CN104357580A - Multiplex RT-PCR (reverse transcription-polymerase chain reaction) method for detecting various viruses of cucurbit plant with two-step method as well as special primer group for method - Google Patents
Multiplex RT-PCR (reverse transcription-polymerase chain reaction) method for detecting various viruses of cucurbit plant with two-step method as well as special primer group for method Download PDFInfo
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Abstract
The invention relates to a multiplex RT-PCR (reverse transcription-polymerase chain reaction) method for detecting six viruses of cucurbit plant with a two-step method as well as a special primer group for the method, and belongs to the field of plant protection. The method comprises steps as follows: the first step, four main viruses including WMV (watermelon mosaic virus), ZYMV (zucchini yellow mosaic virus), CMV (cucumber mosaic virus) and CGMMV (cucumber green mottle mosaic virus) of the cucurbit plant are detected in a PCR; and the second step, two secondary viruses including TMV (tobacco mosaic virus) and PRSV (papaya ringspot virus) are detected in a PCR again. Specially, firstly, specific primers suitable for two-step multiplex PCR are manually screened and synthesized; separately infected disease samples of the six viruses are obtained through analysis, and total RNA (ribonucleic acid) of the plant is extracted; and random primers are subjected reverse transcription, reaction systems and condition of quadruple and double PCRs are integrated respectively, and the multiplex RT-PCR method capable of detecting the six viruses of the cucurbit plant through two-step reaction is established. The method has the advantages of simplicity and convenience in operation, time saving, labor saving, cost saving, reliable result and the like, and has wide application prospect in monitoring and controlling of the virus diseases of the cucurbit plant.
Description
Technical field
The invention belongs to plant virology technical field, refer more particularly to multiplex RT-PCR method and primer special group thereof that a kind of two-step approach detects the multiple virus of ground family crop.
Background technology,
Ground family crop is a kind of important worldwide cash crop, its importance is only second to Gramineae, pulse family and solanaceous crops, about 118 belong to 825 kinds, are mainly distributed in Perenniporia martius, as raise crop to mainly contain watermelon, muskmelon, cucumber, pumpkin, sponge gourd, wax gourd, edible gourd etc. melon.There are 32 genus 154 kinds and 35 mutation in China, mainly be distributed in southern areas, special in economically developed region of Southeast, melon cultivation area is in Fast growth phase, compared with field crop, melon crop type is many, kind is mixed, single variety area is little, the occurrence and harm degree of its disease also heals becomes serious and extensive.The growth having a strong impact on crop of disease and quality, make its output impaired or lose edibleness.Wherein the generation of virus disease is the most serious, viral species is numerous and diverse, strain is numerous, it is general that different virus encroaches on the Combined Infection phenomenon caused simultaneously, it is large to there is change between year in disease, all bring great difficulty to prevention and control and detection, virus disease has become one of main restrictive factor of China's Vegetable produce.
Jiangsu is big agricultural province, and since agricultural structure adjustment development high-efficiency agriculture, rapidly, current multiple cropping area has reached more than 2,000 ten thousand mu (containing western muskmelons) development of growing vegetables area, and in production, virus disease is one of its Major Diseases problem.According to this demand, early stage, we carried out the virus disease tracking monitor of continuous 2 years to each cities and counties, Jiangsu ground family crop, find that the virus infecting each ground family crop mainly contains watermelon mosaic virus (Watermelon mosaic virus, WMV), little zucchini yellow mosaic virus (Zucchini yellow mosaicvirus, ZYMV), cucumber mosaic virus (Cucumber mosaic virus, and cucumber green mottle virus (Cucumber green mottle mosaic virus CMV), CGMMV), the virus detected once in a while has tobacco mosaic virus (TMV) (Tobacco mosaic virus, and prv (Papaya ringspot virus TMV), PRSV), and the phenomenon of multiplicity of infection often can be there is in field.For the pathogenetic feature of our province ground family crop virus, development of new easy, quick, stable, practical and simultaneously can detect the technology of multiple virus, and permanently effective monitoring is carried out to the upper virus disease occurrence dynamics of production, be vital for the prevention and control of ground family crop virus disease.
For now, owing to lacking effective treatment means, the detection of ground family crop virus disease is particularly important in Curcurbitaceae epidemic prevention and control work.Along with biological development, the detection means of a lot of high-throughput, high specificity is come out one after another, the detection of Biological Detection, Electronic Speculum, Serologic detection, RT-PCR detection and nucleic acid hybridization detection etc. are all the common methods being applied to ground family crop Viral diagnosis, wherein the Biological Detection cycle is longer, Electronic Speculum operating skill is higher and expensive, and work is all more concentrated, can not a large amount of sample of disposable process, so these two kinds of methods all also exist significant limitation in the detection of ground family crop virus disease.The feature such as RT-PCR method possesses that specificity is good, susceptibility is high, simple and quick and cost is low, is subject to widespread use in basic level quarantine.But because the cause of disease of Curcurbitaceae virus disease is numerous, infection conditions is complicated, RT-PCR for single virus detects the demand that can not meet testing far away, so in the urgent need to developing the multiplex RT-PCR method that a kind of multiple virus being applicable to different ground family crop detects simultaneously.
Compared with other detection means, multiple RT-PCR technology has many incomparable advantages, and it can amplify multiple goal gene in same PCR system simultaneously, save time, laborsaving, efficiency is high, particularly saves expensive experiment reagent and precious laboratory sample.At present, in life science every field, as medical research, genetics research, the fields such as plant biology research, multiple RT-PCR has all been used widely, and becomes an important and research means for maturation gradually.Therefore, for Jiangsu Province's ground family crop pathogenetic kind of virus and feature, we have invented the multiplex RT-PCR method that a kind of two-step approach detects the multiple virus of ground family crop, carry out permanently effective monitoring to the occurrence dynamics for ground family crop virus disease, thus take prevention and control measure targetedly.
Summary of the invention
The present invention is directed to viral pathogenetic current situation and characteristic on Jiangsu Province's ground family crop, a kind of two-step approach is provided to detect the multiplex RT-PCR method of multiple virus on ground family crop, the first step detects 4 kinds of main viruses (WMV, ZYMV, CMV, CGMMV) in ground family crop in a PCR reaction simultaneously, and second step detects 2 kinds of secondary viruses (TMV and PRSV) again in a PCR reaction simultaneously.According to 6 kinds of virus genome sequence conservative regions design and synthesis a pair Auele Specific Primer respectively, incorporate the primer concentration, the Mg that affect multiplex RT-PCR amplification
2+the optimization of the systems such as concentration, r-Taq archaeal dna polymerase concentration, dNTPs concentration, annealing temperature, establish the method that two-step approach can detect multiple virus on ground family crop, the detection for Curcurbitaceae virus disease is provided a kind of easy, quick, efficient and method of low cost by the foundation of the method.
For achieving the above object, the invention provides following technical scheme: first screen the conservative region in comparison ground family crop virus (WMV, ZYMV, CMV, CGMMV, TMV and PRSV) genome, the Auele Specific Primer of synthetic screening 4 kinds of being applicable to that two steps realize and 2 kinds of viral multiple RT-PCRs; Artificial inoculation purifying obtains the positive that above 6 kinds of virus infects separately; Integrate the concentration of reverse transcription and enzyme, damping fluid, dNTPs, primer etc. needed for PCR reaction, by the optimization of the reaction conditionss such as annealing temperature, elongating temperature, cycle number, set up the multiplex RT-PCR method detecting multiple virus on ground family crop based on two-step approach.
Concrete steps are as follows:
(1) 6 kind of virus-specific nucleotide primer Design and synthesis
Use DNAMAN5.0 software, the each strain of WMV, ZYMV, CMV, CGMMV, TMV and PRSV virus of including in Genbank, each region disconnecting thing whole genome sequence are compared, determine relative conservative region in virus sequence, in selected conservative region, use Oligo5.0 software design primer, and analyzed the specificity guaranteeing primer by BLAST.Because multiplex PCR requires that the annealing temperature of each primer pair is as far as possible close, the size of amplified fragments is as far as possible close when agarose gel electrophoresis can significantly be distinguished, therefore finally in twice PCR reaction, select 6 kinds of viral primers each a pair, spend RNase water dissolution after synthetic for subsequent use to working concentration 10 μMs.
WMV: upstream primer (WMVF) 5 '-CCAGTGGCAAAGGTGATA-3 '
Downstream primer (WMVR) 5 '-TGCTGCGTCTGAGAAATG-3 '
ZYMV: upstream primer (ZYMVF) 5 '-AGCTCCATCATAGCTGAGCAG-3 '
Downstream primer (ZYMVR) 5 '-TAGCTTGCGCTTGCAAACGAC-3 '
CMV: upstream primer (CMVF) 5 '-CACTATTCCCGACTTTGAGACCC-3 '
Downstream primer (CMVR) 5 '-CTCATAAGCGGCATACTCTAACAT-3 '
CGMMV: upstream primer (CGMMVF) 5 '-CCACGAGTTGTTTCCTAATGCTG-3 '
Downstream primer (CGMMVR) 5 '-TTTGCTAGGCGTGATCGGATTGT-3 '
TMV: upstream primer (TMVF) 5 '-TGGATCCGCCGACCCAATAGAGTTA-3 '
Downstream primer (TMVR) 5 '-GTCGAGGTCCAAACTAAACCAGAAG-3 '
PRSV: upstream primer (PRSVF) 5 '-ATGAGGCTGTGGATGCTGGTTTA-3 '
Downstream primer (PRSVR) 5 '-GATTGAGTGGCACGAGTATTAGA-3 '
(2) ground family crop infects the extraction of viral sample total serum IgE
First analyze virus infection ground family crop sample with serological diagnostic kit, find the 6 kinds of samples infected separately by WMV, ZYMV, CMV, CGMMV, TMV and PRSV.Get 4 kinds of viruses (WMV, ZYMV, CMV and CGMMV) and 2 kinds of virus (TMV and PRSV) classical symptom hybrid blade 0.1g respectively, pulverized under liquid nitrogen powdered, extract sample total serum IgE, be finally dissolved in the distilled water that 50 μ l pollute without RNA enzyme ,-70 DEG C save backup.
(3) foundation of two step method multiple RT-PCR method detection system
The primer Oligo (dT) 18 that application is purchased from Takara company carries out reverse transcription to the plant virus RNA extracted and prepares cDNA template, then in applying step (1), synthesis primer pair for subsequent use carries out 4 heavy and 2 heavy PCR reactions, by multiple gradient preliminary experiment, repeatedly grope and adjust system factor and reaction conditions, final acquisition top condition also obtains amplified production.
(4) electrophoresis detection
Agarose gel electrophoresis is carried out to amplified production, obtains virogene amplified production band, judge the viral species infecting ground family crop.
Also can comprise the steps: in aforesaid method
(5) interpretation of result
The target gene band rubber tapping of amplification is reclaimed, be connected with pMD18-T carrier again, transformation of E. coli competent cell, alkaline lysis method of extracting plasmid also checks order, result compares with the size of design virus amplification gene, analyze the homology of gained sequence and reference sequences, thus judge the reliability of multiplex PCR result.
In above-mentioned steps, in step (2), use WMV, ZYMV, CMV, CGMMV, TMV and PRSV diagnostic kit PathoScreen of Agdia company
rwMV, PathoScreen
rzYMV, PathoScreen
rcMV, PathoScreen
rcGMMV, PathoScreen
rtMV and PathoScreen
rpRSV detects the ground family crop sample analyzed and gather, the sample finding 6 kinds of viruses to infect separately by serological method; Application Trizol test kit extracts the total serum IgE of these samples.
In above-mentioned steps, in step (3), 4 pairs of primer concentration ratios of the-step, 4 heavy PCR are preferably WMV primer: ZYMV primer: CMV primer: CGMMV primer=3: 2: 3: 2; 2 pairs of primer concentration ratios of the heavy PCR of second step 2 are preferably TMV primer: PRSV primer=1: 1.
In above-mentioned steps, in step (3) two step multi-PRC reaction systems all preferably r-Taq archaeal dna polymerase concentration be 0.10U/ μ L, Mg
2+concentration is 3.0mmol/L, dNTPs concentration is 0.25mmol/L.
In above-mentioned steps, in step (3) in two step multi-PRC reaction conditions preferably in the first step 4 heavy PCR reaction amplification condition be annealing temperature 53 DEG C, extend time 80s, cycle index 35 times; In the heavy PCR reaction of second step 2, amplification condition is annealing temperature 55 DEG C, extends time 60s, cycle index 30 times.
The invention provides multiplex RT-PCR method and primer special group thereof that a kind of two-step approach detects the multiple virus of ground family crop, the feature of multiple viral Combined Infection is subject to according to producing above ground family crop, optionally carried out two step multiple RT-PCR Screening and Identification to these viruses, the present invention has than single RT-PCR and saves time, reduces costs, the advantage such as to raise the efficiency; Report as compared with the past 5 is heavy and above RT-PCR method interfering factors in actual application is many, sensitivity and the defect such as recall rate is low, and present method more can influence factor in decreasing pollution and reaction, and raising detection sensitivity, reduces undetected probability greatly.In the production of ground family crop, realize the early diagnosis to each viroid, realize in laboratory to virus disease kind quick, efficiently, low-costly and in high volume detect, to controlling the generation of disease in field, reduce financial loss, the fashion forecasting forecast tool of disease is of great significance.
Accompanying drawing explanation
Fig. 1 is 6 kinds of viral substances, 4 heavy and 2 heavy PCR detected results: M.100bp DNA Ladder; 1 is healthy summer squash (CK-); 2-5 is followed successively by the sample that CMV, WMV, CGMMV and ZYMV infect separately; 6 is the sample that CMV, WMV, CGMMV and ZYMV mix; 7-8 is followed successively by the sample that TMV and PRSV infects separately; 9 is the sample that TMV and PRSV mixes.
Fig. 2 is the result that the heavy PCR of the first step 4 detects field sample: M.Marker3; 1 is healthy summer squash (CK-); 2-16 is the pumpkin sample of the watermelon of Dongtai, the watermelon in Nanjing, the summer squash of Hongze, the summer squash of Jiangyan City, the sponge gourd in Gaoyou, the pumpkin in Lianyun Harbour, the pumpkin in Xuzhou, the muskmelon of Jurong, the bottle gourd in Tongzhou, the summer squash of Suqian, the sponge gourd in Suzhou, the muskmelon of Jintan, the cucumber of East Platform, the watermelon of Taixing and Jiangyan City.
Fig. 3 is the result that the heavy PCR of second step 2 detects field sample: M.Marker3; 1 is healthy summer squash (CK-); 2-16 is with the sample of Fig. 2.
Specific implementation method
The present invention is described further with the following Examples, but does not limit the present invention.
Design of primers and preparation
Use DNAMAN 5.0 software, the each strain of WMV, ZYMV, CMV, CGMMV, TMV and PRSV virus of including in Genbank, each region disconnecting thing whole genome sequence are compared, determine relative conservative region in virus sequence, in selected conservative region, use Oligo5.0 software design primer, and analyzed the specificity guaranteeing primer by BLAST.Because multiplex PCR requires that the annealing temperature of each primer pair is as far as possible close, the size of amplified fragments is as far as possible close when agarose gel electrophoresis can significantly be distinguished, therefore finally in twice PCR reaction, select 6 kinds of viral primers each a pair, spend RNase water dissolution after synthetic for subsequent use to working concentration 10 μMs.The information of often kind of primer is as described in Table 1:
The primer information (SEQ ID NO.1-12) of table 1 liang step multiplex RT-PCR amplification virus
Above-mentioned primer is synthesized by the handsome company limited in Shanghai.
Experiment material used in the embodiment of the present invention is as follows:
The isolate of WMV, ZYMV, CMV, CGMMV, TMV and PRSV6 kind virus in 2012 from Nanjing, salt all, Xuzhou, Hongze, the ground such as Huaian morbidity muskmelon, summer squash, watermelon, pumpkin etc. gather, by the sample that 6 kinds of viral serological diagnostic kits find 6 kinds of viruses to infect separately, and on summer squash inoculation reproduction, sample retention is in-70 DEG C of refrigerators;
Total RNA extraction reagent Trizol is purchased from Invitrogen company, r-Taq archaeal dna polymerase, dNTP, BamH I, Sal I are purchased from the precious biotech firm in Dalian, ThermoScript II M-MLV, RNA enzyme inhibitors are purchased from Promega company, sepharose reclaims test kit, intestinal bacteria (E.coli DH5) competent cell, pMD18-T carrier purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, and serological diagnostic kit is purchased from Agdia company.
Embodiment 1, substance RT-PCR
Virus total RNA is extracted
Take each 0.1g of the sick leaf of sample infecting WMV, ZYMV, CMV, CGMMV, TMV and PRSV6 kind virus respectively, fully grind with liquid nitrogen under condition of ice bath, be transferred to rapidly in the 2.0mLEP pipe without RNase, often pipe adds 1mL Trizol Reagent, abundant vibration 1min, room temperature leaves standstill 5min.Add 0.2mL chloroform to turn upside down mixing, room temperature leaves standstill 3min, 4 DEG C, the centrifugal 15min of 12000 × g.Get upper phase to add in another new 1.5mL EP pipe, add 0.5mL Virahol, vibration mixing ,-20 DEG C of standing 30min, 4 DEG C, the centrifugal 10min of 12000 × g, abandons supernatant.With ethanol (preparation of the 0.1%DEPC water) washing precipitation twice of 1mL75%, each 4 DEG C, the centrifugal 5min of 7000 × g, abandons supernatant, seasoning.Finally being dissolved in 50 μ L goes in the DEPC water of RNase, and-70 DEG C save backup.Simultaneously with the total serum IgE of healthy summer squash (being labeled as CK-) for negative control, RNA extraction method is the same.
Reverse transcription (RT) is reacted
With the RNA of 6 kinds of single viruses and CK-for template, oligodT18 primed reverse transcription obtains cDNA first chain, response procedures is with reference to M-MLV specification sheets, 15 μ L reaction systems are as follows: 3 μ L RNA, 1 μ LoligodT18 (10 μm of ol/L), 6 μ L DEPC water, 65 DEG C of sex change 5min, put rapidly 5min on ice; Add 2.5 μ L 5 × RT buffer again, 0.5 μ L dNTP (each 2.5mmol/L), 0.5 μ L RNase Inhibitor (40U/ μ L) and 0.5 μ L M-MLV ThermoScript II (200U/ μ L), of short duration centrifugal rear DEPC water complements to 15 μ L, 42 DEG C of water-bath 1h, 70 DEG C of deactivation 5min, put-20 DEG C stand-by.
Substance PCR reacts
25 μ L PCR standard reaction systems: 15.75 μ L ddH
2o, 2.5 μ L10 × PCR buffer (Mg
2+free), 1.5 μ L MgCl
2(25mmol/L), 2 μ L dNTP Mixture (each 2.5mmol/L), l μ L upstream and downstream primer mixture (each 10 μm of ol/L), 0.25 μ L r-Taq archaeal dna polymerase (5U/ μ L) and 2 μ L cDNA templates.PCR response procedures: 94 DEG C of denaturation 5min; 94 DEG C of sex change 50s, 53 DEG C of annealing 50s, 72 DEG C extend 1min, circulate 35 times; 72 DEG C extend 10min.Get 5 μ L PCR primer through 1% agarose gel electrophoresis, carry out observation analysis through gel imaging system.
Electrophoresis detection
Get 5 μ L PCR primer through 1% agarose gel electrophoresis, in 0.5 × TAE buffered environment, 120V voltage stabilizing electrophoresis 30min, again through gel imaging system observed and recorded result, obtain 6 specific bands respectively, the size of amplified band and primer expect that stripe size is basically identical, see Fig. 1 of Figure of description.
Embodiment 2, multiple RT-PCR
Virus total RNA is extracted
Take equivalent and infect 6 kinds of viral blades respectively, WMV, ZYMV, CMV and CGMMV4 kind blade fully mixes (being labeled as H4), TMV and PRSV2 kind blade fully mixes (being labeled as H2), and healthy summer squash blade is negative control (being labeled as CK-).H4, H2 and CK-respectively get 0.1g, fully grind under condition of ice bath with liquid nitrogen, and be transferred to rapidly in the 2.0mL EP pipe without RNase, often pipe adds 1mLTrizol Reagent, and fully vibrate 1min, and room temperature leaves standstill 5min.Add 0.2mL chloroform to turn upside down mixing, room temperature leaves standstill 3min, 4 DEG C, the centrifugal 15min of 12000 × g.Get upper phase to add in the 1.5mLEP pipe of another-Xin, add 0.5mL Virahol, vibration mixing ,-20 DEG C of standing 30min, 4 DEG C, the centrifugal 10min of 12000 × g, abandons supernatant.With ethanol (preparation of the 0.1%DEPC water) washing precipitation twice of 1mL75%, each 4 DEG C, the centrifugal 5min of 7000 × g, abandons supernatant, seasoning.Finally being dissolved in 50 μ L goes in the DEPC water of RNase, and-70 DEG C save backup.
Reverse transcription (RT) is reacted
With the RNA of 3 samples (H4, H2 and CK-) for template, oligodT18 primed reverse transcription obtains cDNA first chain, response procedures is with reference to M-MLV specification sheets, 15 μ L reaction systems are as follows: 3 μ LRNA, 1 μ L oligodT18 (10 μm of ol/L), 6 μ L DEPC water, 65 DEG C of sex change 5min, put rapidly 5min on ice; Add 2.5 μ L 5 × RT buffer again; 0.5 μ L dNTP (each 2.5mmol/L), 0.5 μ LRNase Inhibitor (40U/ μ L) and 0.5 μ L M-MLV ThermoScript II (200U/ μ L), of short duration centrifugal rear DEPC water complements to 15 μ L, 42 DEG C of water-bath 1h, 70 DEG C of deactivation 5min, put-20 DEG C stand-by
Multi-PRC reaction and system optimization
4 systems that are heavy and 2 heavy PCR reactions are all adjusting on above-mentioned 25 μ L substance PCR standard reaction system bases, forward and reverse primer Mix (10 μm of ol/L each) are replaced by 4 pairs of Auele Specific Primers originally each 0.5 μ L (10 μm of ol/L each) by 4 heavy PCR, finally determine that each primer is than WMV:ZYMV:CMV:CGMMV=3: 2: 3: 2 through repeatedly adjusting, whole primer mixture is 4 μ L; Forward and reverse primer Mix (10 μm of ol/L each) are replaced by 2 pairs of each 0.5 μ L of Auele Specific Primer (10 μm of ol/Leach) by 2 heavy PCR, and determine that each primer is than TMV:PRSV=1: 1 through experiment, whole primer mixture is 2 μ L.
Then, under effective with multiple RT-PCR condition, the principal element affecting multiplex PCR is investigated, when a certain factor is investigated, ceteris paribus.In experimentation, 0.02,0.04,0.06,0.08,0.10 and 0.12U/ μ L 6 process are established respectively to r-Taq archaeal dna polymerase concentration; Mg
2+concentration selects 1.0,2.0,3.0,4.0,5.0 and 6.0mmol/L6 process respectively; DNTPs concentration establishes 0.10,0.25,0.40,0.55,0.70 and 0.85mmol/L6 process respectively; Annealing temperature selects 48 DEG C, 51 DEG C, 53 DEG C, 55 DEG C, 58 DEG C and 60 DEG C respectively; The extension time is respectively 50,60,70,80,90 and 100s; Cycle index selects 20,25,30,35,40 and 45 circulations to carry out the optimization of multiplex PCR system.Finally obtain the optimum reaction condition of two step multiple PCR methods: in 4 heavy and 2 heavy PCR reaction systems, all preferably r-TaqDNA polymerase concentration is 0.10U/ μ L, Mg
2+concentration is 3.0mmol/L, dNTPs concentration is 0.25mmol/L; In the heavy PCR reaction of the first step 4, amplification condition is annealing temperature 53 DEG C, extends time 80s, cycle index 35 times; In the heavy PCR reaction of second step 2, amplification condition is annealing temperature 55 DEG C, extends time 60s, cycle index 30 times.
Electrophoresis detection
Get 5 μ LPCR products through 1% agarose gel electrophoresis, in 0.5 × TAE buffered environment, 120V voltage stabilizing electrophoresis 30min, again through gel imaging system observed and recorded result, H4 and H2 is obtained for comparatively clear and special band, the size of amplified band and primer expect that stripe size is basically identical, see Fig. 1 of Figure of description.
The cloning and sequencing of PCR primer and sequential analysis
6 DNA target fragments of two step multiplex RT-PCR amplification adopt Axygen gel extraction kit (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) to reclaim, and reclaim DNA and are dissolved in the lysate of 30 μ L.Reclaim product to be connected with pMD18-T carrier (see pMD18-T test kit specification sheets), connect product conversion bacillus coli DH 5 alpha competent cell, alkaline lysis method of extracting plasmid, cuts evaluation and screening recombinant plasmid through PCR and enzyme.Each isolate is selected 2 positive colonies and is checked order, and checks order to be completed by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Sequencing result shows, the amplified production sequence of WMV, ZYMV, CMV, CGMMV, TMV and PRSV is made up of 485,1100,389,660,410 and 325 Nucleotide respectively, identical with the PCR primer size of design; Sequence homology rate analytical results shows, in gained sequence and Genbank, the homology of reference sequences reaches 98.25%, 99.01%, 99.93%, 98.22%, 98.30% and 99.12% respectively, demonstrates the reliability of multiple RT-PCR detected result.
Embodiment 3, utilizes two step multiplex RT-PCR methods to detect field sample
Virus total RNA is extracted
Respectively to picking up from the watermelon of Dongtai, the watermelon in Nanjing, the summer squash of Hongze, the summer squash of Jiangyan City, the sponge gourd in Gaoyou, the pumpkin in Lianyun Harbour, the pumpkin in Xuzhou, the muskmelon of Jurong, the bottle gourd in Tongzhou, the summer squash of Suqian, the sponge gourd in Suzhou, the muskmelon of Jintan, the cucumber of East Platform, the watermelon of Taixing, the pumpkin (being labeled as 2-16) of Jiangyan City, healthy summer squash blade is negative control (being labeled as 1).Sample respectively gets 0.1g, fully grinds under condition of ice bath with liquid nitrogen, and be transferred to rapidly in the 2.0mL EP pipe without RNase, often pipe adds 1mLTrizol Reagent, and fully vibrate 1min, and room temperature leaves standstill 5min.Add 0.2mL chloroform to turn upside down mixing, room temperature leaves standstill 3min, 4 DEG C, the centrifugal 15min of 12000 × g.Get upper phase to add in another new 1.5mLEP pipe, add 0.5mL Virahol, vibration mixing ,-20 DEG C of standing 30min, 4 DEG C, the centrifugal 10min of 12000 × g, abandons supernatant.With ethanol (preparation of the 0.1%DEPC water) washing precipitation twice of 1mL 75%, each 4 DEG C, the centrifugal 5min of 7000 × g, abandons supernatant, seasoning.Finally being dissolved in 50 μ L goes in the DEPC water of RNase, and-70 DEG C save backup.
Reverse transcription (RT) is reacted
With the RNA of 16 samples for template, oligodT18 primed reverse transcription obtains cDNA first chain, response procedures is with reference to M-MLV specification sheets, 15 μ L reaction systems are as follows: 3 μ L RNA, 1 μ LoligodT18 (10 μm of ol/L), 6 μ L DEPC water, 65 DEG C of sex change 5min, put rapidly 5min on ice; Add 2.5 μ L5 × RT buffer again, 0.5 μ L dNTP (each 2.5mmol/L), 0.5 μ L RNase Inhibitor (40U/ μ L) and 0.5 μ L M-MLV ThermoScript II (200U/ μ L), of short duration centrifugal rear DEPC water complements to 15 μ L, 42 DEG C of water-bath 1h, 70 DEG C of deactivation 5min, put-20 DEG C stand-by
Multi-PRC reaction
The reaction system first determined with 4 heavy PCR and condition carry out PCR, 25 μ L PCR standard reaction systems: 10.5 μ L ddH
2o, 2.5 μ L10 × PCR buffer (Mg
2+free), 3 μ L MgCl
2(25mmol/L), 2.5 μ L dNTP Mixture (each 2.5mmol/L), 4 pairs of primer mixtures are each 0.6 μ L of 4 μ L:WMV and CMV upstream and downstream primer, the each 0.4 μ L of ZYMV and CGMMV upstream and downstream primer (each 10 μm of ol/L), 0.5 μ L r-Taq archaeal dna polymerase (5U/ μ L) and 2 μ L cDNA templates.PCR response procedures: 94 DEG C of denaturation 5min; 94 DEG C of sex change 50s, 53 DEG C of annealing 50s, 72 DEG C extend 80s, circulate 35 times; 72 DEG C extend 10min.The reaction system determined with 2 heavy PCR again and condition carry out PCR, 25 μ L PCR standard reaction systems: 12.5 μ L ddH
2o, 2.5 μ L10 × PCR buffer (Mg
2+free), 3 μ L MgCl
2(25mmol/L), 2.5 μ L dNTP Mixture (each 2.5mmol/L), 2 pairs of primer mixtures are each 0.5 μ L of 2 μ L:TMV and PRSV upstream and downstream primer (each 10 μm of ol/L), 0.5 μ L r-Taq archaeal dna polymerase (5U/ μ L) and 2 μ L cDNA templates.PCR response procedures: 94 DEG C of denaturation 5min; 94 DEG C of sex change 50s, 55 DEG C of annealing 50s, 72 DEG C extend 60s, circulate 30 times; 72 DEG C extend 10min.Get 5 μ L PCR primer respectively through 1% agarose gel electrophoresis, carry out observation analysis through gel imaging system.
Interpretation of result
Get 4 heavy and 2 heavy PCR primer 5 μ L respectively through 1% agarose gel electrophoresis, in 0.5 × TAE buffered environment, 120V voltage stabilizing electrophoresis 30min, again through gel imaging system observed and recorded result, result all detects comparatively clear and special band, the size of amplified band and virogene to be checked expect that stripe size is basically identical, the viral Combined Infection situation wherein picked up from as seen on each cities and counties, Jiangsu ground family crop is comparatively general, viral species is based on WMV, ZYMV, CMV and CGMMV, and minority infects by TMV and PRSV by crop.See Fig. 2 and Fig. 3 of Figure of description.
Method of the present invention is described by specific embodiment, serves primarily in the work such as early diagnosis and monitoring of field ground family crop virus disease.Those skilled in the art can use for reference the links such as content appropriate change raw material of the present invention, processing condition and realize other object corresponding, relevant change does not all depart from content of the present invention, all similar replacements and change are all apparent to those skilled in the art, are all deemed to be included within scope of the present invention.Above-mentioned enforcement does not limit the present invention in any form.
Claims (4)
1. two-step approach to detect on ground family crop 6 kinds of viral multiplex RT-PCR methods and primer special thereof, it is characterized in that: the first step detects 4 kinds of main viruses (WMV, ZYMV, CMV, CGMMV) in ground family crop in a PCR reaction simultaneously, second step detects 2 kinds of secondary viruses (TMV and PRSV) again in a PCR reaction simultaneously, comprises the steps:
(1) screen WMV, ZYMV, CMV, CGMMV, TMV of including in comparison Genbank and determine relative conservative region with each strain of PRSV virus, each region disconnecting thing whole genome sequence, Oligo5.0 software design primer is used in selected conservative region, and the specificity guaranteeing primer is analyzed by BLAST, the primer 6 that screening synthesis is applicable to two step multiple RT-PCR reactions is right, and described primer sequence is as follows:
WMV: upstream primer (WMVF) 5 '-CCAGTGGCAAAGGTGATA-3 '
Downstream primer (WMVR) 5 '-TGCTGCGTCTGAGAAATG-3 '
ZYMV: upstream primer (ZYMVF) 5 '-AGCTCCATCATAGCTGAGCAG-3 '
Downstream primer (ZYMVR) 5 '-TAGCTTGCGCTTGCAAACGAC-3 '
CMV: upstream primer (CMVF) 5 '-CACTATTCCCGACTTTGAGACCC-3 '
Downstream primer (CMVR) 5 '-CTCATAAGCGGCATACTCTAACAT-3 '
CGMMV: upstream primer (CGMMVF) 5 '-CCACGAGTTGTTTCCTAATGCTG-3 '
Downstream primer (CGMMVR) 5 '-TTTGCTAGGCGTGATCGGATTGT-3 '
TMV: upstream primer (TMVF) 5 '-TGGATCCGCCGACCCAATAGAGTTA-3 '
Downstream primer (TMVR) 5 '-GTCGAGGTCCAAACTAAACCAGAAG-3 '
PRSV: upstream primer (PRSVF) 5 '-ATGAGGCTGTGGATGCTGGTTTA-3 '
Downstream primer (PRSVR) 5 '-GATTGAGTGGCACGAGTATTAGA-3 '
(2) extract the total serum IgE of the ground family crop blade infecting 1-6 kind virus, then carry out reverse transcription, described reverse transcription reaction system is as follows: 3 μ L RNA, 1 μ L oligodT18 (10 μm of ol/L), 6 μ L DEPC water, 65 DEG C of sex change 5min, put rapidly 5min on ice; Add 2.5 μ L5 × RT buffer again, 0.5 μ L dNTP (each 2.5mmol/L), 0.5 μ L RNase Inhibitor (40U/ μ L) and 0.5 μ L M-MLV ThermoScript II (200U/ μ L), of short duration centrifugal rear DEPC water complements to 15 μ L, 42 DEG C of water-bath 1h, 70 DEG C of deactivation 5min, put-20 DEG C stand-by;
(3) in applying step (1), the primer pair reverse transcription product of synthesis carries out 4 heavy and 2 heavy PCR reactions respectively;
(4) agarose gel electrophoresis is carried out to amplified production, obtain virogene amplified production band, judge the viral species infecting ground family crop.
2. method according to claim 1, the method also comprises the steps:
(5) interpretation of result
The target gene band rubber tapping of amplification is reclaimed, be connected with pMD18-T carrier again, transformation of E. coli competent cell, alkaline lysis method of extracting plasmid also checks order, result compares with the size of design virus amplification gene, analyze the homology of gained sequence and reference sequences, thus judge the reliability of multiple RT-PCR result.
3. 4 heavy and 2 heavy PCR method according to claim 1, it is characterized in that, described PCR reaction system is 25 μ L, comprises 2.5 μ L 10 × PCR buffer (Mg
2+free), 3 μ L MgCl
2(25mmol/L), 2.5 μ L dNTP Mixture (each 2.5mmol/L), 0.5 μ L r-Taq archaeal dna polymerase (5U/ μ L), 2 μ L cDNA templates, 4 heavy PCR primer mixtures are each 0.6 μ L of 4 μ L:WMV and CMV upstream and downstream primer, each 0.4 μ L of ZYMV and CGMMV upstream and downstream primer (each 10 μm of ol/L); 2 heavy PCR primer mixtures are each 0.5 μ L of 2 μ L:TMV and PRSV upstream and downstream primer (each 10 μm of ol/L), finally use ddH
2o supplies 25 μ L.
4. 4 heavy and 2 heavy PCR method according to claim 1, is characterized in that, described PCR response procedures: 4 heavy PCR are 94 DEG C of denaturation 5min; 94 DEG C of sex change 50s, 53 DEG C of annealing 50s, 72 DEG C extend 80s, circulate 35 times; 72 DEG C extend 10min; 2 heavy PCR are 94 DEG C of denaturation 5min; 94 DEG C of sex change 50s, 55 DEG C of annealing 50s, 72 DEG C extend 60s, circulate 30 times; 72 DEG C extend 10min.
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