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.

Significance: We describe a highly multiplex PCR approach that can identify 10-fold more targets in current real-time PCR assays without additional enzymes or separate reactions. This single-step, single-tube, homogeneous detection approach, termed MeltArray, is achieved by coupling the 5′-flap endonuclease activity of the Taq DNA polymerase and multiple annealing sites of the molecular beacon reporters. The 5′-flap endonuclease cleaves a probe specifically into a “mediator” primer, and one molecular beacon reporter allows for the extension of multiple “mediator” primers to produce a series of fluorescent hybrids with different melting temperatures unique to each target. The overall number of targets detectable per reaction is equal to the number of the reporters multiplied by the number of mediator primers per reporter.

The target volume of multiplex real-time PCR assays is limited by the number of fluorescent dyes available and the number of fluorescence acquisition channels present in the PCR instrument. We hereby explored a probe labeling strategy that significantly increased the target volume of real-time PCR detection in one reaction. The labeling paradigm, termed “Multicolor Combinatorial Probe Coding” (MCPC), uses a limited number (n) of differently colored fluorophores in various combinations to label each probe, enabling one of 2ⁿ-1 genetic targets to be detected in one reaction. The proof-of-principle of MCPC was validated by identification of one of each possible 15 human papillomavirus types, which is the maximum target number theoretically detectable by MCPC with a 4-color channel instrument, in one reaction. MCPC was then improved from a one-primer-pair setting to a multiple-primer-pair format through Homo-Tag Assisted Non-Dimer (HAND) system to allow multiple primer pairs to be included in one reaction. This improvement was demonstrated via identification of one of the possible 10 foodborne pathogen candidates with 10 pairs of primers included in one reaction, which had limit of detection equivalent to the uniplex PCR. MCPC was further explored in detecting combined genotypes of five β-globin gene mutations where multiple targets were co-amplified. MCPC strategy could expand the scope of real-time PCR assays in applications which are unachievable by current labeling strategy.

Vibrio cholerae is a natural inhabitant of the aquatic environment. However, its toxigenic strains can cause potentially life-threatening diarrhea. A quadruplex real-time PCR assay targeting four genes, the cholera toxin gene ( ctxA ), the hemolysin gene ( hlyA ), O1-specific rfb , and O139-specific rfb , was developed for detection and differentiation of O1, O139, and non-O1, non-O139 strains and for prediction of their toxigenic potential. The specificity of the assay was 100% when tested against 70 strains of V. cholerae and 31 strains of non- V. cholerae organisms. The analytical sensitivity for detection of toxigenic V. cholerae O1 and O139 was 2 CFU per reaction with cells from pure culture. When the assay was tested with inoculated water from bullfrog feeding ponds, 10 CFU/ml could reliably be detected after culture for 3 h. The assay was more sensitive than the immunochromatographic assay and culture method when tested against 89 bullfrog samples and 68 water samples from bullfrog feeding ponds. The applicability of this assay was confirmed in a case study involving 15 bullfrog samples, from which two mixtures of nontoxigenic O1 and toxigenic non-O1/non-O139 strains were detected and differentiated. These data indicate that the quadruplex real-time PCR assay can both rapidly and accurately detect/identify V. cholerae and reliably predict the toxigenic potential of strains detected.