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Fluorescence quantification PCR (Polymerase Chain Reaction) primer, probe and kit for detecting dog leptospira nucleic acid

A leptospira, fluorescence quantitative technology, applied in microorganisms, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of large gene mobility, large differences in gene sequences, and insufficient sensitivity of diagnostic techniques.

Inactive Publication Date: 2014-08-27
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the genes of Leptospira are very mobile, and the gene sequences of different strains are quite different.
Therefore, it is difficult to have a single PCR method for accurate, sensitive and rapid detection of all strains of Leptospira
Existing PCR method fails for rapid and effective molecular diagnosis of Leptospira infection in dogs
It is especially important that patients with Leptospira have low levels of Leptospira in their blood, urine, and diagnostic techniques currently on the market are not sensitive enough

Method used

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  • Fluorescence quantification PCR (Polymerase Chain Reaction) primer, probe and kit for detecting dog leptospira nucleic acid
  • Fluorescence quantification PCR (Polymerase Chain Reaction) primer, probe and kit for detecting dog leptospira nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment -1

[0030] Example-1: PCR Amplification of Pathogenic Dog Leptospira Standard Strain (L. interragans Pomona Pomona)

[0031] The cultured L. interragans Pomona Pomona standard strain (gifted by Dr. Ashutosh Verma, Ross University School of Veterinary Medicine) was used for nucleic acid purification and calculation of the molecular weight of L. interragans Pomona Pomona. The FRET-PCR method of the present invention can detect one copy of LigA gene in a single reaction system.

Embodiment -2

[0032] Example-2: Comparison of Sensitivity and Specificity of Quantitative PCR of the Present Invention and Leptospira Commonly Used in North America

[0033] In the diagnosis of Leptospira, there is a widely used Lip32SYBR technology (Levett PN, Morey RE, Galloway RL, etc. published in J Med Microbiol, 54:45-49 in 2005. The title of the article is: Detection of pathogenic leptospires by real -time quantitative PCR.). We systematically compared the sensitivity of the technology of the present invention and the Lip32SYBR technology in the detection of Leptospira. The cultured Leptospira standard strain was used for nucleic acid purification and molecular number calculation, and then 10-fold dilutions were used for 2 Nucleic acid amplification of a PCR system. It is found by comparison that the present invention has a high degree of repeatability and can detect a single copy of Leptospira nucleic acid, and the recessive control without nucleic acid has always been negative. And...

Embodiment -3

[0034] Example-3. Detection of Dogs Infected with Leptospira Infection

[0035] A dog with typical clinical symptoms, blood and urine were collected for PCR detection of Leptospira. The Lip32SYBR technique detected Leptospira in the urine and blood of affected dogs, while the FRET-PCR of the present invention detected low copies of Leptospira in the urine. According to the diagnostic results of FRET-PCR, the dog was treated effectively.

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Abstract

The invention relates to a fluorescence quantification PCR (Polymerase Chain Reaction) primer, a fluorescence quantification PCR probe and a fluorescence quantification PCR kit for detecting dog leptospira nucleic acid. The primer and the probe are shown as SEQ ID NO. (Sequence Identification Number) 1-4. The kit comprises 22 microliters of 5xFRET (Fluorescence Resonance Energy Transfer)-qPCRStock and 22 microliters of 5xOligo mixture, wherein the Oligo mixture comprises the primer and the probe. With the adoption of the primer and the probe, the pathogenic dog leptospira can be detected quickly, specifically and sensitively.

Description

technical field [0001] The invention relates to fluorescent quantitative PCR detection primers, probes and kits for the diagnosis of pathogenic dog Leptospira. This invention sensitively, specifically, and rapidly amplifies pathogenic Leptospira interrogans Canicola Canicola, L. interrogans Icterohemorrhagiae Icterohemorrhagiae, L. interrogans Pomona Pomona, and L. kerschneri Grippotyphosa Grippotyphosa that infect dogs, without amplifying non- Pathogenic Leptospira (L. biflexa) nucleic acid. Background technique [0002] Leptospira can cause a variety of important zoonotic diseases in animals and humans. The main existing laboratory diagnostic techniques for Leptospira infection include: 1) Clinical diagnosis. The clinical symptoms caused by Leptospira are not specific, and the value in clinical diagnosis is limited; 2) Isolation of pathogens: it takes a long time to develop, and requires specially trained professionals; 3) Serological tests to detect anti-Leptospira Ant...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/01
Inventor 王成明
Owner YANGZHOU UNIV
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