RealHelix™ Superplex qPCR kit [Probe] is designed to perform highly multiple target amplification and detection in a single reaction. Amplification and detection up to 12 targets of DNA in a single reaction by Superplex qPCR kit [Probe] were confirmed using a melting analysis method, FMMA (fluorescence-based multiple melting analysis). The convenient 2x concentrated premix contains a chemically modified hot-start Taq polymerase, Uracil-DNA glycosylase (UDG), dNTPs, buffer, Mg²⁺, and stabilizing agent. The high multiplexing with specificity is achieved by a unique buffer system and optimized hot-start polymerase. Included ROX Dye could be used as a passive reference dye. 

Figure 1. Multiple amplification of Five targets in a reaction by RealHelix^TM Superplex qPCR Kit [Probe]. 10-fold serial dilutions of human genomic DNA (10, 1, 0.1, and 0.01 ng) were analyzed using human GloB(FAM), AAT(α1 antitypsin, HEX), GAPDH(Cy5), GloB(Cy5.5) specific primer and probe sets. And 10-fold serial dilutions of a plasmid DNA were also added to the reaction mixture and analyzed using a gene-specific primer and probe labeled with Texas Red in the reactions. Reactions were done using the RealHelix^TM Superplex qPCR Kit [Probe] on a real-time PCR instrument, Bio-Rad CFX96. 

Figure 2. Twelve-targets multiplex amplification in a reaction by RealHelix^TM Superplex qPCR Kit [Probe]. Using the allele-specific FMMA probes (labeled with FAM, HEX, Texas Red, or Cy5), twelve rice SNP genotypes were identified by a single reaction. A mix of rice genomic DNAs (total 10ng) was used for the FMMA reaction to detect all of the 12 SNPs screened for the determination of rice variants. The multiplex reaction was done by the RealHelix^TM Superplex qPCR Kit [Probe] on a real-time PCR instrument, Bio-Rad CFX96.

Contents 

• Superplex 2x Premix [Probe]
• ROX Dye (25μM) 

Application 

• Real time PCR analysis with labeled-probes
• Multiple target detection and quantification 
• FMCA (Fluorescence melting curve analysis)
• FMMA (Fluorescence multiple melting analysis)
• SNP analysis and Genotyping
• Microbial & viral pathogen detection
• Molecular diagnosis 

Quality control

RealHelix™ Superplex qPCR kit [Probe] was evaluated by real-time PCR using the 10-fold serial-diluted human genomic DNA and a set of human gene-specific primer and dual-labeled probe with ABI 7500 real-time PCR system (Applied Biosystems, USA) and CFX96™ real-time PCR detection system (Bio-Rad, USA).