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Method of multi-gene absolute quantification for PCR array

An absolute quantitative and multi-gene technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve problems such as adverse effects of experimental process testing costs, and achieve the effect of avoiding mutual interference

Pending Publication Date: 2019-05-14
CRACKERBIO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, if you want to use qPCR technology to measure in a 96-well plate, you must put the standard and the sample to be tested in different holes to avoid mutual interference. Therefore, a considerable number of holes must be used. Adverse effects on experimental process and test cost

Method used

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  • Method of multi-gene absolute quantification for PCR array
  • Method of multi-gene absolute quantification for PCR array
  • Method of multi-gene absolute quantification for PCR array

Examples

Experimental program
Comparison scheme
Effect test

experiment example

[0024] Reaction condition test

[0025] Use 5 standard product DNA and its primer set to test the reaction conditions to get the best reaction conditions. pass Green’s fluorescent dyes are used with test carriers with multiple reaction wells for qPCR. The optimal reaction conditions obtained are as follows, which can achieve a PCR efficiency of about 90% to 100%:

[0026] Primer concentration: 0.5μm

[0027] Reaction temperature and time: 95°C for 44 seconds, 60°C for 88 seconds

[0028] Number of reaction cycles: 40 cycles

[0029] Standard DNA Quantification

[0030] The 5 standard DNA storage solutions (stock) (10ng / μl) were serially diluted, and then Green’s fluorescent dye is paired with a test carrier with multiple reaction wells to perform qPCR and digital PCR (digital PCR, dPCR) respectively (125 holes / group, each group has four replicates) to calculate the concentration of 5 standard DNA (copy number / μl). The standard curve and R 2 , The DNA concentration (c...

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Abstract

A method of multi-gene absolute quantification for PCR array is provided, wherein a nucleic acid sample to be tested includes at least one kind of nucleic acid target. Primers for amplifying a plurality of standard DNA or the nucleic acid target to be tested are respectively disposed in a plurality of reaction wells of a test carrier. Afterwards, the plurality of standard DNA with known copy number and the nucleic acid sample are mixed and added into the reaction wells, and qPCR is performed on the standard DNA and the nucleic acid sample. A sequence-specific combination of a forward primer and a reverse primer is designed for each standard DNA, and the same DNA template sequence is presented between regions corresponding to the forward primer and the reverse primer. The primers for amplifying standard DNA and the nucleic acid target to be tested have similar amplification efficiencies.

Description

technical field [0001] The invention relates to an absolute quantitative method, in particular to a multi-gene absolute quantitative method for PCR array. Background technique [0002] With the development of modern biomedical technology and clinical needs, many diagnostic methods require absolute quantification of target genes. For example, monitoring of viral load and course of treatment for infections such as HIV or hepatitis C (HCV), or monitoring of viral load in organ transplant patients. In the prior art, if you want to use qPCR technology to measure in a 96-well plate, you must put the standard and the sample to be tested in different holes to avoid mutual interference. Therefore, a considerable number of holes must be used. The experimental process and the test cost have adverse effects. [0003] Based on the above, for the test carrier with multiple reaction wells, how to improve the smoothness of the experiment and reduce the test cost when using qPCR technology...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851
CPCC12Q1/6851C12Q2537/143C12Q2545/101
Inventor 李铭洲郑尤琇
Owner CRACKERBIO INC