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Haemophilus parasuis detection kit and detection method thereof

A technology of Haemophilus suis and detection kit, applied in the field of molecular biology, can solve the problems of poor specificity, need to improve sensitivity, complicated operation, etc., and achieve the effects of high sensitivity, short time consumption and accurate detection

Active Publication Date: 2015-06-17
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Although conventional physiological and biochemical methods can accurately identify the strains to be tested, the operation is cumbersome and time-consuming
Chinese patent CN102220426A discloses Haemophilus parasuis PCR detection kit, and this kit is the primer designed for traditional 16SrRNA sequence, to other members of Pasteurellaceae and Haemophilus (such as Pasteurella, parabirdophilus Haemobacter, etc.) specificity is not good, and its sensitivity still needs to be improved

Method used

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  • Haemophilus parasuis detection kit and detection method thereof
  • Haemophilus parasuis detection kit and detection method thereof
  • Haemophilus parasuis detection kit and detection method thereof

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Experimental program
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Embodiment 1

[0040] 1. Preparation of DNA template:

[0041] For liquid culture, take Haemophilus parasuis LC strain and centrifuge at 10,000r / min for 5min, discard the supernatant, resuspend in sterilized water, boil in boiling water for 10min, freeze at -20°C, and centrifuge at 10,000r / min after thawing After 5 min, the supernatant was taken as a DNA template.

[0042] Solid culture Pick several colonies and suspend them in 100 μL sterilized water, mix them by blowing and aspiration, boil in boiling water for 10 minutes, freeze at -20°C, after thawing, centrifuge at 10,000 r / min for 5 minutes, and take the supernatant as a DNA template.

[0043] 2. Primer Design

[0044] According to GenBank published HPS mviN gene sequence (CP001321.1), two pairs of specific primers were designed and sent to Shanghai Sangon Bioengineering Technology Service Co., Ltd. for synthesis. The sequences of PCR primer pairs are shown in Table 1.

[0045] Table 1 PCR primer pair sequences

[0046]

[004...

Embodiment 2

[0065] The kit with good specificity and high sensitivity in Example 1 was used to detect Haemophilus parasuis in dead animal tissues and blood samples.

[0066] 1. Preparation of DNA template:

[0067] The dead pig No. 1 that died after the disease and the dead pig No. 2 that was not sick were prepared as DNA templates respectively. The specific preparation method is as follows: take heart, lung, and lymph nodes, add PBS to grind, incubate at room temperature for 10 minutes, centrifuge at 5000 r / min for 5 minutes, and take supernatant for tissue DNA extraction. Blood clots are discarded first, and the liquid is taken for DNA extraction. Add 50 μL of 10% SDS solution and 10 μL of 20ug / ml proteinase K to 500 μL of tissue supernatant or blood, and bathe in water at 56°C for 2 hours; add 500 μL of Tris saturated phenol, mix well, and centrifuge at 12,000 r / min for 15 minutes; take the supernatant, Add an equal amount of Tris saturated phenol: chloroform: isoamyl alcohol (25:24:...

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Abstract

The invention relates to a haemophilus parasuis detection kit and a detection method thereof, and belongs to the technical field of molecular biology. The detection kit comprises a primer pair, PCR Mix, a position control and dd H2O. The haemophilus parasuis detection kit disclosed by the invention has the primer pair designed according to an mviN gene sequence in a high conserved domain, is good in specificity, and can accurately distinguish the haemophilus parasuis strain LC from haemophilus paragallinarum, actinobacillus pleuropneumoniae, pasteurella muhocida, arcanobacterium pyogenes, staphylococcus aureus and streptococcus suis; the detection kit and the detection method provided by the invention are high in sensitivity, short in consumed time, accurate in detection, and important in significance of monitoring haemophilus parasuis reproduction, disease occurrence and prevalence as well as timely control of the disease.

Description

technical field [0001] The invention relates to a kit, in particular to a detection kit for Haemophilus parasuis and a detection method thereof, belonging to the technical field of molecular biology. Background technique [0002] Haemophilus parasuis (HPS) is an NAD-dependent, non-motile, Gram-negative small bacillus belonging to the genus Haemophilus of the Pasteurellaceae family, with 15 serotypes , and 20% of the strain serotypes could not be determined. The bacteria can cause polyserositis, arthritis and meningitis in pigs, and the Haemophilus parasuis disease caused by the bacteria is also called Grass disease (Gl?sser's disease). HPS can affect pigs aged 2 weeks to 4 months, mainly around weaning and in the nursery, usually at 5 to 8 weeks, with an incidence rate of up to 40% and a mortality rate of up to 50% in severe cases. With the development of large-scale pig farms in my country, HPS infection has a tendency to increase significantly, and has become one of the ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
CPCC12Q1/689
Inventor 于江王金宝吴家强张玉玉杜以军李俊陈智
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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