Quick method for testing chain reaction of multiple reverse transcription polymerase of lily virus

A technology of reverse transcription polymerase and chain reaction, which is applied in the field of plant virus detection, can solve the problems of difficult preparation and purchase of monoclonal antibodies, high sensitivity, and many manpower, so as to reduce the difficulty of amplification of large fragment products, high Specificity and amplification efficiency, and the effect of improving detection speed

Inactive Publication Date: 2006-12-06
FLOWER RES INST OF YUNNAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the preparation and purchase of monoclonal antibodies in the serum detection method is not easy, time-consuming and laborious, and it takes at least 2-3 days to obtain the test results, thus limiting the application of this method; although the electron microscope detection method is low in cost, it requires relatively expensive large instruments — Electron microscope, only a few units have the ability to purchase; the sensitivity of the RT-PCR detection method for a single virus is high, and the accurate value can realize the analysis of a very small amount of viral RNA samples, but its detection can only be used for qualitative analysis of a single virus. To detect the three viruses, it will consume more manpower, material and financial resources

Method used

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  • Quick method for testing chain reaction of multiple reverse transcription polymerase of lily virus
  • Quick method for testing chain reaction of multiple reverse transcription polymerase of lily virus
  • Quick method for testing chain reaction of multiple reverse transcription polymerase of lily virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] 1. Virus isolates were collected from the middle leaves of naturally susceptible lily plants at the Unity Base of the Institute of Flowers, Yunnan Academy of Agricultural Sciences. The samples were tested by DAS-ELISA and confirmed to be infected with lily asymptomatic virus (LSV), lily mottled virus (LMoV), cucumber Co-infection with mosaic virus (CMV).

[0055] 2. The total RNA extraction kit was RNAex Reagent SystemIV from Shanghai Huashun Company; reverse transcriptase, cDNA synthesis kit and cloning kit were purchased from Promega Company; Taq enzyme, dNTP and bromophenol blue (6×Loading Dye Solution) were purchased from From TaKaRa Company; other reagents and consumables were purchased from Sangon Company, Huamei Company, etc.

[0056] 3. Commonly used buffers and equipment

[0057] 5×TBE electrophoresis buffer

[0058] Tris 54g

[0059] Boric acid 27.5g

[0060] 0.5mol / L EDTA (pH8.0) 20mL

[0061] Dilute to 1000mL, store at 4°C

[0062] equipment

[0063] ...

Embodiment 2

[0113] 1. The isolates were collected from the outer scales of lily bulbs from Yunnan Longgelan Horticulture Co., Ltd., and the samples were tested by DAS-ELISA and confirmed to be infected with lily asymptomatic virus (LSV), lily mottled virus (LMoV), cucumber flower Co-infection with leaf virus (CMV).

[0114] 2. The total RNA extraction kit was RNAex Reagent SystemIV from Shanghai Huashun Company; reverse transcriptase, cDNA synthesis kit and cloning kit were purchased from Promega Company; Taq enzyme, dNTP and bromophenol blue (6×Loading Dye Solution) were purchased from From TaKaRa Company; other reagents and consumables were purchased from Sangon Company, Huamei Company, etc.

[0115] 3. Commonly used buffers and equipment

[0116] 5×TBE electrophoresis buffer

[0117] Tris 54g

[0118] Boric acid 27.5g

[0119] 0.5mol / L EDTA(PH8.0) 20mL

[0120] Dilute to 1000mL, store at 4°C

[0121] equipment

[0122] High Speed ​​Refrigerated Centrifuge 5417C Eppendorf

[0123]...

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Abstract

This invention relates to a method for rapidly detecting lily virus by multiplex RT-PCR. The method comprises: (1) synthesizing three pairs of primers of lily symptomless virus (LSV), lily mottle virus (LMoV) and Cucumber mosaic virus (CMV) sequences, and optimizing the concentrations, amplification temperatures, amplification time and cycle time; (2) introducing the three pairs of primers to a same reaction system, and performing PCR reaction. The method can detect CMV, LSV and LmoV simultaneously, and has such advantages as rapid detection, high accuracy, high efficiency, high sensitivity, high specificity.

Description

technical field [0001] The invention relates to a detection method of plant virus, in particular to a multiple reverse transcription polymerase chain reaction (RT-PCR) rapid detection method of lily virus. Background technique [0002] Lily is an important economic crop and has a long history of cultivation in my country. With the continuous expansion of cultivation scale in recent years, the phenomenon of lily being infected by viruses has become more and more serious. Studies have shown that there are as many as 16 kinds of viruses infecting lily, wherein cucumber mosaic virus (CMV), lily asymptomatic virus (LSV), lily mottle virus (LMoV) are the main virus pathogens infecting lily, plum necrotic ring spot virus (PNRSV) also occurs in lily. The lily plants after infection showed: yellowing, mosaic, necrotic streaks, chlorotic streaks, bright veins, yellow leathery, deformed, chlorotic mottled, necrotic spots, broken flowers, etc., which seriously affected the fresh-cut fl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/25
Inventor 王丽花王继华瞿素萍杨秀梅苏艳张丽芳陆琳张璐萍
Owner FLOWER RES INST OF YUNNAN ACAD OF AGRI SCI
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