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

摘要

In this study, we established a novel polymerase chain reaction-hybridization chain reaction (PCR-HCR) dual signal amplification assay for sensitive detection of Escherichia coil O157:H7 in milk. In this assay, a blocker forward primer and a biotin reverse primer based on E. coil O157:H7 specific fliC(h7), gene were specifically designed for PCR amplification resulting in a single-double stranded DNA with biotin (ss-dsDNA similar to biotin). These PCR products, ss-dsDNA similar to biotin, were captured by magnetic beads (MBs) decorated with streptavidin (SA) forming the ss-dsDNA similar to 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 x 10(1) CFU/mL. In addition, this assay has been successfully used to detection target bacteria in skim milk spiked samples.