Kit of actinobacillus pleuropneumoniae and use thereof

A technology for porcine pleuropneumonia and detection kit, which is applied to the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of long cycle, unsuitable basic application, inability to detect pathogens, etc., and achieves simple and convenient operation and high specificity. , the effect of high sensitivity

Inactive Publication Date: 2009-12-09
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The etiological diagnosis method requires bacterial isolation, culture and identification, and the cycle is long; the serological diagnosis method includes complement fixation reaction, fluorescent antibody, indirect hemagglutination test and enzyme-linked immunosorbent assay (ELISA), etc. The disadvantages of the serological diagnosis method are It can only detect the antibody of Actinobacillus pleuropneumoniae in animals but cannot detect the pathogen; the molecular biology diagnosis method mainly includes reverse transcription polymerase chain reaction (RT-PCR) and fluorescent nucleic acid probe. This method is fast and sensitive, but requires the use of special instruments, such as PCR machines, and the preparation of fluorescent probes is relatively expensive, which is not suitable for grassroots applications

Method used

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  • Kit of actinobacillus pleuropneumoniae and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1, the preparation of Actinobacillus pleuropneumoniae detection kit

[0059] 1. Synthesis of primers

[0060] Artificially synthesize the following 3 pairs of primers:

[0061] F3: 5'-GACACCTTAAAATTACTGATGTG-3';

[0062] B3: 5'-CGTTGATATTATCATCACCGTC-3';

[0063] FIP: 5′-GACCGAATCCGTATCATGATAACCGTTTTCGGAAGTGAAATTCCGACG-3′;

[0064] BIP: 5′-GGAATTTGCTGACCGCAGTTTTTGCCAAATAATGCCATACCTTG-3′;

[0065] LF: 5'-GAGTAGATAATGACTTAATGTTATTCGG-3';

[0066] LB: 5'-TATAACTCGTGATGAACTAGGTAAA-3'.

[0067] 2. Preparation of LAMP reaction solution

[0068] Each 23 μL LAMP reaction solution contains the following components: 0.5 μmol Tris-HCl, 0.25 μmol KCl, 0.25 μmol (NH 4 ) 2 SO 4 , Tween20 0.025 μL, 0.2 μmol MgSO 4 , 20 μmol betaine (Betaine), four deoxynucleotides (dNTPs) each 0.035umol, 0.04 μmol upstream inner primer (FIP), 0.04 μmol downstream inner primer (BIP), 0.004 μmol upstream outer primer (F3), 0.004 μmol Downstream outer primer (B3), 0.02 μmol upstream...

Embodiment 2

[0071] Embodiment 2, preparation of Actinobacillus pleuropneumoniae detection kit

[0072] 1. Synthesis of primers

[0073] Same as Step 1 of Example 1.

[0074] 2. Preparation of LAMP reaction solution

[0075] Each 23 μL LAMP reaction solution contains the following components: 0.5 μmol Tris-HCl, 0.25 μmol KCl, 0.25 μmol (NH 4 ) 2 SO 4 , Tween20 0.025μL, 20μmol MgSO 4 , 20 μmol betaine (Betaine), four deoxynucleotides (dNTPs) each 0.035umol, 0.06 μmol upstream inner primer (FIP), 0.06 μmol downstream inner primer (BIP), 0.008 μmol upstream outer primer (F3), 0.008 μmol Downstream outer primer (B3), 0.04 μmol upstream circular primer (LF), 0.04 μmol downstream circular primer (LB), 16 U of Bst DNA polymerase, sterile double distilled water.

[0076] 3. Assembly of kit

[0077] Same as Step 3 of Example 1.

Embodiment 3

[0078] Embodiment 3, preparation of Actinobacillus pleuropneumoniae detection kit

[0079] 1. Synthesis of primers

[0080] Same as Step 1 of Example 1.

[0081] 2. Preparation of LAMP reaction solution

[0082] Each 23 μL LAMP reaction solution contains the following components: 0.5 μmol Tris-HCl, 0.25 μmol KCl, 0.25 μmol (NH 4 ) 2 SO 4 , Tween20 0.025μL, 10μmol MgSO 4 , 20 μmol betaine (Betaine), four deoxynucleotides (dNTPs) each 0.035umol, 0.05 μmol upstream inner primer (FIP), 0.05 μmol downstream inner primer (BIP), 0.006 μmol upstream outer primer (F3), 0.006 μmol Downstream outer primer (B3), 0.03 μmol upstream circular primer (LF), 0.03 μmol downstream circular primer (LB), 16 U of Bst DNA polymerase, sterile double distilled water.

[0083] 3. Assembly of kit

[0084] Same as Step 3 of Example 1.

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Abstract

The invention discloses a detection kit for Actinobacillus pleuropneumoniae and its application. The kit provided by the present invention includes three pairs of primers, namely an inner primer pair, an outer primer pair and a circular primer pair combined with the 3' end 1000bp gene in the Actinobacillus pleuropneumoniae Gen Bank Accession Number AF021919 sequence. The kit also includes a loop-mediated isothermal amplification reagent, a positive control, a negative control and a fluorescent chromogenic reagent, and the positive control is the DNA of Actinobacillus pleuropneumoniae. The invention also provides a method for detecting whether the animal carries the Actinobacillus pleuropneumoniae by using the kit of the invention. The kit of the present invention has high detection sensitivity, 6-10 copies can detect the target DNA, is simple and convenient to operate, and is especially suitable for the detection of pathogens carried out at the grassroots level and the detection of Actinobacillus pleuropneumoniae in possibly contaminated animal food .

Description

technical field [0001] The invention relates to a detection kit for Actinobacillus pleuropneumoniae and its application. Background technique [0002] Porcine contagious pleuropneumonia is a fatal respiratory infectious disease of pigs caused by Actinobacillus pleuropneumoniae (APP), characterized by acute hemorrhagic fibrinous pleuropneumonia and chronic focal necrotizing pneumonia, acute cases Most of them die, and subacute and chronic cases are often able to survive, but they can cause growth retardation. It is one of the five major diseases that seriously endanger the pig industry in the world. In the past 20 years, porcine infectious pleuropneumonia has broken out in many regions such as Europe, Asia, and the Americas. Taiwan Province of my country and many provinces in China have reported incidences, and the trend is increasing year by year. Pigs with acute over-tolerance often suffer from chronic lung lesions (cyst formation, abscess and pleural adhesions) and become...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 吴文学李佳禾张跃伟李旭妮黄书林
Owner CHINA AGRI UNIV
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