CN110923358A - RPA detection primer of sweet potato leaf curl virus, detection method and application - Google Patents
RPA detection primer of sweet potato leaf curl virus, detection method and application Download PDFInfo
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Abstract
The invention discloses an RPA detection primer of sweet potato leaf curl virus, a detection method and application, which are specially used for detecting specific molecules of the sweet potato leaf curl virus; the detection primers are as follows: the upstream primer F is shown as SEQ ID No. 1 and the downstream primer R is shown as SEQ ID No. 2; the DNA of a plant infected with the sweet potato leaf curl virus is extracted, RPA constant temperature amplification is carried out for 20min, and agarose gel electrophoresis is carried out after amplification to generate a specific band. The RPA isothermal amplification technology established aiming at the sweet potato leaf curl virus has the advantages of rapid and efficient amplification, good specificity, simple and convenient operation, no need of special instruments and the like, and provides a technical basis for early diagnosis of the sweet potato leaf curl virus.
Description
Technical Field
The invention belongs to the technical field of crop disease detection, identification and prevention and control, and particularly relates to an RPA detection primer of sweet potato leaf curl virus, a detection method and application.
Background
The sweet potato is a vegetative propagation crop which is propagated by root tubers or seedling vines and is often infected by viruses, and the viruses are propagated after infecting the sweet potato, so that the normal growth and development and the germplasm and the seed property degradation of the sweet potato are influenced. Sweet Potato Leaf Curl Virus (SPLCV) is one of the main viruses which can infect Sweet potatoes and is transmitted by whiteflies, and the SPLCV is generated in Sweet potato production areas all over the world every year, thereby bringing serious harm to Sweet potato production, causing Sweet potato dead production seriously and causing huge economic loss.
Ipomoea crispa virus (Geminiviridae) member of the genus Phaseolus viridae (Begomovirus), SPLCV is a typical DNA virus, transmitted in a persistent manner by Bemisia tabaci (Bemisia tabaci). After SPLCV infects sweet potatoes, the sweet potato plants can have symptoms of leaf curling, necrosis, flowering leaves and the like. At present, no specific medicine for preventing and controlling the virus diseases exists in production, so that a high-efficiency, quick and practical virus detection technology is established, the monitoring and diagnosis of the sweet potato virus are enhanced, and the method has important significance for preventing and controlling the sweet potato virus diseases.
At present, the traditional method for detecting geminivirus and the Polymerase Chain Reaction (PCR) molecular detection method are used. The traditional serological detection has high false positive rate, the PCR detection method needs expensive professional instruments, the amplification time is long, and the PCR detection method is not suitable for being applied in the field and basic units.
The loop-mediated isothermal amplification (LAMP) is a rapid, sensitive, specific and visual detection technology, and the technology adopts 4 specific primers to identify 6 specific regions on a target sequence, and utilizes DNA strand displacement polymerase (BstDNA polymerase) to amplify a target gene under a constant temperature condition, so that the technology has the advantages of convenience, rapidness, accuracy and the like, but the LAMP generates aerosol in the detection process and generates false positive.
Recombinase Polymerase Amplification (RPA) is considered as a nucleic acid detection technology that can replace PCR, and is a nucleic acid isothermal Amplification technology developed by twist dx Inc, uk in 2006. In recent years, RPA has been widely used for the detection of many pathogenic bacteria. Boyle (2014) and the like develop a method capable of rapidly and sensitively detecting two target genes of Mycobacterium tuberculosis by utilizing an RPA technology. Daher (2014) develops a real-time RPA detection method which can detect whether a pregnant woman is infected with streptococcus B or not, the detection is completed within 20min, and meanwhile, multiple detections can be realized.
Comparison of RPA with PCR and LAMP detection techniques is as follows:
| PCR | LAMP | RPA | |
| temperature of | Heat cycle system | Constant temperature (65 degree) | Constant temperature (37 degree) |
| Reaction time | 2h | 1h | 20min |
| Instrument for measuring the position of a moving object | PCR instrument | Thermostatic device | No need of special equipment |
| Number of |
1 pair of | 2 pairs of | 1 pair of |
| Availability for downstream applications | Is that | Whether or not | Is that |
| Whether or not it is multiple | Is that | Whether or not | Is that |
| Hard defect | Long time, special equipment | False positive | Is free of |
In conclusion, the RPA detection technology has the advantages of sensitivity, specificity, rapidness (the detection time is shortened to 20min), no need of special instruments and the like, and is widely applied to the detection of animal and plant pathogenic bacteria. The detection of the sweet potato pathogens by applying the technology is not reported, so that the establishment of the sweet potato virus RPA rapid detection technology has wide application prospect in the rapid diagnosis of the sweet potato virus diseases.
Disclosure of Invention
The invention aims to solve the technical problems that the traditional detection method of the sweet potato leafroll virus in the prior art needs long detection period, needs expensive instruments for detection and the like, and provides an RPA detection primer of the sweet potato leafroll virus, a detection method and application.
The invention realizes the technical problems as follows:
an RPA detection primer of sweet potato leaf curl virus comprises the following primer pair:
an upstream primer F: 5'-AGGCTGAACTTCGAGACAGCTATCGTGCCCTAC-3', as shown in SEQ ID No: 1;
a downstream primer R: 5'-AAGACCTGCATTCTATCCCTCAGATCCATTCGGAT-3', as shown in SEQ ID No. 2.
Further, the RPA rapid detection method of sweet potato leaf curl virus by using the RPA detection primer is characterized in that: the method comprises the following steps:
(1) DNA extraction: processing the sample and extracting the DNA of the sample;
(2) amplification of RPA: taking the DNA extracted in the step (1) as a template, and carrying out constant-temperature amplification reaction by adopting the RPA detection primer;
(3) and (3) detection: after the RPA reaction is finished, or the reaction product is detected by agarose gel electrophoresis, the judgment that a 464bp specific band appears is positive, and the judgment that no band appears is negative.
Further, the reaction system of the RPA reaction in the step (2) is 50. mu.L, and 2. mu.L of template DNA, 29.5. mu.L of RehydrationBuffer, 2.4. mu. L, ddH of each of the primers F and R2O 11.2. mu.L, and finally 2.5. mu.L of 280mmol/L magnesium acetate solution, and 50. mu.L of the system was added to the lyophilized enzyme powder.
Further, the RPA reaction in the step (2) is carried out under the conditions of isothermal amplification at 39 ℃ for 20min, and the reaction is stopped on ice.
Further, in step (3), 5. mu.L of the reaction product was detected by 2% agarose gel electrophoresis.
Further, the application of the RPA detection primer of the sweet potato leaf curl virus in the preparation of a sweet potato leaf curl virus detection kit.
The invention has the following advantages:
1. the result is reliable: the RPA detection primer designed by the invention tests and verifies the sweet potato leaf curl viruses collected and separated from different places, thereby fully ensuring the result reliability. 2. The specificity is strong: the RPA primers adopted by the invention are a pair of amplification primers designed aiming at the conserved sequence of the sweet potato leaf curl virus AV1 gene, and simultaneously, positive samples infected with tomato yellow leaf curl virus, sweet potato feathery mottle virus, sweet potato G virus and sweet potato de-greening dwarf virus are amplified without amplifying specific bands, so that the method has stronger specificity. 3. The sensitivity is high: the detection sensitivity of the RPA to the sweet potato leaf curl virus can reach 10pg mu L on the DNA level-1And has high sensitivity. 4. The operation is simple and convenient: the method is carried out under the isothermal condition, only one thermostatic device is needed, and expensive instruments and equipment are not needed. 5. And (3) fast: the detection of the sweet potato leaf curl virus can be finished within 20min, and the detection purpose can be realized by carrying out electrophoresis on the reaction product. The RPA isothermal amplification technology established by the invention aiming at the sweet potato leaf curl virus has the advantages of good amplification specificity, simple and convenient operation, quickness, no need of special instruments and the like, has higher application value and has wide application prospect for diagnosing the sweet potato leaf curl virus.
Drawings
The invention will be further described with reference to the following examples with reference to the accompanying drawings.
FIG. 1 is an electrophoresis chart of the result of the RPA detection according to the first embodiment of the present invention; wherein M is DL2000 DNA marker, 1: RPA product, 2: and (5) negative control.
FIG. 2 is a 2% agarose gel electrophoresis of example two of the present invention; m is DL2000 DNA marker; 1-5 are SPLCV, TYLCD, SPCMV, SPVG, SPCSV, ddH, respectively2O。
FIG. 3 is a 2% agarose gel electrophoresis of example III of the present invention; m is DL2000 DNA marker; 1-6 represent concentrations of 10 ng. mu.L, respectively-1、1ng μL-1、100pg μL-1、10pg μL-1、1pg μL-1、100fg μL-1、10fg μL-1；1fg μL-1The sweet potato leafroll virus DNA of (1).
Detailed Description
The present invention will be further described with reference to the following examples.
Example one
The invention relates to an RPA detection method of sweet potato leaf curl virus, which is characterized in that 1 pair of specific primers are designed by applying primer 5 primer design software according to the conserved AV1 gene sequence of SPLCV published by GenBank as a target gene. The Recombinase Polymerase (recombination Polymerase) is used for rapidly carrying out RPA amplification detection on the target gene under the constant temperature condition.
Wherein, the primer pair specifically comprises:
an upstream primer F: 5'-AGGCTGAACTTCGAGACAGCTATCGTGCCCTAC-3', as shown in SEQ ID No: 1;
a downstream primer R: 5'-AAGACCTGCATTCTATCCCTCAGATCCATTCGGAT-3', as shown in SEQ ID No. 2.
The method comprises the following specific steps:
step 100: according to the conserved AV1 gene sequence of SPLCV published by GenBank as a target gene, primer 5.0 primer design software is used for designing primers and 1 pair of specific primers. And the above-mentioned 1 pair of specific primers were synthesized by Fuzhou Shanghai Biotechnology Ltd.
Step 200: DNA extraction: DNA extraction was performed on tissues infected with SPLCV and healthy, and DNA extracted from healthy tissues was used as a negative control. DNA extraction was performed by CTAB method: placing 100mg of tissue in a mortar, adding liquid nitrogen, grinding into powder, placing the powder in a first centrifuge tube, sucking at 65 deg.CAdding 650 mu L of preheated CTAB solution into a first centrifuge tube, uniformly mixing, putting into a 65 ℃ water bath kettle, and carrying out water bath for 30-60min, and slightly shaking up once every 10 min. After the water bath was complete, 650. mu.L of chloroform-isoamyl alcohol (24: 1) was added. Mixing gently for 5min, standing for 15min to make it react thoroughly. The supernatant was centrifuged (20-25 ℃, 10000rpm, 10min) and placed in a second centrifuge tube (450. mu.L). Add isopropanol 300. mu.L, stir gently and ice-bath for 30 min. The mixture was centrifuged (4 ℃ C., 10000rpm, 5min), and the supernatant and water were discarded. The mixture was dissolved by adding 500. mu.L of TE, and further 250. mu.L of Tris-saturated phenol and 250. mu.L of chloroform-isoamyl alcohol (24: 1) were added. The mixture was centrifuged (4 ℃ C., 12000rpm, 10min) and the supernatant was discarded. Add 800. mu.L 70% ethanol and centrifuge again (4 ℃, 12000rpm, 5 min). Remove water and add 100. mu.L ddH2O dissolves the template DNA.
Step 300: amplification of RPA: the DNA obtained by the treatment in step 200 is used as a template, and the 1 pair of primers obtained in step 100 is used for RPA amplification.
Step 400: the reaction system and reaction procedure for the RPA reaction were as follows: 50 μ L of the DNA template 2 μ L, the regeneration Buffer 29.5 μ L, and the primers F and R each 2.4 μ L, ddH2O11.2. mu.L, and finally adding magnesium acetate solution (MgOAc) 2.5. mu.L (280mmol/L), adding 50. mu.L of the system into the freeze-dried enzyme powder, and reacting at 39 ℃ for 20 min. The reaction was terminated on ice.
Step 500: mu.L of the RPA product was electrophoresed in 2% agarose gel as support medium at 0.5 XTBE and 120V for 40min and observed by UV transilluminator.
The results of the specific experiments are shown in FIG. 1, where the RPA product is a specific band, while the negative control does not produce a band.
Example two
Specificity verification of RPA:
extracting total DNA of a positive sample infected with Sweet Potato Leaf Curl Virus (SPLCV), total DNA of a positive sample infected with tomato yellow leaf curl virus (TYLCD), cDNA of a positive sample infected with sweet potato feathery mottle virus (SPCMV), a positive sample infected with sweet potato G virus (SPVG) and a positive sample infected with sweet potato de-greening dwarfing virus (SPCSV), and performing specific detection on the cDNA by utilizing an RPA reaction system.
The results show that 5 μ L of the product was analyzed by 2% agarose gel electrophoresis, and the specific results are shown in fig. 2, only the detection result of SPLCV had specific bands, and other samples did not produce specific bands, indicating that the established RPA detection method has better specificity for detection of SPLCV.
EXAMPLE III
In order to examine the sensitivity of the RPA detection method of sweet potato leaf curl virus of the present invention, the applicant diluted the extracted DNA of sweet potato leaf curl virus to 10 ng. mu.L each by 10-fold dilution series of the DNA sample in step 200 of example-1、1ng μL-1、100pg μL-1、10pg μL-1、1pg μL-1、100fg μL-1、10fg μL-1；1fg μL-1(ii) a There were 8 different concentration gradients. The RPA reaction was performed as in step 400 of example one.
After the RPA reaction is finished, 5 mul of amplification products are detected by 2% agarose gel electrophoresis, if the characteristic band of the RPA appears, the amplification band is judged to be positive, and if the characteristic band of the RPA does not appear, the amplification band is judged to be negative. The detection results are shown in fig. 3: the sensitivity of the sweet potato leaf curl virus RPA is detected, an RPA characteristic band appears by agarose gel electrophoresis, and the detection sensitivity can reach 10pg mu L-1And has high sensitivity.
In summary, the invention has the following advantages: 1. the result is reliable: the RPA detection primer designed by the invention tests and verifies the sweet potato leaf curl viruses collected and separated from different places, thereby fully ensuring the result reliability. 2. The specificity is strong: the RPA primers adopted by the invention are a pair of amplification primers designed aiming at the conserved sequence of the sweet potato leaf curl virus AV1 gene, and simultaneously, positive samples infected with tomato yellow leaf curl virus, sweet potato feathery mottle virus, sweet potato G virus and sweet potato de-greening dwarf virus are amplified without amplifying specific bands, so that the method has stronger specificity. 3. The sensitivity is high: the detection sensitivity of the RPA to the sweet potato leaf curl virus can reach 10pg mu L on the DNA level-1And has high sensitivity. 4. The operation is simple and convenient: the method is carried out under the isothermal condition, only one thermostatic device is needed, and expensive instruments and equipment are not needed. 5. And (3) fast: to pairThe detection of the sweet potato leaf curl virus can be finished within 20min, and the detection purpose can be realized by carrying out electrophoresis on the reaction product. The RPA isothermal amplification technology established by the invention aiming at the sweet potato leaf curl virus has the advantages of good amplification specificity, simple and convenient operation, quickness, no need of special instruments and the like, has higher application value and has wide application prospect for diagnosing the sweet potato leaf curl virus.
Although specific embodiments of the invention have been described above, it will be understood by those skilled in the art that the specific embodiments described are illustrative only and are not limiting upon the scope of the invention, and that equivalent modifications and variations can be made by those skilled in the art without departing from the spirit of the invention, which is to be limited only by the appended claims.
Sequence listing
<110> institute of agricultural sciences college of Fujian province
<120> RPA detection primer of sweet potato leaf curl virus, detection method and application
<130>100
<160>2
<170>SIPOSequenceListing 1.0
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aagacctgca ttctatccct cagatccatt cggat 35
Claims (6)
1. An RPA detection primer of sweet potato leaf curl virus is characterized in that: the RPA detection primer comprises the following primer pair:
an upstream primer F: 5'-AGGCTGAACTTCGAGACAGCTATCGTGCCCTAC-3', as shown in SEQ ID No: 1;
a downstream primer R: 5'-AAGACCTGCATTCTATCCCTCAGATCCATTCGGAT-3', as shown in SEQ ID No. 2.
2. A rapid detection method for RPA of sweet potato leafroll virus using the RPA detection primer of claim 1, characterized in that: the method comprises the following steps:
(1) DNA extraction: processing the sample and extracting the DNA of the sample;
(2) amplification of RPA: taking the DNA extracted in the step (1) as a template, and carrying out constant-temperature amplification reaction by adopting the RPA detection primer;
(3) and (3) detection: after the RPA reaction is finished, or the reaction product is detected by agarose gel electrophoresis, the judgment that a 464bp specific band appears is positive, and the judgment that no band appears is negative.
3. The RPA rapid detection method of sweet potato leafroll virus according to claim 2, characterized in that: the reaction system of the RPA reaction in the step (2) is 50 mu L, wherein the reaction system contains 2 mu L of template DNA, 29.5 mu L of Rehydration Buffer, and 2.4 mu L, ddH of each of primers F and R2O11.2. mu.L, finally adding 2.5. mu.L of 280mmol/L magnesium acetate solution, and adding 50. mu.L of the system into the freeze-dried recombinant enzyme powder.
4. The RPA rapid detection method of sweet potato leafroll virus according to claim 2, characterized in that: and (3) carrying out the RPA reaction at the constant temperature of 39 ℃ for 20min, and stopping the reaction on ice.
5. The method for detecting the RPA of sweet potato leafroll virus according to claim 2, wherein the method comprises the following steps: in step (3), 5. mu.L of the reaction product was detected by 2% agarose gel electrophoresis.
6. The use of the RPA detection primer of sweet potato leafroll virus of claim 1 in the preparation of sweet potato leafroll virus detection kits.
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| CN111534638A (en) * | 2020-04-30 | 2020-08-14 | 河南科技学院 | Primer, kit, detection method and application for rapidly detecting SPLCV |
| CN113322353A (en) * | 2021-06-15 | 2021-08-31 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | RPA kit for detecting sweet potato pinnate mottle virus and sweet potato chlorotic stunt virus |
| CN113462814A (en) * | 2021-05-21 | 2021-10-01 | 中国热带农业科学院热带生物技术研究所 | RPA primer, probe, kit and detection method for detecting sweet potato golden vein virus |
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| CN113322353A (en) * | 2021-06-15 | 2021-08-31 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | RPA kit for detecting sweet potato pinnate mottle virus and sweet potato chlorotic stunt virus |
| CN113322353B (en) * | 2021-06-15 | 2022-08-26 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | RPA kit for detecting sweet potato pinnate mottle virus and sweet potato chlorotic stunt virus |
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