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Primer probe composition and kit for detecting canine parainfluenza virus, canine adenovirus type II and canine mycoplasma and preparation method of primer probe composition

A technology of canine parainfluenza virus and canine adenovirus, which is applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of unfriendly pathogen identification, poor sensitivity, and low sensitivity, and achieve optimization Reaction system and procedures, avoiding false positive results, and improving the effect of detection throughput

Active Publication Date: 2022-06-03
山东康华生物医疗科技股份有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Separation and culture is the most reliable detection method, but due to the time-consuming, poor sensitivity, and complicated operation of this method, it cannot meet the needs of pathogen detection in pet dogs; serological detection methods are prone to cross-reactions with various pathogens, and are not effective for pathogens. The identification is not friendly enough; the sensitivity of conventional PCR method is not high, it is easy to be polluted by the environment and produce false positives, and it cannot achieve multi-channel detection

Method used

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  • Primer probe composition and kit for detecting canine parainfluenza virus, canine adenovirus type II and canine mycoplasma and preparation method of primer probe composition
  • Primer probe composition and kit for detecting canine parainfluenza virus, canine adenovirus type II and canine mycoplasma and preparation method of primer probe composition
  • Primer probe composition and kit for detecting canine parainfluenza virus, canine adenovirus type II and canine mycoplasma and preparation method of primer probe composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 A primer probe of a nucleic acid detection kit for canine parainfluenza virus, canine adenovirus type II and Mycoplasma canis

[0024] The above primer probes were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

Embodiment 2

[0025] Example 2 Preparation method of nucleic acid detection kit for canine parainfluenza virus, canine adenovirus type II and Mycoplasma canis

Step 1. Prepare negative and positive controls

Negative control is TE Buffer;

Positive control: Insert the CPIV target gene sequence, CAV-II target gene sequence, and MC target gene sequence into the pUC 57 vector plasmid, respectively, and obtain CPIV synthetic plasmid, CAV-II synthetic plasmid, MC through expanded culture, identification and plasmid extraction. Artificially synthesized plasmids. The CPIV target gene sequence is shown in SEQ ID NO.13; the CAV-II target gene sequence is shown in SEQ ID NO.14; the MC target gene sequence is shown in SEQ ID NO.15.

[0026] The CPIV synthetic plasmid, CAV-II synthetic plasmid, and MC synthetic plasmid were quantified to 3 × 10, respectively. 6 After copy / mL, mix them according to the volume ratio of 1:1:1 to make a positive control. In the obtained positive control, the concentrat...

Embodiment 3

[0033] Example 3 Kit Linearity, Sensitivity, Repeatability and Specificity Detection Experiment

Step 1. Template preparation

Insert the CPIV target gene sequence, CAV-Ⅱ target gene sequence, and MC target gene sequence into the pUC 57 vector plasmid respectively, and obtain CPIV synthetic plasmid, CAV-Ⅱ synthetic plasmid and MC synthetic plasmid through expansion culture, identification and plasmid extraction. . The CPIV synthetic plasmid, CAV-II synthetic plasmid, and MC synthetic plasmid were quantified to 1 × 10, respectively. 7 copies / mL.

[0034] The CPIV target gene sequence is shown in SEQ ID NO.13; the CAV-II target gene sequence is shown in SEQ ID NO.14; the MC target gene sequence is shown in SEQ ID NO.15.

[0035] Will 1x10 7 The copies / mL of CPIV synthetic plasmid, CAV-II synthetic plasmid and MC synthetic plasmid were diluted to 1×10 with TE Buffer respectively. 6 Copy / mL, 1×10 5 Copy / mL, 1×10 4 Copy / mL, 1×10 3 Copy / mL, spare.

[0036] Take canine parvo...

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Abstract

The invention provides a primer probe composition and kit for detecting canine parainfluenza virus, canine adenovirus type II and canine mycoplasma and a preparation method of the primer probe composition, the primer probe composition comprises upstream and downstream primers and fluorescent probes designed for the canine parainfluenza virus, and the sequences of the upstream and downstream primers and the fluorescent probes are shown as SEQ ID NO.1-3; the sequences of the upstream and downstream primers and the fluorescent probe designed for the canine adenovirus type II are as shown in SEQ ID NO.4-6; the sequences of the upstream and downstream primers and the fluorescent probe are as shown in SEQ ID NO.7-9, wherein the upstream and downstream primers and the fluorescent probe are designed aiming at the mycoplasma canine; an upstream primer, a downstream primer and a fluorescent probe of an external reference are designed aiming at the arabidopsis thaliana SUC2 gene, and the sequence is shown as SEQ ID NO.10-12. The kit can be used for simultaneously detecting the canine parainfluenza virus, the canine adenovirus type II and the canine mycoplasma, and is high in sensitivity and good in specificity.

Description

technical field [0001] The invention belongs to the technical field of biological detection, in particular to a primer-probe composition, a kit and a preparation method for detecting canine parainfluenza virus, canine adenovirus type II and mycoplasma canis. Background technique [0002] With the improvement of people's living standards, the number of pet dogs has increased significantly. In order to protect the health of pet dogs and dog owners, the demand for pathogen detection of pet dogs is increasing. There are many pathogens that cause respiratory diseases in dogs, and there are co-infections. Common canine respiratory pathogens are canine parainfluenza virus, canine adenovirus type Ⅱ and Mycoplasma canis. [0003] Canine Parainfluenza Virus (CPIV) infection is the main respiratory infectious disease in dogs. CPIV belongs to the family Paramyxoviridae, the genus Paramyxovirus, and the nucleic acid type is single-stranded RNA. Clinical manifestations are fever, runny...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/689C12Q1/6851C12Q1/04C12N15/11
CPCC12Q1/701C12Q1/689C12Q1/6851C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2545/114C12Q2563/107
Inventor 杨帆郑国华田永帅陈欢刘嘉男左雪梅刘万建宋金铃
Owner 山东康华生物医疗科技股份有限公司
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