Narcissus late season yellows virus detection kit and method

A technology for detecting kits and yellowing viruses, which is applied in the direction of biochemical equipment and methods, and the determination/inspection of microorganisms, and can solve the problem of nested RT-PCR technology and molecular detection kits, expensive instruments and equipment for the rapid detection of viruses that have not yet been seen. , port application limitations and other issues, to achieve the effect of avoiding non-specific amplification, high sensitivity, and short detection time

Active Publication Date: 2014-09-24
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Electron microscope observation of virions has the advantages of directness and accuracy, but requires expensive equipment and specialized technicians, and has great limitations in port applications
ELISA detection has the advantages of strong specificity, high sensitivity, and simple operation. It is a widely used serological detection technology at present. The disadvantage of this method is that it needs to have virus antibodies with strong specificity and good stability.
But so far, there are few reports on the molecular biology detection methods of Narcissus Late Season Yellowing Virus (NLSYV), and no nested RT-PCR technology and molecular detection kits specially applied to the rapid detection of this virus have been seen so far.

Method used

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  • Narcissus late season yellows virus detection kit and method
  • Narcissus late season yellows virus detection kit and method
  • Narcissus late season yellows virus detection kit and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Configuration of Narcissus Late Season Yellowing Virus Detection Kit (10 detections)

[0032] 1) Outer upstream primer: 10 μmol / L, 1 tube (30 μL);

[0033] 2) Outer downstream primer: 10 μmol / L, 1 tube (30 μL);

[0034] 3) Inner upstream primer: 10 μmol / L, 1 tube (30 μL);

[0035] 4) Inner downstream primer: 10 μmol / L, 1 tube (30 μL);

[0036] 5) RT Buffer: 5×, 1 tube (30μL);

[0037] 6) RNase inhibitor: 40U / μL, 1 tube (5μL);

[0038] 7) Reverse transcriptase: 200U / μL, 1 tube (5μL);

[0039] 8) dNTPs: 10mmol / L, 1 tube (30μL);

[0040] 9) PCR Buffer: 10×, 1 tube (60μL);

[0041] 10) Mgcl 2 : 25mmol / L, 1 tube (50μL);

[0042] 11) Taq DNA polymerase: 5U / μL, 1 tube (10μL);

[0043] 12) Positive control sample of Narcissus late season yellowing virus, 1 tube (50 μL);

[0044] 13) Negative control sample without narcissus late season yellowing virus, 1 tube (50 μL);

[0045] 14) RNase-free ddH 2 O, 1 tube (1 mL).

Embodiment 2

[0046] Example 2: The detection method of Narcissus late season yellowing virus detection kit

[0047] The detection method of the above-mentioned narcissus late season yellowing virus detection kit comprises the following steps:

[0048] 1) Reverse transcription reaction: Add 2 μL of total RNA of the sample to be tested, 1 μL of the outer downstream primer with a concentration of 10 μmol / L and RNase-free ddH in the PCR tube 2 O 5 μL, 70 °C water bath for 10 min, rapid ice bath for 5 min, then add the following reagents: 5×RTBuffer 2.5 μL, the concentration is 10 mmol / L dNTPs 1 μL, the concentration is 200 U / μL reverse transcriptase 0.5 μL, the concentration is 40 U / μL RNase Inhibitor 0.5 μL. 42°C water bath for 60 minutes, 70°C water bath for 10 minutes, and then naturally cool to room temperature to synthesize cDNA;

[0049] 2) The first round of PCR reaction: take 3 μL of cDNA synthesized in step 1), add 0.5 μL of Taq DNA polymerase at a concentration of 5 U / μL, 0.5 μL of...

Embodiment 3

[0052] Example 3: Specificity determination of Narcissus late season yellowing virus detection kit

[0053] 1) Extraction of total RNA from narcissus samples: Narcissus late season yellow virus (NLSYV), narcissus mosaic virus (NMV), narcissus yellow stripe virus (Narcissus yellow stripe virus, NYSV) and narcissus degradation Narcissus samples of NDV, Narcissus latent virus (NLV), and Arabis mosaic virus (ArMV) were used as materials, and 0.1 g of each was placed in a mortar, and 1 mL of LPBST buffer was added to grind , 4°C, centrifuge at 10000g for 5min, take the supernatant and quickly transfer the supernatant to a sterilized 1.5mL centrifuge tube, add 1mL TrizoL reagent, shake vigorously, let stand at room temperature for 5min; Supernatant; add 300 μL of chloroform, shake vigorously for 15 s, let stand at room temperature for 5 min, centrifuge at 12000 g at 4 °C for 15 min, and take the upper aqueous phase; add an equal volume of isopropanol, invert and mix well, and stand ...

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Abstract

The invention relates to a narcissus late season yellows virus detection kit and method specific for narcissus late season yellows virus detection. The knit comprises an outside upstream primer, an outside downstream primer, an inside upstream primer, an inside downstream primer, an RTBuffer, an RNA enzyme inhibiting factor, reverse transcriptase, dNTPs, a PCR Buffer, MgCl2, TaqDNA polymeras, positive control, negative control and RNase-free ddH2O. Specific primers are designed according to narcissus late season yellows virus gene sequences, and a nested RT-PCR technology is utilized to detect narcissus late season yellows viruses. Firstly, inverse transcription is carried out with extracted RNA as the template to synthesize cDNA, first-round PCR amplification is carried out with the cDNA as the template, second-round PCR amplification is then carried out with first-round PCR products as the template, and agarose gel electrophoresis detection is conducted on amplification products: if a specificity target fragment of the size of 539bp appear, the judgment result indicates that a detected virus is a narcissus late season yellows virus. The narcissus late season yellows virus detection kit and method have the advantages of being strong in specificity, high in sensitivity, good in accuracy and short in detection time and are suitable for quickly detecting and monitoring narcissus late season yellows viruses in entry ports, exit ports and agricultural production.

Description

technical field [0001] The invention relates to a narcissus late-season yellowing virus detection kit and a detection method thereof, which belong to the field of plant quarantine technology and are suitable for rapid detection and monitoring of narcissus late-season yellowing virus in entry and exit and agricultural production. Background technique [0002] Narcissus late season yellows virus (NLSYV) belongs to the family Potyviridae (Potyviridae), a member of the genus Potyvirus (Potyvirus), and the virus has similar characteristics to other potato Y viruses. A typical pinwheel-shaped inclusion body will appear in it. The viral genome is a positive-sense single-stranded RNA with a full length of about 9,651bp. Narcissus late-season yellow virus (NLSYV) was first reported in 1980, and since then people have carried out related studies on its virion structure, host range, hazard symptoms, mode of transmission and serological characteristics. The shape of NLSYV granules is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/6848C12Q1/686C12Q1/70C12Q2549/119C12Q2531/113
Inventor 沈建国于文涛黄振廖富荣蔡伟林双庆
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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