CN116103369B - Potato total RNA rapid extraction method and potato virus RT-PCR detection method - Google Patents
Potato total RNA rapid extraction method and potato virus RT-PCR detection method Download PDFInfo
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Abstract
The invention relates to a potato total RNA rapid extraction method and a potato virus RT-PCR detection method, belonging to the technical field of molecular biology detection. In order to solve the problem that the existing plant total RNA extraction method is difficult to popularize, the invention provides a potato total RNA rapid extraction method, and an RNA extraction test strip is adopted to extract total RNA; on the basis, the extracted potato total RNA sample is taken as a template to obtain cDNA of the sample, and then the cDNA of the sample is taken as the template to perform PCR reaction by using a single potato virus primer pair or multiple PCR reactions by using multiple potato virus primer pairs. The invention reduces the dependence on equipment and reagents, reduces the difficulty of extraction operation, shortens the extraction operation time, achieves the purposes of simple and convenient operation and easy popularization, does not reduce the detection sensitivity, and the newly designed RT-PCR method is faster and accelerates the potato virus detection process.
Description
Technical Field
The invention belongs to the technical field of molecular biology detection, and particularly relates to a potato total RNA rapid extraction method and a potato virus RT-PCR detection method.
Background
The current extraction method of the total RNA of the plant comprises a guanidine isothiocyanate method, a CTAB method, a lithium chloride method, various commercial kits and the like. However, these methods all require specialized equipment such as low-temperature high-speed centrifuges and toxic and harmful reagents such as chloroform, phenol, mercaptoethanol and the like, and require multiple transfer of liquid, and the extraction operation time is generally about 1 hour. Therefore, high requirements are put forward on the professionals of laboratories and personnel, and the popularization of technologies such as molecular detection is limited.
Disclosure of Invention
The invention provides a potato total RNA rapid extraction method and a potato virus RT-PCR detection method, which are used for solving the problem that the existing plant total RNA extraction method is difficult to popularize.
The technical scheme of the invention is as follows:
a method for rapidly extracting total RNA of potatoes, which adopts an RNA extraction test strip for extraction, comprises the following specific extraction steps:
step 1, fully mixing and grinding potato tissues and a lysate to obtain tissue homogenate;
step 2, immersing the nucleic acid binding region of the RNA extraction test strip into the tissue homogenate obtained in the step 1, so that the tissue homogenate fully infiltrates the nucleic acid binding region of the RNA extraction test strip;
step 3, immersing the nucleic acid binding area of the RNA extraction test strip immersed in the tissue homogenate in the step 2 into a cleaning solution for cleaning;
step 4, immersing the nucleic acid binding region of the RNA extraction test strip washed in the step 3 into RNase-free H 2 Soaking in O for 3min to obtain soaking solution which is the total RNA sample of potato.
Furthermore, the RNA extraction test strip is made of slow qualitative filter paper, the two ends of the RNA extraction test strip are respectively provided with a nucleic acid binding area and a wax sealed handle area, and the length ratio of the nucleic acid binding area to the wax sealed handle area is 1:10.
Further, the preparation method of the RNA extraction test strip comprises enzyme removal treatment and wax sealing treatment, wherein the enzyme removal treatment is to soak the slow qualitative filter paper in 0.1% DEPC water solution overnight, take out and sterilize at 121 ℃ for 15min under high pressure and then dry; the wax sealing treatment is to cut the slow qualitative filter paper obtained by enzyme removal into a filter paper strip A with the width of 50mm and a filter paper strip B with the width of 60mm, completely putting the filter paper strip A into melted paraffin, taking out and cooling, clamping the cooled filter paper strip A by using two filter paper strips B, and passing through a thermoplastic machine to obtain a filter paper strip B with a 10 mm-wide non-wax sealed nucleic acid binding area, and cutting according to the size specification of a finished product to obtain the RNA extraction test paper strip.
Further, the lysate in the step 1 is commercial trizol reagent, the production formula is Sieimer feier, and the product number is 15596026.
Further, in the step 1, the mass-volume ratio of the potato tissue to the lysate is 10-20 mg/300 mu L, and the lysis time is 10-20 min.
Further, the washing liquid in the step 3 is RNase-free H 2 O75% ethanol solution.
Further, the specific method for cleaning in the step 3 is as follows: immersing the nucleic acid binding area of the RNA extraction test strip immersed in the lysis buffer solution in the step 2 in 1mL of cleaning solution for 5min, shaking and cleaning for 1-2 seconds, taking out, immersing in 1mL of new cleaning solution again, immersing for 1min, shaking and cleaning for 1-2 seconds, taking out, and lightly throwing or airing until no obvious liquid drops exist.
The potato virus RT-PCR detection method adopts the potato total RNA rapid extraction method provided by the invention to obtain a potato total RNA sample, uses the potato total RNA sample as a template to obtain cDNA of the sample, uses the cDNA of the sample as the template, uses a single potato virus primer pair to carry out PCR reaction or uses multiple potato virus primer pairs to carry out multiple PCR reaction, and carries out agarose electrophoresis identification on the obtained PCR reaction liquid.
Furthermore, the specific method for obtaining the cDNA of the sample by taking the potato total RNA sample as a template comprises the following steps: mu.L of potato total RNA sample was aspirated, 4. Mu.L of 5 XRT MIX was added, and after thorough mixing, incubation was performed at 37℃for 15min and at 85℃for 1min, to obtain sample cDNA.
Further, the method for PCR amplification by using cDNA of the sample as a template comprises the following steps: 1-2. Mu.L of sample cDNA was extracted, 12.5. Mu.L of 2 XPCR MIX was added, and the final concentration of the upstream and downstream primers was adjusted to 0.2. Mu.M, ddH 2 O is added to 25 mu L, namely the PCR reaction liquid; putting the PCR reaction liquid into a PCR instrument, and carrying out PCR reaction at 95 ℃ for 5min, 20s denaturation at 95 ℃, 20s annealing at 54.5 ℃, 20s extension at 72 ℃,30 times of circulation and 5min final extension at 72 ℃;
the upstream and downstream primers comprise the following primers:
potato Y virus: PVY-2F: CCAACCCTTAGGCAAATCA;
PVY-2R：CGTGATGTGACCTCATAAAAGT；
potato virus M: PVM-1F: TCGGCGACCCCATACAAT;
PVM-1R：CAGGTCTGCCTTTTCACTTC；
potato virus a: PVA-2F: CAGGTATGGTCTTCAACGCA;
PVA-2R：TTCCGTCCAGTCCAAACA；
potato virus X: PVX-2F: CAAGCAAAAGATGAGACCCT;
PVX-2R：TTGTGAGAAGGGAATACGC；
potato virus S: XPVS-2F: CAGGGGATAAGCAAAGGTAGGC;
XPVS-2R：GCCAGACCCAGATTACCAAAGAAA；
potato spindle tuber virus: PSTvd-2F: GGGAAACCTGGAGCGAACT;
PSTVd-2R：TAGTAGCYGAAGCGACAGC；
potato leaf curl virus: plrv_f: CCACTCCAACTCCCCAGAAG;
PLRV_R：TACATAGGGACGGCTTGCAT；
reference gene: EF1 α -3F: TCAAGTATGCSTGGGTKC;
EF1α-3R：TCAATAATSAGGACAGCACAG。
the invention has the beneficial effects that:
the rapid extraction method of potato total RNA reduces the dependence on equipment such as a low-temperature centrifuge and toxic reagents such as chloroform, reduces the difficulty of extraction operation, shortens the extraction operation time, reduces the technical threshold of RNA extraction, and achieves the advantages of simple and convenient operation, easy popularization and no reduction of RNA quality and concentration. The rapid potato total RNA extraction method establishes an RT-PCR method for detecting potato viruses, and the newly designed primer PCR reaction program is quicker, so that the potato virus detection process is accelerated.
The potato total RNA rapid extraction method and the potato virus RT-PCR detection method are suitable for the fields of laboratory monitoring, production field monitoring, identification, screening, plant inspection and quarantine and the like of antiviral materials. The extraction method and the monitoring method are applied to early diagnosis of various virus diseases of potato seedlings and seed potatoes, can improve the quality monitoring and virus disease prevention efficiency of the potato seeds and potatoes, and have important application and popularization values.
Drawings
FIG. 1 is a photograph showing the result of agarose electrophoresis identification of the results of PCR amplification in examples 4 to 15 and water control.
Detailed Description
The following embodiments are used for further illustrating the technical scheme of the present invention, but not limited thereto, and all modifications and equivalents of the technical scheme of the present invention are included in the scope of the present invention without departing from the spirit and scope of the technical scheme of the present invention. The process equipment or apparatus not specifically noted in the following examples are all conventional equipment or apparatus in the art, and the raw materials and the like used in the examples of the present invention are commercially available unless otherwise specified; unless specifically indicated, the technical means used in the embodiments of the present invention are conventional means well known to those skilled in the art.
Example 1
The embodiment provides an RNA extraction test strip for extracting total RNA of potatoes and a preparation method thereof.
The RNA extraction test strip used in the embodiment is made of slow qualitative filter paper with the size of 2mm multiplied by 55mm, one end of the test strip is a nucleic acid binding area, and the size of the test strip is 2mm multiplied by 5mm; the other end is a wax sealing area with the size of 2mm multiplied by 50mm.
The preparation method of the RNA extraction test strip in the embodiment is as follows:
first, enzyme removal treatment is performed: the slow qualitative filter paper is soaked in 0.1% DEPC water solution overnight, taken out and autoclaved at 121 ℃ for 15min, and then dried.
And then wax sealing treatment is carried out:
cutting the slow qualitative filter paper obtained by enzyme removal treatment into a filter paper strip A with the width of 50mm and a filter paper strip B with the width of 60mm respectively, wherein the length of the filter paper strip is not strictly limited and can be any length; and (3) completely putting the filter paper strip A into melted paraffin, taking out and cooling, clamping the cooled filter paper strip A by using two filter paper strips B, passing through a thermoplastic machine to obtain the filter paper strip B with a 10mm wide non-wax sealed nucleic acid binding area, and cutting according to the size specification of the finished product to obtain the RNA extraction test paper strip.
Example 2
The embodiment provides a rapid extraction method of total RNA of potatoes, which adopts the RNA extraction test strip prepared in the embodiment 1 for extraction, and comprises the following specific extraction steps:
step 1, 15mg of potato tissues are taken and added into a grinding bag, 300 mu L of lysate is added, a small hammer is utilized for fully grinding, the potato tissues and the lysate are fully mixed, and the potato tissues and the lysate are placed at room temperature for 15min for fully cracking, so that tissue homogenate is obtained;
the cracking liquid used in the step 1 is a trizol reagent which is a commercial reagent, the production formula is Sieimer Feishier, the product number is 15596026, and the specific preparation method comprises the following steps: 500mL of redistilled phenol, 250g of guanidine isothiocyanate, 293mL of distilled water, 17.6mL of 0.75M sodium citrate solution with the pH value more than or equal to 7, 26.4mL of 10% sodium dodecyl sarcosinate solution and 50mL of 2M NaAc solution with the pH value more than or equal to 4 are uniformly mixed.
Step 2, immersing the nucleic acid binding region of the RNA extraction test strip into the tissue homogenate obtained in the step 1, and repeatedly carrying out 3 times of entry and exit to enable the tissue homogenate to fully infiltrate the nucleic acid binding region of the RNA extraction test strip;
step 3, immersing the nucleic acid binding area of the RNA extraction test strip immersed in the tissue homogenate in the step 2 into a cleaning solution for cleaning; the method comprises the following steps: immersing the nucleic acid binding area of the RNA extraction test strip immersed in the lysis buffer solution in the step 2 in 1mL of cleaning solution for 5min, shaking and cleaning for 1-2 seconds, and taking out to separate solid impurities from the test strip; immersing the nucleic acid binding area of the once-cleaned RNA extraction test strip into 1mL of new cleaning solution again, immersing for 1min, shaking and cleaning for 1-2 seconds, taking out, and performing light-weight or air-drying until no obvious liquid drops exist.
The cleaning solution used in the step 3 is RNase-free H 2 O75% ethanol solution.
Step 4: immersing the nucleic acid binding region of the RNA extraction test strip washed in the step 3 into 30 mu LRNase-free H 2 Soaking in O for 3min, taking out the discarded test strip, and obtaining the soaking solution which is the total RNA sample of the potatoes.
Example 3
The embodiment provides a potato virus RT-PCR detection method.
The potato total RNA sample is obtained by adopting the potato total RNA rapid extraction method provided in the embodiment 2, the cDNA of the sample is obtained by taking the potato total RNA sample as a template, then the cDNA of the sample is taken as the template, a single potato virus primer pair is used for carrying out PCR reaction or multiple potato virus primer pairs are used for carrying out multiple PCR reaction, the principle of carrying out multiple PCR reaction is that the product size difference is more than 20bp, and agarose electrophoresis identification is carried out on the obtained PCR reaction liquid.
In this embodiment, the specific method for obtaining the cDNA of the sample by using the potato total RNA sample as the template is as follows:
mu.L of potato total RNA sample was aspirated, 4. Mu.L of 5 XRTMIX was added, and after thorough mixing, incubation was performed at 37℃for 15min and at 85℃for 1min, to obtain sample cDNA.
The 5 XRTMIX used in this example was 5X EasyQuick RT MasterMix, which was a product of century, and contains all reagents required for reverse transcription of cDNA from an RNA template, such as easy quick RT reverse enzyme, RNase Inhibitor, random 6mers, oligo dT Primer, dNTP, EQ-RT Buffer, etc.; wherein the Random primer of Random 6mers has a composition of Random polynucleotide 5'd (N6) 3' [ N=A, C, G, T ] of 6 bases in length.
In this example, the method for PCR amplification using cDNA of the sample as a template comprises: sucking 1-2 mu L of sample cDNA, adding 12.5 mu L of 2 XPCR MIX, adding the final concentration of the upstream and downstream primers to 0.2 mu M, and supplementing ddH2O to 25 mu L to obtain PCR reaction solution; putting the PCR reaction liquid into a PCR instrument, and carrying out PCR reaction at 95 ℃ for 5min, 20s denaturation at 95 ℃, 20s annealing at 54.5 ℃, 20s extension at 72 ℃,30 times of circulation and 5min final extension at 72 ℃;
the upstream and downstream primers comprise the following primers:
potato Y virus: PVY-2F: CCAACCCTTAGGCAAATCA;
PVY-2R：CGTGATGTGACCTCATAAAAGT；
potato virus M: PVM-1F: TCGGCGACCCCATACAAT;
PVM-1R：CAGGTCTGCCTTTTCACTTC；
potato virus a: PVA-2F: CAGGTATGGTCTTCAACGCA;
PVA-2R：TTCCGTCCAGTCCAAACA；
potato virus X: PVX-2F: CAAGCAAAAGATGAGACCCT;
PVX-2R：TTGTGAGAAGGGAATACGC；
potato virus S: XPVS-2F: CAGGGGATAAGCAAAGGTAGGC;
XPVS-2R：GCCAGACCCAGATTACCAAAGAAA；
potato spindle tuber virus: PSTvd-2F: GGGAAACCTGGAGCGAACT;
PSTVd-2R：TAGTAGCYGAAGCGACAGC；
potato leaf curl virus: plrv_f: CCACTCCAACTCCCCAGAAG;
PLRV_R：TACATAGGGACGGCTTGCAT；
reference gene: EF1 α -3F: TCAAGTATGCSTGGGTKC;
EF1α-3R：TCAATAATSAGGACAGCACAG。
example 4
This example uses the methods of example 2 and example 3 to extract total potato RNA and perform virus RT-PCR detection.
In this example, potato virus M, potato spindle tuber virus, potato leaf curl virus and potato virus S were detected, and multiplex PCR detection was performed with 4 pairs of primers as shown in Table 1:
TABLE 1
Example 5
This example uses the methods of example 2 and example 3 to extract total potato RNA and perform virus RT-PCR detection.
In this example, potato virus S and potato virus X were detected, and double PCR detection was performed with 2 pairs of primers as shown in Table 2:
TABLE 2
Example 6
This example uses the methods of example 2 and example 3 to extract total potato RNA and perform virus RT-PCR detection.
In this example, potato virus S and reference genes were detected, and double PCR detection was performed with 2 pairs of primers, the primer pairs being shown in Table 3:
TABLE 3 Table 3
Example 7
This example uses the methods of example 2 and example 3 to extract total potato RNA and perform virus RT-PCR detection.
In this example, potato virus S, potato virus Y and potato leaf curl were detected by multiplex PCR with 3 pairs of primers shown in Table 4:
TABLE 4 Table 4
Example 8
This example uses the methods of example 2 and example 3 to extract total potato RNA and perform virus RT-PCR detection.
In this example, potato virus Y was detected, and PCR detection was performed with 1 pair of primers shown in Table 5:
TABLE 5
Example 9
This example uses the methods of example 2 and example 3 to extract total potato RNA and perform virus RT-PCR detection.
In this example, potato virus A was detected, and PCR was performed with 1 pair of primers as shown in Table 6:
TABLE 6
Example 10
This example uses the methods of example 2 and example 3 to extract total potato RNA and perform virus RT-PCR detection.
In this example, potato virus M was detected, and PCR was performed with 1 pair of primers as shown in Table 7:
TABLE 7
Example 11
This example uses the methods of example 2 and example 3 to extract total potato RNA and perform virus RT-PCR detection.
In this example, potato spindle tuber virus was detected, and PCR detection was performed with 1 pair of primers as shown in Table 8:
TABLE 8
Example 12
This example uses the methods of example 2 and example 3 to extract total potato RNA and perform virus RT-PCR detection.
In this example, potato virus X was detected, and PCR was performed with 1 pair of primers as shown in Table 9:
TABLE 9
Example 13
This example uses the methods of example 2 and example 3 to extract total potato RNA and perform virus RT-PCR detection.
In this example, potato virus S was detected, and PCR detection was performed with 1 pair of primers shown in Table 10:
table 10
Example 14
This example uses the methods of example 2 and example 3 to extract total potato RNA and perform virus RT-PCR detection.
In this example, the reference gene was detected, and 1 pair of primers were PCR-detected, and the primer pairs are shown in Table 11:
TABLE 11
Example 15
This example uses the methods of example 2 and example 3 to extract total potato RNA and perform virus RT-PCR detection.
In this example, potato leaf curl virus was detected, and PCR detection was performed with 1 pair of primers as shown in Table 12:
table 12
FIG. 1 is a photograph showing the results of agarose electrophoresis identification of the results of PCR amplification of example 4-example 15 and water control, lanes: m: marker, from bottom to top, is 100bp, 250bp, 500bp; 1. 2, 3: PVM, PSTVd, PLRV, PVS quadruple PCR; 4.5, 6: PVS and PVX double PCR; 7. 8, 9: PVS and EF-1 alpha double PCR; 10. 11, 12: PVS, PVY, PLRV triple PCR; 13. 14: water control; 15: y virus PCR results; 16: a virus PCR result; 17: m virus PCR results; 18: viroid PCR results; 19: x virus PCR results; 20: s virus PCR results; 21: PCR results of EF-1 alpha reference gene; 22: PLRV virus PCR results.
The result of fig. 1 shows that the rapid extraction method of potato total RNA provided by the invention can rapidly obtain high-concentration and high-quality potato total RNA samples for virus identification. The RT-PCR method for detecting the potato viruses is established according to the potato total RNA rapid extraction method established by the invention, the newly designed primer PCR reaction program is quicker, and clear and accurate identification results can be obtained no matter whether a single potato virus primer pair is used for PCR reaction or multiple potato virus primer pairs are used for multiplex PCR reaction, so that the potato virus detection process is accelerated.
Claims (2)
1. A potato virus RT-PCR detection method is characterized in that a potato total RNA sample is obtained by adopting a potato total RNA rapid extraction method, cDNA of the sample is obtained by taking the potato total RNA sample as a template, then multiple PCR reactions are carried out by taking the cDNA of the sample as the template and a plurality of potato virus primer pairs, and agarose electrophoresis identification is carried out on the obtained PCR reaction liquid;
the method for obtaining the sample cDNA by taking the potato total RNA sample as a template comprises the following steps: sucking 16 mu L of potato total RNA sample, adding 4 mu L of 5 xRT MIX, fully mixing, incubating for 15min at 37 ℃ and incubating for 1min at 85 ℃ to obtain sample cDNA;
the method for PCR amplification by using cDNA of the sample as a template comprises the following steps: 1-2. Mu.L of sample cDNA was extracted, 12.5. Mu.L of 2 XPCR MIX was added, and the final concentration of the upstream and downstream primers was adjusted to 0.2. Mu.M, ddH 2 O is added to 25 mu L, namely the PCR reaction liquid; putting the PCR reaction liquid into a PCR instrument, and carrying out PCR reaction at 95 ℃ for 5min, 20s denaturation at 95 ℃, 20s annealing at 54.5 ℃, 20s extension at 72 ℃,30 times of circulation and 5min final extension at 72 ℃;
the upstream and downstream primers comprise the following primers:
potato Y virus: PVY-2F: CCAACCCTTAGGCAAATCA;
PVY-2R：CGTGATGTGACCTCATAAAAGT；
potato virus M: PVM-1F: TCGGCGACCCCATACAAT;
PVM-1R：CAGGTCTGCCTTTTCACTTC；
potato virus a: PVA-2F: CAGGTATGGTCTTCAACGCA;
PVA-2R：TTCCGTCCAGTCCAAACA；
potato virus X: PVX-2F: CAAGCAAAAGATGAGACCCT;
PVX-2R：TTGTGAGAAGGGAATACGC；
potato virus S: XPVS-2F: CAGGGGATAAGCAAAGGTAGGC;
XPVS-2R：GCCAGACCCAGATTACCAAAGAAA；
potato spindle tuber virus: PSTvd-2F: GGGAAACCTGGAGCGAACT;
PSTVd-2R：TAGTAGCYGAAGCGACAGC；
potato leaf curl virus: plrv_f: CCACTCCAACTCCCCAGAAG;
PLRV_R：TACATAGGGACGGCTTGCAT；
reference gene: EF1 α -3F: TCAAGTATGCSTGGGTKC;
EF1α-3R：TCAATAATSAGGACAGCACAG；
the extraction method adopts an RNA extraction test strip for extraction, and the specific extraction steps are as follows:
step 1, fully mixing and grinding potato tissues and a lysate, wherein the lysate is a commercial trizol reagent, the mass-volume ratio of the potato tissues to the lysate is 10-20 mg:300 mu L, and the lysis time is 10-20 min, so as to obtain tissue homogenate;
step 2, immersing the nucleic acid binding region of the RNA extraction test strip into the tissue homogenate obtained in the step 1, so that the tissue homogenate fully infiltrates the nucleic acid binding region of the RNA extraction test strip;
step 3, immersing the nucleic acid binding area of the RNA extraction test strip immersed in the tissue homogenate in the step 2 into a cleaning solution for cleaning, wherein the cleaning solution is RNase-free H 2 The specific method for cleaning the 75% ethanol solution prepared by O comprises the following steps: immersing the nucleic acid binding area of the RNA extraction test strip immersed in the lysis buffer solution in the step 2 in 1mL of cleaning solution for 5min, shaking and cleaning for 1-2 seconds, taking out, immersing in the 1mL new cleaning solution again for 1min, shaking and cleaning for 1-2 seconds, taking out, and lightly throwing or airing until no obvious liquid drops exist;
step 4, immersing the nucleic acid binding region of the RNA extraction test strip washed in the step 3 into RNase-free H 2 Soaking in O for 3min to obtain soaking solution which is the total RNA sample of potato;
the RNA extraction test strip is made of slow qualitative filter paper, the two ends of the RNA extraction test strip are respectively provided with a nucleic acid binding area and a wax-sealed handle area, and the length ratio of the nucleic acid binding area to the wax-sealed handle area is 1:10;
the preparation method of the RNA extraction test strip comprises enzyme removal treatment and wax sealing treatment, wherein the enzyme removal treatment is to soak the slow qualitative filter paper in 0.1% DEPC water solution overnight, take out and sterilize at 121 ℃ for 15min under high pressure and then dry; the wax sealing treatment is to cut the slow qualitative filter paper obtained by enzyme removal into a filter paper strip A with the width of 50mm and a filter paper strip B with the width of 60mm, completely putting the filter paper strip A into melted paraffin, taking out and cooling, clamping the cooled filter paper strip A by using two filter paper strips B, and passing through a thermoplastic machine to obtain a filter paper strip B with a 10 mm-wide non-wax sealed nucleic acid binding area, and cutting according to the size specification of a finished product to obtain the RNA extraction test paper strip.
2. The potato virus RT-PCR detection method according to claim 1, wherein the specific method for obtaining the cDNA of the sample by taking the potato total RNA sample as a template is as follows: mu.L of potato total RNA sample was aspirated, 4. Mu.L of 5 XRT MIX was added, and after thorough mixing, incubation was performed at 37℃for 15min and at 85℃for 1min, to obtain sample cDNA.
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| CN101210271A (en) * | 2006-12-27 | 2008-07-02 | 河北省农林科学院经济作物研究所 | Potato virus and viroid detecting kit and application thereof |
| CN104178585A (en) * | 2014-08-14 | 2014-12-03 | 福建省农业科学院作物研究所 | Potato virus detection primers and potato virus detection method |
| CN112430687A (en) * | 2020-12-10 | 2021-03-02 | 广西大学 | Method for rapidly detecting potato virus based on RT-PCR (reverse transcription-polymerase chain reaction) of filter paper strip capture virus nucleic acid |
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| CN101210271A (en) * | 2006-12-27 | 2008-07-02 | 河北省农林科学院经济作物研究所 | Potato virus and viroid detecting kit and application thereof |
| CN104178585A (en) * | 2014-08-14 | 2014-12-03 | 福建省农业科学院作物研究所 | Potato virus detection primers and potato virus detection method |
| CN112430687A (en) * | 2020-12-10 | 2021-03-02 | 广西大学 | Method for rapidly detecting potato virus based on RT-PCR (reverse transcription-polymerase chain reaction) of filter paper strip capture virus nucleic acid |
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