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Haemophilus parasuis real-time fluorescent quantitative PCR (polymerase chain reaction) detection method

A real-time fluorescence quantitative, Haemophilus suis technology, applied in the field of fluorescence quantitative PCR detection, can solve the problems of limited anti-interference ability of related bacteria and no effective verification of versatility.

Inactive Publication Date: 2011-09-07
GUANGXI VETERINARY RES INST
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this study is limited to HPS serotype 5, and the universality of other serotypes has not been effectively verified, and its method can only distinguish Haemophilus parasuis from Streptococcus and Pasteurella, and the ability to resist interference from related bacteria is limited

Method used

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  • Haemophilus parasuis real-time fluorescent quantitative PCR (polymerase chain reaction) detection method
  • Haemophilus parasuis real-time fluorescent quantitative PCR (polymerase chain reaction) detection method
  • Haemophilus parasuis real-time fluorescent quantitative PCR (polymerase chain reaction) detection method

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preparation example Construction

[0038] 1.4 Preparation of template DNA

[0039] Pick a single HPS colony and streak it on TSA agar medium containing 5% horse serum and 0.4 mg / mL NAD, place in 5% CO 2 Incubate at 37°C for 36 hours in the environment, elute the bacteria with normal saline, and extract bacterial DNA with a bacterial genome DNA extraction kit, and the extraction method is carried out according to the instructions.

[0040] 1.5 Qualitative PCR amplification

[0041] The designed HPS primers were used to amplify the extracted DNA template by PCR. The PCR reaction system was 50 μL, including 12 μL of PCR Taq Mix, 1 μL of upstream and downstream primers (25 pmol / μL), 5 μL of template DNA, and 31 μL of ultrapure water. The reaction conditions were pre-denaturation at 95°C for 5min; 35 cycles of 95°C for 20S, 60°C for 20S, 72°C for 20S, and finally 72°C for 5min.

[0042] 1.6 Preparation of standard positive plasmid

[0043] After the PCR product was electrophoresed on 2% agarose gel, the PCR produ...

Embodiment

[0065] Example Detection of clinical samples

[0066] The established real-time fluorescent quantitative PCR detection method for Haemophilus parasuis (method A) was used to detect 10 clinically collected pericardial fluids and joint fluids suspected of HPS infection, and they were separated and cultured from bacteria (method B) and conventional PCR (method B) C) for comparison.

[0067] Such as Figure 7 As shown, the specific steps of the real-time fluorescent quantitative PCR detection method for Haemophilus parasuis are as follows:

[0068] DNA sample preparation: take the sample to be tested for extraction and purification of total DNA;

[0069] Pericardial fluid treatment: draw 10 microliters of pericardial fluid into an EP tube containing 100 microliters of ultrapure water, boil for 10 minutes, centrifuge at 12,000 rpm for 5 minutes, and take 3 microliters of the supernatant as a DNA template.

[0070] Joint fluid treatment: pipette 10 microliters of joint fluid int...

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Abstract

The invention discloses a haemophilus parasuis real-time fluorescent quantitative PCR (polymerase chain reaction) detection method which selects the 16S rRNA gene (AB004028) which has high conservation and specificity and is the main target gene of bacteria classification and determination as the amplification target gene, takes a high-conservation area of the gene as an amplification area to design a specificity primer, and establishes a haemophilus parasuis real-time fluorescent quantitative PCR detection method embedding a fluorescent dye SYBR Green I and DNA, wherein the DNA of the initial template of the haemophilus parasuis 16S rRNA gene can be accurately quantified according to the standard curve of the known standard product, and equated to the number of bacteria. The method disclosed by the invention has strong generality and high specificity, can detect many HPS serotypes, is free from influence of related bacteria, and can be applied to quick and accurate quantitative detection of the haemophilus parasuis seriously impairing development of the pig industry.

Description

technical field [0001] The invention relates to fluorescent quantitative PCR detection, in particular to a real-time fluorescent quantitative PCR detection method for Haemophilus parasuis. Background technique [0002] Haemophilus parasuis (HPS) belongs to the genus Haemophilus in the family Pasteurellaceae. HPS usually colonizes the upper respiratory tract of pigs and does not cause disease in pigs under normal conditions. However, when the body is in a state of stress or the immune function declines, it can break through the defense mechanism of the upper respiratory tract and invade multiple organs of the body, causing multiple serositis, joint Severe cases lead to increased body temperature, dyspnea and even death, bringing huge economic losses to the pig industry. [0003] The current diagnosis of HPS is mainly bacterial isolation and culture and conventional PCR methods. The culture conditions of HPS are demanding, requiring NAD, horse serum and 5% CO 2 environment,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/21
Inventor 李军陈泽祥杨威谢宇舟彭昊禤雄标谢永平许力干胡帅马春霞潘艳
Owner GUANGXI VETERINARY RES INST
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