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Real-time fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent and detection method for lupulus masking virus

A technology of RT-PCR and latent viroids, which is applied in the field of real-time fluorescent RT-PCR detection reagents for hop latent viroids, can solve the problems that no public reports have been found in the detection, so as to avoid interference from human factors, eliminate pollution, and improve The effect of sensitivity

Inactive Publication Date: 2012-03-21
中华人民共和国北仑出入境检验检疫局 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, real-time fluorescent PCR technology has been widely used in many fields such as virus detection, bacterial detection, phytoplasma detection, genetically modified product detection, nematode detection, etc., but there is no public report on real-time fluorescent RT-PCR detection of latent viroids in hops

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1, the design of the special-purpose primer and probe that detects hop latent viroid

[0018] Coconut death viroids were retrieved from GenBank ( Cocadviroid ) The gene sequences of four viroids were compared and analyzed using DNAMAN 6.0.40 software to find out the conserved gene sequence of HpLVd as shown in SEQ ID NO: 4. According to the general principles of primer and probe design, use the software Primer Express 3.0 Design primers and TaqMan-MGB probes, then compare the designed primers and probes on NCBI, and finally determine a pair of primers and a probe through conventional screening. The sequence is: Primer HLV-F: 5'-CGTGGAACGG CTCCTTCTT-3'; primer HLV-R: 5'-AGAGTTGTAT CCACCGGGTA GTTT-3', probe HLV-P: 5'-CACCAGCCGG AGTT-3', the 5' end of the probe contains a FAM reporter fluorescent dye, and the 3' end contains A quencher that does not fluoresce and has an MGB molecule.

Embodiment 2

[0019] Embodiment 2, real-time fluorescent RT-PCR detects the specificity test of hop latent viroid

[0020] The specific process includes the following steps:

[0021] 1. Sample source

[0022] One positive sample of hops (sample M17) infected with hop latent viroid; Coleus blumei viroid 1 , CbVd-1), Coleus viroid 5 ( Coleus blumei viroid 5 , CbVd-5) Coleus leaves (sample J) 1; infected with hop dwarf viroid ( Hop stunt viroid , HpSVd), grape yellow spot virus 1 ( Grapevine yellow speckle viroid 1 , GYSVd-1) and grape yellow spot virus 2 ( Grapevine yellow speckle viroid 2 , GYSVd-2) grape total RNA (sample D1) 1 copy; infected with peach latent mosaic virus ( Peach latent mosaic viroid , PLMVd) peach total RNA (sample D2) 1 copy. Samples M17 and J were provided by Li Shifang, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, and samples D1 and D2 were provided by Deng Congliang, Plant Quarantine Laboratory of Beijing Entry-Exit Inspection a...

Embodiment 3

[0027] Embodiment 3, optimization of real-time fluorescence RT-PCR reaction system

[0028] Using the M17 total RNA obtained in step 2 in Example 2 as a template, the Mg in the real-time fluorescent RT-PCR detection system was respectively 2+ Concentration, primer concentration and probe concentration were optimized, the specific steps are as follows:

[0029] 1. Mg 2+ Concentration optimization

[0030] In the system of Step 3 in Example 2, the concentrations of other components were kept constant, and magnesium ions were added to increase the final concentration from 4.0 mM to 7.0 mM by 0.5 mM. The reaction conditions were carried out according to the conditions of Step 3 in Example 2. The results showed that when Mg 2+ When the final concentration was 5.5mM, the △Rn value reached the maximum and the Ct value was the minimum. After three repeated experiments, the optimal final concentration of magnesium ions was determined to be 5.5mM;

[0031] 2. Primer concentration o...

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Abstract

The invention discloses a real-time fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent and a detection method for lupulus masking virus. The real-time RT-PCR detection reagent comprises a pair of special primers and a special fluorescent probe. An amplification target fragment is 61bp in length, and the primers have the HLV-F sequence as follows: CGTGGAACGGCTCCTTCTT; the primers have the HLV-R sequence as follows: AGAGTTGTATCCACCGGGTAGTTT; and the probe has the HLV-P sequence as follows: CACCAGCCGGAGTT, 5' of the probe contains FAM report fluorescent dye, and the nucleotide sequence of the amplification target fragment is shown as SEQIDNO:4. The detection method comprises the steps of total RNA extraction and real-time fluorescent PCR reaction, in the RT-PCR reaction, the amplification of the primers and the detection of the probe are conducted simultaneously, a pipe is closed completely in the whole detection process, the PCR aftertreatment is not needed, the pollution caused by a PCR product is eliminated, the detection steps are reduced, the time is saved, and the detection method is special and sensitive for the lupulus masking virus. The detection reagent and the detection method can rapidly and accurately detect the lupulus masking virus, and has flexible operation.

Description

technical field [0001] The invention relates to a viroid detection technology, in particular to a real-time fluorescent RT-PCR detection reagent and a detection method related to a hop latent viroid. Background technique [0002] Hops latent viroids ( Hop latent viroid , HpLVd) belongs to the Cocadviroid genus, and is distributed on hops in the United Kingdom, Germany, South Korea, Poland, Japan, Czech Republic, Brazil, the United States, Xinjiang, China and other countries and regions. After the virus infects hops, it generally does not show obvious symptoms, but it will change some of its secondary metabolites, thereby affecting the quality of hops and reducing the yield of hops. Adams et al. reported that after different hop varieties were infected by HpLVd, the content of a-acid decreased by 20% to 50%; the yield of hop cones infected by HpLVd decreased by 11%. [0003] At present, the method for detecting latent viroids in hops at home and abroad is mainly molecular h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/94
Inventor 郭立新段维军闻伟刚
Owner 中华人民共和国北仑出入境检验检疫局
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