Viral/bacterial lysate and fluorescent quantitative PCR detection method

A fluorescent quantitative and detection method technology, applied in the field of virus/bacteria lysate and fluorescent quantitative PCR detection, can solve the problems of high cost, cumbersome operation, long purification time, etc., and achieve low cost, simple operation process and operator requirements low effect

Active Publication Date: 2017-07-28
GENFINE BIOTECH BEIJING CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The object of the present invention is to provide a simple, low-cost virus / bacteria lysate and fluorescent quantitative PCR detection method, which can meet the requirements of most clinically common viruses and bacteria directly performing fluorescent quantitative PCR detection without nucleic acid purification. It is used to solve the problems of long purification time, cumbersome operation and high cost in the existing nucleic acid extraction process

Method used

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  • Viral/bacterial lysate and fluorescent quantitative PCR detection method
  • Viral/bacterial lysate and fluorescent quantitative PCR detection method
  • Viral/bacterial lysate and fluorescent quantitative PCR detection method

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Embodiment 1

[0023] Taking the fluorescence quantitative PCR detection of porcine circovirus type 2 (PCV-2) as an example, the application of the lysate of the present invention is illustrated.

[0024] A. Preparation of lysate: add 58.5 grams of sodium chloride, 100 mL of triton X-100 (polyethylene glycol octylphenyl ether), 2 grams of lithium dodecyl sulfate to the container, the concentration is 0.5M, pH 10mL of 8.0 EDTA-2Na solution, then add 600mL of purified water, mix well, transfer to a volumetric flask, and set the volume to 1000mL.

[0025] B. Extraction of virus / bacterial nucleic acid: add 5 μL of the lysate prepared in step A and 5 μL of the serum or plasma sample to be tested to the PCR reaction tube, use a pipette to repeatedly blow and mix 3 times, and let it stand for 10 minutes to obtain bacterial DNA. sample;

[0026] C. PCR amplification: Add 40 μL of the prepared PCR reaction solution to the PCR reaction tube of the DNA sample obtained in step B, place the PCR reaction...

Embodiment 2

[0043] A. Preparation of lysate: add 58.5 grams of sodium chloride, 150 mL of triton X-100, 4 grams of lithium dodecyl sulfate to the container, 16 mL of EDTA-2Na solution with a concentration of 0.5 M and a pH of 8.0, and then add purification Mix 600mL of water, transfer to a volumetric flask, and dilute to 1000mL.

[0044] B. Prepare the sample to be tested: dilute the known concentration of Escherichia coli to 1×10 7 cfu / ml, 1×10 6 cfu / ml, 1×10 5 cfu / ml, 1×10 4 cfu / mL four 10-fold dilution gradient concentration.

[0045] Take multiple PCR reaction tubes, add 5 μL of the lysate prepared in step A respectively, and add 5 μL of the above-mentioned four concentrations of E. coli bacterial solution in turn, use a pipette to repeatedly mix for 3 times, and let stand for 10 minutes to obtain virus lysate system;

[0046] C. PCR amplification: Add 40 μL of the prepared PCR reaction solution to the virus lysis system described in step B, perform amplification and detection acco...

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Abstract

The invention discloses a viral / bacterial lysate and a fluorescent quantitative PCR detection method and belongs to the field of biological detection. The lysate is prepared from NaCl, triton X-100, lithium dodecyl sulfate, EDTA-2Na and purified water through uniform mixing. The PCR reaction process is free of a DNA purification process. The nucleic acid extraction and amplification detection of the sample are carried out in a reaction tube without separation or purification processes such as heating, centrifugation and shocking. The operation is simple and convenient and realizes a low cost. The method has very low requirements on an operator, greatly reduces detection time and is especially suitable for bedside diagnosis of serious diseases and field inspection and quarantine work in the eruption of a major epidemic.

Description

technical field [0001] The invention relates to the technical field of nucleic acid extraction and detection of viruses and bacteria, in particular to a lysate for extracting nucleic acid from viruses and bacteria and a detection method for fluorescent quantitative PCR using the lysate. Background technique [0002] In the prior art, the virus extraction methods in serum and plasma mainly include the following: [0003] 1. Boiling method: Add lysate to virus and bacterial samples such as serum or plasma, boil in a water bath or dry bath, and centrifuge at high speed to take the supernatant as a PCR template. The advantage is that the operation is simple, and the disadvantage is that the extracted nucleic acid has high impurities and is easy Inhibit the PCR amplification in the later stage. In addition, due to the local high pressure generated by boiling, it is easy to cause aerosol pollution, resulting in the indiscrimination of negative and positive test results. [0004] ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/70C12Q1/68C12Q1/06
CPCC12N15/1003C12Q1/6806C12Q1/6851C12Q2527/125C12Q2563/107C12Q2531/113
Inventor 龚小鹏韩典霖杨亮
Owner GENFINE BIOTECH BEIJING CO LTD
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