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Test kit of actinobacillus pleuropneumoniae and use thereof

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

Inactive Publication Date: 2009-02-04
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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  • Test kit of actinobacillus pleuropneumoniae and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1. Preparation of a detection kit for Actinobacillus pleuropneumoniae

[0059] 1. Synthesis of primers

[0060] Synthesize the following 3 pairs of primers:

[0061] F3: 5'-GACACCTTAAAATTTACTGATGTG-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. Prepare LAMP reaction solution

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

Embodiment 2

[0071] Example 2. Preparation of a detection kit for Actinobacillus pleuropneumoniae

[0072] 1. Synthesis of primers

[0073] Same as step one in Example 1.

[0074] 2. Prepare LAMP reaction solution

[0075] Each 23μL LAMP reaction solution contains the following components: 0.5μmol Tris-HCl, 0.25μmol KCl, 0.25μmol (NH 4 ) 2 SO 4 , Tween200.025μL, 20μmol MgSO 4 , 20μmol Betaine (Betaine), four deoxynucleotides (dNTPs) 0.035umol each, 0.06μmol upstream inner primer (FIP), 0.06μmol downstream inner primer (BIP), 0.008μmol upstream outer primer (F3), 0.008μmol Downstream outer primer (B3), 0.04μmol upstream loop primer (LF), 0.04μmol downstream loop primer (LB), 16U Bst DNA polymerase, sterilized double distilled water.

[0076] Third, the assembly of the kit

[0077] Same as the step three of Example 1.

Embodiment 3

[0078] Example 3. Preparation of a detection kit for Actinobacillus pleuropneumoniae

[0079] 1. Synthesis of primers

[0080] Same as step one in Example 1.

[0081] 2. Prepare LAMP reaction solution

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

[0083] Third, the assembly of the kit

[0084] Same as the step three of Example 1.

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Abstract

The invention discloses a test kit for actinobacillus pleuropneumoniae and an application thereof. the kit which is provided by the invention comprises three pairs of primers, namely, the inside primer pair, the outside primer pair and the circular primer pair which are all combined with 3' terminal 1000bp gene combination in an actinobacillus pleuropneumoniae Gen Bank Accession Number AF021919 sequence. The kit also comprises loop-mediated isothermal amplification reagents, positive controls, negative controls and fluorescent chromogenic reagents, and the positive control are actinobacillus pleuropneumoniae DNA. The invention also provides a method for testing whether animals carry the actinobacillus pleuropneumoniae by applying the kit of the invention. The kit of the invention has high testing sensitivity, testing of target DNA by 6 to 10 copies, simple and convenient operation, and is especially suitable for pathogen tests being carried out on spot and the tests of actinobacillus pleuropneumoniae of the possibly-polluted animal food.

Description

Technical field [0001] The invention relates to a detection kit for Actinobacillus pleuropneumoniae and its application. Background technique [0002] Porcine infectious pleuropneumonia is a fatal respiratory infectious disease in pigs caused by Actinobacillus pleuropneumoniae (APP). It is characterized by acute hemorrhagic fibrinous pleuropneumonia and chronic focal necrotizing pneumonia. Acute cases Most of them die. Subacute and chronic cases can often be tolerated, but they can cause growth retardation. It is one of the five major diseases that seriously harm the pig industry in the world. In the past 20 years, porcine infectious pleuropneumonia has outbreaks in many regions such as Europe, Asia, and the Americas. The incidence has been reported in Taiwan Province and many provinces in my country, and it has been increasing year by year. Acutely tolerant pigs often suffer from chronic lung diseases (cyst formation, abscesses and pleural adhesions) and become carriers of bacte...

Claims

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Application Information

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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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