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Novel biological bar code detection method and application thereof

A technology of biological barcode and detection method, which is applied in the simple biological barcode detection method and its application field in biological detection, which can solve the problems of easy cross-contamination, cumbersome operation, false positive and other problems of terminal PCR, and achieve the elimination of unclear boundaries between negative and positive , low equipment requirements, and the effect of simplifying the testing procedures

Inactive Publication Date: 2011-03-16
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] However, it can be seen from the detection process and display system of BCA technology that this technology has obvious defects
In the PCR reaction, due to factors such as the aggregation of templates, reagents, and pyrophosphate molecules, the polymerase reaction is affected, and eventually the PCR reaction no longer proceeds exponentially and enters a "plateau phase", and some reactions have more end products than others , so the quantification of the endpoint PCR reaction method is inaccurate; secondly, the endpoint PCR is prone to cross-contamination, resulting in false positives
The DNA marked by traditional BCA technology is double-stranded, with a length of about 48bp. Not only is the operation cumbersome and the detection process is lengthy, but it also indirectly affects the detection sensitivity. In addition, it is not suitable for real-time fluorescent quantitative PCR detection based on TaqMan probes. Sample templates for precise quantitative analysis
The chip detection of barcode DNA is prone to false positives due to the shortcomings of silver staining reagents, and it is not easy to define negative and positive, and the staining results vary greatly due to time and environmental factors

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  • Novel biological bar code detection method and application thereof
  • Novel biological bar code detection method and application thereof
  • Novel biological bar code detection method and application thereof

Examples

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Effect test

Embodiment 1

[0033] Embodiment 1, the novel biological barcode (BCA) detection of ricin

[0034] This embodiment takes the qualitative and quantitative detection of ricin protein as an example to illustrate how the novel BCA detection method of the present invention is implemented, which specifically includes the following steps:

[0035] (1) Preparation of NP probes

[0036] Barcode DNA chain synthesis: design and synthesize the barcode DNA chain used in the preparation of NP probes in the present invention, its sequence is: 5'-GTG ACT GCA AGT CCA TTG AGG TGA AAT AGC CAG TTC TCG TGG CAT TTGCGT AAT CCT GAT GTA TCG CTC GGT GGT TGC CAA CTA GAG AAC-3' (SEQ ID No: 1 in the sequence listing).

[0037] Preparation of NP probe: Take 10.5 μg polyclonal antibody of the pathogen to be tested (purchased from Novison Biotechnology Co., Ltd.) and add it to 100 μL nano-gold (15nm, 10-40nm is acceptable) solution and mix evenly, then adjust with 0.1M NaOH pH value to 9.5; shake at room temperature for ...

Embodiment 2

[0059] Embodiment 2, the novel biological barcode (BCA) detection of bluetongue virus (BTV)

[0060] Carry out qualitative and quantitative detection to bluetongue virus BTV VP7 protein with novel BCA detection method of the present invention, concrete method comprises the following steps:

[0061] (1) Preparation of NP probes

[0062] Preparation of polyclonal antibody against BTV VP7 protein: Use bluetongue disease virus to immunize New Zealand white rabbits to prepare polyclonal antibody against VP7 protein. The specific methods include the following: before immunization, each male New Zealand white rabbit takes blood from the ear vein 2mL, let stand to separate the serum, store it at -20℃, and use it as a negative control; immunization scheme: for the first immunization, each rabbit is injected with 2mL of emulsified antigen, around the lymph nodes in the paw and cubital fossa, on both sides of the back, under the collar , behind the ear, etc., and the control rabbit was ...

Embodiment 3

[0089] Embodiment 3, the preparation of novel BCA detection kit

[0090] The barcode DNA strands, upstream primers, downstream primers, TaqMan fluorescent probes, gold nanoparticles and magnetic microspheres in the examples are packaged together to obtain a novel BCA detection kit.

[0091]

[0092]

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Abstract

The invention discloses a biological bar code detection method and application thereof. In the biological bar code detection method, a deoxyribonucleic acid (DNA) chain of a marked bar code is improved into a single chain based on the ordinary biological bar code detection method, the length of the DNA chain is increased to a length which is suitable for performing fluorescent quantitation polymerase chain reaction (PCR) / TaqMan fluorescent probe detection, and the DNA chain of a bar code in a sandwich reaction compound does not need dissociating and is directly detected qualitatively and quantitatively by a real-time fluorescent quantitation PCR method or the ordinary PCR method based on a TaqMan fluorescent probe. The method lowers detection cost, simplifies detection processes, can accurately quantify a tested object and has the advantages of high accuracy, high detection speed, low instrument and equipment requirements, low cost and the like. The method plays a greatly practical role in performing high-sensitivity detection on various target proteins and has wide application prospect.

Description

technical field [0001] The invention belongs to a detection method and its application in the field of biotechnology, in particular to a novel simple biological barcode detection method and its application in biological detection. Background technique [0002] Bio-bar codes assay (BCA) is a new type of marker immunoassay technology first reported in 2003 by the research group led by Professor Mirkin of Northwestern University in the United States. 10 of the ELISA method 6 times (Nam JM, Thaxton CS, Mirkin CA. Nano-particle-based bio-bar codes for the ultrasensitive detection of proteins. Science, 2003, 301(5641): 1884-1886). [0003] The basic principle of BCA technology is to use the magnetic microsphere probes (magnetic micropar-ticles, MMPs) labeled with the monoclonal antibody of the test object and the gold nanoparticle probe (nanoparticle, NP) labeled with the polyclonal antibody of the test object and specific DNA double strands. , through the formation of MMP-analy...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/68C12Q1/68
Inventor 尹惠琼章金刚贾茗娴杨姝
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY