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Primer, kit and method for detecting bordetella pertussis BP

A detection reagent, pertussis technology, applied in the field of molecular biology, can solve the problems of difficult to meet the demand, cumbersome detection of pertussis bacillus BP, etc., and achieve the effect of rapid detection, high sensitivity and simple reaction procedure

Inactive Publication Date: 2019-04-26
博迪泰(厦门)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a primer, a kit and a test method for detecting B. pertussis BP, and solve the problem that the detection of B. pertussis BP is cumbersome and difficult to meet the needs in the prior art.

Method used

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  • Primer, kit and method for detecting bordetella pertussis BP
  • Primer, kit and method for detecting bordetella pertussis BP
  • Primer, kit and method for detecting bordetella pertussis BP

Examples

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Embodiment 1

[0032] Embodiment 1: Primer and kit

[0033] 1. Primer design

[0034] The primers were designed according to the pathogenic gene sequence of respiratory tract infection in the NCBI gene database, referring to the TwistDx instructionmanual and Primer-BLAST primer design principles. The primer length is about 30-35nt. Since there is no primer design software for RPA at present, a large number of primers were designed and synthesized in the previous work of the present invention, and the primers with high sensitivity and good specificity were screened out for use in the present invention (Table 1).

[0035] 2. Primer synthesis

[0036]According to the primer sequences shown in Table 1, sequence synthesis was carried out. It is characterized in that the forward primer sequence of the B. pertussis BP gene is shown in SEQ ID NO: 1, modified to add Biotin; the reverse primer sequence is shown in SEQ ID NO: 2, modified to add FITC or Dig. The internal quality control forward prime...

Embodiment 2

[0040] Example 2: Detection of B. pertussis BP

[0041] In this example, the kit described in Example 1 is used to detect B. pertussis BP. The method used for the detection of B. pertussis BP is the recombinase polymerase constant temperature gene amplification method combined with solid-phase chromogenic technology. The specific steps are as follows:

[0042] 1. Clinical sample preparation

[0043] Collect nasal swabs, nasopharyngeal swabs, and throat swabs. The collection conditions are: positive clinical samples (48 cases) confirmed to have B. pertussis BP after laboratory inspection (fluorescent quantitative PCR), and no Negative clinical samples of pertussis BP (52 cases). Clinical samples were stored at -80°C.

[0044] 2. Nucleic acid extraction from samples

[0045] In this embodiment, DNA column chromatography extraction or automatic sample nucleic acid extraction instrument is used to extract nucleic acid from clinical samples.

[0046] 3. Preparation of positive ...

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Abstract

The invention discloses a primer, kit and method for detecting bordetella pertussis (BP), and belongs to the field of molecular biology. The primer is formed by a forward primer with a nucleotide sequence as shown in SEQ ID NO: 1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 2; the forward primer is labeled with biotin; and the reverse primer is labeled with FITC (Fluorescein Isothiocyanate) or Dig. According to the invention, a recombinase-polymerase nucleic acid amplification technology and a solid-phase developing technology are adopted; only isothermal amplification is performed after DNA (Deoxyribonucleic acid) extraction is performed on a clinical sample; a PCR (Polymerase Chain Reaction) instrument does not need to perform a heat cycling reaction; and a detection result can be directly read by naked eyes from a solid-phase developing test strip. The primer disclosed by the invention is strong in specificity for detecting the bordetella pertussis BP, andhigh in amplification efficiency. The kit and the detection method disclosed by the invention are simple in reaction program, capable of effectively and rapidly detecting the bordetella pertussis BP,and suitable for convenient, rapid and accurate detection of pertussis on site.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a primer, a kit and a detection method for detecting bacillus pertussis BP. Background technique [0002] Bacillus pertussis is the pathogenic bacterium of whooping cough in humans, which is mainly transmitted by droplets. The incubation period for whooping cough is 1 to 2 weeks. In the early stage of onset (catarrhal stage), there is only mild cough. Bacteria multiply on the mucous membranes of the trachea and bronchi at this time and are discharged with droplets, which is the most contagious. Paroxysmal spasmodic cough (convulsive phase) occurs after 1 to 2 weeks. At this time, the bacteria release toxins, which cause the cilia of the mucosal epithelial cells to move out of balance, and a large amount of viscous secretions cannot be discharged, which stimulates the receptors in the mucosa to produce a strong spasm. Make a special high-pitched chicken crowing sound. The form...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6844C12Q2521/507C12Q2522/101C12Q2565/625
Inventor 张睿李博安
Owner 博迪泰(厦门)生物科技有限公司
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