A wheat stripe mosaic virus fluorescent rt-pcr detection reagent and method

Abstract

The application belongs to the technical field of molecular biology detection, and particularly relates to a wheat stripe mosaic virus fluorescent Rt-PCR detection reagent and method. The wheat stripe mosaic virus fluorescent Rt-PCR detection reagent adds choline deep eutectic solvents and imidazole ionic liquids in a conventional one-step fluorescent Rt-PCR buffer, constructs a unique "ion-hydrogen bond synergistic" buffer microenvironment in a reaction system, realizes systematic elimination of polysaccharide, polyphenol and humic acid complex inhibitors in a wheat crude extraction sample, and thus realizes rapid detection of WSMV by the fluorescent Rt-PCR under the premise of nucleic acid purification. The application is suitable for port quarantine, field rapid detection and disease-resistant breeding screening, and has outstanding matrix interference resistance and convenient operation.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection technology, specifically relating to a fluorescent Rt-PCR detection reagent and method for wheat streak mosaic virus. Background Technology

[0002] Wheat streak mosaic virus (WSMV) is a Potyviridae virus that seriously harms wheat production. Transmitted by the wheat spider mite, it can cause wheat yield losses of 20%-80%. Traditional detection methods include symptom observation, ELISA, and conventional Rt-PCR, but these methods have low sensitivity and are time-consuming. Fluorescent Rt-PCR (qRt-PCR) has become the mainstream method, but it has significant drawbacks in practical applications.

[0003] Wheat leaves and seed tissues are rich in PCR inhibitors such as polysaccharides (starch, pectin, glucan, etc.), phenols / polyphenols (chlorogenic acid, catechins, etc.), and humic acid (introduced from field soil pollution). These inhibitors work by chelating Mg... 2+ It can bind to nucleic acids / enzymes, increase the viscosity of the reaction system, or interfere with fluorescence signals, leading to low amplification efficiency and high false negative rate, especially in low viral load or complex field samples.

[0004] Current technologies often use conventional buffers with added T4 gp32, BSA, betaine, or DMSO, but these have limited effectiveness in alleviating wheat-specific inhibitors, making direct detection of crude RNA difficult, and resulting in long reaction times and poor stability for multiplex detection. The application of ionic liquids (ILs) or deep eutectic solvents (DES) in PCR is mainly for GC-rich templates or general DNA extraction; there are few systematically optimized designs targeting the complex inhibitors of polysaccharides, phenols, and humic acids in wheat tissue, nor are there any reports of their integration into WSMV one-step multichannel qRT-PCR.

[0005] Therefore, there is an urgent need to develop a robust, sensitive, and easy-to-operate detection technology optimized for wheat tissue-specific inhibitors. Summary of the Invention

[0006] To address the above problems, in a first aspect, the present invention provides a wheat streak mosaic virus fluorescent Rt-PCR detection reagent, which includes a one-step fluorescent Rt-PCR buffer, wherein the buffer contains a composite additive system, the composite additive system being composed of a first additive and a second additive. The first additive is a choline-based deep eutectic solvent; The second additive is an imidazole ionic liquid.

[0007] Furthermore, the choline-based eutectic solvent is formed by the self-assembly of choline chloride and glycerol in a molar ratio of 1:1 to 1:2 via hydrogen bonding.

[0008] Furthermore, the volume fraction of the choline-based eutectic solvent in the wheat streaky mosaic virus fluorescent Rt-PCR detection reagent is 1.5%~3.5% (v / v).

[0009] Furthermore, the imidazole ionic liquid is 1-butyl-3-methylimidazolium chloride.

[0010] Furthermore, the volume fraction of the 1-butyl-3-methylimidazolium chloride in the wheat streaky mosaic virus fluorescent Rt-PCR detection reagent is 0.5%~1.5% (v / v).

[0011] Furthermore, the wheat streaky mosaic virus fluorescent Rt-PCR detection reagent also contains auxiliary additives, which are selected from bovine serum albumin, betaine, and T4 gene 32 protein.

[0012] Further, the concentration of bovine serum albumin in the wheat streak mosaic virus fluorescent Rt-PCR detection reagent is 0.05~0.3 mg / mL, the concentration of betaine in the wheat streak mosaic virus fluorescent Rt-PCR detection reagent is 0.2~0.8 M, and the concentration of T4 gene 32 protein in the wheat streak mosaic virus fluorescent Rt-PCR detection reagent is 0.05~0.2 μg / μL.

[0013] Furthermore, the wheat streaky mosaic virus fluorescent Rt-PCR detection reagent also contains Rt-PCR buffer, MgCl2, and dNTPs.

[0014] Furthermore, the wheat streaky mosaic virus fluorescent Rt-PCR detection reagent also contains 50~100 mM Tris-HCl (pH 8.3~8.8), 3~6 mM MgCl2, and 0.2~0.4 mM dNTPs.

[0015] In a second aspect, the present invention provides a wheat streak mosaic virus fluorescent Rt-PCR detection kit, which comprises wheat streak mosaic virus fluorescent Rt-PCR detection reagents as described herein, reverse transcriptase, DNA polymerase, RNase inhibitor, and specific primer pairs and TaqMan probes for WSMV.

[0016] Furthermore, the nucleotide sequences of the specific primer pair and TaqMan probe targeting WSMV are as follows: Forward primer WSMV-F: 5'-CGTGTTGTGAATAATGCAGGCA-3' (SEQ ID NO:1); Reverse primer WSMV-R: 5'-CGAAGTGATGGCTTCGCAGC-3' (SEQ ID NO:2); TaqMan probe WSMV-P: 5'-VIC-CAATGAGCAGCAACT-MGB-3' (SEQ ID NO:3); The TaqMan probe has a fluorescent reporter group VIC labeled at its 5' end and a non-fluorescent quencher group and an MGB modification group attached thereto at its 3' end.

[0017] Furthermore, the kit also includes plant internal reference primer pairs and probes, exogenous internal control RNA and its corresponding primer pairs and probes.

[0018] Furthermore, the plant internal reference primer pair and probe target the wheat endogenous 18S rRNA gene or the TaEF1-α gene, and the fluorescent reporter group of the probe is labeled as CY5 or ROX.

[0019] Furthermore, the exogenous internal control RNA is an artificially synthesized RNA fragment, and the corresponding probe is labeled with a fluorescent reporter group of ROX or Cy5.

[0020] In a third aspect, the present invention provides a method for detecting wheat streak mosaic virus using a wheat streak mosaic virus fluorescent Rt-PCR detection kit as described herein, characterized in that the method comprises the following steps: (1) Sample lysis treatment: Take the wheat tissue to be tested, add lysis buffer containing 1.0%~2.0% (v / v) choline-based deep eutectic solvent, perform physical grinding or knocking lysis at room temperature, and obtain crude wheat tissue extract containing total RNA after standing. (2) Construction of one-step fluorescent Rt-PCR reaction system: Mix the following in the reaction tube: wheat streak mosaic virus fluorescent Rt-PCR detection reagent, reverse transcriptase, DNA polymerase, RNase inhibitor, specific primer pairs and probes for WSMV, optional plant internal control primer pairs and probes, optional exogenous internal control RNA and its corresponding primer pairs and probes, and 1~5 μL of crude extract liquid obtained in step (1), and make up to a final volume of 10~25 μL with nuclease-free water; (3) Amplification and fluorescence signal acquisition: Place the reaction tube constructed in step (2) in a real-time PCR instrument and perform the following reactions in sequence: reverse transcription at 50°C~52°C for 8~12 min; pre-denaturation at 95°C for 1~3 min; perform PCR amplification for 40~45 cycles, each cycle including denaturation at 95°C for 3~10 s and annealing and extension at 60°C~63°C for 20~30 s, and acquire fluorescence signals simultaneously during the annealing and extension phase of each cycle; (4) Result determination: Based on the Ct value of the fluorescence channel of the WSMV probe, the sample is determined to be WSMV positive or negative.

[0021] Furthermore, the amount of wheat streaky mosaic virus fluorescent Rt-PCR detection reagent added in step (2) is half of the final volume of the reaction system.

[0022] Further, the result judgment criteria for step (4) are as follows: if the fluorescence channel of the WSMV probe shows a typical logarithmic amplification curve and the Ct value is ≤35, and when present, the fluorescence channels corresponding to the plant internal reference probe and the exogenous internal control RNA probe both show normal amplification, then the sample is judged to be WSMV positive.

[0023] In a fourth aspect, the present invention provides the application of the wheat streak mosaic virus fluorescent Rt-PCR detection reagent or wheat streak mosaic virus fluorescent Rt-PCR detection kit as described herein in WSMV port entry and exit seed quarantine, early diagnosis and epidemic survey of viral diseases in the field, and molecular marker-assisted screening of disease-resistant breeding materials.

[0024] Beneficial effects of the present invention The wheat streak mosaic virus (WSMV) fluorescent Rt-PCR detection reagent of the present invention constructs a unique "ion-hydrogen bond synergistic" buffer microenvironment in the reaction system by adding a choline-based eutectic solvent and an imidazole-based ionic liquid to the conventional one-step fluorescent Rt-PCR buffer. This enables the systematic removal of polysaccharides, polyphenols, and humic acid complex inhibitors in crude wheat extract samples, thereby achieving rapid detection of WSMV with high sensitivity, high specificity, and high repeatability through multichannel fluorescent Rt-PCR without the need for nucleic acid purification. Attached Figure Description

[0025] Figure 1 The amplification curve of the enhanced buffer system of the present invention for detecting WSMV positive plasmid standards: 5x10 9 The WSMV positive plasmid (copies / μl) was diluted 100-fold and then serially diluted 10-fold. Rt-PCR amplification was performed using the enhanced buffer system of this invention to obtain the amplification curve.

[0026] Figure 2 The amplification curve of the enhanced buffer system of the present invention for detecting WSMV positive plasmid standards is shown in the figure. Figure 1 Based on this, extreme gradient points with excessively low concentrations (which may produce random errors) were removed, and the amplification curves of the five gradients with the best linear relationship were retained.

[0027] Figure 3 Based on Figure 2 A standard curve plotted from the amplification curves. The vertical axis represents the CT value; the horizontal axis represents the logarithmic copy number. Detailed Implementation

[0028] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0029] Example 1: Synthesis of choline-based DES and preparation of enhanced buffer solution 1.1 Synthesis of choline chloride-glycerol eutectic solvent (ChCl-Glycerol DES) Accurately weigh 139.6 g (1.0 mol) of high-purity anhydrous choline chloride (purity ≥98%, Sigma-Aldrich) and 184.2 g (2.0 mol) of ultrapure glycerol (purity ≥99.5%, Sinopharm Group), and add them to a 500 mL stoppered round-bottom flask at a molar ratio of 1:2. Place the flask in a thermostatically heated magnetically stirred oil bath and stir continuously at 80°C and 400 rpm for 60 min until a completely transparent, colorless, homogeneous viscous liquid is formed. Place the synthesized DES in a vacuum drying oven and vacuum dry at 60°C for 12 h to remove residual moisture. Then transfer it to a desiccator to cool to room temperature, seal and store for later use.

[0030] 1.2 Preparation of Enhanced 2× One-Step Rt-PCR Buffer Prepare enhanced 2× one-step Rt-PCR buffer (with a final volume of 10 mL) in a nuclease-free clean bench according to the following formula: Tris-HCl (pH 8.5, 25°C): 100 mM (final concentration, same below) KCl: 50 mM (NH4)2SO4: 20 mM MgCl2: 4 mM dNTPs (equal molar mixture of dATP, dCTP, dGTP, and dTTP): 0.3 mM each ChCl-Glycerol DES: 2.5% (v / v), i.e., adding 250 μL of DES. [BMIM][Cl] (purity ≥98%, Shanghai Chengjie Chemical): 1.0% (v / v), i.e., add 100 μL of [BMIM][Cl]. BSA (molecular biology grade, DNase / RNase-free): 0.15 mg / mL T4 gp32 (NEB): 0.10 μg / μL Betaine (Sigma): 0.5 M Preparation steps: First, dissolve Tris-HCl, KCl, (NH4)2SO4, MgCl2, and dNTPs in nuclease-free water, stirring until completely dissolved, and adjust the pH to 8.5 (25°C). Then, add betaine, BSA, and T4 gp32 sequentially, gently inverting to mix (avoiding bubble formation). Next, slowly add ChCl-Glycerol DES and [BMIM][Cl] dropwise, stirring gently to ensure complete and uniform dispersion of the ionic liquids and that the solution remains clear and transparent. Finally, aliquot into sterile PCR tubes, 1.0 mL per tube, and store at -20°C protected from light for later use.

[0031] Example 2: Evaluation of amplification performance under the background of wheat complex inhibitor 2.1 Preparation of simulated inhibitor background samples To eliminate the interference of individual differences in real field samples on experimental evaluation, and to accurately control the type and concentration of inhibitors, this experiment used the model of "crude extract of healthy wheat leaves + exogenous addition of a specified concentration of inhibitor + quantitative WSMV positive control RNA" to construct a highly standardized background sample of simulated inhibitors.

[0032] Preparation of crude extract from healthy wheat leaves: 5.0 g of healthy wheat leaves (variety: Zhengmai 9023) that were confirmed negative for WSMV by both RT-PCR and ELISA were cut into small pieces of approximately 0.5 cm × 0.5 cm. 10 mL of sterile PBS buffer (pH 7.4) was added, and the mixture was homogenized thoroughly using a tissue homogenizer (10,000 rpm, 5 min) under ice bath conditions. The homogenate was centrifuged at 3,000 × g for 5 min at 4°C. The supernatant was collected, sterilized by filtration through a 0.22 μm filter, aliquoted, and stored at -80°C for later use. The total RNA concentration of the crude extract was approximately 180 ng / μL, and the total protein concentration was approximately 2.5 mg / mL (Bradford method).

[0033] Preparation of the compound inhibitor: Using sterile water as solvent, prepare a 10× stock solution (100 mL) containing 35 mg / mL pectin (Sigma, from citrus peel), 15 mM chlorogenic acid (Sigma, purity ≥95%), and 800 μg / mL humic acid (Sigma, technical grade), and store at 4°C protected from light.

[0034] Preparation of simulated inhibitor samples: The crude extract of the above healthy leaves was mixed with 10× inhibitor stock solution and WSMV in vitro transcribed RNA standard (10×). 7 Mix the inhibitors (copies / μL) at a volume ratio of 8:1:1 to achieve the following final reaction solution concentrations: pectin 3.5 mg / mL, chlorogenic acid 1.5 mM, humic acid 80 μg / mL, and WSMV RNA template 10 6The concentration of the inhibitor was referenced to the typical range of polysaccharides, phenols and humic acid in actual field wheat samples (References: Rezadoost et al., 3 Biotech, 2016, 6: 61; Liyana-Pathirana & Shahidi, J.Agric. Food Chem., 2006, 54(4): 1256-1264; Matheson et al., The Open EnzymeInhibition Journal, 2010, 3: 38-45).

[0035] 2.2 Experimental Group Design The following 6 experimental groups were set up, with each group undergoing 20 independent replicates (n=20): The present invention group: uses the enhanced buffer system prepared in Example 1 containing 2.5% ChCl-Glycerol DES + 1.0% [BMIM][Cl] + 0.15 mg / mL BSA + 0.10 μg / μL T4 gp32 + 0.5 M betaine.

[0036] Hydrophobic ionic liquid group (control group 1): 2.0% (v / v) 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) was used to replace the DES-IL complex in this invention, and the remaining components were the same as those in this invention group.

[0037] Pyridium-based ionic liquid group (control group 2): 1.5% (v / v) 1-butylpyridine chloride ([BPy][Cl]) was used to replace [BMIM][Cl] in this invention, and 1.0% ChCl-Glycerol DES was added. The remaining components were the same as those in this invention group.

[0038] Quaternary phosphonium-based ionic liquid group (control group 3): 2.0% (v / v) tetrabutylphosphonium chloride ([TBP][Cl]) was used to replace [BMIM][Cl] in this invention, and the remaining components were the same as those in this invention group.

[0039] High-concentration single IL group (control group 4): only 3.0% (v / v) 1-ethyl-3-methylimidazolium chloride ([EMIM][Cl]) was added, without choline DES, and the other components were the same as those in the present invention group.

[0040] The conventional commercial system group (control group 5) used a commercial one-step Rt-PCR buffer (Takara, catalog number RR064A) without any ionic liquids / DES, with only conventional additives added (0.5 mg / mL BSA + 0.4 μg / μL T4 gp32 + 1.0 M betaine).

[0041] 2.3 One-step Rt-PCR reaction procedure One μL of the simulated inhibitor sample was used as a template and added to reaction tubes containing 10 μL of different buffer systems. SuperScript IV reverse transcriptase (Thermo Fisher, 1.5 U / reaction), HotStart Taq DNA polymerase (Takara, 1.25 U / reaction), and Murine RNase Inhibitor (NEB, 20 U / reaction), along with the required primers and probes, were added to prepare a final 20 μL reaction system (the conventional commercial system group already contained the enzymes, so no additional enzymes or RNase inhibitors were added). All groups used the same primers and probes (0.4 μM each for WSMV-F / R, 0.2 μM for WSMV-P) and the same thermal cycling program (52°C reverse transcription for 10 min; 95°C pre-denaturation for 2 min; 95°C denaturation for 5 s; 60°C annealing extension for 20 s, for a total of 40 cycles, with fluorescence signal acquisition at 60°C). Amplification was performed using an ABI 7500 Fast Real-Time PCR instrument.

[0042] The nucleotide sequences of WSMV-F / R and WSMV-P are as follows: WSMV-F: 5'-CGTGTTGTGAATAATGCAGGCA-3' (SEQ ID NO: 1); WSMV-R: 5'-CGAAGTGATGGCTTCGCAGC-3' (SEQ ID NO: 2); WSMV-P: 5'-VIC-CAATGAGCAGCAACT-MGB-3' (SEQ ID NO: 3).

[0043] 2.4 Experimental Results and Data Analysis After amplification, the baseline threshold and Ct value were automatically calculated using the instrument's software. The amplification success rate (the percentage of samples showing an effective logarithmic amplification curve with a Ct value < 38), the average Ct value, and the amplification efficiency (calculated using the slope of the standard curve, E =

[10] ) were recorded. (-1 / 斜率) The results include [-1]×100%, signal-to-noise ratio (endpoint fluorescence intensity / baseline fluorescence intensity), and melting curve peak shape. The results are summarized in Table 1.

[0044] Table 1. Comparison of amplification performance of different buffer systems under the background of composite inhibitors.

[0045] Note: "—" indicates that the amplification success rate is too low or the signal cannot be effectively detected, and the amplification efficiency is not calculated; the only successful reaction in control group 3, Ct=39.1, is close to the detection limit, and its standard deviation cannot be statistically analyzed.

[0046] 2.5 Results Analysis and Conclusions Table 1 shows that under extreme conditions containing high concentrations of the complex inhibitor, the DES-IL complex system of this invention achieved a 100% amplification success rate, successfully eliminating the interference of the polysaccharide-phenol-humic acid complex inhibitor and providing effective protection for both polymerases. This excellent technical effect stems from the positive complementary mechanism between the hydrogen bond network of choline-based DES and the electrostatic-π stacking of [BMIM][Cl] molecules, which cannot be achieved by a single component or similar replacement scheme. Specifically, as shown in Table 1, the amplification efficiency of experimental groups using other types of ionic liquids was significantly worse (control groups 1-3), indicating that the DES-IL complex system of this invention is crucial for eliminating the interference of the polysaccharide-phenol-humic acid complex inhibitor; when only ionic liquids were used without choline-based DES, the amplification success rate was only 15%, indicating that the presence of DES is indispensable; the amplification success rate of control group 5 was 45%, and the amplification efficiency was only 72.5%, indicating that although the traditional additive combination alleviated the inhibitory effect to some extent, it could not completely overcome the synergistic interference of the wheat complex inhibitor, resulting in a high risk of false negatives.

[0047] Example 3: Application of extraction-free rapid detection of blind samples in the field This embodiment uses the enhanced buffer solution of Example 1 and the amplification method of Example 2 to conduct blind sample screening of wheat samples from the field in northern my country, verifying its detection performance and practical application feasibility in real complex sample matrices.

[0048] 3.1 Field sample collection During the wheat grain-filling stage in May and June 2024, a total of 60 wheat leaves (48 leaf samples and 12 seed samples) were collected from five sampling sites in the Huang-Huai-Hai wheat region (Xinxiang, Henan; Jining, Shandong; Xingtai, Hebei) and the Northwest spring wheat region (Wuwei, Gansu; Yongning, Ningxia) of China. The samples exhibited suspected WSMV symptoms such as leaf yellowing, striped mosaic, and stunted growth. Each sample weighed approximately 0.5 g and was placed in a sterile sealed bag, kept at low temperature with ice packs, and transported back to the laboratory for testing within 12 hours. Field symptom grading, variety information, and geographical coordinates were recorded during sampling. All samples were randomly coded with a three-digit code, and the testing personnel remained blind to the sample origin and symptom information.

[0049] 3.2 Extraction-free direct detection method of the present invention Sample pretreatment: Take approximately 0.05 g of wheat leaves and place them in a 1.5 mL low-adsorption centrifuge tube. Add 300 μL of lysis buffer (containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM EDTA, 1.0% (v / v) Triton X-100, 1.5% (v / v) ChCl-Glycerol DES, and 1.0% (v / v) β-mercaptoethanol). Use a disposable polypropylene grinder to rapidly vortex the tissue in the tube for 30 s (approximately 50 times). After standing at room temperature for 3 min, centrifuge at 3,000 rpm for 1 min and collect 2.5 μL of the supernatant as the RT-PCR template.

[0050] Rt-PCR detection: 2.5 μL of Rt-PCR template was added to a 10 μL enhanced 2× one-step Rt-PCR buffer prepared in Example 1. SuperScript IV reverse transcriptase (Thermo Fisher, 1.5 U / reaction), HotStart Taq DNA polymerase (Takara, 1.25 U / reaction), and Murine RNase Inhibitor (NEB, 20 U / reaction), along with the required WSMV-specific primers and probes, were added. An internal control primer pair and probe targeting the wheat endogenous 18S rRNA gene (labeled with CY5 fluorescent reporter group), a synthetically produced RNA fragment, and its corresponding primer pair and probe (labeled with ROX fluorescent reporter group) were also added to prepare a 20 μL reaction system. Amplification was performed according to the thermal cycling procedure in Example 2.3, with three technical replicates for each sample. Simultaneously, a template-free blank control (NTC, using sterile water instead of template) and a WSMV positive standard control (10 μL / reaction) were set on each PCR plate. 6 Copies / Reaction).

[0051] 3.3 Gold Standard Control Method (Column Purification RNA Extraction + Conventional RT-qPCR) Simultaneously, parallel subsamples (approximately 0.05 g) of each sample were purified using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions for RNA extraction via column purification. The elution volume was 50 μL, and RNA concentration and purity (A260 / A280) were determined using a NanoDrop 2000. One μL of purified RNA was used as a template for amplification using a commercial one-step RT-PCR reagent (Takara RR064A) according to the manufacturer's recommended system and procedure. This method was used as the "gold standard" to verify the detection accuracy of the method of this invention.

[0052] 3.4 ChCl-Glycerol DES missing in lysis buffer To verify the pre-clearance and shielding effect of choline-based DES on wheat tissue inhibitors during the sample lysis stage, samples were treated with a lysis buffer (containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM EDTA, 1.0% (v / v) Triton X-100, and 1.0% (v / v) β-mercaptoethanol) that did not contain ChCl-Glycerol DES. The remaining procedures were exactly the same as in Section 3.2.

[0053] 3.5 Test Results Of the 60 samples tested using the method of this invention, 34 samples showed typical logarithmic amplification curves in the VIC channel and Ct values ​​≤35, and were therefore identified as WSMV positive; 26 samples showed no amplification in the VIC channel or Ct values ​​>35, and were therefore identified as WSMV negative. The Ct values ​​of the CY5 channel (plant internal reference 18S rRNA) in all positive samples were in the range of 22–28, and the Ct values ​​of the ROX channel (exogenous internal control) were in the range of 20–26, demonstrating the effectiveness of the reaction system.

[0054] The gold standard control method showed 34 positive and 26 negative results, which were completely consistent with the results of the method of this invention (100% concordance rate). Regarding Ct values, the average Ct value of the extraction-free direct amplification method of this invention (25.1±2.8) was basically the same as the average Ct value of the gold standard purified RNA amplification method (24.9±2.2), with no statistically significant difference (p > 0.05).

[0055] This indicates that the enhanced buffer system of the present invention can perfectly shield matrix interference in crude extracts, achieving the same level of detection sensitivity and accurate amplification efficiency as commercial column purification while completely eliminating the 3.5-hour RNA purification step.

[0056] In the detection of samples lacking ChCl-Glycerol DES in the lysis buffer, 31 positive and 29 negative samples were detected. It can be seen that although the subsequent PCR used the DES-IL enhanced buffer system of this invention, 3 samples still showed false negatives. This may be because these samples had low viral titers. If choline-based DES pre-binding is not performed during the lysis stage, the inhibitor load will exceed the buffer capacity, and the viral single-stranded RNA template will be more sensitive to the highly active nucleases released endogenously from plant tissues, leading to false negatives.

[0057] 3.6 Timeliness Analysis The method of this invention takes approximately 3.2 hours from receiving samples to issuing test reports for all 60 samples (including approximately 0.5 hours for sample pretreatment and approximately 2.7 hours for PCR amplification and result analysis), with an average throughput of approximately 20 samples per hour per person. The gold standard method requires approximately 9.5 hours to complete the testing of the same number of samples (approximately 3.5 hours for RNA extraction and approximately 6.0 hours for RT-PCR amplification and analysis). This invention shortens the testing cycle by approximately 66% and reduces labor costs by approximately 70%.

[0058] Example 4: Establishment of WSMV positive plasmid standard curve and sensitivity analysis 4.1 Construction and Gradient Dilution of Standards The recombinant plasmid standard containing the WSMV-specific target fragment (initial concentration 5 x 10⁻⁶) was used. 9 (Copies / μl) were diluted 100-fold with nuclease-free water, followed by a series of 10-fold dilutions to obtain 5 x 10^5 copies / μl. 7 Eight dilution gradients were applied, up to 5 copies / μl.

[0059] 4.2 qRT-PCR amplification and signal acquisition Using the enhanced buffer system of Example 1.2, 2 μl of each gradient plasmid template was added to each tube, and real-time quantitative amplification was performed according to the reaction procedure of Example 2.3. Three technical replicates were set for each gradient, and amplification curves were obtained (see Appendix). Figure 1 ).

[0060] 4.3 Data Filtering and Standard Curve Plotting Extreme gradient points with extremely low concentrations (significantly affected by Poisson distribution random errors) were removed, and the five core gradients with the best linear relationship (5x10⁻¹⁰) were retained. 7 Up to 5x10 3 Amplification curves of (copies / μl) (see attached) Figure 2 A standard curve was plotted with the average Ct value obtained from each gradient detection on the ordinate and the logarithm of the copy number on the abscissa (see appendix). Figure 3 ).

[0061] The standard curve obtained through automatic fitting by the system is a linear regression equation of y = -3.09x + 39.411, with a linear correlation coefficient R0. 2 =0.999, proving that the buffer system of the present invention also has extremely high quantitative linearity and amplification efficiency for DNA (plasmid) templates.

[0062] It should be noted that while the preferred embodiments of the present invention are given in the specification and accompanying drawings, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are not intended to impose additional limitations on the content of the present invention; their purpose is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. Furthermore, the above-described technical features can be combined with each other to form various embodiments not listed above, all of which are considered to be within the scope of the present invention specification. Moreover, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A fluorescent Rt-PCR detection reagent for wheat streak mosaic virus, characterized in that, The reagent includes a one-step fluorescent Rt-PCR buffer, the buffer containing a composite additive system, the composite additive system being composed of a first additive and a second additive; The first additive is a choline-based deep eutectic solvent; The second additive is an imidazole ionic liquid.

2. The reagent according to claim 1, characterized in that, The choline-based deep eutectic solvent is formed by the self-assembly of choline chloride and glycerol in a molar ratio of 1:1 to 1:2 via hydrogen bonding. Preferably, the volume fraction of the choline-based eutectic solvent in the reagent is 1.5% to 3.5% (v / v).

3. The reagent according to claim 1, characterized in that, The imidazole ionic liquid is 1-butyl-3-methylimidazolium chloride; Preferably, the volume fraction of the 1-butyl-3-methylimidazolium chloride in the reagent is 0.5% to 1.5% (v / v).

4. The reagent according to claim 1, characterized in that, The reagent also contains auxiliary additives selected from bovine serum albumin, betaine, and T4 gene 32 protein. Preferably, the concentration of bovine serum albumin in the reagent is 0.05~0.3 mg / mL, the concentration of betaine in the reagent is 0.2~0.8 M, and the concentration of T4 gene 32 protein in the reagent is 0.05~0.2 μg / μL.

5. The reagent according to claim 1, characterized in that, The reagent also contains Rt-PCR buffer, MgCl2, and dNTPs; Preferably, the reagent further comprises 50-100 mM Tris-HCl (pH 8.3-8.8), 3-6 mM MgCl2, and 0.2-0.4 mM dNTPs.

6. A fluorescent Rt-PCR detection kit for wheat streak mosaic virus, characterized in that, The kit comprises the reagents as described in claim 5, reverse transcriptase, DNA polymerase, RNase inhibitor, and specific primer pairs and TaqMan probes for WSMV. Preferably, the 5' end of the TaqMan probe is labeled with a fluorescent reporter group VIC, and the 3' end is labeled with a non-fluorescent quencher group and an MGB modification group connected thereto.

7. The reagent kit according to claim 6, characterized in that, The kit also includes plant internal reference primer pairs and probes, exogenous internal control RNA and its corresponding primer pairs and probes; Preferably, the plant internal reference primer pair and probe target the wheat endogenous 18S rRNA gene or the TaEF1-α gene, and the fluorescent reporter group of the probe is labeled with CY5 or ROX; The exogenous internal control RNA is an artificially synthesized RNA fragment, and the corresponding probe is labeled with a fluorescent reporter group of ROX or Cy5.

8. A method for detecting wheat streaky mosaic virus using a kit according to any one of claims 6-7, characterized in that, The method includes the following steps: (1) Sample lysis treatment: Take the wheat tissue to be tested, add lysis buffer containing 1.0%~2.0% (v / v) choline-based deep eutectic solvent, perform physical grinding or knocking lysis at room temperature, and obtain crude wheat tissue extract containing total RNA after standing. (2) Construction of one-step fluorescent Rt-PCR reaction system: Mix the reagents, reverse transcriptase, DNA polymerase, RNase inhibitor, specific primer pairs and probes for WSMV, optional plant internal reference primer pairs and probes, optional exogenous internal control RNA and its corresponding primer pairs and probes, and 1~5 μL of crude extract liquid obtained in step (1) in the reaction tube, and make up to a final volume of 10~25 μL with nuclease-free water; (3) Amplification and fluorescence signal acquisition: Place the reaction tube constructed in step (2) in a real-time PCR instrument and perform the following reactions in sequence: reverse transcription at 50°C~52°C for 8~12 min; pre-denaturation at 95°C for 1~3 min; perform PCR amplification for 40~45 cycles, each cycle including denaturation at 95°C for 3~10 s and annealing and extension at 60°C~63°C for 20~30 s, and acquire fluorescence signals simultaneously during the annealing and extension phase of each cycle; (4) Result determination: Based on the Ct value of the fluorescence channel of the WSMV probe, the sample is determined to be WSMV positive or negative.

9. The method according to claim 8, characterized in that, The criteria for judging the results of step (4) are as follows: if the fluorescence channel of the WSMV probe shows a typical logarithmic amplification curve and the Ct value is ≤35, and when present, the fluorescence channels corresponding to the plant internal reference probe and the exogenous internal control RNA probe both show normal amplification, then the sample is judged to be WSMV positive.

10. The application of the wheat streak mosaic virus fluorescent Rt-PCR detection reagent according to any one of claims 1-5 or the wheat streak mosaic virus fluorescent Rt-PCR detection kit according to claim 6 or 7 in WSMV port entry and exit seed quarantine, early diagnosis and epidemic survey of viral diseases in the field, and molecular marker-assisted screening of disease-resistant breeding materials.

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