A primer probe composition for quantitatively detecting wild apricot perforation disease pathogen and application thereof

Abstract

This invention discloses a primer and probe composition for the quantitative detection of the pathogen causing leaf spot disease in wild apricot and its application, belonging to the field of agricultural biotechnology. The primer and probe composition for the quantitative detection of the pathogen causing leaf spot disease in wild apricot is mentioned, wherein the pathogen is *Scutellaria cirrhosa* (*Scutellaria cirrhosa*). Wilsonomyces carpophilus ) and / or clusters of pantothenic acid ( Pantoea agglomerans The primer-probe composition comprises a first primer pair and a first probe W.car-P for detecting *Cyclophorus frutescens*, and a second primer pair and a second probe P.agg-P for detecting *Pantotheca acuminata*. The purpose of this invention is to provide a primer-probe composition for the quantitative detection of the pathogen causing leaf spot disease in wild apricots and its application. This primer-probe composition exhibits high sensitivity, strong specificity, good stability, and high repeatability, enabling rapid and simultaneous detection of *Cyclophorus frutescens* and *Pantotheca acuminata*.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and in particular to a primer-probe composition for the quantitative detection of the pathogen causing leaf spot disease in wild apricots and its application. Background Technology

[0002] Wild apricot is a Class II protected wild plant in China, mainly distributed in the Tianshan Mountains of the Ili region of Xinjiang. It is the origin of cultivated apricots worldwide and plays an irreplaceable role in maintaining regional ecological balance and protecting biodiversity. In recent years, leaf spot disease has become the core cause of death in wild apricot forests in the Tianshan Mountains, posing a serious challenge to the continuation of wild apricot populations. Wild apricot leaf spot disease is mainly caused by two types of pathogens: fungi and bacteria. The main pathogen of fungal leaf spot disease is *Cyclocarya pallida*, which can infect the leaves, fruits, dormant buds, and young branches of wild apricots, causing purplish-brown lesions on leaves that fall off and perforate, and brown sunken spots on fruits. One of the main pathogens of bacterial leaf spot disease is *Panthera solani*, which can damage leaves, fruits, and twigs. Initially, small water-soaked spots appear on the leaves, which later expand into brown perforated lesions, severely affecting fruit yield and quality.

[0003] Currently, the main methods for detecting the pathogen causing leaf spot disease in wild apricots include pathogen isolation, purification, and culture; biological identification; and conventional PCR molecular identification techniques. Among these, biological detection methods require extensive morphological and physiological / biochemical assays, resulting in long detection cycles and cumbersome procedures, making them unsuitable for the timeliness requirements of dynamic disease monitoring. While conventional PCR methods can achieve qualitative identification of the pathogen, they cannot quantitatively analyze the pathogen content in samples, and a single reaction typically detects only a single target pathogen, leading to low detection efficiency and making it difficult to meet the simultaneous detection needs of fungal and bacterial co-infections in wild apricot leaf spot disease.

[0004] Real-time quantitative PCR technology can accurately calculate the initial copy number of target nucleic acids in a sample by monitoring the changes in fluorescence intensity in each amplification cycle in real time, thus enabling quantitative detection of pathogens. However, there is currently no multiplex real-time quantitative PCR detection tool specifically for the two major pathogens of apricot leaf spot—Pantotheca cum Clonella and Scutellarios fructus—making it impossible to achieve simultaneous quantitative detection of these two pathogens. Summary of the Invention

[0005] The purpose of this invention is to provide a primer-probe composition for the quantitative detection of the pathogen causing leaf spot disease in wild apricots and its application. This primer-probe composition has high sensitivity, strong specificity, good stability, and high repeatability, and can rapidly and simultaneously detect *Cyclopyralidus* and *Pantotheca cumulus*.

[0006] To achieve the above objectives, the present invention provides the following solution: A primer-probe composition for the quantitative detection of the pathogen causing leaf spot in wild apricot, the primer-probe composition comprising a first primer pair, a first probe W.car-P, a second primer pair, and a second probe P.agg-P; the first primer pair comprises a first forward primer W.car-F and a first reverse primer W.car-R, the nucleotide sequence of the first forward primer W.car-F is shown in SEQ ID NO.1, the nucleotide sequence of the first reverse primer W.car-R is shown in SEQ ID NO.2; the nucleotide sequence of the first probe W.car-P is shown in SEQ ID NO.3; The second primer pair includes a second forward primer P.agg-F and a second reverse primer P.agg-R. The nucleotide sequence of the second forward primer P.agg-F is shown in SEQ ID NO.4; the nucleotide sequence of the second reverse primer P.agg-R is shown in SEQ ID NO.5; and the nucleotide sequence of the second probe P.agg-P is shown in SEQ ID NO.6.

[0007] Preferably, the pathogen causing the leaf spot disease in wild apricots is *Cypripedium spp.* (also known as *Cypripedium spp.*). Wilsonomyces carpophilus The first primer pair and the first probe W.car-P are used to detect Pantoea agglomerans, and the second primer pair and the second probe P.agg-P are used to detect Pantoea agglomerans.

[0008] Preferably, the first probe W.car-P is labeled with a fluorescent reporter group VIC at its 5' end and a fluorescent quencher group BHQ1 at its 3' end; the second probe P.agg-P is labeled with a fluorescent reporter group FAM at its 5' end and a fluorescent quencher group BHQ1 at its 3' end.

[0009] The present invention also provides the application of the primer-probe composition in the preparation of a kit for the quantitative detection of the pathogen of wild apricot leaf spot.

[0010] The present invention also provides a kit for quantitative detection of the pathogen causing leaf spot disease in wild apricots, the kit comprising the primer and probe composition described above.

[0011] Preferably, the kit further includes qPCR reaction buffer, Taq DNA polymerase, dNTPs, ddH2O, and recombinant standard plasmid.

[0012] Preferably, the recombinant standard plasmid is a fungus containing *Sclerotium falciparum*. tef1 Gene fragments or clusters of pantothenia gyrB Recombinant plasmids of gene fragments.

[0013] The present invention also provides the application of the kit in the quantitative detection of the pathogen causing leaf spot disease in wild apricot.

[0014] Preferably, the pathogen causing the apricot leaf spot disease is *Cyclocarya paliurus* and / or *Pantotheca cumulus*.

[0015] This invention also provides a method for quantitative detection of the pathogen causing leaf spot in wild apricot, comprising the following steps: extracting genomic DNA from the sample to be tested; using the genomic DNA as a template, performing multiplex real-time quantitative PCR amplification using the primer and probe composition; analyzing the PCR amplification products, and based on the amplification curve, achieving quantitative detection of the pathogen causing leaf spot in wild apricot in the sample to be tested.

[0016] The present invention has the following technical effects: This invention discloses a primer-probe composition for the quantitative detection of apricot leaf spot pathogens and its application. Specific primers and probes were designed for the two main pathogens of apricot leaf spot—*Cytosporum falciparum* and *Pantosporum clumps*. Different types of fluorescent reporter groups (FAM for the *Pantosporum clumps* detection channel and VIC for the *Cytosporum falciparum* detection channel) were configured for the two probes. Simultaneous quantitative detection of the two pathogens was achieved in a single tube and single reaction using multiplex real-time quantitative PCR technology. No cross-amplification was observed with more than 10 non-target microorganisms commonly found in the apricot growing environment, such as *Alternaria lobata*, *Syndromea*, and *Alternaria*, effectively avoiding false positives. The composition exhibits high specificity and ensures the accuracy of the detection results. It can be used for the quantitative detection of apricot leaf spot caused by *Cytosporum falciparum* and *Pantosporum clumps* at different stages and at different sites.

[0017] This primer-probe composition is stably amplified and does not interfere with each other in the same multiplex real-time quantitative PCR system, eliminating the need for batch detection. Simultaneous amplification and quantification of dual targets significantly shortens the detection cycle. Combining the technical advantages of real-time quantitative PCR, the correlation between Ct values ​​and initial pathogen concentrations allows for rapid quantification of pathogen load at different disease stages and sites, providing efficient technical support for dynamic monitoring of disease occurrence. The detection efficiency is significantly improved compared to traditional batch-based single-target detection methods. It is simple to operate, has good repeatability, and is applicable to the accurate quantitative monitoring of complex samples such as different diseased tissues of wild apricot plants, field soil samples, and pathogen cultures. This invention is the first to construct a multiplex simultaneous quantitative detection system for the two major pathogens of wild apricot leaf spot, filling the technical gap of lacking simultaneous quantitative detection molecular tools for this disease. It provides reliable technical support for the dynamic monitoring, epidemiological analysis, and scientific control of wild apricot leaf spot multi-infection. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 These are the results of the susceptibility test for *Cyprinus flavophilus*. Figure 2 The results are from the pantothenic acid susceptibility test. Figure 3 TaqMan qPCR standard curve for *Cryptococcus faecalis*; Figure 4 TaqMan qPCR standard curve for pantothenia gravis; Figure 5 This is the result of specificity verification. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Example 1 Select conserved genes in pantothenic bacteria gyrB and conserved genes of Scutellaria fuciformis tef1 As target genes, the target gene sequences of two pathogens (conserved genes of pan-bacterium clumps) were retrieved from the NCBI database. gyrB Gene ID: 66824001, conserved gene of *Scutellaria fuciformis* tef1 (KY905684.1) Sequence homology was performed using NCBI Blast to screen highly conserved regions of each gene. Specific amplification primers and TaqMan fluorescent probes were designed for each conserved region using Primer Express (ABI) software. Primer and probe sequences, base numbers, and modification information are shown in Table 1.

[0026] Table 1 Primer and probe sequences and modification information The first probe, W.car-P, targets *Sclerotium frutescens*. tef1 The conserved gene segment is labeled with a VIC fluorescent reporter group at the 5' end and a BHQ1 fluorescent quencher group at the 3' end, adapted to the VIC detection channel; the second probe, P.agg-P, targets clustered pantothenia. gyrB The conserved gene segment is labeled with the FAM fluorescent reporter group at the 5' end and the BHQ1 fluorescent quencher group at the 3' end, adapted to the FAM detection channel. Two sets of primers and probes are differentially labeled with fluorescent markers, adapted to the FAM channel (for detecting clustered pantothenia) and the VIC channel (for detecting sclerotiorum frutescens) of the real-time PCR instrument, respectively, allowing simultaneous differentiation of the two pathogens in the same reaction system.

[0027] Preparation of standards and establishment of multiplex qPCR standard curves: Construction of recombinant standard plasmids: [The text abruptly shifts to a seemingly unrelated topic about *P gyrB The gene-specific target fragment was inserted into the cloning vector pClone007 Versatile Simple Vector (nucleotide sequence shown in SEQ ID NO.7; the cloning vector was prepared using the DaLing Biopharmaceutical pClone007 Universal TA Cloning Kit, catalog number DLV103) to construct the recombinant plasmid P. agg; *P. aggii* was then inserted into the cloning vector. tef1 Gene-specific target fragments were inserted into the same cloning vector to construct the recombinant plasmid W.car.

[0028]

[0029] Plasmids P. agg and W. car were extracted according to the instructions of the Daling Biomagnetic Bead Plasmid Extraction Kit (catalog number DZN311). All recombinant plasmids were sequenced and verified by Invitrogen to confirm the accuracy of the inserted target gene sequence.

[0030] Plasmid copy number calculation: The OD of the recombinant plasmid was determined using a micro-volume nucleic acid quantification instrument. 260 The plasmid mass concentration is measured, and the initial copy number of the plasmid is calculated using the following formula: ; Where C is the copy number (copies / μL); c is the plasmid concentration (μg / μL); and NA is Avogadro's constant (6.02 × 10⁻⁶). 23 M represents the molecular weight of the plasmid.

[0031] ; The average molecular weight of the single base pairs is 649 Da.

[0032] The plasmid concentration should be controlled between 20-100 ng / μL to ensure accurate and reliable quantitative detection results.

[0033] The recombinant standard plasmid that has passed sequencing verification is serially diluted 10-fold. The specific steps are as follows: Take 10 μL of standard plasmid and add it to 90 μL of water. Vortex mix and dilute 10 times to obtain the first sample A.

[0034] Take 10 μL of sample A and add it to 90 μL of water. Vortex mix and dilute 10 times to obtain the second sample B.

[0035] Take 10 μL of sample B and add it to 90 μL of water. Vortex mix and dilute 10 times to obtain the third sample C.

[0036] This process was repeated stepwise to prepare nine concentration gradients (sample A, sample B, sample C, sample D, sample E, sample F, sample G, sample H, sample I). The amplification curves of the standard plasmid W. car and plasmid P. agg after gradient dilution are shown in the figure. Figure 1 , Figure 2 As shown, this is used for drawing standard curves.

[0037] Depend on Figure 1 and Figure 2 It can be seen that these two primers have good specificity.

[0038] Multiplex TaqMan qPCR reaction system and conditions: Using the serially diluted recombinant plasmids as templates, TaqMan PCR amplification was performed. The reaction system composition is shown in Table 2, and the PCR amplification reaction program is shown in Table 3. During the 60℃ extension phase, FAM and VIC dual-channel fluorescence signals were simultaneously acquired. It can be seen that... Table 2 Composition of the reaction system Table 3 PCR amplification reaction procedure Standard curve fitting: qPCR standard curves for the two pathogens were fitted using the logarithm of the standard plasmid copy number as the x-axis and the corresponding Ct detection value as the y-axis. The curve equation, amplification efficiency, and coefficient of determination R were calculated. 2 And detection sensitivity, etc. Results for Pantothenia gravis clusters are shown in Table 4 and... Figure 3 As shown; the results for *Cyprinus fructus* are shown in Table 5 and... Figure 4 As shown.

[0039] Table 4. Parameters related to the standard curve of pantothenic acid qPCR. From Table 4 and Figure 3 It can be seen that the full-length recombinant plasmid of *Pantotheca acuminata* is 2062 bp, with an initial copy number of 5.15 × 10⁻⁶. 10 copies / μL; the fitted standard curve equation is: Ct = -3.303Log(copy number) + 40.406, with a slope of -3.303, an amplification efficiency of 101%, and R0. 2 The value is 0.9982, and the lowest detection sensitivity can reach 5.15×10. 0 copies / μL.

[0040] Table 5. Parameters related to the qPCR standard curve of *Cypripedium falciparum* Depend on Figure 4 As shown in Table 5, the full-length plasmid of *Cyclophorus fragrans* is 2053 bp, with an initial copy number of 5.24 × 10⁻⁶. 10 copies / μL; the fitted standard curve equation is: Ct = -3.3569Log(copy number) + 41.654, with a slope of -3.3569, an amplification efficiency of 99%, and R0. 2 The value is 0.9991, and the lowest detection sensitivity can reach 5.24 × 10⁻⁶. 0 copies / μL.

[0041] The above results indicate that the dual qPCR detection system for the two pathogens exhibits excellent linearity, with determination coefficients R² both greater than 0.998. The amplification efficiency is within the ideal range of 90%-110%, demonstrating a wide quantitative linear range and high sensitivity, which can meet the requirements for accurate and absolute quantitative detection of pathogens in samples.

[0042] Example 2 To verify the detection specificity of the dual probe primer combination for Pantotheca cumulus and Scutellaria foetida in this invention, a variety of fungi and miscellaneous bacteria commonly found in the wild apricot planting environment and on the plant were selected as control strains, and the established dual TaqMan qPCR system was used for simultaneous detection.

[0043] Positive controls included genomic DNA from *Pantotheca cum Caulis* (strain number 2N-3) and *Cyclophorus* (strain numbers 21 and 23); nine non-target control strains were included, including: *Hylocereus fusiformis* (…). Fusiformiascus favacea strain number 4043-3), *Syngonium* ( Septoria sp. strain number 31-2), Alternaria ( Alternaria alstroemeriae strain number 39-1), Alternaria ( Alternaria sp. strain number RD-1), spurious black disc shell ( Melanconis stilbostoma Strain numbers 4057-1 and 4074-2), Cryptosporidium filamentosa ( Cryptosporella tomentella strain number 4026-1), Curvularia minor ( Eutypella sp. strain number 4038-2), Cryptosporidium biloba ( Cryptosporella betulae strain number 3978-5) and apple black spot pathogen ( Venturia inaequalis (Strain numbers LYX-1 and LYX-4). NTC was selected as a negative control, and the test results are as follows: Figure 5 As shown.

[0044] Depend on Figure 5 It can be seen that only the positive template samples of *Pantotheca cumulus* and *Ceratophyllum demersum* showed specific fluorescence amplification curves with normal fluorescence signal peaks; all non-target control strains showed no fluorescence signal response and the test results were negative.

[0045] The above results demonstrate that the primer-probe combination designed in this invention has high specificity, specifically recognizing only two target pathogenic bacteria—Pantotheca cumulus and Scutellaria foetida—without cross-amplification with other closely related fungi or miscellaneous bacteria, and is not affected by closely related microorganisms or miscellaneous bacteria in the field. It has strong anti-interference ability and can effectively avoid false positives, making it suitable for specific quantitative detection of complex samples in wild apricot fields.

[0046] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A primer probe composition for quantitatively detecting wild apricot perforator pathogen, characterized in that, The primer-probe composition includes a first primer pair, a first probe W.car-P, a second primer pair, and a second probe P.agg-P; the first primer pair includes a first forward primer W.car-F and a first reverse primer W.car-R, the nucleotide sequence of the first forward primer W.car-F is shown in SEQ ID NO.1, and the nucleotide sequence of the first reverse primer W.car-R is shown in SEQ ID NO.2; The nucleotide sequence of the first probe W.car-P is shown in SEQ ID NO.3; The second primer pair includes a second forward primer P.agg-F and a second reverse primer P.agg-R. The nucleotide sequence of the second forward primer P.agg-F is shown in SEQ ID NO.4; the nucleotide sequence of the second reverse primer P.agg-R is shown in SEQ ID NO.5; and the nucleotide sequence of the second probe P.agg-P is shown in SEQ ID NO.

6.

2. The primer-probe composition according to claim 1, characterized in that, The pathogen causing the leaf spot disease in wild apricots is *Cryptospira spp.* (…). Wilsonomyces carpophilus ) and / or clusters of pantothenic acid ( Pantoea agglomerans The first primer pair and the first probe W.car-P are used to detect *Cyclophorus frotherium*, and the second primer pair and the second probe P.agg-P are used to detect *Pantotheca aggregatibacter*.

3. The primer-probe composition according to claim 2, characterized in that, The first probe, W.car-P, is labeled with a fluorescent reporter group VIC at its 5' end and a fluorescent quencher group BHQ1 at its 3' end; the second probe, P.agg-P, is labeled with a fluorescent reporter group FAM at its 5' end and a fluorescent quencher group BHQ1 at its 3' end.

4. The use of the primer-probe composition according to any one of claims 1-3 in the preparation of a kit for quantitative detection of the pathogen of *Apricot spore disease*.

5. A kit for quantitative detection of the pathogen causing leaf spot disease in wild apricots, characterized in that, The kit comprises the primer and probe composition according to any one of claims 1-3.

6. The reagent kit according to claim 5, characterized in that, The kit also includes qPCR reaction buffer, Taq DNA polymerase, dNTPs, ddH2O, and recombinant standard plasmid.

7. The reagent kit according to claim 6, characterized in that, The recombinant standard plasmid contains *Fructus fructus*. tef1 Gene fragments or clusters of pantothenia gyrB Recombinant plasmids of gene fragments.

8. The application of the kit described in any one of claims 5-7 in the quantitative detection of the pathogen causing leaf spot disease in wild apricot.

9. The application according to claim 8, characterized in that, The pathogen causing the leaf spot disease in wild apricots is *Cryptospira spp.* and / or *Pantospira spp.* 10. A method for quantitative detection of the pathogen causing leaf spot disease in wild apricots, characterized in that, Includes the following steps: Genomic DNA is extracted from the sample to be tested; using the genomic DNA as a template, multiplex real-time quantitative PCR amplification is performed using the primer and probe composition according to any one of claims 1-3; By analyzing the PCR amplification products and based on the amplification curve, the pathogen of wild apricot hole disease in the sample to be tested can be quantitatively detected.

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