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Fluorescence quantitative PCR reagent kit and detection method for human respiratory syncytial virus

A fluorescence quantitative and syncytial virus technology, applied in biochemical equipment and methods, microbial measurement/inspection, biological testing, etc., to achieve the effects of avoiding subjectivity, reliable results, and high specificity

Inactive Publication Date: 2004-11-10
WUHAN UNIV +1
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  • Abstract
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  • Application Information

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Problems solved by technology

Development and application of fluorescent quantitative PCR kit for detection of human respiratory syncytial virus

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Configuration of Human Respiratory Syncytial Virus Fluorescent Quantitative PCR Detection Kit

[0032] (1) RNA extract, M-MLV reverse transcriptase (200U / uL), dNTPs (10mM), HotStart TaqDNA polymerase (2.5U / uL), RNase inhibitor (40U / uL), MgCl2 (25mM).

[0033] (2) M-MLV 5×Buffer composition:

[0034] 50mM Tris-HCl (PH8.3, 25°C), 3mM MgCl 2 , 75mM KCl, 10mM DDT;

[0035] (3) Composition of fluorescent PCR 10×Buffer:

[0036] 200mM KCl, 200mM Tris-HCl (PH8.4, 25°C)

[0037] (4) Reverse transcription reaction solution: primer P1 2uL (10umol / L), M-MLV 5×buffer 4uL, dNTPs 3uL (10mmol / L), sterile double distilled water 9uL.

[0038] (5) Fluorescence quantitative reaction solution:

[0039] PCR 10×buffer 2uL, P1, P2 each 0.5uL (10umol / L), fluorescent probe FP 1uL (1umol / L), MgCl 2 2uL (25mmol / L), dNTPs 0.5uL (10mmol / L), sterile double distilled water 8uL.

Embodiment 2

[0040] Example 2: Detection of Respiratory Syncytial Virus with Fluorescent Quantitative PCR Detection Kit

[0041] (1) Take 300 μl of centrifuged cough sputum extract, nasal swab extract or throat swab extract, add 900 μL RNA extract, 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 500ul 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, and centrifuge at 12000g for 3min at 4°C. Discard the supernatant and dry at 37°C for 5 min. The precipitate was dissolved with 18uL reverse transcription reaction solution to obtain viral RNA.

[0042] (2) Add 1uL of RNase inhibitor and 1uL of M-MLV reverse transcriptase. 42°C for 60 minutes, 95°C for 5 minutes to ina...

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Abstract

The invention discloses a Human respiratory syncytial virus (HRSV) fluorescent quantitative PCR reagent box and detecting method, the reagent box including RNA extract, reverse transcription enzyme, RNA enzyme inhibitor, reverse transcription reacting solution, standard positive template, Taq DNA polyase, fluorescent quantitative reacting solution and standard negative reference and control solution. Using the reagent box to firstly extract virus total RNA from the sample to be detected to make reverse transcription into cDNA, where cDNA and the standard positive template act as fluorescent quantitative PCR, and calculating the initial HRSV concentration by the software in fluorescent quantitative PCR apparatus. It has fast detection, convenience and safety of operation, time saving and high efficiency and can implement early diagnosis and effective prevention of HRSV.

Description

technical field [0001] The invention belongs to the technical field of detection of viral nucleic acid, and in particular relates to a human respiratory syncytial virus fluorescent quantitative PCR kit. At the same time, the invention also relates to a fluorescent quantitative PCR kit for detecting human respiratory syncytial virus. PCR is to collect the fluorescent signal in the PCR system through real-time monitoring and quantify the initial template in the sample, which can be widely used in medicine, biology and other corresponding fields. Background technique [0002] Respiratory syncytial virus infection is a common acute respiratory disease second only to influenza virus infection, such as severe pneumonia and bronchiolitis in infants and young children. In recent years, due to the wide application of antibiotics, the rate of bacterial respiratory tract infection has decreased, making more than 90% of human acute respiratory tract infection c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N33/52
Inventor 张楚瑜张其威游上游
Owner WUHAN UNIV
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