Here we report a rapid, low cost, and disposable dipstick-type DNA biosensor that enables multiplex detection in a single assay. The fluorescent probes labeled with different fluorophores were introduced into the lateral flow nucleic acid testing system. In combination with multiple immobilized probes arranged in an array formant on the membrane, a dual-color fluorescent lateral flow DNA biosensor was developed using a portable fluorescence reader. Up to 13 human papillomavirus types could be detected simultaneously by a single-step operation in less than 30 min after linear-after-the-exponential (LATE)-PCR. The sensitivity was determined to be 10-10(2) copies plasmid DNA/μL. The specificity study showed no cross-reactivity among the 31 different common HPV types. In the clinical validation, 95.3% overall agreement showed very good potential for this method in the clinical application when compared to a commercial kit.
Probe-based fluorescence melting curve analysis (FMCA) is a powerful tool for mutation detection based on melting temperature generated by thermal denaturation of the probe-target hybrid. Nevertheless, the color multiplexing, probe design, and cross-platform compatibility remain to be limited by using existing probe chemistries. We hereby explored two dual-labeled, self-quenched probes, TaqMan and shared-stem molecular beacons, in their ability to conduct FMCA. Both probes could be directly used for FMCA and readily integrated with closed-tube amplicon hybridization under asymmetric PCR conditions. Improved flexibility of FMCA by using these probes was illustrated in three representative applications of FMCA: mutation scanning, mutation identification and mutation genotyping, all of which achieved improved color-multiplexing with easy probe design and versatile probe combination and all were validated with a large number of real clinical samples. The universal cross-platform compatibility of these probes-based FMCA was also demonstrated by a 4-color mutation genotyping assay performed on five different real-time PCR instruments. The dual-labeled, self-quenched probes offered unprecedented combined advantage of enhanced multiplexing, improved flexibility in probe design, and expanded cross-platform compatibility, which would substantially improve FMCA in mutation detection of various applications.
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