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Narcissus latent virus detection kit and detection method thereof

A detection kit, latent virus technology, applied in microorganism-based methods, biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as molecular detection kits that have not yet been seen, and achieve good practical application value The effect of saving detection time and high sensitivity

Active Publication Date: 2012-10-17
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 are few reports on the molecular detection method of Narcissus latent virus (NLV), and there is no molecular detection kit specially used for NLV detection.

Method used

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  • Narcissus latent virus detection kit and detection method thereof
  • Narcissus latent virus detection kit and detection method thereof
  • Narcissus latent virus detection kit and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Configuration of Narcissus Latent Virus Detection Kit (10 detections)

[0028] 1) Upstream primer: 10 μmol / L, 1 tube (30 μL);

[0029] 2) Downstream primer: 10 μmol / L, 1 tube (30 μL);

[0030] 3) RT Buffer: 5×, 1 tube (30μL);

[0031] 4) RNase inhibitor: 40U / μL, 1 tube (5μL);

[0032] 5) Reverse transcriptase: 200U / μL, 1 tube (5μL);

[0033] 6) dNTPs: 10mmol / L, 1 tube (30μL);

[0034] 7) PCR Buffer: 10×, 1 tube (30μL);

[0035] 8) Mgcl 2 : 25mmol / L, 1 tube (30μL);

[0036] 9) Taq DNA polymerase: 5U / μL, 1 tube (5μL);

[0037] 10) Positive control sample of narcissus latent virus, 1 tube (20 μL);

[0038] 11) Negative control sample without narcissus latent virus, 1 tube (20 μL);

[0039] 12) RNase-free ddH 2 O, 1 tube (1 mL).

Embodiment 2

[0040]Embodiment 2: the detection method of Narcissus latent virus detection kit

[0041] The detection method of above-mentioned Narcissus latent virus detection kit may further comprise the steps:

[0042] 1) Reverse transcription reaction: Add 2 μL of total RNA of the sample to be tested, 1 μL of downstream primers with a concentration of 10 μmol / L and RNase-free ddH in a 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×RT Buffer 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;

[0043] 2) 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 dNTPs at a concentration of 10 mmol / L, and 2.5 μL of 10×PCR...

Embodiment 3

[0045] Example 3: Specificity Verification of Narcissus Latent Virus Detection Kit

[0046] 1) Extraction of total RNA from narcissus samples: Narcissus latent virus (NLV), narcissus mosaic virus (NMV), narcissus yellow stripe virus (Narcissus yellow stripe virus, NYSV), narcissus late season yellow Narcissus samples of Narcissus late season yellows virus (NLSYV) and Arabis mosaic virus (ArMV) were used as materials, 0.1 g each was placed in a mortar, and 1 mL of PBST buffer was added for grinding, 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; centrifuge at 12000g for 10min at 4°C, take the supernatant; add chloroform 300μL, 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 an equal volume of isopropanol, invert and mix wel...

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Abstract

The invention relates to a narcissus latent virus detection kit and its detecting method that are special for narcissus latent virus detection. The kit comprises: an upstream primer, a downstream primer, an RT Buffer, an RNA enzyme inhibitor, a reverse transcriptase, dNTPs, a PCR Buffer, Mgcl2, Taq DNA polymerase, a positive control, a negative control and RNase-free ddH2O. In the invention, specific primers are designed according to a coat protein CP gene sequence of the narcissus latent virus, which is detected by a reverse transcription-polymerase chain reaction (RT-PCR) technology. With the obvious advantages of rapidity, accuracy, sensitivity, strong operability and simple kit preparation method, the kit and method of the invention are not only suitable for ports of China to conduct rapid detection on the narcissus latent virus of entry and exit narcissus, and also can be used for narcissus latent virus detection and diagnosis, epidemic surveillance as well as early warning and prediction, thus having broad application prospects.

Description

technical field [0001] The invention relates to a narcissus latent virus detection kit and a detection method thereof, belonging to the technical field of plant quarantine, not only applicable to the rapid detection of narcissus latent virus by port inspection and quarantine departments, but also applicable to the detection of narcissus latent virus by agricultural departments Real-time monitoring and early warning and forecasting. Background technique [0002] Narcissus latent virus (NLV) is a member of the family Potyviridae and the genus Maccluravirus. The shape of the virions is straight or slightly curved, and the particle size is about 650nm×13nm. The viral genome is positive-sense single-stranded RNA, the full length of which has not been reported yet. NLV is mainly transmitted by sap contact and non-persistent means of aphids. The virus can infect plants of Amaryllidaceae, Iridaceae, Amaranthaceae, Solanaceae, and Papilionaceae. The reported natural hosts include ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/94
Inventor 沈建国林双庆蔡伟廖富荣闫诚
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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