CN109355430A - A kind of multiple RT-PCR detection method of synchronous detection 3 kinds of pineapples wilting correlated virus and pineapple bacilliform DNA virus - Google Patents

Abstract

The present invention provides a kind of synchronous multiplex RT-PCR method and primer combination for detecting each position of pineapple plant and whether carrying the wilting correlated virus -1 of pineapple, the wilting correlated virus -2 of pineapple, the wilting correlated virus -3 of pineapple and pineapple baculoviral.This method have the characteristics that high specificity, easy to operate, time-consuming shorter, testing result easily distinguish, detection sensitivity and high-efficient, overcome that common molecular detection target is single, take a long time, consumptive material is more, sensitivity is not high, is not suitable for the deficiencies of large-scale promotion application, for pineapple virosis detection and prevention and control early warning a kind of technically reliable, transreplication are provided, be suitable for rapid detection method to department of base and researcher's popularization and application.

Description

A kind of wilting correlated virus of 3 kinds of pineapples of synchronous detection and pineapple bacilliform DNA virus it is more Weight RT-PCR detection method

Technical field

The present invention relates to the multiple RT- of a kind of synchronous detection 3 kinds of pineapples wilting correlated virus and pineapple bacilliform DNA virus PCR detection method belongs to technical field of virus detection.

Background technique

Virosis is one of the important disease of each pineapple main producing region in the world.Pineapple during the growth process, often by virus Harm, virosis seriously affect pineapple fruit yield and quality, have become what each producing region pineapple industry in the world developed in a healthy way at present One of restrictive factor.Pineapple is by after virus infection, and band poison, virus are proliferated in plant body, spread throughout one's life, interferes and destroy evil The normal physiological function of sick plant, plant of causing to fall ill are unable to normal nutrition growth and yield positive results, and last root system is withered, on the ground Portion's shrinkage dehydration, blade gradually redden after chlorisis turns yellow, and the later period, whole strain was wilting, dead, and how abnormal as a result strain growing way decline, fruit be It is shape, abnormal small etc., cause yield decline, fruit quality to deteriorate, variety deterioration, very big economic loss is caused to pineapple industry, to spinach Trailing plants Economical cultivation causes to seriously affect.The asexual propagation materials such as the suction bud of maternal plant pumping of catching an illness, terminal bud, descendants' bud band poison, exists Further spread the potential threat to cause harm.Currently, the pineapple virus that China has found and identifies mainly has: the wilting related diseases of pineapple Poison -1 (Pineapple Mmealybug Wilt-associated Viruse-1, PMWaV-1), the wilting correlated virus -2 of pineapple (Pineapple Mmealybug Wilt-associated Viruse-2, PMWaV-2), the wilting correlated virus -3 of pineapple (Pineapple Mmealybug Wilt-associated Viruse-3, PMWaV-3) and pineapple bacilliform DNA virus 4 kinds of (Pineapple Bacilliform Comosus Virus, PBCoV) etc..Studies have shown that PMWaV-2 and pineapple mealy bug (Dysmicoccus spp.) harm causes pineapple blight (Pineapple Mealybug Wilt, PMW), PMWaV-1 jointly Important role is also play in the development process of PMW with PMWaV-3.

PMW is caused harm, and there are following evident characteristics: first is that the phenomenon that several PMWaVs Combined Infections is usually presented in disease sample, And though PBCoV and PMW fall ill without direct relation, it often appears in PMW jointly with PMWaVs and shows in disease plant.Second is that field Between be commonly present " hidden disease morbidity " phenomenon, false negative rate is higher, and field is difficult to whether take by the not aobvious disease plant of the judgement that observes the symptoms Band virus；Third is that aobvious disease is more unknown when occurring degree and soil water content negative correlation, i.e. soil water content are higher It is aobvious, be easier to aobvious disease when moisture content is relatively low, exist simultaneously part mild, middle disease plant can restore under conditions of moisture abundance The case where " health ", distinguishes for field and increases difficulty；Fourth is that pineapple plant, once virus infection, just band is malicious throughout one's life, and by it Band poison, field can not distinguish the asexual propagation materials such as the suction bud of sprouting, terminal bud, descendants' bud in various degree, and the malicious seedling of band is easily caused to expand Dissipate, propagate, to a certain extent for subsequent disease is a wide range of, quickly diffusion provides convenience, health seedling production is promoted and There are biggish potential threats for industry development.Therefore, it establishes a kind of easy, quick, sensitive, accurately, it is a variety of that detection can be synchronized The multiple RT-PCR detection method of pineapple virus, is respectively organized applied to detection pineapple and seedling whether carries virus and future can The nontoxic seedling breeding of the pineapple of phase seems extremely urgent and important.

Currently, the method for pineapple viral diagnosis mainly has immuno-electron microscope detection, the serology inspection based on elisa technique It surveys, histogenic immunity trace and conventional RT-PCR detect.But all there is biggish limiting factor in these methods, such as immuno-electron microscope is grasped It is required, serological method virion purification difficult and sensitivity not high too high with use cost, histogenic immunity trace is easy There is false positive, conventional RT-PCR is cumbersome, time-consuming, expends high and single can only detect a kind of virus, low efficiency etc..And Pineapple sample detection is examined studies have shown that the phenomenon that field pineapple generally existing several viral Combined Infections using conventional RT-PCR It just needs to be repeated several times to test if surveying a variety of viruses, time and effort consuming, cost is also higher.Multiple RT-PCR is combined by RT-PCR The detection technique that multiple PCR technique (Multiplex Polymerase Chain Reaction, mPCR) is set up, behaviour It is that multipair specific primer is added in a PCR reaction system as method, target fragments a variety of in same system is expanded Increase, and then achieve the purpose that be detected simultaneously by multiple target sequences in same system, compare single RT-PCR, this method has spy It is anisotropic it is strong, time saving it is easy, save cost and sensitivity is unaffected, the advantages that capable of synchronizing detection to a variety of viruses, so Gradually it is widely used in the detection work of animals and plants virosis.Have at present using multiple RT-PCR and its deriving technology to Portugal The report that grape, potato, ground family crop, banana virus disease are detected, but it is not wilting about 3 kinds of pineapples are detected simultaneously The pertinent literature and patent of correlated virus and pineapple baculovirus DNA virus.

Summary of the invention

In view of the deficiencies in the prior art, the present invention provides a kind of wilting correlated virus of 3 kinds of pineapples of synchronous detection and pineapple bar The multiple RT-PCR detection primer of shape DNA virus (PBCoV) combines, and application primer combination carries out multiple RT-PCR detection Method.

The technical solution adopted by the present invention is as follows:

A kind of primer combination for the wilting correlated virus of 3 kinds of pineapples of synchronous detection and pineapple bacilliform DNA virus, it is described to draw Object combines

Wilting -1 (the Pineapple Mmealybug Wilt- of correlated virus of pineapple is detected for specific RT-PCR Associated Viruse-1, PMWaV-1) primer pair I:

PMWaV-1-F:5'-TCCAAAGCTCTGTGGGAGTTGGGT-3'

PMWaV-1-R:5'-ACGTTGTGTTCGCCAGAACCACT-3'；

Wilting -2 (the Pineapple Mmealybug Wilt- of correlated virus of pineapple is detected for specific RT-PCR Associated Viruse-2) primer pair II:

PMWaV-2-F:5'-TGGCAACAATCCCAACGGAAGC-3'

PMWaV-2-R:5'-TCGCCGAATGGTTCATGAGGCT-3'；

Wilting -3 (the Pineapple Mmealybug Wilt- of correlated virus of pineapple is detected for specific RT-PCR Associated Viruse-3) primer pair III:

PMWaV-3-F:5'-AACCATGGGCGCACAAAACCAG-3'

PMWaV-3-R:5'-CGCTCTCTTTACAAGATGCCAA-3'；And

Pineapple bacilliform DNA virus (Pineapple Bacilliform Comosus is detected for specific RT-PCR Virus, PBCoV) primer pair IV:

PBCoV-F 5'-TATCCTTGTATGCGTGCGTCTAC-3'

PBCoV-R 5'-GCAGAGTTTCCAAAGTTTCGTCAC-3'。

The present invention also provides the multiple of a kind of wilting correlated virus of 3 kinds of pineapples of synchronous detection and pineapple bacilliform DNA virus RT-PCR detection method, comprising the following steps:

1) pineapple sample total serum IgE to be measured is extracted, and prepares the first chain cDNA by template reverse transcription of RNA；

2) it using the cDNA obtained in step 1) as template, is combined using above-mentioned primer pair, it is anti-to carry out specific PCR amplification It answers；

3) pcr amplification product is detected with agarose gel electrophoresis；

4) PCR amplification result is analyzed.Under the conditions of gel imaging, if there is obvious band to show that test sample is at 482bp PMWaV-1 is positive, if having obvious band to show that test sample is positive in PMWaV-2 at 612bp, if there is obvious band at 327bp It is positive in PMWaV-3 to show test sample, if there have obvious band to show test sample at 851bp to be positive in PBCoV, otherwise is It is negative.

Preferably, in multiple RT-PCR detection method of the present invention, step 1) specifically: it is total to extract pineapple sample to be measured 5.0 μ L, 10mM random primer of pineapple sample total serum IgE template 1.0 μ L, 10mM are added into the reaction tube of RNase-free by RNA 10.0 μ L, M-MuLV reverse transcriptase of dNTPs 1.0 μ L, 2 × RT of buffer Buffer, 1.0 μ L, RNase inhibitor 0.5 μ L and RNase-free Water of Ribonuclease Inhibitor, 1.0 μ L obtains first in 42 DEG C of incubation 1h Chain cDNA.

Preferably, the 25 μ L PCR reaction systems that multiplex RT-PCR amplification uses are carried out in step 2) are as follows:

First chain cDNA solution, 1 μ L, 5U/ μ L Taq enzyme, 0.25 μ L, 10 × PCR buffer obtained in step 1) (contains Mg²⁺) 2.5 μ L, 2.5mM dNTP mixed liquor, 2.5 upstream and downstream μ L, 10 μm/μ L each 0.5 μ L and ddH of primer₂O 14.75μL。

Preferably, PCR response procedures are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C extend 1min is recycled 35 times；72 DEG C of extension 10min are saved.

Preferably, step 3) specifically: with TAE electrophoretic buffer with 1.5% Ago-Gel of preparation, take 5 μ L RT-PCR Product carries out electrophoresis 30min, determines pineapple viral species according to electrophoretic band.

Compared with prior art, the beneficial effects of the present invention are:

1) test object covers the reported whole pineapple viral species in current China pineapple producing region, can grind in science Study carefully, early warning and monitoring, the relevant departments such as operation instruction and production are widely popularized, applicability is wide；

2) primer that screening obtains combines high specificity, and high sensitivity, between each other without significantly interfering with, each target stripe is big Cell is shown clearly, and electrophoresis detection result is obvious, band is clearly easily distinguished, accuracy is high；

3) it is detected compared to normal serum, multiple RT-PCR detection method established by the present invention has higher sensitive Degree and accuracy realize the purpose of 4 kinds of pineapple virus of synchronous detection in same reaction system, largely improve work Make efficiency, saves testing cost.

4) of the invention is not only that the detection of pineapple virus causing disease provides a kind of quick, simplicity, accurately detection technique means, Pineapple virus disease Fields detection is applied also for select and the cultivation of nontoxic seedling, tissue culture detoxification efficiency with monitoring, healthy maternal plant garden In the work such as evaluation.

Detailed description of the invention

Fig. 1 is the primer combination specific test result figure that screening obtains；Wherein, l is PMWaV-1 positive cDNA template, 2 It is PMWaV-3 positive cDNA template for PMWaV-2 positive cDNA template, 3,4 is PBCoV positive cDNA template, M is DL2000Plus DNA Marker；

Fig. 2 is the result figure of substance RT-PCR amplified production glue recycling；Wherein, 1 it is PMWaV-1 target fragment, 2 is PMWaV-2 target fragment, 3 be PMWaV-3 target fragment, 4 be PBCoV target fragment, M is DL2000Plus DNA Marker；

Fig. 3 is multiple RT-PCR reaction system and response procedures Orthogonal Optimization Test result figure described in the embodiment of the present invention 2； Wherein, 1~16 RT-PCR amplified production handled for 1~No. 16 in table 2, M are DL1000DNA Marker；

Fig. 4 is multiple RT-PCR reaction system and response procedures Orthogonal Optimization Test result figure described in the embodiment of the present invention 2； Wherein, 17~25 RT-PCR amplified production handled for 17~No. 25 in table 2, M are DL1000DNA Marker；

Fig. 5 is to detect field pineapple sample band poison situation using the method that the present invention establishes described in the embodiment of the present invention 3 Test result figure；Wherein, 1 is negative control, and 2~10 be respectively the field sample that number is 1~9, and M is DL2000Plus DNA Marker。

Specific embodiment

Below by specific embodiment, invention is further described in detail.

Experimental method used in the embodiment of the present invention is conventional method unless otherwise specified.

Material used in the embodiment of the present invention, reagent etc., are commercially available unless otherwise specified.

The combination of the multiple RT-PCR detection primer of the wilting correlated virus of 3 kinds of pineapples of embodiment l and bacilliform DNA virus obtains ?

1. design of primers and synthesis

With reference to reported PMWaVs and PBCoV related gene sequence (accession number NC_ in existing literature and GenBank 010178.1、EU769114.1、EU769114.1、AF283103.1、DQ225114.1、EU769116.1、DQ399259.2、 EU372003.1, EF467920.1, EF488753.1, EU377670.1, EU377670.1, EU377670.1), it uses respectively The software screening methods such as DNAMAN, PrimerPrimier 5.0 and Oligo6.0, analysis and design multiple RT-PCR primer pair, mesh Be obtain can be applied to specific amplification China have determined that at this stage be present on pineapple virus target sequence segment, Mutual glitch-free primer pair sequence, and can ensure that can be in 1.5% agarose gel electrophoresis by each target sequence segment Clear, apparent distinguishes.

Finally, design have chosen PMWaV-1 (482bp) in table 1, PMWaV-2 (612bp), PMWaV-3 (327bp) and 4 pairs of primers of PBCoV (851bp) (respectively correspond SEQ ID NO:1 to the SEQ ID NO:8 in sequence table).Use Standard PCR pair It is verified, it can be seen that and to target fragment, primer dimer is unobvious for the amplification that the primer of selection can be clear, special, Without (Fig. 1,2) is significantly interfered between each primer pair, it can be used for the foundation test of next step multiple RT-PCR system.

Table 1 is applied to the multiple RT-PCR detection primer composite sequence of the wilting correlated virus of 3 kinds of pineapples and bacilliform DNA virus And feature

The wilting correlated virus of 2 pineapple of embodiment and pineapple bacilliform DNA virus multiple RT-PCR Establishing and optimization

1. the determination of sampling point and sampling method

In order to make established multiple RT-PCR technology have wider applicability, according to the wilting correlated virus of pineapple (PMWaVs) and it is the polyphenol of characteristic distributions and eyelet stitch of the pineapple bacilliform DNA virus (PBCoV) in pineapple plant, more Sugar, high microsteping feature, determine strain lobus cardiacus, the whole leaf of nearly mature leaf and terminal bud, inhale bud, descendants' bud the whole leaf of whole blades and The aerial stem of plant all can serve as detection sample, with the white young tender part and aerial stem of plant center tender tissue of appropriate to the occasion selection blade base Direct milling and extracting sample total serum IgE is spent, fruit and Gen Yin Total RNAs extraction difficulty are larger, extraction efficiency is low without recommending as inspection Test sample sheet.

2. the extraction of sample total serum IgE

The pineapple leaf base portion white tissues for weighing 0.1g respectively, the polysaccharide polyphenol produced according to Beijing Bioteke company Plant total serum IgE rapidly extracting kit (centrifugal column type) specification step extract pineapple sample total serum IgE, take 5 μ L RNA solutions in The detection of 1.5% agarose gel electrophoresis；It is measured with ND2000 ultraviolet specrophotometer (Thermo NanoDrop company of the U.S.) Concentration, remaining RNA solution are set -80 DEG C and are saved backup.

3. reverse transcription

5.0 μ L, 10mM random primer 1.0 of pineapple sample total serum IgE template is added into the 1.5mL centrifuge tube of RNase-free 10.0 μ L, M-MuLV reverse transcriptase of μ L, 10mM dNTPs 1.0 μ L, 2 × RT of buffer Buffer, 1.0 μ L, RNase inhibitor 0.5 μ L and RNase-free Water of Ribonuclease Inhibitor, 1.0 μ L obtains the first chain in 42 DEG C of incubation 1h CDNA is placed in -20 DEG C and saves backup.

The foundation and optimization of 4 multiplex PCR systems

The cDNA obtained using field pineapple sample reverse transcription is template, in 4 kinds of target pineapple viruses of the single detection of Standard PCR On the basis of, establish 25 μ L multiplex PCR systems.The first step, by single factor experiment for several times, it is determined that optimal 10 × Taq Buffer volume is 2.5 μ L.Second step has determined each factor optimal level, i.e. 25 μ L based on L25 (56) orthogonal test (table 2) Under reaction system: dNTPs (2.5mM) volume is that 2.5 μ L, Taq archaeal dna polymerase (5U/ μ L) volumes are 0.25 μ L, primer combination In each primer volume be respectively PMWaV-1 forward primer (10 μM) 0.5 μ L, PMWaV-1 reverse primer (10 μM) 0.5 μ L, PMWaV-2 forward primer (10 μM) 0.5 μ L, PMWaV-2 reverse primer (10 μM) 0.5 μ L, PMWaV-3 forward primer (10 μM) 0.5 μ L, PMWaV-2 reverse primer (10 μM) 0.5 μ L, PBCoV forward primer (10 μM) 0.5 μ L, PBCoV reverse primer (10 μM) 0.5 μ L and ddH₂14.75 μ L, cDNA template of O, 1 μ L.

Meanwhile the optimal multiple RT-PCR response procedures obtained by orthogonal test are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of changes Property 30s, 54 DEG C of annealing 30s, 72 DEG C of extension 1min, recycle 35 times；72 DEG C of extension 10min are saved.

Table 2 is based on the wilting correlated virus of 3 kinds of pineapples and the multiple RT- of pineapple bacilliform DNA virus of L25 (56) orthogonal design The experimental design table of PCR detection architecture and 6 key factors of response procedures

5. viral diagnosis and result judgement

With 1 × TAE electrophoretic buffer with preparation 1.5% Ago-Gel 20mL (adding 1 μ L Goldview), 5 μ L is taken to react Product is in 1 × TAE buffer, 120V 10A electrophoresis 30min, and the gel of counterband tape is placed in gel imaging system observation, according to Electrophoretic band determines pineapple viral species.If thering is obvious band to show that test sample is positive in PMWaV-1 at 482bp, if 612bp It is positive in PMWaV-2 that place has obvious band to show test sample, if there is obvious band to show test sample in PMWaV- at 327bp 3 is positive, if there is obvious band to show that test sample is positive in PBCoV at 851bp, otherwise is feminine gender.

The band poison detection of 3 field sample of embodiment and specificity verification

To pick up from the pineapple leaf sample in certain orchard as test material, extraction phyllopodium white tender tissue total serum IgE is template, verifying The practicability and feasibility of multiple RT-PCR detection method established by the present invention.

Total tissue RNA, reverse transcription, the multi-PRC reaction system of optimization and program is extracted to describe with embodiment 2.As a result table It is bright, using the detection method that the present invention establishes, 0~4 kind of target pineapple virus not waited can be detected from for sample sheet, The middle sample for carrying two or more virus accounts for the ratio for sample sheet up to 77.78%, and it is universal that this also further demonstrates pineapple virus The phenomenon that there are Combined Infections.In terms of comprehensive, this method detection effect it is good, high-efficient (Fig. 5).

To verify established system to the detection specificity of target viral, each purpose electrophoretic band is sequenced.It surveys Sequence result submits GenBank (http://www.ncbi.nlm.nih.gov/) to carry out BLAST, the results show that each band is corresponding Virus sequence and target viral sequence identity up to 100%, be indicated above the multiplex RT-PCR method that the present invention establishes With high degree of specificity, it is related that relevant departments' development such as scientific research, early warning and monitoring, operation instruction and production can be applied to Research and practical work have wide applicability and practicability.

The above content is specific embodiment is combined, further detailed description of the invention, and it cannot be said that this hair Bright specific implementation is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, it is not taking off Under the premise of from present inventive concept, a number of simple deductions or replacements can also be made.

Sequence table

<110>Tropical Fruit Research Institute, Hainan Academy of Agricultural Sciences

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Claims (8)

1. a kind of primer combination for the wilting correlated virus of 3 kinds of pineapples of synchronous detection and pineapple bacilliform DNA virus, feature exist In the primer combination includes:

Primer pair I for the specific RT-PCR detection wilting correlated virus -1 of pineapple:

PMWaV-1-F:5'-TCCAAAGCTCTGTGGGAGTTGGGT-3',

PMWaV-1-R:5'-ACGTTGTGTTCGCCAGAACCACT-3'；

Primer pair II for the specific RT-PCR detection wilting correlated virus -2 of pineapple:

PMWaV-2-F:5'-TGGCAACAATCCCAACGGAAGC-3',

PMWaV-2-R:5'-TCGCCGAATGGTTCATGAGGCT-3'；

Primer pair III for the specific RT-PCR detection wilting correlated virus -3 of pineapple:

PMWaV-3-F:5'-AACCATGGGCGCACAAAACCAG-3',

PMWaV-3-R:5'-CGCTCTCTTTACAAGATGCCAA-3'；And

Primer pair IV for specific RT-PCR detection pineapple bacilliform DNA virus:

PBCoV-F:5'-TATCCTTGTATGCGTGCGTCTAC-3',

PBCoV-R:5'-GCAGAGTTTCCAAAGTTTCGTCAC-3'.

2. a kind of multiple RT-PCR detection method of synchronous detection 3 kinds of pineapples wilting correlated virus and pineapple bacilliform DNA virus, It is characterized in that, comprising the following steps:

1) pineapple sample total serum IgE to be measured is extracted, and prepares the first chain cDNA by template reverse transcription of RNA；

2) it using the cDNA obtained in step 1) as template, is combined using primer described in claim 1, carries out specific PCR amplification Reaction；

3) pcr amplification product is detected with agarose gel electrophoresis.

3. multiple RT-PCR detection method according to claim 2, which is characterized in that step 1) specifically: extract to be measured 5.0 μ L, 10mM random primer of pineapple sample total serum IgE template is added into the reaction tube of RNase-free for pineapple sample total serum IgE 10.0 μ L, M-MuLV reverse transcriptase of 1.0 μ L, 10mM dNTPs 1.0 μ L, 2 × RT of buffer Buffer, 1.0 μ L, RNA enzyme suppression 0.5 μ L and RNase-free Water of preparation, 1.0 μ L obtains the first chain cDNA in 42 DEG C of incubation 1h.

4. multiple RT-PCR detection method according to claim 2, which is characterized in that carry out multiple RT-PCR in step 2) Expand the 25 μ L PCR reaction systems used are as follows:

2.5 μ L, 2.5mM of first chain cDNA solution, 1 0.25 μ L, 10 × PCR buffer of μ L, 5U/ μ L Taq enzyme obtained by step 1) 2.5 upstream and downstream μ L, 10 μm/μ L each 0.5 μ L and ddH of primer of dNTP mixed liquor₂O 14.75μL。

5. multiple RT-PCR detection method according to claim 4, which is characterized in that PCR response procedures are as follows: 94 DEG C of pre- changes Property 5min；94 DEG C of denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C of extension 1min are recycled 35 times；72 DEG C of extension 10min are saved.

6. multiple RT-PCR detection method according to claim 2, which is characterized in that the step 3) electrophoresis specifically: 5 μ L pcr amplification products are taken to carry out 1.5% agarose gel electrophoresis 30min.

7. multiple RT-PCR detection method according to claim 2, which is characterized in that pineapple sample to be measured is from pineapple Blade, flower, fruit, bud or stem.

8. multiple RT-PCR detection method according to claim 7, which is characterized in that pineapple sample to be measured is from pineapple Blade, bud or the aerial stem of plant.