Visual nucleic acid detection method for simultaneously detecting one or plurality of kinds of target nucleic acids and application of visual nucleic acid detection method

A technology of target nucleic acid and detection method, applied in the field of molecular biology

Active Publication Date: 2021-06-04
THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] Obviously, the detection methods based on EXPAR in the prior art are all limited by the

Method used

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  • Visual nucleic acid detection method for simultaneously detecting one or plurality of kinds of target nucleic acids and application of visual nucleic acid detection method
  • Visual nucleic acid detection method for simultaneously detecting one or plurality of kinds of target nucleic acids and application of visual nucleic acid detection method
  • Visual nucleic acid detection method for simultaneously detecting one or plurality of kinds of target nucleic acids and application of visual nucleic acid detection method

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Experimental program
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Embodiment 1

[0077] Embodiment 1: experimental material and method steps of the present invention

[0078] The materials and primer sequences used in the present invention are shown in Table 1 and Table 2; wherein, the DNA and RNA were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0079] Among them, Bst DNA polymerase (M0275S), Nb.BbvCI (R0631S), Nb.BtsI (R0707S), 10Xbuffer 1.1 (100mM Bis-Tris-HCl pH7, 100mM MgCl 2 , 1mg / mL BSA) etc. were purchased from NEB. HybriDetect test strip detection kit (MGHD 1, MGHD 2) was purchased from Milenia Biotec GmbH (Germany).

[0080] Primers and sequences used in Table 1

[0081]

[0082] *Represents thio modification

[0083] combine figure 1 and figure 2 Described in detail the exemplary experimental steps of the present invention, specifically as follows:

[0084] 1. The primers are dissolved in DEPC water, and then the OD260 value is measured by nanodrop, and the primers are calculated according to the formula A=εbc (A is th...

Embodiment 2

[0094] Embodiment 2: method principle verification of the present invention

[0095] according to figure 1 Principle, the template DNA is Pre-TrigTel, as shown in SEQ ID NO: 1, wherein the 3' end sequence is: CTAACCCTAACCCTAACCCTAA. When configuring the reaction sample, add the following components in order:

[0096] Table 4 Reagents in tube A reaction system

[0097] Added reagent μL / reaction 10X Buffer 1.1 0.5 100nM TrigTel 1 10 μM MB-TrigTel 1 2.5mM dNTPs 1 100nM TS-3 1

[0098] Table 5 Reagents in tube B reaction system

[0099] Added reagent μL / reaction 10X Buffer 1.1 0.5 10U / μL Nb.BbvCI 0.25 8U / μL Bst DNA polymerase 0.125 10U / μL Nb.BtsI 0.25 500ng / μL ET SSB 0.1 water 4.275

[0100] The total volume of each reaction is 10 μL, and the sample is kept at about 4 °C during the preparation of the sample. In order to verify the principle of the scheme of the present inventio...

Embodiment 3

[0101] Example 3: Detection of telomerase extension products using the method of the present invention

[0102] according to figure 1 Principle, the template DNA is Pre-TrigTel, as shown in SEQ ID NO: 1, wherein the 3' end sequence is: CTAACCCTAACCCTAACCCTAA. When configuring the reaction sample, add the following components in order:

[0103] Table 6 Reagents in tube A reaction system

[0104] Added reagent μL / reaction 10X Buffer 1.1 0.5 100nM TrigTel 1 10 μM MB-TrigTel 1 2.5mM dNTPs 1 TS / TS-1 / TS-2 / TS-3 / TS-4 1

[0105] Table 7 Reagents in tube B reaction system

[0106] Added reagent μL / reaction 10X Buffer 1.1 0.5 10U / μL Nb.BbvCI 0.25 8U / μL Bst DNA polymerase 0.125 10U / μL Nb.BtsI 0.25 500ng / μL ET SSB 0.1 water 4.275

[0107] The total volume of each reaction is 10 μL, and the sample is kept at about 4 °C during the preparation of the sample. In the test for the detection o...

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Abstract

The invention provides a visual nucleic acid detection method for simultaneously detecting one or a plurality of kinds of target nucleic acids. The method comprises the following steps of 1) preparing a reaction mixture which comprises one or a plurality of kinds of template DNA, DNA polymerase, DNA nicking enzyme 1, DNA nicking enzyme 2 and one or a plurality of kinds of probes; 2) putting the reaction mixture obtained in the step 1) at a constant temperature, and performing a constant-temperature index amplification reaction; 3) preparing a colloidal gold detection buffer solution; and 4) mixing the reaction product in the step 2) with the buffer solution in the step 3), putting a colloidal gold test strip in the solution for detection, and interpreting the result according to a detection strip. Compared with the prior art, the method provided by the invention has the following advantages that the method provided by the invention uses the test strip for visual reading, the operation is simple, the requirements on professional skills is low, special technical training is not needed, the application range is greatly expanded, and the detection cost is greatly reduced.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a visual nucleic acid detection method for simultaneously detecting one or more target nucleic acids and an application thereof. Background technique [0002] With the rapid development of molecular biology technology, nucleic acid has become an important biomarker in biological research and disease diagnosis, and a large number of analytical methods based on nucleic acid detection have been established. In vitro nucleic acid amplification is an important link in nucleic acid analysis, and it is also the guarantee of important technical parameters such as method sensitivity and specificity. [0003] Polymerase Chain Reaction (PCR) is currently the most common nucleic acid amplification technology, and has become a basic research method in the fields of molecular biology, genomics, and disease diagnosis. The PCR reaction process includes three stages of denaturation, annealing and ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2531/119C12Q2537/1376C12Q2525/207C12Q2521/113C12Q2521/301C12Q2565/625C12Q2563/107
Inventor 李培峰李新敏王胤
Owner THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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