II-type parainfluenza virus (PIV) fluorescence quantitative polymerase chain reaction (PCR) kit and detection method thereof

A technology for virus detection and parainfluenza, applied in the direction of fluorescence/phosphorescence, measurement/inspection of microorganisms, biochemical equipment and methods, etc., to achieve the effects of strong specificity, improved specificity, and high sensitivity

Inactive Publication Date: 2011-05-18
SHANGHAI XINGYAO MED TECH DEV CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] After searching, there is no development and application of fluorescent quantitative PCR kit for detecting type II parainfluenza virus

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Preparation of Type II Parainfluenza Virus Fluorescent Quantitative PCR Detection Kit

[0043] (1) RNA extract, dNTPs (25mM), reverse transcriptase Superscript III (200U / μl), RNase inhibitor (40U / μl), Taq DNA polymerase (5U / μl), MgCl 2 (100mM).

[0044] (2) Composition of fluorescent PCR 5×buffer:

[0045] 50 mM Tris-HCl (PH8.3), 250 mM KCl, 1 mg / ml gelatin.

[0046] (3) Mixed enzyme composition:

[0047] 1.5U / μl reverse transcriptase Superscript III, 1.5U / μl Taq DNA polymerase, 5U / μl RNase inhibitor.

[0048] (4) Fluorescent quantitative PCR reaction solution:

[0049] PCR 5×buffer 6μl, P1 and P2 each 0.3μl (25μM), fluorescent probe FP 0.2μl (25μM), MgCl 2 0.9μl (100mM), dNTPs 0.24μl (25mM), DEPC-treated water 15.86μl.

Embodiment 2

[0050] Embodiment 2: Detection of type II parainfluenza virus with fluorescent quantitative PCR kit

[0051] (1) Take 500 μl of centrifuged sputum or throat swab extract, add 1ml RNA extraction solution, then add 200 μl chloroform, shake for 15 seconds, and incubate at room temperature for 10 minutes. Centrifuge at 12000g for 10min at 4°C, and the RNA is located in the upper layer. Replace the upper layer with a new 1.5ml EP tube, add 500μl isopropanol, incubate at -20°C for 10min, and then centrifuge at 12000g for 10min at room temperature. Discard the supernatant, add 1ml of 75% ethanol to wash the RNA pellet, shake and mix well, and centrifuge at 12000g for 5min at 4°C. The supernatant was discarded and dried at 37°C for 5 minutes to obtain viral RNA.

[0052] (2) Dilute the standard positive template 10 times to 1×10 6 Copy number / μl, 1×10 5 Copy number / μl, 1×10 4 Copy number / μl.

[0053] (3) Take 24 μl of fluorescent quantitative PCR reaction solution, add 2 μl of m...

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PUM

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Abstract

The invention discloses a II-type parainfluenza virus (PIV) detection kit and application thereof. The kit comprises ribonucleic acid (RNA) extract, reverse transcriptase, an RNA enzyme inhibitor, a standard positive template, Taq deoxyribonucleic acid (DNA) polymerase, fluorescence quantitative polymerase chain reaction (PCR) reaction liquid and a standard negative quality control product. A detection method of the kit comprises the following steps of: extracting pathogen RNA of a sample to be tested; performing fluorescence quantitativeRT-PCR on the pathogen RNA, a standard positive quality control product, an inner control template and the negative quality control product; and calculating the starting concentration of the II-type PIV of the sample to be tested according to software of a fluorescence quantitative PCR instrument. The kit is high in detection speed, convenient and safe to operate, time-saving and efficient, and can effectively detect virus RNA of II-type PIV infected patient so as to realize early diagnosis and effectively prevent II-type PIV infection.

Description

technical field [0001] The invention belongs to the field of nucleic acid detection of pathogens, and particularly relates to a type II parainfluenza virus fluorescent quantitative PCR detection kit. At the same time, the invention also relates to a method for detecting type II parainfluenza virus with a fluorescent quantitative PCR kit. Fluorescent quantitative PCR is The fluorescence signal in the PCR system is collected through real-time monitoring, and the initial template in the sample is quantified, which can be widely used in corresponding fields such as medicine and biology. Background technique [0002] Parainfluenza virus (PIV) belongs to the same myxovirus as influenza virus. In 1992, the Fourth International Virus Nomenclature Committee classified it into the Paramyxoviridae family and the Paramyxovirinae subfamily. Most of its members are important pathogens that cause zoonotic diseases. In recent years, some new viruses such as megamyxoviruses (Hendra and Nip...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 沈维祥何剑军吴大治夏懿
Owner SHANGHAI XINGYAO MED TECH DEV CO LTD
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