CN116987820A - qRT-PCR kit for detecting sweet potato virus E - Google Patents
qRT-PCR kit for detecting sweet potato virus E Download PDFInfo
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Abstract
The invention belongs to the technical field of detection of plant pathogenic organism virus molecules, and provides a specific primer group for detecting sweet potato virus E, wherein the nucleotide sequences of forward and reverse primers are respectively shown as SEQ ID NO. 1-2, SEQ ID NO. 1 and SEQ ID NO. 3. The invention also provides a kit for detecting the sweet potato virus E, which comprises the specific primer group, and a method for detecting the sweet potato virus E by using the kit. The specific primer can be used for qPCR, has accurate detection, high efficiency and sensitivity, and can realize diagnosis and early warning of the virus E-carrying condition of the sweet potato viruses in the large Tian Shu seedling, the seed potato seedling and the tissue culture seedling.
Description
Technical Field
The invention belongs to the technical field of detection of plant pathogenic organism virus molecules, and particularly relates to a primer and a method for detecting sweet potato virus E by using a reverse transcription fluorescent quantitative polymerase chain reaction amplification technology.
Background
Sweet potato [Ipomoca batatas(L.) Lam]Plants belonging to the genus sweet potato of the family Convolvulaceae are now widely planted as a plant in more than 100 countries and regions of the world. Sweet potatoes are commonly called sweet potatoes, sweet potatoes and the like in China, are an important grain crop, are processed crops, and can also be used as health food, vegetables and gardening. At present, china is the largest sweet potato planting country in the world, and the yield of the sweet potato planting country accounts for more than half of the world yield.
Sweet potato virus ESweetpotatovirusESPVE) belongs to the potyvirus family (Potyviridae), potyvirus genusPotyvirus) The virus can cause symptoms such as sweet potato leaf flowers She Banbo. The virus was found for the first time in korea in 2019, and the occurrence of the virus in sweet potato planting areas in China is unknown. The potato virus Y such as SPVE and sweet potato pinnate virus, sweet potato virus C and sweet potato virus G have similar gene structures, the symptom characteristics are not obviously different from those of other potato virus Y which infects sweet potato, and the symptoms caused by separate infection are slight, but the potato virus Y can also have the capability of cooperating with SPCSV, so that more serious disease symptoms can be caused.
At present, SPVE is found to occur in China, but the occurrence and development of sweet potato E virus in China are not clear due to the lack of virus detection technology. Because of the characteristic of asexual propagation of the sweet potatoes, viruses can diffuse through the seed potato seedlings for a long distance, and at present, no specific medicament is used for preventing and treating the sweet potato virus diseases temporarily, virus seedlings are shoveled off on a seedbed as soon as possible, and the detoxification of materials by a stem tip detoxification technology becomes an important means for preventing and treating the sweet potato virus diseases, so that the detection of the viruses is enhanced, and the early warning and the inspection of the sweet potato viruses E are carried out, so that the method has important significance for guiding the prevention and the treatment of the sweet potato virus diseases.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a qRT-PCR kit for detecting sweet potato virus E, which has low detection limit and good specificity.
In order to achieve the above purpose, the present invention adopts the following technical scheme.
A specific primer group for detecting sweet potato virus E has the nucleotide sequences of forward and reverse primers shown as SEQ ID NO. 1-2 or SEQ ID NO. 1 and 3 respectively.
A kit for detecting sweet potato virus E, which comprises the specific primer group.
The kit also comprises reagents for performing reverse transcription fluorescent quantitative polymerase chain reaction (qRT-PCR), such as positive molecules, fluorescent dyes, enzymes, substrates, buffers and the like.
Specifically, the reagent includes: RNase inhibitor,5× PrimeScript Buffer, reverse transcriptase, SYBR Green fluorescent quantitative amplification premix, standard positive plasmid template. The above reagents may be prepared or formulated by commercially available or well known methods in the art.
A method of detecting sweet potato virus E comprising the steps of:
(1) Extracting total RNA of a sample to be detected;
(2) Reverse transcription is carried out by taking RNA extracted in the step (1) as a template and taking a sequence shown as SEQ ID NO. 2 or SEQ ID NO. 3 as a primer to obtain cDNA;
(3) Using the cDNA obtained in the step (2) as a template, and using the sequences shown as SEQ ID NO. 1-2 or SEQ ID NO. 1 and SEQ ID NO. 3 as forward and reverse primers to carry out fluorescence quantitative PCR amplification;
(4) When the CT value is less than or equal to 35 and a typical amplification curve appears, the result is positive; when the CT value is >35, no CT value or no amplification curve, the result is negative.
The invention has the following advantages:
the kit and the detection method provided by the invention detect the specific fragment of the sweet potato virus E by using the specific primer, and detect the virus non-amplification curves of other infected sweet potato viruses such as sweet potato chlorosis dwarf virus, sweet potato chlorosis spot virus, sweet potato latent virus, sweet potato virus 2, sweet potato virus C, sweet potato pinnate virus, sweet potato virus G, cucumber mosaic virus and the like, so that the detection is accurate, efficient and sensitive, and the rapid detection and early warning of the virus carrying condition of the sweet potato virus E in large Tian Shumiao and seed potato seedlings or tissue culture seedlings can be realized.
Drawings
FIG. 1 is a graph showing the amplification of qRT-PCR with different primer sets.
FIG. 2 is a bar graph of amplification CT values for different primer sets.
FIG. 3 is a bar graph of qRT-PCR amplification CT values for different reaction temperatures;
FIG. 4 shows qRT-PCR amplification curves for different primer concentrations;
FIG. 5 shows qRT-PCR amplification curves of different sweet potato viruses;
FIG. 6 is a standard curve of E qRT-PCR of sweet potato virus;
FIG. 7 is a SPVE qRT-PCR amplification curve;
FIG. 8 is SPVE PCR sensitivity detection, where M: DL1000 DNA Marker; 1.10 -1 ng/μL；2. 10 - 2 ng/μL；3. 10 -3 ng/μL；4. 10 -4 ng/μL；5. 10 -5 ng/μL；6. 10 -6 ng/μL；7. 10 -7 ng/μL；8. 10 - 8 ng/μL；9.H 2 O。
Detailed Description
The present invention will be further described with reference to examples and drawings, but the present invention is not limited to the examples.
Example 1 design and screening of primers for E qRT-PCR amplification of sweet potato Virus
1. Primer design
Based on the comparison and analysis of the genome sequences of the sweet potato virus E and other sweet potato-infecting viruses (sweet potato virus 2, sweet potato virus C, sweet potato virus G, sweet potato pinnate virus and the like), 2 pairs of primer combinations are designed for PCR amplification and qPCR amplification. Wherein, the specific sequences of the primer groups are shown in Table 1.
TABLE 1 primer set for E qRT-PCR amplification of sweet potato virus
The target sequences of the 2 pairs of primer groups are shown as SEQ ID No. 4.
2. Extraction and reverse transcription of sample RNA
Grinding 0.1. 0.1 g of a positive disease sample of the sweet potato virus E into powder by liquid nitrogen, extracting RNA by FastPure Universal Plant Total RNA Isolation Kit, adding 6 mu L of the extracted RNA into 1 mu L of each of the downstream primers SPVE-R1 and SPVE-R2, placing into 2 PCR tubes, and placing on ice for 2 min after 5 min at 65 ℃. Then Recombinant RNase Inhibitor 0.5.5. Mu.L, 5X PrimeScript Buffer. Mu.L, prime Script was added TM 1. Mu.L of reverse transcriptase, add ddH 2 The cDNA was obtained by supplementing O to 20. Mu.L, 15 min at 50℃and 5s at 85 ℃.
3. qRT-PCR amplification reaction
The total reaction system was 20. Mu.L, and first, a fluorescent quantitative mixture ChamQ Universal SYBR qPCR Master Mix. Mu.L and ddH were added to a fluorescent quantitative PCR tube of 0.2. 0.2 mL 2 O8.4 mu L, respectively adding 10 mu M of SPVE upstream and downstream primer groups SPVE-F1 and SPVE-R1 and SPVE-F1 and SPVE-R2 into samples 1 and 2, respectively adding 1 mu L of cDNA template, and sucking and beating by a pipette to mix uniformly;
placing the prepared reaction system into a fluorescent PCR instrument, and setting conditions: 3 min at 95 ℃; the cycle was repeated 40 times at 95℃for 15s,58℃for 20 s, and 72℃for 20 s.
The dissolution curves and CT values of the different primer sets obtained by fluorescence quantitative PCR are shown in FIG. 1 and FIG. 2, respectively. As can be seen from the graph, the amplification curve of the different primer sets is observed by the fluorescence quantitative PCR instrument software, the amplification of the different primer sets is calculated, and the result is shown in FIG. 1, the amplification curve appears before the primer set 2 in the primer set 1, the amplification CT value of the primer set 1 is 25.60 and is less than the amplification CT value of the primer set 2 by 26.48, so that the primer set 1 is selected for the subsequent experiment.
Example 2 preparation of sweet potato Virus E qRT-PCR kit
1. Preparation of positive plasmid
And (3) carrying out PCR amplification by taking the primer group 1 as a primer and the cDNA obtained in the step (1) as a template, wherein the PCR reaction conditions are as follows: 94 ℃ for 3 min;94℃for 30 s,60℃for 30 s,72℃for 20 s,35 cycles; storing at 72deg.C for 10 min and 4deg.C. The PCR product was ligated into pMD18-T vector by recovery and verified to obtain SPVE positive plasmid.
2. Preparation of the kit
Prime Script is obtained by commercial purchase as Recombinant RNase Inhibitor,5× PrimeScript Buffer TM Reverse transcriptase and 2 XSYBR Green fluorescent quantitative amplification premix (containing HotStart Taq DNA polymerase, dNTP, SYBR Green I dye, buffer and MgCl) 2 ) Then SPVE positive plasmid, primer SPVE-F1 freeze-dried powder, primer SPVE-R1 freeze-dried powder and ddH are added 2 O was placed in each of 1.5 mL EP tubes, and the above-mentioned reagents were assembled into a kit according to the ratio of use.
Example 3 sweet potato Virus E qRT-PCR kit detection condition optimization and specificity and sensitivity detection
1. Optimization of reaction temperature
According to the reaction system of example 1, qRT-PCR reactions were performed with primer set 1 at annealing temperatures of 56.5, 58, 59.5, 61℃using SPVE positive plasmid of 1 ng/. Mu.L as template. As shown in FIG. 3, the amplification CT value was the lowest at a reaction temperature of 59.5℃and found to be 18.06; therefore, the optimum reaction temperature in this experiment was 59.5 ℃.
2. Optimization of primer concentration
According to the reaction system of example 1, the reaction annealing temperature was set at 59.5℃and qRT-PCR reactions were performed with SPVE positive plasmid of 1. 1 ng/. Mu.L as a template at primer set concentrations of 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 and 0.5. Mu. Mol/L, respectively. As shown in FIG. 4, the final concentration of primer set 1 was 0.15. Mu. Mol/L, which resulted in the lowest amplification CT value and significant differences from the other concentrations (p < 0.05).
3. Specific detection
Collecting sweet potato pinnate virus (SPFMV), sweet potato virus 2 (SPV 2), cucumber Mosaic Virus (CMV), sweet Potato Virus C (SPVC), sweet potato chlorosis dwarf virus (SPCSV), sweet Potato Virus G (SPVG), sweet Potato Latent Virus (SPLV), sweet potato chlorosis spot virus (SPCFV) and SPVE positive samples, taking 0.1G, grinding into powder by liquid nitrogen, extracting RNA by FastPure Universal Plant Total RNA Isolation Kit, adding 6 mu L of the extracted RNA into 5X HiScript II Select qRT SuperMix mu L of reverse primer SPVE-R1 1 mu L with the concentration of 10 mu mol/L, placing in a PCR tube at 50 ℃ for 15 min and 85 ℃ for 5s to obtain cDNA;
into a 0.2. 0.2 mL fluorescent quantitative PCR tube was added ChamQ Universal SYBR qPCR Master Mix. Mu.L of a fluorescent quantitative mixture, ddH 2 O8.4 mu L, respectively adding primers SPVE-F1 and SPVE-R1 with concentration of 10 mu mol/L and 0.3 mu L respectively, respectively adding cDNA template 1 mu L, and sucking and beating by a pipette to mix uniformly; placing the prepared reaction system into a fluorescent quantitative PCR instrument ABI Quantum studio TM 6 Flex, reference procedure 95℃for 3 min; amplification was performed by cycling for 40 times at 95℃for 15s,59.5℃for 20 s, and 72℃for 20 s.
As shown in FIG. 5, the SPVE samples showed amplification curves with CT values of 20.00 and less than 35; the result is negative when Ct value of the rest virus sample is > 35. The established detection method is proved to have better specificity for SPVE.
4. Sensitivity detection
Sequentially diluting SPVE positive plasmids in the kit with 10-time concentration gradient to finally dilute into 10 -1 ng/μL，10 -2 ng/μL，10 -3 ng/μL，10 -4 ng/μL，10 -5 ng/μL，10 -6 ng/μL，10 -7 ng/μL，10 -8 The ng/. Mu.L of 8 templates with different concentrations were taken 1. Mu.L each, and fluorescent quantitative mixture ChamQ Universal SYBR qPCR Master Mix. Mu.L and ddH were added 2 O8.4. Mu.L, 10. Mu. Mol/L of each of the primers SPVE-F1/SPVE-R1, 0.3. Mu.L, and the prepared reaction system was placed in a fluorescent quantitative PCR apparatus ABI Quantum studio ™ Flex, see procedure 95℃for 3 min; amplification was performed by cycling for 40 times at 95℃for 15s,59.5℃for 20 s, and 72℃for 20 s.
Will pass the calculation formula: copy number (copy/. Mu.L) = (6.02X10) 23 Copy/mol×plasmid concentration)/(plasmid nucleotide base number×660) calculated 10 -1 ng/. Mu.L plasmid 3.12X10 7 copes/. Mu.L. At 3.12X10 2 -3.12×10 6 Amplifying the copy/mu L divided by 3.12 Log value and the corresponding plasmid as templateCT value, and drawing standard curve. The results are shown in FIG. 6: the slope of the standard curve is-3.4243, the linear regression equation is y= -3.4243x+39.785, R 2 0.9975.
At 10 -1 -10 -8 The ng/. Mu.L of the gradient diluted plasmid is used as a template, the amplification system is 12.5 mu.L of PCR Mix premix (TsingKe, beijing), 1 mu.L of the template and 1 mu.L of each primer group SPVE-F1/SPVE-R1 with the concentration of 10 mu mol/L, and ddH 2 O is added to 25 mu L; amplification was performed on a PCR amplification instrument: 94 ℃ for 3 min;94℃for 30 s,59.5℃for 30 s,72℃for 20 s,35 cycles; storing at 72deg.C for 10 min and 4deg.C. mu.L of the PCR product was separated by electrophoresis on a 1.5% agarose gel, stained with a nucleic acid dye and visualized under an ultraviolet gel imaging system.
As shown in FIG. 7 and FIG. 8, the detection sensitivity of the qRT-PCR method can reach 10 -6 ng/. Mu.L (i.e. 3.12X10) 2 copes/. Mu.L), whereas the ordinary PCR method can detect only 10 -4 ng/. Mu.L (i.e. 3.12X10) 4 copes/. Mu.L), the qRT-PCR method of the present invention is 100-fold more sensitive than conventional PCR in detecting sweetpotato virus E.
The results show that the primer, the kit and the detection method provided by the invention can be used for rapidly identifying the sweet potato virus E, the detection process is simple and convenient, the result is more sensitive and accurate, and services are provided for early diagnosis, identification and the like of the sweet potato virus E.
Claims (6)
1. A specific primer group for detecting sweet potato virus E is characterized in that the nucleotide sequences of forward and reverse primers are respectively shown as SEQ ID NO. 1-2 or SEQ ID NO. 1 and 3.
2. A kit for detecting sweet potato virus E comprising the specific primer set of any one of claim 1.
3. The kit of claim 2, further comprising reagents for performing a reverse transcription fluorescent quantitative polymerase chain reaction.
4. A kit according to claim 3, wherein the reagent is selected from one or more of a positive molecule, a fluorescent dye, an enzyme, a substrate, a buffer.
5. A kit according to claim 3, wherein the reagents comprise: RNase inhibitor,5× PrimeScript Buffer, reverse transcriptase, SYBR Green fluorescent quantitative amplification premix, standard positive plasmid template.
6. A method for detecting sweet potato virus E, comprising the steps of:
(1) Extracting total RNA of a sample to be detected;
(2) Reverse transcription is carried out by taking RNA extracted in the step (1) as a template and taking a sequence shown as SEQ ID NO. 2 or SEQ ID NO. 3 as a primer to obtain cDNA;
(3) Using the cDNA obtained in the step (2) as a template, and using the sequences shown as SEQ ID NO. 1-2 or SEQ ID NO. 1 and SEQ ID NO. 3 as forward and reverse primers to carry out fluorescence quantitative PCR amplification;
(4) When the CT value is less than or equal to 35 and a typical amplification curve appears, the result is positive; when the CT value is >35, no CT value or no amplification curve, the result is negative.
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