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PCR (polymerase chain reaction) primer and kit for detecting pathogenic canine leptospirosis

A kit and a pathogenic technology are used in the field of PCR primers and kits for detecting pathogenic Leptospira canis, which can solve the problem of inability to accurately identify and detect pathogenic and non-pathogenic Leptospira, and achieve specificity. Excellent sensitivity, high specificity, and high specificity

Inactive Publication Date: 2016-06-01
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, both pathogenic and non-pathogenic Leptospira contain the 16srRNA gene. The above-mentioned technical scheme detects Leptospira in dogs and cats by detecting the 16srRNA gene, and cannot accurately identify and detect pathogenic and non-pathogenic Leptospira.

Method used

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  • PCR (polymerase chain reaction) primer and kit for detecting pathogenic canine leptospirosis
  • PCR (polymerase chain reaction) primer and kit for detecting pathogenic canine leptospirosis
  • PCR (polymerase chain reaction) primer and kit for detecting pathogenic canine leptospirosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Primer Design and Synthesis

[0038] According to the published LigB gene sequence in GenBank, primers were designed using Primer Premier 5.0 software, and the primers of the present invention were obtained through screening.

[0039] The screened primers were specifically identified by BLAST in NCBI, as shown in Table 1. Primers were synthesized by Dalian Bao Biological Company.

[0040] Table 1 Universal Primer Sequence

[0041]

Embodiment 2

[0042] The optimization of embodiment 2PCR reaction program

[0043] According to the product instructions of the Bacteria Group DNA Extraction Kit, DNA was extracted from the well-grown Leptoplasma bacteria liquid as a template.

[0044] Using Leptospira DNA as a template, first explore the annealing temperature of the PCR reaction, reaction system and conditions: 10 μL, Taq enzyme 0.1 μL, each of the upstream and downstream primers described in Example 1 0.5 μL, dNTP 0.8 μL, template 1 μL, 10× Buffer1 μL, ddH 2 O make up.

[0045] Reaction conditions: 94°C for 3min; 94°C for 30s, annealing temperature for 30s, 72°C for 30s, 29 cycles; 72°C for 5min. It was carried out on a gradient PCR instrument, and the annealing temperature was set to a temperature gradient, respectively: 56.0°C, 57.0°C, 58.0°C, 59.0°C, 60.0°C. Carry out PCR reaction, and carry out agarose gel electrophoresis to PCR product and observe the result, such as figure 1 shown. Depend on figure 1 It can be...

Embodiment 3

[0046] Embodiment 3 sensitivity test

[0047] The DNA template extracted in Example 2 was quantified as a standard, diluted to 1ng / μL, and then diluted to 1ng / μL-1×10 -6 ng / μL. Use the PCR system and working procedures described in Example 2 to carry out the PCR reaction, and perform agarose gel electrophoresis detection on the PCR products, as shown in 2. Depend on figure 2 It can be seen that the minimum detection concentration is 10 -5 ng / μL.

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Abstract

The invention relates to detection primers and detection kits and particularly discloses a PCR (polymerase chain reaction) primer and a kit for detecting pathogenic canine leptospirosis. The PCR primer includes a forward primer: 5'-CTTGGGATTCTTCTAATMCSGATA-3' and a reverse primer: 5'-GATCCTTGTATTCCACCGATG-3'. The amplified fragment length of the PCR primer is 124bp. The PCR primer and the kit for detecting the pathogenic canine leptospirosis have the advantages that LigB genes are selected as a detection object so as to achieve the purpose of detecting the pathogenic canine leptospirosis; the PCR primer and the kit, which are used for detecting the LigB genes of the canine leptospirosis, are time saving, labor saving and excellent in specificity and sensitivity; the PCR primer and the kit can be used for early diagnosis of the canine leptospirosis during experiments, an established and assembled canine leptospirosis PCR method is high in specificity, and leptospira concentration detection sensitivity can reach 101 copies / microliters.

Description

technical field [0001] The invention relates to a detection primer and a kit, in particular to a PCR primer and a kit for detecting pathogenic canine Leptospira. Background technique [0002] Leptospirosis is a zoonotic disease that spreads all over the world, referred to as leptospirosis, and is especially widespread in developing countries and tropical regions. Dogs are susceptible to the disease, and the clinical manifestations after infection are diverse, making it difficult to diagnose. The pathogen of leptospirosis will continue to exist in the host body, and will further spread to pollute the environment, infect other humans and animals, and cause serious harm. Therefore, after the dog suffers from the disease, while endangering its own health, it will also threaten the safety of its owner. Therefore, it is necessary to diagnose the disease at an early stage and control the disease as early as possible. At present, the methods for diagnosing canine leptospirosis in...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6888C12Q2600/158Y02A50/30
Inventor 曹永国丁壮龚悦金雪敏张文龙吴殿君
Owner JILIN UNIV
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