DirectFast qRT-PCR Kit, a probe-based qRT-PCR kit, is designed for RNA direct real-time amplification from animal tissues, and various clinical samples (including whole blood, serum and swab collections) without any RNA purification processes. This kit allows the fast reaction to complete the qRT-PCR cycles within one hour. The Enzyme Mix in this kit is an optimized blend of Reverse Transcriptase, antibody-coupled Taq DNA polymerase, RNase inhibitor, and a heat-labile Uracil-DNA-glycosylase (HL-UDG). The Reaction Buffer contains all of the required components, including optimized buffer components, Mg2+, dUTP, and dNTPs. The applied HL-UDG/dUTP system eliminates the carryover contamination of PCR products from previous reactions. HL-UDG efficiently removes uracil residues from dU-containing DNA during the PCR mixture setup and handling.

 Figure. Direct and multiplex real-time RT-PCR using the RealHelix™ DirectFast qRT-PCR Kit.   

Swab lysate spiked with the 10⁸ copies of SARS-CoV-2 RNA were prepared following the protocol of this kit. 1μl of lysate (corresponding to approximately 10⁶ copies) and SARS-CoV-2 viral RNA(10⁶,10⁵,10⁴, 10³, 10² copies) were analyzed using three different sets of target-specific primers and probes(S gene, ORF8, and N gene). The probes were labeled with FAM(S gene), HEX(ORF8), Texas Red(N gene). The multiplex reaction was tested using the RealHelixTM DirectFast qRT-PCR Kit on a real-time PCR instrument,Bio-Rad CFX96.

Figure. Direct & Fast real-time RT-PCR using RealHelix™ DirectFast qRT-PCR Kit from mouse tissues.

The lysates from mouse whole blood or liver were prepared following the protocol of this kit. Each 1μl of lysate and purified total RNA(10, 1, 0.1, 0.01ng) were analyzed using a mouse GAPDH gene-specific primers and probe labeled with FAM on a real-time PCR instrument, Bio-Rad CFX9.