Polymerase chain reaction reagent and preserving method for polymerase chain reaction reagent based on porous material

A polymerase chain and preservation method technology, applied in the field of preservation of biochemical reagent compositions, can solve the problems of large energy consumption and inability to use PCR reagents for long-term storage at room temperature, and achieve the effects of reduced storage and transportation costs, low cost and simple process

Inactive Publication Date: 2013-10-23
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The invention patent "preservation method of polymerase chain reaction mixture (ZL 0011250.4)" discloses a method of using a nucleic acid vacuum concentrator to dry the PCR reaction reagent mixture at low temperature to achieve long-term preservation of the PCR reaction reagent mixture, but this method It requires complex equipment such as special nucleic acid vacuum concentrators or vacuum freeze-dryers, and consumes a lot of energy, which is not a very economical method
The invention patent "PCR and RT-PCR full premix reagents containing high concentration of glycerol (ZL 03117011.0)" discloses a method for long-term storage of PCR reagents at low temperature by adding 30-65% (v / v) glycerol, but the method Cannot be used for long-term storage of PCR reagents at room temperature

Method used

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  • Polymerase chain reaction reagent and preserving method for polymerase chain reaction reagent based on porous material
  • Polymerase chain reaction reagent and preserving method for polymerase chain reaction reagent based on porous material
  • Polymerase chain reaction reagent and preserving method for polymerase chain reaction reagent based on porous material

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Analysis of the preservation effect of the PCR reagent mixture after adding porous materials

[0031] 1. Add different concentrations and different types of porous materials to the PCR reaction reagent mixture for preservation

[0032] Mix 22.896mL of the mixture of various PCR reaction reagents except the nucleic acid template (including PCR primer F and primer R 0.4μM each, dATP, dTTP, dGTP and dCTP each 1.4mM, KCl 50mM, Tris / HCl (pH 9.0) 10mM, MgCl 21.5mM, 1%Triton X-100, Taq DNA polymerase 1147.5U) divided into 53 1.5mL centrifuge tubes according to 432μL per tube, according to columns 1, 2 and 3 of Table 1 (a total of 53 rows, including Add porous material to the blank control) at the indicated concentration and type, add porous material to each 1.5mL centrifuge tube and mix well (for the porous material with a lower added amount, it can be pre-prepared to a high concentration of 200 times the final added amount Then shake the suspension and draw it wit...

Embodiment 3

[0052] Example 3 Analysis of the Influence of Porous Material Immobilized Taq DNA Polymerase on PCR Amplification Effect After Long-term Storage

[0053] 1. Add different concentrations and different types of porous materials to immobilize and store Taq DNA polymerase

[0054] Add 30 μL Taq DNA polymerase (1 U / μL) to 51 centrifuge tubes containing 30 μL Taq DNA polymerase (1 U / μL) at the concentration and species shown in columns 1, 2 and 3 of Table 3 (51 rows in total, including the blank control without porous material). Add the porous material to the mixture and mix well (for the porous material with low addition amount, it can be pre-prepared into a high-concentration premix solution 200 times the final addition amount, then shake it to suspend it and draw it with a pipette gun), and then add each 1.5mL The mixture containing Taq DNA polymerase in the centrifuge tube is divided into 18 0.2mL centrifuge tubes according to the amount of 1.25μL, and then each group of 18 0.2m...

Embodiment 4

[0074] Example 4PCR Reaction Kit In the reaction reagent mixture, the preservation effect analysis after the porous material is added

[0075] 1. Assemble the following components into the box of the kit:

[0076] (1) Amplification detection tube, filled with PCR reaction reagent mixture;

[0077] (2) Negative control tube, containing FTA membrane without WSSV nucleic acid;

[0078] (3) Positive control tube, which contains the FTA membrane adsorbed with WSSV nucleic acid;

[0079] The above-mentioned FTA film is a 3 mm square paper piece cut from the sample area on the FTA card of Whatman, UK.

[0080] 2. Add different concentrations and different types of porous materials to the PCR reaction reagent mixture for preservation

[0081] Mix 22.032mL of the mixture of various PCR reaction reagents except the nucleic acid template (including PCR primer F and primer R 0.4μM each, dATP, dTTP, dGTP and dCTP each 1.4mM, KCl 50mM, Tris / HCl (pH 9.0) 10mM, MgCl21 .5mM, 1%Triton X-100...

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Abstract

The invention relates to a polymerase chain reaction reagent and a preserving method for the polymerase chain reaction reagent based on a porous material. According to the preserving method, the porous material is added in components of one or more reaction liquid of a polymerase chain reaction for preserving; the components of the polymerase chain reaction liquid, which are treated by the method disclosed by the invention, can be stored and transported at normal temperature; and the components of the polymerase chain reaction liquid can be directly used without being special treated. The preserving method disclosed by the invention has the characteristics of simple process and low cost. A polymerase chain reaction reagent mixture treated by the preserving method disclosed by the invention has the advantage of stable and durable stability under the convenient storage condition, so that great convenience for actual operation of a polymerase chain reaction is brought while the storage cost of the polymerase chain reaction reagent is greatly reduced. According to the preserving method and the polymerase chain reaction reagent disclosed by the invention, the application of the polymerase chain reaction in the fields of scientific research, medical treatment, check, quarantine and the like and large-scale popularization and application of diagnosing or detecting reagent products based on the technology are bound to be promoted.

Description

technical field [0001] The invention belongs to the technical field of preservation methods for biochemical reagent compositions, and in particular relates to a method for preserving polymerase chain reaction reagents and reaction reagents based on porous materials, specifically a method for preserving polymerase chain reaction reagents and reagent mixtures at room temperature in molecular biology , and the reaction reagents prepared by the method that can be preserved and transported at room temperature. Background technique [0002] In the past 20 years, nucleic acid amplification technology represented by polymerase chain reaction (PCR) has been rapidly developed and applied. Due to the convenience and speed of PCR technology, which can amplify a large number of target nucleic acid fragments in a short period of time, this technology has been widely used in the fields of gene cloning and recombinant expression, biological species identification and classification, species...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 张庆利黄倢李美霞杨冰王崇明许华
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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