Primer group for detecting Bordetella pertussis, detection test kit and detection method

A technology for detection kits and detection methods, applied in biochemical equipment and methods, recombinant DNA technology, microbiological determination/inspection, etc., can solve the problem that children cannot produce normal immune responses to pathogens, are not suitable for early diagnosis, early diagnosis Difficulties and other problems, to achieve the effect of saving testing costs, reducing pollution opportunities, and easy and fast operation

Inactive Publication Date: 2010-11-03
SHENZHEN CHILDRENS HOSPITAL
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  • Summary
  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

However, its sensitivity is low, time-consuming, and work-intensive, so it is not suitable for early diagnosis
The specificity of pertussis toxin antibody detection is high, but the positive rate of detection is generally high in about 10 days of the disease course, so early diagnosis is difficult
And some children with immunodeficiency and immature immune system cannot produce normal immune response to pathogens

Method used

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  • Primer group for detecting Bordetella pertussis, detection test kit and detection method
  • Primer group for detecting Bordetella pertussis, detection test kit and detection method
  • Primer group for detecting Bordetella pertussis, detection test kit and detection method

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Experimental program
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Embodiment 1

[0022] The preparation of embodiment 1 primer set

[0023] By comparing and analyzing B. pertussis IS481 gene and PT gene in Genbank database, design amplification primer pair, IS481 gene primer pair sequence is the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2; PT gene A pair of amplification primers, the sequences of the primer pair are SEQ ID NO: 3 and SEQ ID NO: 4.

Embodiment 2 100

[0024] Example 2 Preparation of Bacillus pertussis double PCR detection kit

[0025] The composition of the kit:

[0026] IS481 gene amplification primers:

[0027] P15'-gatgtcggtggcgctgtt-3' (SEQ ID NO: 1),

[0028] P25'-gagaaactggaaatcgcca-3' (SEQ ID NO: 2);

[0029] PT gene amplification primers:

[0030] P15'-gcatgcgtgcagattcgtcgt-3' (SEQ ID NO: 3),

[0031] P25'-aacgcagagggggaagacg-3' (SEQ ID NO: 4).

[0032] Negative control: high pressure double distilled water

[0033] Positive control: pertussis standard strain DNA (or a plasmid that connects two fragments of IS481 gene and PT gene)

[0034] 10x buffer

[0035] MgCl 2

[0036] dNTP Mixture

[0037] Taq enzyme (can be selected from TaKara's rTaq or Ex Taq or other Taq enzymes with excellent performance).

[0038] The kit described in this implementation, under the premise of not affecting the effect of PCR amplification, can prepare buffers of different multiples according to needs; or mix buffer, MgCl 2 An...

Embodiment 3

[0039] Embodiment 3 Double PCR amplifies IS481 gene and PT gene simultaneously

[0040] The test bacteria is the standard strain of pertussis, sourced from China Institute of Pharmaceutical and Biological Products, number (58001).

[0041] 1. Sample DNA (template) extraction:

[0042] Use Beijing Tiangen Bioengineering Company's Bacterial Genomic DNA Extraction Kit to extract sample DNA, DNA OD 260 / OD 280 In the range of 1.6-2.0, the concentration is 10-100ng / μl.

[0043] 2. Reaction system:

[0044] 10*buffer (buffer solution) 2.5ul

[0045] MgCl 2 1.5mM

[0046] dNTP Mixture (2.5mM each) 2.5ul

[0047] Upstream and downstream primers (10pmol / ul) 0.5ul

[0048] Template 5ul

[0049] Ex Taq (TaKara) 0.125ul

[0050] Add high pressure double distilled water to 25ul

[0051] 3. Carry out double PCR amplification reaction:

[0052] (1) Prepare a 20 μl reaction system using TaKaRa Ex Taq from Dalian Bao Biological Engineering Co., Ltd., and finally ad...

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Abstract

The invention discloses a primer group for detecting Bordetella pertussis, a detection test kit and a detection method. The primer group comprises an IS481 gene amplification pair and/or a PT gene amplification primer pair, wherein the primer pair sequence of the IS481 gene amplification pair is a nucleotide sequence shown as SEQ IDNO:1 and SEQ IDNO:2, and the primer pair sequence of the PT gene amplification primer pair is a nucleotide sequence shown as SEQ IDNO:3 and SEQ IDNO:4. The invention detects two genes simultaneously in the same reaction system, and the two genes can be detected through gel electrophoresis. The specificity of detection is guaranteed, and the sensitivity of detection is increased; meanwhile, detection efficiency is greatly increased, and detection cost is saved.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection, and in particular relates to a primer set capable of accurate nucleic acid detection of B. pertussis by means of a PCR amplification method, a kit and a detection method comprising the primer set. Background technique [0002] The current laboratory testing methods for the diagnosis of pertussis infection mainly include bacterial culture, serological testing and molecular biological testing. The culture method is very specific and is considered the "gold standard" for pertussis diagnosis. However, its sensitivity is low, time-consuming, and work-intensive, so it is not suitable for early diagnosis. The specificity of pertussis toxin antibody detection is high, but the positive rate of detection is generally high at about 10 days after the course of the disease, so early diagnosis is difficult. And some children with immunodeficiency and immature immune system cannot produce norma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 王和平马卓娅邓继岿郑跃杰
Owner SHENZHEN CHILDRENS HOSPITAL
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