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Fluorescence quantitative polymerase chain reaction (PCR) kit for quickly detecting type-16 and type-18 human papilloma viruses (HPVs)

A technology for human papillomavirus and fluorescence quantification, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, and can solve the problems of false positives, poor specificity, and low sensitivity

Inactive Publication Date: 2011-06-29
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 necessary to develop accurate, sensitive, rapid and pollution-free clinical testing methods

Method used

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  • Fluorescence quantitative polymerase chain reaction (PCR) kit for quickly detecting type-16 and type-18 human papilloma viruses (HPVs)
  • Fluorescence quantitative polymerase chain reaction (PCR) kit for quickly detecting type-16 and type-18 human papilloma viruses (HPVs)

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Embodiment 1

[0045] 1. Example 1: Kit composition and preparation

[0046] a. DNA extraction reagent (lysate)

[0047] 50mmol / L NaOH, 10mmol / L Tris-HCl, pH 8.0, 1% TritonX-100 by volume, 1% NP-40 by volume, 0.5mmol / L EDTA pH 8.0.

[0048] b. Fluorescent PCR 10×Buffer composition

[0049] 500 mM KCl, 100 mM Tris-HCl (pH 9.025°C), 1.0% Triton X-100.

[0050] c. Fluorescence quantitative PCR reaction solution contains: PCR 10×buffer 5.0 μl, 10 μmol / L forward primer and 1.5 μl reverse primer each, 5 μmol / L fluorescent probe 1.5 μl, 25 mmol / L MgCl 2 12.5μl, 1.0μl of 10mmol / L dNTPs, 1.0μl of 2.5U / μl Taq DNA polymerase, 26μl of sterile double-distilled water.

[0051] d. Standard positive template stock solution: the concentration is 10 9 copies / ml standard positive template PMD18-T-HPV16&18.

[0052] e. Negative quality control standard: sterile double distilled water.

Embodiment 2

[0053] Example 2: Rapid detection of human papillomavirus type 16 and 18 using a kit

[0054] a. Add 1ml of sterile physiological saline to the specimen test tube, shake it well, transfer it to a 1.5ml centrifuge tube, centrifuge at 10,000rpm for 10min, and repeat the washing once. Add 50 μl of DNA extraction solution directly to the precipitate and mix thoroughly, take a boiling water bath for 10 minutes, centrifuge at 10,000 rpm for 2 minutes, and take 5 μl of the supernatant for PCR reaction;

[0055] 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;

[0056] c. Take 45 μl of the fluorescence quantitative PCR reaction solution (reagent c) respectively, take 5 μl of the HPV16&18 DNA obtained in step a) and 5 μl of the HPV16&18 positive standard template diluted in step b), and set up a negative control, respectively, add different PCR reaction tubes, PCR detect...

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Abstract

The invention discloses a fluorescence quantitative polymerase chain reaction (PCR) kit for quickly detecting type-16 and type-18 HPVs and relates to a genetic detection technique for a pathogen causing human cervical carcinoma and cervical intraepithelial high-grade lesion. The kit comprises DNA lysis solution, fluorescence quantitative PCR reaction solution, HPV16 and HPV18 standard positive templates PMD18-T-HPV16 and PMD18-T-HPV18 and a negative control standard product, wherein the fluorescence quantitative PCR reaction solution container specific primer pairs and fluorescence probes for detecting type-16 and type-18 HPVs. The kit is accurate in quantitation, quick in detection and high in specificity and sensitivity; the using steps of the kit are simple, and the repeatability of the kit is high; and the kit can perform high-throughput sample detection. The kit can be used for quick, qualitative and quantitative detection of type-16 and type-18 HPVs and can be used in place of the conventional culture method which has been used all the time.

Description

technical field [0001] The present invention relates to a kind of pathogenic gene detection technology that causes human cervical cancer and cervical intraepithelial high-grade lesion, especially relates to a kind of rapid detection 16, 18 type human papillomavirus fluorescent quantitative PCR (polymerase chain reaction) kits, suitable for Qualitative and quantitative detection of human papillomavirus types 16 and 18. Background technique [0002] Human papillomavirus (HPV) is a common pathogen that seriously endangers human health and causes various human diseases. The virus is a double-stranded small DNA virus with 7900 base pairs, belonging to the Papovaviridae family. More than 200 subtypes have been identified so far, and 54 of them can infect the genital mucosa. HPV is the most diverse type of papillomavirus. About 30 different types of HPV can infect the epithelial cells of the reproductive system, named "genital" HPV. According to the risk of HPV carcinogenesis, HP...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 唐景峰王业富杨敬镜王维旭汪晓莉
Owner WUHAN BIOTECH GENE ENG
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