Specific detection target PsRrp8 of phytophthora sojae as well as detection primer and application thereof

Abstract

The invention discloses a specific detection target PsRrp8 of phytophthora sojae, a DNA (deoxyribonucleic acid) sequence of the specific detection target PsRrp8 is shown as SEQ ID NO.1. The invention further provides a primer combination for detecting the phytophthora sojae, the primer combination comprises a forward primer and a reverse primer, the sequence of the forward primer PsRrp8-qPCR-F1 is shown as SEQ ID NO.2, and the sequence of the reverse primer PsRrp8-qPCR-R1 is shown as SEQ ID NO.3. The invention further provides a kit for detecting the phytophthora sojae. The lowest detectable DNA concentration of the detection primer combination provided by the invention on the phytophthora sojae (P.sojae) is 0.332 pg.L <-1 >, which indicates that the quantitative detection technology has high sensitivity. The method can be successfully applied to phytophthora detection of phytophthora root rot disease field plants, phytophthora sojae can be rapidly detected, and a new technical means is provided for early warning of diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant pathogen detection, and particularly relates to a specific detection target PsRrp8 of Phytophthora sojae and a detection primer and application thereof. Background Art

[0002] Phytophthora sojae Phytophthora sojae Phytophthora spp. is the primary pathogen causing soybean root rot, causing significant economic losses in major soybean-producing regions worldwide. The bacterium primarily infects the soybean root system and stem base, causing plant wilt and even death. In severe cases, yield losses can reach up to 50%. Therefore, developing a highly sensitive and specific rapid detection system is of great scientific and industrial value for achieving early warning and precise prevention and control of soybean Phytophthora spp. root rot.

[0003] Currently, detection methods for soybean Phytophthora infestans mainly include morphological identification, immunological detection, and molecular biology techniques. Traditional morphological identification relies on pathogen isolation, culture, and microscopic observation. The process is cumbersome (requiring 5-7 days), prone to contamination, and has a low isolation success rate (<60%). Although immunological detection (such as ELISA) shortens the detection time (2-3 hours), it has poor specificity and is inconsistent with closely related species (such as Phytophthora infestans). P. infestans ) have cross-reactivity. Among molecular detection technologies, the loop-mediated isothermal amplification (LAMP) technique developed by Dai et al. can complete detection within 1 hour, making it suitable for rapid field screening but unable to quantitatively analyze pathogens. While the microfluidic chip detection system developed by Chen et al. enables multiplex detection, the equipment is expensive and requires specialized operators. While the most commonly used real-time fluorescence quantitative PCR (qPCR) technique has quantitative capabilities, existing primers (such as those based on the Ypt1 or Cox1 genes) are prone to cross-reactivity between closely related species, and aerosol contamination in the laboratory environment can interfere with test results.

[0004] Therefore, there is an urgent need to develop highly specific PCR detection systems based on new targets to address the limitations of existing technologies. Summary of the Invention

[0005] Aiming at the problems of low sensitivity and few specific detection targets in the prior art for biological detection of Phytophthora sojae, the present invention provides a new Phytophthora sojae ) Specific detection target PsRrp8 and its detection primers and applications.

[0006] In a first aspect, the present invention provides a specific detection target PsRrp8 for Phytophthora sojae. The DNA sequence of the specific detection target PsRrp8 is shown in SEQ ID NO.1.

[0007] In a second aspect, the present invention provides a primer combination comprising a forward primer and a reverse primer, wherein the sequence of the forward primer PsRrp8-qPCR-F1 is shown as SEQ ID NO.2, and the sequence of the reverse primer PsRrp8-qPCR-R1 is shown as SEQ ID NO.3.

[0008] In a third aspect, the present invention provides a kit for detecting Phytophthora sojae, comprising 10 μmol·L -1 A detection solution of the primer combination.

[0009] In a fourth aspect, the present invention provides use of the primer combination or the kit in detecting Phytophthora sojae.

[0010] In a fifth aspect, the present invention provides a qPCR method for detecting Phytophthora sojae, comprising the following steps: taking 1-2 μL of a DNA solution of a test object, adding Hieff UNICON ® Advanced qPCR SYBR Master Mix 8-12 μL, 8-12 μmol·L -1 0.3-0.5 μL of the forward primer PsRrp8-qPCR-F1 and the reverse primer PsRrp8-qPCR-R1 were added, and 0.5-1.5 μL of DMSO was added, and the volume was adjusted to 19-22 μL using RNA-Free H2O for qPCR amplification. The sequence of the forward primer PsRrp8-qPCR-F1 is shown in SEQ ID NO.2, and the sequence of the reverse primer PsRrp8-qPCR-R1 is shown in SEQ ID NO.3.

[0011] In certain embodiments, 2 μL of the DNA solution of the test object is added to the Hieff UNICON ® AdvancedqPCR SYBR Master Mix 10 μL, 10 μmol·L -1 0.4 μL of forward primer PsRrp8-qPCR-F1 and reverse primer PsRrp8-qPCR-R1 were added to 1 μL of DMSO and the volume was adjusted to 20 μL with RNA-Free H2O for qPCR amplification.

[0012] In certain embodiments, the qPCR amplification program is: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 5 s, extension at 60°C for 10 s, for 40 cycles; setting the melting curve to maintain at 65°C for 5 s, extension at 95°C, and cooling to 0.5°C for storage.

[0013] Compared with the existing technology, the present invention provides a highly reliable specific molecular detection target PsRrp8, as well as a qPCR detection primer combination and qPCR detection technology system for specifically detecting the target PsRrp8. The detection primer combination provided by the present invention is used to detect soybean Phytophthora ( P.sojae The lowest detectable DNA concentration was 0.332 pg·µL -1 , indicating that the quantitative detection technology has high sensitivity. The present invention can be successfully applied to the detection of Phytophthora root rot in fields with Phytophthora root rot, and can quickly detect Phytophthora in soybeans, providing a new technical means for early warning of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the specific embodiments.

[0015] Figure 1 The target region of primers F1 / R1 and its sequence alignment among different species.

[0016] Figure 2 The results of qPCR amplification of DNA from 13 pathogens using primers F1 / R1 are shown.

[0017] Figure 3 The results of qPCR amplification of DNA extracted from 10 strains of Phytophthora sojae using primers F1 / R1.

[0018] Figure 4 The amplification results of primers F1 / R1 on different concentration gradients of soybean Phytophthora DNA and the standard curve were drawn.

[0019] Figure 5 Symptoms of soybean root rot in the field and qPCR test results; AB: negative; CG: positive (C: Ct = 34.5; D: Ct = 30.0; E: Ct = 27.3; F: Ct = 26.0; G: Ct = 21.9). DETAILED DESCRIPTION

[0020] The following embodiments of the technical solution of the present invention are described in detail with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and are therefore only provided as examples and are not intended to limit the scope of protection of the present invention. It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0021] Example 1

[0022] The present invention is to use soybean phytophthora ( P.sojaeThe whole-genome protein sequences of 13 soybean pathogens, including 2,477 soybean pathogens (including 2,477 soybean pathogens) and 8 other Phytophthora species, were input into OrthoFinder v2.4.0 software for clustering, resulting in 61,339 homologous protein groups. Analysis revealed that 106 groups contained only one homologous protein in each species, and 19 groups of soybean Phytophthora genes had no sequence polymorphism in intraspecific populations. Subsequently, PsRrp8 was screened as a candidate target from genes with rich polymorphism among different species. The nucleotide sequence of this target is shown in SEQ ID NO.1.

[0023]

[0024] Example 2

[0025] The primers PsRrp8-qPCR-F1 / R1 (hereinafter referred to as F1 / R1) were designed by selecting the polymorphism-rich sequence region of the PsRrp8 gene. The region targeted by this primer pair and the sequence alignment between different species are shown in the figure. Figure 1 shown.

[0026] Based on the new detection target, the detection primers were designed and a quantitative PCR detection method was established. The main pathogens of 9 soybeans were collected ( P.sojae Phytophthora sojae, Pythium ultimum Pythium ultimum, Fusarium oxysporum Fusarium oxysporum, F. solani Fusarium solani, F. equiseti Fusarium equisetum, F. graminearum Fusarium graminearum, Rhizoctonia solani Rhizoctonia solani, Diaporthe longicolla Soybean Phomopsis seed rot pathogen, Colletotrichum truncatum Colletotrichum flatheadensis) and three other species of Phytophthora ( P. infestans Phytophthora infestans, P. capsici Phytophthora capsici, P. nicotianae Nicotiana tabacum). was used as a template to verify the specificity of the detection target.

[0027] The detection primer combination used in the qPCR detection method consists of the forward primer PsRrp8-qPCR-F1 and the reverse primer PsRrp8-qPCR-R1. The primer sequences are as follows: PsRrp8-qPCR-F1: 5'-CGAAGCCTTTGACCCCCG-3' (SEQ ID NO.2) PsRrp8-qPCR-R1: 5'-GAACTGCAATACCATCACCA-3' (SEQ ID NO. 3).

[0028] The genomic DNA of the test strain was amplified using the designed primer combination PsRrp8-qPCR-F1 / R1: Hieff UNICON ® Advanced qPCR SYBR Master Mix 10.0 μL, forward primer PsRrp8-qPCR-F1 and reverse primer PsRrp8-qPCR-R1 (10 μmol·L -1 ), 0.4 μL each of genomic DNA, 2.0 μL of DMSO, and adjust the volume to 20.0 μL with RNA-Free H2O.

[0029] The qPCR reaction program was as follows: 40 cycles of pre-denaturation at 95°C for 30 seconds, denaturation at 95°C for 5 seconds, and extension at 60°C for 10 seconds. The melting curve was set to hold at 65°C for 5 seconds, followed by extension at 95°C and cooling to 0.5°C for storage. After the qPCR reaction, data were analyzed using BioRad CFX Manager.

[0030] The results are as follows Figure 2 As shown in the figure, PsRrp8-qPCR-F1 / R1 could only specifically amplify the melting curve with a melting temperature (Tm value) of 84.0℃ from the soybean P. sojae DNA, while the other non-soybean P. sojae strains and the negative control did not produce amplification, indicating that the primer combination designed based on the new detection target PsRrp8 can achieve the detection of soybean P. sojae ( P.sojae ) specific detection.

[0031] Example 3

[0032] In order to achieve the intraspecies universality of primers F1 / R1 for the specific amplification of P. sojae, genomic DNA was extracted from 10 P. sojae strains of different geographical origins and used as templates. Enzyme-free sterile water was set as a negative control for qPCR amplification.

[0033] The genomic DNA of the test strain was amplified using the designed primer combination PsRrp8-qPCR-F1 / R1: Hieff UNICON ® Advanced qPCR SYBR Master Mix 10.0 μL, forward primer PsRrp8-qPCR-F1 and reverse primer PsRrp8-qPCR-R1 (10 μmol·L -1 ), 0.4 μL each of genomic DNA, 2.0 μL of DMSO, and adjust the volume to 20.0 μL with RNA-Free H2O.

[0034] The qPCR reaction program was as follows: 40 cycles of pre-denaturation at 95°C for 30 seconds, denaturation at 95°C for 5 seconds, and extension at 60°C for 10 seconds. The melting curve was set to hold at 65°C for 5 seconds, followed by extension at 95°C and cooling to 0.5°C for storage. After the PCR reaction, data were analyzed using BioRad CFX Manager.

[0035] The results are as follows Figure 3 As shown in the figure, primers F1 / R1 effectively amplified all 10 strains of P. sojae, with Ct values ​​ranging from 20 to 30 and melting temperatures (Tm values) of 84.0°C. However, no amplification was observed in the negative control, indicating that the target primers have good intraspecies versatility in amplifying P. sojae.

[0036] Example 4

[0037] To evaluate the effect of Phytophthora sojae ( P.sojae The sensitivity of the real-time fluorescence PCR detection system established for the new detection target PsRrp8 was improved by 100 ng·µL -1 The genomic DNA of Phytophthora sojae was serially diluted 10-fold to prepare 100 ng·µL -1 , 10 ng·µL -1 , 1 ng·µL -1 , 100 pg·µL -1 , 10 pg·µL -1 , 1pg·µL -1 g and 100 fg·µL -1 qPCR amplification was performed after seven gradient templates.

[0038] Amplification of different concentrations of soybean Phytophthora strain genomic DNA using the designed primer combination PsRrp8-qPCR-F1 / R1: Hieff UNICON ® Advanced qPCR SYBR Master Mix 10.0 μL, forward primer PsRrp8-qPCR-F1 and reverse primer PsRrp8-qPCR-R1 (10 μmol·L -1 ), 0.4 μL each of genomic DNA, 2.0 μL of DMSO, and adjust the volume to 20.0 μL with RNA-free HO.

[0039] The qPCR reaction program was as follows: 40 cycles of pre-denaturation at 95°C for 30 seconds, denaturation at 95°C for 5 seconds, and extension at 60°C for 10 seconds. The melting curve was set to hold at 65°C for 5 seconds, followed by extension at 95°C and cooling to 0.5°C for storage. After the PCR reaction, data were analyzed using BioRad CFX Manager.

[0040] The results are as follows Figure 4 The concentration is 100 ng·µL -1 , 10 ng·µL -1 , 1 ng·µL -1 , 100 pg·µL -1 , 10pg·µL -1 , 1pg·µL -1 g P.sojae The Ct values ​​of genomic DNA were 17.28, 19.87, 23.43, 26.78, 30.14, and 33.61, respectively. The standard curve was drawn based on the amplification results of the first six template concentrations ( Figure 4The linear relationship between the logarithm of the P. sojae DNA concentration (X) and the Ct value (Y) is: Y = -3.266X + 33.44 (R² = 0.9993). Based on this linear equation, a Ct value of 35 (the conventional detection threshold) corresponds to a DNA concentration of 0.332 pg·µL. -1 , which is the lowest concentration that can be detected by the system, indicating that the quantitative detection technology has high sensitivity.

[0041] Example 5

[0042] To verify that the primers designed for this target can effectively detect the presence of Phytophthora in plants infected with Phytophthora root rot in the field, a survey was conducted on soybean root rot-affected fields. Soybean plants with different disease severity were sampled and their genomes were extracted for F1 / R1-qPCR detection. The designed primer combination PsRrp8-qPCR-F1 / R1 was used to amplify genomic DNA of different concentrations of soybean Phytophthora strains: Hieff UNICON ® Advanced qPCR SYBR Master Mix 10.0 μL, forward primer PsRrp8-qPCR-F1 and reverse primer PsRrp8-qPCR-R1 (10 μmol·L -1 ), 0.4 μL each of genomic DNA, 2.0 μL of DMSO, and adjust the volume to 20.0 μL with RNA-free HO.

[0043] The qPCR reaction program was as follows: 40 cycles of pre-denaturation at 95°C for 30 seconds, denaturation at 95°C for 5 seconds, and extension at 60°C for 10 seconds. The melting curve was set to hold at 65°C for 5 seconds, followed by extension at 95°C and cooling to 0.5°C for storage. After the PCR reaction, data were analyzed using BioRad CFX Manager.

[0044] The results are shown in Figure 5. Soybean plants with Phytophthora root rot can be effectively detected with Phytophthora, and the detection results are highly consistent with the disease severity of the plants. Among them, browning of the stem base and upward extension of lesions are one of the typical symptoms of Phytophthora root rot. The higher the severity of this symptom, the greater the pathogen load detected.

[0045] Unless otherwise specified, the numerical value set forth in these embodiments does not limit the scope of the present invention. In all examples shown and described here, unless otherwise specified, any specific value should be interpreted as merely exemplary, rather than as restriction, and therefore, other examples of exemplary embodiments can have different values.

Claims

1. A primer combination for detecting the soybean phytophthora-specific detection target PsRrp8, characterized in that: The primer combination includes a forward primer and a reverse primer, the forward primer PsRrp8-qPCR-F1 sequence is shown as SEQ ID NO.2, the reverse primer PsRrp8-qPCR-R1 sequence is shown as SEQ ID NO.3, and the DNA sequence of the specific detection target PsRrp8 is shown as SEQ ID NO.

1.

2. A kit for detecting Phytophthora sojae, characterized in that: The kit includes 10 μmol·L -1 A detection solution for the primer combination according to claim 1.

3. Use of the primer combination according to claim 1 or the kit according to claim 2 in detecting Phytophthora sojae.

4. A qPCR method for detecting Phytophthora sojae, characterized in that: The following steps are involved: Take 1-2 μL of the DNA solution of the test object and add Hieff UNICON ® Advanced qPCR SYBR Master Mix 8-12 μL, 8-12 μmol·L -1 0.3-0.5 μL of the forward primer PsRrp8-qPCR-F1 and the reverse primer PsRrp8-qPCR-R1 were added, and 0.5-1.5 μL of DMSO was added, and the volume was adjusted to 19-22 μL using RNA-Free H2O for qPCR amplification. The sequence of the forward primer PsRrp8-qPCR-F1 is shown in SEQ ID NO.2, and the sequence of the reverse primer PsRrp8-qPCR-R1 is shown in SEQ ID NO.

3.

5. The method according to claim 4, characterized in that Take 2 μL of the DNA solution of the test object and add HieffUNICON ® Advanced qPCR SYBR Master Mix 10 μL, 10 μmol·L -1 0.4 μL of forward primer PsRrp8-qPCR-F1, 0.4 μL of reverse primer PsRrp8-qPCR-R1, 1 μL of DMSO were added, and the volume was adjusted to 20 μL with RNA-Free H2O for qPCR amplification.

6. The method according to claim 5, characterized in that The qPCR amplification program was as follows: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 5 s, and extension at 60°C for 10 s, for 40 cycles; the melting curve was set to maintain at 65°C for 5 s, and then cooled to 0.5°C for storage after extension at 95°C.

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