Reagent kit for real time fluorescence quantitative PCR detection of bacillus pyocyaneus

A quantitative detection and Pseudomonas aeruginosa technology, applied in the direction of microbial determination/inspection, biochemical equipment and methods, etc., can solve problems such as false positives, unsuitable for clinical diagnosis, PCR product contamination, etc.

Inactive Publication Date: 2011-06-15
DAAN GENE CO LTD
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AI Technical Summary

Problems solved by technology

[0003] Traditional detection methods mainly rely on the biological characteristics of Pseudomonas aeruginosa (such as Gram-negative bacilli, oxidase-positive, capable of producing pyotoxin, etc.) Although this method has certain specificity and sensitivity, the operation is cumbersome and time-consuming, which is not conducive to the detection of large-scale samples.
Due to the rapid progress of Pseudomonas aeruginosa infection, drug resistance, and high mortality rate, traditional detection methods may delay diagnosis and treatment. Therefore, it is necessary to develop early and rapid detection technology

Method used

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  • Reagent kit for real time fluorescence quantitative PCR detection of bacillus pyocyaneus
  • Reagent kit for real time fluorescence quantitative PCR detection of bacillus pyocyaneus
  • Reagent kit for real time fluorescence quantitative PCR detection of bacillus pyocyaneus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: the development of Pseudomonas aeruginosa detection reagent

[0033] 1. Design of primers and probes: Through sequence comparison and analysis of all Pseudomonas aeruginosa nucleic acid sequences already available in the Genbank database and nucleic acid sequences reported in published literature at home and abroad, the main viruses related to Pseudomonas aeruginosa pathogenicity were analyzed. The force factor exotoxin A (ETA) gene was used as the amplification target site, and a highly conserved segment with no secondary structure was selected. According to the basic principles of primer and probe design, multiple pairs of primers and probes were designed by software and manually.

[0034] 2. Selection of clinical samples: According to relevant domestic and foreign literature reports, secretions or scrapings, sputum, urine, blood, cosmetics, and environmental monitors from clinical wounds, burns, corneal ulcers and other lesion areas can be selected. Whe...

Embodiment 2

[0045] Embodiment 2: Pseudomonas aeruginosa detection kit and its use

[0046] 1. Prepare a kit including the following components: 2 tubes of DNA extraction solution (500 μl / tube), 20 tubes of PCR amplification reaction solution (? μl / tube), 1 tube of negative quality control product (100 μl / tube), 1 tube of positive Control product (50μl / tube) 1 tube, quantitative standard (50μl / tube) 4 tubes.

[0047] 2. Specimen collection, transportation and storage

[0048] (1) Specimen collection: including various clinical specimens, such as blood, urine, sputum, pus, and puncture fluid, etc., as well as various specimens from the hospital environment, such as water, air, object surface sampling, etc., according to clinical Sampling is required for operation; it also includes cosmetic sampling, etc., and is operated in accordance with the national cosmetic microbial inspection standards. Sealed for inspection.

[0049] (2) Specimen storage and transportation: Specimens can be used f...

Embodiment 3

[0057] Example 3: Quantitative detection of clinical samples using Pseudomonas aeruginosa detection kit

[0058] The clinical samples come from the semi-solid culture of Nanjing Children's Hospital, and 50 μl of the upper layer of the culture is directly taken as the sample to be tested. Specimen processing, DNA extraction, PCR reaction and result analysis are carried out with reference to Example 2.

[0059] After the PCR reaction, adjust the analysis parameters according to the amplification curve to make the standard curve under the standard curve (Std curve) window reach the best (ie, the absolute value of the correlation value > 0.97), and then analyze the clinical samples. The test results of clinical samples are attached Figure 5 Shown: The Ct values ​​of the amplification curves of the six clinically positive samples are 8.83, 9.63, 17.02, 19.01, 20.28, and 23.61, respectively. Combined with the obvious exponential growth period of the amplification curves, all of the...

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Abstract

The invention relates to a kit for detecting presence of pathogen pseudomonas aeruginosa in patient samples such as clinical bacterial pneumonia, corneal ulcer, urinary tract infection, septicemia and the like and samples such as cosmetics, environmental monitoring objects and the like, in particular to a kit for early and quickly diagnosing pseudomonas aeruginosa infection by a real-time fluorescence quantitative polymerase chain reaction technique.

Description

technical field [0001] The invention relates to a kit for detecting the presence of Pseudomonas aeruginosa (Pseudomonas aeruginosa) in samples from patients with clinical bacterial pneumonia, corneal ulcer, urinary tract infection, sepsis, etc. A kit for early and rapid diagnosis of Pseudomonas aeruginosa infection by real-time fluorescent quantitative polymerase chain reaction technology. Background technique [0002] Pseudomonas aeruginosa (PA), also known as Pseudomonas aeruginosa, belongs to the genus Pseudomonas and widely exists in nature. Infection is mainly manifested in burns, surgical trauma and postoperative infection. Clinically, it can cause sepsis, respiratory tract infection, endocarditis, urinary tract infection, central nervous system infection, bone and joint infection, ophthalmology, ear, mastoid and sinus infection, skin and soft tissue infection, and digestive tract infection. Because Pseudomonas aeruginosa is widely distributed, adaptable, and easy to...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 邓中平陈娟朱振宇李明程钢何蕴韶
Owner DAAN GENE CO LTD
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