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Composite murine hepatitis virus detection kit, detection method and application

A technology for detecting kits and hepatitis viruses, which is applied in the field of compound detection kits for rat hepatitis viruses, and can solve problems such as the inability to uniformly detect false positive rates

Active Publication Date: 2014-12-24
高忠翔
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is aimed at the above-mentioned defects of the prior art, and provides a kind of method that overcomes the problems such as the inability to uniformly detect and the high false positive rate in a single polymerase chain reaction (PCR) method, and has a high degree of specificity. , sensitivity and accuracy, and a compound detection kit for murine hepatitis virus with easy technical operation

Method used

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  • Composite murine hepatitis virus detection kit, detection method and application
  • Composite murine hepatitis virus detection kit, detection method and application
  • Composite murine hepatitis virus detection kit, detection method and application

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Experimental program
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preparation example Construction

[0094] 3. Preparation of antigenic protein S2:

[0095] (1) Design the antigenic protein construction scheme:

[0096] The antigenic protein used in the present invention is that we obtained 6 main subtypes (MHV-1, MHV-2, MHV-3, JHM, A59 and S) of murine hepatitis virus prevalent at present by retrieval and analysis of the National Biotechnology Information Center of the United States Commonly unique to the stable domain S2 region of structural protein S (gi|298199707). The sequence of the S2 antigen protein is shown as SEQ ID NO.4 in the sequence listing, and the theoretical protein size is 50.4 kDa. The nucleic acid sequence corresponding to the S2 antigen protein sequence is shown in SEQ ID NO.5 in the sequence listing, and the gene size is 1383bp. Primers were designed according to the nucleic acid sequence of the antigenic protein S2. The upstream primer S2-f contained a NcoI restriction endonuclease site, and the sequence was shown in SEQ ID NO.6, 5′-CATGCCATGGCATGGATG...

Embodiment 1

[0115] Embodiment 1: Using the composite kit of the present invention to quickly and accurately detect murine hepatitis virus in mouse blood

[0116] Using mouse blood as the sample to be tested, one-step RT-PCR, rapid indirect ELISA and rapid double-antibody sandwich ELISA were used to detect the presence of murine hepatitis virus and the antibodies produced by the virus in experimental mice Condition. Blood samples were collected from one chronically MHV-infected ICR mouse and two newly infected mice with normal immune function in experimental group 1, one chronically MHV-infected ICR mouse and two newly infected mice in experimental group 2 SCID mice and 2 negative virus-free mice in the control group.

[0117] (1) Sample processing: Collect 5-10 μl of blood from the tail or back vein of each mouse into 50 μl of PBS solution containing 5-10 units of heparin (anticoagulant) and 1 unit / ml DNase I.

[0118] (2) One-step RT-PCR detection of mouse blood samples:

[0119] Reag...

Embodiment 2

[0153] Embodiment 2: Using the composite kit of the present invention to quickly and accurately detect murine hepatitis virus in mouse saliva

[0154] Using mouse saliva as the test sample, one-step RT-PCR and rapid indirect ELISA were used to detect the presence of murine hepatitis virus. Saliva samples were collected from one chronically MHV-infected ICR mouse and two newly infected mice with normal immune function in experimental group 1, one chronically MHV-infected ICR mouse and two newly infected mice in experimental group 2, respectively. SCID mice and 2 negative virus-free mice in the control group.

[0155] (1) Sample treatment: 10 μl of saliva from the above-mentioned mice were collected respectively into 50 μl of PBS solution containing 1 unit / ml DNase I.

[0156] (2) One-step RT-PCR detection of mouse saliva samples:

[0157] Sample number: positive control (No. 11), negative control (No. 12), saliva of 1 chronic MHV-infected ICR mouse in experimental group 1 (No...

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Abstract

The invention discloses a composite murine hepatitis virus detection kit and a composite murine hepatitis virus detection method. Common specific nucleic acid constant regions of main subtypes of murine hepatitis viruses are selected for designing specific nucleic acid probes; gene sequences of stable regions S2 of common specific structural proteins S of six main subtypes (MHV-1, MHV-2, MHV-3, JHM, A59 and S) of the murine hepatitis viruses are selected for designing primers; nucleic acids of the murine hepatitis viruses are detected by a one-step reverse transcription polymerase chain reaction technique; through gene cloning and expression, S2 proteins of the murine hepatitis viruses are biologically synthesized; specific antigens are used for screening a phage antibody library to obtain corresponding monoclonal antibodies; on the basis, antigens of the murine hepatitis viruses and self-generated antibodies of mice are detected by an enzyme-linked immunosorbent technique. The composite murine hepatitis virus detection kit and the composite murine hepatitis virus detection method have the characteristics of accurate detection result, high sensitivity, high specificity, low cost, simple and convenient in operation and the like.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a compound detection kit and detection method for murine hepatitis virus. Background technique [0002] Mouse hepatitis virus (MHV, also known as Murinecoronavirus academically) belongs to the Coronaviridae family and is a ribonucleic acid (RNA) positive virus. The nucleic acid is composed of single-stranded RNA, and its size varies with different animal sources. The general diameter is about 80nm-160nm, and there are many strain types. According to the different tissue-tropic characteristics of murine hepatitis virus, it can be divided into two types: respiratory strain and enterotropic strain. Respiratory strain viruses can spread to various target organs after replicating in nasal epithelial cells of susceptible animals. Common types of viruses include MHV-1, MHV-3, MHV-JHM, MHV-S, and MHV-A59. Enterotropic viruses selectively infect the small intestinal mucosa ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N33/569C12R1/93
CPCC12Q1/706G01N33/56983
Inventor 冯翔石建芳高忠翔唐利军黎炎梅郏志明
Owner 高忠翔
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