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A detection method for Bordetella bronchiseptica in rabbits

A technology of Bordetella blood and bronchi, which is applied in the detection field of Bordetella bronchiseptica in rabbits, can solve the problems of easy misdiagnosis, inability to characterize pathogenic bacteria, and inaccurate diagnosis

Active Publication Date: 2019-06-28
YEBIO BIOENG OF QINGDAO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

None of the above detection methods can directly characterize the isolated pathogenic bacteria, and there is often the influence of the same type of isolated bacteria, which cannot be accurately diagnosed and is prone to misdiagnosis

Method used

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  • A detection method for Bordetella bronchiseptica in rabbits
  • A detection method for Bordetella bronchiseptica in rabbits

Examples

Experimental program
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Effect test

Embodiment 1

[0014] Example 1: Construction of a suspension fluorescent immunoassay kit for detecting Bordetella bronchiseptica in rabbits

[0015] 1.1 Preparation method for single-factor serum antibody against Bordetella bronchiseptica in rabbits

[0016] The main component of the fluorescent immunoassay kit for detecting Bordetella rabbit bronchiseptica is the single-factor serum antibody against the outer model protein of Bordetella rabbit bronchiseptica, which was isolated, identified and preserved by the Technology Center of Qingdao Yibang Bioengineering Co., Ltd. The rabbit Bordetella bronchiseptica QDBb01 strain (preserved in the China Center for Type Culture Collection in Wuhan, China on November 1, 2016, and the preservation number is CCTCC M 2016607) was cultured with 3% TSB, and the bacteria were collected at 4000r / min , and then use PH7.2 Tris-Hcl to resuspend the bacteria, and ultrasonically break the resuspended bacteria, work for 1s, the interval time is 3s, the whole time ...

Embodiment 2

[0017] Example 2 Indirect Suspension Immunofluorescence Antibody Detection Method Operation Steps

[0018] 2.1 collect the rabbit Bordetella bronchiseptica (QDBb01 strain) thalline 1ml of suspension culture (total number of bacteria is 10 6 ~10 7 cells / ml) in a 1.5ml EP tube, centrifuged at 1500r / min for 10min, and discarded the supernatant.

[0019] 2.2 Add 1ml of PBS, gently upside down, 1500r / min, discard the supernatant.

[0020] 2.3 Repeat step 1.2.2 to remove the influence of serum in the culture medium;

[0021] 2.4 Add 1ml of 10% paraformaldehyde, resuspend the bacterial cells, and place at room temperature for 10 minutes; during this period, flick the bottom of the EP tube several times every 2 minutes to make the individual bacterial cells fully contact with the fixative solution; centrifuge at 1500r / min for 10 minutes, discard the supernatant .

[0022] 2.5 Add 1ml of 1% BSA and block at 37°C for 1 hour to block the non-specific antigens on the surface of the ba...

Embodiment 3

[0030] Example 3: Sensitivity and specificity identification of rabbit Bordetella bronchiseptica suspension fluorescence detection test method

[0031] 3.1 The components of the kit used in the fluorescent immunoassay method for suspended Bordetella bronchiseptica in rabbits constructed on the basis of this single-factor serum include:

[0032] 1) Primary antibody: single-factor serum antibody (mouse source) used to detect Bordetella bronchiseptica in rabbits, the working concentration is 100 times diluted;

[0033] 2) Secondary antibody: FITC-labeled goat anti-mouse IgG;

[0034] 3) Positive control sample: Bordetella rabbit bronchiseptica (P13 strain);

[0035] 4) PBS washing solution: Nacl 8.0g / L; Kcl 0.2g / L; NaCl 2 HPO 4 3.58g / L; KH 2 PO 4 0.24g / L to 1L.

[0036] 3.2 Sensitivity, specificity and repeatability test of the suspension fluorescence detection method for detecting Bordetella bronchiseptica in rabbits

[0037] 1) Comparative detection of IFA and PCR

[...

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Abstract

The present invention provides a rabbit bordetella bronchiseptidca suspension fluorescence immunoassay method including a rabbit bordetella bronchiseptidca suspension fluorescence immunoassay kit and a rabbit bordetella bronchiseptidca indirect suspension fluorescence immunoassay method. The kit includes components of a single factor serum antibody used for rabbit bordetella bronchiseptidca detection, FITC labeled goat anti mouse IgG, a positive control sample, a fixed solution and a PBS washing solution; and the single factor serum antibody used for rabbit bordetella bronchiseptidca detection is prepared by immunizing mice with outer membrane protein antigen of rabbit Bordetella bronchiseptica strain QDBb01. The rabbit bordetella bronchiseptidca suspension fluorescence immunoassay method based on the rabbit bordetella bronchiseptidca suspension fluorescence immunoassay kit can be used for clinical detection of antigens in rabbit bordetella bronchiseptidca infected sample tissue to determine whether the tissue is infected by rabbit bordetella bronchiseptidca, and the method has strong specificity, high sensitivity, good repeatability and fast speed of detection.

Description

technical field [0001] The invention belongs to the technical field of livestock virus detection, and in particular relates to a detection method for Bordetella bronchiseptica in rabbits. Background technique [0002] Bordetella bronchiseptica in rabbits occurs frequently in autumn, winter and early spring; the bacteria is mainly transmitted through the air and infected through the respiratory tract. Young rabbits have a high morbidity rate and there have been fatal cases. Adult rabbits are less affected. The contamination rate of this bacterium was 64.4% in group-raised rabbits, and 20% in free-range rabbits. Under natural conditions, this bacterium parasitizes in the upper respiratory tract of various mammals, often causing mutual infection of chronic respiratory diseases. Especially in sudden changes in weather, decreased resistance of rabbits, poor hygiene in the rabbit house, and dirty air, etc., the disease can all be caused. The incubation period of the disease is...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/533
CPCG01N33/533G01N33/56911
Inventor 张恒郭莉莉张会范根成杜元钊
Owner YEBIO BIOENG OF QINGDAO
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