CN102382903B - Real-time fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent and detection method for lupulus masking virus - Google Patents
Real-time fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent and detection method for lupulus masking virus Download PDFInfo
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- CN102382903B CN102382903B CN201110319087.5A CN201110319087A CN102382903B CN 102382903 B CN102382903 B CN 102382903B CN 201110319087 A CN201110319087 A CN 201110319087A CN 102382903 B CN102382903 B CN 102382903B
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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
The present invention relates to the detection technique of viroid, be specifically related to relate to real-time fluorescent RT-PCR detection reagent and the detection method of hop latent viroid.
Background technology
The hop latent viroid (
Hop latent viroid, HpLVd) be under the jurisdiction of Cocadviroid and belong to, be distributed on the hops of countries and regions such as Britain, Germany, Korea S, Poland, Japan, Czech, Brazil, the U.S., Chinese Xinjiang.Generally do not show tangible symptom behind such virus infection hops, but can change its some secondary metabolite compositions, thereby influence the hops quality, reduce hops output.Reports such as Adams, after different hops kinds were infected by HpLVd, the content of a-acid reduced by 20%～50%; The hop cone output that infected by HpLVd reduces by 11%.
The method that detects the hop latent viroid at present both at home and abroad mainly is molecular hybridization; The prerequisite of molecular hybridization is to prepare probe, and is comparatively complicated, and detects complex steps.In recent years, utilizing the TaqMan-MGB probe to carry out the real-time fluorescence RT-PCR reaction is to have added 1 TaqMan-MGB probe in conventional RT-PCR reaction system; The 5' end of probe contains the report fluorophor, and the 3' end contains not fluorescent quenching group and has the MGB molecule.When probe was complete, the fluorescent signal of reporter group emission was absorbed by quenching group, when carrying out pcr amplification,
TaqThe 5'-3' 5 prime excision enzyme activity of archaeal dna polymerase is cut degraded with the probe enzyme, and the report fluorophor is separated with the cancellation fluorophor, sends fluorescent signal, thereby the accumulation of fluorescent signal and PCR product are formed fully synchronously.The real-time fluorescence PCR technology has been widely used in numerous areas such as viral detection, Bacteria Detection, pytoplasma detection, transgenic product detection, nematode detection at present, but open report is not seen in the real-time fluorescence RT-PCR detection of hop latent viroid as yet.
Summary of the invention
Technical problem to be solved by this invention provide a kind of simple to operate, quick, sensitive, detect detection reagent and the method for hop latent viroid accurately.
The present invention solves the problems of the technologies described above one of technical scheme of adopting and provides a kind of real-time fluorescent RT-PCR detection reagent that detects the hop latent viroid, comprise a pair of Auele Specific Primer and a specificity fluorescent probe, amplification target fragment length is 61bp, and primer and probe sequence are:
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 this probe contains FAM report fluorescence dye, and the 3' end contains not fluorescent quenching group and has the MGB molecule; The nucleotide sequence of amplification target fragment is shown in SEQ ID NO:4.
The present invention solve the problems of the technologies described above two of the technical scheme that adopts provide a kind of energy simple, fast, sensitive, detect the method for hop latent viroid exactly, be to be template with the total RNA of hops sample, primer and probe with above-mentioned detection hop latent viroid carry out real-time fluorescence RT-PCR, detect the amplified production fluorescent signal;
Described hops sample can be hops seedling, blade and cone;
In the aforesaid method, as from amplified production, detecting fluorescent signal, illustrate that described test sample contains the hop latent viroid, and can be according to the Ct(cycle threshold) size determine the amount of the starting template in the sample;
Described hops sample extracts test kit (available from Qiagen company) through RNA and extracts the total RNA of plant, obtains total RNA template, and the test kit specification sheets is seen in concrete operations;
The reaction of described real-time fluorescence RT-PCR is that to extract the total RNA of sample be template, Mg in the 25 μ L reaction systems
2+Final concentration is that the final concentration of 5.5mM, primer HLV-F and HLV-R all is that 0.7 μ M, probe HLV-P final concentration are the Mg that 0.5 μ M, 2xQuantiTect Probe RT-PCR Master Mix(contain final concentration 4.0 mM
2+, available from Qiagen company) be that 12.5 μ L, QuantiTect RT Mix(are available from Qiagen company) be 0.25 μ L, RNA(20 ng/ μ L ~ 200 ng/ μ L) be 1 μ L, RNase Free dH
2O mends to 25 μ L; The reaction conditions of described real-time fluorescence RT-PCR is: 50 ℃ of 30 min; 95 ℃ of 15 min; 94 ℃ of 15 s then, 60 ℃ of 60 s, totally 40 circulations.Reaction system also can be 50 μ L etc.
The present invention is directed to shortcomings such as existing hop latent viroid detection method is time-consuming, loaded down with trivial details, provide a kind of simple to operate, quick, sensitive, detect the real-time fluorescent RT-PCR method for detecting of hop latent viroid accurately.The primer special of real-time fluorescent RT-PCR detection reagent and probe have very strong specialization to the hop latent viroid in the inventive method, and by the optimization to reaction system, can not only improve the effect that the hop latent viroid detects, can also carry out quantitative analysis to being subjected to the sample product.
The present invention compared with prior art, have the following advantages: 1, simple fast: as only to need the total RNA of small amount of sample, only need 2 hours just can finish RT reaction and PCR step with the fluorescent PCR instrument, can obtain detected result, avoided the treating processess such as probe preparation in the common molecular detection technology; 2, high specificity: adopting the peculiar primer of a pair of hop latent viroid to carry out the amplification of target gene fragment and one can detect with the fluorescent probe of template complementary pairing, has improved specificity, avoids false positive and false negative effectively; 3, highly sensitive: collect fluorescent signal automatically by the fluorescent PCR instrument, avoid interference from human factor in the interpretation of result, further improve sensitivity, in 25 μ L reaction systems, it is 20pg that total RNA detects lower bound.4, crossed contamination is low: the complete stopped pipe of whole testing process, do not need the PCR aftertreatment, and eliminated the pollution of PCR product.
Embodiment
Describe in further detail below in conjunction with the present invention of embodiment.
Embodiment 1, the primer special that detects the hop latent viroid and the design of probe
From GenBank access cocadviroid (
Cocadviroid) gene orders of four kinds of viroids, utilize DNAMAN 6.0.40 software to compare, find out the conservative gene sequence of the HpLVd shown in SEQ ID NO:4, rule according to primer and probe design, with software Primer Express 3.0 design primer and TaqMan-MGB probes, again primer and the probe of design are compared at NCBI, by the final definite a pair of primer of routine screening and a probe, its sequence is: primer HLV-F:5'-CGTGGAACGG CTCCTTCTT-3'; Primer HLV-R:5'-AGAGTTGTAT CCACCGGGTA GTTT-3', probe HLV-P:5'-CACCAGCCGG AGTT-3', probe 5' end contain FAM report fluorescence dye, and the 3' end contains not fluorescent quenching group and has the MGB molecule.
Embodiment 2, real-time fluorescence RT-PCR detect the specificity test of hop latent viroid
Detailed process may further comprise the steps:
One, sample source
1 part of the hops positive (sample M17) of infection hop latent viroid; Infect coleus blumei viroid 1 No. 1 (
Coleus blumei viroid 1, CbVd-1), No. 5, coleus blumei viroid 1 (
Coleus blumei viroid 5, 1 part in coleus blade CbVd-5) (sample J); Infection hops dwarfing viroid (
Hop stunt viroid, HpSVd), the yellow point of grape viroid No. 1 (
Grapevine yellow speckle viroid 1, GYSVd-1) and the yellow point of grape viroid No. 2 (
Grapevine yellow speckle viroid 2, the total RNA(sample of grape GYSVd-2) D1) and 1 part; Infect peach hide the floral leaf viroid (
Peach latent mosaic viroid, the total RNA(sample of peach PLMVd) D2) and 1 part.Sample M17 and J are provided by the Li Shifang of Scientia Agricultura Sinica research institute plant protection institute, and sample D1 and D2 are very provided by the Plant Quarantine laboratory Deng Cong of Beijing Administration for Entry-Exit Inspection and Quarantine;
Two, the extraction of the total RNA of plant
Sample in the step 1 is extracted test kit (available from Qiagen company) through RNA extract the total RNA of plant, the test kit specification sheets is seen in concrete operations;
Three, specific detection test
The total RNA of plant that obtains with step 1 and step 2 is template, that is: the total RNA of M17, J, D1 and D2, water is blank, carry out the real-time fluorescence RT-PCR reaction, namely in 25 μ L reaction systems, add 12.5 μ L 2xQuantiTect Probe RT-PCR Master Mix, 0.5 μ L primer HLV-F(20 μ mol/L), 0.5 μ L primer HLV-R(20 μ mol/L), 0.25 μ L probe HLV-P(20 μ mol/L), 0.25 μ L QuantiTect RT Mix, 1 μ L RNA(20 ng/ μ L ~ 200 ng/ μ L), RNase Free dH
2O mends to 25 μ L.Reaction conditions is: 50 ℃ of 30 min; 95 ℃ of 15 min; 94 ℃ of 15 s then, 60 ℃ of 60 s, totally 40 circulations.The result shows that designed primer and probe have stronger specificity, (amplification and the target fragment length that detects are 61bp only to detect the hop latent viroid in seven kinds of viroids, nucleotide sequence is shown in SEQ ID NO:4), can not detect CbVd-1, CbVd-5, HpSVd, GYSVd-1, GYSVd-2, PLMVd and water.
The optimization of embodiment 3, real-time fluorescence RT-PCR reaction system
The total RNA of M17 that obtains with step 2 among the embodiment 2 is template, respectively to the Mg in the real-time fluorescence RT-PCR detection architecture
2+Concentration, primer concentration and concentration and probe concentration are optimized, and concrete steps are as follows:
One, Mg
2+Concentration is optimized
Other constituent concentration is constant in the system of step 3 in embodiment 2, adds magnesium ion, and its final concentration is increased progressively with 0.5 mM from 4.0 mM to 7.0 mM.Reaction conditions is undertaken by the condition of step 3 among the embodiment 2.The result shows, works as Mg
2+When final concentration was 5.5mM, △ Rn value reached maximum, the Ct value is minimum, through three repeated experiments, determines that best magnesium ion final concentration is 5.5mM;
Two, primer concentration optimization
Other constituent concentration is constant in the system of step 3 in embodiment 2, and the magnesium ion concentration behind fixing the optimization increases progressively from 0.4 μ M to 1.0 μ M the primer final concentration with 0.1 μ M.Reaction conditions is undertaken by the condition of step 3 among the embodiment 2.The result shows that when the primer final concentration was 0.7 μ M, △ Rn value reached maximum, the Ct value is minimum.Through three repeated experiments, determine that at last 0.7 μ M is the primer optimum concn;
Three, concentration and probe concentration optimization
Other constituent concentration is constant in the system of step 3 in embodiment 2, and magnesium ion and primer concentration behind fixing the optimization increase progressively from 0.1 μ M to 0.8 μ M the probe final concentration with 0.1 μ M.Reaction conditions is undertaken by the condition of step 3 among the embodiment 2.The result shows that when the probe final concentration was 0.7 μ M, △ Rn value was maximum.When the probe final concentration was 0.5 μ M, △ Rn value was taken second place, but this moment, the Ct value was minimum.Through three repeated experiments, under the unconspicuous situation of amplification efficiency difference, consider from economical and practical angle that selected 0.5 μ M is best concentration and probe concentration;
Four, the real-time fluorescence RT-PCR system after the optimization
Optimize through step 1, two and three, determine that optimum real-time fluorescence RT-PCR system is: Mg
2+Final concentration is that the final concentration of 5.5 mM, primer HLV-F and HLV-R all is that 0.7 μ M, probe HLV-P final concentration are 0.5 μ M.
Embodiment 4, real-time fluorescence RT-PCR detect the sensitivity test of hop latent viroid
The total RNA of M17 that obtains with step 2 among the embodiment 2 is template, surveys OD with the nucleic acid-protein analyser
260And OD
280Calculate the concentration of RNA, and be in the 25 μ L reaction systems with its dilution, total RNA is respectively 200ng, 20 ng, 2ng, 200pg, 20pg, 2pg, 0.2pg, 0.02pg, carries out the real-time fluorescence RT-PCR reaction with the optimum system that step 4 among the embodiment 3 obtains.Reaction conditions is undertaken by the condition of step 3 among the embodiment 2.The result shows that real-time fluorescence RT-PCR detects, and it detects lower bound is 20pg.
Embodiment 5, field sample detection
22 different locations collect hops blade and the cone of 6 kinds of different varietiess from Xinjiang, amount to 29 duplicate samples.Extract total RNA by embodiment 2 step 2, carry out the real-time fluorescence RT-PCR reaction with the optimum system that step 4 among the embodiment 3 obtains.Reaction conditions is undertaken by the condition of step 3 among the embodiment 2.The result shows that 29 duplicate samples of collection all detect the hop latent viroid.
＜110〉Beilun Entry-Exit Inspection And Quarantine Bureau, Ningbo Institute of Inspection and Quarantine Science Technology
＜120〉real-time fluorescent RT-PCR detection reagent of hop latent viroid and detection method
<160> 4
<170> PatentIn version 3.1
<210> 1
<211> 19
<212> RNA
＜213〉artificial sequence
<220>
＜223〉according to the Auele Specific Primer of hop latent viroid gene design
<400> 1
CGTGGAACGG CTCCTTCTT 19
<210>2
<211> 24
<212> RNA
＜213〉artificial sequence
<220>
＜223〉according to the Auele Specific Primer of hop latent viroid gene design
<400>2
AGAGTTGTAT CCACCGGGTA GTTT 24
<210>3
<211> 14
<212> RNA
＜213〉artificial sequence
<220>
＜223〉according to the specific probe of hop latent viroid gene design
<400>3
CACCAGCCGG AGTT 14
<210>4
<211> 61
<212> RNA
＜213〉target fragment of amplification hop latent viroid
<400>4
CGTGGAACGG CTCCTTCTTC ACACCAGCCG GAGTTGGAAA 40
CTACCCGGTG GATACAACTC T 61
Claims (2)
1. the real-time fluorescent RT-PCR detection reagent of hop latent viroid comprises a pair of Auele Specific Primer and a specificity fluorescent probe, and amplification target fragment length is 61bp, it is characterized in that primer and probe sequence are:
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 this probe contains FAM report fluorescence dye, and 3' holds to containing not fluorescent quenching group and having the MGB molecule; The nucleotide sequence of amplification target fragment is shown in SEQ ID NO:4.
2. utilize the method for the real-time fluorescent RT-PCR detection reagent detection hop latent viroid of the described hop latent viroid of claim 1, it is characterized in that using RNA to extract test kit hops is carried out the total RNA extraction of plant, the test kit specification sheets is seen in concrete operations; Total RNA is template with the extraction sample, carries out the real-time fluorescence RT-PCR reaction, Mg in the 25 μ L reaction systems
2+Final concentration is that the final concentration of 5.5mM, primer HLV-F and HLV-R all is that 0.7 μ M, probe HLV-P final concentration are 0.5 μ M, 12.5 μ L 2xQuantiTect Probe RT-PCR Master Mix, 0.25 μ L QuantiTect RT Mix; Reaction conditions is: 50 ℃ of 30 min; 95 ℃ of 15 min; 94 ℃ of 15 s then, 60 ℃ of 60 s, totally 40 circulations; Then be the hop latent viroid as if detecting fluorescent signal.
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| CN102732641B (en) * | 2012-06-12 | 2013-09-25 | 中国检验检疫科学研究院 | Chip for screening Pelamoviroid and application thereof |
| CN104031981A (en) * | 2013-12-31 | 2014-09-10 | 陈一强 | Method of inhibiting staphylococcus aureus QS system expression |
| CN104498633B (en) * | 2014-12-29 | 2016-08-17 | 山东出入境检验检疫局检验检疫技术中心 | NASBA primer, test kit and the method for detection Peach latent mosaic viroid |
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| CN101553566A (en) * | 2006-11-30 | 2009-10-07 | 札幌啤酒株式会社 | Method for detection of hop latent virus, primer set for the detection, and kit for the detection |
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