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Nucleotide sequence for detecting Nocardia calcarea and application thereof

A Nocardia, sequence technology, applied in the fields of molecular biology and microbiology, to achieve short-term results

Inactive Publication Date: 2015-01-07
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the application of fluorescent TaqMan PCR technology, the choice of probe is an important factor, because it involves matching with primers and the most critical detection specificity

Method used

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  • Nucleotide sequence for detecting Nocardia calcarea and application thereof
  • Nucleotide sequence for detecting Nocardia calcarea and application thereof
  • Nucleotide sequence for detecting Nocardia calcarea and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The establishment of embodiment 1 Taqman fluorescent quantitative PCR method standard curve

[0044] 1 Preparation of plasmid standards

[0045] ①Using the DNA extracted from Nocardia mallei standard strain DSM43003 as template and rpoB-F and rpoB-R as primers, the target gene rpoB was amplified;

[0046] ② Gel cutting recovery, purification of PCR products;

[0047] ③ connected to the pMD-18T vector;

[0048] ④Introduce JM109-sensitive peptide cells;

[0049]⑤ Screen positive clones, common PCR verification, further sequencing verification;

[0050] ⑥ Extract the plasmid and store it at -20°C, and store the positive bacteria containing the recombinant at -80°C.

[0051] 2 Conversion of plasmid copy number concentration

[0052] The measured plasmid concentration was 34.8ng / ul, and the copy number concentration (copies / ul)=plasmid concentration (ng / ul)×(6.02×10 23 )×10 -9 / (660×plasmid base number), of which 6.02×10 23 is Avogadro's constant, that is, the number...

Embodiment 2

[0059] The specificity evaluation of embodiment 2 method

[0060] Under the same reaction system and reaction conditions as in Example 1, primers were verified with nucleic acids of Legionella pneumophila, Klebsiella pneumoniae, Streptococcus pneumoniae, Bacillus pertussis, Mycobacterium, Yersinia enterica, Clostridium difficile and probe specificity.

[0061] Among the 22 bacterial strains listed in Table 1, all Nocardia (96 strains) had strong fluorescence signals, all had obvious S-type amplification, and the Ct values ​​were all34.

[0062] Table 4 A variety of Nocardia and their corresponding Ct values

[0063]

[0064]

Embodiment 3

[0065] Example 3. Detection of sputum simulated specimen

[0066] 1. Preparation of simulated sputum specimens and nucleic acid extraction: ① Nocardia standard strain DSM43003 was inoculated in brain heart infusion (BHI) liquid culture medium, cultured overnight at 37°C with shaking. Take 50ul of the overnight cultured bacterial solution and inoculate it into 5ml BHI liquid culture solution, shake the bacteria at 37°C until the OD (600nm) value is about 0.8, and the plate count is 2.0×10 8 cfu / ml; ② Serial 10-fold dilution with BHI liquid culture medium to 2.0×10 0 ~2.0×10 7 cfu / ml; ③ Dilute the sterile sputum with normal saline 1:10, take 100ul / tube, add 100ul of BHI liquid culture medium to one of the tubes as the control group, and add 100ul of 10× doubling diluted bacterial solution to the other tubes Made 2.0×10 0 ~2.0×10 7 Cfu / ml gradient sputum simulated samples; ④ Centrifuge at 13000-13500rpm / min for 10min, discard the supernatant, add 180ul of lysozyme with a fina...

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Abstract

The invention provides a nucleotide sequence and application thereof to bacterial differential diagnosis. A probe sequence and the detection method can rapidly, specially and sensitively detect Nocardia calcarea, and effectively prevent non-specificity amplification of other bacteria.

Description

technical field [0001] The invention relates to the application of TaqMan probes in bacterial detection, especially the application in the differential diagnosis of Nocardia, belonging to the fields of molecular biology and microbiology. Background technique [0002] Nocardiaceae (Nocardiaceae) is an aerobic Gram-positive coryneform bacterium. Eppinger reported the first human infection case caused by Nocardiaceae in 1890. Nocardiaceae caused by it is an acute or Chronic purulent or granulomatous lesions. Immunocompromised people are the most susceptible to infection, mainly causing lung diseases, skin diseases and systemic disseminated diseases. The clinical symptoms are complex and diverse, mainly including bacteremia, empyema, pericarditis, infective endocarditis, etc. , can even cause the death of the patient in severe cases. Nocardia is an opportunistic pathogenic bacteria, the chance of endogenous infection is small, and human-to-human transmission rarely occurs. Ac...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689C12Q2561/101C12Q2537/162Y02A50/30
Inventor 李振军孙渭歌
Owner ICDC CHINA CDC
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