PCR enhancer composition, droplet-type reverse transcription digital PCR solution and application

Abstract

The invention provides a PCR enhancer composition. The PCR enhancer composition comprises any one of T4 phage gene 32 coding protein, trehalose and nonidet P-40. The invention further provides a droplet-type reverse transcription digital PCR solution which is characterized by comprising the PCR enhancer composition. The PCR enhancer composition comprises the T4 phage gene 32 coding protein, the trehalose and the nonidet P-40, and preferably, the PCR solution contains 0.05-1 mg / mL T4 phage gene 32 coding protein, 0.1-2 mol / L trehalose and 0.02-1% w / v nonidet P-40. The invention also relates toa kit containing the PCR solution and a method for a droplet-type reverse transcription digital PCR by using the PCR solution. The PCR solution can be used for improving the amplification efficiency of the positive droplet-type reverse transcription digital PCR, generation of false negative is reduced, and accurate quantitative analysis of nucleic acid target molecules is facilitated.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a droplet type reverse transcription digital PCR reaction solution and application thereof. Background technique [0002] Droplet reverse transcription digital PCR (reverse transcription droplet digital polymerase chain reaction, RT-ddPCR) is a method for absolute quantification of target ribonucleic acid (RNA). Compared with quantitative RT-PCR (quantitative RT-PCR, qRT-PCR), it has unparalleled advantages in precision, accuracy and sensitivity; at the same time, RT-ddPCR eliminates the dependence on quantitative standard curves and improves Tolerance to expansion inhibitors. [0003] The working principle of RT-ddPCR is to microdropletize the reaction solution before RT-PCR amplification, that is, to disperse the reaction solution containing template RNA into tens of thousands of nanoliter droplets, and each droplet does not contain or contain one to Sev...

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Problems solved by technology

However, the interfacial effect of nanoliter droplets is significantly enhanced, resulting in hydrophobic reverse transcriptase and DNA polymerase tending to adsorb at the oil-water interface, and protein denaturation may occur at the oil-water interface (Williams R. et al. Amplification of complex gene libraries by emulsionPCR.Nature methods, 2006, 3(7):545-550)

The interface adsorption of reverse transcriptase and DNA polymerase reduces the amplification efficiency of RT-ddPCR, resulting in no fluorescence signal from the microdroplets containing the template RNA, resulting in errors in the absolute quantification of nucleic acid target molecules

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