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Paratuberculosis fluorescence PCR rapid diagnosis kit

A paratuberculosis and nucleic acid sequence technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of lack of standardization and impact of standardization of detection methods and kits, and achieve less chance of cross-contamination and environmental pollution. Not easy to pollute, high accuracy effect

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 paratuberculosis PCR methods, but the lack of standardization of detection methods and kits, standardization is the key to the accuracy of PCR results

Method used

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  • Paratuberculosis fluorescence PCR rapid diagnosis kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Preparation of primers and probes for Mycobacterium paratuberculosis fluorescent PCR rapid diagnostic kit

[0045] According to the specific nucleic acid sequence IS900 insert sequence of Mycobacterium paratuberculosis, select one of the published sequences on NCBI (GeneBank No.S74401), and use the professional primer probe design software Primer express2.0 software to target the conserved region of the IS900 insert sequence Primers were designed, and based on the principle that the annealing temperature of the primers was 60°C and the annealing temperature of the probe was about 10°C higher than the annealing temperature of the primers, the blast software was used for analysis and screening, and finally the primers PB-1U, PB-1R and Probe PB-1P. Nucleic acid sequence homology analysis using blast software showed that the selected primers and probe sequences were consistent with the corresponding sequences of all IS900 insertion sequences in the public databas...

Embodiment 2

[0048] Example 2: Establishment and optimization of the PCR detection reaction system of the Mycobacterium paratuberculosis fluorescent PCR rapid diagnostic kit

[0049] 1. Method

[0050] 1. Optimization of primer and probe concentration

[0051] 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.

[0052] 2. Optimization of Taq DNA polymerase (Taq enzyme)

[0053] 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.

[0054] Requirements for activity: DNA polymerase activity and 5'→3' exonuclease activity, no 3'→5' exonuclease activity and endonuclease activity; thermal stability, 94°C for 1 hour ...

Embodiment 3

[0072] Embodiment 3: paratuberculosis fluorescent PCR rapid diagnostic kit

[0073] The kit includes the following components, and the storage temperature is -20°C

[0074] 1) PCR reaction solution, containing: 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;

[0075] 2) Taq enzyme 5U / μl;

[0076] 3) Negative control: normal saline;

[0077] 4) Positive control: plasmid DNA carrying the amplified product;

[0078] The primer I in the reaction solution is the nucleotide sequence shown in SEQ ID No.1, the primer II is the nucleotide sequence shown in SEQ ID No.2, and the probe is the nucleotide sequence shown in SEQ ID No.3 sequence, the amplified product of the positive control is the nucleotide sequence shown in SEQID No.4,

[0079] The 5' end of the probe is labeled with a FAM fluorescent dye, and the 3' end of the probe is labeled with a TAMARA quenching label.

[0080] Preparation method of positive contro...

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Abstract

The invention discloses a paratuberculosis fluorescence PCR quick diagnosis reagent box, in particular relates to a method and a reagent box for quick detection of the paratuberculosis original bacterium-paratuberculosis mycobacteria, and belongs to the biological technology field. The invention is a group of nucleotide sequences for detecting paratuberculosis virus, and the invention is characterized in that the reagent box is the nucleotide sequences shown from the sequence table SEQ ID No.1 to the sequence table SEQ ID No.4. The invention has the advantages that: firstly, the specificity is good, the template is discriminated through a real-time fluorescence PCR technology and the specificity hybridization of a primer or a probe, the invention has very high accuracy and the false positive is low; secondly, the sensitivity is high, and a sensitive fluorescence detection system is adopted to perform real-time monitoring to the fluorescence signal; thirdly, the operation is simple, the automation degree is high, and the step of the electrophoresis detection of the product of the traditional PCR amplification is not required; fourthly, the detection is not easy to cause pollution, a closed pipe is amplified, a cover opening is not required, and the opportunity of the cross contamination and the environment opportunity is less.

Description

technical field [0001] The invention relates to a paratuberculosis fluorescent PCR rapid diagnosis kit, more specifically a method and a kit for rapidly detecting paratuberculosis pathogenic bacteria-mycobacterium paratuberculosis by using the principle of fluorescent PCR technology, and belongs to the field of biotechnology. Background technique [0002] Paratuberculosis (or Johne's disease) is a consumptive and chronic enteritis mainly in ruminants such as cattle and sheep. It is characterized by persistent diarrhea and chronic emaciation in animals. The mortality rate of herds in infected areas can reach 2%. -10%, because the disease is difficult to eradicate, and its losses to the cattle industry often exceed that of some infectious diseases. Paratuberculosis is widely distributed in all countries in the world, and it is very harmful to the dairy and beef cattle industries. It is listed as a B-type epidemic disease by the International Organization for Animal Health (oie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 刘中勇陈茹高小博杨国海朱道中林志雄
Owner INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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