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Zoonosis tuberculosis fluorescence PCR rapid diagnosis kit

A technology for tuberculosis and animals, applied in the field of fluorescent PCR rapid diagnostic kits for co-infected tuberculosis in humans and animals, can solve the problems of standardization impact, lack of standardization of detection methods and kits, etc.

Inactive Publication Date: 2008-04-30
INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the development of molecular biology technology, many laboratories at home and abroad have carried out research on PCR methods for tuberculosis and paratuberculosis, but the lack of standardization of detection methods and kits is the key to affecting the accuracy of PCR results

Method used

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  • Zoonosis tuberculosis fluorescence PCR rapid diagnosis kit
  • Zoonosis tuberculosis fluorescence PCR rapid diagnosis kit
  • Zoonosis tuberculosis fluorescence PCR rapid diagnosis kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1: Preparation of primers and probes of fluorescent PGR rapid diagnostic kit for tuberculosis in humans and animals

[0071] According to the specific 229bp sequence of Mycobacterium tuberculosis, select one of the sequences (GeneBank No.CP000611), using Primer express2.0 software, according to the annealing temperature of the primer at 60°C and the annealing temperature of the probe is higher than the annealing temperature of the primer Based on the principle of about 10°C, the primers SEQ ID No.1, SEQ ID No.2 and the probe SEQ ID No.3 were selected. Blast homology analysis showed that the selected primers and probes were from the Mycobacterium tuberculosis complex Specific sequence with no homology to other sequences. The length of the primer amplification product SEQ ID No.4 is 91bp.

[0072] According to the specific 12.7kb deletion sequence of Mycobacterium bovis, select one of the sequences (GeneBank No.AM408590), using Primer express2.0 software, accord...

Embodiment 2

[0077] Example 2: Establishment and optimization of the PCR detection reaction system of the fluorescent PCR rapid diagnostic kit for co-infected tuberculosis in humans and animals

[0078] 1. Method

[0079] 1. Optimization of primer and probe concentration

[0080] In the experiment, the primer concentration was increased by 0.1 μmol / L from 0.1 μmol / L to 0.6 μmol / L, and the probe concentration was increased by 0.025 μmol / L from 0.025 μmol / L to 0.2 μmol / L. The matrix method was used to carry out the comparative test, and the other conditions of the comparative test were exactly the same.

[0081] 2. Optimization of Taq DNA polymerase (Taq enzyme)

[0082] The definition of one unit of Taq enzyme: the amount of enzyme required to incorporate 10 μmol / L of dNTPs into acid-soluble substances for 30 minutes at 74°C.

[0083] Requirements for activity: DNA polymerase activity and 5'→3' exonuclease activity, no 3'→5' exonuclease activity and endonuclease activity; thermal stabili...

Embodiment 3

[0101] Embodiment 3: Fluorescent PCR rapid diagnostic kit for co-morbid tuberculosis in humans and animals

[0102] The kit includes the following components, and the storage temperature is -20°C

[0103] 1) Four kinds of PCR reaction solutions (Mycobacterium tuberculosis fluorescent PCR reaction solution, Mycobacterium bovis fluorescent PCR reaction solution, Mycobacterium avium fluorescent PCR reaction solution and Mycobacterium tuberculosis complex fluorescent PCR reaction solution), including: 1 ×PCR buffer; 0.2 μmol / L primer I; 0.2 μmol / L primer II, 0.1 μmol / L probe; 200 μmol / L d NTP, MgCl 2 2.5mmol / L;

[0104] 2) Taq enzyme 2.5U / μl;

[0105] 3) Negative control: sterile saline

[0106] 4) Positive control: plasmid DNA carrying the amplified product

[0107] Preparation method of positive control:

[0108] Using primer SEQ ID No.1 and primer SEQ ID No.2, using the DNA of Mycobacterium tuberculosis as a template, using primer SEQ ID No.5 and primer SEQ ID No.6, using...

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Abstract

The invention discloses a fluorescence PCR quick diagnosis reagent box of the zoonosis tuberculosis, which belongs to the biological technology field. A group of nucleotide sequence for detecting the zoonosis tuberculosis is the nucleotide sequence shown from the sequence table SEQ ID No.1 to the sequence table SEQ ID No.12. The content disclosed by the invention comprises a fluorescence PCR primer and a probe sequence for a mycobacterium tuberculosis composite group, mycobacterium tuberculosis, cow mycobacterium and fowl mycobacterium, a fluorescence PCR reaction system, a detection program, a reaction parameter and a judging method of various reagent boxes, and the preliminary treatment and the nucleic acid extraction method of various clinic samples of bacteria fluid, milk sample, blood sample, sputum, fecal sample and lymph nodes. The invention provides diagnosis reagent for the zoonosis tuberculosis diagnosis of which the technology method is special, sensitive and quick and the quality is reliable. The adoption of the reagent box provided by the invention can accomplish the whole process from the sample treatment to the fluorescence PCR detection within a day, and the detection sensitivity can reach the single gene copy or the signal bacterial cell.

Description

technical field [0001] The present invention relates to a rapid fluorescent PCR diagnostic kit for co-infected tuberculosis in humans and animals, more specifically to provide real-time fluorescent PCR rapid detection methods and diagnostic reagents for various pathogenic bacteria or pathogenic bacteria complexes that cause human, livestock, and poultry tuberculosis, The invention relates to a real-time fluorescent PCR method and reagents for detecting Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium and Mycobacterium tuberculosis complex, belonging to the field of biotechnology. Background technique [0002] Tuberculosis is a major infectious disease that threatens human and animal health. The World Health Organization (WHO) specially released the Global Tuberculosis Control Strategy, and designated March 24 every year as World Tuberculosis Day. Mycobacterium tuberculosis complex (Mycobacterium tuberculosis complex, MTC) is the pathogenic bacteria of tu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 陈茹高小博杨国海刘中勇曾碧健林志雄
Owner INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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