CN111455109A - Method for synchronously detecting ZYMV, PRSV and ZTMV - Google Patents
Method for synchronously detecting ZYMV, PRSV and ZTMV Download PDFInfo
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Abstract
The invention relates to a method for synchronously detecting ZYMV, PRSV and ZTMV, belonging to the field of plant protection, which adopts a CTAB method to extract total nucleic acid of a disease sample as a template and utilizes a one-step method multiple RT-PCR technology to realize simultaneous detection of ZYMV, PRSV and ZTMV 3 viruses by designing specific detection primers of ZYMV, PRSV and ZTMV 3 viruses. Through optimization of a reaction system and reaction conditions, the method can efficiently and accurately realize specificity detection of ZYMV, PRSV and ZTMV 3 viruses, has higher efficiency compared with the traditional RT-PCR detection process in which only a single virus can be detected, reduces the detection cost, can also ensure the accuracy of the detection result, and has wide application prospect in detection of the 3 viruses.
Description
Technical Field
The invention belongs to the field of plant protection, and particularly relates to a method for synchronously detecting ZYMV, PRSV and ZTMV.
Background
The cucurbitaceae Zymvirus detection method is characterized in that 26 cucurbitaceae crops are distributed in the north and south of China, wherein cucurbitaceae crops such as cucumber (Cucumis), pumpkin (Cucurbita), cucurbitaceae (L agenaria) and the like are cultivated widely after human domestication, the cucurbitaceae crops are wide in application range and can be eaten by human beings, appreciated, medicinal and the like, with the continuous expansion of vegetable planting areas in China and the continuous development of facility agriculture, the planting areas of cucurbitaceae are enlarged, the occurrence and damage of diseases are more serious, the virus diseases are usually the diseases which cause the most serious loss and are most difficult to control of cucurbitaceae crops, at least 59 viruses which can infect cucurbitaceae crops are reported at present, the cucurbitaceae mosaic virus (zucchini yellow mosaic virus, ZYMV), cucumber mosaic virus (cucumber mosaic virus, CMV), watermelon mosaic virus (WMV, PRymvirus, MRSV, TMSV and TMSV are distributed in the field and the important field virus detection methods for detecting the cucurbitaceae crops such as the cucurbitaceae Zymvirus, the TMSV, the cucurbitaceae virus, the TMSV, the TMmosaic virus, the TMSV, the TMV.
The detection and identification technologies of plant viruses are more, and mainly comprise conventional biological detection, serological detection, electron microscope detection technology and molecular biological detection technology. When the ZTMV infects pumpkin, pumpkin and cucumber, yellow-green alternate stripes appear on the leaves; the symptoms of PRSV infection of melon vegetable crops are characterized by severe leaf blister spots, shrinkage and deformity; ZYMV-infected melon vegetable crops mainly show slight shrinkage, yellowing and flowering and leafing of leaves. According to the difference of the symptom expression, the cucurbitaceae vegetable samples caused by 3 viruses can be initially classified in field investigation, but the identification of the final virus type needs further indoor identification for verification under the influence of factors such as environment, variety and the like.
When the electron microscope technology is used for identifying plant viruses, virus types are mainly identified according to the shapes of virus granules, and ZYMV, PRSV and ZTMV belong to potyviruses, and the shapes and sizes of the granules are similar, so that accurate identification cannot be carried out according to shape observation results, and the technology has high requirements on equipment and high cost. The serological detection has high accuracy compared with the biological and electron microscope technology, but serological cross reaction is easy to occur for viruses with close relativity, and the detection result of the viruses is influenced.
The RT-PCR technology has high detection sensitivity, short detection time and high accuracy, and is widely applied to the detection of plant viruses at present, but the conventional two-step RT-PCR reaction can only detect one virus at a time, multiple PCR reactions are often required when plants with various viruses are detected, the experiment operation is complex, and the time consumption is long. The multiple RT-PCR can realize the simultaneous detection of various viruses, reduce the experiment cost and save the time. However, when multiple RT-PCR techniques are used to detect multiple viruses simultaneously, multiple pairs of primers need to be amplified in the same reaction system, and the amplification effect is affected by many factors that affect the amplification effect during the reaction process, and primer dimers and hairpin structures, mismatches between primers, competitive amplification between primers, etc. may occur during the reaction. In addition, the concentration of each reaction component in the reaction system, the annealing temperature of the reaction, the number of cycles, and the like all have an influence on the amplification effect. In the multiplex RT-PCR technology, the design of primers is key, and particularly for viruses with relatively close relativity, the problem of competitive amplification among the primers needs to be overcome.
Disclosure of Invention
In order to overcome the problems in the background technology, the invention establishes a method for synchronously detecting ZYMV, PRSV and ZTMV 3 viruses, and ensures that the virus detection is more economical, efficient and rapid on the premise of ensuring the accuracy and sensitivity of pathogen identification.
In order to realize the purpose, the invention is realized by the following technical scheme:
the method for synchronously detecting ZYMV, PRSV and ZTMV comprises the following steps:
1) designing and synthesizing a primer:
designing and synthesizing specific detection primers for detecting ZYMV, PRSV and ZTMV, wherein the primer sequences are as follows:
ZYMV upstream primer (ZYMVdF)5 '-3': GAACATGTCGAGTTGGAGAG
Downstream primer (ZYMVdR)5 '-3': CGCTTGCTCTGTGTTTCAAC
PRSV upstream primer (PRSVdF)5 '-3': CTAGCACAAAAACTGGAGAGAG
Downstream primer (PRSVdR)5 '-3': CTCTCATTCYAAGAGGCTC
Forward primer (ZTMVdF)5 '-3': GCATTAAGTTTACGACAGCGC
Downstream primer (ZTMPdR) 5 '-3': CAATCACTCTTGTCRGTGTC
(2) Extracting total nucleic acid of infected plant tissues by a CTAB method to be used as an RT-PCR amplification template;
(3) one-step multiplex RT-PCR amplification;
4) and detecting the amplification result by agarose gel electrophoresis.
Further preferably, in step (1), the amplified fragment size of ZYMV detection primer pair ZYMVdF/ZYMVdR is 755bp, the amplified fragment size of PRSV detection primer pair PRSVdF/PRSVdR is 939bp, and the amplified fragment size of ZTMV detection primer pair ZTMD F/ZTMD is 631 bp.
Preferably, the CTAB method for extracting total nucleic acid from the infected plant tissue comprises the steps of adding 1 large and 5 small sterilized steel balls into a 2M L sterilized centrifuge tube, weighing 0.1g of the infected leaves into the centrifuge tube, adding 1.2M L CTAB buffer (2% CTAB, 2% PVP-40, 100mM Tris-HCl, pH 8.0, 1.4M NaCl, 20mM EDTA, 0.2% mercaptoethanol), precooling in a 80 ℃ refrigerator for 3-5 min, grinding for 2 times in a high-throughput tissue grinder (60 Hz and 90 s), bathing at 65 ℃ for 45-60 min, centrifuging at 13000r/min for 10min, taking 650 mu L supernatant into a new centrifuge tube of 1.5M L, adding equal volume of chloroform/isoamyl alcohol (24:1), vortexing and mixing for 30s, centrifuging at 13000r/min for 10min, carefully transferring 500 mu L supernatant into a new centrifuge tube of 1.5M 6778, adding 350 mu isopropanol, fully preserving at equal volume, placing in a vortex for 30s, centrifuging at 3500 r/min, sucking off the supernatant, centrifuging at room temperature for 10min, adding 3500 mu 35 mu ethanol, and pouring off the supernatant into a pipette, and precipitating at room temperature, and repeatedly, adding 3500 h, precipitating at room temperature, centrifuging for 3500 h, and dissolving the supernatant of 3500 mu 30-30 min, and precipitating, adding 3515 mu 30min, and centrifuging at room temperature, and centrifuging at 3515 mu of a small pipette, and adding 30min, and precipitating, and centrifuging at room temperature.
Further preferably, the reaction system of the one-step multiplex RT-PCR is 7.5 mu L2 × 1step Buffer, 4.5 mu L RNase Free ddH2O, 0.6 mu L
10. mu.M upstream and downstream primers for 1Step Enzyme Mix, ZYMV and PRSV, 0.25. mu. L each, 10. mu.M upstream and downstream primers, 0.4. mu. L template, 0.6. mu. L, and 15. mu. L total.
Further preferably, the one-step multiplex RT-PCR reaction conditions are: 30min at 50 ℃; pre-denaturation at 94 ℃ for 2min, at 94 ℃ for 30sec, at 56 ℃ for 30sec, and at 72 ℃ for 1min, and continuously performing 35 cycles; keeping at 72 deg.C for 10min, and cooling at 4 deg.C.
The invention has the beneficial effects that:
1. the specific detection primers for the 3 viruses designed by the invention can singly or compositely detect the 3 potyviruses ZYMV, PRSV and ZTMV infecting plants, have good detection effect and detection specificity, avoid competitive amplification among the primers, and have single amplified target band and high identification degree.
2. The invention optimizes the concentration of 3 pairs of detection primers and the reaction annealing temperature in the system on the basis of establishing a preliminary detection system, finally determines the concentration of the detection primers of the one-step method multiple detection system to be that the concentration of the upstream primers and the downstream primers of ZYMV and PRSV 10 MuM are respectively 0.25 Mu L and the concentration of the upstream primers and the downstream primers of ZYMV and PRSV 10 MuM are respectively 0.4 Mu L templates 0.6 Mu L, the optimal annealing temperature is 56 ℃, and the lowest nucleic acid detection concentration of the established one-step method multiple RT-PCR detection technology for detecting ZYMV, PRSV and ZTMV 3 viruses is 10-5A nucleic acid stock solution.
3. The invention can detect ZYMV, PRSV and ZTMV 3 viruses at the same time, can detect the above 3 viruses efficiently, quickly and accurately, and the one-step method multiple RT-PCR technology does not need to carry out reaction again when detecting one virus like the conventional RT-PCR technology, and realizes synchronous detection of various viruses only by carrying out reaction once, thereby greatly reducing the repeated freezing and melting times and using times of sample nucleic acid and reagent, and avoiding the degradation and pollution of nucleic acid samples; meanwhile, the use of experimental reagents is reduced, and the detection cost is saved.
Drawings
FIG. 1 shows the detection effect of a preliminarily established one-step-method multiple detection system; m: DNA molecular weight standard; 1-7: melon crop virus disease samples;
FIG. 2 is a diagram showing the detection effect of the optimized one-step multiple detection system; m: DNA molecular weight standard; 1: infecting a PRSV + ZTMV + ZYMV virus sample;
FIG. 3 shows the results of one-step multiplex RT-PCR detection of ZYMV, PRSV and ZTMV specificity; m: DNA molecular weight standard; 1: PRSV + ZTMV + ZYMV; 2: ZTMV; 3: ZYMV; 4: PRSV; 5: PVY; 6: TVDV; 7: healthy pumpkin seedlings.
FIG. 4 shows the results of one-step multiplex RT-PCR detection of PRSV, ZTMV, and ZYMV in field melon crop samples; wherein, M: DNA molecular weight standard; NC: negative control; 1-23: melon crop field samples.
FIG. 5 shows the sensitivity of one-step multiplex RT-PCR; wherein, M: DNA molecular weight standard; 1-6: in turn is diluted by 10-0、10-1、10-2、10-3、10-4、10-5The diluted nucleic acid solution of (1).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, preferred embodiments of the present invention will be described in detail below to facilitate understanding of the skilled person.
Example one-step method establishment and optimization of multiplex RT-PCR detection System
1. Experimental Material
Field samples infected with ZYMV, PRSV and ZTMV alone, and samples infected with ZYMV, PRSV and ZTMV 3 viruses in combination.
2. Designing and synthesizing a primer:
(a) designing and synthesizing specific detection primers for detecting ZYMV, PRSV and ZTMV, wherein the primer sequences are as follows:
ZYMV upstream primer (ZYMVdF)5 '-3': GAACATGTCGAGTTGGAGAG
Downstream primer (ZYMVdR)5 '-3': CGCTTGCTCTGTGTTTCAAC
PRSV upstream primer (PRSVdF)5 '-3': CTAGCACAAAAACTGGAGAGAG
Downstream primer (PRSVdR)5 '-3': CTCTCATTCYAAGAGGCTC
Forward primer (ZTMVdF)5 '-3': GCATTAAGTTTACGACAGCGC
Downstream primer (ZTMPdR) 5 '-3': CAATCACTCTTGTCRGTGTC
(b) Each pair of primer amplified fragments and the virus to be detected are as follows:
3. the CTAB method is used for extracting total nucleic acid of infected plant tissues as an RT-PCR amplification template:
adding 1-size and 5-size sterilized steel balls into a sterilized centrifugal tube of 2m L,weighing about 0.1g of diseased leaves into a centrifuge tube, adding 1.2M L CTAB buffer solution (2% CTAB, 2% PVP-40, 100mM Tris-HCl, pH 8.0, 1.4M NaCl, 20mM EDTA, 0.2% mercaptoethanol), precooling in a refrigerator at 80 ℃ for 3-5 min, grinding for 2 times in a high-throughput tissue grinder 60Hz and 90s, bathing at 65 ℃ for 45-60 min, centrifuging for 10min at 13000r/min, taking 650 mu L supernatant into a new centrifuge tube of 1.5M L, adding equal volume of chloroform/isoamyl alcohol (24:1), vortex shaking and mixing for 30s, centrifuging for 13000r/min for 10min, transferring 500 mu L supernatant into a new centrifuge tube of 1.5M L, adding 350 mu isopropanol, slightly inverting to suck out, fully mixing the liquid, placing the centrifuge tube at room temperature for 10min, 13000r/min, centrifuging for 15min, centrifuging the supernatant by a micropipette, adding 500 mu L% ethanol, precipitating at 13000 r/75 min, precipitating at 38710 mu 3910 min, adding supernatant, centrifuging at room temperature, and drying by a micropipetition pump, and drying2Placing O on ice for 30min, repeatedly blowing liquid with micropipette to dissolve precipitate, and storing at-20 deg.C for use.
4. Preliminary establishment of one-step method multiple RT-PCR detection system
Identifying the field collected melon crop samples infected with PRSV, ZTMV and ZYMV by using conventional RT-PCR, and then mixing two by two and three kinds of single toxicity sources. According to
One Step RT-PCR KitVer.2 kit instruction designs One-Step multiple detection initial system of 7.5 mu L2 × 1Step Buffer, 4.5 mu L RNase Free ddH2O，0.6μL
one Step Enzyme Mix, PRSV, ZTMV, and ZYMV, upstream and downstream primers 10. mu.M each 0.3. mu. L template 0.6. mu. L initial reaction conditions 50 ℃ reverse transcription 30min, 94 ℃ 2min, then 94 ℃ 30sec, 55 ℃ 30sec, 72 ℃ 1min for 35 cycles, 72 ℃ 10min, 4 ℃ cold storage.
5. Optimization of one-step method multiple RT-PCR detection system
Adjusting the concentration of the primer: based on a preliminarily established one-step method multiple RT-PCR detection system, primer concentration combinations of 3 viruses in the system are set, and a mixed sample of the 3 viruses is amplified to obtain the system with the optimal primer concentration combination.
(a) Primer concentration combination of one-step multiplex RT-PCR:
and (3) adjusting annealing temperature: and optimizing the annealing temperature of the PCR reaction based on the optimal primer concentration system. According to the fact that the Tm values of all the primers are 55 ℃, annealing temperature gradients are set to be 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃ and 59 ℃ for 6 gradients, and the optimal annealing temperature is obtained.
6. Agarose gel electrophoresis detection
The PCR product was electrophoresed in 7.5. mu. L using 1.2% agarose gel at 100V in 0.5 × TBE buffer for 45min, and the results were observed.
7. Analysis of detection results
Designing an initial system for one-step multiplex RT-PCR detection, carrying out gel electrophoresis identification on an amplification result, wherein the sizes of amplification fragments corresponding to PRSV, ZTMV and ZYMV 3 viruses are 939bp, 631bp and 755 bp. respectively, and the phenomenon that amplification bands of PRSV and ZYMV are brighter and amplification bands of ZTMV are weaker in a part of samples exists in the initial reaction system (figure 1).
In summary, the one-step multiplex detection line for 3 viruses was determined to be 7.5. mu. L2 × 1step Buffer, 4.5. mu. L RNase Free ddH2O，0.6μL
1Step Enzyme Mix, ZYMV and PRSV upstream and downstream primers 10 μ M0.25 μ L each upstream and downstream primers 10 μ M0.4 μ L template 0.6 μ L total 15 μ L reaction conditions of 50 ℃ 30min, 94 ℃ pre-denaturation 2min, 94 ℃ 30sec, 56 ℃ 30sec, 72 ℃ 1min continuous 35 cycles, 72 ℃ 10min, 4 ℃ cold storage, the system can effectively amplify the target fragment (figure 2), thereby further ensuring the amplification effect of the one-Step multiplex RT-PCR technology.
EXAMPLE detection of two different combinations of viruses
1. Experimental Material
Field samples infected with ZYMV, PRSV and ZTMV alone, samples infected with ZYMV, PRSV and ZTMV 3 viruses in combination, negative control samples infected with PVY and TVDV alone, and healthy pumpkin seedlings.
2. Primer design Synthesis
(c) Designing and synthesizing specific detection primers for detecting ZYMV, PRSV and ZTMV, wherein the primer sequences are as follows:
ZYMV upstream primer (ZYMVdF)5 '-3': GAACATGTCGAGTTGGAGAG
Downstream primer (ZYMVdR)5 '-3': CGCTTGCTCTGTGTTTCAAC
PRSV upstream primer (PRSVdF)5 '-3': CTAGCACAAAAACTGGAGAGAG
Downstream primer (PRSVdR)5 '-3': CTCTCATTCYAAGAGGCTC
Forward primer (ZTMVdF)5 '-3': GCATTAAGTTTACGACAGCGC
Downstream primer (ZTMPdR) 5 '-3': CAATCACTCTTGTCRGTGTC
(d) Each pair of primer amplified fragments and the virus to be detected are as follows:
3. the CTAB method is used for extracting total nucleic acid of infected plant tissues as an RT-PCR amplification template:
adding 1 size and 5 small sterilized steel balls into 2m L sterilized centrifuge tube, weighing about 0.1g diseased leaf into the centrifuge tube, adding 1.2m L CTAB buffer solution (2% CTAB, 2%)PVP-40, 100mM Tris-HCl, pH 8.0, 1.4M NaCl, 20mM EDTA, 0.2% mercaptoethanol), pre-cooling in a refrigerator at 80 ℃ for 3-5 min, grinding for 2 times in a high-throughput tissue grinder at 60Hz and 90s, warm bathing at 65 ℃ for 45-60 min, centrifuging at 13000r/min for 10min, taking 650 mu L supernatant to a new 1.5M L centrifugal tube, adding equal volume chloroform/isoamyl alcohol (24:1), shaking, whirling, mixing uniformly for 30s, centrifuging at 13000r/min for 10min, carefully transferring 500 mu L supernatant to a new 1.5M L centrifugal tube, adding 350 mu L isopropanol, slightly reversing the centrifugal tube, fully mixing the liquid, standing at room temperature for 10min, centrifuging at 13000r/min for 15min, sucking supernatant by a micropipette carefully, adding 500 mu L75% ethanol, 13000r/min, centrifuging at 13000r/min for 10min, carefully drying supernatant by a micropipetition, naturally precipitating at room temperature for 10min, adding L-83H 832Placing O on ice for 30min, repeatedly blowing liquid with micropipette to dissolve precipitate, and storing at-20 deg.C for use.
4. One-step multiplex RT-PCR amplification
The reaction system of the one-step multiplex RT-PCR is 7.5 mu L2 × 1step Buffer, 4.5 mu L RNaseFree ddH2O，0.6μL
1Step Enzyme Mix, ZYMV and PRSV upstream and downstream primers 10. mu.M 0.25. mu. L each upstream and downstream primers 10. mu.M 0.4. mu. L each template 0.6. mu. L at 15. mu. L, the reaction conditions were 50 ℃ for 30min, 94 ℃ for 2min, 94 ℃ for 30sec, 56 ℃ for 30sec, 72 ℃ for 1min, 35 cycles were continued, 72 ℃ for 10min, and 4 ℃ for cold storage.
5. Agarose gel electrophoresis detection
The PCR product was electrophoresed in 7.5. mu. L using 1.2% agarose gel at 100V in 0.5 × TBE buffer for 45min, and the results were observed.
6. Analysis of detection results
The established one-step multiplex RT-PCR detection system is used for detection, and the result shows that 3 characteristic bands with the same size as expected can be simultaneously amplified in a sample compositely infected by 3 viruses, wherein the sizes of the characteristic bands are 631bp, 939bp and 755bp and respectively correspond to ZYMV, PRSV and ZTMV; the materials which are independently infected with ZYMV, PRSV and ZTMV only respectively amplify corresponding specific target bands of 631bp, 939bp or 755bp, and do not amplify other bands; no corresponding bands were amplified from plants infected with PVY or TVDV alone and healthy pumpkin seedlings (FIG. 3).
Example detection of field melon samples by triple multiplex RT-PCR
1. Experimental Material
23 field melon crop samples suspected of being infected with viruses such as ZYMV, PRSV, ZTMV and the like are collected from Jiangcuan area of Yuxi city, Yunnan province.
2. Primer design Synthesis
(a) Designing and synthesizing specific detection primers for detecting ZYMV, PRSV and ZTMV, wherein the primer sequences are as follows:
ZYMV upstream primer (ZYMVdF)5 '-3': GAACATGTCGAGTTGGAGAG
Downstream primer (ZYMVdR)5 '-3': CGCTTGCTCTGTGTTTCAAC
PRSV upstream primer (PRSVdF)5 '-3': CTAGCACAAAAACTGGAGAGAG
Downstream primer (PRSVdR)5 '-3': CTCTCATTCYAAGAGGCTC
Forward primer (ZTMVdF)5 '-3': GCATTAAGTTTACGACAGCGC
Downstream primer (ZTMPdR) 5 '-3': CAATCACTCTTGTCRGTGTC
(b) Each pair of primer amplified fragments and the virus to be detected are as follows:
3. CTAB method for extracting total nucleic acid of infected plant tissue as RT-PCR amplification template
Adding 1-size and 5-size sterilized steel balls into a 2M L sterilized centrifuge tube, weighing about 0.1g of diseased leaves into the centrifuge tube, adding 1.2M L CTAB buffer solution (2% CTAB, 2% PVP-40, 100mM Tris-HCl, pH 8.0, 1.4M NaCl, 20mM EDTA, 0.2% mercaptoethanol), pre-cooling in a 80 ℃ refrigerator for 3-5 min, grinding for 2 times in a high-throughput tissue grinder (60 Hz and 90 s), warm bathing at 65 ℃ for 45-60 min, centrifuging at 13000r/min for 10min, collecting 650 mu L supernatant to a new centrifuge tube with the thickness of 1.5M L, adding equal volume of chloroform/isoamylol (24:1), vortexing and mixing for 30s, 1Centrifuging at 3000r/min for 10min, transferring 500 μ L supernatant to a new centrifuge tube of 1.5m L, adding 350 μ L isopropanol, slightly inverting the centrifuge tube, mixing the liquid, standing at room temperature for 10min, centrifuging at 13000r/min for 15min, carefully sucking out the supernatant with a micropipette, adding 500 μ L75% ethanol into the precipitate, centrifuging at 13000r/min for 10min, carefully sucking out the supernatant with a micropipette, naturally drying the precipitate at room temperature for 10-15 min, adding 100 μ L ddH2Placing O on ice for 30min, repeatedly blowing liquid with micropipette to dissolve precipitate, and storing at-20 deg.C for use.
4. Single RT-PCR detection verification of field samples
And respectively carrying out virus species identification on the 23 suspected virus disease samples by using a conventional single RT-PCR technology, carrying out single RT-PCR detection by using designed and synthesized specific detection primers of ZYMV, PRSV and ZTMV, and determining the infection conditions of the ZYMV, PRSV and ZTMV in the samples so as to verify the accuracy of the one-step multiple RT-PCR detection result.
5. One-step multiplex RT-PCR amplification
The reaction system of the one-step multiplex RT-PCR is 7.5 mu L2 × 1step Buffer, 4.5 mu L RNaseFree ddH2O，0.6μL
1Step Enzyme Mix, ZYMV and PRSV upstream and downstream primers 10. mu.M 0.25. mu. L each upstream and downstream primers 10. mu.M 0.4. mu. L each template 0.6. mu. L at 15. mu. L, the reaction conditions were 50 ℃ for 30min, 94 ℃ for 2min, 94 ℃ for 30sec, 56 ℃ for 30sec, 72 ℃ for 1min, 35 cycles were continued, 72 ℃ for 10min, and 4 ℃ for cold storage.
6. Agarose gel electrophoresis detection
The PCR product was electrophoresed in 7.5. mu. L using 1.2% agarose gel at 100V in 0.5 × TBE buffer for 45min, and the results were observed.
7. Analysis of detection results
The established one-step multiplex RT-PCR detection system is used for detecting 3 viruses of PRSV, ZTMV and ZYMV on 23 field melon crop samples collected from Jiangxi area of Yuxi city, Yunnan province, the result can show that the detected target strip is single, only one sample in 23 detected samples is not infected with the viruses, the rest samples are infected with at least 1 virus, 4 samples are compositely infected with the PRSV, ZTMV and ZYMV 3 viruses, 4 samples are compositely infected with the ZTMV and ZYMV, 4 samples are compositely infected with the PRSV and ZYMV, 3 samples are compositely infected with the PRSV and ZTMV, 2 samples are infected with the PRSV, 3 samples are infected with the ZYMV, and the rest 2 samples are infected with the ZTMV. Therefore, the detection efficiency, the detection effect and the detection specificity of the multiple detection system are good, and the multiple detection system can be used for field sample detection of PRSV, ZTMV and ZYMV 3 viruses (figure 4).
Example four multiplex RT-PCR detection sensitivity test
1. Experimental Material
Selecting a sample infected by ZYMV, PRSV and ZTMV 3 viruses in the field, and diluting nucleic acid according to a gradient of 10 times, wherein the dilution time is 10-0、10-1、10-2、10-3、10-4、10-5The nucleic acid template is used.
2. Designing and synthesizing a primer:
(a) designing and synthesizing specific detection primers for detecting ZYMV, PRSV and ZTMV, wherein the primer sequences are as follows:
ZYMV upstream primer (ZYMVdF)5 '-3': GAACATGTCGAGTTGGAGAG
Downstream primer (ZYMVdR)5 '-3': CGCTTGCTCTGTGTTTCAAC
PRSV upstream primer (PRSVdF)5 '-3': CTAGCACAAAAACTGGAGAGAG
Downstream primer (PRSVdR)5 '-3': CTCTCATTCYAAGAGGCTC
Forward primer (ZTMVdF)5 '-3': GCATTAAGTTTACGACAGCGC
Downstream primer (ZTMPdR) 5 '-3': CAATCACTCTTGTCRGTGTC
(b) Each pair of primer amplified fragments and the virus to be detected are as follows:
3. CTAB method for extracting total nucleic acid of infected plant tissue as RT-PCR amplification template
Adding 1-size-5-size sterilized steel balls into a 2M L sterilized centrifuge tube, weighing 0.1g of left and right diseased leaves into the centrifuge tube, adding 1.2M L CTAB buffer solution (2% CTAB, 2% PVP-40, 100mM Tris-HCl, pH 8.0, 1.4M NaCl, 20mM EDTA, 0.2% mercaptoethanol), pre-cooling in a 80 ℃ refrigerator for 3-5 min, grinding for 2 times in a high-throughput tissue grinder 60Hz and 90s, warm bathing at 65 ℃ for 45-60 min, centrifuging at 13000 r/387min for 10min, taking 650 mu 2 supernatant to a new 1.5M L, adding equal volume of chloroform/isoamyl alcohol (24:1), shaking and mixing for 30s, centrifuging at 13000r/min for 10min, transferring 500 mu L supernatant to a new 1.5M L centrifuge tube, adding 350 mu L isopropanol, slightly inverting, mixing the supernatant, placing the mixture at room temperature for 10min, centrifuging at 13000r/min for 15min, sucking 500 mu L min, centrifuging at 13000 mu 35 min, adding 500 mu 35 mu 75 mu ethanol, adding 500 mu 75 mu ethanol into the supernatant, centrifuging at room temperature, adding 100 mu 10 mu H, adding 500 mu 75 mu ethanol, and precipitating2Placing O on ice for 30min, repeatedly blowing liquid with micropipette to dissolve precipitate, and storing at-20 deg.C for use.
4. One-step multiplex RT-PCR amplification
The reaction system of the one-step multiplex RT-PCR is 7.5 mu L2 × 1step Buffer, 4.5 mu L RNaseFree ddH2O，0.6μL
1Step Enzyme Mix, ZYMV and PRSV upstream and downstream primers 10. mu.M 0.25. mu. L each upstream and downstream primers 10. mu.M 0.4. mu. L each template 0.6. mu. L at 15. mu. L, the reaction conditions were 50 ℃ for 30min, 94 ℃ for 2min, 94 ℃ for 30sec, 56 ℃ for 30sec, 72 ℃ for 1min, 35 cycles were continued, 72 ℃ for 10min, and 4 ℃ for cold storage.
5. Agarose gel electrophoresis detection
The PCR product was electrophoresed in 7.5. mu. L using 1.2% agarose gel at 100V in 0.5 × TBE buffer for 45min, and the results were observed.
6. Analysis of detection results
As a result, it was found that when the nucleic acid was diluted to 10-4The system can effectively amplify 3 viruses when diluted to 10-5Although PRSV bands are weak, 3 viruses can be detected, so that the method can detect the viruses with the minimum nucleic acid concentration of 10-5Nucleic acid stock solution (FIG. 5).
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (5)
1. A method for simultaneously detecting ZYMV, PRSV and ZTMV, comprising: the method comprises the following steps:
(1) designing and synthesizing a primer:
designing and synthesizing specific detection primers for detecting ZYMV, PRSV and ZTMV, wherein the primer sequences are as follows:
ZYMV upstream primer (ZYMVdF)5 '-3': GAACATGTCGAGTTGGAGAG
Downstream primer (ZYMVdR)5 '-3': CGCTTGCTCTGTGTTTCAAC
PRSV upstream primer (PRSVdF)5 '-3': CTAGCACAAAAACTGGAGAGAG
Downstream primer (PRSVdR)5 '-3': CTCTCATTCYAAGAGGCTC
Forward primer (ZTMVdF)5 '-3': GCATTAAGTTTACGACAGCGC
Downstream primer (ZTMPdR) 5 '-3': CAATCACTCTTGTCRGTGTC
(2) Extracting total nucleic acid of infected plant tissues by a CTAB method to be used as an RT-PCR amplification template;
(3) one-step multiplex RT-PCR amplification;
(4) and detecting the amplification result by agarose gel electrophoresis.
2. A method for simultaneous detection of ZYMV, PRSV and ZTMV as defined in claim 1, wherein: in the step (1), the amplified fragment size of the primer pair ZYMVdF/ZYMVdR for detecting ZYMV is 755bp, the amplified fragment size of the primer pair PRSVdF/PRSVdR for detecting PRSV is 939bp, and the amplified fragment size of the primer pair ZTMVdF/ZTMVdR for detecting ZTMV is 631 bp.
3. The method for synchronously detecting ZYMV, PRSV and ZTMV according to claim 1 is characterized in that the CTAB method for extracting total nucleic acid of an infected plant tissue comprises the steps of taking a 2M L sterilized centrifuge tube, adding 1-5-inch sterilized steel balls, weighing about 0.1g of infected leaves into the centrifuge tube, adding 1.2M L CTAB buffer solution (2% CTAB, 2% PVP-40, 100mM Tris-HCl, pH 8.0, 1.4M NaCl, 20mM EDTA, 0.2% mercaptoethanol), precooling in a 80 ℃ refrigerator for 3-5 min, grinding for 2 times in a high-throughput tissue grinder 60Hz and 90s, bathing for 45-60 min at 65 ℃, centrifuging for 10min at 13000r/min, taking a fresh supernatant of 650 μ L to 1.5M L, adding chloroform/isoamyl alcohol (24:1) with the same volume, vortexing and mixing for 30s, 13000r/min, centrifuging for 10min, transferring the supernatant of 500 μ L min for standby, adding 355 μ M supernatant of supernatant fluid, centrifuging for 355 min, adding 355 μ M supernatant fluid, adding a micropipetitioning, adding ethanol, shaking and dissolving the supernatant of supernatant fluid, centrifuging for 3515 μ 5min, adding 3515 μ M supernatant fluid, adding a small pipette, shaking and mixing thoroughly, adding a supernatant fluid of a small pipette, shaking for 3515-30 min, shaking, dissolving, and dissolving, shaking, and mixing, and stirring at room temperature, and stirring, wherein the supernatant fluid is carried out.
4. The method for synchronously detecting ZYMV, PRSV and ZTMV according to claim 1 or 2, characterized in that the reaction system of the one-Step method multiple RT-PCR is 7.5 μ L2 × 1Step Buffer, 4.5 μ L RNase Free ddH2O, 0.6 μ L PrimeScript 1 Steel Enzyme Mix, 0.25 μ L of the upper and lower primers 10 μ M of ZYMV and PRSV respectively, 0.4 μ L template 0.6 μ L of the upper and lower primers 10 μ M of 0.25 μ L, and 15 μ L in total.
5. A method for the simultaneous detection of ZYMV, PRSV and ZTMV, according to claim 1 or 4, wherein: the one-step method multiple RT-PCR reaction conditions are as follows: 30min at 50 ℃; pre-denaturation at 94 ℃ for 2min, at 94 ℃ for 30sec, at 56 ℃ for 30sec, and at 72 ℃ for 1min, and continuously performing 35 cycles; keeping at 72 deg.C for 10min, and cooling at 4 deg.C.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116574851A (en) * | 2023-07-04 | 2023-08-11 | 广东省农业科学院植物保护研究所 | Method and kit for detecting six kinds of cucurbits viruses by multiplex PCR |
| CN116769977A (en) * | 2023-08-18 | 2023-09-19 | 云南农业大学 | PEMV-1, BYMV and BrYV one-step method multiplex RT-PCR detection kit and detection method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102230027A (en) * | 2011-06-10 | 2011-11-02 | 云南农业大学 | Method for synchronous detection of sweet potato feathery mottle virus (SPFMV), sweet potato virus C (SPVC), sweet potato virus G (SPVG) and sweet potato virus 2 (SPV2) |
| CN104357580A (en) * | 2014-10-09 | 2015-02-18 | 江苏省农业科学院 | 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 |
| WO2016148074A1 (en) * | 2015-03-19 | 2016-09-22 | 国立研究開発法人農業生物資源研究所 | Polynucleotide having potyvirus resistance, protein, and use of same |
| CN106702029A (en) * | 2017-03-16 | 2017-05-24 | 中国农业科学院植物保护研究所 | Multiple RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method for synchronously detecting five watermelon viruses and application of method |
-
2020
- 2020-04-15 CN CN202010294114.7A patent/CN111455109B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102230027A (en) * | 2011-06-10 | 2011-11-02 | 云南农业大学 | Method for synchronous detection of sweet potato feathery mottle virus (SPFMV), sweet potato virus C (SPVC), sweet potato virus G (SPVG) and sweet potato virus 2 (SPV2) |
| CN104357580A (en) * | 2014-10-09 | 2015-02-18 | 江苏省农业科学院 | 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 |
| WO2016148074A1 (en) * | 2015-03-19 | 2016-09-22 | 国立研究開発法人農業生物資源研究所 | Polynucleotide having potyvirus resistance, protein, and use of same |
| CN106702029A (en) * | 2017-03-16 | 2017-05-24 | 中国农业科学院植物保护研究所 | Multiple RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method for synchronously detecting five watermelon viruses and application of method |
Non-Patent Citations (3)
| Title |
|---|
| CRAIG G. WEBSTER 等: "Development and Evaluation of ELISA and qRT-PCR for Identification of Squash vein yellowing virus in Cucurbits", 《PLANT DISEASE》 * |
| 赵红庆 等: "多重PCR技术在病原检测中的应用", 《生物技术通讯》 * |
| 赵芹 等: "侵染节瓜的3种病毒多重PCR检测体系的建立", 《园艺学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116574851A (en) * | 2023-07-04 | 2023-08-11 | 广东省农业科学院植物保护研究所 | Method and kit for detecting six kinds of cucurbits viruses by multiplex PCR |
| CN116574851B (en) * | 2023-07-04 | 2023-10-31 | 广东省农业科学院植物保护研究所 | Method and kit for detecting six kinds of cucurbits viruses by multiplex PCR |
| CN116769977A (en) * | 2023-08-18 | 2023-09-19 | 云南农业大学 | PEMV-1, BYMV and BrYV one-step method multiplex RT-PCR detection kit and detection method |
| CN116769977B (en) * | 2023-08-18 | 2023-11-03 | 云南农业大学 | PEMV-1, BYMV and BrYV one-step method multiplex RT-PCR detection kit and detection method |
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