CN105755173A

CN105755173A - Method for synchronously detecting three viruses of lily by immunocapture triple RT-PCR (reverse transcription-polymerase chain reaction)

Info

Publication number: CN105755173A
Application number: CN201610210127.5A
Authority: CN
Inventors: 张玉宝; 王亚军; 谢忠奎; 王若愚; 郭志鸿; 杨果; 邱阳
Original assignee: Xuzhou Muyang Biotechnology Development Co ltd; Cold and Arid Regions Environmental and Engineering Research Institute of CAS
Current assignee: XUZHOU MUYANG BIOTECHNOLOGY DEVELOPMENT Co.,Ltd.; Northwest Institute of Eco Environment and Resources of CAS
Priority date: 2016-04-07
Filing date: 2016-04-07
Publication date: 2016-07-13
Anticipated expiration: 2036-04-07
Also published as: CN105755173B

Abstract

The invention provides a method for synchronously detecting a latent symptom virus, a mottle virus and a cucumber mosaic virus of lily by immunocapture triple RT-PCR (reverse transcription-polymerase chain reaction). The method comprises the following steps: specifically capturing LSV, LMoV and CMV particles by polyclonal antibodies of LSV, LMoV and CMV in the same PCR reaction tube; and carrying out triple RT-PCR and agarose gel electrophoresis detection on a target segment by the specific primers of the LSV, the LMoV and the CMV. According to the method, by combination of ELISA and RT-PCR, existing immunocapture triple RT-PCR is improved; and synchronous detection of three lily viruses LSV, LMoV and CMV by immunocapture triple RT-PCR is achieved, so that the immunocapture RT-PCR technology is perfected.

Description

By immunocapture three kinds of viral methods of triple RT-PCR synchronous detecting lily

Technical field

The present invention relates to the hidden syndrome virus of a kind of synchronous detecting lily, mottle virus and the method for cucumber mosaic virus, more enter One step relates to one immunocapture triple RT-PCR technology hidden syndrome virus of synchronous detecting lily, mottle virus and cucumber mosaic virus The method of poison.

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 lily (Lily symptomless Virus, LSV), lily mottle virus (Lily mottle virus, LMoV) and cucumber melon mosaic virus (Cucumber Mosaic virus, CMV) be occur relatively universal, endanger serious 3 kind virus.

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 ORFs (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.

CMV is typical case's one-tenth of Bromoviridae (Bromoviridae) Cucumovirus (Cucumovirus) Member, the most all have distribution, host range is dispersed throughout dicotyledon and monocotyledon, illustrated the most up to More than 1000 kinds；CMV virion molecular weight is between 5000～67000kDa, and virion rna content accounts for 18%, and protein contains Amount 82%, capsid protein molecules amount is 24.5KDa；A260/A280 about 1.7；Virion is that positive 20 bodies are spherical, diameter 28 ～30nm；RNA is made up of four RNA molecule, RNA1 and RNA2 is coated respectively, RNA3 and RNA4 is the most coated in same clothing In shell.

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 synchronizing to detect multiple virus；Although RT-PCR can be to multiple disease Poison realizes synchronizing detection, but detection must extract high-quality RNA, lily and the bulb flowers kind ball such as narcissus, tulip Rich in polyphenol and polysaccharide, the RNA of extraction remains polyphenol and polysaccharide can have a strong impact on the sensitivity of detection with specific；Immunity is caught Obtain RT-PCR or IC-RT-PCR and combine the advantage of ELISA and RT-PCR another process, there is 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, is especially suitable for lily, water Celestial, the Viral diagnosis of tulip seed balls；About IC-RT-PCR, the most by the method detection various plants virus Relevant report, but the detection architecture set up all has been only capable of the detection of a kind of single virus, and it is same by the method that so far there are no The relevant report of step detection two or more virus.

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, mottle virus and cucumber mosaic virus；Specifically exist The most single immune response is improved to the immune response being combined, i.e. PCR pipe by the Part I of IC-RT-PCR reaction Capture LSV, LMoV and CMV particle on wall simultaneously；The Improved synthesis of the first chain cDNA is the first chain cDNAs mixing by Part II The synthesis of thing；Single PCR reaction is improved in order to triple PCR detects by Part III.

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 Triple RT-PCR detect the method for LSV, LMoV and CMV simultaneously.

Whole detection method eliminates RNA and extracts process, utilizes the specific of three kinds of antiviral antibodies, rapidly three 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 method synchronizing to detect LSV, LMoV and CMV, it is characterised in that comprise the following steps:

1. during the polyclonal antibody of LSV, LMoV and CMV is coated on same PCR reaction tube, efficient special capture LSV, LMoV With CMV particle；

2. the specific primer utilizing LSV, LMoV and CMV carries out triple inverse transcription polymerase chain reaction RT-PCR；

3. agarose gel electrophoresis testing goal fragment.

The above-mentioned specific primer that primer is LSV, LMoV and CMV carrying out triple anti-RT-PCR.

Use the product that 2. agarose gel electrophoresis detecting step obtains, the target electrophoresis strip of LSV, LMoV and CMV Band respectively appears in 198,395 and 248 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, LMoV and CMV particle in LSV, LMoV and CMV Anti-TNF-α physical efficiency specific Acquisition Detection sample on tube wall, plays Concentrate and the effect of purified virus, utilize again round pcr to be exaggerated the sensitivity of detection, can be used for susceptible sample low concentration virus Detection；This invention eliminates the tedious steps extracting total serum IgE in conventional RT-PCR, greatly reduces testing cost；The opposing party Face, due to the combination of antigen Yu antibody, improves the specific of detection；Utilize immunocapture PCR can also avoid napiform root class flowers In kind of ball rich in polysaccharide and the polyphenol interference to RT-PCR, thus this technology is especially suitable for lily ball bulb LSV, LMoV Detect accurately with CMV, thus from kind of source, strictly control LSV, LMoV and CMV harm to lily industry, reduce warp Ji loss.

Accompanying drawing explanation

Fig. 1 is the electrophoresis detection figure of the hidden syndrome virus of embodiment of the present invention lily, mottle virus and cucumber mosaic virus

In figure:

M:600bp Marker；

1: negative control；

The susceptible tissue of 2:LSV+LMoV+CMV；

3: positive control.

Detailed description of the invention

Embodiments of the invention employing following steps:

1. design of primers

CP gene order according to LSV, LMoV and CMV, devises specific forward and the reverse primer of LSV, LMoV and CMV, Primer sequence is: LSV-F(5 '-TATGGGCTTCCA ATACAAC-3 ')/LSV-R(5 '-TATTCGGTTTCCAGGTTC- 3 '), LMoV-F(5 '-TGGCACCTCACCAAATGTA-3 ')/LMoV-R(5 '-CATCATCTGCTGTATGCCTCT- 3 '), CMV-F(5 '-CTTTGTAGGGAGTGAACGCTGTA-3 ')/CMV-R(5 '-AGATGGCGGCAACGGATA-3 '), The size of amplified production is respectively 198bp, 395bp and 248bp;

2. immunocapture

1) take the tissue of 100mg MOI LSV+LMoV+CMV, add and after 1mL PBST grinds, lapping liquid is proceeded to 1.5mL sterilizing In centrifuge tube, 4000rpm is centrifuged 2min, supernatant the most susceptible tissue crude extract；

2) mix after LMoV, LSV and CMV polyclonal antibody being diluted with the carbonate buffer solution of pH9.6, make LMoV, LSV and The final concentration of tri-kinds of antibody of CMV is respectively 1 μ g/mL, 10 μ g/mL and 50 μ g/mL；The mixed antibody taking the 200 above-mentioned dilutions of μ L adds Enter PCR pipe, hatch 2h for 37 DEG C；

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 raffinate；

The most triple RT-PCR

1) in the PCR pipe being coated, add concentration and be LSV, LMoV and CMV specific reverse primers of 10 μm ol/L The each 2 μ L of LSV-R, LMoV-R and CMV-R, 6 μ L ddH₂O, is incubated 10min, afterwards rapid ice bath 2 min in 70 DEG C after mixing；

2) triple RT: add reagent, after mixing, 42 DEG C of water-bath 1h, 70 DEG C of insulations 15 by following system in above-mentioned PCR 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 transcriptase (200U/μ L) 0.70 μ L

DEPC-H₂O 0.62μL

3) triple PCR: take a new PCR pipe, adds each reagent by following system and carries out double PCR:

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

CMV-F (10 μm ol/L) 0.5 μ L

CMV-R (10 μm ol/L) 0.5 μ L

Taq archaeal dna polymerase (5U/ μ L) 0.3 μ L

ddH₂O 12.2μL

The reaction condition of triple PCR is: 94 DEG C of denaturations 4min, 94 DEG C of sex change 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 normal temperature；

4. electrophoresis detection

Triple 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, LMoV or CMV 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 only exists the nucleic acid fragment of 248bp, illustrate in sample containing CMV；

If gel exists the nucleic acid fragment of 198bp and 395bp simultaneously, then explanation sample contains LSV and LMoV simultaneously；

If gel exists the nucleic acid fragment of 198bp and 248bp simultaneously, then explanation sample contains LSV and CMV simultaneously；

If gel exists the nucleic acid fragment of 198bp, 395bp and 248bp simultaneously, then explanation sample contains LSV, LMoV simultaneously And CMV.

According to said method, LSV, LMoV and the CMV on Lanzhou edible lily and flower lily sample, electricity after testing Such as the fragment containing 198bp in swimming testing result, illustrate in sample containing the hidden syndrome virus of lily；Such as the fragment containing 395bp, say Containing lily mottle virus in bright sample；Such as the fragment containing 248bp, illustrate in sample containing cucumber mosaic virus.

Claims

1. one kind with immunocapture triple RT-PCR hidden syndrome virus of synchronous detecting lily, mottle virus and the side of cucumber mosaic virus Method, it is characterised in that comprise the following steps:

1. design of primers

CP gene order according to LSV, LMoV and CMV, the specific forward of design LSV, LMoV and CMV and reverse primer；

2. immunocapture

2) mix after LMoV, LSV and CMV polyclonal antibody being diluted with the carbonate buffer solution of pH 9.6, make LMoV, LSV and The final concentration of tri-kinds of antibody of CMV is respectively 1 μ g/mL, 10 μ g/mL and 50 μ g/mL；The mixed antibody taking the 200 above-mentioned dilutions of μ L adds Enter PCR pipe, hatch 2h for 37 DEG C；

The most triple RT-PCR

1) in the PCR pipe being coated, add the LSV specific reverse primers LSV-R 2 μ L of 10 μm ol/L, 10 μm ol/L LMoV specific reverse primers LMoV-R 2 μ L, CMV specific reverse primers CMV-R 2 μ L and ddH of 10 μm ol/L₂O 6 μ L, are incubated 10min, afterwards rapid ice bath 2 min in 70 DEG C after mixing；

2) triple RT: be separately added in above-mentioned PCR 5 × M-MLV buffer 4 μ L, 10mmol/L dNTP mixture 2 μ L, M-MLV reverse transcriptase 0.70 μ L and DEPC-H of RNase inhibitor 0.68 μ L, 200U of 30U/ μ L/μ L₂O 0.62 μ L, After mixing, 42 DEG C of water-bath 1h, 70 DEG C of insulation 15 min, obtain cDNAs mixture；

3) triple 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/L₂The dNTP mixture 2.5 μ L of 2.5 μ L, 2.5mmol/L, the specific forward of LSV of 10 μm ol/L 0.5 μ L each with reverse primer LSV-F and LSV-R, the specific forward of LMoV of 10 μm ol/L and reverse primer LMoV-F and The each 0.5 μ L of LMoV-R, the specific forward of CMV of 10 μm ol/L and each 0.5 μ L of reverse primer CMV-F and CMV-R, 5U/ μ L Taq DNA polymerase 0.3 μ L and ddH₂O 12.2 μ L, carries out triple PCR after mixing；The reaction of above-mentioned triple PCR Condition is: 94 DEG C of denaturations 4min, 94 DEG C of sex change 30s, and 50 DEG C～58 DEG C annealing 45s, 72 DEG C extend 1min, expand 30～35 Individual circulation, last 72 DEG C extend 7min, are down to normal temperature；

4. triple 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. according to the presence or absence of amplified band on running gel and size thereof judge the sample detected whether infect LSV, LMoV or CMV, if only existing the nucleic acid fragment of 198bp in gel, illustrates in sample containing LSV；If gel only exists the core of 395bp Acid fragment then illustrates in sample containing LMoV；If gel only exists the nucleic acid fragment of 248bp, illustrate in sample containing CMV； If gel exists the nucleic acid fragment of 198bp and 395bp simultaneously, then explanation sample contains LSV and LMoV simultaneously；If in gel There is the nucleic acid fragment of 198bp and 248bp simultaneously, then explanation sample contains LSV and CMV simultaneously；If gel exists simultaneously The nucleic acid fragment of 198bp, 395bp and 248bp, then contain LSV, LMoV and CMV in explanation sample simultaneously.

The hidden syndrome virus of synchronous detecting lily the most according to claim 1, mottle virus and the method for cucumber mosaic virus, institute State LSV, LMoV and CMV and be respectively the hidden syndrome virus of lily, lily mottle virus and cucumber mosaic virus.