Fluorescence quantitative kit PCR for quick testing fine

A fluorescence quantitative, ureaplasma technology, applied in the qualitative and quantitative detection of small Ureaplasma, the rapid detection of small Ureaplasma fluorescence quantitative PCR kits, can solve the problems of time-consuming, laborious, inaccurate quantification, low sensitivity, etc.

Inactive Publication Date: 2006-12-06
WUHAN BIOTECH GENE ENG
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004] The traditional culture method and serological method in clinical detection have the disadvantages of low sensitivity, poor specificity, false positive, time-consuming and labor-intensive; although the conventional PCR method has the advantages of simplicity, rapidity and sensitivity, it has the disadvantages of inaccurate quantification and PCR post-processing. False positives caused by pollution; therefore, it is necessary to develop accurate, sensitive, fast, and pollution-free clinical testing methods

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: kit composition and preparation

[0042] a. DNA extraction reagent (lysate)

[0043] Prepare for yourself, lysate: 50mmol / L NaOH, 10mmol / L Tris-HCl, pH 8.0, volume fraction 1% Triton X-100, volume fraction 1% NP-40, 0.5mmol / L EDTA pH 8.0.

[0044] b. Composition of fluorescent PCR 10×Buffer:

[0045] 500mM KCl, 100mM Tris-HCl (PH9.0 25℃),

[0046] 1.0% Triton X-100;

[0047] c. Fluorescent quantitative PCR reaction solution: PCR 10×buffer 2μl, forward primer and reverse primer 0.8μl (10μmol / L), fluorescent probe 0.8μl (5μmol / L), MgCl 2 2.5μl (25mmol / L), dNTPs 0.4μl (10mmol / L), HOTSTART Taq DNA polymerase 0.4μl (2.5U / μl), sterile double distilled water 10.3μl. Fluorescent probes were used at a concentration of 5 μmol / L.

[0048] d. Standard positive template stock solution: the concentration is 10 9 Copy / μl standard positive template pU-UP.

[0049] e. Negative quality control standard: sterile double distilled water

Embodiment 2

[0050] Implementation Example 2: Rapid detection of U. parvum using a kit

[0051] a. Add 1ml of sterile normal saline to the specimen test tube, shake well, transfer to a 1.5ml centrifuge tube, centrifuge at 10000g for 5min, and repeat washing once. Add 50 μl of DNA extraction solution directly to the precipitate, mix thoroughly, bathe in boiling water for 10 minutes, centrifuge at 10,000 g for 5 minutes, and take 2 μl of the supernatant for PCR reaction.

[0052] b. Serially dilute the positive standard template (reagent d) to 10 8 copies / μl, 10 7 copies / μl, 10 6 copies / μl, 10 5 copies / μl, 10 4 copies / μl, 10 3 copies / μl.

[0053] c. Take 18 μl of fluorescent quantitative PCR reaction solution (reagent c) respectively, take 2 μl each of UPDNA obtained in step a) and UP positive standard template diluted in step b), and set up a negative control, add to different PCR reaction tubes respectively, Perform PCR detection in parallel on a fluorescence quantitative detector. ...

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Abstract

This invention discloses a fluorescent PCR test kit for quantitatively detecting Ureaplasma parvum, which is composed of DNA lysis solution, fluorescent quantitative PCR reaction solution, positive standard template pU-UP and negative standard sample. The fluorescent quantitative PCR reaction solution comprises a pair of specific primers of Ureaplasma parvum and fluorescent probes. The test kit has such advantages as high specificity, high sensitivity, high accuracy, simple operation and good repeatability. The test kit can be used for detecting four serotypes of Ureaplasma parvum quantitatively and qualitatively, and can differentiate Ureaplasma parvum and Ureaplasma urealyticum.

Description

technical field [0001] The invention relates to a gene detection technology of sexually transmitted disease pathogens, in particular to a rapid detection U. parvum fluorescent quantitative PCR (polymerase chain reaction) kit, which is suitable for qualitative and quantitative detection of U. parvum. Background technique [0002] Ureaplasma urealyticum (Ureaplasma urealyticum) is a common commensal microorganism in the human genitourinary tract. In sexually transmitted diseases, Ureaplasma urealyticum is related to nongonococcal urethritis (NGU) and epididymitis; it is related to prostatitis and female urethral symptoms , pyelonephritis, and pelvic inflammatory disease are significantly related or suggest an etiological role. NGU is one of the eight kinds of venereal diseases controlled in our country, and its incidence is increasing day by day. Ureaplasma urealyticum is divided into two biological groups (Biovar) and 14 serotypes. These two groups of organisms can be ident...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 曹轩王业富
Owner WUHAN BIOTECH GENE ENG
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