Detection primer and probe for detecting pathogenic bacteria of basal stem rot of crops and chip digital PCR detection method

Abstract

The application discloses a detection primer and probe for detecting a pathogenic bacterium of a crop stem base rot and a chip digital PCR detection method. TEF-1 alpha The application takes a pathogenic bacterium of a crop stem base rot as a target, and the pathogenic bacterium is a Fusarium pseudograminearum Tri Core gene cluster nucleic acid fragments are respectively designed to obtain high-specificity detection primer pairs and probes, and a double-chip digital PCR quantitative detection method for a dominant pathogenic bacterium of a wheat stem base rot is established according to the same. The correlation coefficients of ideal copy numbers and actual copy numbers of target samples detected by the double-chip digital PCR quantitative method are all greater than 0.9998, the target DNA can be more accurately detected, the double-chip digital PCR quantitative method shows higher accuracy and sensitivity than fluorescence quantitative PCR in a complex rhizosphere soil sample, and the double-chip digital PCR quantitative method has application prospects in accurate quantification and detection of nucleic acids of the dominant pathogenic bacterium of the wheat stem base rot.

Description

Technical Field

[0001] This invention relates to specific primers, probes, and detection methods for pathogen detection, and particularly to specific primers, probes, and a chip digital PCR (cdPCR) detection method for detecting pathogens of crop stem base rot, belonging to the field of molecular detection of crop stem base rot. Background Technology

[0002] Wheat stem rot ( Fusarium Wheat crown rot (FCR) is a common disease that occurs widely in major wheat-growing areas and is showing a trend of increasing severity. This disease is caused by various Fusarium species (FCR). Fusarium Caused by *Fusarium pseudogracilis* (…), primarily by *Fusarium pseudogracilis* (…). F. pseudograminearum Caused by . Fusarium oxysporum ( F. culmorum This disease is mainly distributed in cool, humid regions, and surveys have shown that it can also cause wheat stem rot in Xinjiang, China. Wheat stem rot is a typical soil-borne disease; the pathogen survives in the soil for extended periods in the form of mycelium, chlamydospores, or conidia. The disease primarily affects the base of the wheat stem, causing symptoms such as stem rot and root rot, leading to premature death of seedlings or whiteheads in later stages. Infected wheat grains also produce toxins, which can severely reduce yield or even cause total crop failure.

[0003] Because Fusarium fungi share common characteristics in morphology, ecology, metabolism, genetics, and pathogenicity, while also exhibiting high diversity and adaptability, the pathogens causing wheat stem rot are morphologically similar to other pathogens. Traditional methods are insufficient to accurately distinguish between *Fusarium graminearum* and *Fusarium graminearum*, and currently, there are no reports in the literature on the effective simultaneous detection of these two pathogens.

[0004] With the rapid development of molecular biology techniques, the methods for identifying the pathogen of wheat stem rot have evolved from traditional morphological identification to high-precision molecular detection, significantly improving the accuracy, efficiency, and sensitivity of pathogen identification. Traditional identification methods relying on culture characteristics and microscopic observation are not only time-consuming but also struggle to distinguish closely related species, easily leading to misidentification. The introduction of molecular biology techniques has ushered in a completely new stage for pathogen identification.

[0005] The application of early molecular biology techniques, such as PCR (polymerase chain reaction), greatly improved the efficiency and accuracy of pathogen identification. This was achieved by amplifying specific gene fragments of pathogens (such as...). ITS , TEF-1α or β-tubulinand sequence alignment. However, the conventional PCR technique is not sensitive enough for the detection of low-concentration pathogenic bacteria in practical applications, and real-time fluorescent quantitative PCR (qPCR) technology has been introduced into the field of pathogenic bacteria identification. qPCR is the most commonly used pathogenic bacteria detection technology. This technology adds fluorescent dyes or probes to the PCR reaction system, and the entire amplification process is monitored in real time through the change of fluorescent signal. Finally, the relative content of the target nucleic acid fragment in the sample is determined based on the Ct (threshold cycle) value of the sample. If absolute quantification is performed, a standard curve needs to be prepared by using standard samples with known absolute concentrations to obtain the nucleic acid content of the actual unknown sample. There are the following problems when using qPCR technology to perform absolute quantification of actual samples: (1) Especially when extracting DNA from complex soil samples, impurities may be introduced, which may contain PCR inhibitors, resulting in a decrease in actual amplification efficiency. Therefore, when using a standard curve constructed with standard samples for quantification, the actual DNA concentration of the sample will be underestimated. (2) When the content of the target nucleic acid fragment in the sample is very low, the PCR inhibitors contained therein can easily cause "false negatives" in the qPCR reaction. The above reasons make it difficult to accurately quantify nucleic acid samples based on qPCR technology.

[0006] Digital PCR (dPCR), the third generation of PCR technology, as a new nucleic acid quantitative molecular detection technology, has gradually become a leading tool for pathogenic bacteria identification. Compared with the first generation of traditional PCR and the second generation of qPCR, digital PCR divides the nucleic acid sample into a large number of independent, parallel micro-reaction units (nL, nanoliter level), so that each reaction unit contains as many template molecules as possible, and then performs amplification, detection and statistical distribution. The determination of the results can not depend on the standard curve, avoiding the interference between PCR inhibitors and different nucleic acid molecules, and being more suitable for the detection of target molecules in complex samples such as field soil, realizing the absolute counting of target molecules, and having extremely high sensitivity and accuracy.

[0007] There are mainly two forms of dPCR commonly seen on the market: droplet dPCR (ddPCR) and chip dPCR (cdPCR) technology. No matter which technology route is adopted, the main purpose is to distinguish target genes through different detection channels. Common detection channels include FAM, HEX, TAMRA, ROX, CY5, etc. When designing primers and probes, a fluorescent group corresponding to the detection channel is added to the 5' end of the probe, and a quenching group is added to the 3' end. When the probe binds to the target sequence and is amplified, the fluorescent signal is released. During detection, different products are determined according to the color of the fluorescence.

[0008] If qPCR is used to quantify trace amounts and impurity-containing DNA samples, the quantification is inaccurate, and even "false negatives" can occur. Based on the dPCR technology, it will be evenly distributed to a large number of independent reaction units for amplification reaction, and the copy number of specific nucleic acid fragments will be quantified according to the Poisson distribution and the proportion of positive signals, which has higher sensitivity and accuracy for detecting nucleic acid fragment copy number in soil samples. Therefore, the specific dPCR quantification method for the dominant pathogen of wheat stem base rot is established, which can realize the absolute quantification of the dominant pathogen in soil samples and be used for early pathogen diagnosis. SUMMARY

[0009] One of the purposes of the present application is to provide a PCR primer pair and a probe for detecting the pathogenic fungi Fusarium pseudograminearum and Fusarium graminearum;

[0010] The second purpose of the present application is to provide a cdPCR kit for simultaneously detecting the pathogenic fungi Fusarium pseudograminearum and Fusarium graminearum.

[0011] The third purpose of the present application is a cdPCR detection method for simultaneously detecting the pathogenic fungi Fusarium pseudograminearum and Fusarium graminearum.

[0012] To achieve the above-mentioned purposes, the main technical solutions adopted by the present application include:

[0013] One aspect of the present application provides a PCR primer pair and a probe for detecting the pathogenic fungi Fusarium pseudograminearum and Fusarium graminearum; wherein the PCR primer pair for detecting the pathogenic fungi Fusarium pseudograminearum is composed of an upstream primer shown in SEQ ID NO. 1 and a downstream primer shown in SEQ ID NO. 2, and the 8th cytosine of the upstream primer shown in SEQ ID NO. 1 is a locked nucleic acid modified cytosine; the nucleotide sequence of the probe for detecting the pathogenic fungi Fusarium pseudograminearum is shown in SEQ ID NO. 3, a fluorescent group corresponding to the detection channel is connected to the 5' end of the probe shown in SEQ ID NO. 3, and a quenching group is connected to the 3' end thereof, and the 3rd thymine and the 15th cytosine of the probe shown in SEQ ID NO. 3 are a locked nucleic acid modified thymine and a locked nucleic acid modified cytosine, respectively, counted from the 5' end.

[0014] The PCR primer pair for detecting the pathogenic fungi Fusarium graminearum is composed of an upstream primer shown in SEQ ID NO. 16 and a downstream primer shown in SEQ ID NO. 17, and the nucleotide sequence of the probe for detecting the pathogenic fungi Fusarium graminearum is shown in SEQ ID NO. 18, a fluorescent group corresponding to the detection channel is connected to the 5' end of the probe shown in SEQ ID NO. 18, and a quenching group is connected to the 3' end thereof.

[0015] In a preferred embodiment of the present application, the fluorescent group linked to the probe shown in SEQ ID NO. 3 is FAM, and the quenching group linked to the probe shown in SEQ ID NO. 3 is BHQ1; the fluorescent group linked to the probe shown in SEQ ID NO. 18 is Cy5, and the quenching group linked to the probe shown in SEQ ID NO. 18 is MGB.

[0016] Another aspect of the present application provides a cdPCR kit for simultaneously detecting the pathogenic fungi Fusarium pseudograminearum and Fusarium culmorum, comprising: Probe PCR Master Mix, PCR amplification primers and probes; wherein the PCR amplification primers are a primer pair consisting of the upstream primer shown in SEQ ID NO. 1 and the downstream primer shown in SEQ ID NO. 2 or a primer pair consisting of the upstream primer shown in SEQ ID NO. 16 and the downstream primer shown in SEQ ID NO. 17; the nucleotide sequence of the probe is shown in SEQ ID NO. 3 or SEQ ID NO. 18; wherein a fluorescent group corresponding to the detection channel is linked to the 5' end of the probe, and a quenching group is linked to the 3' end of the probe.

[0017] Still another aspect of the present application provides a cdPCR detection method for simultaneously detecting the pathogenic fungi Fusarium pseudograminearum and Fusarium culmorum, comprising: (1) extracting DNA from a sample to be detected; (2) establishing a cdPCR amplification system with the extracted DNA and a primer pair for detecting the pathogenic fungi Fusarium pseudograminearum or Fusarium culmorum and a probe to perform cdPCR amplification; and (3) determining whether the pathogenic fungi Fusarium pseudograminearum or Fusarium culmorum is contained according to the fluorescent color of the amplification product.

[0018] In a preferred embodiment of the present application, the cdPCR reaction system is as follows: 4x Probe PCR Master Mix 10 μL, each of the upstream and downstream primers for detecting the pathogenic fungi Fusarium pseudograminearum 1 μL, each of the upstream and downstream primers for detecting the pathogenic fungi Fusarium culmorum 1 μL, the probe for detecting the pathogenic fungi Fusarium pseudograminearum 1 μL, the probe for detecting the pathogenic fungi Fusarium culmorum 1 μL, each of the DNA of the sample to be detected 2 μL, and the rest is sterilized water, with a total reaction volume of 40 μL; wherein the concentration of the primers for detecting the pathogenic fungi Fusarium pseudograminearum or Fusarium culmorum is preferably 500 nmol / L, and the concentration of the probes for detecting the pathogenic fungi Fusarium pseudograminearum or Fusarium culmorum is preferably 250 nmol / L.

[0019] The amplification procedure of the cdPCR reaction is preferably as follows: pre-denaturation: 95℃, 2 min, 1 cycle; annealing and extension: 95℃, 15 s; 58℃, 30 s; 45 cycles.

[0020] To rapidly and accurately distinguish and identify two important plant pathogenic fungi, *Fusarium graminearum* and *Fusarium graminearum*, this invention is based on the characteristics of *Fusarium graminearum*. TEF Gene encodes translation elongation factor 1-α ( TEF-1α Design and screen highly specific primer pairs and probes based on Fusarium oxysporum. Tri Highly specific primer pairs and probes were designed and screened. Based on this, the present invention established a cdPCR quantitative detection method for pathogenic fungi *Fusarium graminearum* and *Fusarium graminearum*. Compared with qPCR detection methods, the cdPCR quantitative detection method established in this invention has higher sensitivity and accuracy. The correlation coefficient between the ideal copy number and the actual copy number of the target sample detected by this cdPCR quantitative detection method is greater than 0.9998. This achieves high sensitivity and high specificity in the detection of these two pathogens, providing strong technical support for the early detection, diagnosis, and precise control of pathogens in the soil for wheat stem rot, and providing technical support for the effective control of wheat stem rot. Attached Figure Description

[0021] Figure 1 This is a 1D diagram validating the specificity of the primer-probe set of *Fusarium graminearum*; A in the diagram represents... TEF -Fw1 / Rv1 / Probe1 primer and probe set amplification 1D diagram; B in the diagram is TEF -1D diagram of Fw2 / Rv2 / Probe2 primer and probe set amplification; C in the diagram represents TEF -Fw3 / Rv3 / Probe3 primer and probe set amplification 1D diagram; D in the diagram represents TEF -Fw4 / Rv4 / Probe4 primer and probe set amplification 1D diagram; E in the diagram is ITS -1D diagram of Fw5 / Rv5 / Probe5 primer and probe set amplification.

[0022] Figure 2 A 1D diagram validating the specificity of the primer-probe set for Fusarium oxysporum; A in the diagram represents... Tri -Fw1 / Rv1 / Probe1 primer and probe set amplification 1D diagram; B in the diagram is Tri -1D diagram of Fw2 / Rv2 / Probe2 primer and probe set amplification; C in the diagram represents Tri -Fw3 / Rv3 / Probe3 primer and probe set amplification 1D diagram; D in the diagram represents ITS -Fw4 / Rv4 / Probe4 primer and probe set amplification 1D diagram; E in the diagram is TEF -1D diagram of Fw5 / Rv5 / Probe5 primer and probe set amplification.

[0023] Figure 3 The standard curve of double cdPCR.

[0024] Figure 4 The detection results of the two kinds of PCR (qPCR and cdPCR) for detecting Fusarium pseudograminearum and Fusarium graminearum, respectively. DETAILED DESCRIPTION

[0025] The advantages and features of the present application will become more apparent from the following specific test examples. However, it should be understood that the test examples are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications or replacements all fall within the protection scope of the present application.

[0026] Test Example 1: Establishment of cdPCR quantitative detection method for detecting pathogenic bacteria Fusarium pseudograminearum and Fusarium graminearum

[0027] 1. Design of specific primer pairs and probe sequences

[0028] The endogenous Internally Transcribed Spacer (ITS), translation elongation factor (TEF), and core gene cluster nucleic acid fragments of the pathogenic bacteria Fusarium pseudograminearum (F. pseudograminearum) and Fusarium graminearum (F. graminearum) were selected. Fusarium pseudograminearum F. culmorum ITS TEF-1α Tri A plurality of species-specific primer sets and probe sequences were designed for F. pseudograminearum and F. graminearum, respectively, based on the nucleotide sequences of the ITS, TEF, and core gene cluster nucleic acid fragments, as shown in Table 1:

[0029] Table 1: Information of primer sets and probe sequences

[0030]

[0031] [N] represents a base modified as a locked nucleic acid.

[0032] 2. Specificity verification

[0033] Other pathogenic bacteria of the Fusarium genus, such as Fusarium solani (F. solani), Fusarium oxysporum (F. oxysporum), Fusarium sporotrichioides (F. sporotrichioides), and Fusarium proliferatum (F. proliferatum), were selected. F. solani (Fs) F. oxysporum (Fo) F. sporotrichioides (Fsp) F. proliferatum (Fpro) ​​​​​​​Fusarium falciforme F. falciforme (Ffal) Fusarium venenatum F. redolens (Fred) Fusarium graminearum F. commune (Fcomm) Fusarium compactum F. compactum (Fcomp) Fusarium negundi F. nygamai (Fnyg) ; and other genus pathogen Pythium gramineum Bipolaris sorokiniana (Bs) Microdochium nivale Microdochium nivale (Mn) Cryphonectria parasitica Coprinopsis psychromorbida (Cop) Ustilaginoidea virens Ceratobasidium cereale (Cer) Gaeumannomyces graminis Gaeumannomyces graminis (Gae) Magnaporthe grisea Nigrospora oryzae (Nig) and Chaetomium globosum Chaetomium globosum (Cha) DNA was extracted and used as a template, and the specificity of the above 10 primer probe combinations was verified by cdPCR system.

[0034] According to the target gene TEF-1α, ITS , the primer probe group was designed to amplify the pathogenic fungus Fusarium pseudograminearum, and the results are shown in Figure 1 ; Figure 1 In A, Fw1 / Rv1 / Probe1 primer probe group designed according to the target gene TEF-1α amplifies the pathogenic fungus Fusarium pseudograminearum 1D, only amplifies the target pathogenic fungus, showing excellent specificity; Figure 1 In B, C, D, E, Fw2 / Rv2 / Probe2, Fw3 / Rv3 / Probe3, Fw4 / Rv4 / Probe4, Fw5 / Rv5 / Probe5 primer probe group designed according to the target gene TEF-1α and ITS amplifies the pathogenic fungus Fusarium pseudograminearum 1D, and non-specific amplification occurs in other species Fc, Fsp, Fo, Fs, Ffal of the same genus, and there is cross reaction. Therefore, among the many primer probe groups designed according to the different target genes of the pathogenic fungus Fusarium pseudograminearum, only the Fw1 / Rv1 / Probe1 primer probe group has good specificity and can specifically detect the pathogenic fungus Fusarium pseudograminearum. Accordingly, the Fw1 / Rv1 / Probe1 primer probe group designed according to the target gene TEF was selected for subsequent cdPCR specificity quantitative detection of Fusarium pseudograminearum.

[0035] According to the target gene Tri , ITS, TEF-1α , the primer probe group was designed to amplify the specificity of the pathogenic fungus Fusarium graminearum, and the results are shown in Figure 2 ; wherein, Figure 2 In A, B, C, Fw1 / Rv1 / Probe1, Fw2 / Rv2 / Probe2, Fw3 / Rv3 / Probe3 primer probe group designed according to the target gene TriThe designed primer probe set Fw1 / Rv1 / Probe1 amplified 1D of the pathogenic fungus Fusarium graminearum, and only the target pathogenic fungus was amplified, showing good specificity. However, the amplification results of Fw2 / Rv2 / Probe2 and Fw3 / Rv3 / Probe3 showed non-specific amplification, and could not specifically detect Fusarium graminearum. Figure 2 D is according to the target gene ITS The designed Fw4 / Rv4 / Probe4 primer probe set amplified 1D of the pathogenic fungus Fusarium graminearum, and non-specific amplification occurred, showing cross-reaction. Fo, Fs, Fsp, Fpg, Ffal Non-specific amplification occurred, and cross-reaction existed. Figure 2 E is according to the target gene TEF The designed Fw5 / Rv5 / Probe5 primer probe set amplified 1D of the pathogenic fungus Fusarium graminearum, and the amplification effect was not ideal, non-specific amplification occurred, and cross-reaction existed. Therefore, among the many primer probe sets designed according to different target genes of the pathogenic fungus Fusarium graminearum, only the Fw1 / Rv1 / Probe1 primer probe set had good specificity and could specifically detect the pathogenic fungus Fusarium graminearum. Subsequently, the Fw1 / Rv1 / Probe1 primer probe set was designed according to the target gene Tri The designed Fw1 / Rv1 / Probe1 primer probe set was used for cdPCR specific quantitative detection of Fusarium graminearum.

[0036] 3. Amplification system and conditions

[0037] Fungal DNA extraction: The fungal genomic DNA extraction kit (Beijing Solabio Technology Co., Ltd.) was used, and the DNA was extracted from the cultured fungi according to the kit and instrument operation instructions.

[0038] Wheat root DNA extraction: CTAB classic DNA extraction method was used.

[0039] After optimization of the primer and probe concentration, the final concentration of the selected primer and probe was 500 nmol / L and 250 nmol / L.

[0040] This experiment was performed on QIAcuity dPCR System (QIAGEN), and 24-well QIAcuity nanosheets of 26K were used. A 24-well nanosheet of 26K contains 24 wells, each with 26000 partitions, meaning that 26000 cdPCR reactions can occur in each well. In the reaction system, 4x Probe PCR Master Mix 10 μL was added, 2 μL of each of the two pairs of primers, 1 μL of each of the two probes, 4 μL of the mixed pathogenic fungus DNA with a mass ratio of 1:1, and sterilized water was added to 40 μL. cdPCR amplification was performed. The amplification conditions are shown in Table 2:

[0041] Table 2 cdPCR amplification conditions

[0042]

[0043] 4 cdPCR quantitative accuracy linear regression verification

[0044] The linear regression curve of cdPCR was plotted with ideal copy number as the abscissa (X-axis) and actual copy number as the ordinate (Y-axis) to evaluate the accuracy of double cdPCR in nucleic acid quantification.

[0045] The linear regression results of cdPCR quantitative accuracy are shown in Table 2. Figure 3 As shown in Table 2, the standard curve correlation coefficient of double cdPCR for F. pseudograminearum was 0.9999, and for F. culmorum was 0.9998, indicating that the error range between actual copy number and ideal copy number measured by the double cdPCR method established in this experiment was small. The cdPCR quantitative detection method established in this experiment can be used as an effective means for accurate quantification of nucleic acids of F. pseudograminearum and F. culmorum.

[0046] Test Example 2 Verification test of detection of pathogenic fungi F. pseudograminearum and F. culmorum in wheat rhizosphere soil samples using cdPCR quantitative detection method

[0047] To evaluate the detection performance of the two PCR methods, 97 samples of wheat rhizosphere soil were detected.

[0048] qPCR was performed on LightCycler 480, and the primer probe set was the same as that in the cdPCR detection system. In the reaction system, 2x Probe qPCR Master Mix 10 μL, two pairs of primers each 1 μL, two probes each 0.5 μL, and DNA 2 μL were added, and sterilized water was added to make up to 20 μL. The amplification program was optimized as follows: pre-denaturation: 95℃, 10 min, 1 cycle; annealing extension: 94℃, 30 s; 58℃, 1 min; 45 cycles.

[0049] The detection rate of the pathogen Fusarium pseudograminearum in the samples was 17.6% by qPCR and 44.4% by cdPCR. The detection rate of the pathogen Fusarium culmorum in the samples was 37.1% by qPCR and 43.3% by cdPCR. The statistical results are shown in Table 3. The total proportion of positive samples detected by cdPCR was 52.6%, while the total proportion of positive samples detected by qPCR was only 39.2%. Due to the interference of inhibitors and competitive amplification in rhizosphere soil, the signal of low-abundance pathogens may be masked or underestimated, resulting in "false negative" of qPCR. The cdPCR divides the reaction system into tens of thousands of independent microdroplets or micropores, which significantly reduces the influence of background noise and inhibitors. Meanwhile, the data comparison also confirms that cdPCR has higher sensitivity.

[0050] Table 3 qPCR, cdPCR statistics of rhizosphere soil samples

[0051]

[0052] To evaluate the accuracy of qPCR and cdPCR in detecting pathogens, the copy number of Fusarium pseudograminearum and Fusarium culmorum in rhizosphere soil samples that were positive by both methods was compared. Due to the rich inhibitors in rhizosphere soil DNA extract, cdPCR can more accurately detect target DNA due to its stronger tolerance to inhibitors and lower background noise. qPCR is easily interfered by inhibitors, resulting in underestimated copy number. As shown in Table 4, the concentration of the two pathogens detected by cdPCR in the same sample was higher than that detected by qPCR, indicating that cdPCR has higher accuracy in complex rhizosphere soil environment. Figure 4 Table 4 Comparison of the copy number of pathogens detected by qPCR and cdPCR

Claims

1. Detection of the pathogen *Fusarium oxysporum* (Fusarium oxysporum) Fusarium pseudograminearum PCR primer pairs and probes, characterized in that, The PCR primer pair for detecting the pathogenic fungus Fusarium pseudograminearum consists of an upstream primer shown in SEQ ID NO. 1 and a downstream primer shown in SEQ ID NO. 2; the nucleotide sequence of the probe for detecting the pathogenic fungus Fusarium pseudograminearum is shown in SEQ ID NO. 3; The 8th cytosine of the upstream primer shown in SEQ ID NO. 1 is a locked nucleic acid modified cytosine, counted from the 5' end; the probe shown in SEQ ID NO. 3 is connected with a fluorescent group corresponding to the detection channel at its 5' end and with a quenching group at its 3' end; the 3rd thymine and the 15th cytosine of the probe shown in SEQ ID NO. 3 are a locked nucleic acid modified thymine and a locked nucleic acid modified cytosine, respectively, counted from the 5' end.

2. The PCR primer pair and probe according to claim 1, characterized in that, The fluorescent group connected to the probe shown in SEQ ID NO. 3 is FAM, and the quenching group connected to the probe shown in SEQ ID NO. 3 is BHQ1.

3. Use of the PCR primer pair and the probe according to claim 1 or 2 in detecting the pathogenic fungus Fusarium pseudograminearum.

4. A chip digital PCR kit for detecting the pathogenic fungus Fusarium pseudograminearum, comprising: Probe PCR MasterMix, PCR primer pair and probe; characterized in that the PCR primer pair and the probe are the PCR primer pair and the probe according to claim 1 or 2.

5. A chip digital PCR detection method for detecting the pathogenic fungus Fusarium pseudograminearum, characterized in that, It comprises: (1) extracting DNA from the sample to be detected; (2) establishing a chip digital PCR amplification system with the extracted DNA and the PCR primer pair and the probe for detecting the pathogenic fungus Fusarium pseudograminearum to perform chip digital PCR amplification; and (3) determining whether the pathogenic fungus Fusarium pseudograminearum is contained according to the fluorescence signal of the amplification product; the PCR primer pair and the probe for detecting the pathogenic fungus Fusarium pseudograminearum are the PCR primer pair and the probe according to claim 1.

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