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Direct real-time quantitative PCR method of throat swab sample or nasopharyngeal swab sample

A nasopharyngeal swab and real-time quantitative technology, applied in the field of PCR, can solve problems such as pollution and nucleic acid loss, and achieve the effects of simplifying the operation process, preventing pollution, saving time and cost

Inactive Publication Date: 2017-05-10
SHAOXING INGENIGEN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When performing fluorescent quantitative PCR detection, the nucleic acid extraction of the sample must be carried out first. At present, the nucleic acid extraction methods mainly include alkaline lysis method, chromatography column method and magnetic bead method. Equipment; the magnetic bead method is easy to operate, but there are multiple elution operations that cause nucleic acid loss and contamination risks. Therefore, it is necessary to find a direct PCR method that does not require nucleic acid extraction, which is of great significance to improve the quality of detection

Method used

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  • Direct real-time quantitative PCR method of throat swab sample or nasopharyngeal swab sample
  • Direct real-time quantitative PCR method of throat swab sample or nasopharyngeal swab sample
  • Direct real-time quantitative PCR method of throat swab sample or nasopharyngeal swab sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The method of the present invention is applied to the real-time fluorescent quantitative PCR detection of clinical samples of stream B

[0025] (1) Prepare 100ml of lysate containing 1MNaCl, 0.01% Triton-X and 0.01MKCl, add 0.5ml of magnetic beads to the prepared lysate according to the volume ratio of magnetic beads and lysate of 1:200, and mix well. 4 identical dedicated PCR tubes, each containing 80ul of prepared lysate mixed with magnetic beads;

[0026] (2) Take 20ul from the four samples of pharyngeal swabs of 2 cases of B-type positive and 2 cases of B-type negative provided by Shaoxing Center for Disease Control and Prevention, and add them to the four PCR tubes in step (1) respectively as sample No. 1 , No. 2 sample, No. 3 sample and No. 4 sample, cover the tube cap;

[0027] (3) Heat the four PCR tubes in step (2) at 95°C for 5 minutes and place them at room temperature (18-28°C) for 10 minutes;

[0028] (4) Put the four PCR tubes in step (3) into the magnet...

Embodiment 2

[0034] Sensitivity Test of the Method of the Invention

[0035] Use the negative B-flow throat swab sample in the step (2) of Example 1 as a diluent, and the B-flow minimum detection limit reference product S2 2.0×10 in the A-B flow national standard of China Institute for the Control of Pharmaceutical and Biological Products 6 TCID 50 / L for dilution to obtain 2.0×10 4 TCID 50 / L, 2.0×10 3 TCID 50 / L, 2.0×10 2 TCID 50 / L, 2.0×10 1 TCID 50 / L of four samples, using the same method as in Implementation 1, real-time fluorescent PCR detection was performed on the above four diluted samples (ABI7500 was selected for PCR amplification).

[0036] Experimental results such as figure 2 Shown: The four curves labeled 1, 2, 3 and 4 in the figure represent the concentration of 2.0×10 4 TCID 50 / L, 2.0×10 3 TCID 50 / L, 2.0×10 2 TCID 50 / L, 2.0×10 1 TCID 50 The experimental result of the sample of / L, the result shows, the minimum concentration of the B flow that the prese...

Embodiment 3

[0038] The repeatability test of the fluorescent PCR detection of the inventive method

[0039] Using the negative B flow throat swab sample in Example 1 as a diluent, the B flow minimum detection limit reference product S2 2.0×10 in the A and B flow national standard of China Institute for the Control of Pharmaceutical and Biological Products 6 TCID 50 / L for dilution to obtain 2.0×10 3 TCID 50 / L samples. Utilize the same method as implementing 1 to carry out 5 wells reexamination real-time fluorescent PCR detection (Bio-RAD CFX96 is amplified) to the sample, and the five curves labeled 1, 2, 3, 4 and 5 in the figure are respectively in 5 wells Experimental results for samples.

[0040] Test results such as image 3 and the following table:

[0041]

[0042]

[0043] The results show that the detection of the method of the present invention can be repeated.

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Abstract

The invention discloses a direct real-time quantitative PCR method of a throat swab sample or a nasopharyngeal swab sample. The method comprises the following steps: (1) lysate and a PCR reaction solution are prepared; (2) the throat swab sample or the nasopharyngeal swab sample are added into a PCR amplification tube containing the lysate in the step (1), magnetic beads are added into the PCR amplification tube, and vibration, uniform shaking and mixing are carried out; (3) the PCR amplification tube in the step (2) is treated at a high temperature 80-100 DEG C for 10 minutes, cooling is carried out to a room temperature, and a reaction is carried out at 18-28 DEG C for 5-10 minutes; (4) the PCR amplification tube is instantly centrifuged and placed into a magnetic rack, after the magnetic beads are absorbed to one side of a magnetic rack, supernatant is sucked out; (5) the PCR amplification tube is added into a prepared PCR reaction solution, and the real time quantitative PCR reaction is carried out; the lysate comprises the following components 0-2M NaCl, 0-0.3% Triton-X and 0-0.2M KCl, the magnetic beads are hydroxyl magnetic beads sold on the market for extracting nucleic acid, the throat swab sample detection is rapid, simple, high-efficient and practical, nucleic acid extraction process and centrifuge and other equipment are not needed, operation processes are simplified, time and cost are saved, a large amount of samples can be processed, and during operation and detection, pollution is prevented.

Description

technical field [0001] The invention belongs to the technical field of molecular diagnostic biology, and in particular relates to a direct real-time quantitative PCR method for throat swab samples or nasopharyngeal swab samples. Background technique [0002] Polymerase Chain Reaction (Polymerase Chain Reaction PCR) is an enzymatic synthesis reaction technology that relies on DNA polymerase in the presence of template DNA, primers and four deoxyribonucleotides. Since the technology was invented in 1958 by Kary Mullis and colleagues in the human genetics laboratory of Cetus Corporation in the United States, related derivative technologies have been developed rapidly and have been widely used in the fields of microbiology, genetics and forensic medicine. Especially in the detection of pathogens, when a gene of a certain pathogen is known, specific primers are designed according to the gene and PCR amplification is carried out to make it reach the detection amount, and then thro...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12N15/1013C12Q1/6806C12Q1/6851C12Q1/686C12Q1/689C12Q1/701C12Q1/705C12Q2600/16C12Q2561/113C12Q2563/143C12Q2563/149C12Q2531/113C12Q2563/107C12Q2537/143C12Q2521/107C12Q2545/114
Inventor 胡彬刘杰陶施芳潘玲玲
Owner SHAOXING INGENIGEN BIOTECH CO LTD
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