A Nested PCR Digital Quantification Method

Abstract

The invention provides a nested PCR digital quantitative method which is characterized in that the method is an improved digital PCR method of dispersing nested PCR and a sample to limited units by 10-fold diluting gradient. The method comprises the following steps: adding mineral oil into each reaction unit in advance; adding 1-5 microliters of a small sized first round PCR mixed liquid under the mineral oil, wherein the mixed liquid comprises an isometric 2* reaction liquid and a sample liquid with continuous 10-fold diluting gradient, and carrying out first round amplification by using an outer primer at a first annealing temperature; then, adding a second round PCR reaction liquid which is 10-20 times of the first round PCR volume to the first round amplification product, and carrying out second amplification by using an inner primer at a second annealing temperature; and finally, detecting amplification products for counting and quantifying. The invention further provides a gene detection kit for the nested PCR digital quantitative method.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid amplification in the field of molecular biology and molecular testing, and specifically relates to the field of digital amplification quantitative method for performing 10-fold dilution gradient of samples by improving nested PCR operation to eliminate non-specific amplification of primers. Background technique [0002] The so-called digital quantitative detection or digital diagnosis refers to diluting and dispersing the sample to a large number of micro-separated units under the condition that the detection signal of a single positive molecule or more than one molecule is determined as 1, and the negative signal of absolutely no reaction is determined as 0. Each detection unit contains 0-1 target molecule, and the target molecule is quantified by counting the reaction signal 1 of the detection unit, so as to facilitate the automatic operation of the computer in 0 or 1 mode, but the premise m...

AI Technical Summary

Problems solved by technology

Due to the large content span of the target molecule in the sample, the low concentration is less than 10 o =1copy / ml, the concentration is higher than 10 1o Copies / ml, concentrated samples often require millions of reaction units, that is, picoliter volume microfluidic or microdrop devices, to be able to distribute less than or equal to one nucleic acid template per reaction unit. This pursuit of the ultimate response unit will lead to Application popularization is limited

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