Specific detection primer and kit for sarcococca ruscifolia tail anthracnose fungus and application thereof

Abstract

The present application belongs to the technical field of plant pathogenic bacteria detection, and particularly relates to Cercospora cf. sigesbeckiae specific detection primers, a kit and application thereof. The detection primers or the kit can realize specific detection of Cercospora cf. sigesbeckiae and have good intraspecies strain universality. Meanwhile, through sensitivity experiments, it is known that the detection system and the detection method established by the present application have a minimum detection concentration of 0.021 pg muL for Cercospora cf. sigesbeckiae. ‑1 For detection of soybean grains showing purple blight symptoms in the field, the detection system of the present application can also quickly and accurately identify whether the soybean plants carry the pathogenic bacteria Cercospora cf. sigesbeckiae. Therefore, the detection primers and the kit of the present application have good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of plant pathogen detection technology, specifically relating to primers, kits and their applications for the specific detection of Siegesbeckia orientalis. Background Technology

[0002] The fungus *Cercospora cf. sigesbeckiae* causes two diseases in soybeans: Purple Seed Stain (PSS) and Cercospora Leaf Blight (CLB). Both diseases occur in soybean-producing regions worldwide and can infect multiple parts of the soybean plant. Purple Seed Stain primarily affects the seeds, causing irregular, purplish-brown to dark purple patches on the seed coat. In severe cases, the entire seed turns purple, significantly reducing the appearance and commercial value of the soybean. Cercospora Leaf Blight primarily affects the leaves, initially causing brown to reddish-brown spots with yellow halos around the edges. Later, the spots expand and merge, leading to premature leaf death and abscission. Severe outbreaks can result in yield losses of nearly 30%.

[0003] As an important food and economic crop in my country, soybean production safety is a major concern. Therefore, establishing an accurate and rapid detection system for Siegesbeckia orientalis pathogens is of significant scientific importance and practical value for achieving early warning and precise control of this disease.

[0004] Currently, there is no mature and specific detection system for *Cercospora siegescens*. Detection of *Cercospora* fungi mainly relies on morphological identification and molecular biology techniques. Traditional morphological identification requires the isolation and culture of pathogens, mainly based on the colony morphology, conidiophores, and microscopic characteristics of conidia. However, this method is time-consuming, difficult to distinguish between morphologically similar species, and easily influenced by subjective experience. Molecular biology detection techniques have become the focus of current research and application due to their high specificity, high sensitivity, and speed. Conventional PCR identification based on specific primers is widely used, but its sensitivity is low. Loop-mediated isothermal amplification and recombinase polymerase amplification techniques have been successfully applied to the detection of *Cercospora sojina*, suitable for rapid screening, but cannot achieve quantitative analysis of pathogens, and both pose a risk of aerosol contamination. Costa et al. established a real-time fluorescence quantitative PCR method for *Cercospora kikuchii* based on the CTB6 gene, but its target gene still has certain limitations in terms of intraspecific conservation.

[0005] Therefore, it is of great significance to develop a qPCR detection system based on highly specific detection targets of Siegesbeckia orientalis. Summary of the Invention

[0006] To address the technical problems in the prior art, this invention provides specific detection primers and kits for Siegesbeckia orientalis and their applications. Using the detection primers or kits of this invention, specific detection of Siegesbeckia orientalis can be achieved, while also having good versatility with strains within the same species.

[0007] In a first aspect, the present invention provides primers for the specific detection of *Cercospora cf. sigesbeckiae*, wherein the target gene detected by the primers is Ccs11137, the nucleotide sequence of which is shown in SEQ ID NO.1. The primers include a forward primer Ccs-F1 and a reverse primer Ccs-R1, the sequence of which is shown in SEQ ID NO.2, and the sequence of which is shown in SEQ ID NO.3.

[0008] In this application, a novel specific detection target gene Ccs11137 for Siegesbeckia orientalis is provided, and corresponding quantitative detection primers are developed based on this target gene.

[0009] Secondly, the present invention also provides a kit for detecting Siegesbeckia orientalis, the kit comprising the aforementioned forward primer Ccs-F1 and reverse primer Ccs-R1.

[0010] Furthermore, the concentrations of the forward primer Ccs-F1 and the reverse primer Ccs-R1 are 8-12 μmol·L⁻¹, respectively. -1 Preferably, the concentrations are 8 μmol·L⁻¹. -1 9 μmol·L -1 10 μmol·L -1 11 μmol·L -1 12 μmol·L -1 .

[0011] Furthermore, the kit also includes DNA polymerase, fluorescent dye, dNTPs, and Mg. 2+ and buffer solution.

[0012] Thirdly, the present invention also provides the application of the primers or the kits described herein in the detection of Siegesbeckia orientalis.

[0013] Fourthly, the present invention also provides a qPCR method for detecting Siegesbeckia orientalis, the method comprising the step of performing qPCR amplification on the genomic solution of the target using the primers or the kit described above.

[0014] Further, the step includes taking 1-2 μL of the DNA solution of the target sample and adding Hieff UNICON. ®Advanced qPCR SYBR Master Mix 8-12 μL, 8-12 μmol·L -1 0.3-0.5 μL each of the forward primer Ccs-F1 and the reverse primer Ccs-R1, 0.5-1.5 μL of DMSO, and bring the volume to 19-22 μL using RNA-Free H2O for qPCR amplification.

[0015] Further, the step includes taking 2 μL of the DNA solution of the target sample and adding Hieff UNICON. ® Advanced qPCR SYBR Master Mix 10 μL, 10 μmol·L -1 0.4 μL each of the forward primer Ccs-F1 and the reverse primer Ccs-R1, 1 μL of DMSO, and RNA-Free H2O were added to bring the volume to 20 μL for qPCR amplification.

[0016] Furthermore, the qPCR amplification program is as follows: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, 60℃ extension for 10 s, for 40 cycles; the melting curve is set to 65℃ for 5 s, 95℃ extension followed by cooling to 0.5℃ for storage.

[0017] Compared to existing technologies, this invention provides a specific detection target gene Ccs11137 for *Cercospora cf. sigesbeckiae*, and establishes quantitative detection primers and a quantitative PCR method based on this target. The detection primers of this invention can achieve specific detection of *Cercospora cf. sigesbeckiae* across different genera, and also possess good versatility within the same species. Furthermore, sensitivity experiments show that the detection system and method established in this invention achieve a minimum detection concentration of 0.021 pg·µL for *Cercospora cf. sigesbeckiae*. -1 The detection system of this invention can quickly and accurately identify whether soybean plants carry the pathogen Siegesbeckia orientalis when testing soybean grains exhibiting symptoms of purple blotch in the field, and has great application prospects. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below.

[0019] Figure 1 The target regions of primers Ccs-F1 / Ccs-R1 and their sequence alignments across different species are shown.

[0020] Figure 2The results show the qPCR amplification of DNA from 13 pathogenic bacteria using primers Ccs-F1 / Ccs-R1.

[0021] Figure 3 The results of PCR amplification of DNA extracted from six strains of Siegesbeckia orientalis using primers Ccs-F1 / Ccs-R1.

[0022] Figure 4 The amplification results and standard curves of Siegesbeckia orientalis DNA with different concentration gradients using primers Ccs-F1 / Ccs-R1 are plotted; where A is the melting curve, B is the amplification curve, and C is the standard curve.

[0023] Figure 5 Symptoms of soybean purple spot disease and qPCR test results. Detailed Implementation

[0024] The embodiments of the technical solution of the present invention will now be described in detail with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art to which this invention pertains.

[0025] Example 1

[0026]

[0027] Table 1. Species genomic information used for homology clustering analysis

[0028]

[0029] Example 2

[0030] Primers Ccs11137-qPCR-F1 / R1 (hereinafter referred to as Ccs-F1 / Ccs-R1) were designed based on polymorphic sequence regions in the target gene Ccs11137. The targeted regions of the primers and the sequence alignment results between different species are as follows: Figure 1 As shown.

[0031] Based on the aforementioned new detection targets, primers were designed to establish a quantitative PCR detection method. Genomic DNA from four Cercospora species (C. cf. sigesbeckiae, C. cf. flagellaris, C. fagopyri, and C. kikuchii) and eight major soybean pathogens (F. equiseti, F. oxysporum, Diaporthe longicolla, Alternaria alternatea, Colletotrichum truncatum, Rhizoctonia solani, Pythium ultimum, and Phytophthora sojae) and one Pseudocerospora kaki was used as templates to verify the specificity of the detection system.

[0032] The detection primer composition used in the qPCR detection method for C. cf. sigesbeckiae consists of the forward primer Ccs11137-qPCR-F1 and the reverse primer Ccs11137-qPCR-R1. The primer sequences are as follows:

[0033] Ccs11137-qPCR-F1: 5'-ACAGGCCTTGAAATGGCGTA-3' (SEQ ID NO. 2);

[0034] Ccs11137-qPCR-R1: 5'-CCGCCATCTGTTCGAGCTAA-3' (SEQ ID NO. 3).

[0035] Genomic DNA of the tested strain, HieffUNICON, was amplified using the designed primer combination Ccs11137-qPCR-F1 / R1. ® Advanced qPCR SYBR Master Mix 10.0 μL, forward and reverse primers (10 μmol·L⁻¹) -1 Add 0.4 μL of each of the following: genomic DNA, 2.0 μL of DMSO, and 1.0 μL of RNA-free H2O to a final volume of 20.0 μL.

[0036] The qPCR reaction program was as follows: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, and 60℃ extension for 10 s, for 40 cycles; the melting curve was set to 65℃ for 5 s, followed by 95℃ extension and then cooling to 0.5℃ for storage. After the qPCR reaction was completed, data analysis was performed in BioRad CFX Manager.

[0037] The results are as follows Figure 2 As shown, Ccs11137-qPCR-F1 / R1 specifically amplified a single product from the DNA of *C. cf. sigesbeckiae*, with a melting curve Tm value of 79.0 ℃. No effective amplification was observed in any of the other tested strains or the negative control. These results indicate that the primer combination designed based on the novel detection target Ccs11137 possesses high specificity for the target species.

[0038] Example 3

[0039] To verify the intraspecific universality of primers Ccs-F1 / Ccs-R1, six C. cf. sigesbeckiae strains from different geographical origins were selected and named CS-1 to CS-6, respectively. Their genomic DNA was extracted and used as templates. Simultaneously, enzyme-free sterile water was set as a negative control for PCR amplification.

[0040] Genomic DNA of the tested strain was amplified using the designed primer combination Ccs11137-qPCR-F1 / R1: 12.5 μL of 2 × RapidTaq Plus Master Mix (Dye Plus) and upstream and downstream primers (10 μmol·L⁻¹). -1 Add 1.0 μL of each of the following: genomic DNA and RNA-free H2O to a final volume of 25.0 μL.

[0041] The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 35 cycles of 95℃ denaturation for 30 s, 60℃ annealing for 10 s, and 72℃ extension for 20 s; final extension at 72℃ for 7 min; and storage at 12℃. After the reaction, the amplified products were verified by agarose gel electrophoresis.

[0042] The results are as follows Figure 3 As shown, all six C. cf. sigesbeckiae strains were able to amplify the expected bands using primers Ccs-F1 / Ccs-R1, while the negative controls showed no amplification. These results indicate that Ccs-F1 / Ccs-R1 has good intraspecific universality in the corresponding target species.

[0043] Example 4

[0044] To evaluate the sensitivity of the real-time fluorescence PCR detection system established based on the novel detection target Ccs11137, the detection limit of genomic DNA of C. cf. sigesbeckiae was analyzed. The initial concentration was 10 ng·µL. -1 Genomic DNA of C. cf. sigesbeckiae was serially diluted 10-fold to obtain a concentration of 10 ng·µL. -1 1 ng·µL -1 100 pg·µL -1 10 pg·µL -1 1 pg·µL -1 and 100 fg·µL -1 A series of templates were used for qPCR amplification.

[0045] Genomic DNA of C. cf. sigesbeckiae at different concentrations was amplified using the designed primer combination Ccs11137-qPCR-F1 / R1: Hieff UNICON ® Advanced qPCR SYBR Master Mix 10.0 μL, forward and reverse primers (10 μmol·L⁻¹) -1 Add 0.4 μL of each of the following: genomic DNA, 2.0 μL of DMSO, and 1.0 μL of RNA-free H2O to a final volume of 20.0 μL.

[0046] The qPCR reaction program was as follows: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, 60℃ extension for 10 s, for 40 cycles; the melting curve was set to 65℃ for 5 s, followed by 95℃ extension and then cooling to 0.5℃ for storage. After the PCR reaction was completed, data analysis was performed in BioRad CFX Manager.

[0047] like Figure 4 As shown, for the detection system of C. cf. sigesbeckiae, the template concentration ranged from 10 ng·µL. -1 Up to 1 pg·µL -1 All six gradients achieved stable amplification (e.g. Figure 4 (See Figure A in the image), where the Ct values ​​are 16.20, 19.23, 22.57, 25.83, 29.26, and 33.00 respectively (e.g., ...). Figure 4 (The amplification curve in Figure B). Use this to plot a standard curve (e.g., Figure 4 (See Figure C). The linear relationship between the logarithm of DNA concentration (X) and the Ct value (Y) is: Y = -3.353X + 29.38 (R²). 2 =0.9990). According to this equation, when the Ct value is 35 (the conventional detection threshold), the corresponding DNA concentration is 0.021 pg·µL. -1 This refers to the lowest concentration that the system can detect.

[0048] The above results indicate that the real-time fluorescence PCR detection system established based on the new target gene Ccs11137 has high sensitivity.

[0049] Example 5

[0050] To verify the effectiveness of the above detection system in detecting the presence of *Siegesbeckiae*, a pathogenic fungus in soybean plants, healthy seeds of the Nannong 47 variety and purple-spotted soybean seeds from different origins (Heinong 113, Heinong 143, Heinong 327, Heinong 531, Hefeng 47, Nannong 47, and Gongxiadou 13) were selected, and the seed coat genomic DNA was extracted for detection. Soybean genomic DNA carrying *C. cf. sigesbeckiae* was amplified using the designed primer combination Ccs11137-qPCR-F1 / R1: Hieff UNICON ® Advanced qPCR SYBRMaster Mix 10.0 μL, forward and reverse primers (10 μmol·L⁻¹) -1 Add 0.4 μL of each of the following: genomic DNA, 2.0 μL of DMSO, and 1.0 μL of RNA-free H2O to a final volume of 20.0 μL.

[0051] The qPCR reaction program was as follows: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, and 60℃ extension for 10 s, for 40 cycles. The melting curve was set to 65℃ for 5 s, followed by 95℃ extension and then cooling to 0.5℃ for storage. After the PCR reaction, data analysis was performed in BioRad CFX Manager, and the results are as follows. Figure 5 show.

[0052] When testing soybean grains exhibiting symptoms of purple blotch (Heinong 113, Heinong 143, Heinong 327, Heinong 531, Hefeng 47, Nannong 47, and Gongxiadou 13), the above-mentioned qPCR system was able to effectively and specifically detect infection with Siegesbeckia orientalis (see...). Figure 5 (Ct value in the middle table).

[0053] Subsequent pathogen isolation, culture, and phylogenetic identification of soybean grains that tested positive by qPCR yielded results consistent with the detection conclusions, further confirming the reliability of the detection system. This indicates that the real-time fluorescent PCR detection system constructed based on the target gene Ccs11137 can rapidly and accurately identify whether soybean plants carry the pathogen Siegesbeckia orientalis.

[0054] Unless otherwise specifically stated, the numerical values ​​set forth in these embodiments do not limit the scope of the invention. In all examples shown and described herein, any specific value should be interpreted as merely exemplary and not as a limitation, unless otherwise specified; therefore, other examples of exemplary embodiments may have different values.

Claims

1. Primers specifically for the detection of Cercospora cf. Sigesbeckiae, characterized in that, The target gene detected by the primers is Ccs11137, the nucleotide sequence of which is shown in SEQ ID NO.

1. The primers include a forward primer Ccs-F1 and a reverse primer Ccs-R1, the sequence of which is shown in SEQ ID NO.2 and the sequence of which is shown in SEQ ID NO.

3.

2. A kit for detecting Siegesbeckia orientalis, characterized in that, The kit includes the forward primer Ccs-F1 and the reverse primer Ccs-R1 as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The concentrations of the forward primer Ccs-F1 and the reverse primer Ccs-R1 are 8-12 μmol·L⁻¹. -1 .

4. The reagent kit according to claim 3, characterized in that, The kit also includes DNA polymerase, fluorescent dye, dNTPs, and Mg. 2+ and buffer solution.

5. The use of the primers of claim 1 or the kits of any one of claims 2-4 in the detection of Siegesbeckia orientalis.

6. A qPCR method for detecting Siegesbeckia orientalis, characterized in that, The method includes the step of performing qPCR amplification on the genomic solution of the target object using the primers of claim 1 or the kits of any one of claims 2-4.

7. The method according to claim 6, characterized in that, The qPCR amplification program is as follows: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, 60℃ extension for 10 s, for 40 cycles; the melting curve is set to 65℃ for 5 s, 95℃ extension followed by cooling to 0.5℃ for storage.

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