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Double-temperature multiple RT-PCR kit for simultaneously detecting two orchid viruses and preparation method thereof

An RT-PCR, two-temperature technology, applied in the field of two-temperature multiplex RT-PCR kits and preparation, can solve the problems of high cost and low sensitivity, and achieve the effects of reducing cost, high detection sensitivity, and reducing the number of reactions

Inactive Publication Date: 2010-10-27
厦门华侨亚热带植物引种园
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the problems of low sensitivity, need for multiple detection, and high cost in the existing virus detection technology, and provide a two-temperature multiplex RT-PCR reagent that can detect two kinds of orchid viruses simultaneously in one detection process Box and how to use it

Method used

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  • Double-temperature multiple RT-PCR kit for simultaneously detecting two orchid viruses and preparation method thereof
  • Double-temperature multiple RT-PCR kit for simultaneously detecting two orchid viruses and preparation method thereof
  • Double-temperature multiple RT-PCR kit for simultaneously detecting two orchid viruses and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 2

[0036] Example 1 Application of two-temperature multiplex RT-PCR kit in the screening of non-toxic Phalaenopsis female parent

[0037] According to the requirements of actual application, before the virus detection, the collection of Phalaenopsis samples was completed, and the sample RNA was extracted with Trizol reagent for future use. This embodiment is only for demonstration, and the number of collected samples may include 3 samples, and at the same time, more or fewer samples may be detected according to different requirements.

[0038] 1. cDNA synthesis: 1 μL of total RNA extracted by Trizol reagent was pre-denatured at 94°C for 5 min, then placed in an ice bath for testing, and 23.5 μL of reagent A and 0.5 μL of reagent B were added. cDNA was synthesized by reacting in a water bath at 37°C for 1 hour.

[0039] 2. Amplification detection: the total volume is 25 μL, of which cDNA 1 μL PCR reaction solution 23.5 μL reagent D 0.5 μL Reaction conditions: 94 ° C for 5 min; th...

Embodiment 2 2

[0042] Application of Example 2 Two-temperature Multiplex RT-PCR Kit in the Detection of Imported Phalaenopsis Viruses

[0043] According to the requirements of actual application, before the virus detection, the collection of Phalaenopsis samples for isolation test was completed, and the sample RNA was extracted with Trizol reagent for future use. This embodiment is only for demonstration, and the number of collected samples may include 3 samples, and at the same time, more or fewer samples may be detected according to different requirements.

[0044] 1. Synthesis of cDNA: Take 1 μL of total RNA extracted with Trizol reagent, pre-denature at 94°C for 5 minutes, and place it in an ice bath for testing. Add 23.5 μL of reagent A and 0.5 μL of reagent B. cDNA was synthesized by reacting in a water bath at 37°C for 1 hour.

[0045] 2. Amplification detection: the total volume is 25 μL, including 1 μL of cDNA, 23.5 μL of PCR reaction solution, and 0.5 μL of reagent D. Reaction co...

Embodiment 3 2

[0048] Example 3 Application of two-temperature multiplex RT-PCR kit in orchid virus investigation

[0049] According to the requirements of actual application, before the virus detection, the collection of orchid samples in greenhouses was completed, and the sample RNA was extracted with Trizol reagent for future use. This embodiment is only for demonstration, and the number of collected samples may include 33 samples, and at the same time, more or fewer samples may be detected according to different requirements.

[0050] 1. cDNA synthesis: 1 μL of total RNA extracted by Trizol reagent was pre-denatured at 94°C for 5 minutes, then placed in an ice bath for testing, and 23.5 μL of reagent A and 0.5 μL of reagent B were added. cDNA was synthesized by reacting in a water bath at 37°C for 1 hour.

[0051] 2. Amplification detection: the total volume is 25 μL, including 1 μL of cDNA, 23.5 μL of PCR reaction solution, and 0.5 μL of reagent D. Reaction conditions Denaturation at ...

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Abstract

The invention discloses a double-temperature multiple RT-PCR kit for simultaneously detecting two orchid viruses and a preparation method thereof, relates to a plant virus detecting kit, and provides a double-temperature multiple RT-PCR kit capable of simultaneously detecting two orchid viruses in one detection process and an application method thereof. The kit comprises a reagent A, a reagent B, a reagent C, a reagent D and a reagent E, wherein the reagent A is an RT reaction liquid which contains an M-MuLV reverse transcriptase buffer solution, random primers and dNTP; the reagent B is an M-MuLV reverse transcriptase; the reagent C is a PCR reaction liquid which contains a PCR buffer solution, MgCl2, dNTP and detection primers; the reagent D is a Taq polymerase; and the reagent E is a reference substance. Both positive control and negative control are established for each detection, and the application method comprises the following steps: carrying out reverse transcription; carrying out amplification detection, and determining the sizes of amplified bands according to the conventional agarose gel electrophoresis by taking 5 mu L of reaction products. The invention is especially applicable to the use by orchid enterprises, port quarantine authorities and related scientific research institutions.

Description

technical field [0001] The invention relates to a plant virus detection kit, in particular to a two-temperature multiplex RT-PCR kit and a preparation method for simultaneously detecting two orchid viruses. Background technique [0002] There are more than 20 kinds of orchid viruses, among which Cymbidium mosaic virus (CyMV) and Odontoglossum ringspot virus (ORSV) are the most common and the most harmful. Since orchid virus cannot be controlled by chemicals, how to detect orchid virus as early as possible and eliminate the diseased orchids has become the most important way of prevention and control. [0003] Chinese patent applications with publication numbers CN1975427, CN1975428, CN1975425, CN1975426, and CN101294962 respectively adopt serological techniques such as dot enzyme-linked enzymes, double-antibody sandwich enzyme-linked enzymes, and colloidal gold test strips to establish the detection technology of Jianlan mosaic virus and tooth ringspot virus However, the det...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 郑国华明艳林陈良华郑志忠吴美爱童庆宣
Owner 厦门华侨亚热带植物引种园
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