Multiple touchdown PCR (polymerase chain reaction) detection kit of haemophilus influenzae

A technology of Haemophilus influenzae and detection kit, which is applied in the field of molecular diagnosis of Haemophilus influenzae infection, and achieves the effect of high sensitivity

Inactive Publication Date: 2012-09-05
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But in countries where vaccination is not promoted, hundreds of thousands of children can still die each year from diseases caused by Haemophilus influenzae infection
Currently, there is no report on the multiplex PCR of Haemophilus influenzae omp P6, frdB, and 16S rRNA genes to detect Haemophilus influenzae

Method used

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  • Multiple touchdown PCR (polymerase chain reaction) detection kit of haemophilus influenzae
  • Multiple touchdown PCR (polymerase chain reaction) detection kit of haemophilus influenzae
  • Multiple touchdown PCR (polymerase chain reaction) detection kit of haemophilus influenzae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Pretreatment of sputum specimens: the sputum specimens should be washed twice with normal saline, added with an equal amount of 1% trypsin (prepared and used on the same day) and digested at 37°C for 90 minutes.

[0034] Bacterial DNA extraction from sputum samples: Take 1.5ml of digested sputum samples and use Takara minibest Bacterial Genome Extraction Kit (Takara, Baosheng, Dalian) to extract bacterial DNA from sputum samples. The extracted DNA was directly used for PCR amplification or stored at -20°C for future use.

[0035]PCR amplification total system 25μl, add 12.55μl double distilled water, 2.5μl buffer (10×PCR), BSA (10mg / ml) 0.25μl, primer Hi_omp P6-F (10μmol / L) 0.5 μl, Hi_omp P6-R (10 μmol / L) 0.5 μl, Hi_frdB-F (10 μmol / L) 1 μl, Hi_frdB-R (10 μmol / L) 1 μl, Hi_16S rRNA-F (10 μmol / L) 1 μl, Hi_16S rRNA-R ( 10μmol / L) 1μl, 2μl MgCl 2 (25mM), 0.5μl dNTP (10mM), 0.2μl Taq DNA polymerase (5U / μl), 2μl genomic DNA extracted from sputum samples. At the same time, a ...

Embodiment 2

[0043] Specific detection by multiplex touchdown PCR.

[0044] Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Moraxella catarrhalis, Enterococcus faecalis, Mycobacterium smegmatis, Streptococcus pneumoniae, Genomic DNA of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis BCG, Haemophilus influenzae and Haemophilus influenzae type b, the pure culture of the above bacteria was extracted with Takaraminibest Bacterial Genome Extraction Kit (Takara, Baosheng, Dalian) ) to extract its genomic DNA. The extracted DNA was directly used for PCR amplification or stored at -20°C for future use.

[0045] PCR amplification total system 25μl, add 12.55μl double distilled water, 2.5μl buffer (10×PCR), BSA (10mg / ml) 0.25μl, primer Hi_omp P6-F (10μmol / L) 0.5 μl, Hi_omp P6-R (10 μmol / L) 0.5 μl, Hi_frdB-F (10 μmol / L) 1 μl, Hi_frdB-R (10 μmol / L) 1 μl, Hi_16S rRNA-F (10 μmol / L) 1 μl, Hi_16S rRNA-R ( 10μmol / L) 1μ...

Embodiment 3

[0053] The detection limit of the multiplex touchdown PCR was determined.

[0054] Genomic DNA extraction of Haemophilus influenzae type b: Take 1.5 ml of pure culture solution of Haemophilus influenzae type b and use Takaraminibest Bacterial Genome Extraction Kit (Takara, Baosheng, Dalian) to extract its genomic DNA. The extracted DNA was directly used for PCR amplification or stored at -20°C for future use.

[0055] PCR amplification total system 25μl, add 12.55μl double distilled water, 2.5μl buffer (10×PCR), BSA (10mg / ml) 0.25μl, primer Hi_omp P6-F (10μmol / L) 0.5 μl, Hi_omp P6-R (10 μmol / L) 0.5 μl, Hi_frdB-F (10 μmol / L) 1 μl, Hi_frdB-R (10 μmol / L) 1 μl, Hi_16S rRNA-F (10 μmol / L) 1 μl, Hi_16S rRNA-R ( 10μmol / L) 1μl, 2μl MgCl 2 (25mM), 0.5μl dNTP (10mM), 0.2μl Taq DNA polymerase (5U / μl), the template is 2μl extracted Haemophilus influenzae type b genomic DNA or 10 -1~-6 Dilute the template. At the same time, a negative control (double distilled water as a template) was s...

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Abstract

The invention discloses a multiple touchdown PCR (polymerase chain reaction) detection kit capable of quickly detecting haemophilus influenzae in lower respiratory tract specimens, belongs to the technical field of molecular diagnosis of infectious diseases of lower respiratory tracts and aims to solve quick diagnosis of haemophilus influenzae in lower respiratory tract specimens. The kit comprises 10*PCR buffer solution, MgCl2, dNTP, TaqDNA polymerase, BSA (bovine serum albumin), haemophilus influenzae positive control DNA and three pairs of haemophilus influenzae primers. The invention discloses the multiple touchdown PCR detection kit and a detection method capable of detecting haemophilus influenzae and adopts agarose gel electrophoresis to detect the PCR products. The kit and the method have the advantages of quickness, simpleness and convenience, specificity and sensitivity and can be used for quick diagnosis and epidemiological survey of haemophilus influenzae infection.

Description

technical field [0001] The invention relates to the technical field of molecular diagnosis of Haemophilus influenzae infection, in particular to the application of a multiple touchdown PCR detection method in the detection of Haemophilus influenzae in lower respiratory tract specimens. Background technique [0002] Haemophilus influenzae is the main pathogen causing community-acquired pneumonia. It is a fastidious bacterium, and its culture requires V and X factors. It mainly causes pneumonia and meningitis, causing serious illness in about 3 million people and killing about 386,000 people every year. Most patients are children under 5 years of age. Most of the deaths from H. influenzae occurred in developing countries, where the H. influenzae vaccine has not been included in the routine immunization program (China is also currently unscheduled). [0003] Risk factors for invasive H. influenzae pneumonia in adults include immunocompromised persons, COPD, diabetes, cancer, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 邵世和罗欲承杜蓬侯艳娇邵晨潘红
Owner JIANGSU UNIV
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