Specific detection target Ps54945515 of phytophthora syringae and application of specific detection target Ps54945515

Abstract

The invention belongs to the field of molecular detection, and discloses a specific detection target Ps54945515 of phytophthora syringae as well as a PCR (Polymerase Chain Reaction) detection method, a fluorescence detection method and a nucleic acid test strip detection method which are established based on the target. The PCR detection method provided by the invention can amplify a 770 bp specific band aiming at the target Ps54945515, the real-time fluorescence detection method provided by the invention can realize the specific detection of the target Ps54945515, and meanwhile, the invention also establishes a rapid, specific, sensitive and portable phytophthora syringae detection method based on recombinase polymerase amplification and a nucleic acid test strip by taking the Ps54945515 as the target. Sensitivity experiments show that the genomic DNA containing 1.28 pg / mu L of phytophthora syringae generates obvious detection strips, and the sensitivity of real-time fluorescent PCR is also 1.28 pg / mu L, which indicates that the sensitivity of a new detection target is high. Therefore, the rapid detection method for the phytophthora syringae established by the invention plays an important role in developing early diagnosis of diseases caused by the phytophthora syringae through specificity and sensitivity evaluation.

Description

Technical Field

[0001] The present invention belongs to the field of molecular detection, and particularly relates to a specific detection target Ps54945515 of Phytophthora syringae and its application. Background Art

[0002] Phytophthora syringae ( Phytophthora syringae , PS) is a quarantine fungal disease in the List of Quarantine Pests of Imported Plants of the People's Republic of China. Its natural hosts mainly include Syringa, Castanea henryi, Sargassum, Pyracantha fortuneana, Citrus, Foeniculum vulgare, Malus pumila, Prunus armeniaca, Prunus avium, Prunus persica, Pyrus pyrifolia and Pyrus plants. It can also inoculate hosts such as Aesculus hippocastanum, Alnus cremastogyne, Alnus virginiana, Corylus heterophylla, Crataegus pinnatifida, Jasminum nudiflorum, Ligustrum lucidum, Prunus salicina and Quercus plants to produce pathogenicity. The harms caused are: (1) causing the withering and blight of Syringa branches; (2) causing fruit rot and ear rot of woody Rosaceae plants such as apples and pears; (3) causing root rot of sweet chestnuts and beech; (4) causing fruit brown rot, branch and flower blight of citrus. Phytophthora syringae is mainly transmitted through the soil and is common in orchard soil. It can still persist on the ground for 15 years after orchard plowing. Even if stored in swamp soil for more than 2 years, Phytophthora syringae still has pathogenicity. Phytophthora syringae is also transmitted through water and spreads in irrigation canals and reservoirs that supply water to citrus orchards in California in winter and spring. Summary of the Invention

[0004] Aiming at the deficiencies in the current prior art, the present invention provides a specific detection target Ps54945515 of Phytophthora syringae, and based on this target, a PCR detection method, a fluorescence detection method and a nucleic acid test strip detection method are established. The rapid detection method of Phytophthora syringae established by the present invention plays an important role in the early diagnosis of diseases caused by the development of Phytophthora syringae through specificity and sensitivity evaluation.

[0005] In order to achieve the above invention purpose, the present invention provides the following technical solutions.

[0006] On the one hand, the present invention provides a specific detection target Ps54945515 of Phytophthora syringae ( Phytophthora syringae ), and the nucleotide sequence of the specific detection target Ps54945515 is shown as SEQ ID No: 1.

[0007] On the other hand, the present invention also provides a PCR detection primer group for the specific detection target Ps54945515, the PCR detection primers include a forward primer and a reverse primer, and the sequences of the forward primer and the reverse primer are shown as SEQ ID No: 2 and SEQ ID No: 3 respectively.

[0008] On the other hand, the present invention also provides a real-time fluorescence PCR detection primer and probe combination for specifically detecting the target Ps54945515. The real-time fluorescence PCR detection primer includes a forward primer and a reverse primer. The sequences of the forward primer and the reverse primer are shown in SEQ ID No: 4 and SEQ ID No: 5 respectively, and the probe primer sequence is shown in SEQ ID No: 6. The 5' end of the probe is connected with a FAM modification, and the 3' end is connected with an MGB modification.

[0009] On the other hand, the present invention also provides a primer and probe combination for detecting the specific detection target Ps54945515 based on the nucleic acid test strip technology. The primer includes a forward primer and a reverse primer. The sequences of the forward primer and the reverse primer are shown in SEQ ID No: 7 and SEQ ID No: 8 respectively, and the probe sequence is shown in SEQ ID No: 9.

[0010] In some embodiments, the 5' end of the reverse primer is connected with a Biotin modification; the 5' end of the probe is connected with a FAM modification, the 3' end is connected with a C3 Space modification, and a dSpacer is connected between the 31st and 32nd nucleotides.

[0011] On the other hand, the present invention also provides a kit for detecting Phytophthora syringae ( Phytophthora syringae ), and the kit includes the above-mentioned PCR detection primer group, the above-mentioned real-time fluorescence PCR detection primer and probe combination, or the above-mentioned primer and probe combination.

[0012] In some embodiments, the kit includes the above-mentioned primer and probe combination, and also includes a reaction unit tube containing a lyophilized enzyme mixture, a buffer solution, an initiator, detection water, and a nucleic acid test strip.

[0013] On the other hand, the present invention also provides the above-mentioned PCR detection primer group, the above-mentioned real-time fluorescence PCR detection primer and probe combination, the above-mentioned primer and probe combination, or any one of the above-mentioned kits for use in detecting Phytophthora syringae ( Phytophthora syringae ).

[0014] In some embodiments, the use is as follows 1) Extract the genomic DNA of the sample to be tested, 2) Perform PCR amplification using the above-mentioned PCR detection primer group or the above-mentioned kit. When a specific band of 770 bp appears in the amplification product, it indicates that Phytophthora syringae ( Phytophthora syringae ) is detected.

[0015] In some embodiments, the use is as follows 1) Extract the genomic DNA of the sample to be tested; 2) Using the genomic DNA as a template, perform RPA amplification with the primer and probe combination or the kit described above; 3) Detect the RPA amplification product using a nucleic acid test strip; when two bands appear on the test strip, one in the quality control area and one in the detection area, the result is positive, indicating that the sample contains Phytophthora syringae ( Phytophthora syringae ); when only one band appears in the quality control area and no band appears in the detection area on the test strip, the result is negative, indicating that the sample does not contain Phytophthora syringae ( Phytophthora syringae ).

[0016] Compared with the prior art, the present invention has the following technical effects: 1) The present invention provides a highly reliable specific molecular detection target Ps54945515 for Phytophthora syringae ( Phytophthora syringae ), and based on the new target, a sensitive and specific molecular detection method is established, which plays an important role in the development of early diagnosis of diseases caused by Phytophthora syringae.

[0017] 2) The fluorescence quantitative PCR method and the isothermal amplification / nucleic acid test strip portable detection method established by the present invention can significantly detect 1.28 pg / μL (15.6 copies / μL) of Phytophthora syringae genomic DNA, indicating that the new detection target Ps54945515 of Phytophthora syringae can be used to develop highly sensitive detection technologies. Description of the Drawings

[0018] Figure 1 For P. syringae Gel electrophoresis diagram of PCR primer combination for detecting fungal genomic DNA based on the specific detection target Ps54945515. Among them, from left to right are: M, Maker; 1. Phytophthora syringae Phytophthora syringae ; 2. Phytophthora hibernalis Phytophthora hibernalis ; 3. Phytophthora capsici Phytophthora capsici ; 4. Phytophthora sojae Phytophthorasojae ; 5. Phytophthora cinnamomi Phytophthora cinnamomi ; 6. Phytophthora palmivora Phytophthorapalmivora ; 7. Phytophthora citrophthora Phytophthora citrophthora ; 8. Pythium oligandrum Pythiumoligandrum ; 9. Phytophthora hydropathica Phytophthora gonapodyides ; 10. Phytophthora cactorum Phytophthoracactorum ; 11. Phytophthora infestans Phytophthora infestans ; 12. Pythium aphanidermatum Pythium aphanidermatum ; 13. Phytophthora nicotianae Phytophthora nicotianae ; 14. Negative control.

[0019] Figure 2 For the real-time fluorescence curve of detecting fungal genomic DNA with the real-time fluorescence PCR primer-probe combination specifically detecting the target Ps54945515. 1. Phytophthora syringae P. syringae ; 2. Phytophthora hibernalis Phytophthora syringae ; 3. Phytophthora capsici Phytophthora hibernalis ; 4. Phytophthora sojae Phytophthora capsici ; 5. Phytophthora cinnamomi Phytophthorasojae ; 6. Phytophthora palmivora Phytophthora cinnamomi ; 7. Phytophthora citrophthora Phytophthorapalmivora ; 8. Pythium oligandrum Phytophthora citrophthora ; 9. Phytophthora hydropathica Pythiumoligandrum ; 10. Phytophthora cactorum Phytophthora gonapodyides ; 11. Phytophthora infestans Phytophthoracactorum ; 12. Pythium aphanidermatum Phytophthora infestans ; 13. Phytophthora nicotianae Pythium aphanidermatum ; 14. Negative control Phytophthora nicotianae ; 14. Negative control

[0020] Figure 3 For the test strip detection result of detecting fungal genomic DNA with the isothermal nucleic acid amplification test strip method specifically detecting the target Ps54945515. Among them, 1. Phytophthora syringae P. syringae ; 2. Phytophthora hibernalis Phytophthora syringae ; 3. Phytophthora capsici Phytophthora hibernalis ; 4. Phytophthora sojae Phytophthora capsici ; 5. Phytophthora cinnamomi Phytophthorasojae ; 6. Phytophthora palmivora Phytophthora cinnamomi ; 7. Phytophthora citrophthora Phytophthorapalmivora ; 8. Pythium oligandrum Phytophthora citrophthora ; 9. Phytophthora hydropathica Pythiumoligandrum ; 10. Phytophthora cactorum Phytophthora gonapodyides ; 11. Phytophthora infestans Phytophthoracactorum ; 12. Pythium aphanidermatum Phytophthora infestans ; 13. Phytophthora nicotianae Pythium aphanidermatum ; 13. Phytophthora nicotianae Phytophthora nicotianae ; 14. Negative control

[0021] Figure 4 For the real-time fluorescence curve of detecting different concentrations of P. syringae fungal genomic DNA with the real-time fluorescence PCR primer-probe combination specifically detecting the target Ps54945515. Among them, 1, 1.28 ng / μL; 2, 0.128 ng / μL; 3, 12.8 pg / μL; 4, 1.28 pg / μL; 5, 0.128 pg / μL P. syringae ; 13. Phytophthora nicotianae P. syringae; 6, Negative control.

[0022] Figure 5 is based on P. syringae The results of strip detection of genomic DNA at different concentrations by the isothermal amplification nucleic acid strip method for specifically detecting the target Ps54945515. Among them, 1, 1.28 ng / μL; 2, 0.128 ng / μL; 3, 12.8 pg / μL; 4, 1.28 pg / μL; 5, 0.128 pg / μL P. syringae ; 6, Negative control. P. syringae ; 6, Negative control. Specific embodiments

[0023] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description progresses. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications and replacements all fall within the protection scope of the present invention.

[0024] Example 1

[0025] The present invention performs a whole-genome alignment on the genomic data of the fungi of the genus recorded in NCBI GenBank Phytophthora to obtain 162 specific gene fragments of Phytophthora syringae ), and predicts and analyzes the sequence conservation by calculating the test factor, and selects the highly conserved specific detection target Ps54945515 with a test factor of 0.946, and its DNA sequence is shown in SEQ ID No: 1.

[0026]

[0027] Example 2

[0028] Based on this new detection target, a common PCR detection method was established, and genomic DNAs of fungi belonging to the same genus but different species or having the same / close hosts were used as templates to verify the specificity of the detection target.

[0029] The detection primer composition used in the PCR detection method consists of a forward primer Ps-PCR-F and a reverse primer Ps-PCR-R. The primer sequences are as follows: Ps-PCR-F: 5'- TGGATATGACAGTGGGAACGTC-3' (SEQ ID No: 2) Ps-PCR-R: 5'- CACCATGACATCACAGTAGTCG-3' (SEQ ID No: 3) The DNA of the test bacteria was amplified by the designed primer combination Ps-PCR-F / Ps-PCR-R: 1.0 μL of each of the forward and reverse primers (10 mM), 10 μL of 2× Taq PCR MasterMix Ⅱ, 6 μL of sterilized deionized water, and 2 μL of genomic DNA were taken, and the total volume was 20 μL.

[0030] The PCR amplification program was as follows: denaturation at 94 °C for 3 minutes, 94 °C for 3 min; 94 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min, for 30 cycles; 72 °C for 5 min. After the reaction ended, 5 μL of the amplification product was electrophoresed on a 1.5% agarose gel for 30 min, and after staining, it was detected and photographed with a gel imaging system.

[0031] The results were as Figure 1 shown. Ps-PCR-F / Ps-PCR-R could specifically amplify a 770-bp band only from the DNA of Phytophthora syringae strains, which was consistent with the designed band, while the other control bacteria did not produce the target band, indicating that the primer combination designed based on the new detection target Ps54945515 could achieve specific detection of Phytophthora syringae ( P. syringae )

[0032] Example 3

[0033] To verify the specificity of the new detection target Ps54945515 of Phytophthora syringae ( P. syringae ), a set of forward and reverse primers and TaqMan probes were designed in this example for detecting the genomic DNA of the plant pathogenic bacteria in Example 2 by real-time fluorescence PCR method.

[0034] The detection primer composition used in this real-time fluorescence PCR detection method consists of a forward primer Ps-qPCR-F, a reverse primer Ps-qPCR-R, and a probe. The primer and probe sequences are as follows: Ps-qPCR-F: 5'-GGATATGACAGTGGGAACG-3' (SEQ ID No: 4) Ps-qPCR-R: 5'-TTCCTTGTGTAGACAGACGCTGACACCC-3' (SEQ ID No: 5) Ps-qPCR-Pb: 5'-CTGAAGAATAACGCCAACTGGACCCCC-3' (SEQ ID No: 6). The 5' end of the probe Ps-qPCR-Pb is linked with a FAM modification, and the 3' end is linked with an MGB modification.

[0035] Detect fungal genomic DNA through the designed primer-probe combination Ps-qPCR-F / Ps-qPCR-R / Ps-qPCR-Pb: Take 0.5 μL each of the forward and reverse primers and the probe (10 mM), 10 μL of PerfectStart ® Ⅱ Probe qPCR SuperMix (TransGen Biotech Co., Ltd.), 6.5 μL of sterilized deionized water, and 2 μL of genomic DNA. The total volume is 20 μL. Reaction conditions: Pre-denaturation at 50°C for 2 min, denaturation at 95°C for 10 min; enter 40 cycles, 95°C for 15 s, 60°C for 1 min.

[0036] The results are as Figure 2 , the Ct value of Phytophthora syringae of sample No. 1 is 25.02, judged as positive; while the Ct values of other fungal genomic DNAs (strains No. 2 - 13 are the same as those in Example 1, strains No. 2 - 13) are all greater than 35, judged as negative, indicating that the primer-probe combination Ps-qPCR-F / Ps-qPCR-R / Ps-qPCR-Pb designed based on the new detection target Ps54945515 can achieve fluorescence detection of Phytophthora syringae ( P. syringae ) and has specificity.

[0037] Example 4

[0038] In order to achieve rapid and specific on-site detection of Phytophthora syringae ( P. syringae ), based on P. syringaeA sensitive, accurate, rapid and on-site available isothermal amplification strip detection technology system has been established for the new detection target Ps54945515. The detection primer and probe composition used in this detection technology system includes: forward primer Ps-RPA-F, reverse primer Ps-RPA-R and probe Ps-RPA-Pb, and the specific sequences are as follows: Ps-RPA-F: 5'-CCGCCGTCAAATGCTATCTGAAGGAAATCTACG-3' (SEQ ID No: 7) Ps-RPA-R: 5'- CCACCACCATGACATCACAGTAGTCGCTCAG-3' (SEQ ID No: 8), and the 5' end of the reverse primer is linked with a Biotin modification.

[0039] Ps-RPA-Pb: 5'-TTCGCCTGTCAAGAACTCGCTGGACCACCAGGCAACGCACGCTCGTC-3' (SEQID No: 9), the 5' end of the probe Ps-RPA-Pb is linked with a FAM modification, the 3' end is linked with a C3 Space modification, and a dSpacer is linked between the 31st and 32nd nucleotides.

[0040] The detection method for detecting the genomic DNA of Phytophthora syringae using this primer-probe composition is as follows: After mixing 29.4 μL of buffer A, 11.4 μL of sterile water, 1.7 μL of 10 μM forward primer, 2.0 μL of 10 μM reverse primer and 0.3 μL of 10 μM probe solution, add it to a 0.2 mL reaction unit tube containing freeze-dried enzyme powder), add 2 μL of sample DNA, mix well and then add 2.5 μL of buffer B (280 mM magnesium acetate), and place the reaction tube in a 39°C constant temperature heater for reaction for 20 min.

[0041] After the reaction is completed, take out 5 μL of the reaction solution, add 95 μL of deionized water and mix well, then put in a nucleic acid test strip, and the result is as Figure 3 shown. Only Phytophthora syringae has a positive detection band, while other fungal genomic DNAs (strains numbered 2-13 are the same as those in Example 1) have no detection band, indicating that the isothermal amplification strip detection technology can specifically detect Phytophthora syringae.

[0042] Example 5

[0043] In order to evaluate the sensitivity of the real-time fluorescence PCR detection system established based on P. syringae the new detection target Ps54945515, take P. syringae the genomic DNA nucleic acid of 1.28 ng / μL (1.56×10 4(copies / μL), perform 10-fold serial dilutions to obtain genomic DNA with concentrations of 0.128 ng / μL (1.56×10 3 copies / μL), 12.8 pg / μL (1.56×10 2 copies / μL), 1.28 pg / μL (1.56×10 1 copies / μL), 0.128 pg / μL (1.56×10 0 copies / μL). Detect the diluted DNA nucleic acid by real-time fluorescence PCR.

[0044] Detect fungal genomic DNA using the designed primer-probe combination Ps-qPCR-F / Ps-qPCR-R / Ps-qPCR-Pb: Take 0.5 μL each of the forward and reverse primers and the probe (10 mM), 10 μL of PerfectStart®Ⅱ Probe qPCR SuperMix (TransGen Biotech Co., Ltd., Beijing), 6.5 μL of sterilized deionized water, and 2 μL of genomic DNA, with a total volume of 20 μL. Reaction conditions: Pre-denaturation at 50 °C for 2 min, denaturation at 95 °C for 10 min; enter 40 cycles, 95 °C for 15 s, 60 °C for 1 min.

[0045] The results are as Figure 4 shown. The Ct values of genomic DNA at 1.28 ng / μL, 0.128 ng / μL, 12.8 pg / μL, and 1.28 pg / μL are 26.05, 28.44, 30.63, and 33.01 respectively, while the Ct value of genomic DNA at 0.128 pg / μL P. syringae is greater than 35, which is determined to be negative, indicating that the sensitivity of the qPCR primer-probe designed based on the new detection target Ps54945515 is 1.28 pg / μL of genomic DNA, demonstrating that the isothermal amplification test strip technology has high sensitivity. P. syringae Example 6

[0046] To evaluate the sensitivity of the isothermal amplification test strip detection technology system established based on the new detection target Ps54945515, take the genomic DNA nucleic acid at 1.28 ng / μL (1.56×10

[0047] of P. syringae and perform 10-fold serial dilutions to obtain concentrations of 0.128 ng / μL (1.56×10 P. syringae copies / μL), 12.8 pg / μL (1.56×10 4 copies / μL), 0.128 ng / μL (1.56×10 3 copies / μL), 12.8 pg / μL (1.56×102 copies / μL), 1.28 pg / μL (1.56×10 1 copies / μL), 0.128 pg / μL (1.56×10 0 copies / μL) of genomic DNA, and the diluted DNA nucleic acid was detected by the isothermal amplification test strip detection technique.

[0048] After mixing 29.4 μL of buffer A, 11.4 μL of sterile water, 1.7 μL of 10 μM forward primer, 2.0 μL of 10 μM reverse primer, and 0.3 μL of 10 μM probe solution, add them to a 0.2 mL reaction unit tube containing freeze-dried enzyme powder. Add 2 μL of sample DNA, mix well, and then add 2.5 μL of buffer B (280 mM magnesium acetate). Place the reaction tube in a 39°C constant temperature heater and react for 20 min. After the reaction is completed, take out 5 μL of the reaction solution, mix it with 95 μL of deionized water, and put in a nucleic acid test strip.

[0049] The results are as Figure 5 shown. The genomic DNA of 1.28 pg / μL (15.6 copies / μL) P. syringae produced a test strip band different from the negative control, indicating that the isothermal amplification test strip technology has high sensitivity and can detect 15.6 copies of genomic DNA.

[0050] Unless otherwise specifically stated, the numerical values set forth in these examples do not limit the scope of the present invention. In all examples shown and described herein, any specific value should be construed as merely exemplary and not as a limitation. Thus, other examples of the exemplary embodiments may have different values.

Claims

1. A specific detection target Ps54945515 of Phytophthora syringae Phytophthora syringae ), characterized in that The nucleotide sequence of the specific detection target Ps54945515 is as shown in SEQ ID No:

1.

2. A PCR detection primer set for specifically detecting the target Ps54945515 described in claim 1, characterized in that, The PCR detection primers include a forward primer and a reverse primer, and the sequences of the forward primer and the reverse primer are as shown in SEQ ID No: 2 and SEQ ID No: 3 respectively.

3. A real-time fluorescence PCR detection primer and probe combination for specifically detecting the target Ps54945515 as described in claim 1, characterized in that, The real-time fluorescence PCR detection primers include a forward primer and a reverse primer, and the sequences of the forward primer and the reverse primer are as shown in SEQ ID No: 4 and SEQ ID No: 5 respectively. The probe primer sequence is as shown in SEQ ID No:

6. The 5' end of the probe is linked with FAM modification, and the 3' end is linked with MGB modification.

4. A primer and probe combination for detecting the specific detection target Ps54945515 recited in claim 1 based on nucleic acid test strip technology, characterized in that, The primers include a forward primer and a reverse primer, and the sequences of the forward primer and the reverse primer are as shown in SEQ ID No: 7 and SEQ ID No: 8 respectively. The probe sequence is as shown in SEQ ID No:

9.

5. The primer and probe combination for specifically detecting the target Ps54945515 according to claim 4, characterized in that, The 5' end of the reverse primer is linked with Biotin modification; the 5' end of the probe is linked with FAM modification, the 3' end is linked with C3Spacer modification, and a dSpacer is linked between the 31st and 32nd nucleotides.

6. A kit for detecting Phytophthora syringae ( Phytophthora syringae ), characterized in that The kit includes the PCR detection primer set according to claim 2, the real-time fluorescence PCR detection primers and probe combination according to claim 3, or the primers and probe combination according to claim 4.

7. The kit according to claim 6, characterized in that, The kit includes the primers and probe combination according to claim 5, and also includes a reaction unit tube containing a freeze-dried enzyme mixture, a buffer, an initiator, detection water, and a nucleic acid detection test strip.

8. The use of the PCR detection primer set according to claim 2, the real-time fluorescence PCR detection primer and probe combination according to claim 3, the primer and probe combination according to claim 5, or the kit according to any one of claims 6-7 in detecting Phytophthora syringae ( Phytophthora syringae ).

9. The application according to claim 8, characterized in that 1) Extract the genomic DNA of the sample to be tested. 2) Perform PCR amplification using the PCR detection primer set described in claim 2 or the kit described in claim 6. When a specific band of 770 bp appears in the amplification product, it indicates the detection of Phytophthora syringae ( Phytophthora syringae ).

10. The application according to claim 8, characterized in that 1) Extract the genomic DNA of the sample to be tested. 2) Using the genomic DNA as a template, perform RPA amplification with the primers and probe combination according to claim 5 or the kit according to claim 7. 3) Use a nucleic acid test strip to detect the RPA amplification product; when two bands appear on the test strip, one in the control region and one in the test region, the result is positive, indicating that the sample contains Phytophthora syringae ( Phytophthora syringae ); when only one band appears in the control region of the test strip and no band appears in the test region, the result is negative, indicating that the sample does not contain Phytophthora syringae ( Phytophthora syringae ).

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