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Multi-cross isothermal amplification and nanogold biosensing combined nucleic acid detection technology

A constant temperature amplification and biosensor technology, which is applied in the determination/testing of microorganisms, recombinant DNA technology, biochemical equipment and methods, etc., and can solve problems such as expensive, time-consuming and labor-consuming, and poor diagnostic sensitivity

Active Publication Date: 2017-02-15
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the detection of Shigella and Salmonella mainly relies on traditional enrichment culture and biochemical identification. This method takes about 5 to 7 days, including enrichment, selective culture and subsequent biochemical identification. The disadvantage is that it is time-consuming The interpretation of biochemical results depends on human subjective judgment, resulting in poor repeatability of results and easy misjudgment
With the rapid development of nucleic acid diagnostic technology, some PCR-based diagnostic technologies (such as ordinary PCR technology, fluorescent PCR technology) are used for the rapid detection of Shigella and Salmonella, but these methods rely on expensive equipment, Requires subsequent electrophoresis, expensive probe synthesis, and skilled operators
For some laboratories with backward conditions, it cannot be carried out, thus limiting the application of these technologies
At present, the PCR method and Real-time PCR method that use these detection technologies to diagnose Salmonella and Shigella have poor diagnostic sensitivity and a long detection process, which is not conducive to rapid detection and emergency detection.

Method used

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  • Multi-cross isothermal amplification and nanogold biosensing combined nucleic acid detection technology
  • Multi-cross isothermal amplification and nanogold biosensing combined nucleic acid detection technology
  • Multi-cross isothermal amplification and nanogold biosensing combined nucleic acid detection technology

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1 standard MCDA-LFB detects

[0063] 1. Standard MCDA reaction system

[0064] The concentration of cross primers CP1 and CP2 was 60 pmol, the concentration of displacement primers F1 and F2 was 10 pmol, the concentration of amplification primers R1, R2, D1 (D1*) and D2 (D2*) was 30 pmol, and the concentration of amplification primer C1 (C1* ) and C2 (C2*) at a concentration of 20pmol, Betain of 10mM, MgSO of 6mM 4 , 1 mM dNTP, 12.5 μL of 10×Bst DNA polymerase buffer, 10 U of strand-displacing DNA polymerase, 1 μL of template, and add deionized water to 25 μL. The entire reaction was kept at 65°C for 1 hour, and the reaction was terminated at 80°C for 5 minutes.

[0065] 2. Result detection

[0066] (1) Visible color change method: MCDA produces a large amount of pyrophosphate ions while synthesizing DNA, which can capture the manganese ions combined with calcein, so that calcein returns to a free state and fluoresces. The luminescent mixture can combine ...

Embodiment 2

[0075] Embodiment 2 multiple ET-MCDA reaction system:

[0076] ET-MCDA, that is, endonuclease-mediated real-time multiple cross-displacement nucleic acid amplification technology, see CN105755134A for details, and the present invention takes this patent document as a part of the disclosure content of the present invention by citing it.

[0077] The concentration of cross primers Sal-CP1 and Sal-E-CP1 is 30pmol, the concentration of Sal-CP2 is 60pmol, the concentration of displacement primers Sal-F1 and Sal-F2 is 10pmol, and the concentration of amplification primers Sal-R1, Sal-R2, Sal -The concentration of D1 and Sal-D2 is 30pmol, the concentration of amplification primers Sal-C1 and Sal-C2 is 20pmol, the concentration of cross primer Shi-CP1 and Shi-E-CP1 is 8pmol, and the concentration of Shi-CP2 is 16pmol, The concentration of displacement primers Shi-F1 and Shi-F2 is 10pmol, the concentration of amplification primers Shi-R1, Shi-R2, Shi-D1 and Shi-D2 is 8pmol, and the con...

Embodiment 3

[0079] Embodiment 3 multiple MCDA reaction system:

[0080] 1. Multiple MCDA reaction system

[0081] The concentration of cross primer Sal-CP1 and Sal-CP2 is 60pmol, the concentration of displacement primer Sal-F1 and Sal-F2 is 10pmol, and the concentration of amplification primer Sal-R1, Sal-R2, Sal-D1* and Sal-D2 is 30pmol, the concentration of amplification primers Sal-C1* and Sal-C2 is 20pmol, the concentration of cross primer Shi-CP1 and Shi-CP2 is 16pmol, the concentration of displacement primer Shi-F1 and Shi-F2 is 10pmol, the concentration of amplification primer Shi -The concentration of R1, Shi-R2, Shi-D1 and Shi-D2* is 8pmol, the concentration of amplification primers Shi-C1 and Shi-C2* is 10pmol, 10mM Betain, 6mM MgSO 4 , 1 mM dNTP, 12.5 μL of 10×Bst DNA polymerase buffer, 10 U of strand displacement DNA polymerase, 1 μL of templates for Salmonella and Shigella, and added deionized water to 25 μL. The entire reaction was kept at 65°C for 1 hour, and the reaction...

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Abstract

The invention discloses a multi-cross isothermal amplification and gold nanogold biosensing combined nucleic acid detection technology. A biotin is marked at a 5'-end of an amplification primer C1 in multi-cross replacement isothermal amplification, and a hapten is marked at a 5'-end of D1, so that a double-mark product can be generated and can be visually detected by a nanogold biosensor. The technology is convenient, quick, sensitive and specific and is suitable for detection of various nucleotide fragments and especially can be used for simultaneous detection of various target gene fragments.

Description

technical field [0001] The invention discloses a nucleic acid amplification technology and its application, belonging to the fields of microbiology 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, biological research, clinical diagnosis, epidemiological research, archaeological research, transgenic research and other fields. Among the established nucleic acid amplification technologies, polymerase chain reaction (PCR) is the first established in vitro nucleic acid amplification technology, which has epoch-making significance and has been widely used in biology, medicine and other 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 complicated and requires a se...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/10C12N15/11
CPCC12Q1/6804C12Q1/689C12Q2600/16C12Q2537/1376C12Q2563/131C12Q2565/607C12Q2537/143
Inventor 叶长芸王毅王艳
Owner ICDC CHINA CDC
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