Loop-mediated isothermal amplification-based nucleic acid test method for Riemerella anatipestifer

A duck plague Riemer's, ring-mediated isothermal technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of expensive fluorescent quantitative PCR instruments, limited popularization and use, cumbersome operation process, etc., to achieve Ease of application and promotion, simple equipment requirements, and the effect of reducing pollution

Active Publication Date: 2012-04-18
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, because the fluorescent quantitative PCR technology requires expensive instruments and cumbersome operating procedures, the popularization and use of this technology in grassroots laboratories and quarantine institutions has been limited.

Method used

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  • Loop-mediated isothermal amplification-based nucleic acid test method for Riemerella anatipestifer
  • Loop-mediated isothermal amplification-based nucleic acid test method for Riemerella anatipestifer
  • Loop-mediated isothermal amplification-based nucleic acid test method for Riemerella anatipestifer

Examples

Experimental program
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Effect test

example 1

[0022] 1. Test samples: Riemerella anatipestifer ATCC 11845, Escherichia coli O46, Staphylococcus aureus ATCC6538, Pasteurella multocida CVCC493, Salmonella CMCC 50083.

[0023] 2. Nucleic acid extraction: In the present invention, the sample DNA extraction methods include two methods.

[0024] Method 1: Phenol imitation extraction hair method

[0025] (1) Riemerella anatipestifer was cultured in tryptic soybean broth at 37°C for 24 hours with shaking in a water bath, and Escherichia coli, Staphylococcus aureus, Pasteurella multocida, and Salmonella were cultured in LB broth for 24 hours with shaking in a water bath at 37°C;

[0026] (2) Take 500 ul of the bacterial suspension from the above-mentioned bacterial suspension and put it in the EP tube (the throat swab is soaked with 500 ul of normal saline to get the immersion solution, and various tissues of dead ducks are homogenized with 500 ul of normal saline to get the homogenate) Centrifuge at 1000rpm for 5min, discard the...

example 2

[0046] 1. The Riemerella anatipestifer ATCC 11845 bacterium liquid of overnight culture is diluted 10 times to (10 1 -10 8 ), take each 100ul plate count of each dilution bacterial solution. The number of colonies per milliliter of the stock solution was measured to be 4.0x10 8 CFU / mL, after 10-fold dilution, nucleic acid was extracted according to the pyrolysis method in Method 2 above, and 1ul of the supernatant was used as a template for LAMP amplification.

[0047] 2. The reaction composition system of Riemerella anatipestifer LAMP detection is as follows:

[0048] 2xU-LAMP reaction mixture (2xU-LAMP reaction mixture is a purchased kit, which consists of: 40mM Tris-HCl (pH 8.8), 20mM KCl, 20mM (NH4) 2 SO 4 , 10mM MgSO 4 , 0.2% Triton X-100, 2.4mM dNTPs, 1.6M betaine;) 10ul;

[0049] 10x primer mixture 1ul, its composition is: primer F3 is 2uM, primer B3 is 2uM, primer BIP is 12uM, primer FIP is 12uM;

[0050] Template DNA 1ul, Bst polymerase 0.75ul, water up to 20ul...

example 3

[0054] 1. Take 10 livers of ducks artificially infected with Riemerella anatipestifer and 20 heart, liver and brain tissues of suspected Riemerella anatipestifer sent for inspection from different regions in Sichuan, Chongqing, Shandong, and Henan provinces for implementation Nucleic acid extraction method one (phenol imitation extraction hair method) method extracts nucleic acid in the example 1, is compared with the same treatment of normal duck liver tissue simultaneously;

[0055] 2. The reaction composition system of Riemerella anatipestifer LAMP detection is as follows:

[0056] 2xU-LAMP reaction mixture (2xU-LAMP reaction mixture is a purchased kit, which consists of: 40mM Tris-HCl (pH 8.8), 20mM KCl, 20mM (NH4) 2 SO 4 , 10mM MgSO 4 , 0.2% Triton X-100, 2.4mM dNTPs, 1.6M betaine;) 10ul;

[0057] 10x primer mixture 1ul, its composition is: primer F3 is 2uM, primer B3 is 2uM, primer BIP is 12uM, primer FIP is 12uM;

[0058] Template DNA 1ul, Bst polymerase 0.75ul, wat...

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Abstract

The invention discloses a loop-mediated isothermal amplification-based nucleic acid test method for Riemerella anatipestifer. The method includes the following steps: a specific primer sequence is designed according to the gyrB gene of the Riemerella anatipestifer, the DNA (deoxyribonucleic acid) of a sample to be tested is extracted as a template, and LAMP (loop-mediated isothermal amplification) reaction is carried out; an LAMP result is analyzed, and if the LAMP amplification result is positive, then the sample to be tested contains the Riemerella anatipestifer. The test method aimed at the Riemerella anatipestifer, which is provided by the invention, is simple, fast, specific, sensitive and economic, the result judgement method is simple, and the test method is suitable for use in clinic or grass-roots labs, and has a broad application prospect.

Description

technical field [0001] The invention relates to a nucleic acid detection method for loop-mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) of Riemerella anatipestifer. Background technique [0002] Riemerella anatipestifer (RA) disease is a contagious disease mainly affecting ducks, turkeys and other poultry. The main pathological changes of the disease are fibrinous pericarditis, perihepatitis, air sac inflammation, meningitis, caseous salpingitis, etc. The incidence rate is high, and the mortality rate is also high. Most of the ducks that are resistant to overcrowding become stiff ducks, and their growth is retarded. cause huge economic losses. [0003] Since Reiemer first reported the disease in 1904 to the present, the disease is prevalent in the world, and the morbidity and mortality are generally 10-30%, but some duck farms reach more than 95%. The harm of the disease to the aquaculture industry not only directly causes animal disease a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 程安春王雪平汪铭书朱德康陈孝跃
Owner SICHUAN AGRI UNIV
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