A primer set for simultaneous detection of multiple soybean quarantine pathogens and detection method thereof

Abstract

The present invention discloses a primer set and a detection method thereof for synchronously detecting a plurality of quarantine pathogens of soybean. The primer set is composed of a detection primer pair SoqF / SoqR for Phytophthora sojae, a detection primer pair DPM1F / DPM1R for southern soybean stem canker pathogen, a detection primer pair DPC2F / DPC2R for northern soybean stem canker pathogen, and a detection primer pair FV3F / FV3R for North American soybean sudden death syndrome pathogen. The primer set is added to the same PCR reaction system, and specific amplification products of different sizes are obtained by multiple PCR amplification and agarose gel electrophoresis. The primer set and the detection method of the present invention have strong specificity and high sensitivity, and can quickly and accurately synchronously detect a plurality of quarantine pathogens in soybean in a system. The primer set can be used for port inspection and quarantine, early diagnosis of field diseases, and monitoring and identification of pathogens.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural pathogen detection, and specifically relates to a primer set and a detection method for synchronously detecting multiple soybean quarantine pathogens, and more specifically, to a simple, rapid, specific and sensitive multiple PCR primer and detection method for synchronously detecting multiple pathogens of soybean Phytophthora, southern soybean stem canker, northern soybean stem canker and North American soybean sudden death syndrome. Background Art

[0002] Soybean is an important oil crop and food crop in my country. It is also the main source of edible oil and vegetable protein in the world and occupies an important position in my country's national economy. At present, my country's soybean production ranks fourth in the world, but it still cannot meet the needs of domestic vegetable oil and feed protein processing. As the amount of soybean imported by my country increases year by year, the risk of various diseases that harm soybean production is also gradually increasing. There are hundreds of pests and diseases that can harm soybeans. The soybean phytophthora, southern soybean stem canker, northern soybean stem canker and North American soybean sudden death syndrome have been listed as quarantine pests in China and can cause serious harm to production. These four soybean pathogens are currently pathogens that seriously affect soybean production and trade, and have important quarantine importance and economic importance.

[0003] Phytophthora sojae Kaufmann & Gerdemann infects soybeans, causing soybean Phytophthora root rot. The pathogen is listed as a list of imported plant pests in my country. The disease was first discovered in Heilongjiang Province in 1991. Since then, the disease has been expanding year by year. At present, the disease has posed a threat to my country's soybean production. Soybean Phytophthora root rot is a soil-borne pathogen. The pathogen can spread from the diseased area to the non-diseased area through rain, soil, diseased residues, etc., and it has a strong infection ability. The diseases caused by it are mostly multi-cycle diseases, which can cause plant death in a short period of time. Therefore, it is of great significance to carry out soybean Phytophthora root rot detection, prevent it from entering from the port or spreading from the diseased area to the non-diseased area, and conduct diagnostic detection in the early stage of its onset, as well as rapid detection and identification of customs entry and exit, for the spread and control of soybean Phytophthora root rot.

[0004] Diaporthe phaseolorum var.meridionalis (DPM), the disease of soybean, was first reported in the United States in 1973. It has been included in the quarantine list of my country and is an important quarantine pathogen in my country. It seriously affects soybean production and trade and has quarantine and economic importance. The stem canker pathogen overwinters on the residues of soybean plants with hyphae and ascocarps, and has strong stress resistance. The diseased residues can survive for 14 months at -18℃ to 15℃. It can be spread over long distances through seeds and diseased residues mixed in soybeans. Seeds carrying bacteria are one of the factors for disease transmission. It is currently distributed in the United States, Brazil, Argentina, Bolivia, Paraguay and other countries, and is an important disease in foreign soybean production. Due to the high risk of introduction, most areas in my country are suitable for the growth of the pathogen, so there is an urgent need for a quick and simple method to deal with the inspection of goods entering and leaving the port.

[0005] Soybean northern stem canker (Diaporthe phaseolorum var.caulivora, DPC), currently seriously affects soybean production and trade, and has quarantine importance and economic importance. Soybean northern stem canker was first discovered in Iowa, USA in 1950. It infects soybeans and causes serious diseases, and seriously affects soybean production, with a loss rate of up to 100% in some areas. It is currently mainly distributed in the Americas such as Brazil, Bolivia, Paraguay, the United States, Argentina, and is also distributed in parts of Europe such as Bulgaria and Italy. So far, soybean stem canker has not been found in my country, but from relevant reports in the literature, the epidemic is still spreading, seriously affecting soybean production. Due to the high risk of introduction and the fact that most areas in my country are suitable for the growth of the pathogen, it has been listed as a quarantine pest in my country, and a quick and simple method is urgently needed to deal with the inspection of goods entering and leaving the port.

[0006] Sudden Death Syndorme of Soybean, or SDS, is a relatively new fungal disease caused by Fusarium virguliforme that has occurred on soybeans in the United States and other countries in the past two decades. The disease was first discovered in Arkansas, USA in 1971. By 1982, the disease caused a serious decline in soybean production in the state, which attracted people's attention. Based on its rapid onset, it was named soybean crushing death syndrome. Soybean crushing death syndrome is very harmful and can cause 100% loss under suitable conditions.

[0007] At present, pathogens such as soybean phytophthora, soybean southern stem canker, soybean northern stem canker and North American soybean sudden death syndrome are important entry quarantine pathogens in my country and have been listed in the list of quarantine pests of plants entering the People's Republic of China. Among them, the four soybean quarantine pathogens involved in the present invention are diseases that currently seriously affect soybean production and trade, and have quarantine importance and economic importance. Common detection methods for soybean phytophthora, soybean southern stem canker, soybean northern stem canker and North American soybean sudden death syndrome include traditional separation and identification, immunological methods, molecular biology methods, etc. The detection process of traditional separation and identification and immunological methods is time-consuming and laborious, and the sensitivity is low. With the development of biotechnology, multiple detection technologies based on nucleic acids have played an increasingly important role in the field of nucleic acid diagnosis, mainly including multiple nucleic acid detection technologies based on multiple PCR, nucleic acid isothermal amplification and gene chips, which can detect multiple targets simultaneously, and have the characteristics of rapidity, high throughput and low sample consumption. Multiplex PCR is a PCR-derived method. Its basic principle, reaction reagents and operation are the same as those of conventional PCR. The difference is that the multiplex PCR system contains two or more pairs of primers, which amplify different templates respectively, and can detect multiple targets simultaneously. Multiplex PCR (multiplex polymerase chain reaction) is a PCR technology that amplifies multiple target fragments from multiple DNA templates or different regions of the same template in the same reaction system. It can detect multiple pathogens at one time. Its characteristics are that it takes a short time while improving accuracy and sensitivity. Multiplex PCR technology has the advantages of saving time, reducing costs and improving efficiency. It has broad application prospects in the identification and detection of plant pathogens, especially in the detection of crops carrying multiple pathogens or complex infection of pathogens. Summary of the invention

[0008] In view of the shortcomings of the prior art, the present invention provides a primer set and a detection method thereof for synchronously detecting multiple soybean quarantine pathogens. The multiplex PCR detection primer combination of the present invention has strong specificity and high sensitivity, the detection method is simple and rapid, and the results are reliable.

[0009] The scheme of the present invention includes the following contents:

[0010] Specific detection primers were designed for specific targets of Phytophthora spp., southern soybean stem canker pathogen, northern soybean stem canker pathogen, and North American soybean sudden death syndrome pathogen. Among them, the specific detection target sequence of Phytophthora spp. (PHYSODRAFT_299276) is shown in SEQ ID NO: 1, the specific detection target sequence of southern soybean stem canker pathogen (MS-SSC91) scaffold_2633 is shown in SEQ ID NO: 2, the specific detection target sequence of northern soybean stem canker pathogen (EF-1α sequence) is shown in SEQ ID NO: 3, and the specific detection target sequence of North American soybean sudden death syndrome pathogen (AEYB01000435.1) is shown in SEQ ID NO: 4.

[0011] A primer set for synchronously detecting multiple soybean quarantine pathogens, comprising at least two of the following primer pairs;

[0012] a. Primer pair SoqF / SoqR for detecting Phytophthora sojae, wherein the nucleotide sequence is SoqF: 5'-CACTTGTGGCTCTGTAG-3', SoqR: 5'-GCAGCTTTCTCTAGGTAG-3'; (SEQ ID NO: 5-6)

[0013] b. Primer pair DPM1F / DPM1R for detecting southern soybean stem canker pathogen, the nucleotide sequence of which is DPM1F: 5'-GAATCCTTGTGGGTATTTG-3', DPM1R: 5'-GTCAATATGCTATGGTCAC-3'; (SEQ ID NO: 7-8)

[0014] c. Primer pair DPC2F / DPC2R for detecting soybean stem canker pathogen, the nucleotide sequence of which is DPC2F: 5'-ACTCCCATCCCAGTGCTA-3', DPC2R: 5'-AGAAGAGGTCAGCATCATGC-3'; (SEQ ID NO: 9-10)

[0015] d. The primer pair FV3F / FV3R for detecting the North American soybean sudden death syndrome pathogen has the nucleotide sequence of FV3F: 5'-ATCGCTCATCTCTCTTCC-3', FV3R: 5'-TCGTCTCTTTCTCGCTTAG-3'. (SEQ ID NO: 11-12)

[0016] Furthermore, the present invention provides a multiplex PCR dedicated primer set for synchronously detecting soybean Phytophthora, southern soybean stem canker pathogen, northern soybean stem canker pathogen and North American soybean sudden death syndrome pathogen, which consists of a primer pair SoqF / SoqR, a primer pair DPM1F / DPM1R, a primer pair DPC2F / DPC2R and a primer pair FV3F / FV3R.

[0017] The invention relates to the application of the primer set in synchronously detecting a variety of soybean quarantine pathogens.

[0018] The invention relates to the application of the primer set in preparing a reagent kit for synchronously detecting a plurality of soybean quarantine pathogens.

[0019] On the other hand, the present invention further provides a method for synchronously detecting multiple soybean quarantine pathogens using the primer set, comprising the following steps:

[0020] Step 1: Using the DNA of the sample to be tested as a template, multiplex PCR reaction is performed using the designed specific primers;

[0021] Step 2: Perform electrophoresis on the products of multiplex PCR amplification to observe whether 1310bp, 678bp, 279bp, and 112bp bands appear; if the 1310bp band appears, it is judged to be positive for soybean phytophthora; if the 678p band appears, it is judged to be positive for soybean southern stem canker; if the 279p band appears, it is judged to be positive for soybean northern stem canker; if the 112bp band appears, it is judged to be positive for North American soybean sudden death syndrome; if the corresponding size bands do not appear, it is judged to be negative for the corresponding pathogen.

[0022] Preferably, each 20 μL multiplex PCR reaction system includes: 10 μL of 2×Taq PCR Master Mix II, 0.25 μL of each 10 μM detection primer pair, 50 μg / mL -1 1 μL DNA template, dd.HO 2 Make up to 20 μL with HO.

[0023] Preferably, the multiplex PCR reaction program is: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, for a total of 30 cycles, and extension at 72°C for 5 min.

[0024] Effects achieved by the present invention:

[0025] In summary, the present invention analyzes the complete genome sequence of the pathogen, finds its specific target site, designs a soybean quarantine pathogen specific target site PCR primer pair for the specific target, and the primers of the present invention have very good specificity. The PCR primer pair of the present invention is used for multi-target synchronous detection of soybean phytophthora, soybean southern stem canker, soybean northern stem canker, and North American soybean sudden death syndrome pathogen. The detection method has important practical value for port inspection and quarantine and the prevention and control of diseases caused by agricultural production.

[0026] 1. High specificity. The present invention obtains specific target sites by analyzing the whole genome sequence of pathogens, and designs specific detection primer combinations for detection. Other different closely related species from different pathogens have been verified, and the results show strong specificity;

[0027] 2. Simple and fast operation. The method of the present invention can be used to extract the DNA of the pathogen, perform PCR amplification and perform conventional agarose gel electrophoresis to determine the result. There is no need to perform restriction endonuclease digestion on the amplified product, and no need for expensive instruments and equipment and complicated operations. Generally, the entire detection process can be completed in 1-2 hours, which improves the interception rate of imported soybean epidemics and the ability to prevent the invasion of foreign pests, and has important practicality and technicality.

[0028] 3. Save time and effort, and low detection cost. The multi-target synchronous detection method of the present invention can simultaneously detect four important quarantine pathogens on soybeans, greatly improving the speed of customs port inspection and quarantine; especially in recent years, when the import volume of soybeans has increased, it provides a fast, sensitive and specific detection method, which can shorten the traditional detection time, speed up the customs clearance of imported and exported goods at my country's ports, and reduce enterprise costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Multiplex PCR specificity test results of Example 3.

[0030] Wherein, M: standard DNA molecular weight 2000; 1: soybean phytophthora; 2: soybean southern stem canker pathogen; 3: soybean northern stem canker pathogen; 4: soybean North American sudden death syndrome pathogen; 5: soybean phytophthora + soybean southern stem canker pathogen; 6: soybean phytophthora + soybean northern stem canker pathogen; 7: soybean phytophthora + soybean North American sudden death syndrome pathogen; 8: soybean southern stem canker pathogen + soybean northern stem canker pathogen; 9: soybean southern stem canker pathogen + soybean North American sudden death syndrome pathogen; 10: soybean northern stem canker pathogen + soybean North American Sudden death syndrome pathogen; 11: soybean phytophthora + soybean southern stem canker pathogen + soybean northern stem canker pathogen; 12: soybean phytophthora + soybean southern stem canker pathogen + soybean North American sudden death syndrome pathogen; 13: soybean phytophthora + soybean northern stem canker pathogen + soybean North American sudden death syndrome pathogen; 14: soybean southern stem canker pathogen + soybean northern stem canker pathogen + soybean North American sudden death syndrome pathogen; 15: soybean phytophthora + soybean southern stem canker pathogen + soybean northern stem canker pathogen + soybean North American sudden death syndrome pathogen; 16: negative control (H 2 O); 17: Phytophthora of cowpea; 18: Phytophthora of cucumber; 19: Phytophthora infestans; 20: Phytophthora of palm; 21: Phytophthora parasiticus; 22: Phytophthora of digging; 23: Phytophthora of pepper; 24: Phytophthora of camphor; 25: Phytophthora of taro; 26: Phytophthora of evil; 27: Phytophthora of litchi; 28: Phomopsis spp.; 29: Colletotrichum lychee; 30: Colletotrichum pakchoi; 31: Colletotrichum pepper; 32: Colletotrichum edamame; 33: Fusarium oxysporum; 34: Fusarium multifilamentum; 35: Fusarium oxysporum; 36: Fusarium solani; 37: Fusarium graminearum.

[0031] Figure 2 : Multiplex PCR sensitivity test results of Example 4. Wherein, M: standard DNA molecular weight 2000; 1-8: the concentration of the mixture of soybean phytophthora, soybean northern stem canker, and North American soybean sudden death syndrome pathogen was diluted in sequence to 100ng / μL, 10ng / μL, 1ng / μL, 0.1ng / μL, 0.01ng / μL, 0.001ng / μL, 0.0001ng / μL, 0.00001ng / μL; 9: distilled water (negative control) DETAILED DESCRIPTION

[0032] In order to better understand the technical content of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0033] Example 1 Establishment of single-plex PCR reaction system and reaction conditions for soybean pathogens

[0034] 1. Primer design and screening

[0035] First, the genome-wide specific target sites of four soybean pathogens, namely, Phytophthora sojae, southern soybean stem canker, northern soybean stem canker, and North American soybean sudden death syndrome, were analyzed to find the unique base sites of each detection target sequence. A primer pair with the following nucleic acid sequence was designed;

[0036] Specific primer pairs for detection of soybean Phytophthora:

[0037] SoqF: 5'-CACTTGTGGCTCTGTAG-3'

[0038] SoqR: 5'-GCAGCTTTCTCTAGGTAG-3';

[0039] Specific primer pairs for detecting southern soybean stem canker pathogen:

[0040] DPM1F:5'-GAATCCTTGTGGGTATTTG-3',

[0041] DPM1R: 5'-GTCAATATGCTATGGTCAC-3'

[0042] Specific primer pairs for detecting soybean northern stem canker pathogen:

[0043] DPC2F: 5'-ACTCCCATCCCAGTGCTA-3'

[0044] DPC2R: 5'-AGAAGAGGTCAGCATCATGC-3'

[0045] Specific primer pairs for detecting North American soybean sudden death syndrome pathogen:

[0046] FV3F: 5'-ATCGCTCATCTTCTCTTCC-3'

[0047] FV3R: 5'-TCGTCTCTTTCTCGCTTAG-3'

[0048] 2. Establishment of PCR reaction system and reaction conditions

[0049] In this study, the single-plex PCR detection reaction system and reaction conditions for soybean Phytophthora spp., southern soybean stem canker pathogen, northern soybean stem canker pathogen, and North American soybean sudden death syndrome pathogen were optimized, and the optimal reaction system and reaction conditions were determined.

[0050] The optimal PCR reaction system is (calculated based on 20 μL of PCR final reaction solution):

[0051] 2×Taq PCR MasterMix 10μL, 0.25μM of each PCR primer pair, 1μL of each of the four soybean pathogen template DNAs, ddH 2 O to make up to 20 μL;

[0052] The optimal PCR reaction conditions for soybean phytophthora were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; 56℃ annealing for 30 s; 72℃ extension for 40 s, 30 cycles; 72℃ extension for 5 min;

[0053] The optimal PCR reaction conditions for southern soybean stem canker pathogen were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; 56℃ annealing for 30 s; 72℃ extension for 30 s, 30 cycles; 72℃ extension for 5 min;

[0054] The optimal PCR reaction conditions for soybean northern stem canker pathogen were: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s; 56℃ annealing for 30s; 72℃ extension for 30s, 30 cycles; 72℃ extension for 5min;

[0055] The optimal PCR reaction conditions for North American soybean sudden death syndrome pathogen were: pre-denaturation at 95℃ for 5min; denaturation at 95℃ for 30s; annealing at 56℃ for 30s; extension at 72℃ for 30s, 30 cycles; and extension at 72℃ for 5min.

[0056] Example 2: Establishment of multiplex PCR reaction system and reaction conditions for soybean pathogens

[0057] Based on the reaction system and reaction conditions of single-plex PCR detection, the present invention optimizes various parameters in multiplex PCR detection and determines the best reaction system and reaction conditions.

[0058] 2×Taq PCR MasterMix 10μL, 0.25μM of each PCR primer pair, 1μL of each of the four soybean pathogen template DNAs, ddH 2 O to make up to 20 μL;

[0059] The reaction conditions of the above multiplex PCR reaction are: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 56°C for 30 s; extension at 72°C for 30 s, 30 cycles; and extension at 72°C for 5 min.

[0060] Example 3: Verification of the detection specificity of primer sets for multiplex PCR detection of soybean pathogens

[0061] Before testing, each strain used in the experiment was cultured, genomic DNA was extracted, and the DNA concentration was detected using an ultra-micro spectrophotometer NanoDrop 2000. PCR amplification was performed using DNA with a concentration of 10 ng / μL as a template.

[0062] The multiplex PCR reaction system and reaction conditions established in Example 2 were used to perform multiplex PCR detection, and the PCR products were analyzed by electrophoresis. Figure 1 As shown, only soybean phytophthora, southern soybean stem canker, northern soybean stem canker, North American soybean sudden death syndrome pathogen and their mixtures can amplify specific target fragments, while the others are all negative. The results are consistent with expectations, indicating that the multiplex PCR detection system of the present invention has good specificity.

[0063] Example 4: Verification of the detection sensitivity of the primer set for multiplex PCR detection of soybean pathogens

[0064] Before the test, the strains used in the experiment were cultured, genomic DNA was extracted, and the DNA concentration was detected by ultra-micro spectrophotometer NanoDrop 2000. A 10-fold gradient dilution was performed, and PCR amplification was performed using the primer pairs shown in Example 1 with different concentrations of DNA as templates. The other reaction systems and reaction conditions were as described in Example 2, and the PCR products were analyzed by electrophoresis. The results are shown in FIG. Figure 2 As shown, the sensitivity of the soybean Phytophthora multiplex PCR method of the present invention is 1 ng / μL.

[0065] The PCR method of the present invention is used to detect soybean phytophthora, soybean southern stem canker, soybean northern stem canker, and North American soybean sudden death syndrome pathogen, which can greatly improve the sensitivity and specificity of detection; and compared with traditional bacterial isolation methods, it greatly shortens the detection cycle and improves the detection sensitivity.

[0066] The above description is only a preferred embodiment of the present invention and does not limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A primer set for synchronous detection of multiple soybean quarantine pathogens, characterized in that: It consists of the following primer pairs; a. Primer pair SoqF / SoqR for detecting Phytophthora sojae, the nucleotide sequence of which is SoqF: 5'-CACTTGTGGCTCTGTAG-3', SoqR: 5'-GCAGCTTTCTCTAGGTAG-3'; b. The primer pair DPM1F / DPM1R for detecting southern soybean stem canker pathogen, the nucleotide sequence of which is DPM1F: 5'-GAATCCTTGTGGGTATTTG -3', DPM1R: 5'-GTCAATATGCTATGGTCAC -3'; c. Primer pair DPC2F / DPC2R for detecting northern soybean stem canker pathogen, the nucleotide sequence of which is DPC2F: 5'-ACTCCCATCCCAGTGCTA-3', DPC2R: 5'-AGAAGAGGTCAGCATCATGC -3'; d. The primer pair FV3F / FV3R used for detecting the North American soybean sudden death syndrome pathogen has the nucleotide sequences of FV3F: 5'-ATCGCTCATCTCTCTTCC-3', FV3R: 5'-TCGTCTCTTTCTCGCTTAG -3'.

2. Use of the primer set described in claim 1 in the simultaneous detection of multiple soybean quarantine pathogens.

3. Use of the primer set according to claim 1 in preparing a kit for synchronously detecting multiple soybean quarantine pathogens.

4. A method for synchronously detecting multiple soybean quarantine pathogens using the primer set of claim 1, characterized in that: The steps include: Step 1: Using the DNA of the sample to be tested as a template, a multiplex PCR reaction is performed using the designed specific primers; Step 2: Perform electrophoresis on the products of multiplex PCR amplification to observe whether 1310bp, 679bp, 279bp, and 112bp bands appear; if a 1310bp band appears, it is judged to be positive for soybean phytophthora; if a 679bp band appears, it is judged to be positive for soybean southern stem canker; if a 279bp band appears, it is judged to be positive for soybean northern stem canker; if a 112bp band appears, it is judged to be positive for North American soybean sudden death syndrome; if no bands of corresponding size appear, it is judged to be negative for the corresponding pathogen.

5. The method according to claim 4, characterized in that Each 20 μL multiplex PCR reaction system includes: 2×TaqPCRMasterMixⅡ 10 μL, 0.25 μL of each 10 μM detection primer pair, 1 μL of 50 μg / mL DNA template, and ddH2O to make up to 20 μL.

6. The method according to claim 4, characterized in that The multiplex PCR reaction program was as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, for a total of 30 cycles, and extension at 72°C for 5 min.

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