Nucleic acid gold-labeled rapid detection method and kit for pathogen

A detection method and pathogen technology, applied in the field of medical biology, can solve problems such as detection difficulties, biological safety hazards, unfavorable detection and large-scale general surveys

Active Publication Date: 2011-11-16
蓝十字生物药业(北京)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although almost all blood donation sources have to go through HBV screening, since the current screening of HBV in my country is still based on the detection of pathogen antigens, for those in the "window period" ("window period" is after the virus infection During the period until the body produces specific antibodies, because both the virus content and the titer of the antigen in the body are very low, the detection is very difficult and often missed) patients, chronic carriers of occult infection, antigen or antibody titers in the serum The accuracy is lower than the current detection level and patients infected with mutant strains, but the current detection method may cause miss

Method used

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  • Nucleic acid gold-labeled rapid detection method and kit for pathogen
  • Nucleic acid gold-labeled rapid detection method and kit for pathogen
  • Nucleic acid gold-labeled rapid detection method and kit for pathogen

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1. The design of primer, probe

[0059] Step 1. Design of HBV nucleic acid amplification primers

[0060] According to the homologous comparison of 818 gene sequences of 8 genotypes of HBVA-H, and the analysis of their inter-type and intra-type conservation and specificity, the relatively conserved S region of the gene sequence was selected as the PCR amplification region, and two pairs were designed. PCR primer, its sequence is as follows:

[0061] Outer Primer1: 5-TCACCATATTCTTGGGAACAAGA-3

[0062] Outer Primer2: 5-CGAACCACTGAACAAATGGC-3

[0063] Inner Primer1: 5-AGRTRGGAGYGGGAGCATTCGG-3

[0064] Inner Primer2: 5-GGCACTAGTAAARTGAGCCA-3 (R is A or G; Y is C or T).

[0065] The PCR amplification of the sample is carried out by the nested PCR method, which ensures the sensitivity and specificity of the PCR amplification. Step 2. Design and labeling of HBV oligonucleotide probes

[0066] According to the homologous comparison of the gene sequences of 8 ge...

Embodiment 2

[0071] Embodiment 2. preparation detection test paper

[0072] Step 1 Preparation of specific monoclonal antibodies against hapten biotin and digoxin

[0073] 1) Immunization of animals and determination of serum antibody titers

[0074] Biotin and digoxin coupled with poly-lysine were used as antigens to immunize pure-line BALB / C female mice aged 8-12 weeks, respectively, and blood was collected from the orbit 3 days after the immunization, and enzyme-linked immunosorbent assay was used to (ELISA) was used to determine the serum antibody titer to determine the immune effect. A booster immunization was performed 3 days before fusion.

[0075] 2) Take out the spleen of the effectively immunized mouse under aseptic conditions, prepare and count the spleen cell suspension. The myeloma cells SP2 / 0 with a survival rate of not less than 95% and a good growth state were used for counting. Under the fusion-promoting effect of polyethylene glycol, it promotes the fusion of splenocy...

Embodiment 4

[0123] Embodiment 4 detection of hepatitis B virus

[0124] 1) Test sample processing and preparation

[0125]20 positive serums and 10 negative control serums confirmed to contain different hepatitis B virus loads through fluorescent quantitative PCR and five detections of hepatitis B are the specificity of the inspection method of the present invention and the designed primers and probes for testing specimen verification, each Take 200 μl of each portion, and extract viral nucleic acid with a high-purity viral nucleic acid extraction kit (High Pure viral nucleic acid kit, Roche Diagnostics, Mannheim, Germany), and dissolve the extracted viral nucleic acid in 50 μl of nucleic acid-free double-distilled water at -80 Store at ℃ for later use.

[0126] 2) Nested PCR (tested PCR) amplifies the S region fragment of HBV.

[0127] 10 μl DNA template

[0128] 5μl 10×buffer

[0129] 0.4 μl 10 mM dNTPs

[0130] 10pmol primer

[0131] 1U Taq enzyme

[0132] wxya 2 O make up 50μl...

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Abstract

The invention discloses a nucleic acid gold-labeled rapid detection method and kit for a pathogen, belonging to the technical field of medical biotechnology, wherein the advantages of a colloidal gold-labeling technology and a nucleic acid PCR (Polymerase Chain Reaction) detection technology are combined; no instrument is relied except a PCR amplifier; the method and kit have an important significance to the large-scale screening and the blood product screening with the outbreak and prevalence of infectious diseases; in particular, the method and kit detect the early infection of an HBV virus, screen the potential HBV carriers in a blood donation source, ensure the safety of blood products, and realize a purpose of early, specifically, sensitively and economically detect the pathogen.

Description

technical field [0001] The invention relates to medical biotechnology, in particular to a nucleic acid gold standard rapid detection method for pathogens. Background technique [0002] Immunogold labeling technique (Immunogold labeling technique) is a solid-phase labeling immunoassay technique developed in the 1980s after the three major labeling techniques of fluorescein, radioisotope and enzyme. This technology mainly utilizes the characteristics of high electron density of gold particles. When these markers accumulate in a large amount at the corresponding ligands, red or pink spots visible to the naked eye are formed, so they are used in qualitative or semi-quantitative rapid immunoassay methods. middle. Rapid diagnostic test strips are a new type of in vitro diagnostic technology developed on the basis of monoclonal antibody technology, colloidal gold immunochromatography technology and new chromatographic materials since the 1990s. It has developed rapidly in recent y...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/576C12Q1/70C12Q1/68
Inventor 郑建张君
Owner 蓝十字生物药业(北京)有限公司
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