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Multiple touchdown PCR (polymerase chain reaction) detection kit of acinetobacter baumannii

A technology of Acinetobacter baumannii and a detection kit, which is applied to the determination/testing of microorganisms, microorganisms, biochemical equipment and methods, etc., to achieve the effect of high sensitivity

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

AI Technical Summary

Problems solved by technology

At present, there is no simultaneously targeted Acinetobacter baumannii gltA, gyrB, bla OXA-51-like Gene to detect Acinetobacter baumannii multiplex PCR reporter

Method used

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  • Multiple touchdown PCR (polymerase chain reaction) detection kit of acinetobacter baumannii
  • Multiple touchdown PCR (polymerase chain reaction) detection kit of acinetobacter baumannii
  • Multiple touchdown PCR (polymerase chain reaction) detection kit of acinetobacter baumannii

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 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.

[0033] 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.

[0034] The total PCR amplification system is 25 μl, add 12.55 μl double distilled water, 2.5 μl buffer (10×PCR), 0.25 μl BSA (10 mg / ml), 0.5 μl primer AB_gltA-F (10 μmol / L) into a 200 μl PCR small tube, AB_gltA-R (10 μmol / L) 0.5 μl, AB_gyrB-F (10 μmol / L) 1 μl, AB_gyrB-R (10 μmol / L) 1 μl, AB_OXA-F (10 μmol / L) 1 μl, AB_OXA-R (10 μmol / L) 1 μl , 2 μl MgCl 2(25mM), 0.5μl dNTP (10mM), 0.2μl Taq DNA polymerase (5U / μl), 2μl genomic DNA extracted from sputum...

Embodiment 2

[0042] Specific detection by multiplex touchdown PCR.

[0043] Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Moraxella catarrhalis, Enterococcus faecalis, Haemophilus influenzae, Mycobacterium smegmatis, Streptococcus pneumoniae, b Genomic DNA of Haemophilus influenzae type, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis BCG, Acinetobacter baumannii, the pure culture of the above bacteria was extracted with Takara minibest bacterial genome extraction kit (Takara, Dalian Baosheng) to extract its genomic DNA. The extracted DNA was directly used for PCR amplification or stored at -20°C for future use.

[0044] The total PCR amplification system is 25 μl, add 12.55 μl double distilled water, 2.5 μl buffer (10×PCR), 0.25 μl BSA (10 mg / ml), 0.5 μl primer AB_gltA-F (10 μmol / L) into a 200 μl PCR small tube, AB_gltA-R (10 μmol / L) 0.5 μl, AB_gyrB-F (10 μmol / L) 1 μl, AB_gyrB-R (10 μmol / L) 1 μl, AB_OXA-F (10 μmol / L) 1 μl, A...

Embodiment 3

[0052] The detection limit of the multiplex touchdown PCR was determined.

[0053] Genomic DNA extraction of Acinetobacter baumannii: Take 1.5ml of pure culture solution of Acinetobacter baumannii and use Takara minibest 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.

[0054] The total PCR amplification system is 25 μl, add 12.55 μl double distilled water, 2.5 μl buffer (10×PCR), 0.25 μl BSA (10 mg / ml), 0.5 μl primer AB_gltA-F (10 μmol / L) into a 200 μl PCR small tube, AB_gltA-R (10 μmol / L) 0.5 μl, AB_gyrB-F (10 μmol / L) 1 μl, AB_gyrB-R (10 μmol / L) 1 μl, AB_OXA-F (10 μmol / L) 1 μl, AB_OXA-R (10 μmol / L) 1 μl , 2 μl MgCl 2 (25mM), 0.5μl dNTP (10mM), 0.2μl Taq DNA polymerase (5U / μl), the template is 2μl extracted Acinetobacter baumannii genomic DNA or its 10 -1~-6 Dilute the template. At the same time, a negative control (double distilled water as ...

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Abstract

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

Description

technical field [0001] The invention relates to the technical field of molecular diagnosis of Acinetobacter baumannii infection, in particular to the application of a multiple touchdown PCR detection method in the detection of Acinetobacter baumannii in lower respiratory tract specimens. Background technique [0002] Acinetobacter baumannii is an important nosocomial infection pathogen, widely exists in nature, and has tenacious vitality. In the hospital environment, Acinetobacter baumannii is widely found in various medical devices and patients' bedding, and can also be detected in the hands, work clothes, and stethoscopes of medical staff. In recent years, its importance in nosocomial infections has been increasing, mainly involving critically ill patients (such as IUC patients), and can cause respiratory infections, sepsis and urinary system infections, etc. important pathogens of machine-associated pneumonia (VAP). [0003] bla OXA-51-like The detection results of the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/01
Inventor 邵世和罗欲承杜蓬侯艳娇潘红钱静漪陈珵
Owner JIANGSU UNIV
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