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Fluorescence quantitative kit PCR for quick testing chlamydia trachomatis

A Chlamydia trachomatis and fluorescence quantitative technology, which is applied to the determination/testing of microorganisms, biochemical equipment and methods, etc., can solve the problems of poor specificity, time-consuming and laborious, and inaccurate quantification

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

AI Technical Summary

Problems solved by technology

[0005] 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 still necessary to develop accurate, sensitive, rapid, and pollution-free clinical testing methods

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: Chlamydia trachomatis fluorescent quantitative PCR kit composition and preparation

[0041] Reagent composition:

[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. Fluorescence quantitative PCR reaction solution: PCR 10×buffer 2μl, forward primer and reverse primer 1.0μl (10μmol / L), fluorescent probe 0.8μl (5μmol / L), MgCl 2 2μl (25mmol / L), dNTPs 0.4μl (10mmol / L), HOTSTART Taq DNA polymerase 0.4μl (2.5U / μl), sterile double distilled water 10.4μl. 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-CT.

[0049] e. Negative quality ...

Embodiment 2

[0050] Implementation Example 2: Detection of Chlamydia trachomatis with a fluorescent quantitative PCR kit for rapid quantitative detection of Chlamydia trachomatis

[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 the fluorescent quantitative PCR reaction solution (reagent c) respectively, take 2 μl each of the DNA obtained in step a) and the diluted positive standard template in step b), and set up a negative control, and add them to different P...

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PUM

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Abstract

This invention discloses a fluorescent PCR test kit for quantitatively detecting Chlamydia trachomatis, which is composed of DNA lysis solution, fluorescent quantitative PCR reaction solution, positive standard template and negative standard sample. The fluorescent quantitative PCR reaction solution comprises forward and reverse primers and fluorescent probes. The test kit has such advantages as high specificity, high sensitivity, high accuracy, simple operation and good repeatability.

Description

technical field [0001] The invention relates to a genetic detection technology of sexually transmitted disease pathogens, in particular to a fluorescent quantitative PCR (polymerase chain reaction) kit for rapid detection of Chlamydia trachomatis (CT), which is suitable for qualitative and quantitative detection of CT. Background technique [0002] CT is a pathogenic microorganism, neither bacteria nor virus. It can cause urinary tract infections and is a common sexually transmitted disease other than gonorrhea. Typical manifestations of this type of urethritis are itching of the urethra, painful urination, and difficulty urinating. In women, there may also be vaginitis, cervicitis, and even pelvic inflammatory disease at the same time. [0003] Women infected with this disease can also lead to infertility and ectopic pregnancy. Pregnant women are sick, and their newborns can also induce conjunctivitis or pneumonia through birth canal infection. Chlamydia trachomatis inf...

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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