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Kit and method for fluorescent quantitative detection of Bordetella pertussis

A Bordetella pertussis, fluorescence quantitative technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. problems, to achieve the effect of improving the utilization rate of the instrument, solving the problem of throughput, and reducing the cost of testing

Inactive Publication Date: 2017-10-20
BEIJING FUANHUA BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The kits for the detection of Bordetella pertussis based on fluorescence quantitative methods in the market generally use IS481 and ptxS1 gene sequence as the target sequence, but the IS481 gene sequence exists in both Bordetella pertussis and Bordetella hallii and Bordetella bronchiseptica (Karen B etc.2006), the ptxS1 gene sequence exists in both Bordetella pertussis and Bordetella parapertussis (Fengxiang Gao etc.2013), so these kits Does not specifically detect Bordetella pertussis
Some people use the IS481 and IS1001 gene sequence combination to detect Bordetella pertussis and Bordetella parapertussis, but this combination requires two reactions to complete the detection, which increases the cost of detection

Method used

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  • Kit and method for fluorescent quantitative detection of Bordetella pertussis
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  • Kit and method for fluorescent quantitative detection of Bordetella pertussis

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Example 1. Real-time fluorescence quantitative detection kit for Bordetella pertussis and its special primers

[0042] 1. Design and synthesis of special primers for real-time fluorescence quantitative detection of Bordetella pertussis

[0043] By comparing and analyzing the gene sequences of Bordetella pertussis, Bordetella parapertussis, Bordetella hormones and Bordetella bronchiseptica, a 187bp section of Bordetella pertussis was specifically conserved. Sequence, this sequence is sequence 1.

[0044] Design primer sequence as follows:

[0045] The sequence of the upstream amplification primer for Bordetella pertussis is: 5'-TTTCACTATGGGCTGTCGTG-3' (sequence 2);

[0046] The downstream amplification primer sequence of Bordetella pertussis is 5'-TGCGCTATTGCCGGATT-3' (sequence 3);

[0047] The fluorescent probe sequence of Bordetella pertussis is: 5'-AATCCGGCAATAGCGCA-3' (sequence 4), the 5'end is labeled with FAM fluorescent group, and the 3'end is labeled with TAMRA quenching g...

Embodiment 2

[0080] Example 2. Application of Bordetella pertussis real-time fluorescence quantitative detection kit

[0081] 1. Sensitivity detection

[0082] Set the concentration to 10 5 The copies / ul Bordetella pertussis standard products are respectively 10 5 , 10 4 , 10 3 , 10 2 , 10copies / ul gradient dilution, and then use them as the sample DNA to be tested respectively, and detect according to the second fluorescence quantitative method in Example 1.

[0083] The result is figure 2 As shown, it can be seen that the kit can detect at least about 10 copies / ul copy, with high sensitivity.

[0084] 2. Specificity test

[0085] To common other lung pathogenic bacteria Bordetella pertussis, Bordetella parapertussis, Bordetella holmanii, Bordetella bronchiseptica, Legionella pneumophila, Streptococcus pneumoniae, Influenza Haemophilus, Mycoplasma pneumoniae (Isolation and identification of rabbit Bordetella bronchiseptica and some biological characteristics research, Fan Zhiyu target sequence en...

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Abstract

The invention discloses a kit and a method for fluorescent quantitative detection of Bordetella pertussis, and provides a fluorescent quantitative reagent for detecting the Bordetella pertussis. The fluorescent quantitative reagent comprises a primer 1 for detecting the Bordetella pertussis, a primer 2 for detecting the Bordetella pertussis and a probe 1 for detecting the Bordetella pertussis. Experimental results prove that the kit and the detection method is based on Taqman fluorescent quantitative detection technique, pathogens of infection can be rapidly defined, detection expense is decreased, use efficiency of instruments is improved, and the method solves the problem of flux of clinical respiratory fungus infection pathogen detection and has important guide significance for clinical early diagnosis and treatment.

Description

Technical field [0001] The invention belongs to the technical field of nucleic acid detection, and particularly relates to a kit and a method for fluorescent quantitative detection of Bordetella pertussis. Background technique [0002] Whooping cough is an acute respiratory infection caused by Bordetella pertussis. Its clinical features are paroxysmal convulsive cough accompanied by a deep "cock crow"-like inspiratory roar. If not treated in time and effective, the course of the disease can reach several months. The disease is very contagious. The younger the child is, the more severe the condition, and death due to concurrent pneumonia and encephalopathy. [0003] Most cases (80-98%) are caused by the small gram-negative bacterium Bordetella pertussis (B.pertussis), but a few cases (2-20%) are caused by Bordetella parapertussis (B.parapertussis) (Mattoo etc.2005). Symptoms of whooping cough caused by B. parapertussis (B. parapertussis) are generally milder than those caused by ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06C12N15/11
CPCC12Q1/6851C12Q1/689C12Q2600/166C12Q2545/101C12Q2561/101
Inventor 乔阳余倩葛猛王宏伟
Owner BEIJING FUANHUA BIOLOGICAL TECH CO LTD
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