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Real-time reverse transcription-polymerase chain reaction (RT-PCR) detection kit and method for detecting alfalfa mosaic virus (AMV)

A detection kit and leaf virus technology, applied in the biological field, can solve problems affecting crop yield, economic loss, etc., and achieve high consistency, good stability, and strong specificity

Active Publication Date: 2018-12-11
兰州海关技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The virus can be mixed with other viruses, affecting crop yield and causing serious economic losses

Method used

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  • Real-time reverse transcription-polymerase chain reaction (RT-PCR) detection kit and method for detecting alfalfa mosaic virus (AMV)
  • Real-time reverse transcription-polymerase chain reaction (RT-PCR) detection kit and method for detecting alfalfa mosaic virus (AMV)
  • Real-time reverse transcription-polymerase chain reaction (RT-PCR) detection kit and method for detecting alfalfa mosaic virus (AMV)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: a kind of alfalfa mosaic virus real-time RT-PCR detection kit, detection pathogen is alfalfa mosaic virus, and it comprises following components:

[0030] RT-PCR buffer, 2×RT-PCR buffer, 250 μL; Ex Taq HS 5U / μL, 20 μL; Prime Script RTEnzyme Mix, 20 μL; Alfalfa mosaic virus AMV upstream primer, concentration 10 μmol / L, 30 μL; Alfalfa mosaic virus AMV downstream primer, the concentration is 10 μmol / L, 30 μL; alfalfa mosaic virus AMV probe 10 μmol / L, 30 μL; RNase-free deionized water, 1 mL; alfalfa mosaic virus AMV non-infective positive control 60 μL.

[0031] RT-PCR amplification primers and probes are:

[0032] The upstream primer of alfalfa mosaic virus AMV is: 5'-GCTACAGGAGAGTTAGTG-3';

[0033] The downstream primer of alfalfa mosaic virus AMV is: 5′-GACAGCAGAGTTCATATTG-3′;

[0034] The probe sequence of alfalfa mosaic virus AMV is: 5'-TGTTGACACCGACCATGATGCTA-3'.

Embodiment 2

[0035] Embodiment 2: a kind of alfalfa mosaic virus real-time RT-PCR detection kit, alfalfa pathogen is alfalfa mosaic virus, and it comprises the following components:

[0036] RT-PCR buffer, 2×RT-PCR buffer, 40μL; Ex Taq HS 5U / μL, 1.5 μL; Prime Script RTEnzyme Mix, 1.5 μL; alfalfa mosaic virus AMV upstream primer, concentration 10 μmol / L, 1.5 μL; alfalfa mosaic virus AMV downstream primer, concentration 10 μmol / L, 1.5 μL; Alfalfa mosaic virus AMV probe 10 μmol / L, 1.5 μL; RNase-free deionized water, 20 μL; alfalfa mosaic virus AMV non-infective positive control 6 μL.

[0037] RT-PCR amplification primers and probes are:

[0038] The upstream primer of alfalfa mosaic virus AMV is: 5'-GCTACAGGAGAGTTAGTG-3';

[0039] The downstream primer of alfalfa mosaic virus AMV is: 5′-GACAGCAGAGTTCATATTG-3′;

[0040] The probe sequence of alfalfa mosaic virus AMV is: 5'-TGTTGACACCGACCATGATGCTA-3'.

Embodiment 3

[0041] Embodiment 3: a kind of real-time RT-PCR detection method of alfalfa mosaic virus, specifically:

[0042] 1) RNA extraction of samples to be tested;

[0043] 2) The RT-PCR reaction system is 20 μL, including 10 μL of 2×one step RT-PCR buffer, Ex TaqHS 5U / μL 0.4μL, Prime Script RT Enzyme Mix 0.4μL, alfalfa mosaic virus AMV upstream and downstream primers 10μmol / L 0.4μL each, alfalfa mosaic virus AMV probe 10μmol / L 0.8μL, total RNA template 2μL, RNase-free deionized water to make up the total reaction volume;

[0044] 3) RT-PCR reaction conditions are:

[0045] cDNA synthesis: 5 min at 42°C, 1 cycle;

[0046] PCR amplification: 95°C for 10 s; 95°C for 5 s, 59°C for 34 s, 40 cycles;

[0047] 4) Use a real-time fluorescent PCR detector for PCR amplification and result analysis. Through fluorescent quantitative detection, judge the result according to the Ct value of the fluorescent detection. If the fluorescent RT-PCR reaction is positive, the sample to be tested conta...

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Abstract

The invention relates to a real-time reverse transcription-polymerase chain reaction (RT-PCR) detection kit and a method for detecting alfalfa mosaic virus (AMV). The kit comprises the following components: 40-250mu L of RT-PCR buffer, 40-250mu L of 2*RT-PCR buffer, 1.5-20mu L of Ex Taq HS, 1.5-20mu L of Prime Script RT Enzyme Mix, 1.5-30mu L of an AMV upstream primer with concentration of 10mu mol / L, 1.5-30mu L of an AMV downstream primer with concentration of 10mu mol / L, 1.5-30mu L of an AMV probe with concentration of 10mu mol / L, 20muL-1mL of deionized water without RNA enzyme, and 6-60mu Lof a positive control without infection activity for the AMV. The detection method provided by the invention can be used for specifically amplifying gene segments of the AMV, and is good in detecting stability, strong in specificity, high in consistency and short in detection period.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a method for detecting alfalfa diseases, which is mainly aimed at the detection of alfalfa mosaic virus. Background technique [0002] by alfalfa mosaic virus ( Alfalfa mosaic virus , AMV) caused by mosaic, first reported in 1931, is currently occurring all over the world. Alfalfa mosaic virus belongs to the family Bromoviridae (Bromoviridae) and the genus Alfalfa mosaic virus ( Alfamovirus ) of the definite species. The virus has a wide range of hosts, naturally occurring on many herbaceous and woody plants, and can infect 430 species of dicotyledonous plants in 51 families. Among them, important hosts with economic value include alfalfa, potato, soybean, cowpea, red bean, and mung bean , sweet clover, clover, vetch, lupin, sweet pea, chickpea, pea, broad bean, lentil, kidney bean, tobacco, pepper, tomato, hops, celery, etc. [0003] Alfalfa mosaic virus can be transmitted by aphi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12R1/94
CPCC12Q1/686C12Q1/701C12Q2563/107
Inventor 文朝慧周小平李儒王军平王溪桥尤佳满岳左佳妮
Owner 兰州海关技术中心
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