CN105296626A - Method for rapidly detecting TMV carried by diseased residue in soil - Google Patents

Method for rapidly detecting TMV carried by diseased residue in soil Download PDF

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Publication number
CN105296626A
CN105296626A CN201510737364.2A CN201510737364A CN105296626A CN 105296626 A CN105296626 A CN 105296626A CN 201510737364 A CN201510737364 A CN 201510737364A CN 105296626 A CN105296626 A CN 105296626A
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tmv
soil
detection
cdna
lamp
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刘世超
孙现超
许博文
彭浩然
朱虹
青玲
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Southwest University
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Southwest University
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    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • 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

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Abstract

The invention discloses a method for rapidly detecting TMV carried by diseased residues in soil. The method comprises steps of extraction of total RNA of diseased residue TMV, reverse transcription to prepare a cDNA template, establishment and optimization of an LAMP technical system, electrophoresis detection, specification, sensitivity detection and sample detection. By improving E.Z.N.A.TM Soil RNA Kit and reverse transcription systems and optimizing conditions of the use amount, the reaction time, the reaction time and the like of dNTPmix, MgSO4, buffer, BstDNA polymerase and cDNA in LAMP, optimal reaction of the LAMP technique for detecting TMV in the diseased residues in tobacco field can be achieved. The method disclosed by the invention is rapid and accurate, high in specificity, high in field operability and applicable to rapid detection on field samples and has certain instruction function on prevention and treatment on TMV diseases of tobacco fields, the sensitivity is 100 times higher than that of ordinary RT-PCR, no expensive instrument is needed, and only a thermostat water bath is necessary to complete the detection.

Description

In a kind of rapid detection soil, invalid body carries TMV method
Technical field
The present invention relates to a kind of plant virus detection technique, particularly relate to invalid body in a kind of rapid detection soil and carry TMV method.
Background technology
Tobacco virus is one of important disease of tobacco.The tobacco virus that current China has reported has 16 kinds, and main viral species has tobacco mosaic viruses (TMV), tobacco cucumber mosaic virus (CMV), tobacco potato Y virus (PVY), marmor erodens (TEV), tobacco potato X virus (PVX) and nepovirus (TRSV).That cause heavy losses is TMV, CMV, PVY, and tobacco virus causes production loss to reach 30%-50%, and some ground piece even has no harvest.After Tobacco Infected virus disease, tobacco leaf degree declines, product qualitative change is bad, but clear and definite conclusion is not had so far for the source of Causal Viruses of Tobacco, therefore, the viral source of specifying tobacco mosaic virus disease has become the key effectively preventing Tobacco mosaic disease, sets up differential high efficient and detects Tobacco mosaic disease pathogen, and especially the method for TMV has become the upper problem in the urgent need to address of production.
Summary of the invention
Object of the present invention is just to provide to solve the problem invalid body in a kind of rapid detection soil to carry TMV method.
The present invention is achieved through the following technical solutions above-mentioned purpose:
The present invention includes following steps:
(1) invalid body TMV total serum IgE in soil is extracted;
(2) with the total serum IgE extracted for template, reverse transcription obtains cDNA;
(3) take cDNA as template, carry out LAMP amplification with following two pairs of primers, obtain amplified production; 1st pair of primer:
TMV-FIP5'-GCTGTGACTAACGGATCTAATACCG-AGGTTCCCTGACAGTGAC-3'
TMV-BIP5'-GAAGTTGAAAATCAGGCGAACCC-TGCGTCGTCTACTCTACG-3'
2nd pair of primer:
TMV-F3：5'-GGAAACCTTCACCACAAGT-3'
TMV-B3：5'-TATAGCGCTCCTTATGGC-3'
(4) prepare 2% sepharose with TAE electrophoretic buffer, get 5 μ LLAMP amplified productions and carry out electrophoresis in Horizontal electrophoresis tank, 110V/100mA electrophoresis 35 points of min, obtain the waterfall discrete type band of target TMV amplified production;
Further, the test kit described in steps A uses, and takes the 0.5g pedotheque in 0.2g pedotheque replacement specification sheets; Process of reverse-transcription described in step (2), according to the downstream primer of TMV coat protein design pair of primers, replaces universal primer oligDT; Foundation described in step (3) also optimizes LAMP system, 4 μ LFIP BIP (10 μMs), 0.5 μ LF3 B3 (10 μMs), 1.0 μ LMgSO 4(100 μMs), 1.0 μ LdNTPmix (10 μMs), 2.5 μ L10 × PCRbuffer (Mg 2+-free), 1.0 μ LBSTDNAploymerase (8U), 5.0 μ L reverse transcription products are as cDNA template, and all the other use ddH 2o is supplemented to 25 μ L; Response procedures is: 62 DEG C of thermostat water baths, isothermal amplification reactions 1h, and 80 DEG C of reaction 10min, are used for detecting TMV in vega soil invalid body.
Beneficial effect of the present invention is:
The present invention is that in a kind of rapid detection soil, invalid body carries TMV method, compared with prior art, the present invention includes the extraction of the total serum IgE of invalid body TMV in vega soil, reverse transcription reaction prepares cDNA template, LAMP detection technique Establishment and optimization and electrophoresis detection determine virus, detection method quick and precisely, without the need to some expensive equipment, only need thermostat water bath, field is workable, high specificity, highly sensitive, be applicable to the rapid detection of field sample, have certain directive function to the prevention and management of vega TMV virus disease.
Accompanying drawing explanation
Fig. 1 is vega soil invalid body Total RNAs extraction result;
Fig. 2 is that the LAMP of TMV in vega soil invalid body detects preliminary experiment;
Fig. 3 is LAMP reaction system: Mg 2+, dNTPs, BstDNAploymerase, cDNA optimization;
Fig. 4 is LAMP reaction conditions: the optimization of time, temperature;
Fig. 5 is the specific detection of LAMP reaction;
Fig. 6 is RT-PCR detection sensitivity and LAMP detection sensitivity contrast experiment;
Fig. 7 is the pedotheque LAMP detected result of three different areas.
Embodiment
Below in conjunction with accompanying drawing, the invention will be further described:
(1) vega soil invalid body virus total RNA is extracted
Get the pedotheque collected a little, be contained in culture dish, 25 DEG C of constant incubators are dried, and stir 1 time, until soil sample complete drying every about 1 hour.Take 0.2g dry soil, be fully ground to fine powdered fast in liquid nitrogen, be contained in the centrifuge tube of 15mL, extract soil total serum IgE according to E.Z.N.A.TMSoilRNAKit operation steps; As shown in Figure 1.
(2) reverse transcription of RNA
With the total serum IgE extracted for template, adopt Reverse Transcription box (PrimeScriptRTreagentKit, TaKaRa company) synthesize the first chain cDNA, reverse transcription system is 10 μ L:2.5 μ L total serum IgE, 2 μ L5 × PrimeScriptBuffer, 0.5 μ LTB, 0.5 μ LRandom6mers, 0.5 μ LPrimeScriptRTEnzymeMixI, 4.0 μ LRNaseFreeddH 2o, response procedures is: 37 DEG C of 15min, 85 DEG C of 20s;
(3) LAMP preliminary experiment
LAMP reaction system: 1.6 μMs of FIP BIP, 0.2 μM of F3 B3 (10 μMs), 6mMMgSO 4, 1.0mMdNTPmix, 1 × PCRbuffer (Mg 2+-free), 8UBstDNAploymerase, 1.0 μ L reverse transcription products as cDNA template, ddH 2o supplies 25 μ L.Reaction conditions: 62 DEG C of water-bath 1h, 80 DEG C stop 10min.Prepare 2% sepharose with TAE electrophoretic buffer, get 5 μ LLAMP products and in Horizontal electrophoresis tank, carry out electrophoresis, 110V/100mA electrophoresis 40min; As shown in Figure 2;
(4) optimization of LAMP reaction system
Mg 2+: add 0 μ L respectively in (3) system, 0.5 μ L, 1.0 μ L, 1.5 μ L, 2.0 μ L, the MgSO of 2.5 μ L 4(100mM), after amplified reaction, as Fig. 3-A, optimum amount is selected to be 1.0 μ L according to electrophorogram.
DNTPs: respectively to adding 0 μ L in system, the dNTPs (10mM) of 1 μ L, 2 μ L, 3 μ L, 4 μ L, 5 μ L, after amplified reaction, as Fig. 3-B, select optimum amount to be 1.0 μ L according to electrophorogram.
BstDNAploymerase: add 0 μ L respectively in (3) system, 0.3 μ L, 0.5 μ L, 0.8 μ L, the BstDNAploymerase (8U) of 1.0 μ L, 1.2 μ L, 1.5 μ L, after amplified reaction, as Fig. 3-C, optimum amount is selected to be 1.0 μ L according to electrophorogram.
CDNA: add 0.5 μ L respectively in (3) system, 1.0 μ L, 2.5 μ L, 4.0 μ L, 5.0 μ L, 7.5 μ L, the cDNA of 10.0 μ L, after amplified reaction, as Fig. 3-D, select optimum amount 5.0 μ L according to electrophorogram.Comprehensive above experiment, obtains optimum system.
(5) optimization of LAMP reaction conditions
With the cDNA of tobacco mosaic viruses for template, according to the optimum system obtained in (4), increase under 55 DEG C, 57 DEG C, 59 DEG C, 61 DEG C, 63 DEG C, 65 DEG C 6 temperature condition respectively, the reaction times is 1h; In conjunction with above preliminary experiment and all optimization experiment results, optimal reaction temperature is selected to be 62 DEG C; Fig. 4-A
With the cDNA of tobacco mosaic viruses for template, according to the optimum system obtained in (4), increase under 0min, 20min, 30min, 50min, 60min and 70min6 time conditions respectively, temperature of reaction is 62 DEG C, Fig. 4-B, final optimum response of selecting is tested as 60min;
(6) specificity of LAMP and sensitivity test
With the cDNA of TMV, CMV, PVX, TRV, TuMV for template, optimum system and the condition of pressing (4) and (5) carry out LAMP reaction.Contrast with health tobacco.As Fig. 5, result display only have TMV to occur waterfall discrete type becomes clear band, specificity is remarkable;
With the cDNA of TMV for template, cDNA concentration is 80.2 μ g/ μ L, and 10 times of gradient dilutions, to 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, react according to LAMP system in (6).LAMP product 2% agarose gel electrophoresis detects; Meanwhile, this series concentration gradient is carried out common PCR reaction, reaction system: 12.5 μ LPrimixTap (TaKaRa), 0.5 μ LT-A, 0.5 μ LT-B, 10.0 μ LddH 2o.Reaction conditions is: 94 DEG C of 3min, then 94 DEG C of 45s, 56 DEG C of 45s, 72 DEG C of 1.5min, totally 29 circulations, 72 DEG C of 10min.PCR primer 1% agarose gel electrophoresis detects, as Fig. 6.Comparative result shows, and LAMP remolding sensitivity RT-PCR sensitivity exceeds 100 orders of magnitude, as Fig. 6.In Fig. 6: M (on): marker V; M (under): marker I; 1:1 μ g; 2:100ng; 3:10ng; 4:1ng; 5:100pg; 6:10pg; 7:1pg; 8:100fg; 9:10fg; 10: negative control (7) field sample detection
Take three different areas field soil samples, extract total serum IgE, adopt above LAMP method 4 μ LFIP BIP (10 μMs) set up, 0.5 μ LF3 B3 (10 μMs), 1.0 μ LMgSO 4(100 μMs), 1.0 μ LdNTPmix (10 μMs), 2.5 μ L10 × PCRbuffer (Mg 2+-free), 1.0 μ LBstDNAploymerase (8U), 5.0 μ L reverse transcription products are as cDNA template, and all the other use ddH 2o is supplemented to 25 μ L; Response procedures is: 62 DEG C of thermostat water baths, isothermal amplification reactions 1h, 80 DEG C of reaction 10min, carry out augmentation detection, check its field practical function, as Fig. 7 display, LAMP detected result is obvious, and the faint band that RT-PCR detects, LAMP method test strip becomes clear, consuming time short, easy and simple to handle, can be used for Fields detection.In Fig. 7: M (on): marker V; M (under): marker I; 1-5: the first regional vega soil sample; 6-10: the second regional vega soil sample; 11-14: the three regional vega soil sample; 15: positive control; 16: negative control.
More than show and describe ultimate principle of the present invention and principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.

Claims (4)

1. in rapid detection soil, invalid body carries a TMV method, it is characterized in that, comprises the following steps:
(1) invalid body TMV total serum IgE in soil is extracted;
(2) with the total serum IgE extracted for template, reverse transcription obtains cDNA;
(3) take cDNA as template, carry out LAMP amplification with following two pairs of primers, obtain amplified production; 1st pair of primer:
TMV-FIP5'-GCTGTGACTAACGGATCTAATACCG-AGGTTCCCTGACAGTGAC-3'
TMV-BIP5'-GAAGTTGAAAATCAGGCGAACCC-TGCGTCGTCTACTCTACG-3' the 2nd pair of primer:
TMV-F3：5'-GGAAACCTTCACCACAAGT-3'
TMV-B3：5'-TATAGCGCTCCTTATGGC-3'
(4) prepare 2% sepharose with TAE electrophoretic buffer, get 5 μ LLAMP amplified productions and in Horizontal electrophoresis tank, carry out electrophoresis, 110V/100mA electrophoresis 35min, obtain the waterfall discrete type band of target TMV.
2. in rapid detection soil according to claim 1, invalid body carries TMV method, it is characterized in that: the test kit described in steps A uses, and takes the 0.5g pedotheque in 0.2g pedotheque replacement specification sheets.
3. in rapid detection soil according to claim 1, invalid body carries TMV method, it is characterized in that: the process of reverse-transcription described in step (2), according to the downstream primer of TMV coat protein design pair of primers, replaces universal primer oligDT.
4. in rapid detection soil according to claim 1, invalid body carries TMV method, it is characterized in that: the foundation described in step (3) also optimizes LAMP system, 4 μ LFIP BIP (10 μMs), 0.5 μ LF3 B3 (10 μMs), 1.0 μ LMgSO 4(100 μMs), 1.0 μ LdNTPmix (10 μMs), 2.5 μ L10 × PCRbuffer (Mg 2+-free), 1.0 μ LBSTDNAploymerase (8U), 5.0 μ L reverse transcription products are as cDNA template, and all the other use ddH 2o is supplemented to 25 μ L; Response procedures is: 62 DEG C of thermostat water baths, isothermal amplification reactions 1h, and 80 DEG C of reaction 10min, are used for detecting TMV in vega soil invalid body.
CN201510737364.2A 2015-11-03 2015-11-03 Method for rapidly detecting TMV carried by diseased residue in soil Pending CN105296626A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110257559A (en) * 2019-06-19 2019-09-20 湖南省动物疫病预防控制中心 A method of whether African swine fever virus is carried in detection pig farm soil

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102286637A (en) * 2011-07-21 2011-12-21 陕西省烟草研究所 Method for detecting tobacco viruses by reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) technology
US20140024014A1 (en) * 2012-07-23 2014-01-23 National Cheng Kung University Primer set, method and kit for detecting pathogen in animals or plants

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102286637A (en) * 2011-07-21 2011-12-21 陕西省烟草研究所 Method for detecting tobacco viruses by reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) technology
US20140024014A1 (en) * 2012-07-23 2014-01-23 National Cheng Kung University Primer set, method and kit for detecting pathogen in animals or plants

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
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YANHUA LIU,ET AL: "Rapid detection of tobacco mosaic virus using the reverse transcription loop-mediated isothermal amplification method", 《ARCHIVES OF VIROLOGY》 *
刘世超等: "建立并优化检测土壤中病残体重TMV的LAMP技术", 《中国植物病理学会2015年学术年会论文集》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110257559A (en) * 2019-06-19 2019-09-20 湖南省动物疫病预防控制中心 A method of whether African swine fever virus is carried in detection pig farm soil

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Application publication date: 20160203