dsDNA~biotin, were captured by magnetic beads (MBs) decorated with streptavidin (SA) forming the ss-dsDNA~biotin+SA~MBs. The ssDNA on the PCR products opened the hairpin probe (H1 and H2) to trigger the HCR for secondary signal amplification. This is the first to use HCR to directly amplify PCR products through a novel blocker primer. With the help of two nucleic acid dual signal amplification techniques, PCR-HCR analysis can effectively improve the detection sensitivity and the limit of detection is 7.2 × 101 CFU/mL. In addition, this assay has been successfully used to detection target bacteria in skim milk spiked samples.