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Rubella virus fluorescence quantitative polymerase chain reaction (PCR) kit and detection method thereof

A rubella virus and fluorescence quantitative technology is applied in the field of pathogen nucleic acid detection to achieve the effects of improving specificity, strong 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

After searching, there is no development and application of fluorescent quantitative PCR kit for detecting rubella virus

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Preparation of rubella virus fluorescent quantitative PCR detection kit

[0042] (1) RNA extract, dNTPs (25mM), reverse transcriptase Superscript III (200U / μl), RNase inhibitor (40U / μl), Taq DNA polymerase (5U / μl), MgCl 2 (100mM).

[0043] (2) Composition of fluorescent PCR 5×buffer:

[0044] 50 mM Tris-HCl (PH8.3), 250 mM KCl, 1 mg / ml gelatin.

[0045] (3) Mixed enzyme composition:

[0046] 1.5U / μl reverse transcriptase Superscript III, 1.5U / μl Taq DNA polymerase, 5U / μl RNase inhibitor.

[0047] (4) Fluorescent quantitative PCR reaction solution:

[0048] 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

[0049] Example 2: Detection of rubella virus with fluorescent quantitative PCR kit

[0050] (1) Take 500 μl of centrifuged gargle 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.

[0051] (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.

[0052] (3) Take 24 μl of fluorescent quantitative PCR reaction solution, add 2 μl of mixed enzymes resp...

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PUM

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Abstract

The invention discloses a Rubella virus (RV) detection kit and an application thereof. The kit contains RNA extraction solution, reverse transcriptase (RT), RNA enzyme inhibitor, a standard positive template, Taq DNA polymerase, fluorescence quantitative polymerase chain reaction (PCR) reaction solution and a standard negative quality control product. The detection method comprises the following steps: the kit is used to extract the virus DNAs of a sample to be detected, then the virus DNAs along with the standard positive quality control product and the standard negative quality control product perform fluorescence quantitative RT-PCR, and the software of the fluorescence quantitative PCR instrument is utilized to calculate the initial concentration of the RV in the sample. The invention has the advantages of high detection speed, and short time and high efficiency, and is convenient and safe to operate, thus the virus RNA detection can be effectively performed to the patient with RV infection and the early diagnosis and effective prevention of RV infection can be realized.

Description

technical field [0001] The invention belongs to the field of nucleic acid detection of pathogens, and in particular relates to a rubella virus fluorescent quantitative PCR detection kit. At the same time, the invention also relates to a method for detecting rubella virus with a fluorescent quantitative PCR kit. Fluorescent quantitative PCR collects PCR systems through real-time monitoring The fluorescent signal in the sample can be used to quantify the initial template in the sample, which can be widely used in corresponding fields such as medicine and biology. Background technique [0002] Rubella virus (rubella virus, RV) is the only member of the Rubellavirus genus in the Togaviridae family. It is a clinically common respiratory tract infection and one of the pathogens that cause rashes (sometimes asymptomatic). Humans are its only host. Rubella virus infection in pregnant women, especially in early pregnancy, can lead to fetal congenital rubella syndrome (CRS). At prese...

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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