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Fluorescence quantitative RT-PCR(Reverse Transcription-Polymerase Chain Reaction) kit and application thereof for detecting NDV(Newcastle Disease Virus)

A technology of RT-PCR and detection kit, which is applied in the direction of fluorescence/phosphorescence, measurement/inspection of microorganisms, biochemical equipment and methods, etc. It can solve the problems of low repeatability and environmental pollution, and achieve low false positive rate, The effect of avoiding false positives and reducing background fluorescent signal

Active Publication Date: 2012-04-11
WUHAN CHOPPER BIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the conventional methods of molecular biology can make up for the deficiencies of these methods in some aspects, the disadvantages of false positives, environmental pollution, and low repeatability limit their popularization and application.

Method used

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  • Fluorescence quantitative RT-PCR(Reverse Transcription-Polymerase Chain Reaction) kit and application thereof for detecting NDV(Newcastle Disease Virus)
  • Fluorescence quantitative RT-PCR(Reverse Transcription-Polymerase Chain Reaction) kit and application thereof for detecting NDV(Newcastle Disease Virus)
  • Fluorescence quantitative RT-PCR(Reverse Transcription-Polymerase Chain Reaction) kit and application thereof for detecting NDV(Newcastle Disease Virus)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Composition and Reagent Preparation of the Newcastle Disease Virus Fluorescence Quantitative RT-PCR Rapid Detection Kit

[0046] 1. NDV RT-PCR reaction solution: each reaction includes 10×RT-PCR Buffer 2.5μL, 25mM dNTP Mix 1μL, 10μM TaqMan probe PB 0.25μL, 10μM upstream primer PF 0.5μL, 10μM downstream primer PR 0.5μL, DEPC water 16.25μL, 21μL in total. A total of 1008μL of 48 reactions were divided into 1 tube.

[0047] 2. RNA extraction solution: 30mL / bottle, aliquot into brown bottles and store at 4°C.

[0048] 3. DEPC water: add diethyl pyrocarbonate with a final concentration of 0.1% to deionized water, overnight at room temperature, 15 pounds of high pressure for 20 minutes, and aliquot into 1.5 mL centrifuge tubes, 1 mL / tube.

[0049] 4. DNA polymerase: Taq DNA polymerase (5U / μL) 25μL / tube aliquoted and stored at -20°C.

[0050] 5. Reverse transcriptase: AMV reverse transcriptase (5U / μL) 25μL / tube aliquoted and stored at -20℃.

[0051] 6. Working standard: The working sta...

Embodiment 2

[0055] Application method of newcastle disease virus fluorescent quantitative RT-PCR rapid detection kit

[0056] 1 Sample nucleic acid extraction

[0057] 1.1 Take several 1.5mL sterilized centrifuge tubes (depending on the number of samples to be tested) without RNase contamination and mark them. Add 600μL of RNA extraction solution to each, and then add 200μL each of the sample and the negative control in the kit, pipette and mix; then add 200μL of chloroform, shake and mix. Let it stand for 5 minutes and centrifuge at 12,000 rpm for 10 minutes.

[0058] 1.2 Take the upper liquid phase in each tube (do not suck the lower liquid) and transfer it to a new 1.5ml centrifuge tube without RNase contamination, add 200μL of isopropanol pre-cooled at -20℃, mark it, and mix upside down uniform. Let it stand for 5 minutes and centrifuge at 12,000 rpm for 10 minutes.

[0059] 1.3 Gently pour off the supernatant, place it on absorbent paper, and dry the liquid; add 600 μL of 75% ethanol and ...

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Abstract

The invention provides a group of specific nucleotide sequences for detecting subtypes of Newcastle disease virus (NDV), and a fluorescence quantitative RT-PCR(Reverse Transcription-Polymerase Chain Reaction) quick detection kit for detecting NDV infection. The kit is used in the clinical and scientific research fields, including quick qualitative and quantitative detection of NDV infection, and monitoring of NDV in chickens and poultry products. The invention also provides a fluorescence quantitative RT-PCR method for detecting NDV infection of chicken not for diagnosis.

Description

Technical field [0001] The invention relates to a fluorescent quantitative RT-PCR rapid detection kit for specifically detecting chicken Newcastle disease virus (NDV) infection and an application method thereof, belonging to the field of viral nucleic acid detection, and suitable for rapid qualitative and scientific research on chicken Newcastle disease virus in clinical and scientific research. Quantitative detection and auxiliary diagnosis for chicken Newcastle disease virus infection. Background technique [0002] Newcastle Disease (ND), also known as Asian chicken plague or pseudo-avian plague, is an acute and highly contagious infectious disease of chickens, pigeons and turkeys caused by avian type I Paramyxoviridae Newcastle Disease Virus (NDV). The virulent strains can destroy the entire flock of chickens, while the weakly virulent strains only cause respiratory infections and decreased egg production in the flocks, but the loss caused by the reduction in production perfor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 薛霜陈其兵孙庆歌朱薇谢红玲漆世华温文生
Owner WUHAN CHOPPER BIOLOGY
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