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A PCR primer used by a lily symptomless virus test method and a test kit thereof

A technology for detection of lily asymptomatic virus and primers, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of low detection sensitivity, time-consuming and labor-intensive, easy cross-contamination, etc., and achieve simple and convenient experimental operation , high detection sensitivity, and the effect of preventing cross-contamination

Inactive Publication Date: 2009-09-16
INSPECTION AND QUARANTINE TECHNOLOGY CENTER ZHONGSHAN ENTRY EXIT INSPECTION AND QUARANTINE +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, some people use PCR to detect lily asymptomatic virus, but the two-step method used, that is, the reverse transcription RT step and the PCR amplification step are carried out separately, which is not only very easy to cross-contamination, but also time-consuming and laborious, and the detection sensitivity is not high.

Method used

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  • A PCR primer used by a lily symptomless virus test method and a test kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 1. Extraction of lily total nucleic acid

[0033] (1) Take 0.1-0.2g of tender leaves to be tested, place them in a 1.5mL centrifuge tube, and freeze them with liquid nitrogen;

[0034] (2) Vigorously oscillate by an oscillator, and grind the blade with a glass rod;

[0035] (3) Add 0.6mL CTAB extraction buffer homogenate + 2% 2-ME, shake and mix;

[0036] (4) Incubate at 65°C for 30 minutes; then add 0.5 mL of chloroform and mix for 5 minutes;

[0037] (5) Centrifuge at 12000rpm for 5min, and transfer the upper aqueous phase to a new centrifuge tube;

[0038] (6) Add 1× volume of isopropanol, mix gently, and place at -20°C for 30 minutes;

[0039] (7) centrifuge at 12000rpm for 10min, discard the supernatant;

[0040] (8) Wash the precipitate with 70% ethanol, centrifuge at 12000rpm for 2min; then wash once with 100% ethyl alcohol;

[0041] (9) Discard the supernatant, and vacuum-dry for 5 minutes;

[0042] (10) The precipitate was dissolved in 50 uLTE (pH 8.0) an...

Embodiment 2

[0071] Embodiment two: the preparation of lily asymptomatic virus PCR kit

[0072] The composition of the kit is as follows:

[0073] 1. Enzyme Mix 100μl

[0074] 2. Buffer 625μl×2

[0075] 3. LSV upstream primer (LSV-1) 50 μl (concentration is 1 μm)

[0076] 4. 50 μl of LSV downstream primer (LSV-2) (concentration is 1 μm)

[0077] 5. Lily asymptomatic virus positive control 50μl (50μg)

[0078] 6. RNase Free deionized water (RNase Free dH20) 1000μl

[0079] The above is the amount of the RT-PCR reaction system for 50 times, each time 50 μl.

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Abstract

The invention provides an RT-PCR primer used by a lily symptomless virus test method and a test kit thereof; wherein an upstream primer LSV-P1 is: 5'-GAY GAR YTY AAR ATG AAR GT-3'; a downstream primer LSV-P2 is: 5'-ARY TGY TTR TGY GCR TTR TG-3'; wherein R is A or G; and Y is C or T. Using the test kit of the invention to test can greatly reduce the possibility of cross contamination, improve the test efficiency to a large extent, ensure high test sensitivity and simple and convenient operation, and prevent cross contamination.

Description

technical field [0001] The invention relates to a primer for detecting lily asymptomatic virus and a detection kit thereof. Background technique [0002] The scientific name of lily symptomless virus is: Lily symptomless virus (LSV), which is one of the main viruses that seriously affect the production of lily flowers. The virus belongs to the genus Carlavirus and is widely distributed in Sweden, the United Kingdom, Germany, Denmark, the Netherlands, Belgium, Italy, Latvia, the United States, Canada, Japan and other countries. [0003] The virus mainly infects Liliaceae plants, most of which are systemic infections, and generally do not cause obvious symptoms, but it will cause early yellowing of the lower leaves of lilies after harvesting, affecting the life of vases, and co-infects with Cucumber Mosaic Virus (CMV) Sometimes, it can cause bright pulse, mosaic, deformity, necrotic spots and other symptoms. [0004] Under natural conditions, it is transmitted non-persistent...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
Inventor 陈定虎王龙贵杨雷亮管维
Owner INSPECTION AND QUARANTINE TECHNOLOGY CENTER ZHONGSHAN ENTRY EXIT INSPECTION AND QUARANTINE
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