Fluorescent quantitative primer and kit for detecting ditylenchus destructor in soil and application of fluorescent quantitative primer and kit

Abstract

The invention belongs to the technical field of biological detection, and provides fluorescent quantitative primers and a kit for detecting ditylenchus destructor in soil and application of the fluorescent quantitative primers and the kit. The kit comprises a TaqMan qPCR primer and a probe for specifically detecting the ditylenchus destructor, nucleotide sequences of the TaqMan qPCR primer and the probe are respectively shown as SEQ ID NO: 1-3, and two ends of the probe respectively comprise a fluorophore and a quenching group. According to the TaqMan qPCR kit for detecting the ditylenchus destructor in the soil and the application of the TaqMan qPCR kit, the total DNA of a soil sample serves as a sample, operation processes such as nematode separation, morphological identification under a microscope and ditylenchus destructor picking are not needed to be carried out on the soil to be detected, only about 3 hours are needed from extraction of the total DNA of the soil to obtaining of a detection result, time consumption is short, and operation is easy. The kit for qPCR quantitative detection of ditylenchus destructor is suitable for qPCR quantitative detection of ditylenchus destructor in soil, and has the characteristics of strong specificity, high sensitivity and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a primer, a kit and an application thereof for detecting stem nematodes in soil. Background Art

[0002] Ditylenchus destructans ( Ditylenchus destructor Thorne, also known as potato rot stem nematode and sweet potato rot stem nematode, is a polyphagous plant parasitic nematode that mainly harms tubers, corms and fleshy root crops such as sweet potatoes, potatoes, angelica and garlic. It is reported that the nematode can infect more than 120 crops and weeds and feed on more than 70 fungi. It has now been listed as a national agricultural plant quarantine pest.

[0003] The decaying stem nematode survives adverse environments in the soil as eggs, larvae or adults. At present, all nematodes in the soil are usually separated by shallow plate method, Baermann funnel method or sucrose flotation centrifugation method, and then the decaying stem nematodes are identified and counted under a microscope based on morphological characteristics. The identification process is cumbersome and mainly depends on the operator's experience, and the identification results are somewhat subjective. With the development of molecular biology, DNA molecular detection technology has gradually become the most commonly used means of nematode detection. Based on PCR detection technology, Peng Deliang et al. (patent CN103740857A) developed PCR-specific detection primers for Ditylenchus rottenensis; based on loop-mediated isothermal amplification technology, Jian Heng et al. and Xu Chunling et al. developed a kit for detecting Ditylenchus rottenensis by loop-mediated isothermal technology amplification (patents CN102260746A and CN107988383A); based on recombinant recombinase polymerase amplification technology, Gao Bo et al. and Ma Jukui et al. developed recombinase polymerase amplification technology primer pairs and kits for identifying Ditylenchus rottenensis (patents CN110863058A and CN111206106A). However, these technologies are all for detecting whether Ditylenchus rottenensis exists in soil or samples, and cannot specifically predict the content of Ditylenchus rottenensis in soil, resulting in huge difficulties in predicting and forecasting diseases caused by Ditylenchus rottenensis. The initial density of nematodes in the soil is closely related to the degree of crop damage, which directly affects the growth and yield of crops. Therefore, there is an urgent need for a detection technology that can specifically detect the content of Ditylenchus destructans in the soil to lay the foundation for formulating scientific preventive measures and reducing the occurrence of diseases.

[0004] Real-time fluorescence quantitative PCR (TaqMan qPCR) technology is a nucleic acid quantitative technology developed on the basis of ordinary PCR qualitative analysis. It can not only conduct qualitative research on nematodes, but also quickly, accurately and sensitively quantify nematode templates. By directly extracting total DNA from the soil and combining it with real-time fluorescence quantitative PCR technology to quantitatively detect plant pathogenic nematodes in the soil, it is faster and more accurate than traditional morphological observation and identification. This technology has been widely used in the quantitative detection of plant pathogenic nematodes. Zhao et al. separated the nematodes in the soil by sucrose flotation centrifugation and used Real-time PCR technology to identify southern root-knot nematodes ( Meloidogyne incognita ) was quantitatively tested (Zhao YL, Ruan WB, Yu L, et al. Combining max Ratio analysis with real-time PCR and its potential application for the prediction of Meloidogyne incognita in field samples[J].Journal of nematology, 2010, 42(2): 166.); Deych et al. established a quantitative detection technology of three important nematode species from the genus Ditylenchus by Real-time qPCR (Deych KO, Pridannikov MV, Nikitin MM, et al. Simultaneous detection of three plant-parasitic nematode species from thegenus Ditylenchus Using a real-time qPCR matrix-based technology[J]. Russian Journal of Nematology, 2018, 26(2): 97-107.). However, in these studies, the pathogenic nematodes were first isolated and then quantitatively detected, which could not detect dormant nematodes and eggs in the soil, and often underestimated the number of nematodes in the soil.

[0005] By directly extracting DNA from the soil, the real-time fluorescence quantitative PCR method can accurately quantify the number of Pratylenchus penetrans in the soil ( Pratylenchus penetran ) (Sato E, Suga Y, Kisaki C, et al. Quantification of Pratylenchus penetransin radish fields using a combination method of soil compaction and real-time PCR to determine the economic threshold[J]. Soil science and plant nutrition, 2011, 57(2): 213-220.); Min et al. established a real-time fluorescence quantitative PCR technology for detecting southern root-knot nematodes by directly extracting soil DNA, and applied this technology to monitor nematodes in sweet potato planting areas in Tokushima Prefecture, Japan; Watanabe et al. established a southern root-knot nematode ( Meloidogyne incognita ) and northern root-knot nematode ( M. hapla ) and applied this technology to evaluate the correlation between eggplant yield loss and initial nematode density (Watanabe T, Masumura H, Kioka Y, et al. Development of a direct quantitative detection method for Meloidogyne incognita and M. hapla in andosol and analysis of relationship between the initial population of Meloidogyne spp. and yield of eggplant in an andosol[J]. Nematological Research (Japanese Journal of Nematology), 2013, 43(2):21-29.). Jian et al. established a real-time fluorescence quantitative PCR method based on TaqMan-MGB probe and successfully applied it to Philip cyst nematode ( Heterodera filipjevi ) quantitative analysis (Jian JZ, Huang WK, Kong LA, et al. Molecular diagnosis and direct quantification of cereal cyst nematode ( Heterodera filipjevi) from field soil using TaqMan real-time PCR[J]. Journal of Integrative Agriculture, 2023, 22(8): 2591-2601.). A large number of previous studies have shown the feasibility of directly extracting DNA from soil and combining it with real-time fluorescence quantitative PCR technology for early warning and monitoring of nematode diseases. However, there is currently no report on the direct application of real-time fluorescence quantitative PCR technology to quantitative detection of stem nematodes in soil. Summary of the invention

[0006] In view of the problems in the prior art, the present invention provides a fluorescent quantitative kit for detecting stem nematodes in soil, which can specifically, quickly, simply and quantitatively detect stem nematodes in soil, and can provide services for early diagnosis and auxiliary identification of stem nematode diseases.

[0007] To achieve the above purpose, the present invention adopts the following technical solution.

[0008] A specific detection of Ditylenchus destructor ( Ditylenchus destructor Thorne) TaqMan qPCR primers and probes, including upstream primers, downstream primers and probes whose nucleotides are shown in SEQ ID NOs: 1-3 respectively; The two ends of the probe respectively contain a fluorescent group and a quenching group.

[0009] Preferably, the 5' end of the probe contains a fluorescent group, and the 3' end contains a quencher group. Preferably, the fluorescent group is FAM, and the quencher group is BHQ.

[0010] A kit comprising the above-mentioned specific primers and probes.

[0011] The kit also includes a positive molecule, a polymerase, a substrate, a buffer and the like.

[0012] A method for detecting the decay nematode Ditylenchus destructor ( Ditylenchus destructor Thorne's method, comprising the following steps: (1) Extract DNA from the sample to be tested as a template and perform TaqMan qPCR reaction using the above primers and probes; (2) Determine the results of the sample to be tested based on the Ct value and amplification curve of the TaqMan qPCR reaction.

[0013] Preferably, in step (1), the sample to be tested is soil.

[0014] Preferably, in step (1), the TaqMan qPCR reaction system includes: 2×AceQ Universal U + Probe Master Mix 10μL, 0.4μL each of 10μM upstream primer and downstream primer, 0.2μL of 10μM probe, 1μL DNA template, 8μL H2O.

[0015] Preferably, in step (1), the conditions for the TaqMan qPCR reaction are: 37°C for 2 min, 95°C for 5 min, and then 40 cycles of 95°C for 10 s and 60°C for 30 s were performed, and the fluorescence signal was collected in each cycle.

[0016] Preferably, the above method further comprises the step of preparing a standard curve: (1a) DNA was extracted from standard samples spiked with different amounts of Ditylenchus destructans, and TaqMan qPCR reaction was performed using the above primers and probes; (2a) Establish a linear relationship between the Ct value and the logarithm of the number of Ditylenchus destructans.

[0017] Preferably, in step (2), the result determination criteria are: When the Ct value is ≤ threshold a and there is an obvious amplification curve, the result is judged as positive, indicating that the decay stem nematode is present in the sample to be tested; When threshold a < Ct value ≤ threshold b, and there is an obvious amplification curve, it means that the test has failed and needs to be retested; When the Ct value is greater than the threshold value b or there is no value, and there is no obvious amplification curve, the result is judged as negative, indicating that there is no Ditylenchus destructor in the sample to be tested; The threshold a is the intercept of the linear equation between the Ct value and the logarithm of the number of stem nematodes, and the threshold b is 36.

[0018] More preferably, in the above method, when the result is positive, the number of Ditylenchus destructans in the sample can be calculated according to the standard curve.

[0019] The present invention has the following advantages: The invention provides a primer combination for detecting a destructive nematode in soil by TaqMan qPCR. The primer combination is designed and screened according to the determination result of an ITS sequence of the destructive nematode, has strong specificity, and has a fluorescent signal only for the detection result of a soil sample containing the destructive nematode.

[0020] The present invention provides a detection method and application of TaqMan qPCR for specifically detecting stem nematodes in soil. The total DNA extraction of soil samples is simple and rapid, and there is no need to perform nematode separation, morphological identification under a microscope, and worm picking on the soil to be tested. The total DNA of the soil is directly extracted for rapid testing. It only takes about 3 hours from extracting the total DNA of the soil to obtaining the test results, which is time-saving and simple to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The primers and probes are used for specific detection. 1-6 are stem nematodes, 7-11 are root-knot nematodes incognita, root-knot nematodes of elephant-ear beans, soybean cyst nematodes, cereal cyst nematodes, rice stem-tip nematodes, coffee nematodes, fallen nematodes, avenae true sliding nematodes, and longling hair pad nematodes, and 12 is ddH2O. Figure 2 For the optimized TaqMan qPCR system sensitivity detection, A is the use of Ditylenchus destructor genomic DNA as template (concentration gradient 100 ng / μL, 10 ng / μL, 1 ng / μL, 10 - 1 ng / μL, 10 -2 ng / μL, 10 -3 ng / μL, 10 -4 ng / μL, ddH2O); B is a single female Ditylenchus destructor DNA as template (concentration gradient 10 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , ddH2O); C is a single male DNA of Ditylenchus destructor as template (concentration gradient 10 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , ddH2O); D is a single larva of Ditylenchus destructans as a template (concentration gradient 4 0 , 4 -1 , 4 -2 , 4 -3 , 4 -4 , 4 -5 , 4 -6 , ddH2O); Figure 3It is the standard curve for quantitative detection of the relationship between the number of stem nematodes in soil and the Ct value; Figure 4 The correlation between the microscopic counting method and the TaqMan qPCR quantitative detection method of Ditylenchus destructans in soil. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited by the following embodiments.

[0023] Example 1 Obtaining TaqMan qPCR Primers and Probes (1) Extraction of DNA from a single Ditylenchus destructor A single D. destructans was picked and placed in a 200 μL PCR tube containing 10 μL lysis buffer (8 μL ddH2O and 1 μL 10×PCR buffer), followed by freezing in liquid nitrogen for 2 min, 65°C water bath for 2 min, and repeated 3 times. Then 1 μL 10 mg / mL proteinase K was added to the PCR tube, incubated at 65°C for 1.5 h, treated at 95°C for 10 min, and centrifuged at 14000 rpm for 1 min to obtain a single D. destructans DNA template.

[0024] (2) Amplification of ITS region sequences of Ditylenchus destructor Using the reported Ditylenchus destructor PCR method, the above-mentioned single Ditylenchus destructor DNA was used as a template, and PCR amplification was performed using primers ITSP1 / ITSP2 (5'-CGTAACAAGGTAGCTGTAG-3' / 5'-TTTCACTCGCCGTTACTAAGG-3') with nucleotide sequences as shown in SEQ ID NO: 5 and 6. The obtained PCR amplification product was sent to Sangon Biotech (Shanghai) Co., Ltd. for bidirectional sequencing to obtain the sequencing results.

[0025] (3) TaqMan qPCR primer design for Ditylenchus destructor According to the sequencing results of the ITS region sequence of Ditylenchus destructor, primers and probes were designed using PrimerQuest Tool; the sequences of the primers and probes are as follows: DD-F: 5'-TCCTCAAAGGTGGCATGCTT-3' (SEQ ID NO: 1); DD-R: 5'-GAAGCATACAACTGCTCAGG-3' (SEQ ID NO: 2); DD-Probe: 5'-CCGTTTTTCGCCCACAAATTAGCC-3' (SEQ ID NO: 3).

[0026] The ITS region sequence of the destructive nematode is shown in SEQ ID NO: 4.

[0027] (4) Specificity detection of primers and probes Collect the southern root-knot nematode ( Meloidogyne incognita ), Elephant ear bean root knot nematode ( M. enterolobii )、Soybean cyst nematode ( Heterodera glycines )、Grain cyst nematode ( H. avenae )、Rice stem nematode( Aphelenchoides besseyi ), Pratylenchus coffeeensis ( Pratylenchus coffeae )、The defeated Brachycera elegans( P. neglectus ), Aphelenchoidea avenae ( Aphelenchus avenae )、Changling hair-blade nematode( Trichotylenchus changlingensis ), and their DNA was extracted as templates for qPCR detection together with the DNA template of Ditylenchus destructor to verify the specificity of primers and probes in the TaqMan qPCR detection method of Ditylenchus destructor.

[0028] TaqMan qPCR reaction system total 20 μL, including 2×AceQ Universal U + Probe Master Mix (Nanjing Novozymes Biotech Co., Ltd.) 10 μL, 10 μM upstream primer Dd-F and downstream primer Dd-R 0.4 μL each, 10 μM probe Dd-Probe 0.2 μL, DNA template 1 μL, ddH2O 8 μL.

[0029] The TaqMan qPCR amplification reaction program is preferably 37°C for 2 min, 95°C for 5 min, [95°C for 10 s, 60°C for 30 s, 40 cycles], and the fluorescence signal is collected at the end of each extension.

[0030] Figure 1 The fluorescence data collected show that only the decay stem nematode can show a typical amplification curve, while other plant nematodes have no amplification curve, with good specificity.

[0031] (5) Sensitivity testing of primers and probes The Baermann funnel method was used to collect the rotten stem nematodes and put them into a 1.5 mL centrifuge tube. After centrifugation at 5000 rpm for 10 minutes, the water in the tube was removed as much as possible using a pipette. The centrifuge tube was placed in liquid nitrogen for quick freezing for 30 seconds and then ground with a pestle. The homogenate after grinding was used to extract the nematode gDNA using the Animal Genomic DNA Rapid Extraction Kit (Shanghai Sangon Biotech Co., Ltd.). The gDNA concentration was determined using Nanodrop 2000 and diluted to 100 ng / μL, 10 ng / μL, 1 ng / μL, 10 -1 ng / μL, 10 -2 ng / μL, 10 -3 ng / μL, 10 -4 ng / μL for future use.

[0032] The extraction method of Example 1 (1) was used to extract DNA templates of single female and male Ditylenchus destructor (10 0 and diluted in 10-fold gradients to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 4 nematode DNA templates were used as spare parts; at the same time, a single larvae of the decaying stem nematode DNA template was extracted (4 0 ), and then diluted in 4-fold gradient to 4 -1 , 4 -2 , 4 -3 , 4 -4 , 4 -5 , 4 -6 The larvae DNA template is used as a backup.

[0033] TaqMan qPCR amplification was performed with different concentrations of Ditylenchus destructor DNA template according to the method in Example 2 to verify the sensitivity of primers and probes in the TaqMan qPCR detection method for Ditylenchus destructor. The amplification results are shown in Figure 2 As shown in Figure 2, the detection threshold of the primers and probe for gDNA of Ditylenchus destructor was 10 -3 ng / μL( Figure 2 A), the detection threshold of a single female DNA is 10 -2 strip( Figure 2 B), the detection threshold of a single male DNA is 10 -1 strip( Figure 2 C) and the detection threshold for single larval DNA was 4 -2 strip( Figure 2 D).

[0034] Example 2 Application of TaqMan qPCR Detection Method for Ditylenchus destructans in Soil (1) Establishment of standard curve Under a stereomicroscope, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, and 10000 stem nematodes were picked up and added to 20 g of dry soil. The soil samples were ground using a planetary ball mill. After sufficient grinding and mixing, 0.5 g of soil sample was taken for DNA extraction using the Fast DNA Spin Kit for Soil (MP Biomedicals) and set aside.

[0035] Using the extracted soil DNA samples, TaqMan qPCR reaction was carried out according to the method in Example 1, and a standard curve was established based on the linear relationship between the Ct value of the fluorescence data collected in the TaqMan qPCR amplification and the logarithm of the number of Ditylenchus destructor.

[0036] The results are as follows Figure 3 As shown in Figure 2, the linear relationship between the logarithmic value of nematodes in the soil (x) and the corresponding Ct value (y) in TaqMan qPCR is y=kx+a, where k=-3.3885 and a=34.421 (R 2 =0.9711). That is, the result is judged with 34.421 as the threshold.

[0037] Therefore, the following result criteria can be determined: When the Ct value is ≤34.421 and there is an obvious amplification curve, the result is judged as positive, indicating that the decay stem nematode is present in the sample to be tested; When 34.421<Ct value≤36, and there is an obvious amplification curve, it means that the test has failed and needs to be retested; When the Ct value is greater than 36 or has no value, and there is no obvious amplification curve, the result is judged as negative, indicating that there is no destructive stem nematode in the sample to be tested.

[0038] At the same time, when the result is judged as positive, according to the formula X=10 (34.421-Ct值) / 3.3885 Calculate the number X of stem nematodes in the sample to be tested.

[0039] (2) Quantitative detection of Ditylenchus destructans in sweet potato field soil Twelve soil samples were collected from different diseased plots, and the Baermann funnel method was used for separation, microscope counting and TaqMan qPCR for quantitative detection of Ditylenchus destructans in soil samples.

[0040] Table 1 Quantitative detection of the number of Ditylenchus destructans in soil by microscope counting and TaqMan qPCR The results are shown in Table 1: The number of nematodes in the soil calculated by TaqMan qPCR and the Ct value of the amplification curve was significantly correlated with the count observed under the microscope, R 2 = 0.7875, p <0.01.

[0041] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A specific method for detecting stem nematodes ( Ditylenchus destructor Thorne) TaqMan qPCR primers and probes, characterized in that, comprising an upstream primer, a downstream primer and a probe whose nucleotides are respectively shown as SEQ ID NOs: 1-3; The two ends of the probe respectively contain a fluorescent group and a quenching group.

2. The TaqMan qPCR primer and probe according to claim 1, characterized in that: The 5' end of the probe contains a fluorescent group, and the 3' end contains a quenching group.

3. The TaqMan qPCR primer and probe according to claim 1, characterized in that: The fluorescent group of the probe is FAM, and the quenching group is BHQ.

4. A kit comprising the specific primers and probes according to any one of claims 1 to 3.

5. The kit according to claim 4, characterized in that The kit further comprises at least one of a positive molecule, a polymerase, a substrate and a buffer.

6. A method for detecting stem nematodes ( Ditylenchus destructor Thorne's method, characterized in that The following steps are involved: (1) Extract DNA from the sample to be tested as a template and perform TaqMan qPCR reaction using the above primers and probes; (2) Determine the results of the sample to be tested based on the Ct value and amplification curve of the TaqMan qPCR reaction.

7. The method according to claim 6, characterized in that In step (2), the result judgment criteria are: When the Ct value is ≤ threshold a and there is an obvious amplification curve, the result is judged as positive, indicating that the decay stem nematode is present in the sample to be tested; When threshold a < Ct value ≤ threshold b, and there is an obvious amplification curve, it means that the test has failed and needs to be retested; When the Ct value is greater than the threshold value b or there is no value, and there is no obvious amplification curve, the result is judged as negative, indicating that there is no Ditylenchus destructor in the sample to be tested; The threshold a is the intercept of the linear equation between the Ct value and the logarithm of the number of stem nematodes, and the threshold b is 36.

8. The method according to claim 7, characterized in that When the result is positive, the number of Ditylenchus destructans in the sample can be calculated according to the linear equation.

9. The method according to claim 6, characterized in that It also includes the steps for preparing a standard curve: (1a) DNA was extracted from standard samples spiked with different amounts of Ditylenchus destructans, and TaqMan qPCR reaction was performed using the above primers and probes; (2a) Establish a linear relationship between the Ct value and the logarithm of the number of Ditylenchus destructans.

10. The method according to claim 6, characterized in that In step (1), the sample to be tested is soil; The TaqMan qPCR reaction system includes: 2×AceQ Universal U + Probe Master Mix 10 μL, 10 μM upstream primer and downstream primer 0.4 μL each, 10 μM probe 0.2 μL, DNA template 1 μL, H2O 8 μL; The conditions for the TaqMan qPCR reaction were: 37°C for 2 min, 95°C for 5 min, and then 40 cycles of 95°C for 10 s and 60°C for 30 s were performed, and the fluorescence signal was collected in each cycle.

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