Narcissus retrovirus nested RT-PCR detection kit and its detection method

A Narcissus degenerated virus, RT-PCR technology, applied in biochemical equipment and methods, methods based on microorganisms, measurement/testing of microorganisms, etc., can solve problems such as nested RT-PCR detection kits for rapid detection of viruses that have not yet been seen , to achieve the effect of high sensitivity, strong specificity and short cycle

Active Publication Date: 2016-03-16
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But so far, there are few reports about the nested RT-PCR detection method of Narcissus degenerate virus (NDV), and there is no nested RT-PCR detection kit specially used for the rapid detection of this virus so far.

Method used

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  • Narcissus retrovirus nested RT-PCR detection kit and its detection method
  • Narcissus retrovirus nested RT-PCR detection kit and its detection method
  • Narcissus retrovirus nested RT-PCR detection kit and its detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: the configuration of narcissus retrovirus nested RT-PCR detection kit (10 detections)

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

[0034] 2) Outer reverse primer: 10 μmol / L, 1 tube (30 μL);

[0035] 3) Inner forward primer: 10 μmol / L, 1 tube (30 μL);

[0036] 4) Inner reverse primer: 10 μmol / L, 1 tube (30 μL);

[0037] 5) RTBuffer: 5×, 1 tube (30 μL);

[0038] 6) RNase inhibitor: 40U / μL, 1 tube (5μL);

[0039] 7) Reverse transcriptase: 200U / μL, 1 tube (5μL);

[0040] 8) dNTPs: 10mmol / L, 1 tube (30μL);

[0041] 9) PCRBuffer: 10×, 1 tube (60 μL);

[0042] 10) Mgcl 2 : 25mmol / L, 1 tube (50μL);

[0043] 11) TaqDNA polymerase: 5U / μL, 1 tube (10μL);

[0044] 12) Positive control sample of Narcissus retrograde virus, 1 tube (50 μL);

[0045] 13) Negative control sample without narcissus retrovirus, 1 tube (50 μL);

[0046] 14) RNase-freeddH 2 0, 1 tube (1 mL).

Embodiment 2

[0047] Embodiment 2: the detection method of Narcissus retrograde virus nested RT-PCR detection kit

[0048] The detection method of above-mentioned narcissus retrovirus nested RT-PCR detection kit comprises the following steps:

[0049] 1) Reverse transcription reaction: Add 3 μL of the total RNA of the sample to be tested, 1 μL of the outer reverse primer with a concentration of 10 μmol / L and RNase-freedH in the PCR tube 2 O7μL, 70℃ water bath for 10min, rapid ice bath for 5min, then add the following reagents: 5×RTBuffer 2.5μL, concentration 10mmol / LdNTPs 1μL, concentration 200U / μL reverse transcriptase 0.5μL, concentration 40U / μL RNase inhibitor 0.5 μL. 42°C water bath for 60 minutes, 70°C water bath for 10 minutes, synthesize cDNA;

[0050] 2) The first round of PCR reaction: take 3 μL of cDNA synthesized in step 1), add 0.5 μL of TaqDNA polymerase at a concentration of 5 U / μL, 0.5 μL of 10 mmol / LdNTPs, 2.5 μL of 10×PCRBuffer, and a concentration of 25 mmol / LMgCl into e...

Embodiment 3

[0053] Example 3: Specificity determination of Narcissus retrovirus nested RT-PCR detection kit

[0054] 1) Extraction of total RNA from Narcissus samples: Narcissus degenerate virus (NDV), Narcissus late season yellowing virus (NLSYV), Narcissus mosaic virus (NMV), Narcissus yellow stripe virus (Narcissus yellowstripevirus, NYSV), Narcissus latent virus (Narcissus latent virus, NLV) and Arabismosaic virus (Arabismosaicvirus, ArMV) narcissus samples were used as materials, each 0.1g was placed in a mortar, 1mL of LPBST buffer was added to grind, 4°C, centrifuged at 10000g for 5min, and 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; centrifuge at 12000g at 4°C for 10min, take the supernatant; add 300μL chloroform, shake vigorously for 15s , let stand at room temperature for 5min, centrifuge at 12000g at 4°C for 15min, take the upper aqueous phase; add a...

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Abstract

The invention relates to a nest type RT-PCR detection kit and a detection method for narcissus degeneration virus, which are specially used for detecting narcissus degeneration virus. The kit comprises an outer-side forward primer, an outer-side reverse primer, an inner-side forward primer, an inner-side reverse primer, an RT Buffer, an RNA enzyme inhibiting factor, a reverse transcriptase, dNTPs, a PCR Buffer, MgCl2, Taq DNA polymerase, a positive control, a negative control and RNase-free ddH2O. The nest type RT-PCR detection kit and the detection method conduct detection to the narcissus degeneration virus by adopting nest type RT-PCR technology, and have the advantages of being strong in specificity, high in sensitivity, simple and convenient to operate, and fast in detection, and are applicable to fast detection, epidemic situation monitoring and early-warning and forecasting on the narcissus degeneration virus in China export and import quarantine and agricultural production, thus being wide in application prospects.

Description

technical field [0001] The invention relates to a nested RT-PCR detection kit for daffodil degeneration virus and a detection method thereof, belonging to the technical field of plant quarantine, and is suitable for rapid detection and monitoring of daffodil degeneration virus in entry and exit and agricultural production. Background technique [0002] Narcissus degeneration virus (NDV) is a member of the Potyviridae family Potyviridae. The full length of the virus genome is about 9,816bp. It is a positive-sense single-stranded RNA without a polyA tail. The typical pinwheel-shaped inclusions were produced in the mesophyll cells of Narcissus. The virus was first discovered on the British multiflora narcissus variety (Narcissustazettac v. Grand Soleild'Or), and the symptoms of chlorotic stripes appeared on the leaves of infected narcissus. The host of NDV experiments is narrow, mainly limited to several plants of Amaryllidaceae (Amaryllidaceae), and the plants that can be ino...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/94
CPCC12Q1/6848C12Q1/70C12Q2531/113C12Q2549/119
Inventor 沈建国蔡伟金晶廖富荣高芳銮林双庆
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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