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.