Multiplex real-time fluorescent PCR detection method and kit for six quarantine pathogens of soybean

Abstract

This invention relates to a primer and probe set and method for detecting six quarantine pathogens in soybeans. The primer and probe set includes specific primers and probes for detecting Southern Bean Mosaic Virus, Tomato Ringspot Virus, Tobacco Ringspot Disease, Bean Pod Mottle, Tomato Leaf Spot Virus, and Ergot Scab. The method of this invention has advantages such as high detection sensitivity, strong specificity, good repeatability, low requirements for equipment, simple operation, and short time requirement, and has significant application value.

Description

Technical Field

[0001] This invention relates to the field of agricultural pathogen detection technology, specifically to a method and kit for detecting six quarantine pathogens in soybeans using multiplex real-time fluorescence PCR. Background Technology

[0002] Soybeans are the world's major oilseed crop, accounting for nearly 50% of the total oilseed area and production. Globally, they are primarily used as a source of protein (60%) and oil (30%). Popular for their adaptability to different climate zones, soybeans have become the fourth most important crop globally, after wheat, rice, and corn. The United States ranks first in soybean planting area, production, and trade.

[0003] Soybean diseases have always been one of the major factors restricting soybean production. Common soybean pathogens include Southern bean mosaic virus (SBMV), Tomato ringspot virus (ToSRV), Tobacco ringspot virus (TRSV), Beanpod mottle virus (BPMV), Tobacco spotted wilt virus (TSWV), and Claviceps purpurea (Cp). These pathogens can severely reduce soybean yield and quality, causing huge economic losses to agricultural production. They are of quarantine importance and are also the main source of inoculum in viral ecology and epidemiology. Timely and accurate detection of pathogens is crucial for disease control. Traditional biological and serological detection methods are cumbersome and inefficient, making it difficult to meet the needs of large-scale testing. Real-time quantitative PCR technology, due to its high specificity, high sensitivity, and high degree of automation, has become the mainstream method for plant virus detection. However, there is still a lack of multiplex real-time fluorescent PCR detection methods and kits for various soybean viruses.

[0004] To improve the interception capacity of quarantine pests in imported soybeans and provide a reference for imported soybean quarantine work and other related work, there is an urgent need in this field for a multiplex real-time fluorescent PCR detection method and kit that can simultaneously detect six major soybean viruses. This will help improve the efficiency and accuracy of soybean virus detection and provide important technical support for the integrated management of soybean diseases. Summary of the Invention

[0005] The purpose of this invention is to establish a high-throughput, high-specificity multiplex detection system for soybean viruses, enabling rapid and accurate detection of six important soybean pathogens, including SBMV, ToSRV, and TRSV, thus providing a powerful technical means for soybean cultivation and pest and disease control.

[0006] A first aspect of the present invention provides a primer-probe set for detecting six soybean quarantine pathogens, the primer-probe set comprising: (1) A first primer and probe set for Southern Common Bean Mosaic Virus, the first primer and probe set including the upstream primer shown in SEQ ID NO:1, the downstream primer shown in SEQ ID NO:2 and the probe shown in SEQ ID NO:3; (2) A second primer and probe set for tomato ringspot virus, comprising the upstream primer shown in SEQ ID NO:4, the downstream primers shown in SEQ ID NO:5 and 6, and the probe shown in SEQ ID NO:7; (3) A third primer and probe set for tobacco ringspot virus, the third primer and probe set comprising the upstream primer shown in SEQ ID NO:8, the downstream primer shown in SEQ ID NO:9, and the probe shown in SEQ ID NO:10; (4) A fourth primer and probe set for bean pod mottle virus, the fourth primer and probe set comprising the upstream primer shown in SEQ ID NO:11, the downstream primer shown in SEQ ID NO:12, and the probe shown in SEQ ID NO:13; (5) A fifth primer-probe set for tomato spotted wilt virus, the fifth primer-probe set comprising the upstream primer shown in SEQ ID NO:14, the downstream primer shown in SEQ ID NO:15, and the probe shown in SEQ ID NO:16; and (6) A sixth primer-probe set for ergot, the sixth primer-probe set comprising the upstream primer shown in SEQ ID NO:17, the downstream primer shown in SEQ ID NO:18, and the probe shown in SEQ ID NO:19.

[0007] In another preferred embodiment, the primer-probe set further includes a primer-probe group for an internal reference.

[0008] In another preferred embodiment, the primer and probe set further includes an internal reference primer and probe set targeting the soybean Lectin gene.

[0009] In another preferred embodiment, the primer and probe set further includes (7) an internal reference primer and probe set for the soybean Lectin gene, the internal reference primer and probe set including the upstream primer shown in SEQ ID NO:20, the downstream primer shown in SEQ ID NO:21, and the probe shown in SEQ ID NO:22.

[0010] In another preferred embodiment, the probe is modified with a fluorescent group and a quenching group.

[0011] In another preferred embodiment, the fluorescent group is selected from the group consisting of ATTO425, FAM, VIC / HEX, ROX, CY5, CY5.5, CY7, AF405, or combinations thereof.

[0012] In another preferred embodiment, the quenching group is selected from the group consisting of BHQ1, BHQ2, BHQ3, or combinations thereof.

[0013] In another preferred embodiment, the fluorescent group is modified at the 5' end of the probe; the quenching group is modified at the 3' end of the probe.

[0014] In another preferred embodiment, the fluorescent groups modified on the probe may be the same or different.

[0015] In another preferred embodiment, the primer-probe set includes: In a second aspect, the present invention provides a PCR amplification system, comprising a buffer system for amplification and a primer and probe set as described in the first aspect of the present invention.

[0016] In another preferred embodiment, the PCR amplification system includes AccurSTART U+ One Step RT-qPCRSuper PreMix (ONE TUBE) reaction solution and DEPC water.

[0017] In another preferred embodiment, the concentration of each upstream primer is 100-1000 nM, the concentration of each downstream primer is 100-1000 nM, and the concentration of each probe is 100-1000 nM.

[0018] In another preferred embodiment, the concentration of each upstream primer is 200-800 nM, the concentration of each downstream primer is 200-800 nM, and the concentration of each probe is 200-800 nM.

[0019] In another preferred embodiment, the concentration of each upstream primer is 200-600 nm, the concentration of each downstream primer is 200-600 nm, and the concentration of each probe is 200-600 nm.

[0020] In a third aspect, the present invention provides a kit comprising a container and a primer-probe set as described in the first aspect of the present invention or a PCR amplification system as described in the second aspect of the present invention, located within the container.

[0021] In another preferred embodiment, the kit further includes positive and / or negative standards.

[0022] In another preferred embodiment, the positive standard comprises a plasmid containing any of the nucleotide sequences shown in SEQ ID NO:23-29.

[0023] In another preferred embodiment, the negative standard is a sodium chloride solution or water.

[0024] In a fourth aspect, the present invention provides a method for detecting six quarantine pathogens in soybeans, the method comprising the steps of: (s1) Provide the nucleic acid from the sample to be tested; (s2) Using the primer and probe set as described in the first aspect of the present invention, the PCR amplification system as described in the second aspect of the present invention, or the kit as described in the third aspect of the present invention, the nucleic acid of the sample to be tested is subjected to a nucleic acid amplification reaction, and the fluorescence signal is detected to obtain a fluorescent PCR amplification curve; and (s3) Analyze the fluorescence PCR amplification curve to determine whether there are six soybean quarantine pathogens in the sample to be tested; The six quarantine pathogens of soybeans are Southern Bean Mosaic Virus, Tomato Ringspot Virus, Tobacco Ringspot Virus, Bean Pod Mottle Virus, Tomato Spotted Wilt Virus, and Ergot.

[0025] In another preferred embodiment, the reaction procedure for the nucleic acid amplification reaction is: reverse transcription at 50°C for 5 min; pre-denaturation at 95°C for 90 s; denaturation at 95°C for 5 s; annealing / extension at 55°C for 30 s; 42 cycles.

[0026] In another preferred embodiment, in step (s2), the fluorescence signal is detected during the annealing / extension stage.

[0027] In another preferred embodiment, the criterion for determining the method is: (1) The internal reference (Lectin gene) Ct value is ≤38, and the negative control group and template-free control group have no Ct value; if the condition is not met, multiplex real-time quantitative PCR detection must be performed again, or nucleic acid must be extracted again for multiplex real-time quantitative PCR detection. (2) The amplification curve is a standard “S” shape with no abnormal fluctuations. The pathogen Ct value is ≤38. If 38 < Ct value ≤40, a retest is required. If the retest results are consistent, the result is considered positive.

[0028] In another preferred embodiment, the sample includes soybean seeds, leaves, etc.

[0029] In another preferred embodiment, the detection limit of the method is ≤100 copies / mL, more preferably ≤80 copies / mL, and even more preferably ≤50 copies / mL.

[0030] In another preferred embodiment, the method is in vitro.

[0031] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0032] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0033] Figure 1 The results of the screening of primers and probes for tomato ringspot virus are shown.

[0034] Figure 2 The detection and sensitivity verification of the Southern Common Bean Mosaic Virus plasmid were demonstrated.

[0035] Figure 3 The detection and sensitivity verification of tomato ringspot virus plasmid were demonstrated.

[0036] Figure 4 The detection and sensitivity verification of tobacco ringspot virus plasmid were demonstrated.

[0037] Figure 5 The detection and sensitivity verification of the bean pod mottle virus plasmid were shown.

[0038] Figure 6 The detection and sensitivity verification of tomato spotted wilt virus plasmid were demonstrated.

[0039] Figure 7 The detection and sensitivity verification of ergot plasmids were shown.

[0040] Figure 8 The detection and sensitivity verification of the internal reference Lectin plasmid are shown.

[0041] Figure 9 The results are shown with a 0.9% sodium chloride solution as a negative control.

[0042] Figure 10 The image shows the effect of using soybean samples with negative nucleic acid as a negative control.

[0043] Figure 11 The results of screening primers and probes for Southern Common Bean Mosaic Virus are shown. A: Detection results of positive standard samples; B: Detection results of multiple targets combined. Detailed Implementation

[0044] Through extensive and in-depth research, and after numerous experiments and screenings, the inventors unexpectedly discovered for the first time a primer-probe set for detecting six soybean quarantine pathogens. This set includes specific primers and probes for detecting Southern beanmosaic virus (SBMV), Tomato ringspot virus (ToSRV), Tobacco ringspot virus (TRSV), Bean pod mottle virus (BPMV), Tobacco spotted wilt virus (TSWV), and Claviceps purpurea (Cp). When using the primer-probe combination of this invention, multiplex quantitative PCR is employed, simultaneously using specific primers and probes for the soybean lectin gene and other conventional reagents for multiplex quantitative PCR. The method of this invention has advantages such as high detection sensitivity, strong specificity, good repeatability, low requirements for equipment, simple operation, and short time requirement, thus possessing significant application value. This invention was completed based on this discovery.

[0045] the term To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0046] As used herein, the term “comprising” or its variations such as “including” or “comprising” are understood to include the said element or component without excluding other elements or other components.

[0047] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0048] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0049] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0050] The primer probe set of the present invention The primer and probe set of this invention is a combination of specific primers and probes for detecting Southern Bean Mosaic Virus, Tomato Ringspot Virus, Tobacco Ringspot Disease, Bean Pod Mottle Disease, Tomato Spotted Wilt Virus, and Ergot Scirpus. It includes... (1) A first primer and probe set for Southern Common Bean Mosaic Virus, the first primer and probe set including the upstream primer shown in SEQ ID NO:1, the downstream primer shown in SEQ ID NO:2 and the probe shown in SEQ ID NO:3; (2) A second primer and probe set for tomato ringspot virus, comprising the upstream primer shown in SEQ ID NO:4, the downstream primers shown in SEQ ID NO:5 and 6, and the probe shown in SEQ ID NO:7; (3) A third primer and probe set for tobacco ringspot virus, the third primer and probe set comprising the upstream primer shown in SEQ ID NO:8, the downstream primer shown in SEQ ID NO:9, and the probe shown in SEQ ID NO:10; (4) A fourth primer and probe set for bean pod mottle virus, the fourth primer and probe set comprising the upstream primer shown in SEQ ID NO:11, the downstream primer shown in SEQ ID NO:12, and the probe shown in SEQ ID NO:13; (5) A fifth primer-probe set for tomato spotted wilt virus, the fifth primer-probe set comprising the upstream primer shown in SEQ ID NO:14, the downstream primer shown in SEQ ID NO:15, and the probe shown in SEQ ID NO:16; and (6) A sixth primer-probe set for ergot, the sixth primer-probe set comprising the upstream primer shown in SEQ ID NO:17, the downstream primer shown in SEQ ID NO:18, and the probe shown in SEQ ID NO:19.

[0051] The tomato ringspot virus exhibits significant interspecies variation, resulting in low coverage with a single primer pair or degenerate primers. The inventors overcame this problem by designing two downstream primers.

[0052] In a preferred embodiment, the primer and probe set further includes (7) an internal reference primer and probe set for the soybean Lectin gene, the internal reference primer and probe set including the upstream primer shown in SEQ ID NO:20, the downstream primer shown in SEQ ID NO:21, and the probe shown in SEQ ID NO:22.

[0053] In another preferred embodiment, the primer-probe set includes: In a preferred embodiment, the primer and probe set includes a first subset and a second subset, the first subset including primer and probe sets SEQ ID No. 1-10, and the second subset including primer and probe sets SEQ ID No. 11-22.

[0054] The reagent kit of the present invention The kit of the present invention is used to detect six soybean quarantine pathogens. The kit includes a container and a primer and probe set as described in the first aspect of the present invention or a PCR amplification system as described in the second aspect of the present invention located in the container.

[0055] In a preferred embodiment, the kit further includes positive and / or negative standards. The positive standards comprise plasmids containing the nucleotide sequences shown in any of SEQ ID NO:23-29. The negative standards are sodium chloride solution or water.

[0056] In a preferred embodiment, the primer probe sets in the primer probe set are located in the same or different containers.

[0057] The method of the present invention This invention provides a method for detecting six quarantine pathogens in soybeans, the method comprising the following steps: (s1) Provide the nucleic acid from the sample to be tested; (s2) Using the primer and probe set as described in the first aspect of the present invention, the PCR amplification system as described in the second aspect of the present invention, or the kit as described in the third aspect of the present invention, the nucleic acid of the sample to be tested is subjected to a nucleic acid amplification reaction, and the fluorescence signal is detected to obtain a fluorescent PCR amplification curve; and (s3) Analyze the fluorescence PCR amplification curve to determine whether there are six soybean quarantine viruses in the sample to be tested; The six quarantine pathogens of soybeans are Southern Bean Mosaic Virus, Tomato Ringspot Virus, Tobacco Ringspot Virus, Bean Pod Mottle Virus, Tomato Spotted Wilt Virus, and Ergot.

[0058] In a preferred embodiment, the reaction program for the nucleic acid amplification reaction is: reverse transcription at 50°C for 5 min; pre-denaturation at 95°C for 90 s; denaturation at 95°C for 5 s; annealing / extension at 55°C for 30 s; 42 cycles.

[0059] In a preferred embodiment, in step (s2), the fluorescence signal is detected during the annealing / extension stage.

[0060] In a preferred embodiment, the criterion for determining the method is: (1) The internal reference (Lectin gene) Ct value is ≤38, and the negative control group and template-free control group have no Ct value; if the condition is not met, multiplex real-time quantitative PCR detection must be performed again, or nucleic acid must be extracted again for multiplex real-time quantitative PCR detection. (2) The amplification curve is a standard “S” shape with no abnormal fluctuations. The pathogen Ct value is ≤38. If 38 < Ct value ≤40, a retest is required. If the retest results are consistent, the result is considered positive.

[0061] In a preferred embodiment, the sample includes soybean seeds, leaves, etc.

[0062] In one exemplary implementation, the method includes the steps of: (1) Extraction of nucleic acid (DNA) from samples, such as seeds and leaves; (2) Using the nucleic acid extracted in step (1) as a template, specific primers and probes targeting 6 pathogens (the nucleotide sequences of the specific primers and probes for detecting 6 pathogens and internal reference genes are shown in SEQ ID NO:1 and SEQ ID NO:19) were used, with the Lectin gene as an internal reference (the nucleotide sequences of the specific primers and probes are shown in SEQ ID NO:20 and SEQ ID NO:22). The results were determined based on the Ct value. The amplification reaction system for detecting pathogens using multiplex real-time PCR detection reagents is as follows: 5 μL of AccurSTART U+ OneStep RT-qPCR Super PreMix (ONE TUBE), 4 μL of a mixture of upstream and downstream primers and probes for 6 pathogens and the internal control Lectin gene, 5 μL of DNA template, and ddH2O to a final volume of 25 μL. The reaction procedure was as follows: 95℃ pre-denaturation for 90s; 95℃ denaturation for 5s; annealing and extension at 55℃ for 15s, for 45 cycles. Fluorescence signals were collected during the annealing and extension phase of each cycle. Specific primers and probes for detecting Southern Bean Mosaic Virus, Tomato Ringspot Virus, Tobacco Ringspot Virus, Bean Pod Mottle Virus, Tomato Spotted Wilt Virus, and Ergot Scirpus were used simultaneously. The soybean Lectin gene was used as an internal control.

[0063] (3) The interpretation of a positive test result is as follows: a) The internal reference (Lectin gene) Ct value is ≤38, and the negative control group and template-free control group have no Ct value; if the condition is not met, multiplex real-time quantitative PCR detection must be performed again, or nucleic acid must be re-extracted for multiplex real-time quantitative PCR detection. b) The curve shows a standard "S" shape with no abnormal fluctuations, and the pathogen Ct value is ≤38. If 38 < Ct value ≤40, a retest is required. If the retest results are consistent, the result is considered positive.

[0064] The main advantages of this invention include: 1. The method and kit for detecting six quarantine pathogens in soybeans by multiplex real-time fluorescence PCR of the present invention can detect six plant pathogens at the same time, avoiding the cumbersome steps of performing multiple reactions separately, greatly simplifying the operation process, improving work efficiency, and quickly meeting the customs' needs for monitoring agricultural products upon entry, and rapidly identifying soybean samples with potential biological threats.

[0065] 2. This invention uses the soybean lectin gene, which is stably expressed at all stages of soybean growth, as an internal control. This allows for monitoring of the entire process of sample collection, nucleic acid extraction, amplification, and detection, avoiding interference from endogenous plant substances, significantly reducing misjudgments caused by false negatives, and improving the accuracy of detection results. Simultaneously, the use of an internal standard avoids the inconvenience of using external standards for quantification, reduces operational complexity, and saves costs.

[0066] 3. The primer combination for multiplex quantitative PCR provided by this invention was applied to multiplex quantitative PCR detection. Experimental results showed that the primer and probe combination of this invention had no cross-reaction with other pathogens and had good specificity. Sensitivity evaluation revealed that the detection limit for Southern Bean Mosaic Virus, Tomato Ringspot Virus, Tobacco Ringspot Virus, Bean Pod Mottle Virus, and Tomato Spotted Wilt Virus reached the level of 50 copies / mL, and the detection limit for Ergot worms reached the level of 100 copies / mL, indicating very high sensitivity.

[0067] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0068] Example 1: Reagent Kit Design and Usage The method and kit for detecting six soybean quarantine pathogens by multiplex real-time fluorescence quantitative PCR in this embodiment include reaction solutions for the six soybean quarantine pathogens, positive controls, and negative controls.

[0069] The reaction solutions for the six soybean quarantine pathogens include: upstream primers for Southern Bean Mosaic Virus (SVM), downstream primers for SVM, Southern Bean Mosaic Virus probe, downstream primer 1 for Tomato Ringspot Virus (TVM), downstream primer 2 for Tomato Ringspot Virus (TVM), downstream primer for Tomato Ringspot Virus (TVM), probe for Tomato Ringspot Virus (TVM), upstream primers for Tobacco Ringspot Virus (TVM), downstream primers for Tobacco Ringspot Virus (TVM), probe for Tobacco Ringspot Virus (TVM), upstream primers for Bean Pod Mottle Virus (BVM), downstream primers for Bean Pod Mottle Virus (BVM), probe for Bean Pod Mottle Virus (BVM), upstream primers for Tomato Spotted Wilt Virus (BVM), downstream primers for Tomato Spotted Wilt Virus (BVM), probe for Tomato Spotted Wilt Virus (BVM), upstream primers for Ergot (Citric Acid), downstream primers for Ergot (Citric Acid), probe for Ergot (Citric Acid), upstream primers for Soybean Lectin internal standard, downstream primers for Soybean Lectin internal standard, and probe for Soybean Lectin internal standard. The reaction solutions for these six soybean quarantine pathogens also include a nucleotide mixture, AccurSTART U+ One Step RT-qPCR Super PreMix (ONE TUBE) reaction solution, and DEPC water. Table 1 shows the specific formulations of reaction solutions for six soybean quarantine pathogens.

[0070] Table 1 The positive control solution includes a mixture of Southern Common Bean Mosaic Virus (SCNV) virus particles, Tomato Ringspot Virus (TUVV) virus particles, Tobacco Ringspot Virus (TBV) virus particles, Common Bean Pod Mottle Virus (TBV) virus particles, Tomato Spotted Wilt Virus (TBV) virus particles, Ergot Scirpus virus particles (any one of the above six types of virus particles), Soybean Lectin gene internal standard virus particles, and TE Buffer. The negative control solution is a 0.9% sodium chloride solution.

[0071] 1. Primer and probe design Download reference sequences of six pathogen genes from the NCBI (National Center for Biotechnology Information) website, compare the nucleotide sequences, and design primers and probes. The primers should have a TM value between 55-60℃, a GC content between 30-80%, a TM value difference between the two primers of less than 5℃, an amplification length between 50-150bp, a 3' end mismatch of less than 5bp, and no specific binding site in the non-target sequence, or a specific binding region greater than 1000bp. The probes should have a TM value between 60-75℃, a GC content of 30-80%, the first base at the 5' end of the probe should not be G, and the probe should not have hairpin structures greater than 5bp.

[0072] The primer and probe sequences for nucleic acid detection targeting six soybean quarantine pathogens are shown in Table 2: SEQ ID No. 1 to SEQ ID No. 19.

[0073] Preferably, the sequences of the internal reference primer probes are shown in Table 2: SEQ ID No. 20 to SEQ ID No. 22.

[0074] The internal control in this embodiment detects the presence of a large number of lectin genes naturally present in soybean samples. Since the internal control gene is independent of other pathogens, it will be positive regardless of whether the soybean sample contains the six pathogens detected by the kit. This internal control participates in the process from sample collection to RT-PCR, and can be used to monitor sample collection, preservation, transportation, and nucleic acid extraction and amplification processes, avoiding false negative results.

[0075] Table 2 2. Plasmid construction The specific sequences of six pathogens and the sequence of the internal reference gene lectin were ligated into the pUC57 vector to synthesize plasmid standards. For each pathogen, plasmids for Southern Bean Mosaic Virus, Tomato Ringspot Virus, Tobacco Ringspot Virus, Bean Pod Mottle Virus, Tomato Spotted Fusarium Virus, and Ergot Scirpus, along with the internal reference lectin, were synthesized separately. Plasmid construction was performed by Jiangsu Saisofe Biotechnology Co., Ltd., and the copy number of the plasmids was quantitatively calculated using a standard curve. The specific sequences of each pathogen are shown in Table 3.

[0076] Table 3 3. Instructions for using the reagent kit The usage method of the multiplex real-time fluorescence PCR detection kit for six soybean quarantine pathogens includes the following steps: ① Pretreatment: Take out the positive control and negative control separately, thaw them at room temperature, shake them thoroughly to mix, and then centrifuge them instantly; ② Nucleic acid extraction: Nucleic acid extraction was performed simultaneously on the sample to be tested, along with the positive and negative controls; ③ Sample addition: Take out the pre-dispensed eight-tube containing the reaction solution of 6 kinds of soybean quarantine pathogens, add 5 μL each of the test sample, positive control and negative control nucleic acid, tighten the cap, shake well and then centrifuge briefly; ④ Amplification and fluorescence signal detection: Place the PCR reaction tubes obtained in step ③ into a real-time PCR instrument for amplification and fluorescence signal detection; ⑤ Combine the results of internal reference testing to determine the sample test results.

[0077] The configuration information of the fluorescence detection channel in step ④ is shown in Table 4.

[0078] Table 4 The amplification and fluorescence signal detection cycle parameters of the real-time PCR instrument in step ④ are shown in Table 5.

[0079] Table 5 Real-time fluorescence PCR (RTPCR) technology is employed. This technology involves adding one or more probes and primers with different fluorescent labels to the same reaction system (FAM for Southern Bean Mosaic Virus, Tomato Ringspot Virus, Tobacco Ringspot Virus, Bean Pod Mottle Virus, Tomato Spotted Wilt Virus, and Ergot Scab; CY5 for the soybean lectin internal standard). This allows for a single fluorescent PCR amplification operation to detect infection with six pathogens that can cause soybean diseases. An internal control is used to monitor the entire process and avoid false negatives. The probes are oligonucleotides containing a 5' fluorescent reporter group and a 3' quencher group. When the probe is intact, the quencher group, being close to the reporter group, significantly inhibits the reporter group's fluorescence. During primer extension, the probe bound to the template is cleaved by Taq enzyme (5'→3' exonuclease activity), separating the reporter group from the quencher group and generating a fluorescent signal, thereby enabling the detection of Southern Bean Mosaic Virus, Tomato Ringspot Virus, Bean Pod Mottle Virus, Tobacco Ringspot Virus, Tomato Spotted Wilt Virus, and Ergot at the nucleic acid level.

[0080] During the PCR reaction, if the sample to be tested contains one or more pathogens from Southern Bean Mosaic Virus, Tomato Ringspot Virus, Bean Pod Mottle Virus, Tobacco Ringspot Virus, Tomato Spotted Wilt Virus, and Ergot, the probe labeled with FAM will produce a fluorescent signal. In addition, each reaction contains an internal reference gene, and the probe labeled with CY5 should produce a fluorescent signal to detect instrument malfunctions, reagent factors, polymerase activity factors, etc.

[0081] Example 2: Screening of primers and probes in the kit of the present invention 2.1 Screening of primers and probes for tomato ringspot virus Positive standard samples were tested using the primer and probe combinations shown in Table 6 according to the method described in Example 1 to screen primer and probe combinations against tomato ringspot virus.

[0082] Table 6 The results are as follows Figure 1As shown, the average Ct value for combination 1 was 27.07, the average Ct value for combination 2 was 25.15, and the average Ct value for combination 3, with its dual downstream primer strategy, was as low as 21.99. Compared to the traditional single downstream primer combination 1, combination 3 advanced the amplification threshold cycle by approximately 5 cycles, exhibited superior amplification kinetics, lower baseline interference, significantly higher fluorescence response signal during the plateau phase, and significantly improved detection sensitivity and amplification stability. These results indicate that the dual reverse primer synergistic amplification strategy can significantly improve the amplification efficiency of target nucleic acids, which is presumably related to the effective primer targeting binding efficiency and the advantages of primer synergistic amplification kinetics within the system.

[0083] 2.2 Screening of primers and probes against Southern Common Bean Mosaic Virus Positive standard samples were tested using the primer and probe combinations shown in Table 7 according to the method described in Example 1 to screen primer and probe combinations against Southern Common Bean Mosaic Virus.

[0084] Table 7 Two primer-probe combinations (combination 1 and combination 2) were designed to target Southern Common Bean Mosaic Virus (SBMV). These combinations were screened and validated against SBMV-positive standard samples under identical reaction conditions. Both combinations specifically amplified the target sequence, with no significant difference in Ct values, and both demonstrated good amplification efficiency. Combination 1 showed higher fluorescence signal intensity and a better signal-to-noise ratio, but it was prone to non-specific cross-reactions with other targets in multi-target detection systems. Combination 2, while showing a slightly lower fluorescence signal than Combination 1, exhibited stronger specificity and showed no non-specific amplification signals in multi-target systems, making it a more suitable preferred combination for multiplex detection systems. Therefore, Combination 2 was ultimately selected as the primer-probe combination for SBMV detection.

[0085] 2.3 Other primers and probes For the four target pathogens—tobacco ringspot virus, bean pod mottle virus, tomato spotted wilt virus, and ergot—the standardized primer and probe screening process described above was followed: multiple sets of candidate primer and probe combinations were designed based on the conserved gene sequences of each virus; Ct values, amplification curve kinetics, and amplification efficiency were evaluated using quantitative real-time PCR amplification of positive reference samples; then, specificity verification experiments were conducted to eliminate non-specific amplification and cross-reaction interference; by integrating orthogonal experimental data from multiple systems, the reaction conditions and concentration ratios of each primer and probe combination were systematically optimized and compatibility adjusted; finally, a set of primer and probe combinations with high specificity, high sensitivity, and no mutual interference was screened and determined, achieving seamless compatibility with soybean lectin internal control; and finally, the reaction solution formulations for the six soybean quarantine pathogens were determined, ensuring that each detection target has independent, specific, and efficient amplification capabilities in a single reaction tube.

[0086] Example 3: qPCR amplification performance test 1. Pathogen detection and sensitivity test Southern bean mosaic virus, tomato ringspot virus, tobacco ringspot virus, bean pod mottle virus, tomato wilt virus, ergot fungus, and the internal control lectin plasmid were formulated to a final concentration of 10. 7 A positive control of copies / mL was prepared using serial dilutions, with a total of 7 dilution gradients of 10 copies / mL. 6 10 5 10 4 10 3 Quantities of 250, 100, and 50 copies / mL were used. Multiplex quantitative PCR was employed to analyze the gradients of 10... 7 10 6 10 5 10 4 10 3 Plasmid templates of 250, 100, and 50 copies / mL were used for detection to determine the lowest plasmid concentration detectable by multiplex quantitative PCR. The results are as follows: Figure 2-8 As shown.

[0087] from Figure 2-6 It can be seen that the detection limit for Southern Bean Mosaic Virus, Tomato Ringspot Virus, Tobacco Ringspot Virus, Bean Pod Mottle Virus, and Tomato Spotted Wilt Virus reaches the level of 50 copies / mL.

[0088] Figure 7 The results showed that the detection limit for ergot reached the level of 100 copies / mL.

[0089] Figure 8 The results showed that the detection limit of the internal reference Lectin gene reached the level of 50 copies / mL.

[0090] 2. Multiplex qPCR specificity assay Using 0.9% sodium chloride solution and negative nucleic acid from soybean samples (soybean gene extract, virus-free) as controls, the multiplex nucleic acid detection system of this invention has no cross-contamination and no light crosstalk, ensuring that the detection results of the six target sequences and internal reference genes do not affect each other.

[0091] The results are as follows Figure 9 and Figure 10 As shown.

[0092] discuss Compared with existing technologies, the beneficial effects of this technical solution are as follows: 1. The multiplex real-time fluorescent PCR detection method and kit for six soybean quarantine pathogens of this invention can simultaneously detect six plant pathogens, avoiding the cumbersome steps of performing multiple reactions separately, greatly simplifying the operation process, improving work efficiency, and quickly meeting the customs' needs for monitoring imported agricultural products. 2. This invention uses soybean internal standards, avoiding interference from endogenous plant substances and improving the accuracy of detection results. Simultaneously, the use of internal standards avoids the inconvenience of using external standards for quantification, reducing operational complexity and saving costs. 3. The multiplex real-time fluorescent PCR detection method and kit for six soybean quarantine pathogens of this invention has the advantages of high sensitivity, high specificity, and rapid and simple operation, enabling rapid and accurate detection of plant pathogens, meeting the customs' needs for monitoring imported agricultural products, and has broad application prospects.

[0093] Due to the advanced nature of this technical solution, it has wide applications in fields such as biotechnology, molecular biology, and plant protection. Firstly, in the field of biotechnology, this invention develops a multiplex real-time fluorescent PCR detection method and kit for six soybean quarantine pathogens. This method enables single-tube detection of six plant pathogens without genotyping, greatly simplifying the operation process and improving detection efficiency. Simultaneously, the method uses soybean internal standards for quantification, avoiding the use of external standards and reducing operational complexity and cost. Therefore, this invention has broad application prospects in the field of biotechnology. Secondly, in the field of molecular biology, the real-time fluorescent PCR technology employed in this invention has advantages such as high sensitivity, high specificity, and rapid and convenient operation, enabling rapid and accurate detection of plant pathogens. Furthermore, this method can simultaneously detect multiple pathogens, significantly shortening detection time and improving work efficiency. Therefore, this invention has broad application prospects in the field of molecular biology. Finally, in the field of plant protection, the multiplex real-time fluorescent PCR detection method and kit for six soybean quarantine pathogens of this invention can meet the customs' requirements for monitoring imported agricultural products, which is of great significance for ensuring the safety and stability of agricultural production. Meanwhile, this method can rapidly and accurately detect plant pathogens, playing a crucial role in the prevention and control of plant epidemics. Therefore, this invention has broad application prospects in the field of plant protection. In summary, the multiplex real-time fluorescent PCR detection method and kit for six soybean quarantine pathogens of this invention is essentially a key tool for transforming cutting-edge biotechnology into national biosecurity defense capabilities and agricultural productivity assurance. Due to its high efficiency, accuracy, and speed, it can not only meet the customs' needs for monitoring imported agricultural products, but also has broad application prospects in fields such as biotechnology, molecular biology, and plant protection. Its significance goes far beyond "detection" itself; it is a concentrated embodiment of the modern plant protection concept of "proactive defense, source control, and scientific management." It protects not only soybean crops, but also the nation's oil security, farmers' incomes, international trade, and the long-term health of the agricultural ecological environment. Market demand is huge, and it has good commercial value.

[0094] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A primer and probe set for detecting six quarantine pathogens in soybeans, characterized in that, The primer and probe set includes: (1) A first primer and probe set for Southern Common Bean Mosaic Virus, comprising the upstream primer shown in SEQ ID NO:1, the downstream primer shown in SEQ ID NO:2, and the probe shown in SEQ ID NO:3; (2) A second primer and probe set for tomato ringspot virus, comprising the upstream primer shown in SEQ ID NO:4, the downstream primers shown in SEQ ID NO:5 and 6, and the probe shown in SEQ ID NO:7; (3) A third primer and probe set for tobacco ringspot virus, the third primer and probe set comprising the upstream primer shown in SEQ ID NO:8, the downstream primer shown in SEQ ID NO:9, and the probe shown in SEQ ID NO:10; (4) A fourth primer and probe set for bean pod mottle virus, the fourth primer and probe set comprising the upstream primer shown in SEQ ID NO:11, the downstream primer shown in SEQ ID NO:12 and the probe shown in SEQ ID NO:13; (5) A fifth primer-probe set for tomato spotted wilt virus, the fifth primer-probe set comprising the upstream primer shown in SEQ ID NO:14, the downstream primer shown in SEQ ID NO:15, and the probe shown in SEQ ID NO:16; and (6) A sixth primer-probe set for ergot, the sixth primer-probe set comprising the upstream primer shown in SEQ ID NO:17, the downstream primer shown in SEQ ID NO:18, and the probe shown in SEQ ID NO:

19.

2. The primer-probe set as described in claim 1, characterized in that, The probe is modified with fluorescent groups and quenching groups.

3. A PCR amplification system, characterized in that, The PCR amplification system includes a buffer system for amplification and a primer and probe set as described in claim 1.

4. The PCR amplification system as described in claim 3, characterized in that, The concentrations of each upstream primer and each downstream primer are 100-1000 nM, and the concentrations of each probe are 100-1000 nM.

5. The PCR amplification system as described in claim 3, characterized in that, The concentrations of each upstream primer and each downstream primer are 200-800 nM, and the concentrations of each probe are 200-800 nM.

6. A reagent kit, characterized in that, The kit includes a container and a primer and probe set as described in claim 1 or a PCR amplification system as described in claim 3, located within the container.

7. A method for detecting six quarantine pathogens in soybeans, characterized in that, The method includes the following steps: (s1) Provide the nucleic acid from the sample to be tested; (s2) Using the primer and probe set as described in claim 1, the PCR amplification system as described in claim 3, or the kit as described in claim 5, perform a nucleic acid amplification reaction on the sample to be tested, and detect the fluorescence signal to obtain a fluorescent PCR amplification curve; and (s3) Analyze the fluorescence PCR amplification curve to determine whether there are six soybean quarantine pathogens in the sample to be tested; The six quarantine pathogens of soybeans are Southern Bean Mosaic Virus, Tomato Ringspot Virus, Tobacco Ringspot Virus, Bean Pod Mottle Virus, Tomato Spotted Wilt Virus, and Ergot.

8. The method as described in claim 7, characterized in that, The reaction procedure for the nucleic acid amplification reaction is as follows: reverse transcription at 50℃ for 5 min; pre-denaturation at 95℃ for 90 s; denaturation at 95℃ for 5 s; annealing / extension at 55℃ for 30 s; 42 cycles.

9. The method as described in claim 7, characterized in that, In step (s2), the fluorescence signal is detected during the annealing / extension stage.

10. The method as described in claim 7, characterized in that, The judgment criteria for the method are as follows: (1) The internal reference (Lectin gene) Ct value is ≤38, and the negative control group and template-free control group have no Ct value; if the condition is not met, multiplex real-time quantitative PCR detection must be performed again, or nucleic acid must be extracted again for multiplex real-time quantitative PCR detection. (2) The amplification curve is a standard "S" shape with no abnormal fluctuations. The pathogen Ct value is ≤38. If 38 < Ct value ≤40, a retest is required. If the retest results are consistent, the result is considered positive.

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