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Hybridoma cell strains for detecting canine distemper virus and canine parvovirus as well as dual-detection test paper card for canine distemper virus and canine parvovirus

A hybridoma cell line and canine distemper virus technology, applied in the biological field, can solve the problems of simultaneous detection of two viruses, single colloidal gold detection method, low detection sensitivity, etc., achieve high accuracy, improve the level of prevention and control, The effect of high sensitivity

Active Publication Date: 2020-06-26
北京市动物疫病预防控制中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are also colloidal gold test strips on the market that can detect canine distemper virus and canine parvovirus alone, but their detection sensitivity is low, and a high concentration of virus is required to detect it, resulting in missed detection and false negative results during the detection process; And there is only a single colloidal gold detection method, which cannot detect two viruses at the same time, and the cost of use is still high

Method used

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  • Hybridoma cell strains for detecting canine distemper virus and canine parvovirus as well as dual-detection test paper card for canine distemper virus and canine parvovirus
  • Hybridoma cell strains for detecting canine distemper virus and canine parvovirus as well as dual-detection test paper card for canine distemper virus and canine parvovirus
  • Hybridoma cell strains for detecting canine distemper virus and canine parvovirus as well as dual-detection test paper card for canine distemper virus and canine parvovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Obtaining hybridoma cell T1 that continuously and stably secretes monoclonal antibody against canine distemper virus

[0069] The hybridoma cell line that continuously and stably secretes monoclonal antibodies against canine distemper virus obtained by immunizing mice with canine distemper virus as an immunogen is named T1.

[0070] The method for obtaining hybridoma cell line T1 comprises the following steps:

[0071] 1. Animal immunity

[0072] 1) Basic immunity: Canine distemper virus (provided by Beijing Animal Disease Prevention and Control Center) was used as the immunogen, determined by sucrose density gradient centrifugation method, the purity was ≥85%, and the concentration was 10-1000 μg / mL. If necessary, the virus antigen can be concentrated in an ultrafiltration tube to increase the virus content. After concentration, the antigen is mixed with Freund's complete adjuvant (purchased from Sigma Company) in equal volumes and fully emulsified, and the ...

Embodiment 2

[0102] Example 2, detection of canine distemper virus and canine parvovirus with gold-labeled paper chromatography test card prepared by monoclonal antibodies 1F2 and 6D6

[0103] 1. Preparation of gold standard paper chromatography test paper

[0104] Such as figure 1 , 2 Shown (in the figure 1 is the longitudinal section structure diagram of gold-labeled paper chromatography test paper, among the figure 2 is the front structure diagram of gold-labeled paper,), the gold-labeled paper chromatography test paper for detecting canine distemper virus is made of water-absorbing pad ( 1), nitrocellulose membrane is composed of NC membrane (2), glass fiber membrane sample pad (3), glass cellulose membrane conjugate release pad (4), the nitrocellulose membrane (2) is equipped with detection line T1 line (6) and T2 line (7) and quality control line C line (8).

[0105] The preparation method of gold label paper chromatography test paper comprises the following steps:

[0106] 1) Co...

Embodiment 3

[0120] 1. Specificity

[0121] The colloidal gold test strip was used to detect canine parvovirus, canine distemper virus, canine coronavirus (CCV), canine infectious hepatitis virus (ICHV) and canine parainfluenza virus (CPIV). The results showed that canine distemper virus was detected Line T1, canine parvovirus detection line T2, other viruses did not appear, the results are as follows Figure 5 shown.

[0122] 2. Sensitivity

[0123] Dilute the recombinant plasmids of canine distemper virus and canine parvovirus 10 times to 1.3×10 8 ~1.3×10 3 copies / μL, and then detected by PCR method. The results showed that the lower limit of the positive DNA template for canine distemper virus in the PCR detection sample was 1.3×10 3 Copy / μL (results such as Figure 6 shown), and the lower limit of colloidal gold test strips for the detection of canine distemper virus DNA template is 1.3×10 4 Copy / μL (results such as Figure 7 shown). The lower limit of the positive DNA template ...

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Abstract

The invention discloses hybridoma cell strains for detecting canine distemper virus and canine parvovirus as well as a dual-detection test paper card for the canine distemper virus and canine parvovirus. Four hybridoma cell strains T1-T4 (two strains 1F2 and 6D6 are selected for collection) and monoclonal antibodies secreted by the hybridoma cell strains are obtained through experiments, and the obtained monoclonal antibodies are used for preparing the dual-detection test paper card for the canine distemper virus and the canine parvovirus. The prepared dual-detection test paper card for the canine distemper virus and the canine parvovirus has high sensitivity, high specificity and high accuracy, can simultaneously obtain the canine distemper virus and canine parvovirus in a sample in one step, and has the advantages that the operation is convenient and rapid, results can be judged by naked eyes and are easy to store, special instruments (equipment) and reagents are not required and thelike.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a hybridoma cell strain of canine distemper virus and canine parvovirus and a double detection test paper card thereof. Background technique [0002] Canine distemper virus disease and canine parvovirus disease are currently the most important diseases that seriously endanger the health of dogs. The clinical symptoms caused by the two diseases in the early stage of the disease are similar and often mixed infections. It is often difficult to distinguish the specific disease based on clinical symptoms alone. What kind of virus infection must rely on laboratory testing methods. There are three commonly used detection methods stipulated in the current national standards, namely immunoenzyme method, immunohistochemical method and RT-PCR method. Clinically, there is only one colloidal gold technology that can detect only one virus. [0003] Applying the method in the national...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/10C07K16/08G01N33/577G01N33/569G01N33/558C12R1/91
CPCC07K16/081C07K16/10G01N33/558G01N33/56983G01N33/577
Inventor 邓柏林郑雪莹郑金来宋彦军陈会玲郭俊林刘庆斌韦海涛李志军
Owner 北京市动物疫病预防控制中心