CN105713994A - Method for synchronously detecting LSV (lily symptomless viruse) and LMoV (lily mottle viruse) with immuno-capture double RT-PCR (reverse transcription-polymerase chain reaction) techniques - Google Patents

Method for synchronously detecting LSV (lily symptomless viruse) and LMoV (lily mottle viruse) with immuno-capture double RT-PCR (reverse transcription-polymerase chain reaction) techniques Download PDF

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CN105713994A
CN105713994A CN201610210126.0A CN201610210126A CN105713994A CN 105713994 A CN105713994 A CN 105713994A CN 201610210126 A CN201610210126 A CN 201610210126A CN 105713994 A CN105713994 A CN 105713994A
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lsv
lmov
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CN105713994B (en
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张玉宝
王亚军
谢忠奎
王若愚
杨果
郭志鸿
王乐
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Northwest Institute of Eco Environment and Resources of CAS
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Abstract

The invention provides a method for synchronously detecting the LSV (lily symptomless viruse) and the LMoV (lily mottle viruse) with immuno-capture double RT-PCR (reverse transcription-polymerase chain reaction) techniques. The method comprises steps as follows: polyclonal antibodies of the LSV and the LMoV are used for specifically capturing LSV particles and LMoV particles in one PCR reaction tube; specific primers of the LSV and the LMoV are used for the RT-PCR; agarose gel electrophoresis is used for detecting a target fragment. According to the method, ELISA (enzyme-linked immuno sorbent assay) and the RT-PCR are combined, an existing immuno-capture RT-PCR is improved, two kinds of lily viruses including the LSV and the LMoV are synchronously detected through immuno-capture RT-PRC, and accordingly, the immuno-capture RT-PCR technique is improved.

Description

With the immunocapture hidden syndrome virus of dual RT-PCR synchronous detecting lily and mottle virus Method
Technical field
The present invention relates to the hidden syndrome virus of a kind of synchronous detecting lily and the method for mottle virus, further relate to a kind of use The immunocapture dual RT-PCR hidden syndrome virus of technology synchronous detecting lily and the method for lily mottle virus.
Background technology
Lily virus is since the forties in 20th century is found, it has also become harm lily ball produces and Fresh Cutting flower quality Worldwide disease；So far it is found to infect lily virus and has kind more than 20, the wherein hidden syndrome virus of Bulbus Lilii (Lily symptomless Virus, LSV) and lily mottle virus (Lily mottle virus, LMoV) be occur relatively universal, endanger serious 2 kind disease Poison.
LSV belongs to Carnation Latent Virus In China and belongs to (Carlavirus), is sense single stranded rna virus；LSV genome is Not segmentation RNA, total length is 8394 bp, and 5 ' ends have polyA tail containing cap sequence, 3 ' ends, have 6 open reading frame (ORF), encode 4 albumen, be separately encoded according to 5 ' to 3 ': the RNA polymerase (RdRp) that RNA relies on, three gene linkages Structure (TGBp1-3), coat protein (CP) and nucleic acid binding protein；The ORF1 of LSV is RdRp, encodes an albumen (220 KD), replicate relevant in plant with virus；ORF2, ORF3 and ORF4 are separately encoded TGBp1, TGBp2 and TGBp3 (25 kD, 12 kD, 7 kD), are the virus motor protein (MP) that 3 gene orders of motion overlap each other in host； ORF5 is CP, and coding is unique participates in the protein (32kD) that Morphology of Virions is constituted；It is nucleic acid binding protein (16 that ORF6 speculates KD), this albumen contains the Zinc finger domain of a CysZ-CysZ structure.
LMoV belongs to marmor upsilon section, Potyvirus (Potyvirus), virion be bending and in Helical symmetry, size (650～900) nm × (11～15) nm；Viral genome is unimolecule positive chain RNA, about 9.4kb, has Potyvirus belongs to the Exemplary gene group architectural feature of member, including a Poly(A) tail, encode one by 3095 amino The polyprotein that molecular weight is 351.0 kDa of acid composition；Polyprotein is by forming coat protein (CP) etc. after self splicing 10 different size of functional proteins；LMoV CP subunit is made up of 274 aa, and size is about 30kDa, is assembled into shell parcel Viral RNA.
The preventing and treating more effective means of lily viral diseases are to use nontoxic propagating materials or carry out detoxifying fast breeding, and this depends on Sensitive, quick and reliable detection technique；Enzyme linked immunosorbent assay based on virus capsid protein antigen antibody reaction (ELISA) It is the method for the most relatively broad detection virus with RT-PCR based on viral nucleic acid detection；But, ELISA detects one instead System is answered to can be only done the detection of a kind of virus, it is impossible to realize synchronous detecting two or more virus；Although RT-PCR can be right Two or more virus realizes synchronous detecting, but detection must extract high-quality RNA, Bulbus Lilii and narcissus, Flos Tulipae Gesnerianae etc. Bulb flowers kind ball rich in polyphenol and polysaccharide, the RNA of extraction remains polyphenol and polysaccharide can have a strong impact on detection susceptiveness and Specificity；IC-RT-PCR or IC-RT-PCR combine the advantage of ELISA and RT-PCR another process, have higher Sensitivity, and more simple and effective, it is often more important that need not extract RNA, testing cost is low, is not affected by polysaccharide polyphenol, non- Often it is suitable for Bulbus Lilii, narcissus, the Viral diagnosis of tulip seed balls；
About IC-RT-PCR, the most by the relevant report of the method detection various plants virus, but set up Detection system be all only capable of the detection of a kind of single virus, so far there are no by the method synchronous detecting two or more virus Relevant report.
In order to improve detection efficiency further, reducing experimentation cost, existing IC-RT-PCR is reacted by the present invention Improved, it is achieved that with the hidden syndrome virus of the method synchronous detecting lily and lily mottle virus；Specifically at immunocapture The most single immunoreation is improved to the immunoreation being combined by the Part I of RT-PCR reaction, i.e. on PCR pipe wall simultaneously Capture LSV and LMoV particle；Part II is by synthesis that the Improved synthesis of the first chain cDNA is the first chain cDNAs mixture；The Single PCR reaction is improved in order to double PCR detects by three parts.
Summary of the invention
It is an object of the invention to overcome the most methodical deficiency, it is provided that one uses immunocapture quickly, accurately, easily Dual RT-PCR detects the method for LSV and LMoV simultaneously.
Whole detection method eliminates RNA and extracts process, utilizes the specificity of two-strain antibody, rapidly two kinds of mesh of capture Virus, thus efficiently quickly obtain purpose fragment, the accurately detection for corm kind crop virus provides the most special Method, thus perfect IC-RT-PCR technology.
It is an object of the invention to be achieved through the following technical solutions:
A kind of synchronous detecting LSV and the method for LMoV, it is characterised in that comprise the following steps:
1. during the polyclonal antibody of LSV and LMoV is coated on same PCR reaction tube, efficient special capture LSV and LMoV grain Son；
2. the specific primer utilizing LSV and LMoV carries out double inverse transcription polymerase chain reaction RT-PCR；
3. agarose gel electrophoresis testing goal fragment.
The above-mentioned specific primer that primer is LSV and LMoV carrying out dual RT-PCR.
Using the product that 2. agarose gel electrophoresis detecting step obtains, the target electrophoretic band of LSV and LMoV divides Do not occur in 198 and 395 base pair (bp) places.
The present invention is immunology detection technology and molecular Biological Detection technology organically to be combined, and absorption is at PCR LSV and the LMoV particle in LSV and LMoV Anti-TNF-α physical ability specificity Acquisition Detection sample on tube wall, plays concentration with pure Change the effect of virus, utilize again round pcr to be exaggerated the sensitivity of detection, can be used for the detection of susceptible sample low concentration virus； This invention eliminates the tedious steps extracting total serum IgE in conventional RT-PCR, greatly reduces testing cost；On the other hand due to anti- The former combination with antibody, improves the specificity of detection；Utilize immunocapture PCR can also avoid in napiform root class flowers kind ball rich The polysaccharide contained and the polyphenol interference to RT-PCR, thus this technology is especially suitable for lily ball bulb LSV and LMoV is carried out standard True detection, thus strict control LSV and the LMoV harm to Bulbus Lilii industry from kind of source, reduce economic loss.
Accompanying drawing explanation
Fig. 1 is the electrophoresis detection figure of the hidden syndrome virus of embodiment of the present invention Bulbus Lilii and mottle virus
In figure:
M, 600bp Marker；
1, negative control；
2, LSV+LMoV susceptible tissues；
3, positive control.
Detailed description of the invention
Embodiments of the invention technical scheme is as follows:
1. design of primers
CP gene order according to LSV and LMoV, devises specificity forward and reverse primer, the primer sequence of LSV and LMoV For: LSV-F(5 '-TATGGGCTTCCAATACAAC-3 ')/LSV-R(5 '-TATTCGGTTTCCAGGTTC-3 '), LMoV-F (5 '-TGGCACCTCACCAAATGTA-3 ')/LMoV-R(5 '-CATCATCTGCTGTATGCCTCT-3 '), amplified production Size is respectively 198bp and 395bp;
2. immunocapture
1) take the tissue of 100mg MOI LSV+LMoV, after adding 1mL PBST grinding, lapping liquid is proceeded to 1.5mL sterilizing and be centrifuged Guan Zhong, 4000rpm are centrifuged 2min, supernatant the most susceptible tissue crude extract；
2) mix after LMoV and LSV polyclonal antibody being diluted with the carbonate buffer solution of pH9.6, make LMoV and LSV two kinds resist The final concentration of body is respectively 1 μ g/mL and 10 μ g/mL；The mixed antibody taking the 200 above-mentioned dilutions of μ L adds PCR pipe, hatches for 37 DEG C 2h；
3) discard and be coated liquid, wash 3 times with PBST, each 3min;Add 200 μ L susceptible tissue crude extract, hatch 2h for 37 DEG C；
4) discard antigen liquid, wash 3 times with PBST, ddH2O washes 1 time, of short duration centrifugal, sucks residual liquid；
3. dual RT-PCR
1) in the PCR pipe being coated, add concentration and be each 2 μ L of LSV and LMoV downstream primer R, 8 μ of 10 μm ol/L LddH2O, is incubated 10min, afterwards rapid ice bath 2 min in 70 DEG C after mixing；
2) dual RT: add reagent, after mix homogeneously, 42 DEG C of water-bath 1h, 70 DEG C of insulations 15 by following system in above-mentioned PCR pipe Min, obtains cDNAs mixture；
M-MLV buffer (5 ×) 4 μ L
DNTP mixture (each 10mmol/L) 2 μ L
RNase inhibitor (30U/ μ L) 0.68 μ L
M-MLV reverse transcription (200U/μ L) 0.70 μ L
DEPC-H2O 0.62μL
3) double PCR: take a new PCR pipe, adds each reagent by following system and carries out double PCR:
Above-mentioned cDNAs mixture 2.0 μ L
10×PCR buffer 2.5μL
MgCl2(25 mmol/L) 2.5 μ L
DNTP mixture (each 2.5mmol/L) 2.5 μ L
LSV-F (10 μm ol/L) 0.5 μ L
LSV-R (10 μm ol/L) 0.5 μ L
LMoV-F (10 μm ol/L) 0.5 μ L
LMoV-R (10 μm ol/L) 0.5 μ L
Taq archaeal dna polymerase (5U/ μ L) 0.3 μ L
ddH2O 13.2μL
The reaction condition of double PCR is: 94 DEG C of denaturations 4min, 94 DEG C of degeneration 30s, and 50 DEG C～58 DEG C annealing 45s, 72 DEG C are prolonged Stretching 1min, expand 30～35 circulations, 72 DEG C extend 7min, are down to room temperature；
4. electrophoresis detection
Double PCR reaction takes 10 μ L product after terminating and carries out 2.0% agarose gel electrophoresis, in 1 × tbe buffer pendular ring border In, 100V voltage stabilizing electrophoresis 40min, then with gel imaging system and observe and record result；
5. interpretation of result
Judge whether the sample detected infects LSV or LMoV according to the presence or absence of amplified band on running gel and size thereof:
If gel only exists the nucleic acid fragment of 198bp, illustrate in sample containing LSV；
If gel only exists the nucleic acid fragment of 395bp, illustrate in sample containing LMoV；
If gel exists the nucleic acid fragment of 198bp and 395bp simultaneously, then explanation sample contains LSV and LMoV simultaneously.
According to said method, LSV and LMoV on Lanzhou edible lily and flower lily sample after testing, electrophoresis is examined Such as the fragment containing 198bp in survey result, illustrate in sample containing the hidden syndrome virus of Bulbus Lilii；Such as the fragment containing 395bp, sample is described Containing lily mottle virus in product.

Claims (2)

1., by the immunocapture hidden syndrome virus of dual RT-PCR synchronous detecting lily and a method for mottle virus, its feature exists In, comprise the following steps:
1. design of primers
CP gene order according to LSV and LMoV, the specificity forward of design LSV and LMoV and reverse primer；
2. immunocapture
1) take the tissue of 100mg MOI LSV+LMoV, after adding 1mL PBST grinding, lapping liquid is proceeded to 1.5mL sterilizing and be centrifuged Guan Zhong, 4000rpm are centrifuged 2min, supernatant the most susceptible tissue crude extract；
2) mix after LMoV and LSV polyclonal antibody being diluted with the carbonate buffer solution of pH9.6, make LMoV and LSV two kinds resist The final concentration of body is respectively 1 μ g/mL and 10 μ g/mL；The mixed antibody taking the 200 above-mentioned dilutions of μ L adds PCR pipe, hatches for 37 DEG C 2h；
3) discard and be coated liquid, wash 3 times with PBST, each 3min;Add 200 μ L susceptible tissue crude extract, hatch 3h for 37 DEG C；
4) discard antigen liquid, wash 3 times with PBST, ddH2O washes 1 time, of short duration centrifugal, sucks residual liquid；
3. dual RT-PCR
1) in the PCR pipe being coated, it is separately added into LSV specific reverse primers LSV-R 2 μ L, 10 μ of 10 μm ol/L LMoV specific reverse primers LMoV-R 2 μ L and ddH of mol/L2O 8 μ L, is incubated 10min in 70 DEG C after mixing, the most fast Speed ice bath 2 min；
2) dual RT: be separately added into dNTP mixture 2 μ of 5 × M-MLV buffer 4 μ L, 10mmol/L in above-mentioned PCR pipe M-MLV reverse transcription 0.70 μ L and DEPC-H of RNase inhibitor 0.68 μ L, 200U of L, 30U/ μ L/μ L2O 0.62μ L, after mix homogeneously, 42 DEG C of water-bath 1h, 70 DEG C of insulation 15 min, obtain cDNAs mixture；
3) double PCR: take a new PCR pipe, is separately added into above-mentioned cDNAs mixture 2.0 μ L, 10 × PCR buffer 2.5 μ L, the MgCl of 25 mmol/L2 The dNTP mixture 2.5 μ L of 2.5 μ L, 2.5mmol/L, the LSV specificity forward of 10 μm ol/L 0.5 μ L each with reverse primer LSV-F and LSV-R, the LMoV specificity forward of 10 μm ol/L and reverse primer LMoV-F and The each 0.5 μ L of LMoV-R, Taq archaeal dna polymerase 0.3 μ L and ddH of 5U/ μ L2O 13.2 μ L, carries out dual after mix homogeneously PCR；The reaction condition of above-mentioned double PCR is: 94 DEG C of denaturations 4min, 94 DEG C of degeneration 30s, 50 DEG C～58 DEG C annealing 45s, 72 DEG C extend 1min, expand 30～35 circulations, 72 DEG C of extension 7min, are down to room temperature；
4. double PCR reaction takes 10 μ L product and carries out 2.0% agarose gel electrophoresis, at 1 × tbe buffer pendular ring after terminating In border, 100V voltage stabilizing electrophoresis 40min, then with gel imaging system and observe and record result；
5. judge whether the sample detected infects LSV or LMoV according to presence or absence and the size thereof of amplified band on running gel, if The nucleic acid fragment only existing 198bp in gel then illustrates in sample containing LSV；If gel only exists the nucleic acid fragment of 395bp Then containing LMoV in explanation sample；If gel exists the nucleic acid fragment of 198bp and 395bp simultaneously, then in explanation sample simultaneously Containing LSV and LMoV.
The hidden syndrome virus of synchronous detecting lily the most according to claim 1 and the method for mottle virus, described LSV and LMoV It is respectively the hidden syndrome virus of Bulbus Lilii and lily mottle virus.
CN201610210126.0A 2016-04-07 2016-04-07 Method for synchronously detecting lily latent virus and lily mottle virus by using double composite immunocapture RT-PCR (reverse transcription-polymerase chain reaction) Active CN105713994B (en)

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Cited By (2)

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CN108754025A (en) * 2018-06-21 2018-11-06 中国科学院寒区旱区环境与工程研究所 A kind of kit and its detection method of the hidden syndrome virus of specific detection lily
CN108796127A (en) * 2018-06-21 2018-11-13 中国科学院寒区旱区环境与工程研究所 A kind of kit and its detection method of specific detection lily mottle virus

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Publication number Priority date Publication date Assignee Title
CN108754025A (en) * 2018-06-21 2018-11-06 中国科学院寒区旱区环境与工程研究所 A kind of kit and its detection method of the hidden syndrome virus of specific detection lily
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