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A detection kit for Haemophilus parasuis and its detection method

A technology of Haemophilus suis and a detection kit, which is applied in the field of molecular biology, can solve problems such as poor specificity, sensitivity needs to be improved, and cumbersome operation, and achieve high sensitivity, short time-consuming, and accurate detection

Active Publication Date: 2017-04-26
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 the Haemophilus parasuis PCR detection kit, which is a primer designed for the traditional 16SrRNA sequence, and other members of Pasteurellaceae and Haemophilus (such as Pasteurella, parabirdophilus Haemobacter, etc.) the specificity effect is not good, and its sensitivity still needs to be improved

Method used

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  • A detection kit for Haemophilus parasuis and its detection method
  • A detection kit for Haemophilus parasuis and its detection method
  • A detection kit for Haemophilus parasuis and its detection method

Examples

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Effect test

Embodiment 1

[0040] 1. Preparation of DNA template:

[0041] The liquid culture was centrifuged at 10000r / min for 5min of the liquid culture of Haemophilus parasuis LC strain, discarded the supernatant, resuspended in sterile water, boiled in boiling water for 10min, frozen at -20℃, thawed, centrifuged at 10000r / min 5min, take the supernatant as DNA template.

[0042] Solid culture: Pick several colonies and suspend them in 100μL of sterilized water. After pipetting and mixing, boil for 10min in boiling water, freeze at -20℃, after thawing, centrifuge at 10000r / min for 5min, and take the supernatant as DNA template.

[0043] 2. Primer design

[0044] According to GenBank HPS mviN gene sequence (CP001321.1), design two pairs of specific primers, and send them to Shanghai Shenggong Biological Engineering Technology Service Co., Ltd. for synthesis. The PCR primer pair sequence is shown in Table 1.

[0045] Table 1 Sequence of PCR primer pair

[0046]

[0047] 2. PCR amplification

[0048] The DNA tem...

Embodiment 2

[0065] The kit of Example 1 with good specificity and high sensitivity 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 did not suffer from the disease were separately prepared for DNA templates. The specific preparation method is as follows: take the heart, lungs, and lymph nodes into PBS for grinding, incubate at room temperature for 10 minutes, centrifuge at 5000 r / min for 5 minutes, and take the supernatant for tissue DNA extraction. The blood first discards the clot and takes the liquid for DNA extraction. Add 50µL of 10% SDS solution and 10µL of 20ug / ml proteinase K to 500µL of tissue supernatant or blood, and water bath at 56°C for 2h; add 500µL of saturated phenol, mix well, centrifuge at 12000r / min for 15min; take the supernatant, Add the same amount of Tris saturated phenol: chloroform: isoamyl alcohol (25:24:1), m...

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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, and belongs to the technical field of molecular biology. Background technique [0002] Haemophlius parasuis (HPS) is a NAD-dependent, non-moving, gram-negative microbacteria belonging to the Pasteurellacea family (Haemophilus), with 15 serotypes , There are still 20% of strains whose serotype cannot be determined. This bacterium can cause multiple serositis, arthritis and meningitis in pigs. Haemophilus parasuis disease caused by this bacterium is also called Glässer's disease (Glässer's disease). HPS can affect pigs at the age of 2 weeks to 4 months. The disease mainly occurs before and after weaning and in the nursery. It is common at 5 to 8 weeks of age. The incidence rate can reach 40%, and the mortality rate can reach 50% in severe cases. With the development of large-scale pig farms in China, HPS infection has a significant incre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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