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Gene diagnosis kit and detection method for major cattle pathogenic bacteria-Pasteurella multocida

A pasteurella and detection method technology, applied in the field of biological science, can solve the problems of limited application and development, long detection time, troublesome preparation, etc., achieve high practical value, avoid the spread of germs, and ensure the effect of rapidity

Active Publication Date: 2014-10-01
KUNMING DIANLONG BIOLOGICAL MEDICINE SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, some of these detection methods have high technical requirements and long detection time.
The preparation of some required materials is troublesome and expensive, and some methods have low sensitivity and specificity. Therefore, the techniques mastered by veterinarians and cattle breeding owners are very difficult and difficult to promote, which limits the application and development of these techniques.

Method used

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  • Gene diagnosis kit and detection method for major cattle pathogenic bacteria-Pasteurella multocida
  • Gene diagnosis kit and detection method for major cattle pathogenic bacteria-Pasteurella multocida
  • Gene diagnosis kit and detection method for major cattle pathogenic bacteria-Pasteurella multocida

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] A genetic diagnostic kit for a serious bovine pathogen-Pasteurella multocida, which includes liquid A, liquid B, liquid C, a box and a foam board;

[0037] Said liquid A is a sample diluent, filled with sterile physiological saline, 5 tubes, 5ml / tube;

[0038]The B solution is the PCR reaction solution, 1 tube, containing 192 μl PCR amplification reaction solution, including 168 μl ddH 2 O, 14.4μl containing Mg 2+ 10×Buffer [Tris-HCl 100mmol / L, pH 9.0, KCl 500mmol / L, MgCl 2 15mmol / L,

[0039] Triton-X100 1%], 3.2 μl dNTP with a concentration of 250 μmol / L, 2.4 μl primer PmF with a concentration of 0.5 μmol / L, 2.4 μl primer PmR with a concentration of 0.5 μmol / L, and 1.6 μl TaqE with an enzyme activity unit of 8 U; PmF is: 5'-ACGCTTTGCCTT GCAAGATAC-3', PmR is: 5'-GATTCACGTAACAAACCAGGC-3'; the C solution is a positive control solution, 1 tube, containing 50 μl of Pasteurella multocida positive template;

[0040] The C solution is a positive control solution, 1 t...

Embodiment 2

[0054] A method for detecting a major bovine pathogen-Pasteurella multocida, using the kit of Example 1, carried out according to the following steps:

[0055] Step (1), take 0.05g of the lung tissue of the acutely ill dead cattle of the test specimen, add the sample diluent, dilute 10 times, and homogenize in an ice bath in a sterile homogenizer;

[0056] Step (2), centrifuge the homogenized sample at a speed of 3000r / min for 5min to obtain the supernatant of the centrifuged sample;

[0057] Step (3), boil the supernatant of 200 μl centrifuged sample for 10 minutes, then immediately place it on ice for 5 minutes to cool;

[0058] Step (4), centrifuge the cooled sample at a speed of 3000r / min for 5min, and take the supernatant as a PCR template;

[0059] In step (5), take the PCR template and C solution with a volume ratio of 1:1, respectively, and add them to the PCR reaction solution. The volume ratio of the PCR template to the PCR reaction solution is 1:24, and the volume ...

Embodiment 3

[0063] A method for detecting a major bovine pathogen-Pasteurella multocida, using the kit of Example 1, carried out according to the following steps:

[0064] Step (1), take 0.05g of fresh nasal swab fluid of the tested specimen, add sample diluent, dilute 20 times, and homogenize in an ice bath in a sterile homogenizer;

[0065] Step (2), centrifuge the homogenized sample at a speed of 4000r / min for 10min to obtain the supernatant of the centrifuged sample;

[0066] Step (3), boil the supernatant of 200 μl centrifuged sample for 10 minutes, then immediately place it on ice for 10 minutes to cool down;

[0067] Step (4), centrifuge the cooled sample at a speed of 4000r / min for 10min, and take the supernatant as a PCR template;

[0068] In step (5), take the PCR template and C solution with a volume ratio of 1:1, respectively, and add them to the PCR reaction solution. The volume ratio of the PCR template to the PCR reaction solution is 1:24, and the volume of the C solution ...

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Abstract

The invention provides a gene diagnosis kit and detection method for major cattle pathogenic bacteria-Pasteurella multocida and belongs to the technical field of bioscience. The kit and the detection method are designed by taking a pair of primers as the main body, and the primers are designed according to the Pasteurella multocida gene conservative area sequence. According to the invention, the polymerase chain reaction (PCR) technology is adopted for qualitatively detecting the specificity DNA fragments of cattle pathogenic bacteria-Pasteurella multocida simply, conveniently and quickly, the specificity is good and the sensitivity is high; the kit and the detection method can be used for clinical detection of cattle pasteurellosis, bacteria tracking and detecting in all cattle farming periods and monitoring of the cattle farming environment and avoid pathogen transmission and outbreak, thereby achieving a very high utility value.

Description

[0001] technical field [0002] The invention belongs to the technical field of biological sciences, and in particular relates to a diagnostic kit and a detection method for major bovine pathogenic bacteria, mainly for Pasteurella multocida ( Pasteurella multocida ) kits and detection methods for genetic diagnosis. Background technique [0003] Pasteurellosis of cattle, also known as bovine hemorrhagic septicemia, is an acute, febrile infectious disease of cattle, characterized by high fever, pneumonia and extensive hemorrhage of internal organs. The pathogen is Pasteurella multocida, the bacteria are small, club-shaped or short rod-shaped bacteria with blunt ends, the size is 1-1.5μm×0.3-0.6μm, mostly scattered, non-motile, and do not form spores , negative Gram stain. Pasteurella multocida is pathogenic to various livestock and wild animals, and can occur regardless of age. Cattle are susceptible to the disease with cattle and dairy cows, among which young and middle-ag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
CPCC12Q1/686C12Q2531/113
Inventor 邓先余林连兵夏雪山
Owner KUNMING DIANLONG BIOLOGICAL MEDICINE SCI & TECH
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