Taqman-MGB NanoPCR For Highly Specific Detection Of Single-Base Mutations

Background and ObjectiveDetection of single-base mutations is of great significance for real-time monitoring of tumor progression,treatment efficacy and drug resistance.However,detection of specific single-base mutations from an excess of wild-type background sequences using conventional PCR techniques remains challenging.This is because the conventionally designed primers and probes are hard to distinguish single-base mismatch,resulting in the output mutant signal normally masked by large amount of wild-type signal.Currently,sequencing and digital PCR(d PCR)methods are often used to detect single-base mutations,but they are costly and unsuitable for the detection of a large number of samples.In contrast,real-time fluorescent quantitative PCR(q PCR)is the most classic and commonly used technique in clinical practice.but due to the low specificity of conventional q PCR,it is rarely used for mutation detection,especially single-base mutations.In recent years,with the development of nanotechnology,various nanomaterials have been used to improve the amplification yield,amplification efficiency,and amplification specificity of PCR.Inspired by this,we propose a single-base mutation detection method using gold nanoparticles(AuNPs)to improve the specificity of q PCR.Methods1.A mutation at position 2369 in the base sequence of the epidermal growth factor receptor(EGFR T790M)was used as a model for the study,and AuNPs were added to Taqman-MGB probe-based q PCR to improve the specificity of amplification.2.AuNPs with a particle size of 13 nm were prepared by sodium citrate reduction method.The prepared AuNPs were characterized(TEM,DLS,UV-VIS,zeta potential,etc.)and the changes of the nanoparticles before and after the amplification were recorded.Meanwhile,the optimum concentration to improve the specificity of amplification was explored by adding different concentrations of Au NP.3.Single-stranded wild-type and mutant templates were incubated with probes for 30minutes at 60?,and then SYBR Green?was added to reflect the effect of AuNPs on template/probe binding.Based on the above results,a mechanism for AuNPs to improve amplification specificity was proposed.4.50 n M of AuNPs were added to templates of different concentration ranges(mutant,wild type)to investigate the range of their improved specificity for single base mutation detection.5.Genomic DNA was extracted from normal human whole blood as wild-type template,its concentration was fixed,and mixed with mutant plasmid DNA to configure template sequences with different mutation abundance,and two specific probes(VIC probe and FAM probe were paired perfectly complementary to the two mutant templates and wild-type template,respectively)were added to construct the amplification system.Results1.The prepared AuNPs were well dispersed and homogeneous in size to fully meet the amplification requirements.Using EGFR T790M as a template,50 n M AuNPs produced significant suppression of the background noise of the wild-type sequence while barely affecting the detection signal generated by the mutant sequence,improving the specificity of single-base mutation detection.2.Comparing the changes of AuNPs before and after amplification,we found that the UV-visible absorption spectra of the amplified AuNPs were red-shifted,and the dynamic light scattering and zeta potential results were also changed,indicating that the components of the q PCR system(enzyme,primer,template,etc.)were non-specifically adsorbed on the surface of the AuNPs.Observing the change of fluorescence intensity of SYBR Green I after amplification,we proposed a theory based on the energy barrier,i.e.,successful pairing between primers and templates requires crossing the barrier,and the addition of AuNPs can adjust the barrier.Under optimal concentration conditions,AuNPs allow mutant sequences to complementarily pair with the probe to form a double strand to generate a fluorescent signal,while wild-type sequences cannot fully complementarily pair with the probe to form a stable double-stranded structure even with a single base mismatch.3.It was also confirmed that Taqman-MGB nano PCR can improve specificity in the target sequence concentration range of 10^-9?M to 10?M.Compared with traditional Taqman-MGB q PCR and existing PCR methods,the Taqman-MGB nano PCR was able to detect as low as 0.95%mutation abundance in spiked samples.ConclusionThe experimental results confirmed that mismatches between template and probe can be effectively suppressed using AuNPs,resulting in highly specific single-base mutation analysis.This method outperforms conventional Taqman-MGB q PCR in terms of specificity and sensitivity.considering that only a small amount of AuNPs are required in these assays and that AuNPs are inexpensive and readily available,current laboratory instrumentation and assay protocols can be readily adapted for single-base mutation detection.We believe that the developed Taqman-MGB probe-based nanoparticle-assisted PCR is expected to be a powerful tool for diagnosing various genetic mutation diseases.