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AUDG (antarctic thermal sensitive uracil deoxyribonucleic acid glycosylase) mediated multiple cross displacement amplification and biosensing combined nucleic acid testing technique

A biosensing and biosensor technology, applied in the field of microorganisms and molecular biology, can solve problems such as cross-contamination, and achieve the effects of eliminating pollutants, high sensitivity, and convenient methods

Active Publication Date: 2017-09-15
ICDC CHINA CDC
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, in order to apply the amplified product to the detection of biosensing technology, opening the reaction tube is a necessary step, which will cause a large amount of amplified product to volatilize in the form of aerosol, resulting in cross-contamination

Method used

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  • AUDG (antarctic thermal sensitive uracil deoxyribonucleic acid glycosylase) mediated multiple cross displacement amplification and biosensing combined nucleic acid testing technique
  • AUDG (antarctic thermal sensitive uracil deoxyribonucleic acid glycosylase) mediated multiple cross displacement amplification and biosensing combined nucleic acid testing technique
  • AUDG (antarctic thermal sensitive uracil deoxyribonucleic acid glycosylase) mediated multiple cross displacement amplification and biosensing combined nucleic acid testing technique

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Experimental program
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Effect test

Embodiment 1

[0060] Embodiment 1.MCDA amplification

[0061] 1. Construction of detectable products by MCDA amplification

[0062] The MCDA reaction system includes 10 primers, which recognize 10 regions of the target sequence, including 2 internal cross primers, namely CP1 and CP2 (Cross Primer, CP), 2 displacement primers (Displacement Primer), namely F1 and F2, and 6 Amplification primers, namely D1, C1, R1, D2, C2 and R2. In order to construct a detectable product, select any one of the 10 primers, label the hapten (fluorescein or digoxin) at the 5' end, and the newly labeled primers are named F1*, F2*, CP1*, CP2*, C1*, C2*, D1*, D2*, R1* and R2*. In the present invention, CP1* is taken as an example to illustrate the principle of the present invention.

[0063] Under the given constant temperature conditions, the double-stranded DNA is in a dynamic equilibrium state of half-dissociation and half-binding. When any primer performs base pairing extension to the complementary part of t...

Embodiment 2

[0073] Embodiment 2. measure the optimal reaction temperature of MCDA technology

[0074] Under standard reaction system conditions, add the corresponding MCDA primers designed for HPV16 and HPV18 DNA templates, and the template concentration is 5×10 4 copies / µl. The reaction was carried out under constant temperature conditions (60-67°C), and the results were detected by a real-time turbidimeter, and different dynamic curves were obtained at different temperatures, see Figure 7 with Figure 8 . 61-64°C is recommended as the optimal reaction temperature for the two sets of MCDA primers. In the follow-up verification of the present invention, 63° C. was selected as the constant temperature condition for MCDA amplification. Figure 7 Represents the temperature dynamic curve of the MCDA primers designed for the detection of HPV16 against the E7 gene; Figure 8 It shows the temperature dynamic curve of the MCDA primer designed for detecting HPV18 aimed at the L1 gene.

Embodiment 3

[0075] Embodiment 3.AUDG-MCDA-LFB detects the sensitivity of single target

[0076] Use the serially diluted plasmid (pMD18-T-HPV16: 5×10 5 , 5×10 4 , 5×10 3 , 5×10 2 , 5×10 1, 5×10 0 and 5×10 -1 copy / microliter) after performing standard MCDA amplification reaction, the result is displayed by LFB detection. For HPV16 detection, the detection range of MCDA-LFB is 5×10 5 ~5×10 0 copies / microliter, LFB appears red lines in TL1 and CL regions ( Figure 9 1-6 in A). When the amount of genome template in the reaction system is reduced to 5×10 0 When copying below, LFB only appears a red line in the CL area, indicating a negative result ( Figure 9 7-8 in A). Figure 9 A is to use LFB to visualize and read the MCDA amplification results: Figure 9 1 to 6 in A indicate that the template amount of pMD18-T-HPV16 is 5×10 5 , 5×10 4 , 5×10 3 , 5×10 2 , 5×10 1 , 5×10 0 copies / µl, Figure 9 7-8 in A respectively represent that the template amount of pMD18-T-HPV16 is 5×1...

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Abstract

The invention discloses a multiple cross isothermal amplification and macromolecular nano biosensing combined target gene testing method. According to the method, a half antigen is marked at a 5' terminal of a cross primer CP1 or CP2 in multiple cross displacement amplification, and antarctic thermal sensitive uracil deoxyribonucleic acid glycosylase and biotinylated deoxyuridine are introduced into an amplification system to test amplification products on the basis of multiple cross displacement amplification combined with macromolecular nano biosensing. According to the method, amplification products of HPV16 type E7 gene or HPV18 type L1 gene can be visually tested by a macromolecular nano biosensor. The method is convenient, fast, sensitive, specific and suitable for testing of various nucleotide fragments.

Description

technical field [0001] The invention discloses a method for AUDG-mediated multi-crossover displacement amplification combined with biosensing to detect microbial target genes, and belongs to the technical field of microorganisms and molecular biology. Background technique [0002] In the field of modern medicine and biology, nucleic acid amplification is an indispensable technology, widely used in basic research, clinical diagnosis, archaeological research, epidemiological research, transgenic research and other fields. Among the nucleic acid amplification technologies that have been developed, PCR is the first established in vitro nucleic acid amplification technology, which has epoch-making significance and has been widely used in biological related fields. However, when PCR is used for nucleic acid amplification, it is limited by laboratory conditions and relies on complex and expensive thermocycling instruments. In addition, the detection of PCR products is relatively c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q2531/119C12Q2537/143C12Q2537/1376C12Q2565/607
Inventor 叶长芸王毅王艳
Owner ICDC CHINA CDC
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