TaqMan real-time fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection fluorescent probe for corn necrosis stripe virus, kit and application

Abstract

The invention discloses a TaqMan real-time fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection fluorescent probe and kit for corn necrosis stripe virus and application, and relates to the technical field of virus detection. The nucleotide sequence of the fluorescent probe provided by the invention is 5 '-FAM-CGCTGGTACATAGGATGGCAAC-TAMRA-3', and the nucleotide sequence of the fluorescent probe provided by the invention is shown in the description. The nucleotide sequence of the primer is as follows: an upstream primer: 5 '-CCTGCTCCTCTGCTATTCG-3'; a downstream primer: 5 '- The downstream primer is 5 '-CAGACTTCCCTTGGCAGTGT-3', and the downstream primer is 5 '- The kit at least comprises the fluorescent probe and the primer. The TaqMan real-time fluorescent quantitative RT-PCR detection method established by the invention is simple and convenient to operate, strong in specificity, high in sensitivity and good in repeatability, is not only suitable for qualitative identification, but also can realize accurate quantitative analysis; the sensitivity is at least 100 times higher than that of a common RT-PCR detection method. The problem that in the prior art, a TaqMan real-time fluorescent quantitative RT-PCR detection method for detecting the corn necrotic stripe virus is lacked can be solved.

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, and particularly to a TaqMan real-time fluorescence quantitative RT-PCR detection fluorescent probe, kit and application for Maize Necrotic Streak Virus. Background Art

[0002] Maize is a major food crop in China, with its planting area and total output ranking third after wheat and rice. Maize virus diseases are widely distributed, and their harm degree is second only to diseases caused by fungi and bacteria. Compared with other types of diseases, viral diseases show the characteristics of concealment and long-term nature, making prevention and control measures very complex and difficult to implement.

[0003] Maize Necrotic Streak Virus (MNeSV), a single-stranded positive-sense RNA virus, is a member of the Tombusvirus family and Zeavirus genus. The virus contains 5 open reading frames, encoding p89 protein, p30 protein, capsid protein, p21 protein and p19 protein respectively. MNeSV was first discovered in the United States. After infection, it will cause obvious symptoms such as light green or yellow spots and stripes on maize leaves. As the plant grows, these symptoms will gradually worsen, seriously affecting the photosynthesis ability and overall health of the plant. The virus not only reduces crop yields but may also cause plant death, bringing huge economic losses to farmers.

[0004] Therefore, there is an urgent need to provide a convenient, fast and highly sensitive probe and kit for detecting Maize Necrotic Streak Virus. Summary of the Invention

[0005] The object of the present invention is to provide a TaqMan real-time fluorescence quantitative RT-PCR detection fluorescent probe, kit and application for Maize Necrotic Streak Virus, and solve the technical problem that there is a lack of a fluorescence RT-PCR detection method for detecting the nucleic acid of Maize Necrotic Streak Virus in the prior art.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] The present invention provides a TaqMan real-time fluorescence quantitative RT-PCR detection fluorescent probe for Maize Necrotic Streak Virus, and the nucleotide sequence of the fluorescent probe is:

[0008] MNeSP1: 5'-FAM-CGCTGGTACACTAGGATGGCAAC-TAMRA-3'.

[0009] Preferably, the nucleotide sequence of the primer used in combination with the fluorescent probe is:

[0010] Upstream primer MNeSF1: 5’-CCTGCTCCCTCTGCTATTCG-3’;

[0011] Downstream primer MNeSR1: 5’-CAGACTCTCCCTTGGCAGTGT-3’.

[0012] The present invention provides a TaqMan real-time fluorescence quantitative RT-PCR detection kit for maize necrotic streak virus, which at least includes the above-mentioned fluorescence probe and the above-mentioned primers.

[0013] The present invention provides the application of the above-mentioned fluorescence probe and primers or the above-mentioned kit in the preparation of reagents for detecting maize necrotic streak virus.

[0014] Preferably, the TaqMan real-time fluorescence quantitative RT-PCR detection method for maize necrotic streak virus is as follows: performing real-time fluorescence PCR amplification by using the above-mentioned fluorescence probe and primers or the above-mentioned kit, including the following steps:

[0015] Step 1: Extract the RNA of the sample to be tested and reverse-transcribe it into a cDNA template;

[0016] Step 2: Prepare an amplification reaction system and perform real-time fluorescence PCR amplification through an amplification reaction program to obtain an amplification curve. The amplification reaction system at least includes the cDNA template in Step 1, the above-mentioned fluorescence probe and the above-mentioned primers;

[0017] Step 3: Analyze the amplification curve and make a judgment.

[0018] Preferably, the amplification reaction system in Step 2 is specifically: 12.5 uL of the premix for TaqMan real-time fluorescence PCR, 0.5 uL each of the upstream and downstream primers at 10 uM, 0.2 uL of the fluorescence probe at 10 uM, 3.0 uL of the cDNA template, and supplemented with ddH2O to a total volume of 25 uL.

[0019] Preferably, the amplification reaction program in Step 2 is: 95°C for 30 s, 95°C for 5 s, 60°C for 30 s, for 40 cycles.

[0020] Preferably, the rules for the judgment in Step 3 are specifically:

[0021] When the Ct value ≤ 35 and a standard S-shaped amplification curve appears, the sample result is judged to be positive for maize necrotic streak virus;

[0022] When there is no Ct value, the Ct value > 40 or there is no standard S-shaped curve amplification curve, the sample result is judged to be negative for maize necrotic streak virus;

[0023] When 35 < Ct value ≤ 40 and a standard S-shaped curve appears, the sample result is judged as suspicious and needs to be retested; if the Ct value of the sample to be tested ≤ 35 and a standard S-shaped amplification curve appears, it is positive for maize necrotic streak virus, otherwise it is negative for maize necrotic streak virus.

[0024] In summary, compared with the prior art, the solution of the present invention has the following beneficial effects:

[0025] A TaqMan real-time fluorescence quantitative RT-PCR detection fluorescence probe or kit for maize necrotic streak virus provided by the present invention is applicable to fluorescence quantitative PCR amplification, has good specificity and high sensitivity, and is suitable for wide promotion and application in the field of plant disease diagnosis. The real-time fluorescence RT-PCR detection method established based on the fluorescence probe or kit of the present invention can detect up to 10 3 fg / μL concentration level, which is at least 100 times more sensitive than the ordinary RT-PCR method; and through experiments, it is verified that this detection method can accurately identify maize necrotic streak virus MNesv from maize necrotic streak virus MNesv, maize chlorotic mottle virus MCMV, potato virus Y MDMV and wheat mosaic virus WSMV. Description of the Drawings

[0026] Figure 1 are the real-time fluorescence quantitative RT-PCR amplification curves of three groups of different primers and probes in the present invention (P1, P2 and P3 are the amplification curves of different primers and probes respectively, NC is the negative control; 1-1, 1-2 are the repetitions of the same experiment);

[0027] Figure 2 is the specific test result diagram of the real-time fluorescence quantitative RT-PCR detection method established by the present invention (Mnesv is maize necrotic streak virus, MCMV is maize chlorotic mottle virus, MDM is potato virus Y, WSMV is wheat mosaic virus, NC is the negative control);

[0028] Figure 3 is the sensitivity test result diagram of the RT-PCR detection method (M is DNA Maker2000, total RNA of maize leaves infected with Mnesv, concentrations are 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 fg / uL and 10 2 fg / uL, NC is the negative control);

[0029] Figure 4It is the sensitivity test result diagram of the real-time fluorescence quantitative RT-PCR detection method established by the present invention (2-8 are the logarithms of RNA concentration, and the corresponding concentrations are 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 fg / uL and 10 2 fg / uL; NC is the negative control);

[0030] Figure 5 It is the standard curve of the real-time fluorescence quantitative RT-PCR detection method established by the present invention;

[0031] Figure 6 It is the result of detecting maize leaf samples suspected of being infected with MNesv in different regions by using the real-time fluorescence quantitative RT-PCR detection method established by the present invention (LX is Linxia, L is Lanzhou, JC is Jinchang, WW is Wuwei, BY is Baiyin, DX is Dingxi, GN is Gannan, JQ is Jiuquan, ZY is Zhangye, TS is Tianshui, LN is Longnan, QY is Qingyang, CK is the positive control, NC is the negative control). Detailed implementation manners

[0032] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments and drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0033] Example 1

[0034] This example describes the TaqMan real-time fluorescence quantitative RT-PCR detection process of the present invention for maize necrotic streak virus

[0035] I. Test reagents:

[0036] The positive sample was collected from Yuzhong area of Gansu Province, with relatively severe disease, and the Mnesv virus was detected by high-throughput sequencing in the early stage. The cDNA of this sample was used as the template for the positive control (CK) in Examples 2-4 and the test examples.

[0037] RNA extraction reagent: Coolaber (RE611-50T); reverse transcription reagent: Takara (6210A); TaqMan fluorescence quantitative reagent: Takara (RR390A); RT-PCR reagent: Kangwei Century (CW2849M).

[0038] Design fluorescent probes and primers using Primer Express 3.0 software, and design RT-PCR primers using Primer 3input.

[0039] Second, a TaqMan real-time fluorescence quantitative RT-PCR detection method for maize necrotic streak virus, comprising the following steps:

[0040] 1. RNA extraction

[0041] Extract RNA from plant tissues suspected of being infected with MNesv virus using a universal RNA extraction kit (Universal RNA Extraction Kit, RE611, Coolaber), and the specific operation is as follows:

[0042] (1) Sample preparation: Take 100 mg of plant tissue and place it in a mortar. Add an appropriate amount of liquid nitrogen and quickly grind it into a fine powder. Then add 1 mL of lysis buffer CZ and continue to grind until a uniform slurry is formed. Transfer this homogenate to a 1.5 mL centrifuge tube and let it stand at room temperature for 5 minutes.

[0043] (2) Separation and purification:

[0044] Add 200 μL of chloroform to the above mixture, mix it well by vigorous shaking for about 15 seconds, and incubate it at room temperature for another 3 minutes.

[0045] After centrifuging at 12,000 revolutions per minute at 4 °C for 10 minutes, carefully aspirate the upper clear liquid (about 500 μL) and transfer it to a new 1.5 mL centrifuge tube.

[0046] Add 250 μL of absolute ethanol to this supernatant, gently invert it several times to mix well, and then transfer all the liquid and any possible precipitate to the RNA adsorption column. Centrifuge again at 12,000 revolutions per minute at 4 °C for 1 minute, discard the permeate, and place the adsorption column back into the original collection tube.

[0047] Add 500 μL of RNA washing buffer to the adsorption column, repeat the above centrifugation process once, and then perform the same washing step again. Finally, let the adsorption column spin dry for 2 minutes to completely remove the residual ethanol.

[0048] (3) Elution and storage:

[0049] Remove the old collection tube and replace it with a new 1.5 mL RNase-free centrifuge tube as the receiving container. Add 30 μL of sterile distilled water to the center of the adsorption membrane, let it stand at room temperature for two minutes, and then centrifuge at 4°C and 12,000 rpm for 1 minute to elute the RNA. The obtained RNA solution should be immediately stored in an -80°C refrigerator for later use.

[0050] 2. cDNA synthesis:

[0051] Reverse transcribe the RNA into cDNA using a kit (6210A, Takara). The specific operations are as follows:

[0052] (1) Mix the reaction system: 1 μL of Oligo dT primer (50 μM), 1 μL of dNTP Mix (10 mM), 2 μL of template RNA, and 6 μL of RNase Free dH2O. After mixing the above components, incubate at 65°C for 5 minutes, and then quickly cool on ice.

[0053] (2) Add the remaining components and start the reverse transcription reaction: Add the following components to the above denatured reaction solution: 4 μL of 5×PrimeScript II Buffer, 0.5 μL of RNase Inhibitor (40 U / μL), 1 μL of PrimeScript II RTase (200 U / μL), and 4.5 μL of RNase Free dH2O.

[0054] (3) Conduct the reverse transcription reaction: Incubate at 42°C for 45 minutes, and then heat at 72°C for 15 minutes to inactivate the enzyme activity. After the reaction, store the obtained cDNA in a -20°C refrigerator for later use.

[0055] 3. Real-time fluorescence quantitative PCR detection

[0056] Perform real-time fluorescence quantitative PCR detection of MNesv using a kit (RR390A, Takara). In a 25 μL reaction system, the components of the optimal system are as follows: 12.5 μL of Primix Ex Taq Cprobe qPCR (2×), 0.5 μL of upstream primer (10 μM), 0.5 μL of downstream primer (10 μM); 0.2 μL of TaqMan probe (10 μM); 2 μL of template cDNA; 9.3 μL of RNase free H2O. The specific reaction procedure is as follows: First, pre-denature at 95°C for 30 seconds, and then enter 40 cycles, each cycle including denaturation at 95°C for 5 seconds, annealing and extension at 60°C for 30 seconds.

[0057] Example 2

[0058] This example describes the primers and fluorescent probes for TaqMan real-time fluorescence quantitative RT-PCR detection of maize necrotic streak virus in the present invention.

[0059] (1) Design of primers and fluorescent probes

[0060] Using Primer Express 3.0 software, three sets of primers and probes were designed for the replicase protease and capsid protein sequences of the MNesv virus respectively (see Table 1). Among them, the 5' end of the TaqMan probe is labeled with the FAM fluorescent reporter group, and the 3' end is labeled with the TAMRA fluorescent quenching group. After the design was completed, all primers and probes were sent to Tsingke Biotechnology Co., Ltd. for synthesis.

[0061] Table 1 Primers and probes for three sets of real-time fluorescence primer quantitative RT-PCR detection

[0062]

[0063] Referring to Example 1, real-time fluorescence quantitative PCR detection was performed on 3 sets of probes and primers. Each experiment included two replicates and a negative control (NC), where the negative control used RNase free H2O to replace the template. The above three sets of primers and probes were used to perform real-time fluorescence quantitative PCR on MNesv positive samples respectively. The results showed that the Ct values of the negative controls of the three sets of primers and probes were all higher than 40, indicating no non-specific amplification (see Figure 1 ). Since the Ct value of the probes and primers in Group P1 was the lowest, and Group P1 was located on the replicase-related protein sequence of MNesv, therefore, the present invention selected the fluorescent probe and primer in Group P1 for subsequent experiments.

[0064] Example 3

[0065] This example describes the specificity of the TaqMan real-time fluorescence quantitative RT-PCR detection method for maize necrotic streak virus in the present invention.

[0066] In this example, the fluorescent probe and primer in Group P1 in Example 2 were used to perform real-time fluorescence quantitative PCR detection on different virus samples, and the specificity of the primers, probes designed by the present invention and the established TaqMan real-time fluorescence quantitative RT-PCR detection method for maize necrotic streak virus was evaluated.

[0067] The "replicase-related protein" sequences of the following viruses were synthesized respectively: MCMV (maize chlorotic mottle virus), MDMV (potato virus Y), WSMV (wheat mosaic virus). Using these synthesized sequences as templates, fluorescence quantitative PCR detection was performed with reference to the detection method in Example 1, and a negative control (NC) was set at the same time.

[0068] The results showed that, except for MNesv, the Ct values of other viruses were all greater than 35, and there was no standard "S-shaped" amplification curve, indicating that the primers and fluorescent probes designed in this declaration for the MNesv virus had high specificity. At the same time, it also verified the accuracy and reliability of the primers and fluorescent probes designed in this declaration for the MNesv virus when detecting different viruses (see Figure 2 ).

[0069] Example 4

[0070] This example illustrates the sensitivity of the TaqMan real-time fluorescence quantitative RT-PCR detection method for maize necrotic streak virus of the present invention.

[0071] By comparing the sensitivities of RT-PCR and real-time fluorescence quantitative PCR detection methods, the application potential of the two methods in actual detection was comprehensively evaluated.

[0072] (1) Sensitivity evaluation of RT-PCR

[0073] The total RNA of maize leaves infected with MNesv was diluted to different concentrations: 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 fg / μL and 10 2 fg / μL, and a negative control (NC) was set at the same time. For the primers designed in Example 2, RT-PCR amplification primers were designed (see Table 2). The RT-PCR detection was carried out using a (CW2849L, Kangwei Reagent Company) kit. The sensitivity of this method was evaluated.

[0074] Table 2 RT-PCR primers

[0075]

[0076] The results showed that obvious amplification bands could be seen when the RNA concentration was 10 8 fg / μL, 10 7 fg / μL, and 10 6 fg / μL. However, when the RNA concentration dropped to 10 5 fg / μL, only weak amplification bands could be observed. Therefore, the lowest RNA concentration of MNesv that RT-PCR could detect was 10 5 fg / μL, which also indicated that RT-PCR had a certain detection ability in high-concentration samples (see Figure 3 ).

[0077] (2) Sensitivity evaluation and standard curve construction of real-time fluorescence quantitative PCR

[0078] Dilute the total RNA of maize leaves infected with MNesv to different concentrations: 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 fg / μL and 10 2 fg / μL, and set a negative control (NC) at the same time. Refer to the detection method in Example 1 to perform real-time fluorescence quantitative PCR detection on templates with different concentrations, evaluate the sensitivity of the primers, probes designed by the present invention and the established TaqMan real-time fluorescence quantitative RT-PCR detection method for maize necrotic streak virus, and construct a standard curve for fluorescence quantitative PCR amplification.

[0079] The results show that when the RNA concentration is greater than 10 3 fg / μL, the corresponding Ct value is less than 35; while when the RNA concentration is 10 2 fg / μL or lower, the corresponding Ct value is greater than 35, and there is no standard "S-shaped" amplification curve. At this time, it is considered that the MNesv virus cannot be detected. Therefore, the lowest RNA concentration of MNesv that can be detected by fluorescence quantitative PCR is 10 3 fg / μL, indicating that the sensitivity of the TaqMan real-time fluorescence quantitative RT-PCR detection method for maize necrotic streak virus established by the present invention is 100 times that of the RT-PCR method (see Figure 4 ).

[0080] Construct a standard curve using the logarithm of the RNA concentration and its corresponding Ct value, and obtain a standard curve with R 2 = 0.9816. The standard equation of the curve is y = -2.136*X + 42.45. The above experiments verify the high sensitivity and accuracy of the TaqMan real-time fluorescence quantitative RT-PCR detection method for maize necrotic streak virus established by the present invention in low-concentration samples (see Figure 5 ).

[0081] Test example:

[0082] Collect suspected diseased MNesv samples from different regions for fluorescence quantitative PCR detection

[0083] Taking the municipal units as the division, corn leaf samples suspected of being infected with MNesv were collected from 12 cities in Gansu Province (Lanzhou, Tianshui, Wuwei, Zhangye, Pingliang, Jiuquan, Qingyang, Dingxi, Longnan, Linxia, Gannan, Jinchang). After preliminary screening and processing, these samples were detected using real-time fluorescence quantitative PCR technology. Through these experiments, the infection status of MNesv in each region was evaluated, and the distribution characteristics of the virus in different regions were analyzed.

[0084] The results showed that the content of MNesv virus was the highest in the samples from the Gannan region, followed by the Dingxi region; in addition, the presence of MNesv virus was also detected in the samples from the Longnan region (see Figure 6 ). These results indicate that there are significant differences in the distribution of MNesv in different regions, suggesting that corresponding prevention and control measures need to be taken according to the specific conditions of different regions. This finding is of great significance for understanding the transmission mechanism of MNesv and formulating effective prevention and control strategies.

[0085] The above-described embodiments only represent the preferred embodiments of the present invention, and the description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. A fluorescent probe for TaqMan real-time fluorescence quantitative RT-PCR detection of maize necrotic streak virus, characterized in that: The nucleotide sequence of the fluorescent probe is: MNeSP1: 5'-FAM-CGCTGGTACACTAGGATGGCAAC-TAMRA-3'.

2. A fluorescent probe for detecting maize necrotic streak virus TaqMan real-time fluorescence quantitative RT-PCR according to claim 1, characterized in that: The nucleotide sequence of the primer used in conjunction with the fluorescent probe is: Upstream primer MNeSF1: 5′-CCTGCTCCCTCTGCTATTCG-3′; Downstream primer MNeSR1: 5′-CAGACTCTCCCTTGGCAGTGT-3′.

3. A TaqMan real-time fluorescence quantitative RT-PCR detection kit for corn necrotic streak virus, characterized in that: At least comprises the fluorescent probe according to claim 1 and the primer according to claim 2.

4. Use of the fluorescent probe for detecting corn necrotic streak virus TaqMan real-time fluorescence quantitative RT-PCR according to claim 2 or the kit according to claim 3 in the preparation of a reagent for detecting corn necrotic streak virus.

5. The use according to claim 4, characterized in that The maize necrotic streak virus TaqMan real-time fluorescence quantitative RT-PCR detection method is: using the fluorescent probe described in claim 2 or the kit described in claim 3 to perform real-time fluorescence PCR amplification, comprising the following steps: Step 1, extracting RNA from the sample to be tested and reversely transcribing it into a cDNA template; Step 2, preparing an amplification reaction system to perform real-time fluorescent PCR amplification through an amplification reaction program to obtain an amplification curve, wherein the amplification reaction system at least includes the cDNA template in step 1, the fluorescent probe according to claim 1, and the primers according to claim 2; Step 3: Analyze the amplification curve and make a judgment.

6. The use according to claim 5, characterized in that The amplification reaction system described in step 2 is specifically: 12.5uL of TaqMan real-time fluorescent PCR premix, 0.5uL of 10uM upstream and downstream primers, 0.2uL of 10uM fluorescent probe, 3.0uL of cDNA template, and supplemented with ddH2O to a total volume of 25uL.

7. The use according to claim 6, characterized in that The amplification reaction program in step 2 is: 95°C for 30s, 95°C for 5s, 60°C for 30s, for 40 cycles.

8. The use according to claims 5 to 7, characterized in that: The specific judgment rules in step 3 are: When the Ct value is ≤35 and a standard S-shaped amplification curve appears, the sample result is judged to be positive for maize necrotic streak virus; When there is no Ct value, Ct value>40, or there is no standard S-curve amplification curve, the sample result is judged to be negative for maize necrotic streak virus; When 35<Ct value≤40, and a standard S-shaped curve appears, the sample result is judged to be suspicious and needs to be retested; if the Ct value of the sample to be tested is ≤35, and a standard S-shaped amplification curve appears, it is positive for corn necrotic streak virus, otherwise it is negative for corn necrotic streak virus.

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