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Narcissus yellow stripe virus (NYSV) and narcissus mosaic virus (NMV) multiplex fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) kit and detection method

A multiple fluorescence quantitative and daffodil technology, applied in biochemical equipment and methods, microorganism-based methods, microbial measurement/inspection, etc., can solve the problem of multiple fluorescence quantitative RT-PCR detection kits and simultaneous detection that have not yet been seen Problems such as multiple fluorescent quantitative RT-PCR detection methods, to achieve the effects of saving detection samples, shortening the detection cycle, and high sensitivity

Inactive Publication Date: 2017-06-20
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there is no multiple fluorescent quantitative RT-PCR detection method specially used for simultaneous detection of NYSV and NMV, let alone a dedicated multiple fluorescent quantitative RT-PCR detection kit

Method used

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  • Narcissus yellow stripe virus (NYSV) and narcissus mosaic virus (NMV) multiplex fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) kit and detection method
  • Narcissus yellow stripe virus (NYSV) and narcissus mosaic virus (NMV) multiplex fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) kit and detection method
  • Narcissus yellow stripe virus (NYSV) and narcissus mosaic virus (NMV) multiplex fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Configuration of Narcissus Yellow Streak Virus and Narcissus Mosaic Virus Multiplex Fluorescent Quantitative RT-PCR Kit (10 detections)

[0031] 1) NYSV-forward primer: 10 μmol / L, 1 tube (20 μL);

[0032] 2) NYSV-reverse primer: 10 μmol / L, 1 tube (20 μL);

[0033] 3) NYSV-fluorescent probe: 10 μmol / L, 1 tube (20 μL);

[0034] 4) NMV-forward primer: 10 μmol / L, 1 tube (20 μL);

[0035] 5) NMV-reverse primer: 10 μmol / L, 1 tube (20 μL);

[0036] 6) NMV-fluorescent probe: 10 μmol / L, 1 tube (20 μL);

[0037] 7) Random primer: 100μmol / L, 1 tube (20μL);

[0038] 8) RT Buffer: 5×, 1 tube (100 μL);

[0039] 9) RNase inhibitor: 40U / μL, 1 tube (50μL);

[0040] 10) Reverse transcriptase: 200U / μL, 1 tube (20μL);

[0041] 11) dNTPs: 10mmol / L, 1 tube (20μL);

[0042] 12) TaqMan PCR Master Mix: 2×, 1 tube (150μL);

[0043] 13) Positive control samples of Narcissus Yellow Streak Virus and Narcissus Mosaic Virus, 1 tube (50 μL);

[0044] 14) Negative control samples wit...

Embodiment 2

[0046] Example 2: Detection method of Narcissus yellow streak virus and Narcissus mosaic virus multiplex fluorescent quantitative RT-PCR kit

[0047] The detection method of above-mentioned narcissus yellow streak virus and narcissus mosaic virus multiple fluorescent quantitative RT-PCR kit comprises the following steps:

[0048] 1) Reverse transcription reaction: Add 3 μL of total RNA of the sample to be tested, 1 μL of random primers with a concentration of 100 μmol / L and RNase-free ddH in a PCR tube 2 O 7 μL, 70 °C water bath for 10 min, rapid ice bath for 5 min, then add the following reagents: 5 × RTBuffer 5 μL, concentration 10mmol / L dNTPs 2 μL, concentration 200U / μL reverse transcriptase 1 μL, concentration 40U / μL RNase inhibition Factor 1 μL. 42°C water bath for 60 minutes, 70°C water bath for 10 minutes, then cool to room temperature to synthesize cDNA;

[0049] 2) Fluorescent quantitative PCR reaction: Take 3 μL of the cDNA synthesized in step 1), and add 10 μmol / L...

Embodiment 3

[0050] Example 3: Specificity determination of Narcissus yellow streak virus and Narcissus mosaic virus multiplex fluorescent quantitative RT-PCR detection kit

[0051] 1) RNA extraction: Take 0.1 g of the sample and place it in a mortar, add 1 mL of PBST buffer to grind, centrifuge at 10,000 g for 5 min at 4°C, take the supernatant and quickly transfer the supernatant to a sterilized 1.5 mL centrifuge tube, add 1mL TrizoL reagent, shake vigorously, stand at room temperature for 5min; centrifuge at 12000g for 10min at 4°C, take the supernatant; add 300μL of chloroform, shake vigorously for 15s, let stand at room temperature for 5min, centrifuge at 12000g for 15min at 4°C, take the upper aqueous phase; add Equal volume of isopropanol, invert and mix well, let stand at room temperature for 15min, centrifuge at 12000g for 10min at 4°C, discard the supernatant; add 1mL of 75% ethanol to wash the precipitate twice, centrifuge at 7500g for 3min at 4°C each time, discard Supernatant;...

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Abstract

The invention relates to a narcissus yellow stripe virus (NYSV) and narcissus mosaic virus (NMV) multiplex fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) kit and a detection method, used for simultaneously and rapidly detecting NYSV and NMV. The kit comprises an NYSV-forward primer, an NYSV-reverse primer, an NYSV-fluorescence probe, an NMV-forward primer, an NMV-reverse primer, an NMV-fluorescence probe, a random primer, RT Buffer, an RNA enzyme inhibitor, reverse transcriptase, dNTPs, TaqMan, PCR Master Mix, positive control, negative control and RNase-free ddH2O. The detection method disclosed by the invention comprises the steps: respectively designing specific primers and fluorescence probes aiming at conserved sequences of NYSV and NMV genes, and simultaneously detecting the NYSV and NMV by utilizing a multiplex fluorescent quantitative RT-PCR technology. The kit disclosed by the invention has obvious advantages of high sensitivity, high specificity and high accuracy, and is very suitable for inspection and quarantine at entry-exit ports and rapid detection of the NYSV and NMV in agricultural production.

Description

technical field [0001] The invention relates to a multiple fluorescent quantitative RT-PCR detection kit for Narcissus yellow streak virus and Narcissus mosaic virus and a detection method thereof, which belongs to the field of plant quarantine technology and is suitable for entry and exit port inspection and quarantine, agricultural production of Narcissus yellow streak virus and Rapid detection of narcissus mosaic virus. Background technique [0002] Narcissus is a perennial herbaceous plant of the genus Narcissus. It has a long history of cultivation in my country and is a famous traditional export flower in my country. Virus disease is an important disease on narcissus. Virus infection of narcissus can cause symptoms such as smaller bulbs, smaller flower arrows, weaker aroma, degeneration of bulbs, and dwarfing of plants, resulting in a decline in the yield and quality of narcissus. Narcissus Yellow Streak Virus (NYSV) and Narcissus Mosaic Virus (NMV) are two viruses th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2521/107C12Q2537/143C12Q2563/107
Inventor 沈建国高芳銮王宇金玲吴祖建
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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