Fluorescent quantitative PCR (polymerase chain reaction) kit for quickly detecting gonococcus

A fluorescent quantitative, gonococcal technology, applied in the rapid detection of gonococcus fluorescent quantitative PCR detection method and its kit, the field of qualitative and quantitative detection of gonococcus, can solve the problems of poor specificity, time-consuming and laborious, and inability to accurately quantify

Inactive Publication Date: 2011-06-29
WUHAN BIOTECH GENE ENG
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  • 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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  • Fluorescent quantitative PCR (polymerase chain reaction) kit for quickly detecting gonococcus
  • Fluorescent quantitative PCR (polymerase chain reaction) kit for quickly detecting gonococcus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: kit composition and preparation

[0041] a. DNA extraction reagent

[0042] DNA lysate composition:

[0043] Lysis solution I: prepare 0.01M PBS (28.94g Na 2 HPO 4 12H 2 O, 2.6 g KH 2 PO 4 2H 2 O add 1000ml ultra-clean water and mix well, adjust the pH to 7.4).

[0044]Lysis Solution II: In pH7.4 Tris-EDTA buffer (pH7.610mM Tris.HCL, pH8.01mMEDTA), add NP-40 according to the ratio of 1:100 and mix well to complete the preparation. Divide the extract II into 5ml cryopreservation tubes (transparent tubes with lids). The filling volume is 5ml / tube.

[0045] b. Composition of fluorescent PCR 10×Buffer:

[0046] 500mM KCl, 100mM Tris-HCl (PH9.025°C), 1.0% Triton X-100;

[0047] c. Fluorescent quantitative PCR reaction solution: PCR 10×buffer 5.0 μl, 10 μmol / L forward primer and reverse primer 1.5 μl each, 5 μmol / L fluorescent probe 1.5 μl, 25 mmol / L MgCl 2 12.5μl, 10mmol / L dNTPs 1.0μl, 2.5U / μl Taq DNA polymerase 1.0μl, sterile double distilled water...

Embodiment 2

[0050] Example 2: Rapid detection of Neisseria gonorrhoeae using a kit

[0051] a. DNA extraction process:

[0052] ① Transfer 500μl of the liquid in the centrifuge tube to a 1.5ml centrifuge tube, and centrifuge at 12000rpm for 5min;

[0053] ②Discard the supernatant, add 500μl extract solution I, and centrifuge at 12000rpm for 5min;

[0054] ③Discard the supernatant, add 200μl extract solution I, and centrifuge at 12000rpm for 5min;

[0055] ④ Discard the supernatant and add 50 μl of extraction solution II;

[0056] ⑤ Oscillating and mixing, 100 ℃ constant temperature treatment for 10 minutes;

[0057] ⑥ Centrifuge at 12,000 rpm for 5 minutes, and take 5 μl of the supernatant for PCR amplification.

[0058] b. Serially dilute the positive standard template (reagent d) to 10 8 copies / ml, 10 7 copies / ml, 10 6 copies / ml, 10 5 copies / ml, 10 4 copies / ml, 10 3 copy / ml.

[0059] c. Take 45 μl each of the fluorescent quantitative PCR reaction solution (reagent c), 5 μl ea...

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Abstract

The invention discloses a fluorescent quantitative PCR (polymerase chain reaction) kit for quickly detecting gonococcus, relates to a technique for detecting a pathogen gene causing various human diseases, and is applicable to qualitative / quantitative detection of gonococcus. The kit comprises a DNA (deoxyribonucleic acid) lysate, a fluorescent quantitative PCR reaction solution, a standard positive template PMD (polarization mode dispersion) 18-T-NG and a negative quality control standard substance, wherein the fluorescent quantitative PCR reaction solution contains a gonococcus specific primer pair and a fluorescent probe. The kit disclosed by the invention has the advantages of accurate quantification, high detection speed, high specificity, high sensitivity, simple operating steps and high repeatability. The kit can be used for quickly detecting gonococcus qualitatively / quantitatively and can be substituted for the traditional culturing method which is still in use until today.

Description

technical field [0001] The invention relates to a gene detection technology of pathogens causing various human diseases, in particular to a rapid detection method of gonorrhea by fluorescent quantitative PCR (polymerase chain reaction) and a kit thereof, which are suitable for qualitative and quantitative detection of gonorrhea. Background technique [0002] Sexually transmitted disease (Sexually Transmitted Disease, STD) is a variety of infectious diseases that are mainly transmitted through sexual contact, which seriously endangers human physical and mental health. Among them, gonorrhea caused by Neisseria gonorrhea (NG) (gonorrhea) is the most common sexually transmitted disease. Gonorrhea is mainly transmitted through sexual behavior, and may also be infected through contact. It belongs to a class of genitourinary system infection diseases. Clinically, it is mainly manifested as acute and chronic suppurative inflammation of the urethra and genitals. Its onset The mechani...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 王业富唐景峰王维旭杨敬镜汪晓莉
Owner WUHAN BIOTECH GENE ENG
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