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Primer-probe composition, kit and preparation method for detecting canine parainfluenza virus, canine adenovirus type Ⅱ and Mycoplasma canis

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

Active Publication Date: 2022-07-15
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, kit and preparation method for detecting canine parainfluenza virus, canine adenovirus type Ⅱ and Mycoplasma canis
  • Primer-probe composition, kit and preparation method for detecting canine parainfluenza virus, canine adenovirus type Ⅱ and Mycoplasma canis
  • Primer-probe composition, kit and preparation method for detecting canine parainfluenza virus, canine adenovirus type Ⅱ and Mycoplasma canis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 A primer probe of a nucleic acid detection kit for canine parainfluenza virus, canine adenovirus type II and Mycoplasma canis

[0048] The gene sequences of CPIV, CAV-II, MC and Arabidopsis thaliana SUC2 were obtained according to the NCBI GeneBank database, the gene sequences were compared, and the following primer probes were designed:

[0049] SEQ ID NO.1: the upstream primer designed for canine parainfluenza virus, the sequence of this upstream primer is 5'-cgatggctttgaggagggatc-3'; CPIV-F1 for short;

[0050] SEQ ID NO.2: the downstream primer designed for canine parainfluenza virus, the sequence of this downstream primer is 5'-tgtcaggtagatcttctgcaagtg-3'; CPIV-R1 for short;

[0051] SEQ ID NO.3: A fluorescent probe designed for canine parainfluenza virus, the sequence of the fluorescent probe is 5'-cataggcattgatctctccacggctcatacc-3'; CPIV-P1 for short; the 5' end of the fluorescent probe is labeled as a reporter group 6-FAM, the 3' end is marked with th...

Embodiment 2

[0062] Example 2 Preparation method of nucleic acid detection kit for canine parainfluenza virus, canine adenovirus type II and Mycoplasma canis

[0063] Step 1. Prepare negative and positive controls

[0064] Negative control is TE Buffer;

[0065] 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.

[0066] 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...

Embodiment 3

[0079] Example 3 Kit Linearity, Sensitivity, Repeatability and Specificity Detection Experiment

[0080] Step 1. Template preparation

[0081] 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 expanded 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.

[0082] 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.

[0083] 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.

[0084] Ta...

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Abstract

The present invention provides a primer-probe composition, a kit and a preparation method for detecting canine parainfluenza virus, canine adenovirus type II and Mycoplasma canis, and the primer-probe composition comprises: Downstream primers and fluorescent probes, whose sequences are shown in SEQ ID NO.1-3; upstream and downstream primers and fluorescent probes designed for canine adenovirus type II, whose sequences are shown in SEQ ID NO.4-6; for Mycoplasma canis The designed upstream and downstream primers and fluorescent probes, the sequences are shown in SEQ ID NO.7-9; the upstream and downstream primers and fluorescent probes of the external reference are designed for the Arabidopsis SUC2 gene, and the sequences are shown in SEQ ID NO.10-12 Show. The invention can simultaneously detect canine parainfluenza virus, canine adenovirus type II and mycoplasma canis, and has high sensitivity and good 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 Patents(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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