Multiplex Q-PCR (Quantitative Polymerase Chain Reaction) detection method for detecting fungal diseases carried in barley and wheat

Abstract

The invention relates to an application of a PCR (polymerase chain reaction) detection method in food detection, in particular to an application in identifying and detecting fungal diseases in cereal barley and wheat, and more particularly relates to a multiple Q-PCR detection method for detecting fungal diseases carried in barley and wheat. The detection kit can be used for simultaneously detecting wheat basal rot pathogen, wheat leaf blight pathogen, wheat shell polycystic leaf spot pathogen and phyllosticta costata in barley and wheat. Meanwhile, the detection does not need complex pretreatment, a detection template can be obtained only through a nucleic acid releasing agent, and detection of different germs is achieved on an iFlashdx Q16S portable detection platform.

Description

Technical Field

[0001] This invention relates to the application of PCR detection methods in food testing, particularly in the identification and detection of fungal diseases in barley and wheat, and more specifically to a multiplex Q-PCR detection method for detecting fungal diseases in barley and wheat. Background Technology

[0002] Barley and wheat are important raw materials for the production of alcoholic beverages, food, and feed in my country, and are the main forage for livestock, poultry, and aquaculture in my country's agricultural and pastoral areas.

[0003] Barley and wheat infected with fungal viruses are the main pathways for the long-distance transmission of diseases. Therefore, strengthening the detection and identification of fungal diseases in imported barley and wheat is of great significance.

[0004] Currently, in the import inspection of barley and wheat, we pay close attention to wheat basal rot pathogens (… Pseudocercosporella herpotrichoides Wheat leaf blight fungus ( Alternaria triticina ), wheat hull multicellular spot disease ( Stagonospora avenae f. sp. Triticea ), *Pseudomonas spp.* leaf spot pathogen ( Ramularia collo-cygni Four main fungal diseases, including , , and .

[0005] Wheat basal rot fungus ( Pseudocercosporella herpotrichoides It first occurred in France in 1912, and was reported in Oregon and Washington in the United States in 1929. It has since spread to parts of Europe, the Americas, Africa, New Zealand, and Australia. It was reported in Japan in 1982.

[0006] wheat leaf blight fungus ( Alternaria triticina Leaf spot is one of the most important foliar diseases of wheat and barley, ultimately leading to leaf death. This disease has long been a serious problem for wheat in India, with yield losses exceeding 60% when the disease is severe. If susceptible varieties are infected during the booting stage, yields are almost completely lost.

[0007] Multicellular spot disease of wheat husk ( Stagonospora avena e f. sp. Triticea This fungus can infest wheat, barley, and other gramineous plants, causing leaf spot disease. In the later stages of growth, it can cause necrosis, leading to severe yield reduction in wheat and other crops. This fungus has gradually become a global disease, distributed in Africa, the Americas, Asia, Europe, and Oceania. It is listed in my country's list of quarantine pests for imported plants and is an important quarantine fungus.

[0008] Cylindrica spp. leaf spot pathogen ( Ramularia collo-cygni*Barley spp.* is a major pathogen affecting wheat and barley, primarily causing barley leaf spot, leading to yield loss and quality decline. It is distributed in Europe, the Americas, and Australia, but has not yet been recorded in China. *Barley spp.* is a quarantine pest of concern in the Protocol on Phytosanitary Requirements for Barley Exports from Finland, the United States, and Uruguay to China.

[0009] Current detection and identification methods for the four diseases mentioned above mainly include traditional morphology, immunofluorescence staining, mass spectrometry, and conventional PCR. However, all of these methods require samples to be sent to a specialized laboratory for processing. This not only increases the sample processing time but also results in a considerable waiting period.

[0010] Therefore, how to provide a rapid detection and identification method that can be completed on-site has become a hot research topic for those skilled in the art. Summary of the Invention

[0011] The purpose of this invention is to address the above-mentioned problems by providing a rapid detection method that can be performed on-site.

[0012] To achieve this objective, this invention discloses a multiplex Q-PCR detection method for detecting fungal diseases in barley and wheat. The method targets *Wheat basal rot*, *Wheat leaf blight*, *wheat shell multicellular spot*, and *Parasiticum columnaris* in barley and wheat. It utilizes four sets of primers and probes, employing a Q-PCR reaction to detect the presence of one or more of these fungi. The primer and probe set includes: The upstream primer PAtub2-F with the nucleotide sequence shown in SEQ ID NO.1, the downstream primer PAtub2-R with the nucleotide sequence shown in SEQ ID NO.2, and the probe PAtub2-P with the nucleotide sequence shown in SEQ ID NO.3; The upstream primer OF1 with the nucleotide sequence shown in SEQ ID NO.4, the downstream primer OR2 with the nucleotide sequence shown in SEQ ID NO.5, and the probe P-OY with the nucleotide sequence shown in SEQ ID NO.6; The upstream primer Alt-F4 with the nucleotide sequence shown in SEQ ID NO.7, the downstream primer Alt-R5 with the nucleotide sequence shown in SEQ ID NO.8, and the probe Alt-P2 with the nucleotide sequence shown in SEQ ID NO.9; The upstream primer RAMF6 with nucleotide sequence as shown in SEQ ID NO.10, the downstream primer RAMR6 with nucleotide sequence as shown in SEQ ID NO.11, and the probe RAMP6 with nucleotide sequence as shown in SEQ ID NO.12.

[0013] Meanwhile, the 5' end of the probe is labeled with different fluorescent groups, including but not limited to FAM, HEX, VIC, ROX or Cy5, and the 3' end of the probe is labeled with quenching groups, including but not limited to BHQ1, BHQ2 or TAMRA.

[0014] The different fluorescent groups labeled on the 5' end of the probes mentioned in this invention refer to the selection of different fluorescent groups as reporter groups at the 5' end of probes PAtub2-P, P-OY, Alt-P2, and RAMP6, in order to distinguish different analytes.

[0015] Specifically, the multiplex Q-PCR detection method for detecting fungal diseases in barley and wheat includes the following steps: (1) Obtain nucleic acid from the sample; (2) Using the total nucleic acid extracted in step (1) as a template, a one-step Q-PCR amplification reaction was performed using the primer and probe set; (3) Determine whether the amplification curve contains wheat basal rot fungus, wheat leaf blight fungus, wheat shell multicellular spot fungus, and / or columnar spore leaf spot fungus. If amplification occurs, it indicates the presence of a specific pathogen; If there is no amplification, it means that the specific pathogen is not present.

[0016] In one specific implementation, the iFlashdx Q16S portable detection platform was used for Q-PCR.

[0017] The iFlashdx Q16S portable detection platform mentioned here refers to the Q16S fluorescence PCR instrument.

[0018] In one specific implementation, the Q-PCR amplification program is as follows: 52℃ for 3 min, 95℃ for 5 min, 95℃ for 5 s, 60℃ for 15 s (collect fluorescence signal, select FAM channel), 45 cycles.

[0019] Furthermore, this invention also discloses the primers and probes used to achieve this detection, as described above, including four sets of primers and probes, namely: Primer and probe set for detecting multicellular spot disease of wheat husk: upstream primer PAtub2-F with nucleotide sequence as shown in SEQ ID NO.1, downstream primer PAtub2-R with nucleotide sequence as shown in SEQ ID NO.2, and probe PAtub2-P with nucleotide sequence as shown in SEQ ID NO.3; Primer and probe set used to detect wheat basal rot fungus: upstream primer OF1 with nucleotide sequence as shown in SEQ ID NO.4, downstream primer OR2 with nucleotide sequence as shown in SEQ ID NO.5, and probe P-OY with nucleotide sequence as shown in SEQ ID NO.6; Primer and probe set for detecting wheat leaf blight: upstream primer Alt-F4 with nucleotide sequence as shown in SEQ ID NO.7, downstream primer Alt-R5 with nucleotide sequence as shown in SEQ ID NO.8, and probe Alt-P2 with nucleotide sequence as shown in SEQ ID NO.9; The primer and probe set used to detect the leaf spot pathogen *Sphaerocera columnare* consists of the upstream primer RAMF6 (nucleotide sequence shown in SEQ ID NO. 10), the downstream primer RAMR6 (nucleotide sequence shown in SEQ ID NO. 11), and the probe RAMP6 (nucleotide sequence shown in SEQ ID NO. 12).

[0020] On the other hand, the present invention also discloses a kit containing the above-mentioned primers and probes.

[0021] In one specific implementation, the kit contains a plant-based extract-free nucleic acid release agent.

[0022] This invention discloses a multiplex Q-PCR detection method for detecting fungal diseases in barley and wheat, capable of simultaneously detecting *Wheat basal rot*, *Wheat leaf blight*, *Triticum aestivum*, and *Triticum cytogenes* leaf spot pathogens in both barley and wheat. Furthermore, this detection requires no complex pretreatment; only a nucleic acid release agent is needed to obtain the detection template, and the detection of different pathogens can be achieved on the iFlashdxQ16S portable detection platform. The kit prepared using the aforementioned primers and probes enables rapid detection, significantly improving detection efficiency. Attached Figure Description

[0023] Figure 1 This is a schematic diagram showing the results of Q-PCR testing of a simulated sample template and a negative control using the iFlashdx Q16S portable detection platform in Example 1.

[0024] Figure 2 This is a schematic diagram showing the detection results of the positive control of wheat basal rot after Q-PCR detection using the iFlashdx Q16S portable detection platform in Example 1.

[0025] Figure 3 This is a schematic diagram showing the detection results of the positive control of wheat leaf blight after Q-PCR detection using the iFlashdx Q16S portable detection platform in Example 1.

[0026] Figure 4 This is a schematic diagram showing the detection results of the positive control of multicellular spot disease of wheat husk after Q-PCR detection using the iFlashdx Q16S portable detection platform in Example 1.

[0027] Figure 5 This is a schematic diagram showing the detection results of the positive control of *Schistosporum cuspidatum* after Q-PCR detection using the iFlashdx Q16S portable detection platform in Example 1. Detailed Implementation

[0028] To better understand this invention, we will further elaborate on it below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional experimental procedures; the materials and reagents used, unless otherwise specified, are commercially available. Example 1

[0029] Positive strains of wheat basal rot, wheat leaf blight, wheat shell multicellular spot, and *Sphaerocera columnaris* leaf spot were directly added to a plant-derived nucleic acid release agent (containing 0.05% SDS detergent, 0.1% Tween-20 surfactant, 0.2% Triton X-100 surfactant, 0.4% BSA PCR stabilizer, 1% glycerol, and 0.005% Proclin 300 preservative). The mixture was then shaken upside down for 1 minute to serve as a simulated sample template.

[0030] Meanwhile, as positive controls, positive strains of wheat basal rot, wheat leaf blight, wheat shell multicellular spot, and *Sphaerocera columnaris* leaf spot were added to a plant-derived nucleic acid release agent (containing 0.05% SDS detergent, 0.1% Tween-20 surfactant, 0.2% Triton X-100 surfactant, 0.4% BSA PCR stabilizer, 1% glycerol, and 0.005% Proclin 300 preservative) to prepare positive control templates containing a single pathogen.

[0031] In this example, we used purified water as a blank control.

[0032] The simulated sample template, four positive control templates, and the blank control sample were amplified using the following method: Dilute the primers and probes shown in Table 1 with sterile purified water to a final concentration of 10 μmol / μL. In this example, we have labeled the 5' end of each probe with a different fluorescent group and the 3' end with a corresponding quenching group. Prepare the amplification system (20 μL) according to the instructions of the plant extraction-free high-sensitivity detection kit, as shown in Table 2. Table 1: Primer and probe sets

[0033] Table 2: Amplification System

[0034] Amplification program: 52℃ for 3 min, 95℃ for 5 min, 95℃ for 5 s, 60℃ for 15 s (collect fluorescence signal, select FAM channel), 45 cycles. Mix well, centrifuge briefly, and then amplify using a Q16S fluorescence PCR instrument.

[0035] Experimental results are as follows Figures 1 to 5 As shown.

[0036] Figure 1 The red curve represents the amplification curve of wheat basal rot fungus, the blue line represents the amplification curve of wheat leaf blight fungus, the green line represents the amplification curve of wheat hull multicellular spot fungus, and the magenta line represents the amplification curve of columnar spore leaf spot fungus.

[0037] Depend on Figure 1 It can be seen that the designed specific primers and probes can specifically amplify barley grains carrying pathogens, while the blank control shows no amplification and is negative. This indicates that the established rapid on-site detection method can effectively detect wheat hull multicellular spot pathogen, *Synapticus cylindrica* leaf spot pathogen, *Brachystomata basalis*, and *Brachystomata thunbergii*.

[0038] At the same time, combined Figures 2 to 5 amplification curves of single pathogens and Figure 1 Based on the amplification curves of various pathogens in the mixed sample, the specific primers and probes disclosed in this invention do not interfere with each other, making it a reliable multiplex Q-PCR detection method that can simultaneously detect wheat basal rot fungus, wheat leaf blight fungus, wheat shell multicellular spot fungus, and columnar spore leaf spot fungus in barley and wheat.

[0039] The above describes specific embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A multiplex Q-PCR detection method for detecting fungal diseases in barley and wheat, characterized by: The detection method targets wheat basal rot fungus, wheat leaf blight fungus, wheat shell multicellular spot fungus, and *Septoria cylindrica* leaf spot fungus in barley and wheat. It uses four sets of primers and probes and employs Q-PCR to detect whether the fungus carries one or more of these fungi. The primer and probe set includes: The upstream primer PAtub2-F with the nucleotide sequence shown in SEQ ID NO.1, the downstream primer PAtub2-R with the nucleotide sequence shown in SEQ ID NO.2, and the probe PAtub2-P with the nucleotide sequence shown in SEQ ID NO.3; The upstream primer OF1 with the nucleotide sequence shown in SEQ ID NO.4, the downstream primer OR2 with the nucleotide sequence shown in SEQ ID NO.5, and the probe P-OY with the nucleotide sequence shown in SEQ ID NO.6; The upstream primer Alt-F4 with the nucleotide sequence shown in SEQ ID NO.7, the downstream primer Alt-R5 with the nucleotide sequence shown in SEQ ID NO.8, and the probe Alt-P2 with the nucleotide sequence shown in SEQ ID NO.9; The upstream primer RAMF6 with nucleotide sequence as shown in SEQ ID NO.10, the downstream primer RAMR6 with nucleotide sequence as shown in SEQ ID NO.11, and the probe RAMP6 with nucleotide sequence as shown in SEQ ID NO.

12.

2. The multiplex Q-PCR detection method for detecting fungal diseases in barley and wheat according to claim 1, characterized in that: The 5' end of the probe is labeled with different fluorescent groups, and the 3' end of the probe is labeled with a quenching group.

3. The multiplex Q-PCR detection method for detecting fungal diseases in barley and wheat according to claim 1, characterized in that, Includes the following steps: (1) Obtain nucleic acid from the sample; (2) Using the total nucleic acid extracted in step (1) as a template, a one-step Q-PCR amplification reaction was performed using the primer and probe set; (3) Determine whether the amplification curve contains wheat basal rot fungus, wheat leaf blight fungus, wheat shell multicellular spot fungus and / or columnar spore leaf spot fungus. If amplification occurs, it indicates the presence of a specific pathogen; If there is no amplification, it means that the specific pathogen is not present.

4. The multiplex Q-PCR detection method for detecting fungal diseases in barley and wheat according to claim 1, characterized in that: Q-PCR was performed using the iFlashdx Q16S portable detection platform.

5. The multiplex Q-PCR detection method for detecting fungal diseases in barley and wheat according to claim 4, characterized in that, The Q-PCR amplification program is as follows: 52℃ for 3 min, 95℃ for 5 min, 95℃ for 5 s, 60℃ for 15 s (collecting fluorescence signal), 45 cycles.

6. Primers and probes for a multiplex Q-PCR detection method for detecting fungal diseases in barley and wheat, characterized in that, It includes four sets of primers and probes, namely: Primer and probe set for detecting multicellular spot disease of wheat husk: upstream primer PAtub2-F with nucleotide sequence as shown in SEQ ID NO.1, downstream primer PAtub2-R with nucleotide sequence as shown in SEQ ID NO.2, and probe PAtub2-P with nucleotide sequence as shown in SEQ ID NO.3; Primer and probe set used to detect wheat basal rot fungus: upstream primer OF1 with nucleotide sequence as shown in SEQ ID NO.4, downstream primer OR2 with nucleotide sequence as shown in SEQ ID NO.5, and probe P-OY with nucleotide sequence as shown in SEQ ID NO.6; Primer and probe set for detecting wheat leaf blight: upstream primer Alt-F4 with nucleotide sequence as shown in SEQ ID NO.7, downstream primer Alt-R5 with nucleotide sequence as shown in SEQ ID NO.8, and probe Alt-P2 with nucleotide sequence as shown in SEQ ID NO.9; The primer and probe set used to detect the leaf spot pathogen *Sphaerocera columnare* consists of the upstream primer RAMF6 (nucleotide sequence shown in SEQ ID NO. 10), the downstream primer RAMR6 (nucleotide sequence shown in SEQ ID NO. 11), and the probe RAMP6 (nucleotide sequence shown in SEQ ID NO. 12).

7. A kit comprising the primers and probes of claim 6.

8. The kit according to claim 7, characterized in that: The kit contains a plant-based nucleic acid release agent that does not require extraction.

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