Primer probe combination, kit and detection method for detecting longibrachiatum and application thereof

Abstract

This application belongs to the field of biological detection, specifically relating to primer-probe combinations, kits, detection methods, and applications for detecting *Verticillium longissima*. Through comparative genomic analysis of *Verticillium longissima* and its close relative *Verticillium dahliae*, this application identified the specific genomic sequence VL-spefic1 of *Verticillium longissima*, which can accurately distinguish between *Verticillium dahliae* and *Verticillium longissima*. Based on this specific sequence, DNA primers and RNA probes, as well as an EmDEA rapid detection method, were designed. This method is simple to operate, requires low experimental conditions, is quick, and the reaction reagents are easy to preserve. It can meet the detection requirements under different conditions, specifically detects *Verticillium longissima* from different sources, shows no positive results when detecting other similar species, and has high sensitivity. It is very suitable for routine import and export detection at ports and field monitoring needs, and has good application prospects.

Description

Technical Field

[0001] This application belongs to the field of biological detection, specifically relating to primer-probe combinations, kits, detection methods, and applications for detecting Verticillium longissima. Background Technology

[0002] Verticillium longisporum is a highly pathogenic plant fungus, currently mainly distributed in Sweden, Germany, the United Kingdom, Belgium, and Russia in Europe; the United States and Canada in North America; and parts of Japan and China in Asia. Verticillium longisporum has recently emerged in some parts of my country and is extremely harmful; therefore, the detection of Verticillium longisporum in imported samples is crucial.

[0003] In recent years, molecular biology methods have played a crucial role in the detection of plant pathogens, with common methods including qPCR, LAMP, and RCA. However, qPCR requires complex temperature changes, placing high demands on equipment and limiting its application. LAMP, on the other hand, suffers from non-specific amplification. Recombinase-mediated amplification (RAA) and recombinase polymerase amplification (RPA) technologies, developed in recent years, offer the advantage of low-temperature (37℃-42℃) amplification and can produce results in just 10-20 minutes. The entire process is rapid, efficient, simple to operate, and uses inexpensive equipment, making it a good alternative to qPCR.

[0004] With the development of molecular biology techniques, DNA sequence analysis technology has been widely used in fungal classification and identification. Among them, ITS sequences are the most widely used. However, ITS still has the disadvantages of insufficient variability and low resolution. In many cases, ITS cannot distinguish species at the species level.

[0005] In species identification, besides ITS, commonly used conserved sequences include mitochondrial genes (such as COI and cytb), ribosomal RNA genes (such as 16S, 18S, and 28S), and other functional genes (such as RPB1). Mitochondrial genes (such as COI) are the first choice for species identification due to their moderate mutation rate and strong primer universality, but they all suffer from insufficient resolution at the species level. Among ribosomal RNA genes, 16S is suitable for microbial classification, but its interspecific differentiation is limited, while 18S is used for higher-order eukaryotic classification. However, all of these marker genes suffer from low resolution at the species level.

[0006] Currently, the industry standard only includes a quantitative real-time PCR method for detecting *Verticillium*. This method significantly reduces detection time, but it requires sophisticated experimental conditions and high-quality equipment. Furthermore, within the *Verticillium* genus, the ITS sequence cannot differentiate between pathogens such as *Verticillium dahliae* and *Verticillium longissima* at the species level. A phylogenetic tree constructed based on the ITS sequence shows that *Verticillium longissima* has a bootstrap support value of only 24, making accurate classification impossible. Traditional methods primarily differentiate *Verticillium longissima* by isolating and culturing the organisms and then observing the conidia under a microscope. However, traditional isolation and culture methods are time-consuming, impacting subsequent processing and resulting in significant manpower and economic losses. Moreover, traditional isolation methods are prone to isolating large amounts of other microorganisms, affecting experimental results, and require highly skilled operators.

[0007] Therefore, there is an urgent need in this field for a simple, rapid, sensitive, and specific method for detecting Verticillium longissima at the species level. Summary of the Invention

[0008] To address the problems existing in the prior art, the purpose of this application is to provide a primer-probe combination, kit, detection method, and application for detecting *Verticillium longissima*. By using the described primer-probe combination, kit, and detection method, *Verticillium longissima* can be detected simply and rapidly at the species level, with high sensitivity and specificity.

[0009] The gene sequences of elongation factors (actins) are also commonly used for pathogen detection at the species level. For example, Duan et al. (2023) discovered a 16 bp sequence, CTCGACATGATCTTTG, in EF, which is unique to *Verticillium longissima* and could therefore be used as a TaqMan probe for specific identification of *Verticillium longissima*. However, with the increase in *Verticillium* actin sequences in NCBI, we re-analyzed all *Verticillium* actin sequences (a total of 483) and found that 29.67% of *Verticillium longissima* have a 16 bp specific sequence, CTCATCATGATCTTTG, which is consistent with the specific sequence of *Verticillium dahliae*. Therefore, these *Verticillium longissima* cannot be identified using the aforementioned TaqMan probe.

[0010] This application identified the specific genomic sequence VL-spefic1 of *Verticillium longissima* (L.) through comparative genomic analysis of *Verticillium dahliae*, a closely related species. The sequence is shown in SEQ ID NO. 1. Alignment of this sequence with the NCBI nucleic acid database revealed that it only aligned with *Verticillium longissima*, indicating that this sequence can accurately distinguish between *Verticillium dahliae* and *Verticillium longissima*. Based on the specific genomic sequence VL-spefic1, DNA primers and RNA probes were designed. Studies showed that using this combination of DNA primers and RNA probes can specifically detect *Verticillium longissima* from different sources, with no positive results when detecting other similar species. Furthermore, the sensitivity is very high, making it suitable for routine field detection of *Verticillium longissima*.

[0011] In a first aspect, this application provides a primer-probe combination, wherein the primer comprises a forward primer selected from any of the nucleotide sequences described in SEQ ID NO. 2-7 and a reverse primer selected from any of the nucleotide sequences described in SEQ ID NO. 14-19, and the probe is an RNA fluorescent probe.

[0012] In some embodiments, the RNA fluorescent probe is an RNA fluorescent probe selected from any of the nucleotide sequences described in SEQ ID NO. 8-13, with a reporter group labeled at the 5' end and a quencher group labeled at the 3' end. More preferably, the RNA fluorescent probe is an RNA fluorescent probe with the nucleotide sequence described in SEQ ID NO. 12.

[0013] The reporter group is selected from at least one of FAM, 6-FAM, VIC, HEX, TRT, CY3, CY5, ROX, JOE, FITC, TET, NED, TAMRA, LC RED640, LC RED705, Quasar705, or Texas Red. The quenching group is selected from at least one of TAMRA, BHQ1, BHQ2, BHQ3, MGB, and Dabcy1. Preferably, the reporter group is FAM, and the quenching group is BHQ1.

[0014] In some embodiments, the primer comprises a forward primer with the nucleotide sequence as described in SEQ ID NO. 3 and a reverse primer selected from any of the nucleotide sequences described in SEQ ID NO. 14-19, and the probe is an RNA fluorescent probe. More preferably, the RNA fluorescent probe is an RNA fluorescent probe with the nucleotide sequence as described in SEQ ID NO. 12.

[0015] In some embodiments, the primer comprises a forward primer selected from any of the nucleotide sequences described in SEQ ID NO. 2-7 and a reverse primer selected from the nucleotide sequences described in SEQ ID NO. 18, and the probe is an RNA fluorescent probe. More preferably, the RNA fluorescent probe is an RNA fluorescent probe with the nucleotide sequence described in SEQ ID NO. 12.

[0016] In some embodiments, the primer-probe combination comprises a forward primer with a nucleotide sequence as described in SEQ ID NO. 3 and a reverse primer with a nucleotide sequence as described in SEQ ID NO. 18, and an RNA fluorescent probe with a nucleotide sequence as described in SEQ ID NO. 12. Such a primer-probe combination offers superior detection sensitivity. Preferably, the 5' end of the probe is labeled with a reporter group, and the 3' end of the probe is labeled with a quencher group. More preferably, the reporter group is FAM, and the quencher group is BHQ1.

[0017] In a second aspect, this application provides a kit containing the primer-probe combination described in the first aspect.

[0018] In some embodiments, the kit further contains detection reagents required for enzyme-mediated double exponential amplification (DEA) nucleic acid detection. The detection reagents include a basic dry powder and an activation solution. The basic dry powder includes lyophilized products of the enzymes required for detection: DNA recombinase, shrimp alkaline phosphatase, T7 RNA polymerase, reverse transcriptase, and signal amplification enzyme, as well as a lyophilization protectant. The activation solution contains NTPs (nucleoside triphosphates) and a buffer solution.

[0019] In this application, the basic dry powder and activation solution are commercially available products. Specifically, the basic dry powder and activation solution used in this application are both purchased from the Basic Fluorescence Isothermal Amplification Detection Kit of Suzhou Jingrui Biotechnology Co., Ltd., catalog number RR032.

[0020] In some embodiments, the kit may also contain a DNA extraction reagent. The DNA extraction reagent is used to extract DNA (such as genomic DNA) from the sample to be tested.

[0021] In some embodiments, the primer-probe combination and various reagent components contained in the kit may be present in separate containers or may be partially pre-assembled into a reagent mixture if the detection requirements are met.

[0022] In some embodiments, the kit may also include a readable carrier describing the detection method for *Verticillium longissima* as described in this application. The readable carrier may be a kit instruction manual (e.g., a printed instruction manual) or a computer-readable medium (e.g., a floppy disk, CD, etc.) on which information has been recorded.

[0023] In a third aspect, this application provides a method for detecting Verticillium longissima, which detects whether the DNA of the sample to be tested contains a gene fragment as shown in SEQ ID NO.1. If the gene fragment as shown in SEQ ID NO.1 is present, then Verticillium longissima is present in the sample to be tested.

[0024] In one embodiment, the detection method includes:

[0025] (1) Extract DNA from the sample to be tested;

[0026] (2) Using the DNA of the sample to be tested as a template, the primer and probe combination described in the first aspect or the kit described in the second aspect are used to perform enzyme-mediated double exponential amplification (EmDEA) nucleic acid detection to form an amplification curve;

[0027] (3) Analyze the results based on the detected amplification curves (such as Ct value and fluorescence signal value) to determine whether Verticillium longissima is present in the sample to be tested.

[0028] If no Ct value is found, the test result is considered negative; if a Ct value is found, the test result is considered positive. The smaller the Ct value and the larger the fluorescence signal value, the better the detection effect or the higher the amount of *Verticillium laurentii* in the sample. Preferably, the criteria for judgment are: if an amplification curve appears in the sample and the Ct value is ≤20, then *Verticillium laurentii* is found in the sample; if no amplification curve appears in the sample or the Ct value is >20, then *Verticillium laurentii* is not found in the sample.

[0029] In some embodiments, the reaction procedure for enzyme-mediated double exponential amplification (DEA) nucleic acid detection is as follows: temperature 40–45°C, one cycle every 0.5–2 min, fluorescence signal detection once per cycle, and 20–35 cycles. Preferably, the reaction procedure for enzyme-mediated DEA nucleic acid detection is as follows: temperature 42°C, one cycle every 1 min, fluorescence signal detection once per cycle, and 30 cycles. In this application, the detection of fluorescence signals in the sample is preferably performed using fluorescence detection equipment well-known in the art or commercially available. In this application, a quantitative real-time PCR instrument or a constant-temperature fluorescence detector is preferably used to detect fluorescence signals in the sample. In this application, FAM fluorescence signals are acquired in each cycle, and the illumination of other fluorescence channels is turned off. The baseline period is preferably set to start at the beginning of the first cycle and end at the end of the second cycle, with signal acquisition performed once per cycle.

[0030] In practical implementation, those skilled in the art can configure the primers and probes required in the reaction system for enzyme-mediated double exponential amplification nucleic acid detection according to the detection purpose. Simultaneously, those skilled in the art can, based on common knowledge, set the other components involved in the reaction system for enzyme-mediated double exponential amplification nucleic acid detection and their specific amounts, all of which can achieve superior detection results in this application.

[0031] In some embodiments, the reaction system for enzyme-mediated double exponential amplification nucleic acid detection includes the following components: basic dry powder, forward primer 0.1-1 μM (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 μM), reverse primer 0.1-1 μM (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 μM), RNA fluorescent probe 2-3 ng / μL (e.g., 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3 ng / μL), DNA from the sample to be tested 1-8 μL (e.g., 1, 2, 3, 4, 5, 6, 7, 8 μL), and activation solution 10-15 μL (e.g., 10, 11, 12, 13, 14, 15 μL). Preferably, the reaction system is 20 μL, which can be made up with nuclease-free water. Preferably, the reaction system for enzyme-mediated double exponential amplification nucleic acid detection comprises the following components: basic dry powder, 0.5 μM forward primer, 0.5 μM reverse primer, 2.5 ng / μL RNA fluorescent probe, 5 μL DNA of the sample to be tested, and 12 μL activation solution. In this application, the reaction system is preferably prepared by adding the primer and probe combination, optional nuclease-free water, DNA of the sample to be tested, and activation solution to the basic dry powder and mixing thoroughly.

[0032] In some embodiments, the methods and steps for extracting DNA from the sample to be tested are well known in the art and are not particularly limited herein. The DNA extraction reagent in the kit described in the second aspect can be used to extract DNA from the sample to be tested.

[0033] In a fourth aspect, this application provides for the use of the primer-probe combination described in the first aspect or the kit described in the second aspect in any of the following:

[0034] 1) Application in the detection of Verticillium longissima;

[0035] 2) Application in the preparation of products for the detection of Verticillium longissima;

[0036] 3) Application in the identification or auxiliary identification of Verticillium longissima;

[0037] 4) Application in the preparation of products for the identification or auxiliary identification of Verticillium longissima;

[0038] 5) Application in identifying or assisting in the identification of whether a sample contains Verticillium longissima;

[0039] 6) Application in the preparation of products for identifying or assisting in the identification of whether a sample to be tested contains Verticillium longissima.

[0040] In a fifth aspect, this application provides the application of the detection method described in the third aspect in any of the following:

[0041] 1) Application in the detection of Verticillium longissima;

[0042] 2) Application in the identification or auxiliary identification of Verticillium longissima;

[0043] 3) Application in identifying or assisting in the identification of whether a sample to be tested contains Verticillium longissima.

[0044] In this application, the sample to be tested may be a host plant, an organ or tissue of a host plant, a fruiting body and / or sclerotium of a microorganism, or a microbial culture.

[0045] Those skilled in the art can combine the above-mentioned embodiments with common sense to obtain more embodiments of the method of this application. For example, features described or illustrated as part of some embodiments can be used in other embodiments to produce further embodiments.

[0046] Research has revealed that, compared to existing technologies, this application offers at least the following advantages:

[0047] (1) This application identified the specific genome sequence VL-spefic1 of Verticillium longisporum and its close relative Verticillium dahliae through comparative genomic analysis. This sequence can accurately distinguish Verticillium dahliae from Verticillium longisporum.

[0048] (2) This application designs specific DNA primer pairs and RNA probes, along with a rapid EmDEA detection method, based on the analysis of the VL-spefic1 specific genomic sequence of *Verticillium longissima*. This method is simple to operate, requires minimal experimental instruments and conditions, and can shorten the detection time to 0.5 hours, significantly saving manpower and resources. Furthermore, the reaction reagents and kits can be stored at 4°C, making storage conditions relatively simple. Therefore, the method described in this application can meet the detection requirements under different conditions and is highly suitable for routine import and export inspections at ports of entry and field monitoring needs, showing promising application prospects.

[0049] (3) This application determined the optimal primer-probe combination for the EmDEA detection method through cross-experiments with primers and probes. Specificity experiments showed that the primer-probe combination described in this application can distinguish *Verticillium longissima* from different sources, and showed no positive results when detecting other similar species, indicating good specificity and suitability for routine examination of *Verticillium longissima*. Sensitivity tests determined that the minimum detection limit of the method described in this application reaches 0.5 pg / μl. Attached Figure Description

[0050] To more clearly illustrate the technical solutions of the specific embodiments of this application, the drawings used in the description of the specific embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0051] Figure 1 EmDEA detection results for screening RNA probes for Verticillium longissima.

[0052] Figure 2 EmDEA detection results for reverse primer screening of Verticillium longissima DNA.

[0053] Figure 3 EmDEA detection results for forward primer screening of Verticillium longissima DNA.

[0054] Figure 4 This is a specific detection result for Verticillium longissima EmDEA.

[0055] Figure 5 The results show the sensitivity test results for EmDEA, the pathogen of *Lycopodium clavatum*. Detailed Implementation

[0056] The specific embodiments of this application are described in detail below. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit this application.

[0057] Unless otherwise stated, all terms used in the disclosure of this application (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. Further guidance is provided below for a better understanding of the teachings of this application. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0058] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0059] The terms “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, and do not exclude additional, uncited members, elements, or method steps.

[0060] The range of values ​​represented by endpoints in this application includes all values ​​and fractions contained within that range, as well as the endpoints referenced.

[0061] The concentration values ​​mentioned in this application include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% may fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are allowed. For example, 100mM may fluctuate within the ranges of ±1%, ±2%, ±5%, etc. Molecular weight fluctuations are allowed to include ±10%.

[0062] In this application, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0063] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0064] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0065] In this application, "optionally," "optionally," "optionally," "optionally," "optionally," and "optional" mean that they are optional, that is, they are selected from either "with" or "without." If a technical solution has multiple instances of "optional" or "optional," unless otherwise specified, and there are no contradictions or mutual constraints, then each "optional" or "optional" is independent.

[0066] In this application, *Verticillium longisporum* is a newly emerging and highly damaging plant pathogenic fungus in some parts of my country. It belongs to the kingdom Fungi, phylum Ascomycota, subphylum Pezizomycotina, class Sordariomycetes, subclass Ilypocreomycetidae, order Glomerellales, family Plectosphaerellaceae, and genus *Verticillium*. It has a wide host range and can infect common vegetable crops such as broccoli, radish, beet, cabbage, turnip, tomato, and pepper, causing various plant diseases, such as black heart disease, stem rot, and root rot in radishes.

[0067] In this application, the enzyme-mediated dual exponential amplification (EmDEA) nucleic acid detection technology organically integrates nucleic acid exponential amplification and signal exponential amplification into a single reaction tube. Through multi-enzyme collaboration, it performs dual amplification of target nucleic acid and fluorescent probe, achieving extremely high detection sensitivity and specificity in a short time. It can stably detect single-copy-level target nucleic acid under isothermal conditions for 20-30 minutes, combining the advantages of RAA and RPA. Furthermore, the kit can be stably stored at 4°C, making it highly potential for application in lower-tier markets.

[0068] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0069] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0070] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0071] I. Experimental Materials

[0072] 1.1 Test strains

[0073] The experimental strains were standard strains of *Verticillium longissima*, purchased from the DSMZ strain bank in Germany, with strain numbers DSM12231, 12232 and DSM 12233. These strains are available to the public from the applicant. The resulting biological materials can only be used for experimental verification in this application and cannot be used for other purposes.

[0074] 1.2 Instruments and Reagents

[0075] Nucleic acid extraction kit: DNAsecure plant kit, a novel plant genomic DNA extraction kit, is a product of Tiangen Biotech (Beijing) Co., Ltd., Cat No. DP320-03.

[0076] The Basic Fluorescent Isothermal Amplification Detection Kit (RR032) is a product of Suzhou Jingrui Biotechnology Co., Ltd., Cat No. 613046.

[0077] The real-time fluorescence PCR instrument was the CFX96 Real-time system manufactured by ABI Pharmaceuticals, Inc.

[0078] II. Test Methods

[0079] 2.1 DNA Extraction

[0080] The standard strain of *Verticillium longissima* was inoculated onto plates containing PDA medium and cultured. Mycelia of the strain cultured on the PDA plates for approximately 10 days were scraped using a sterilized pipette tip, and DNA was extracted according to the DNAsecure plant kit instructions. The extracted DNA concentration was determined using a spectrophotometer and then stored at -20°C for later use.

[0081] 2.2 EmDEA Detection

[0082] Nucleic acid amplification experiments were performed using the Basic Fluorescent Isothermal Amplification Detection Kit (catalog number: RR032). The specific reaction system contained the following components: 0.5 μL (20 μmol / L) of forward primer (DNA-F), 0.5 μL (20 μmol / L) of reverse primer (DNA-R), 0.2 μL (250 ng / μL) of fluorescent probe (Probe), 1.8 μL of deionized water (ddH2O, prepared using a Milli-Q ultrapure water system), 5.0 μL of DNA extract from the sample to be tested, 12.0 μL of the kit's activation solution, and one tube of the basic dry powder reaction system included in the kit.

[0083] Add 0.5 μL each of 20 μmol / L forward and reverse primers, 0.2 μL of 250 ng / μL RNA probe, and 1.8 μL of ddH2O to an eight-tube strip of the Basic Fluorescent Isothermal Amplification Detection Kit containing the basic dry powder (including the lyophilized products of the enzymes required for detection: DNA recombinase, shrimp alkaline phosphatase, T7 RNA polymerase, reverse transcriptase, and signal amplification enzyme, and the lyophilization protectant), and a total of 3.0 μL. Centrifuge briefly. After centrifugation, add 5.0 μL of template DNA along the lower part of the tube wall, centrifuge briefly, and incubate at room temperature for 2 min. Add 12.0 μL of activation solution (a solution composed of NTP (nucleoside triphosphate) and buffer, a component of the Basic Fluorescent Isothermal Amplification Detection Kit) to the lower part of the tube wall, cap the tube, vortex for 10 s, centrifuge briefly, and then perform the detection.

[0084] ddH2O was used as the amplification template for the blank control group. The only difference between the blank control group and the experimental group was the presence or absence of sample DNA template. The blank control was set up in three replicates, and the results were averaged.

[0085] The real-time fluorescence PCR instrument was set to 42℃, with one cycle lasting 1 minute, for a total of 30 cycles. Signal acquisition was performed once per cycle, with the reporter group set to FAM for real-time detection.

[0086] Example 1: Primer screening and establishment of a rapid detection method for enzyme-mediated double-exponential amplification nucleic acid detection of *Verticillium longissima*.

[0087] 1.1 Design and Synthesis of Primers and Probes

[0088] Comparative genomic analysis of *Verticillium longissima* DSM 12233 and its close relative *Verticillium dahliae* identified the *Verticillium longissima*-specific genomic sequence VL-spefic1, whose sequence is SEQ ID NO. 1:

[0089]

[0090] The sequence was compared with the nucleic acid database in NCBI and it was found that the sequence could accurately distinguish between Verticillium longissima and Verticillium dahliae.

[0091] Based on the *Verticillium longissima*-specific sequence VL-spefic1 obtained from comparative genomics analysis, DNA primers and RNA probes were designed. The DNA primers were synthesized by BGI Genomics, and the RNA probes were provided by Suzhou Jingrui Biotechnology Co., Ltd. The specific sequences of the primers and probes are shown in Table 1. The RNA probe was labeled with the reporter group FAM at its 5' end and the quencher group BHQ1 at its 3' end.

[0092] Table 1. Specific sequences of primers and probes.

[0093]

[0094]

[0095] 1.2 RNA probe screening

[0096] DNA from DSM 12233 was used as the amplification template (template DNA) for the experimental group, with a DNA concentration of 50 ng / μL. DNA primers VL-F2, VL-R3, VL-F4, and VL-R5 were cross-combined to obtain four DNA primer combinations: F2R3, F2R5, F4R3, and F4R5. Two RNA probes, VL-RNA3 and VL-RNA5, were added to each primer combination, resulting in a total of eight combinations. Three blank controls were included in each combination. The reaction system and procedure were followed according to the experimental methods section.

[0097] By comparing the groups with the same primer pair, the RNA probe with the lowest CT value and the highest endpoint fluorescence value was selected as the RNA probe for subsequent studies.

[0098] Table 2 Grouping of DNA primer pairs and RNA probe screening

[0099] F2R3 F2R5 F4R3 F4R5 RNA3 RNA3F2R3 RNA3F2R5 RNA3F4R3 RNA3F4R5 RNA5 RNA5F2R3 RNA5F2R5 RNA5F4R3 RNA5F4R5

[0100] Note: In the table, RNA represents RNA probe, F represents forward primer, and R represents reverse primer.

[0101] The results of RNA probe screening are as follows: Figure 1 As shown, the RNA5F4R5 group had a lower CT value of 6.27 and a higher fluorescence value, while the corresponding blank control showed no signal. Therefore, the RNA probe VL-RNA5 was selected for subsequent experimental screening.

[0102] 1.3 DNA Reverse Primer Screening

[0103] The experiment was divided into an experimental group (using strain DNA as amplification template) and a blank control group (using water as amplification template). DSM 12233 strain DNA was used as the DNA template, VL-RNA5 as the RNA probe, VL-F4 as the forward primer, and VL-R1, VL-R2, VL-R3, VL-R4, VL-R5, and VL-R6 as the reverse primers. The reaction system and procedure were followed according to the experimental methods section. Each experimental group had three replicates, and a blank control was included.

[0104] The results of DNA reverse primer screening are as follows: Figure 2 As shown, the CT value is relatively low (9.27) when the reverse primer is VL-R5. Therefore, VL-RNA4 and VL-R5 were selected for subsequent screening. That is, a primer-probe combination containing probe RNA4 and reverse primer R5 was used for subsequent screening.

[0105] 1.4 DNA forward primer screening

[0106] The experiment was divided into an experimental group (using bacterial DNA as amplification template) and a blank control group (using water as amplification template). DSM 12233 bacterial DNA was used as the DNA template, RNA5 as the RNA probe, and VL-R5 as the reverse primer. VL-F1, VL-F2, VL-F3, VL-F4, VL-F5, and VL-F6 were used as the forward primers. The reaction system and procedure were followed according to the experimental methods section. Each experimental group had three replicates, and a blank control was included.

[0107] The results of DNA forward primer screening are as follows: Figure 3 As shown, the CT value of the RNA5F2R5 group is relatively low (8.59), and the fluorescence intensity is relatively high. Therefore, the primer-probe combination containing RNA probe VL-RNA5, forward primer VL-F2, and reverse primer VL-R5 was determined to be more effective and was used for subsequent screening.

[0108] 1.5 Specificity verification of primer-probe combinations

[0109] Using the DNA of the tested strain as a template and water as a blank control, EmDEA detection was performed. DNA was partially extracted from the strain according to the experimental method, and the detection was performed according to the reaction system and procedure.

[0110] Results of specific detection, such as Figure 4As shown, all three standard strains of *Verticillium longissima* could be detected by the RNA5F2R5 primer-probe combination, while other soil-borne diseases, including *Fusarium wilt* (FOP), *Cladosporium pubescens* (PB1), *Verticillium dahliae* (FS), and *Verticillium rot* (XCA), could not be detected. This indicates that RNA5F2R5 has excellent species specificity and can specifically detect *Verticillium longissima* disease in actual testing without interference from other non-*Verticillium longissima* fungi.

[0111] 1.6 Sensitivity Verification of Primer-Probe Combinations

[0112] Using DSM12233 DNA as a template, the extracted DNA concentration was 20 ng / μL. Six 10-fold serial dilutions were performed using RNase-free ddH2O, with three replicates for each concentration. Real-time fluorescent PCR was performed using the primer-probe combination RNA5F2R5, following the reaction system and procedure described in the assay section, to determine the sensitivity.

[0113] The results of the sensitivity verification are as follows Figure 5 As shown, the minimum amount of DNA that can be detected in the reaction system is 10 pg, which means that the sensitivity of this group is 0.5 pg / μL.

[0114] This application identified the specific genomic sequence of *Verticillium longissima* through comparative genomic analysis and designed corresponding primers and probes, thus developing an EmDEA rapid detection method for *Verticillium longissima*. Through cross-experiments with primers and probes, the optimal primer-probe combination for the EmDEA method was determined, enabling specific detection of *Verticillium longissima* without interference from other non-*Verticillium longissima* species. Sensitivity tests determined the limit of detection to be 0.5 pg / μL.

[0115] This application describes the detection of Verticillium longissima using the EmDEA method. The method is simple to operate, requires few experimental instruments, has low experimental condition requirements, and is quick, reducing the detection time to 0.5 hours. It can meet the detection requirements under different conditions, greatly saving manpower and resources. Furthermore, since the kit can be stored at 4°C, the storage conditions are relatively simple, making it suitable for daily import and export inspections at ports and on-site monitoring needs, and it has good application prospects.

[0116] The embodiments described above are merely one implementation method of this application, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these modifications and improvements all fall within the protection scope of this application.

Claims

1. A primer-probe combination, characterized in that, The primers comprise a forward primer selected from any of the nucleotide sequences described in SEQ ID NO. 2-7 and a reverse primer selected from any of the nucleotide sequences described in SEQ ID NO. 14-19, and the probe is an RNA fluorescent probe; preferably, the RNA fluorescent probe is an RNA fluorescent probe selected from any of the nucleotide sequences described in SEQ ID NO. 8-13, with a reporter group labeled at the 5' end of the probe and a quencher group labeled at the 3' end of the probe.

2. The primer-probe combination according to claim 1, characterized in that, The primers comprise a forward primer with a nucleotide sequence as described in SEQ ID NO.3 and a reverse primer with a nucleotide sequence as described in SEQ ID NO.18, and the probe is an RNA fluorescent probe with a nucleotide sequence as described in SEQ ID NO.12; preferably, the reporter group is FAM and the quencher group is BHQ1.

3. A reagent kit, characterized in that, It contains the primer-probe combination as described in claim 1 or 2.

4. The reagent kit according to claim 3, characterized in that, The kit also contains detection reagents required for enzyme-mediated double exponential amplification nucleic acid detection; preferably, the detection reagents include basic dry powder and activation solution; more preferably, the basic dry powder includes lyophilized products of the enzymes required for detection: DNA recombinase, shrimp alkaline phosphatase, T7 RNA polymerase, reverse transcriptase and signal amplification enzyme, as well as a lyophilization protectant, and the activation solution contains NTP (nucleoside triphosphate) and buffer solution.

5. The kit according to claim 3 or 4, characterized in that, The kit also contains DNA extraction reagents.

6. A method for detecting *Verticillium longissima*, characterized in that, It detects whether the DNA of the sample to be tested contains a gene fragment as shown in SEQ ID NO.

1. If it contains a gene fragment as shown in SEQ ID NO.1, then Verticillium longissima is present in the sample to be tested.

7. The detection method according to claim 6, characterized in that, include: (1) Extract DNA from the sample to be tested; (2) Using the DNA of the sample to be tested as a template, the primer and probe combination described in claim 1 or 2, or the kit described in any one of claims 3-5, is used to perform enzyme-mediated double exponential amplification nucleic acid detection to form an amplification curve; (3) Analyze the results based on the detected amplification curve to determine whether Verticillium longissima is present in the sample to be tested.

8. The detection method according to claim 7, characterized in that, The reaction procedure for enzyme-mediated double exponential amplification nucleic acid detection is as follows: temperature 40–45℃, one cycle every 0.5–2 min, fluorescence signal detection once per cycle, and 20–35 cycles; and / or The reaction system for enzyme-mediated double exponential amplification nucleic acid detection comprises the following components: basic dry powder, 0.1-1 μM forward primer, 0.1-1 μM reverse primer, 2-3 ng / μL RNA fluorescent probe, 1-8 μL DNA of the sample to be tested, and 10-15 μL activation solution; preferably, the reaction system is 20 μL, which can be made up with nuclease-free water.

9. The use of the primer-probe combination of claim 1 or 2, or the kit of any one of claims 3-5, in any of the following: 1) Application in the detection of Verticillium longissima; 2) Application in the preparation of products for the detection of Verticillium longissima; 3) Application in the identification or auxiliary identification of Verticillium longissima; 4) Application in the preparation of products for the identification or auxiliary identification of Verticillium longissima; 5) Application in identifying or assisting in the identification of whether a sample contains Verticillium longissima; 6) Application in the preparation of products for identifying or assisting in the identification of whether a sample to be tested contains Verticillium longissima.

10. The application of the detection method according to any one of claims 6-8 in any of the following: 1) Application in the detection of Verticillium longissima; 2) Application in the identification or auxiliary identification of Verticillium longissima; 3) Application in identifying or assisting in the identification of whether a sample to be tested contains Verticillium longissima.

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