Quantitative PCR kit and detection method for South American soybean sudden death syndrome pathogen

Abstract

The present invention belongs to the field of biological detection technology, and relates to a quantitative PCR kit for South American soybean sudden death syndrome pathogen and a detection method thereof. The kit comprises a first primer pair for amplifying the ITS gene of the ribosomal internal transcribed spacer region of the South American soybean sudden death syndrome pathogen, and a first probe that specifically binds to the amplified product of the first primer pair, the sequence of the forward primer of the first primer pair is shown as SEQ ID NO: 1, the sequence of the reverse primer is shown as SEQ ID NO: 2, and the sequence of the first probe is shown as SEQ ID NO: 3. The quantitative PCR kit for South American soybean sudden death syndrome pathogen and the detection method thereof of the present invention can detect South American soybean sudden death syndrome pathogen with high specificity, high sensitivity and high stability.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and relates to a quantitative PCR kit for South American soybean sudden death syndrome pathogen and a detection method thereof. Background Art

[0002] The South American soybean sudden death syndrome pathogen is a pathogen that affects soybean growth and is extremely harmful. It not only causes the beans of soybean plants to become smaller, but also seriously affects the yield of soybeans. In the United States, the occurrence of soybean sudden death syndrome has become common, especially in years with low temperatures and heavy rains, and the loss may even reach 100%. As a major soybean producer, my country cannot ignore the potential threat of soybean sudden death syndrome. my country needs to import a large amount of soybeans from abroad every year, and many of the major exporting countries are epidemic areas of soybean sudden death syndrome, which increases the risk of pests invading China.

[0003] The South American soybean sudden death syndrome pathogen is mainly spread over long distances through flowing water, soil movement, and agricultural operations, and is mainly spread over short distances through soil and diseased residues carried in soybean seedlings and seeds. Species that are morphologically similar to the South American soybean sudden death syndrome pathogen include North American soybean sudden death syndrome pathogen (Fusariumvirguliform), Brazilian soybean sudden death syndrome pathogen (Fusariumbrasiliense), wedge-beak bean root rot pathogen (Fusariumcuneirostrum), bean root rot pathogen (Fusariumphaseoli) and Matt Fusarium (Fusariummartii).

[0004] The current standard for the detection of South American soybean sudden death syndrome pathogen in my country is GB / T31796-2015 "Quarantine Identification Methods for South American Soybean Sudden Death Syndrome Pathogen and North American Soybean Sudden Death Syndrome Pathogen", which records that the detection methods for South American soybean sudden death syndrome pathogen and North American soybean sudden death syndrome pathogen are mainly morphological and molecular biological methods, among which morphological identification has problems such as long identification cycle and poor timeliness; while the molecular biological detection of South American soybean sudden death syndrome pathogen adopts fluorescent PCR detection method, but this method may miss detection of low-abundance pathogens in complex mixed samples, and may produce non-specific amplification for similar species of South American soybean sudden death syndrome pathogen (such as bean root rot pathogen), resulting in false positives, etc. In addition, the current standard uses fluorescent quantitative PCR method to determine negative and positive samples as Cq≤36, and the test results of 36≤Cq≤40 cannot determine the "negative or positive" of the sample, so the sample needs to be tested repeatedly or even after repeated testing, it cannot be determined. Moreover, missed detection of true positive samples may lead to diseases caused by the “spillover” of pathogens. Therefore, it is urgent to develop a molecular detection quantification and evaluation method with high sensitivity, high accuracy, and high tolerance to inhibitor interference, so as to reduce the spread of pests and diseases caused by the “missed detection” of “false negative” samples.

[0005] The main principle of digital PCR technology (digital PCR, dPCR) is to randomly disperse the sample into thousands of tiny reaction units of equal volume (also called droplets), and then perform PCR amplification on the reaction units. After the amplification, the fluorescence signal of each reaction unit is read and the data is analyzed according to the principle of Poisson distribution. The total number of droplets formed needs to reach more than 10,000 to meet the requirements of Poisson distribution. This technology can accurately detect low-abundance targets, targets in complex mixtures, and small multiple differences in target genes, and can be applied to a wide range of sample types and application fields. In order to effectively improve the specificity, sensitivity and stability of the detection of South American soybean sudden death syndrome pathogens, it is very necessary to provide a detection method based on digital PCR to meet the increasingly sophisticated needs of harmful biological quarantine. Summary of the invention

[0006] The primary purpose of the present invention is to provide a quantitative PCR kit for detecting the South American soybean sudden death syndrome pathogen, which can be used for detecting the South American soybean sudden death syndrome pathogen with high specificity, high sensitivity and high stability.

[0007] To achieve this object, in a basic embodiment, the present invention provides a quantitative PCR kit for South American soybean sudden death syndrome pathogen, the kit comprising a first primer pair for amplifying the ribosomal internal transcribed spacer ITS gene of South American soybean sudden death syndrome pathogen, and a first probe that specifically binds to the amplification product of the first primer pair,

[0008] The sequence of the forward primer of the first primer pair is shown in SEQ ID NO: 1, and the sequence of the reverse primer is shown in SEQ ID NO: 2.

[0009] The sequence of the first probe is shown in SEQ ID NO:3.

[0010] In a preferred embodiment, the present invention provides a quantitative PCR kit for South American soybean sudden death syndrome pathogen, wherein the 5' end of the first probe is connected to a FAM fluorescent reporter group, and the 3' end is connected to a BHQ1 fluorescent quencher group.

[0011] In a preferred embodiment, the present invention provides a quantitative PCR kit for the South American soybean sudden death syndrome pathogen, wherein the kit further comprises a second primer pair for amplifying the soybean endogenous gene Lectin, and a second probe that specifically binds to the amplification product of the second primer pair.

[0012] The sequence of the forward primer of the second primer pair is shown in SEQ ID NO: 4, and the sequence of the reverse primer is shown in SEQ ID NO: 5.

[0013] The sequence of the second probe is shown in SEQ ID NO:6.

[0014] In a preferred embodiment, the present invention provides a quantitative PCR kit for South American soybean sudden death syndrome pathogen, wherein the 5' end of the second probe is connected to a Cy5 fluorescent reporter group, and the 3' end is connected to a BHQ3 fluorescent quencher group.

[0015] In a preferred embodiment, the present invention provides a quantitative PCR kit for detecting the South American soybean sudden death syndrome pathogen, wherein the kit is a digital PCR kit.

[0016] In a preferred embodiment, the present invention provides a quantitative PCR kit for detecting the South American soybean sudden death syndrome pathogen, wherein the primer and probe sequences for detecting the South American soybean sudden death syndrome pathogen ITS gene and the soybean internal reference Lectin gene are shown in Table 1 below:

[0017] Table 1

[0018]

[0019] In a preferred embodiment, the present invention provides a quantitative PCR kit for South American soybean sudden death syndrome pathogen, wherein the digital PCR kit comprises a digital PCR reaction solution and a digital PCR premix solution,

[0020] The digital PCR reaction solution contains primers and probes.

[0021] The digital PCR premix contains multiple qPCR DNA amplification enzyme mixture, fluorescein sodium salt, and endonuclease HhaI.

[0022] Among them, the endonuclease HhaI can specifically recognize and cut the GCGC sequence in the South American soybean sudden death syndrome pathogen, the wedge-shaped bean root rot pathogen and the bean root rot pathogen.

[0023] In a preferred embodiment, the present invention provides a quantitative PCR kit for the South American soybean sudden death syndrome pathogen, wherein the concentration of each primer in the digital PCR reaction solution is independently 5-20 μmol / L, and the concentration of each probe is independently 3-8 μmol / L.

[0024] In a preferred embodiment, the present invention provides a quantitative PCR kit for the South American soybean sudden death syndrome pathogen, wherein the kit further comprises a negative control and a positive control, wherein the negative control is ddH2O, and the positive control is a solution of a plasmid containing a segment of a gene amplified by a primer pair.

[0025] In a preferred embodiment, the present invention provides a quantitative PCR kit for detecting the South American soybean sudden death syndrome pathogen, wherein the positive control contains a mixture of two plasmid DNAs, wherein the plasmid DNAs are: a segment plasmid of the ITS gene and a segment plasmid of the Lectin gene.

[0026] In a preferred embodiment, the present invention provides a quantitative PCR kit for the South American soybean sudden death syndrome pathogen, wherein the composition of the kit is shown in Table 2 below:

[0027] Table 2

[0028]

[0029] The second object of the present invention is to provide a detection method of the kit as described above, so as to detect the South American soybean sudden death syndrome pathogen with high specificity, high sensitivity and high stability.

[0030] To achieve this object, in a basic embodiment, the present invention provides a detection method of the kit as described above, the detection method comprising the following steps:

[0031] (1) Extracting DNA template for PCR amplification from soybean;

[0032] (2) Amplifying and quantifying the DNA template using the kit.

[0033] In a preferred embodiment, the present invention provides a detection method of the kit as described above, wherein in step (1), CTAB lysis buffer is used to extract the DNA template.

[0034] In a preferred embodiment, the present invention provides a detection method of the kit as described above, wherein the specific operation method of step (1) is: using CTAB lysis solution to extract DNA from samples collected from different parts of soybeans suspected of being infected, and the DNA template obtained after extraction is used for subsequent detection process.

[0035] In a preferred embodiment, the present invention provides a detection method of the kit as described above, wherein in step (2), the PCR chip used is a Ruby chip, and the PCR reaction procedure corresponding to the PCR chip is as follows:

[0036] Droplet generation step: temperature 25°C;

[0037] Pre-denaturation step: temperature 95°C, time 5 minutes;

[0038] PCR reaction step: denaturation temperature 95°C for 15 seconds, annealing temperature 58°C for 30 seconds, wherein the PCR reaction step is cycled multiple times;

[0039] Pressure release step: temperature 25°C;

[0040] Signal detection: Each droplet will contain 0, 1 or more South American soybean sudden death syndrome pathogen DNA, and will also contain 0, 1 or more soybean endogenous Lectin gene DNA; after PCR amplification, the droplets containing South American soybean sudden death syndrome pathogen DNA or soybean endogenous Lectin gene DNA will generate strong fluorescence signals after PCR amplification; the fluorescence signal is detected using a fluorescence detection device;

[0041] Data analysis: The detected fluorescence signals are respectively brought into specific fluorescence thresholds and fluorescence compensation parameters to obtain the copy number concentration of each target and interpret the results.

[0042] The beneficial effect of the present invention is that the South American soybean sudden death syndrome pathogen quantitative PCR kit and the detection method thereof of the present invention can detect the South American soybean sudden death syndrome pathogen with high specificity, high sensitivity and high stability.

[0043] The kit and the detection method of the present invention do not rely on standard products or other reference products, are suitable for detecting low-concentration South American soybean sudden death syndrome pathogens, can achieve absolute quantification of South American soybean sudden death syndrome pathogens, and have higher sensitivity and accuracy.

[0044] The present invention first verifies the correctness of the principle, feasibility, accuracy and precision of the kit, and determines the optimal PCR amplification system through verification, which can detect the nucleic acid of the South American soybean sudden death syndrome pathogen to the greatest extent in both simple and complex samples.

[0045] In the present invention, the kit can detect and determine the trace nucleic acid of the South American soybean sudden death syndrome pathogen, proving that the digital PCR kit is a kit with higher detection sensitivity than the qPCR kit, can detect the South American soybean sudden death syndrome pathogen earlier, and can accurately determine the load of the South American soybean sudden death syndrome pathogen.

[0046] The kit of the present invention comprises accurate quality control samples of two positive plasmids, and the positive conformity rate thereof is 100%.

[0047] The kit of the present invention can detect DNA extracted from the South American soybean sudden death syndrome pathogen, and the minimum detection amount reaches 0.30 copies / μL.

[0048] The kit of the present invention can detect each precision quality control product in the corresponding reaction solution by repeating the test three times, and the CV value is lower than 5%.

[0049] Compared with the prior art, the detection method of the kit of the present invention solves the problem that the "positive" samples with cq values ​​between 36-40 in the fluorescent quantitative PCR test results cannot be accurately judged, greatly improves the detection ability of trace nucleic acids, and can stably and accurately detect 1.55 copies / μL of South American soybean sudden death syndrome pathogen nucleic acid, and the minimum detection concentration can reach 0.30 copies / μL. The technology can be used to quantitatively detect South American soybean sudden death syndrome pathogen nucleic acid samples with extremely low abundance, and avoid the overflow of harmful pathogen samples due to "missed detection" as much as possible, resulting in the spread of pests and diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a one-dimensional graph of the amplification of the South American soybean sudden death syndrome pathogen DNA in Example 1. Legend: The horizontal axis is the sample number, and numbers 1-3 are three replicate wells of one sample; the vertical axis is the intensity of the blue channel fluorescence signal, and the middle straight line is the threshold line. The droplets above the threshold line contain the South American soybean sudden death syndrome pathogen DNA, and the droplets below the threshold line do not contain the South American soybean sudden death syndrome pathogen DNA.

[0051] Figure 2This is a one-dimensional graph of the amplification of the soybean endogenous gene lectin in Example 1. Legend: The horizontal axis is the sample number, and numbers 1-3 are three replicate wells of one sample; the vertical axis is the intensity of the red channel fluorescence signal, and the middle curve is the threshold line. The droplets above the threshold line are positive droplets containing the soybean endogenous gene Lectin, and the droplets below the threshold line are negative droplets that do not contain the soybean endogenous gene Lectin.

[0052] Figure 3 It is a one-dimensional graph of specific verification amplification of primer and probe combination 1 in Example 2. Legend: the horizontal axis is the sample number, and numbers 1-6 are DNA of South American soybean sudden death syndrome pathogen, North American soybean sudden death syndrome pathogen, wedge-shaped bean root rot pathogen DNA, bean root rot pathogen DNA, Brazilian soybean sudden death syndrome pathogen, and Fusarium mattii DNA respectively; the vertical axis is the fluorescence signal intensity of the blue channel, the middle straight line is the threshold line, and the positive droplets above the threshold line contain the South American soybean sudden death syndrome pathogen DNA, and the negative droplets below the threshold line do not contain the South American soybean sudden death syndrome pathogen DNA.

[0053] Figure 4 It is a one-dimensional graph of specific verification amplification of primer and probe combination 2 in Example 2. Legend: the horizontal axis is the sample number, and numbers 1-6 are respectively the DNA of South American soybean sudden death syndrome pathogen, North American soybean sudden death syndrome pathogen, wedge-shaped bean root rot pathogen DNA, bean root rot pathogen DNA, Brazilian soybean sudden death syndrome pathogen, and Fusarium mattii DNA; the vertical axis is the fluorescence signal intensity of the blue channel, the middle straight line is the threshold line, the positive droplets containing South American soybean sudden death syndrome pathogen DNA are above the threshold line, and the negative droplets containing no South American soybean sudden death syndrome pathogen DNA are below the threshold line.

[0054] Figure 5 It is a one-dimensional graph of specific verification amplification of primer and probe combination 3 in Example 2. Legend: the horizontal axis is the sample number, and numbers 1-6 are respectively the DNA of South American soybean sudden death syndrome pathogen, North American soybean sudden death syndrome pathogen, wedge-shaped bean root rot pathogen DNA, bean root rot pathogen DNA, Brazilian soybean sudden death syndrome pathogen, and Fusarium mattii DNA; the vertical axis is the blue channel fluorescence signal intensity, the middle straight line is the threshold line, the positive droplets containing South American soybean sudden death syndrome pathogen DNA are above the threshold line, and the negative droplets containing no South American soybean sudden death syndrome pathogen DNA are below the threshold line.

[0055] Figure 6This is a one-dimensional graph of the amplification of the digital PCR master mix 1 in Example 3. Legend: The horizontal axis is the sample number, and numbers 1-3 represent three replicate wells; the vertical axis is the fluorescence signal intensity of the blue channel, and the middle straight line is the threshold line. The droplets above the threshold line contain the DNA of the South American soybean sudden death syndrome pathogen, and the droplets below the threshold line do not contain the DNA of the South American soybean sudden death syndrome pathogen.

[0056] Figure 7 This is a picture of droplets formed by the digital PCR premix 1 in Example 3. Legend: The droplets on the black background in the field of view are flat, uniform in number and size, and a large number of positive droplets (bright spots) are present after amplification, and the software identification is normal.

[0057] Figure 8 This is a droplet image formed by the digital PCR premix 2 in Example 3. Legend: There is a large dark area on the right side of the droplet image, with no droplets or positive droplets (bright spots) after amplification. The number of positive droplets (bright spots) after amplification in the left area is relatively small, and the software identification is abnormal.

[0058] Fig. 9 This is a picture of droplets formed by the digital PCR premix 3 in Example 3. Legend: The size of the droplets under the black background in the field of view is very uneven, the total number of droplets and the number of positive droplets (bright spots) after amplification are basically 0, and the software identification is abnormal.

[0059] Fig.10 This is a one-dimensional graph of the linear range amplification of the South American soybean sudden death syndrome pathogen in Example 4. Legend: The horizontal axis is the sample number, No. 1-3 is a sample diluted 1 times, No. 4-6 is a sample diluted 10 times, No. 7-9 is a sample diluted 100 times, No. 10-12 is a sample diluted 1000 times, No. 13-15 is a sample diluted 1×10 4 times, numbers 16-18 are samples diluted 2×10 4 times, No. 19-21 is sample dilution 4×10 4 times, and numbers 22-24 are samples diluted 8×10 4 times, and numbers 25-27 are samples diluted 1.6×10 5 times the plasmid DNA of the South American soybean sudden death syndrome pathogen; the ordinate is the fluorescence signal intensity of the blue channel, the middle straight line is the threshold line, the droplets above the threshold line are positive droplets containing the South American soybean sudden death syndrome pathogen DNA, and the droplets below the threshold line are negative droplets without the South American soybean sudden death syndrome pathogen DNA.

[0060] Fig.11This is a fitting curve diagram of the linear range of the detection of South American soybean sudden death syndrome pathogens in Example 4. Legend: The horizontal axis is the logarithmic value of the dilution multiple; the vertical axis is the logarithmic value of the copy number concentration. DETAILED DESCRIPTION

[0061] In order to better understand the technical solutions and advantages of the present invention, the present invention is further described below through embodiments in conjunction with the accompanying drawings.

[0062] The general detection method in the following embodiments is as follows:

[0063] 1. Positive reference

[0064] Positive samples of South American soybean sudden death syndrome pathogen and North American soybean sudden death syndrome pathogen that were seized by customs and confirmed through relevant inspections.

[0065] 2. Preparation of positive plasmid samples (positive control)

[0066] The artificially synthesized ITS gene of South American soybean sudden death syndrome pathogen (GenBank accession number AY220201.1) and the soybean lectin internal reference Lectin gene (GenBank accession number K00821.1) were respectively transferred into plasmid pUC57 to construct a positive plasmid sample (positive control).

[0067] Similarly, positive plasmid samples containing ITS genes of North American soybean sudden death syndrome pathogen (Genbank accession number AY220212.1), wedge-beak bean root rot pathogen (Genbank accession number MG967563.1), bean root rot pathogen (Genbank accession number AY220194.1), Brazilian soybean sudden death syndrome pathogen (Genbank accession number FJ919501.1), and Fusarium solani (Genbank accession number OP099552.1) can be prepared.

[0068] 3. Digital PCR Procedure

[0069] (1) Primer and probe dilution

[0070] The forward primer, reverse primer and probe were first diluted to 100 μmol / L respectively. Then, all forward primers and reverse primers were prepared at a concentration of 10 μmol / L, and all probes were prepared at a concentration of 5 μmol / L to prepare the primer-probe mixture for use, and then the digital PCR reaction system was prepared. The preparation of the reaction system is shown in Table 3.

[0071] Table 3 Digital PCR reaction system

[0072] Components Single reaction well (μL) Digital PCR Master Mix 1 Primer probe mixture 0.3 Positive reference or positive plasmid sample 3.7 Total volume 5

[0073] The digital PCR premix includes multiplex qPCR DNA amplification enzyme mixture (5×) (5×PerfeCTaMultiPlex qPCR ToughMix, QUANTABIO, USA), fluorescein sodium salt (1 μmol / L), and endonuclease HhaI (96 U).

[0074] (2) Digital PCR uses the naica fully automatic droplet chip digital PCR system (Stilla). The PCR chip is a Ruby chip, which is an integrated microfluidic chip. The PCR reaction solution forms a large number of circular droplet-state microreactors in the Ruby chip. Each reactor contains only a small amount or a single DNA molecule. Each reactor performs PCR amplification independently to achieve quantitative detection of the target gene.

[0075] (3) The digital PCR reaction procedure is shown in Table 4.

[0076] Table 4 Digital PCR reaction program

[0077]

[0078] (4) After the digital PCR reaction is completed, place the chip on the Naica TM In the prism3 droplet reading and analysis system, fluorescence signals are collected and the results are automatically saved.

[0079] Example 1: Preliminary test

[0080] The primer and probe combination in Table 1 was used to conduct a preliminary detection test of positive reference materials of South American soybean sudden death syndrome pathogen. The test results are shown in Table 5 below.

[0081] Table 5 Detection results of positive droplet number and copy number of positive reference

[0082]

[0083] like Figure 1 As shown in the figure, the horizontal axis is the sample number, the vertical axis is the intensity of the blue channel fluorescence signal, the middle straight line is the fluorescence value threshold line, the positive droplets above the threshold line contain the South American soybean sudden death syndrome pathogen DNA, and the negative droplets below the threshold line do not contain the South American soybean sudden death syndrome pathogen DNA; Figure 2 As shown, the abscissa is the sample number, the ordinate is the intensity of the red channel fluorescence signal, the middle line is the threshold line, the droplets above the threshold line are positive droplets containing the soybean endogenous gene Lectin, and the droplets below the threshold line are negative droplets not containing the soybean endogenous gene Lectin.

[0084] Example 2: Primer and probe optimization test (specificity test)

[0085] By comparing the DNA sequences of South American soybean sudden death syndrome pathogen, North American soybean sudden death syndrome pathogen, wedge-shaped bean root rot pathogen, bean root rot pathogen, Brazilian soybean sudden death syndrome pathogen, and Fusarium mattii, different primer and probe combinations for detecting South American soybean sudden death syndrome pathogen as shown in Table 6 below were designed (the detection primers and probes of soybean endogenous gene Lectin were the same in each group), which were used for the detection of different positive references or positive controls, respectively, and the results were shown in Tables 7-9 below. The digital PCR amplification system in each reaction well is composed of multiple qPCR DNA amplification enzyme mixture (PerfeCTaMultiPlex qPCR ToughMix (0.8×), QUANTABIO, USA), fluorescein sodium salt (0.2 μmol / L), primers (0.6 μmol / L), probes (0.3 μmol / L), and endonuclease HhaI (2U).

[0086] Table 6 Designed primer and probe combinations

[0087]

[0088]

[0089] Table 7 Detection results of primer and probe combination 1

[0090]

[0091] Table 8 Detection results of primer and probe combination 2

[0092]

[0093]

[0094] Table 9 Detection results of primer and probe combination 3

[0095]

[0096]

[0097] As can be seen from the results of Tables 7-9 above, primer and probe combination 1 only detects the copy number concentration in the DNA sample of South American soybean sudden death syndrome pathogen, indicating that the primer and probe combination can accurately distinguish South American soybean sudden death syndrome pathogen from North American soybean sudden death syndrome pathogen, wedge-shaped bean root rot pathogen, bean root rot pathogen, Brazilian soybean sudden death syndrome pathogen, and Fusarium mattii and can perform accurate quantitative analysis. However, primer and probe combinations 2 and 3 detect the copy number concentration in the DNA sample of South American soybean sudden death syndrome pathogen and bean root rot pathogen, indicating that primer and probe combinations 2 and 3 have poor specificity and cannot distinguish South American soybean sudden death syndrome pathogen from bean root rot pathogen.

[0098] like Figure 3-5 As shown, the horizontal axis is the sample number, the vertical axis is the intensity of the blue channel fluorescence signal, the middle line is the threshold line, the positive droplets above the threshold line contain the nucleic acid of the South American soybean sudden death syndrome pathogen, and the negative droplets below the threshold line do not contain the nucleic acid of the South American soybean sudden death syndrome pathogen. For primer and probe combination 1: Figure 3 As shown, the horizontal axis is the sample number, the vertical axis is the intensity of the blue channel fluorescence signal, the middle line is the threshold line, the positive droplets above the threshold line contain the nucleic acid of the South American soybean sudden death syndrome pathogen, and the negative droplets below the threshold line do not contain the nucleic acid of the South American soybean sudden death syndrome pathogen. Only No. 1 is a positive droplet detected in the South American soybean sudden death syndrome pathogen, and no positive droplets are detected in other reaction wells. For primer and probe combinations 2 and 3: Figure 4 and 5 As shown, positive droplets were detected in No. 1 (South American soybean sudden death syndrome pathogen) and No. 4 (bean root rot pathogen), further indicating that the specificity of primer probe combinations 2 and 3 is poor.

[0099] Therefore, the first set of primers and probes are preferably used for subsequent detection in specific detection, and the specific sequence is:

[0100] Nan-f1: 5'-AGGGTATAGGGTAGTGGTA-3' (SEQ ID NO: 1)

[0101] Nan-r1: 5'-GTACCTACCCTGTACCTTC-3' (SEQ ID NO: 2)

[0102] Nan-p1: 5'-CTTGGTGAAATTCTGCCATGCTCC-3' (SEQ ID NO: 3)

[0103] Example 3: Digital PCR premix optimization test

[0104] The following digital PCR premix was used respectively, and the digital PCR premix was mixed with commercially available DNA amplification enzyme premix, fluorescein sodium salt and endonuclease HhaI. The combination 1 of primers (0.6 μmol / L) and probes (0.3 μmol / L) was used to detect the ITS gene positive plasmid sample of the South American soybean sudden death syndrome pathogen, and the detection was repeated three times.

[0105] Digital PCR premix 1: The DNA amplification enzyme premix contained therein is PerfeCTa Multiplex qPCRToughMix (5×) (QUANTABIO, USA), the Chinese name is: Multiplex qPCR DNA amplification enzyme mixture (5×), and also contains fluorescein sodium salt (1 μmol / L) and endonuclease HhaI (96U).

[0106] Digital PCR premix 2: The DNA amplification enzyme premix contained therein is APTA genotyping Master (5×) (Roche, Switzerland), and its Chinese name is: APTA genotyping DNA amplification enzyme mixture (5×); it also contains fluorescein sodium salt (1 μmol / L) and endonuclease HhaI (96U).

[0107] Digital PCR premix 3: The DNA amplification enzyme premix contained in it is

[0108] Multiplex Probe ARMS-qPCR SuperMix (UDG) (Suzhou Jinan Protein Technology Co., Ltd.), the Chinese name is: Multiplex Probe ARMS-qPCR Amplification Enzyme Mixture (UDG); it also contains fluorescein sodium salt (1μmol / L) and endonuclease HhaI (96U).

[0109] Test results such as Figure 6-9 As shown in Tables 10-12.

[0110] Table 10 Digital PCR premix 1 test results

[0111]

[0112]

[0113] Table 11 Digital PCR premix 2 test results

[0114]

[0115] Table 12 Digital PCR premix 3 test results

[0116]

[0117]

[0118] From the results in Tables 10-12 above, it can be seen that the total number of droplets formed after amplification using digital PCR premixes 2 and 3 is very small, and the correct copy number calculation cannot be performed; only digital PCR premix 1 can form a normal number of stable droplets and calculate the copy number concentration of the positive control of the South American soybean sudden death syndrome pathogen. The average detection concentration of digital PCR premix 1 is 579.5 copies / μL, and the coefficient of variation is 1.08%.

[0119] like Figure 6-9 As shown, they are the amplification graph and droplet graph formed by digital PCR premix 1, the droplet graph formed by digital PCR premix 2, and the droplet graph formed by digital PCR premix 3. The amplification result of digital PCR premix 1 is shown in Figure 6 The positive droplets and negative droplets are clearly distinguished, and there are no scattered droplets containing fluorescent signals on both sides of the threshold line; the droplet diagram is shown in Figure 7 As shown in the figure, the droplets formed are uniform in size and there is no large-scale fusion of droplets. The results after testing digital PCR premix 2 and digital PCR premix 3 are as follows Figure 8 and Fig. 9 As shown in the droplet diagram, the droplets formed large-area fusion and very uneven droplets, making it impossible to obtain a normal number of droplets, and therefore the number of nucleic acid copies detected. Therefore, only digital PCR premix 1, which consists of multiple qPCR DNA amplification enzyme mixture (5×), fluorescein sodium salt (1μmol / L), and endonuclease HhaI (96U), can perform normal digital PCR amplification and obtain copy number results.

[0120] The composition of the quantitative PCR kit for the South American soybean sudden death syndrome pathogen finally determined by the above Examples 1-3 is shown in Table 2, and is used for the research of the following Examples 4-7.

[0121] Example 4: Sensitivity and linear range verification test

[0122] The sample used in this embodiment is a South American soybean sudden death syndrome pathogen ITS gene positive plasmid sample, the primer and probe combination used is the above-mentioned primer and probe combination 1, and the digital PCR premix used is the above-mentioned digital PCR premix 1.

[0123] The ITS gene positive plasmid sample of the South American soybean sudden death syndrome pathogen was first diluted in a 10-fold gradient concentration, and then diluted in a 2-fold gradient concentration to obtain a plasmid sample with a final concentration range of 0-40000 copies / μL and subjected to digital PCR detection. Each gradient was tested three times. The test results are as follows Fig.10 , Fig.11 As shown in Table 13.

[0124] Table 13 Sensitivity test results of South American soybean sudden death syndrome pathogen

[0125]

[0126]

[0127] From the above results, it can be seen that the detection range of digital PCR for the ITS gene of the South American soybean sudden death syndrome pathogen is five orders of magnitude, and it is linear in the concentration range of 1.20-43103.0 copies / μL (r=0.9983≥0.98), with RSD<25%.

[0128] Example 5: Limit of Quantitation Test

[0129] The sample used in this embodiment is a South American soybean sudden death syndrome pathogen ITS gene positive plasmid sample, the primer and probe combination used is the above-mentioned primer and probe combination 1, and the digital PCR premix used is the above-mentioned digital PCR premix 1.

[0130] The ITS gene positive plasmid sample of South American soybean sudden death syndrome pathogen with a concentration of 1.20 copies / μL was tested 10 times with repeated detection. The test results are shown in Table 14.

[0131] Table 14 Quantitative detection limit test results

[0132]

[0133] From the results in Table 14, it can be seen that the average value of the test results of 10 parallel repeated tests is 1.55 copies / μL, and the RSD is 23.84% (required to be less than 25%), which shows that the kit of the present invention can perform absolute quantification on low-concentration South American soybean sudden death syndrome pathogen samples.

[0134] Example 6: Minimum detection limit test

[0135] The sample used in this embodiment is a South American soybean sudden death syndrome pathogen ITS gene positive plasmid sample, the primer and probe combination used is the above-mentioned primer and probe combination 1, and the digital PCR premix used is the above-mentioned digital PCR premix 1.

[0136] The ITS gene positive plasmid sample of the South American soybean sudden death syndrome pathogen with a detection concentration of 0.30 copies / μL in Example 4 was tested in parallel 10 times, and the test results are shown in Table 15.

[0137] Table 15 Minimum detection limit test results

[0138]

[0139]

[0140] As can be seen from the results in Table 15, the positive detection rate was 9 in 10 parallel test results, which met the requirement of the minimum detection limit (not less than 9), indicating that the kit of the present invention can stably detect samples of South American soybean sudden death syndrome pathogens with a concentration as low as 0.30 copies / μL.

[0141] In summary, the present invention has developed a dual digital PCR detection method for the South American soybean sudden death syndrome pathogen based on digital PCR technology, which solves the positive uncertainty situation between cq values ​​of 36-40 and greatly improves the trace detection capability. The kit and method are suitable for the detection of low-concentration South American soybean sudden death syndrome pathogen templates, do not rely on standard products or other reference products, can achieve absolute quantification of South American soybean sudden death syndrome pathogens, can accurately detect 1.55 copies / μL of South American soybean sudden death syndrome pathogens, and can detect a minimum of 0.30 copies / μL of South American soybean sudden death syndrome pathogens, with higher sensitivity and accuracy.

[0142] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these changes and variations. The above embodiments or implementations are merely illustrative of the present invention, and the present invention may also be implemented in other specific ways or other specific forms without departing from the gist or essential features of the present invention. Therefore, the described implementations should be considered illustrative rather than restrictive in any respect. The scope of the present invention should be described by the appended claims, and any changes equivalent to the intent and scope of the claims should also be included within the scope of the present invention.

Claims

1. A quantitative PCR kit for South American soybean sudden death syndrome pathogen, characterized in that: The kit is a digital PCR kit, which comprises a first primer pair for amplifying the ribosomal internal transcribed spacer ITS gene of the South American soybean sudden death syndrome pathogen, and a first probe that specifically binds to the amplification product of the first primer pair. The sequence of the forward primer of the first primer pair is shown in SEQ ID NO: 1, and the sequence of the reverse primer is shown in SEQ ID NO:

2. The sequence of the first probe is shown in SEQ ID NO: 3, The digital PCR kit comprises a digital PCR reaction solution and a digital PCR premix solution. The digital PCR reaction solution contains primers and probes. The digital PCR premix contains multiple qPCR DNA amplification enzyme mixture, fluorescein sodium salt, and endonuclease HhaI.

2. The kit according to claim 1, characterized in that: The 5' end of the first probe is connected to a FAM fluorescent reporter group, and the 3' end is connected to a BHQ1 fluorescent quencher group.

3. The kit according to claim 1, characterized in that: The kit also comprises a second primer pair for amplifying the soybean endogenous gene Lectin, and a second probe that specifically binds to the amplification product of the second primer pair. The sequence of the forward primer of the second primer pair is shown in SEQ ID NO: 4, and the sequence of the reverse primer is shown in SEQ ID NO:

5. The sequence of the second probe is shown in SEQ ID NO:

6.

4. The kit according to claim 3, characterized in that: The 5' end of the second probe is connected to a Cy5 fluorescent reporter group, and the 3' end is connected to a BHQ3 fluorescent quencher group.

5. The kit according to claim 1, characterized in that: The concentration of each primer in the digital PCR reaction solution is independently 5-20 μmol / L, and the concentration of each probe is independently 3-8 μmol / L.

6. The kit according to claim 1, characterized in that: The kit also contains a negative control substance and a positive control substance. The negative control substance is ddH2O, and the positive control substance is a solution of a plasmid containing a segment of a gene amplified by a primer pair.

7. A detection method of the kit according to any one of claims 1 to 6, characterized in that: The detection method comprises the following steps: (1) Extracting DNA template for PCR amplification from soybean; (2) Amplifying and quantifying the DNA template using the kit.

8. The detection method according to claim 7, characterized in that: In step (1), the DNA template is extracted using CTAB lysis buffer.

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