CN105483284B - A kind of RT-qPCR kits of three kinds of citrus virus of synchronous detection - Google Patents

A kind of RT-qPCR kits of three kinds of citrus virus of synchronous detection Download PDF

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CN105483284B
CN105483284B CN201510906257.8A CN201510906257A CN105483284B CN 105483284 B CN105483284 B CN 105483284B CN 201510906257 A CN201510906257 A CN 201510906257A CN 105483284 B CN105483284 B CN 105483284B
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ctv
qpcr
cyvcv
ctlv
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CN105483284A (en
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宋震
刘科宏
陈洪明
李中安
周常勇
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Southwest University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Abstract

The invention discloses a kind of real time fluorescent quantitative RT qRT kits of synchronous detection CYVCV, CTV and CTLV, including following 3 pairs of virus-specifics RT qPCR primers：SEQ ID NO：1, SEQ ID NO：2, SEQ ID NO：3, SEQ ID NO：4, SEQ ID NO：5, SEQ ID NO：6.The present invention can realize synchronous detection and the Viral Quantification of CYVCV, CTV, CTLV by step RT qPCR reactions.The method of the present invention is compared with conventional RT PCR detection methods, improve 100 times of CYVCV, CTLV and CTV detection sensitivity or more, detection time saves 3 times or more, pollute probability reduce by more than 30%, diagnosis disease occur and monitor plant disease epidemic and safeguard Citrus Industry safety etc. have a good application prospect.

Description

A kind of RT-qPCR kits of three kinds of citrus virus of synchronous detection
Technical field
The invention belongs to plant virus diagnostic techniques field more particularly to a kind of synchronous detection CYVCV, CTV and CTLV The RT-qPCR kits of three kinds of citrus virus.
Background technology
Citrus is the fifth-largest trade agricultural product of the largest fruit in the world and the whole world.2013, China's citriculture area 36330000 mu, 33,210,000 tons of yield ranks first in the world.In recent years, with the fast development of Citrus Industry, citrus occurs more The situation of cause of disease mixed infection and secondary infection is more and more common.Citrus yellow vein virus, citrus tristeza virus and the broken leaf disease of citrus Epidemic Scopes are wide in production, it is larger to cause harm, and there is a situation where mixed infection for these three malicious viruses, and China's Citrus Industry is pacified It constitutes a threat to entirely.Citrus yellow vein disease caused by citrus yellow vein clearing virus is a kind of disease occurred recently.Lemon and bitter orange sense After contaminating CYVCV, tree vigo(u)r weakens, yield reduces, and seriously affects its economic value.It was found for the first time in Yunnan Province of China Ruili from 2009 Since, citrus yellow vein disease also has generation on Anyue Sichuan, Chongqing and other places, and the lemon industry of some areas is produced than more serious Influence.CYVCV is α linear virals section (Alphaflexiviridae) India's citrus Tobamovirus (Mandarivirus) virus, Except propagation can be connect, it is also possible to by aleyrodid and aphis propagation, thus with large area spread and epidemic risk, to Citrus Industry especially It is lemon composition of industry serious threat.Citrus decline caused by citrus tristeza virus is a kind of great economic weight in world wide The disease for the property wanted caused seriously to cause harm, still be threaten at present using bitter orange as the citrus of anvil and to CTV to world's Citrus Industry The production of the grape fruit of stem trapping spot type strain sensitivity, shaddock and sweet orange.It is wide although being mostly used disease-resistant or resistance to sick stock in China's production The general various velogen strains for being dispersed with CTV and strength medium brown citrus aphid, with the great development of sweet orange etc., the strong strain of CTV stem trapping spot types Cause harm and be on the rise.The broken leaf disease of citrus caused by the broken mosaic virus of citrus is one of important disease for threatening Orange Producing. There are Japan, the U.S., South Africa, Australia and China in the country that the broken leaf disease of citrus occurs for report, wherein being occurred with China and Japan It is the most universal.CTLV has resulted in more serious cause harm in some areas on the ground such as the Zhejiang in China, Hunan, Fujian and Guangxi. The method for being presently used for above-mentioned single viral diagnosis is more, such as the observation of indicator plant Testing and appraisal, Electronic Speculum, enzyme linked immunological (ELISA), direct interlacing point is immunized (DTBIA), the technologies such as molecular probe hybridization, and is widely applied in production.With The development of molecular biology, RT-PCR detection method are favored with the features such as its speed is fast, high sensitivity, good specificity.Nest Formula RT-PCR, Immunocapture RT-PCR etc. then further improve detection sensitivity or convenience.
But time-consuming and is limited by all many conditions such as environment for biological detection method such as Indexing by indicator plants, it is time-consuming to take Power；Serological method especially DTBIA, it is easy to operate, it is of low cost, field sample is suitble to detect on a large scale, but its sensitivity and Accuracy is to be improved；Molecular biology method such as RT-qPCR detection methods are quick, sensitive, special, are the main inspections used at present One of survey method, but there is also multiple partitivirus troublesome operation is needed, time cost is high, and pollution probability is larger, and needs to connect Touch noxious material.
Invention content
The purpose of the present invention is to provide a kind of RT-qPCR kits of three kinds of citrus virus of synchronous detection, it is intended to solve Biological detection method is limited by environmental condition, time-consuming and laborious；Serological method sensitivity and accuracy are relatively low；Molecular biology There is the multiple partitivirus troublesome operation of needs in method, time cost is high, and pollution probability is larger, need to contact asking for noxious material Topic；And further improve detection sensitivity.
The invention is realized in this way a kind of RT-qPCR kits of three kinds of citrus virus of synchronous detection, the synchronous inspection The RT-qPCR kits for surveying three kinds of citrus virus are hybrid packed with managing to tri- kinds of viral special primers of CYVCV, CTV, CTLV, The viral upstream and downstream primer concentration of tri- kinds of CYVCV, CTV, CTLV are respectively：CYVCV1.3μM、CTV1.0μM、CTLV2.7μM； Tri- kinds of virus amplification Product characteristics Tm value ranges of CYVCV, CTV, CTLV：CYVCV is 86.25 ± 0.35 DEG C, CTLV be 84.00 ± 0.25 DEG C, CTV be 81.25 ± 0.25 DEG C；
Tri- kinds of virus-specific RT-qPCR primer sequences of CYVCV, CTV, CTLV are as follows：
CYVCV-F：TCAAACCTCCAACGCACAAAC；
CYVCV-R：CATTGTTGTGGGTTTTTGCTTC；
CTV-F：GTGTGCAGATTTCTTGACCG；
CTV-R：TCCCAAGCTGCCTGACATT；
CTLV-F：AGTTTGGAAGACGTGCTTCA；
CTLV-R：TGCAGAGAAGAAGGTAAAGCTC.
Further, the RT-qPCR kits of three kinds of citrus viruses of the synchronous detection further include：RT-qPCR reaction solutions, RT-Taq enzymes, positive reference substance, negative controls, standard items and free nucleic acid ultra-pure water.
Further, the fluorescent quantitation RT-qPCR reaction solutions take off comprising fluorescent dye EvaGreen, magnesium chloride and triphosphoric acid Oxygen ribonucleotide.
Further, the positive reference substance is：Containing CYVCV, CTV and CTLV segment recombinant plasmid melange, concentration is 200ng/μl。
Further, the negative controls are：Healthy Citrus leaf total nucleic acid extracting solution.
Further, the quantitative RT-qPCR standard items by following 3 kinds of standard item groups into：CYVCV standard items, CTV standards Product, CTLV standard items.
Further, the RT-qPCR kits of three kinds of citrus viruses of the synchronous detection are kept in dark place in -20 degree.
Another object of the present invention is to provide a kind of use of the RT-qPCR kits of three kinds of citrus virus of synchronous detection Method, the application method include：
Sample total nucleic acid is extracted：Total core is extracted from sample to be tested blade using conventional method or nucleic acid extraction kit Acid, you can as RT-qPCR templates；
One-step method multiple real time fluorescence RT-qPCR：2.0 μ l templates, positive reference substance, negative controls are taken respectively, successively 0.3 μ l, CYVCV, CTV and CTLV specific primers mixture of RT-qPCR 12.5 μ l, RT-Taq enzymes of reaction solution, 1.5 μ l are added in, are added ddH2O to reaction system total volume be 25 μ l；Response parameter is：45℃30min；95℃5min；95 DEG C of 15s, 60 DEG C of 1min, 40 A cycle；Melting curve program is 95 DEG C of 15s, 60 DEG C of 1min, 95 DEG C of 15s；Fluorescence signal is collected since 60 DEG C；
Standard curve making：CYVCV, CTV and CTLV standard items is taken to carry out 10 times of gradient series with aseptic double-distilled water respectively It is diluted to 1 × 100~1 × 107Then Copies/ μ l carry out real time fluorescent quantitative RT-qPCR reactions, amplification using the above method After the completion, using Ct values as ordinate, log10X is abscissa, makes standard curve；
As a result judge：Under the premise of expected from positive negative control meeting, by melting curve peak value height and whether there is to sentence Whether regular inspection sample is positive, if generating special melting peakss at the corresponding Tm values of solubility curve CYVCV, CTLV and CTV, i.e., CYVCV is 86.25 ± 0.35 DEG C, CTLV is 84.00 ± 0.25 DEG C, CTV is 81.25 ± 0.25 DEG C, then corresponding virus is detection The positive, i.e. sample to be tested carry corresponding virus, conversely, it is then negative for detection, i.e., without carrying corresponding virus；If testing result For the positive, according to the standard curve and sample to be tested Ct values obtained, sample to be tested virus concentration is calculated.
The present invention, using the specific primer of CYVCV, CTLV and CTV, is opened with real time fluorescent quantitative RT-qPCR technologies The one-step method for sending out a kind of synchronous detection citrus yellow vein virus, citrus tristeza virus and the broken mosaic virus of citrus synchronizes three kinds of mandarin oranges of detection The RT-qPCR kits of tangerine virus.The present invention by RT-qPCR reaction can simultaneously be detected from sample CYVCV, The presence of CTLV and CTV, and accurate quantitative analysis can be carried out to virus to be detected.Compared with traditional common RT-qPCR, this hair It is bright to improve 100 times of CYVCV, CTLV and CTV detection sensitivity or more；Step is only needed to complete detection, operation more it is easy simultaneously Pollution, which can be reduced, leads to false positive probability 30%；Need not individually reverse transcription and electrophoresis, time saving 3 times or more；In diagnosis disease hair It gives birth to and monitors plant disease epidemic and safeguard that Citrus Industry safety etc. has a good application prospect.
Description of the drawings
Fig. 1 is the RT-qPCR kit application method streams of three kinds of citrus virus of synchronous detection provided in an embodiment of the present invention Cheng Tu.
Fig. 2 is that three kinds of virus (CYVCV, CTLV and CTV) mixed infection citrus sample provided in an embodiment of the present invention carries out Synchronous real time fluorescent quantitative RT-qPCR detection example schematic diagrames.
Fig. 3 is standard curve and Viral Quantification schematic diagram provided in an embodiment of the present invention.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to Limit the present invention.
The application principle of the present invention is explained in detail below in conjunction with the accompanying drawings.
It is special that the RT-qPCR kits of three kinds of citrus virus of synchronous detection of the embodiment of the present invention include following 3 pairs of virus Property RT-qPCR primers, sequence are as follows：
SEQ ID NO：1, CYVCV-F：TCAAACCTCCAACGCACAAAC；
SEQ ID NO：2, CYVCV-R：CATTGTTGTGGGTTTTTGCTTC；
SEQ ID NO：3, CTV-F：GTGTGCAGATTTCTTGACCG；
SEQ ID NO：4, CTV-R：TCCCAAGCTGCCTGACATT；
SEQ ID NO：5, CTLV-F：AGTTTGGAAGACGTGCTTCA；
SEQ ID NO：6, CTLV-R：TGCAGAGAAGAAGGTAAAGCTC.
The kit, tri- kinds of viral special primers of CYVCV, CTV, CTLV are hybrid packed with managing, each virus upstream and downstream Primer concentration is：CYVCV1.3μM、CTV1.0μM、CTLV2.7μM.
The kit, each virus amplification Product characteristics Tm value ranges：CYVCV is 86.25 ± 0.35 DEG C, CTLV is 84.00 ± 0.25 DEG C, CTV be 81.25 ± 0.25 DEG C.
The kit further includes：A) RT-qPCR reaction solutions, b) positive reference substance, c) negative controls, d) standard items, E) RT-Taq enzymes and f) free nucleic acid ultra-pure water.
A) the fluorescent quantitation RT-qPCR reaction solutions include fluorescent dye EvaGreen, magnesium chloride and triphosphoric acid deoxidation core Ribotide；
B) positive reference substance is：Containing CYVCV, CTV and CTLV segment recombinant plasmid melange (concentration about 200ng/ μl)；
C), the negative controls are：Healthy Citrus leaf total nucleic acid extracting solution；
D) standard items by following 3 kinds of standard item groups into：CYVCV standard items, CTV standard items, CTLV standard items.
The CYVCV standard items are CYVCV coat protein gene cloning plasmids, and sequence is SEQ ID NO：7：
1ATGAGCTTCG ACTACACCCA CCCTCTCTAC CGCAGCTATC CATTTCCACA CTACTGCGAG
61TTCGACCGGC ACCAACTCTG CGACCATCAT CCAGTACTCA AACCTCCAAC GCACAAACCC
121AGCGCCCCGA ACTCTCTCAT GTCTACCGAC GACAACAAGG GCAAACAACC ACTTCACCCG
181 ACACCTTCGG GCCCTAACGA CACGACCCCG AAACCTATCC CTGTACCCAC TCCCTCAGTT
241 ACGCCTACAG CTGCAGGTAA GGAAAGCCAA GAGCCCATCG AAAAGCGTAT CACACACGCT
301 TTCCACGCTG AAGCAAAAAC CCACAACAAT GGGGTCTCTC CACCTGCCTT CAACCCGAAC
361AACATGAATG CTGTGCCGCT GAACCTGCTC AACCTCAACC TAAGATACTC ACCGGTCACT
421AACTCCATAG CTAACCCTAA ACAGACCGAG GCTATCGGGA AAGCTTGGGT CCGCATCTTG
481AACATCGATC CTGCCAACGT GTTCTTATAC GCCATCGACC TCGCCAGAGC TTGCGCCGAC
541GCGGGCTCCT CCCCTGAAGC TGATATTATT GGAGCGAACG AAGATCTCAA CCCCGTTGTT
601GAACGAAACG CATTGGCCCT AGTGGTTAGG GATTTCTGCC CGCTGCGCGC TTTTTGCGCT
661TACTACTCTC GAGTGGTATG GAACCTCATG ATCAAGGCGG ACCAGCCTCC GGCCAACTGG
721ATGAAATCCG GGGTAGACGA GAACGCGAAA TTCGCGGCAT TCGACTTCTT CCATGGTATC
781CTCTCGCCCG CTTCCCTGTA TGTGCCCCTA GAGAGACACC CTACTTCCGC GGAGAGGATC
841GCAAATCAGG CCATGTTCGC TGTGAAAATT GCCAACGCTC CAGGAAATGG CACGGACCTC
901ACGATGGACC ACATTGCCTT CACCAAAGGA AGGACTACCC AGCACTCCGG CCTTCGCCCG
961ACCCCTTTCA ACATCTAA
The CTV standard items be CTV coat protein gene cloning plasmids, SEQ ID NO：8 sequences are：
1ATGGACGACG AAACAAAGAA ATTGAAGAAC AAAAACAAGG AAACGAAAGA AGGCGACGAT
61GTTGTTGCCG CTGAGTCTTC TTTCGGTTCC TTAAACTTAC ACATCGATCC AACTCTGATA
121GCGATGAATG ACGTGCGTCA GTTGGGTACC CAACAGAATG CTGCTTTGAA TAGAGATTTG
181TTTCTCACCT TGAAAGGGAA GTATCCTAAC TTACCTGACA AAGATAAAGA CCTTCACTTA
241GCTATGATGT TATATCGTAA AGCAGTTAAG AGTTCATCAT TACAAAGCGA TGACGATACT
301ACGGGTATAA CGTACACTCG GGAGGGTGTT GAAGTGGATT TGCTTGACAA ACTTTGGACT
361GACGTCGTGT TTAATTCCCA GGGTATTGGC AACCGTACTA ACGCCCTTCG AGTTTGGGGT
421AGAACTAACG ATGCCCTTTA CTTAGCTTTT TGTAGACAAA ATCGCAATTT GAGTTATGGC
481GGACGTCCGC TAGATGCAGG GATTCCGGCC GGGTATCATT ACCTGTGTGC AGATTTCTTG
541ACCGGAGCTG GCTTGACTGA TTTAGAATGT GCTGTGTACA TACAAGCTAA AGAACAATTG
601TTGAAGAAGC GAGGAGCTGA TGAAGTTGTA GTTACCAATG TCAGGCAGCT TGGGAAATTC
661AACACACGTT GA
The CTLV standard items be CTLV parts ORF1 sequence cloned plasmids, SEQ ID NO：9 its sequence are：
1TCAAAGCTGC AAGAAAAGAG AGGATTTAGG TCCCTCTCAG CTAGAATTGA AAGATTTGGG
61GAAAATGAGT TTGGAAGACG TGCTTCAACA AGCGAGGCGC CACCGGGTAG GAGTGTATCT
121CTGGAAGACT CACATAGACC CGGCAAAGGA ACTTTTGACG GTTCCTCCCC CTGAAGGATT
181CAAAGAAGGT GAAAGCTTTG AAGGCAGAGA GCTTTACCTT CTTCTCTGCA ATCACTATTG
241TAAATATTTA TTTGGTAATA TTGCTGTTTT TGGGTCTTCT GATAAGACCC AGTTTCCCGC
301TGTCGGTTTT GATACTCCAC CAGTTCATTA CA
The kit is kept in dark place in -20 degree.
As shown in Figure 1, the application method of the RT-qPCR kits of three kinds of citrus virus of synchronous detection of the embodiment of the present invention It specifically can be as follows：
S101：Sample total nucleic acid is extracted：With nucleic acid extraction kit MiniBEST Viral RNA/DNA Extraction Kit Ver.4.0 (TAKARA) extract viral nucleic acid according to products instruction from sample blade；
S102：Multiple real time fluorescence RT-qPCR：2.0 μ l templates, positive reference substance, negative controls are taken respectively, are added successively Enter 12.5 μ l, RT-Taq enzyme of RT-qPCR reaction solutions, 0.3 μ l, CYVCV, CTV and CTLV specific primers mixture, 1.5 μ l, add ddH2O to reaction system total volume be 25 μ l；Response parameter is：45℃30min；95℃5min；95 DEG C of 15s, 60 DEG C of 1min, 40 A cycle；Melting curve program is 95 DEG C of 15s, 60 DEG C of 1min, 95 DEG C of 15s；Fluorescence signal is collected since 60 DEG C；
S103：Standard curve making：CYVCV, CTV and CTLV standard items is taken to carry out 10 times of gradients with aseptic double-distilled water respectively It is serially diluted to 1 × 100~1 × 107Then Copies/ μ l carry out real-time quantitative fluorescence RT-qPCR reactions using the above method. After the completion of amplification, using Ct values as ordinate, log10X (X is standard items series concentration) is abscissa, makes standard curve.
S104：Under the premise of expected from positive negative control meeting, by melting curve peak value height and whether there is to judge to examine Whether sample is positive.If solubility curve generates the special melting peakss of CYVCV, CTLV and CTV, i.e., corresponding Tm values (CYVCV For 86.25 ± 0.35 DEG C, CTLV be 84.00 ± 0.25 DEG C, CTV is 81.25 ± 0.25 DEG C), then corresponding virus is positive for detection, I.e. sample to be tested carries corresponding virus, conversely, it is then negative for detection, i.e., without carrying corresponding virus；If testing result is sun Property, according to the standard curve and sample to be tested Ct values obtained, calculate sample to be tested virus concentration (Copies/ μ l).
The application principle of the present invention is further described with reference to specific embodiment.
Embodiment 1, the present invention is to the real-time fluorescence RT-qPCR detections of CYVCV, CTLV and CTV and quantitative example
1st, sample total nucleic acid is extracted：The Citrus leaf middle arteries portion of CYVCV, CTLV and CTV mixed infection is taken with sterile razor blade 0.1mg is divided to be placed in 1.5ml centrifuge tubes, with nucleic acid extraction kit MiniBEST Viral RNA/DNA Extraction Kit Ver.4.0 (TAKARA) extract viral nucleic acid according to products instruction from sample blade.
2nd, multiple fluorescence quantitative RT-qPCR：It is protected from light on ice and prepares real-time fluorescence RT-qPCR reaction mixtures, wherein RT- 12.5 μ l, RT-Taq enzyme of qPCR reaction solutions, 0.3 μ l, CYVCV, CTV and CTLV specific primers mixture, 1.5 μ l, add ddH2O is extremely Reaction system total volume is 23 μ l；Positive reference substance, negative controls and each 2 μ l to RT- of above-mentioned total nucleic acid to be measured are taken respectively QPCR is managed, and respectively plus each 23 μ l mixings of reaction mixture, 5000rmp, 1min are centrifuged.Reaction 7000 fluorescent quantitation instruments of ABI into Row, response parameter are：45℃30min；95℃5min；95 DEG C of 15s, 60 DEG C of 1min, 40 cycles；Melting curve program is 95 DEG C 15s, 60 DEG C of 1min, 95 DEG C of 15s；Fluorescence signal is collected since 60 DEG C.
4th, test results report：
Under the premise of expected from positive negative control meeting, by melting curve peak value height and whether there is to judge to detect sample Whether it is positive.If solubility curve generates the special melting peakss of CYVCV, CTLV and CTV, i.e., (CYVCV is for corresponding Tm values 86.25 ± 0.35 DEG C, CTLV be 84.00 ± 0.25 DEG C, CTV is 81.25 ± 0.25 DEG C), then corresponding virus is positive for detection, i.e., Sample to be tested carries corresponding virus, conversely, it is then negative for detection, i.e., without carrying corresponding virus.
The result of the test of the present embodiment see Fig. 2, positive control and sample to be tested solubility curve 86.50 DEG C, 84.00 DEG C, There are characteristic peaks respectively at 81.00 DEG C, show corresponding CYVCV, CTLV, CTV as the positive, and negative control is in the no spy in corresponding position Levy peak value.This show kit of the present invention by real-time fluorescence RT-qPCR reaction can synchronize detection CYVCV, CTLV and Tri- kinds of viruses of CTV, and, by the difference of Tm values, these three viruses can effectively be distinguished on melting curve.
5th, viral accurate quantitative analysis
1) standard curve making：CYVCV, CTLV and CTV standard items is taken to carry out 10 times of gradient systems with aseptic double-distilled water respectively Row are diluted to 1 × 102~1 × 107Copies/μl.Then using the above method to various concentration standard items and the same stepping of product to be tested Row real-time quantitative fluorescence RT-qPCR reacts.After the completion of amplification, using various concentration standard items Ct values as ordinate, log10(X is mark to X Quasi- product series concentration) it is abscissa, make standard curve.The present embodiment standard curve result (Fig. 3) display, pair of template concentrations There is good linear relationship, related coefficient (R between numerical value and its Ct value2) it is 1.000, amplification efficiency (E) is 99.97%, Linear equation is y=-3.28lg (x)+35.01.
2) Viral Quantification：Ct values according to virus to be measured can be to viral accurate fixed using linear equation obtained by standard curve Amount.The Ct=15 of sample to be tested (※) in the present embodiment, can by linear equation for y=-3.208lg (x)+35.01 quickly It is 1 × 10 to calculate its virus concentration in the sample6.5Copies/μl。
The application effect of the present invention is described further with reference to experiment.
The specificity experiments of the present invention detection CYVCV, CTLV and CTV
Only carrying citrus yellow vein disease, citrus decline, the broken leaf disease of citrus, Satsuma dwarf virus, citrus is extracted respectively to split Skin disease, the citrus sample total nucleic acid of citrus mosaic disease, citrus psorosis take 2.0 μ l to carry out real-time fluorescence RT- as template respectively QPCR reacts.
Specific test result is shown：On solubility curve, only CYVCV, CTLV and CTV have in its corresponding Tm values position Specific peak generates；Other disease samples are without apparent amplification curve, and solubility curve is without purpose characteristic peak.This explanation application present invention Detect the high specificity of CYVCV, CTLV and CTV.
The stability experiment of the present invention detection CYVCV, CTLV and CTV
Respectively with CYVCV, CTLV and CTV standard items of 1000 times of dilution and a negative controls (DEPC-H20) for mould Plate, carry out batch in, batch between repeat to test.It is that above-mentioned template is heavy in a real-time fluorescence RT-qPCR that experiment is repeated in criticizing It is 3 times multiple；It is that (interval 1 day) carries out real time fluorescent quantitative RT-qPCR in the experiment of 3 different times that experiment is repeated between batch.Inspection Result is surveyed to show：Batch between, batch in repeat experiment average coefficient of variation CV values be respectively less than 2%, illustrate using the present invention detect CTV, CTLV and CYVCV are with good stability.
The sensitivity experiment of the present invention detection CYVCV, CTLV and CTV
CYVCV, CTLV and CTV standard items (1.0 × 10 being serially diluted respectively with 10 times7~1.0 × 100Copies/ μ L) as template, each dilution sets up 3 repetitions, carries out real time fluorescent quantitative RT-qPCR with kit of the present invention respectively, and Parallel progress conventional RT-PCR detection, compares detection sensitivity.Result of the test shows that the present invention can detect that 102Copies/μl Virus quantity, and it is 10 that conventional RT-PCR, which detects minimum virus quantity,4Copies/ μ l, it can be seen that the spirit of present invention detection virus Sensitivity is higher than about 100 times of conventional RT-PCR.
The present invention and the comparative experiments of conventional RT-PCR detection CYVCV, CTLV and CTV
Sample to be tested 90 carries out the detection of CYVCV, CTLV and CTV with common RT-qPCR and the method for the present invention respectively. The former for each virus be required for reverse transcription, amplification, glue, electrophoresis, dyeing and etc., 15 hours of used time.The latter only needs Single stepping can obtain data, about 4 time saving 3 times of hours of used time or more；The former is due to 3, false positive sample caused by pollution Sick sample.The sick sample of the latter 1, false positive probability reduce by more than 30%.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention All any modification, equivalent and improvement made within refreshing and principle etc., should all be included in the protection scope of the present invention.

Claims (7)

  1. A kind of 1. RT-qPCR kits of three kinds of citrus virus of synchronous detection, which is characterized in that three kinds of citruses of the synchronous detection The RT-qPCR kits of virus are hybrid packed with managing to tri- kinds of viral special primers of CYVCV, CTV, CTLV, CYVCV, CTV, The viral upstream and downstream primer concentration of tri- kinds of CTLV are respectively：CYVCV1.3μM、CTV1.0μM、CTLV2.7μM；CYVCV、CTV、CTLV Three kinds of virus amplification Product characteristics Tm value ranges：CYVCV is 86.25 ± 0.35 DEG C, CTLV is 84.00 ± 0.25 DEG C, CTV is 81.25±0.25℃；
    Tri- kinds of virus-specific RT-qPCR primer sequences of CYVCV, CTV, CTLV are as follows：
    CYVCV-F：TCAAACCTCCAACGCACAAAC；
    CYVCV-R：CATTGTTGTGGGTTTTTGCTTC；
    CTV-F：GTGTGCAGATTTCTTGACCG；
    CTV-R：TCCCAAGCTGCCTGACATT；
    CTLV-F：AGTTTGGAAGACGTGCTTCA；
    CTLV-R：TGCAGAGAAGAAGGTAAAGCTC.
  2. 2. the RT-qPCR kits of three kinds of citrus virus of synchronous detection as described in claim 1, which is characterized in that described same The RT-qPCR kits of step three kinds of citrus viruses of detection further include：RT-qPCR reaction solutions, RT-Taq enzymes, positive reference substance, the moon Property reference substance, standard items and free nucleic acid ultra-pure water.
  3. 3. the RT-qPCR kits of three kinds of citrus virus of synchronous detection as claimed in claim 2, which is characterized in that RT-qPCR Reaction solution is fluorescent quantitation RT-qPCR reaction solutions；The fluorescent quantitation RT-qPCR reaction solutions include fluorescent dye EvaGreen, Magnesium chloride and triphosphate deoxyribose nucleotide.
  4. 4. the RT-qPCR kits of three kinds of citrus virus of synchronous detection as claimed in claim 2, which is characterized in that the sun Property reference substance is：Containing CYVCV, CTV and CTLV segment recombinant plasmid melange, concentration is 200ng/ μ l.
  5. 5. the RT-qPCR kits of three kinds of citrus virus of synchronous detection as claimed in claim 2, which is characterized in that described the moon Property reference substance is：Healthy Citrus leaf total nucleic acid extracting solution.
  6. 6. the RT-qPCR kits of three kinds of citrus virus of synchronous detection as claimed in claim 2, which is characterized in that RT-qPCR The quantitative RT-qPCR standard items of reaction solution by following 3 kinds of standard item groups into：CYVCV standard items, CTV standard items, CTLV standards Product；
    The CYVCV standard items be CYVCV coat protein gene cloning plasmids, sequence SEQIDNO：7：
    1ATGAGCTTCGACTACACCCACCCTCTCTACCGCAGCTATC CATTTCCACACTACTGCGAG
    61TTCGACCGGCACCAACTCTGCGACCATCATCCAGTACTCA AACCTCCAACGCACAAACCC
    121AGCGCCCCGAACTCTCTCATGTCTACCGACGACAACAAGG GCAAACAACCACTTCACCCG
    181ACACCTTCGGGCCCTAACGACACGACCCCGAAACCTATCC CTGTACCCACTCCCTCAGTT
    241ACGCCTACAGCTGCAGGTAAGGAAAGCCAAGAGCCCATCG AAAAGCGTATCACACACGCT
    301TTCCACGCTGAAGCAAAAACCCACAACAATGGGGTCTCTC CACCTGCCTTCAACCCGAAC
    361AACATGAATGCTGTGCCGCTGAACCTGCTCAACCTCAACC TAAGATACTCACCGGTCACT
    421AACTCCATAGCTAACCCTAAACAGACCGAGGCTATCGGGA AAGCTTGGGTCCGCATCTTG
    481AACATCGATCCTGCCAACGTGTTCTTATACGCCATCGACC TCGCCAGAGCTTGCGCCGAC
    541GCGGGCTCCTCCCCTGAAGCTGATATTATTGGAGCGAACG AAGATCTCAACCCCGTTGTT
    601GAACGAAACGCATTGGCCCTAGTGGTTAGGGATTTCTGCC CGCTGCGCGCTTTTTGCGCT
    661TACTACTCTCGAGTGGTATGGAACCTCATGATCAAGGCGG ACCAGCCTCCGGCCAACTGG
    721ATGAAATCCGGGGTAGACGAGAACGCGAAATTCGCGGCAT TCGACTTCTTCCATGGTATC
    781CTCTCGCCCGCTTCCCTGTATGTGCCCCTAGAGAGACACC CTACTTCCGCGGAGAGGATC
    841GCAAATCAGGCCATGTTCGCTGTGAAAATTGCCAACGCTC CAGGAAATGGCACGGACCTC
    901ACGATGGACCACATTGCCTTCACCAAAGGAAGGACTACCC AGCACTCCGGCCTTCGCCCG
    961ACCCCTTTCAACATCTAA
    The CTV standard items be CTV coat protein gene cloning plasmids, SEQIDNO：8 sequences are：
    1ATGGACGACGAAACAAAGAAATTGAAGAACAAAAACAAGG AAACGAAAGAAGGCGACGAT
    61GTTGTTGCCGCTGAGTCTTCTTTCGGTTCCTTAAACTTAC ACATCGATCCAACTCTGATA
    121GCGATGAATGACGTGCGTCAGTTGGGTACCCAACAGAATG CTGCTTTGAATAGAGATTTG
    181TTTCTCACCTTGAAAGGGAAGTATCCTAACTTACCTGACA AAGATAAAGACCTTCACTTA
    241GCTATGATGTTATATCGTAAAGCAGTTAAGAGTTCATCAT TACAAAGCGATGACGATACT
    301ACGGGTATAACGTACACTCGGGAGGGTGTTGAAGTGGATT TGCTTGACAAACTTTGGACT
    361GACGTCGTGTTTAATTCCCAGGGTATTGGCAACCGTACTA ACGCCCTTCGAGTTTGGGGT
    421AGAACTAACGATGCCCTTTACTTAGCTTTTTGTAGACAAA ATCGCAATTTGAGTTATGGC
    481GGACGTCCGCTAGATGCAGGGATTCCGGCCGGGTATCATT ACCTGTGTGCAGATTTCTTG
    541ACCGGAGCTGGCTTGACTGATTTAGAATGTGCTGTGTACA TACAAGCTAAAGAACAATTG
    601TTGAAGAAGCGAGGAGCTGATGAAGTTGTAGTTACCAATG TCAGGCAGCTTGGGAAATTC
    661AACACACGTTGA
    The CTLV standard items be CTLV parts ORF1 sequence cloned plasmids, SEQIDNO：9 its sequence are：
    1TCAAAGCTGCAAGAAAAGAGAGGATTTAGGTCCCTCTCAG CTAGAATTGAAAGATTTGGG
    61GAAAATGAGTTTGGAAGACGTGCTTCAACAAGCGAGGCGC CACCGGGTAGGAGTGTATCT
    121CTGGAAGACTCACATAGACCCGGCAAAGGAACTTTTGACG GTTCCTCCCCCTGAAGGATT
    181CAAAGAAGGTGAAAGCTTTGAAGGCAGAGAGCTTTACCTT CTTCTCTGCAATCACTATTG
    241TAAATATTTATTTGGTAATATTGCTGTTTTTGGGTCTTCT GATAAGACCCAGTTTCCCGC
    301TGTCGGTTTTGATACTCCACCAGTTCATTACA。
  7. 7. the RT-qPCR kits of three kinds of citrus virus of synchronous detection as described in claim 1, which is characterized in that described same The RT-qPCR kits of step three kinds of citrus viruses of detection are kept in dark place in -20 degree.
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CN112251543A (en) * 2020-11-26 2021-01-22 西南大学 Specific primer, kit and method for detecting citrus yellow vein clearing virus RT-RPA

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