Quantitative determination process for enterovirus in environment water body

A quantitative detection method, enterovirus technology, applied in the field of quantitative detection of enterovirus in environmental water, achieving high precision, good specificity, and accurate quantification

Inactive Publication Date: 2008-12-31
XI'AN UNIVERSITY OF ARCHITECTURE AND TECHNOLOGY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] In view of the defects or deficiencies in the above-mentioned prior art, the purpose of the present invention is to provide a quantitative detection method for enteroviruses in environmental water, to solve the problem of quantitative detection, and to improve the sensitivity and specificity of detection

Method used

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  • Quantitative determination process for enterovirus in environment water body
  • Quantitative determination process for enterovirus in environment water body
  • Quantitative determination process for enterovirus in environment water body

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specific Embodiment

[0035] 1. Equipment and reagents

[0036] (1) vacuum filter;

[0037] (2) High-speed refrigerated centrifuge with a maximum speed of 14000r / min;

[0038] (3) Centrifuge tube, 50mL, 1.5mL;

[0039] (4) UV spectrophotometer;

[0040] (5) Ultraviolet viewer;

[0041] (6) Constant temperature water bath box

[0042] (7) Real-time fluorescent quantitative PCR instrument;

[0043] (8) Poliovirus type 1;

[0044] (9) Vero cells;

[0045] (10) pMD18-T vector;

[0046] (11) Competent Escherichia coli XL1-blue:

[0047] (12) Mixed cellulose ester microporous filter membrane, with a pore size of 0.22 μm and a diameter of 50 mm;

[0048] (13) 2.5mol / L MgCl 2 solution;

[0049] (14) 1mol / L hydrochloric acid;

[0050] (15) 3% beef extract-0.05mol / L glycine buffer solution (pH=9.5);

[0051] (16) polyethylene glycol, molecular weight 6000;

[0052] (17) 5mol / L NaCl solution;

[0053] (18) IPTG / X-gal / Amp plate;

[0054] (19) LB liquid medium;

[0055] (20) total RNA extractio...

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Abstract

The invention discloses a quantitative detection method of enteroviruses in environmental water, according to the homology of a conserved sequence at an RNA5` non-coding region of enteroviruses and on the basis of the existing primers of EV1 and EV2, the method further designs a pair of enterovirus universal primers EVN, prepares a series of recombinant plasmids adopting gradient dilution as standard substances, and builds a real-time fluorescence quantitative PCR reaction system applying the enterovirus universal primers EVN, so as to quantitatively detect the enterovirus content in the environmental water. The method has advantages of accurate quantification, greatly improving the sensitivity of the enterovirus qualitative RT-PCR detection technique in the current environmental water, broad linearity range, high precision, good specificity, and having no cross reaction with other common pathogenic microorganisms in the environment.

Description

technical field [0001] The invention relates to a quantitative detection method for enteroviruses in environmental water bodies. The method uses a real-time fluorescent quantitative RT-PCR method to detect enteroviruses, and is suitable for quantitative determination of various viruses of the genus Enterovirus in environmental water bodies. Background technique [0002] Reverse transcription-polymerase chain reaction (RT-PCR) technology uses RNA as a template to reverse transcribe the first strand of complementary DNA, and then use this strand as a template to perform PCR reaction, so that a very small amount of RNA can be synthesized within a few hours The internal specificity is amplified by millions of times. Real-time fluorescent quantitative RT-PCR technology adds fluorescent groups to the reaction system, uses fluorescent signals to monitor the entire PCR process in real time, and finally uses the standard curve to quantitatively analyze the concentration of unknown te...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
CPCY02A50/30
Inventor 张崇淼王晓昌刘永军
Owner XI'AN UNIVERSITY OF ARCHITECTURE AND TECHNOLOGY
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