3.1 Principle of the colorimetric assay
The principle of the colorimetric assay for multiple mutations of exon 19 from EGFR gene is shown in Fig. 1. The colorimetric method was described in Fig. 1. We known that the different mutations of exon 19 from EGFR gene (Take T1 – T7 as an example) have the same biological significance, so we don’t need to distinguish type of the mutations. The universal primers (F-Primer and R-Primer) and upstream probe (U-Probe) were designed for all of mutation sites and the downstream probes (dProbes) were specially designed for every mutation site. Usually, Tm of primers (F-Primer and R-Primer) is 72°C, Tm of U-probe is 68°C, Tm of dProbes is 63°C, and the Tm of silica- GNPs is 55°C, so when DNA amplification reaction is in progress under highly anealing temperature, invasive reaction and GNPs aggreggation can’t occur at the high temperature. The target DNA was quickly amplified by DNA amplification reaction with F-Primer and R-Primer, furtherly, the complicon was discriminated and hybridized with U-probe and dProbe (dP1 – dP7), which was cut by Afu endonuclease, then the flap was released. Flap hybridized with hairpin probe (H- probe), and the H- probe was cut. The intact H-probe could hybridize with Silica-GNPs and Mg2+-induced aggregation would occur, but the cut H- probe could not. When target DNA exists in the reaction, the reaction mixture present deep red because hairpin probe was cut off and silica- GNPs were dispersed after hybrization reaction, conversely, the reaction result presents purple and the supernatant is colourless because silica- GNPs hybridizing with the intact H- probe were not stable against.
3.2 Sensitivity of colorimetric assay with every dProbe
To evaluate the sensitivity of this colorimetric assay with every dProbe for seven mutation sites in exon 19 of EGFR gene, the synthesized plasmid DNA (template T1-T7) was detected. As shown in Fig. 2(a-g), the giving sensitivity is about 10 copies, indicating the sensitivity of this assay is approximately same with that of qPCR, but in comparison to reported colorimetric assay, this assay is able to measure the template with a 200- fold higher detection limit for plasmid DNA.
3.3 Specificity of the colorimetric assay with dProbes
To evaluate the detecting specificity for the mixture with every dProbe we detected the mutations with gradient dilution. The samples (template T1-T7) with a concentration of 10%, 1%, 0.1%, 0.01% and 0% were used (Fig. 3a-g). The results show the specificity of this method can be 0.1%. Furtherly, the detecting specificity for the mixture with all of dProbes was investigated by adding various amounts of DNA template (series of samples: 10%, 1%, 0.1%, 0.01% and 0% of mutant/wild-type ratio), which were prepared by spiking the plasmids of the mutated template (T1-T7) into that of wild-type (Fig. 4a-g). The results show the specificity can be 0.1% as same as the specificity for the mixture with every dProbe. Accordingly, this method provides a good specificity and capatibility of multiple mutation sites.
3.4 Detection of the colorimetric assay with dProbes
Template T1-T7 was respectively added to the mixtures with different dProbe to evaluate the detecting specificity of this colorimetric assay, and the result was showed as Fig. 5(a-g). For every mixture with different dProbe, only the detecting result of the template complementary with the dProbe was positive, and the other reaction is negative. Furtherly, different templates were detected by using the mixtures with different dProbes (dP1-dP7), and the results from Figure S1 suggested that different mixtures with the different pannel of dProbes from dP1 to dP7 have a good specificity for differently multiple target detection. These results suggested the colorimetric assay for the samples (template T1-T7) was specific and could be used to specifically detect the target DNA.
3.5 Analysis of sample
Our method’s feasibility in clinical application was extensively evaluated by detecting somatic mutation in tissue samples, 104 clinical samples from non-small cell lung cancer patients were detected, and Fig. 6 shows typical results. As can be seen, 29 cases of all samples gave positive results, and then the positive samples were analyzed ulteriorly by ARMS-PCR. Twenty-eight cases of all positive samples were verified by ARMS-PCR, only a weakly positive result of our method was not verified by ARMS-PCR, since the ARMS-PCR’s result was negative. These suggested that our proposed method has a good performance in detecting mutants in exon 19 of EGFR gene from clinical samples, and has a higher specificity than ARMS-PCR.