Method for synchronously detecting four viruses of carnation

A technology for simultaneous detection and detection methods, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., to achieve the effects of appropriate fragment length differences, clear bands, and low total cost.

Inactive Publication Date: 2012-11-21
BEIJING FORESTRY UNIVERSITY
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Problems solved by technology

[0003] After years of continuous improvement, the current PCR technology has developed into a general-purpose technology. Multiplex RT-PCR can provide amplification information of several conventional reactions in one reaction at the same time. It is of great significance in the rapid breeding and promotion of virus-free seedlings, but there is no report on the simultaneous detection of multiple carnation viruses using this technology

Method used

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  • Method for synchronously detecting four viruses of carnation
  • Method for synchronously detecting four viruses of carnation
  • Method for synchronously detecting four viruses of carnation

Examples

Experimental program
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Effect test

Embodiment 1 4

[0040] Example 1 Quadruple compound RT-MPCR technique detects carnation CarMV, CNFV, CRSV, CLV four kinds of viruses

[0041] 1.1 Primer design for CarMV, CNFV, CRSV, CLV

[0042] By designing primers with Primer 5.0 software, four pairs of specific viral primers with good compatibility were screened out, and the sequences are shown in the table below.

[0043] The primer sequence of table 1 CarMV, CNFV, CRSV, CLV

[0044]

[0045] 1.2 RNA extraction

[0046] (1) Weigh 0.1 g of young leaves or petals of Carnation 'Mast', 'Yafen' and 'Freedom' (purchased from Yunnan Academy of Agricultural Sciences) into a mortar, and grind into a very fine powder under liquid nitrogen, The powder was quickly transferred to a pre-cooled 1.5mL centrifuge tube, 1mL Trizol (Invitrogen) was added, mixed thoroughly with a vortex mixer, and allowed to stand at room temperature for 5min to fully lyse.

[0047] (2) Centrifuge at 13,000 rpm at 4°C for 10 min, absorb the supernatant, add an equal v...

Embodiment 2

[0062] The RT-MPCR system optimization of embodiment 2 carnation four kinds of virus detection

[0063] In order to optimize the RT-MPCR system of the present invention, based on the RT-MPCR system established above, the components and concentrations in the PCR system were studied. The basic principles of optimizing the PCR system are: obtaining clear and stable target-specific products; low cost; short reaction time, etc.

[0064] The main factors affecting multiplex PCR include: the size of the fragment amplified by each pair of primers, primer design, pairing and competitive amplification between primers, annealing temperature and primer concentration.

[0065] 2.1 Optimization of annealing temperature

[0066] The annealing temperature (annealing temperature) has a very important influence on the success or failure of the PCR test.

[0067] In this experiment, TC-512 gradient PCR instrument from Techne Company was used to explore the annealing temperature of the reaction...

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Abstract

The invention relates to a method for synchronously detecting four viruses of carnation, in particular to a method for synchronously detecting carnation mottle virus, latent virus, ringspot virus and necrotic spot virus. The invention discloses primers for synchronously detecting the four viruses, and the sequences of the primers are shown as SEQ ID No.1-8. The invention also discloses a method for synchronously detecting the four viruses, which comprises the following steps of: 1) extracting total RNA of the carnation; and 2) performing reverse transcription-multiplex polymerase chain reaction: performing reverse transcription of the RNA obtained in the step 1), performing synchronous amplification on a reverse transcription production by using four pairs of primers, and synchronously detecting the four viruses. The primers have good compatibility, and belts are clear and easy to identify; and by the method, the detection cost is reduced, the detection speed is improved, and the synchronization, high efficiency and sensitivity for detecting the viruses of the carnation are realized.

Description

technical field [0001] The invention belongs to the field of plant virus detection, and in particular relates to a method for synchronous detection of four carnation viruses. Background technique [0002] In the RT-PCR detection technology of carnation virus, people such as Kong Baohua (Kong Baohua, Cai Hong et al. Identification and PT-PCR detection of carnation mottle virus [J]. Plant Protection, 2002, 2, 28 (1): 5 -8) according to the RNA sequence design primer of carnation mottle virus CarMV, can dilute 10 from RNA 4 Carnation mottled virus was detected in the crude RNA extract of Zhao Shan et al. (Zhao Shan, Wang Jihua, Wang Lihua, Yang Xiumei. Identification and detection of carnation necrotic spot virus [J]. Southwest Agricultural Journal, 2010, 1: 103-106) by designing primers from carnation necrotic spot virus CNFV The CNFV virus was detected in the susceptible carnation tissue, and the sensitivity test results showed that the specific primer RT-PCR could be dilut...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 吕英民程堂仁王舒藜张启翔
Owner BEIJING FORESTRY UNIVERSITY
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