PCR kit for fluorescence quantitative detecting aspergilli

A fluorescence quantitative, Aspergillus technology, applied in fluorescence/phosphorescence, microbial determination/inspection, biochemical equipment and methods, etc., can solve problems such as high cost and different, and achieve high specificity.

Inactive Publication Date: 2007-09-19
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The World Intellectual Property Organization published a "Molecular Identification of Aspergillus Species" invention patent application (WO03/097815) on November 27, 2003, which discloses a sequence that utilizes real-time PCR to amplify the five regions of ITS1 to identify and Detecting Aspergillus, it is also disclo

Method used

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  • PCR kit for fluorescence quantitative detecting aspergilli
  • PCR kit for fluorescence quantitative detecting aspergilli
  • PCR kit for fluorescence quantitative detecting aspergilli

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1: the preparation of kit of the present invention

[0040] 1. The composition of Aspergillus fluorescent quantitative PCR kit:

[0041] Upstream primer: 5'-CGGAAGGATCATTACCGAGTGA-3'(SEQ NO.1), 5pmol / μl;

[0042] Downstream primer: 5'-CCCGCCGAAGCAACAAG-3' (SEQ NO.2), 5pmol / μl;

[0043] Fluorescent probe: 5'FAM-CCAACCTCCCACCCGTGTCTATYGT-BHQ-1 3'(SEQ NO.3), 10pmol / μl;

[0044] Quantitative positive template: pMD19-T recombinant vector containing DNA fragment SEQ NO.4, the concentration is 10 5 copy / ml.

[0045] DNA polymerase: TaqDNA polymerase, purchased from Huayin Biogene Co., Ltd.;

[0046] PCR Mix: purchased from Huayin Biogene Co., Ltd.;

[0047] The above nucleic acids were all synthesized by Guangzhou Huayin Biotechnology Co., Ltd.

[0048] 2. The preparation method of the quantitative positive template is:

[0049] (1) Using the total DNA extracted from Aspergillus as a template, use the primers whose sequences are SEQ NO.5 an...

Embodiment 2

[0083] Embodiment 2: in vitro detection experiment

[0084] 1. Preparation of template DNA:

[0085] a. Reagents: Nucleic acid extraction kit, purchased from Guangzhou Huayin Biotechnology Co., Ltd. and synthesized.

[0086] b. Cultivation and preparation of standard strains:

[0087] Aspergillus standard strains: Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus niger, Aspergillus nidulans, provided by Nanfang Hospital;

[0088] Samples to be tested: several Aspergillus species isolated clinically;

[0089] Negative control strains: Candida glabrata, Candida tropicalis, Cryptococcus, Staphylococcus aureus, Pseudomonas aeruginosa.

[0090] c. DNA extraction: Take 0.5ml of the bacterial suspension, centrifuge at 13000r / min for 6min, discard the supernatant, add 0.2ml of Tris (100mmol / L, pH7.4) buffer, 5U of lyticase, and bathe in water at 37°C for 1h. Take 50 μl, and extract DNA with a nucleic acid extraction kit, with a f...

Embodiment 3

[0104]Embodiment 3: clinical experiment

[0105] 1. Experimental method:

[0106] 1) Collection of clinical specimens:

[0107] From April 2006 to February 2007, a total of 71 highly suspected cases of Aspergillus infection were collected in Guangzhou Nanfang Hospital, with 232 specimens. Take 2ml of patient's venous blood with common biochemical tube, centrifuge at 4000r / min for 8min, take 300μl and 400~600μl of serum into sterile 1.5ml EP tubes, register the number, and store in -20℃ refrigerator.

[0108] 2) Main instruments

[0109] MJ opticon2 fluorescent quantitative PCR instrument MJ company (USA)

[0110] Biological Safety Cabinet Haier Company (China)

[0111] High Speed ​​Centrifuge TG16-WS(China)

[0112] Water Bath Jiangsu Taicang Experimental Equipment Factory

[0113] Ultra-clean workbench Suzhou Purification Equipment Factory

[0114] Vortex shaker Thermolyne M63210-26 (USA)

[0115] Automatic enzyme label analyzer BIO-RAD (USA)

[0116] 3) Inclusion an...

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Abstract

The present invention provides a PCR reagent kit used for detecting quantificationally fluorescence of aspergillus. Said reagent kit contains primers such as 5'-CGGAAGGATCATTACCGAGTGA-3' (SEQ NO.1), 5'-CCCGCCGAAGCAACAAG-3' (SEQ NO.2) and a fluorescence probe 5'FAM-CCAACCTCCCACCCGTGTCTATYGT-BHQ-1 3' (SEQ NO.3). Said reagent kit in accordance with the present invention amplifies specifically gene in the interzone ITS I of aspergillus and generates a production with a size of 79 bp, is capable of quickly and correctly detecting a variety of common pathogenic aspergillus (such as aspergillus fumigatus, aspergillus flavus, aspergillus terreus, aspergillus niger and aspergillus nidulans) with a detection limit of 5-10 CFU/ml, and then is applied for a clinical diagnoses of invasive aspergillus infection with excellent sensitivity and specificity.

Description

technical field [0001] The invention relates to a composition for testing microorganisms, in particular to a composition for testing Aspergillus, especially a kit for testing a nucleic acid sequence in the gene of Aspergillus. Background technique [0002] At present, the common clinical deep pathogenic bacteria are Candida, Aspergillus, Cryptococcus neoformans, etc., among which Candida accounts for about 70%. In recent years, the rate of fungal infection has increased significantly. In patients with neutropenia and bone marrow transplantation, the infection rate of invasive aspergillosis is as high as 50%, and the fatality rate is as high as 40%. Survival mainly depends on early diagnosis and appropriate antifungal therapy However, the traditional laboratory diagnosis takes a long time and lacks sensitivity, so exploring a fast and reliable method for detecting Aspergillus has become a hot issue in clinical experimental research. [0003] There are 13 populations of patho...

Claims

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Application Information

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IPC IPC(8): G01N21/64C12Q1/04
Inventor 王前张梦宇
Owner SOUTHERN MEDICAL UNIVERSITY
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