DNA fragment, primer, probe and kit for detecting four kinds of abalone bacteria and specifically detecting pertussis abalone bacteria and application

A technology of primer probe and whooping cough, which is applied in the field of microbial detection, can solve the problems of false negative, low sensitivity, and long test period, and achieve the effect of strong specificity and high sensitivity

Active Publication Date: 2021-11-30
SHANGHAI BIOGERM MEDICAL TECH CO LTD BEIJING BRANCH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is highly specific, fast and economical, and is currently one of the reliable methods for diagnosing Bordetella pertussis infection. However, the sensitivity is low, and the cultivation of Bordetella pertussis is difficult and prone to false negatives.
[0007] (2) Western blot method: using anti-pertussis toxin monoclonal antibody to detect pertussis toxin in nasopharyngeal secretions of pertussis patients with high specificity , but the operation steps are complex and cumbersome
[0008] (3) Monoclonal antibody colony imprinting method: use anti-Bordetella pertussis lipopolysaccharide and filamentous hemagglutinin monoclonal antibody colony imprinting ELISA to detect Bordetella pertussis Bacteria, 48h can appear clear blue spot positive blot reaction on the nitrocellulose membrane, this method has a long test cycle, laborious and time-consuming detection, and is not convenient for large-scale detection and promotion
However, the IS481 gene is not specific for the detection of Bordetella pertussis. In addition to being able to detect Bordetella pertussis, there is crossover between the detection of Bordetella pertussis, resulting in positive detections of Bordetella hallii and Bordetella bronchus.
There is currently no sequencing and typing method for the 4 different Bordetella species

Method used

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  • DNA fragment, primer, probe and kit for detecting four kinds of abalone bacteria and specifically detecting pertussis abalone bacteria and application
  • DNA fragment, primer, probe and kit for detecting four kinds of abalone bacteria and specifically detecting pertussis abalone bacteria and application
  • DNA fragment, primer, probe and kit for detecting four kinds of abalone bacteria and specifically detecting pertussis abalone bacteria and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Example 1 The selection of detection target and the design of primers and probes

[0114] 1. Selection of four Bordetella sequencing targets and primer design

[0115] Download all the genome sequences of Bordetella pertussis, Bordetella parapertussis, Bordetella hallii and Bordetella bronchus from Genebank, and find out the difference regions of the genomes of these 4 pathogens through multiple alignment and analysis. The region was screened and verified, and finally the region where the BP2463 gene was located at the 2574316th to 2576520th position of the genome was determined to be the best detection region. In the above region, there were four conserved regions of Bordetia, and there were also specificities of each Bordetia Area.

[0116] Several target genes for screening are listed below, including the final determined optimal target gene BP2463 region and BP3771 gene. Primers and probes are designed for these target gene sequences, and fluorescent quantitative P...

Embodiment 2

[0129] Example 2 The kit for Bordetella detection

[0130] This embodiment provides a kit for detecting Bordetella, which includes the following components: nucleic acid amplification reaction solution, specific primers for detecting Bordetella (SEQ ID NO.1-2) , Bordetella pertussis reaction liquid, positive control, negative control.

[0131] Wherein, the components and concentrations in the nucleic acid amplification reaction solution are shown in Table 3.

[0132] The composition of table 3 nucleic acid amplification reaction solution

[0133] Reagent name concentration Buffer Buffer 5× dN(U)TP Mix 20mM UDG 2U / μL MgCl 2 the solution

200mM DNA polymerase 5U / μL PCR enhancer 30％

[0134] Wherein, the composition and concentration of the PCR enhancer are shown in Table 4.

[0135] Table 4 Composition of PCR enhancer

[0136] Reagent name Concentration(g / L) Manganese chloride 2.0 SSB single ch...

Embodiment 3

[0140] Example 3 Method for specific detection of Bordetella pertussis and simultaneous detection of 4 different Bordetella species

[0141] This embodiment provides a method for the specific detection of Bordetella pertussis and the simultaneous detection of four different Bordetella pertussis using the kit in Example 2, including the following steps:

[0142] (1) Reagent pretreatment: Take out the nucleic acid amplification reaction solution, pertussis reaction solution, sequencing primers (SEQ ID NO.1-2), positive control and negative control respectively, equilibrate to room temperature, shake and mix well, and centrifuge instantly;

[0143] (2) Preparation of PCR reaction solution for sequencing and typing of Bordetella: Prepare nucleic acid amplification reaction solution and sequencing primers in proportion, 20 μL in each PCR reaction tube, of which, 16 μL of nucleic acid amplification reaction solution per person , Sequencing primer 4μL / person;

[0144] (3) Preparatio...

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Abstract

The invention relates to the technical field of microbiological detection, and particularly relates to a DNA fragment, primer, probe and kit for detecting four kinds of abalone bacteria and specifically detecting pertussis abalone bacteria and application. The DNA fragment and specific primer for detecting the pertussis abalone bacteria, the abalone bacteria and the bordetella bronchiseptica can achieve sequencing and typing detection of the four kinds of abalone bacteria and have high specificity and accuracy. The invention further provides a primer probe set for specifically detecting the pertussis abalone bacteria. The pertussis abalone bacteria can be specifically detected through a multiple fluorescence PCR method, high sensitivity, specificity and repeatability are achieved, and an effective tool and method are provided for detecting the abalone bacteria.

Description

technical field [0001] The invention relates to the technical field of microbial detection, in particular to DNA fragments, primers, probes and kits for detecting four kinds of Bordetella and specific detection of Bordetella pertussis and applications thereof. Background technique [0002] Bordetella pertussis (BP) is a bacterium of the genus Bordetella, a small Gram-negative bacillus, and the pathogen of whooping cough in humans. The host can cause acute respiratory infectious diseases, and the crowd is generally susceptible, and it is more common in infants and young children. It is one of the main infectious diseases that seriously threaten human health. The clinical manifestations of this bacterial infection are paroxysmal spasmodic cough, vomiting, and the course of the disease usually lasts for several weeks. Infants are prone to concurrent pneumonia and epilepsy, resulting in death. Timely diagnosis and early treatment of Bordetella pertussis infection are very impor...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12N15/11C12R1/01
CPCC12Q1/689C12Q1/686C12Q2600/112C12Q2563/107Y02A50/30
Inventor 李春明赵百慧汤志强
Owner SHANGHAI BIOGERM MEDICAL TECH CO LTD BEIJING BRANCH
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