Fluorescent probe, kit and application for detecting maize necrotic streak virus by TaqMan real-time fluorescent quantitative RT-PCR

Abstract

The application discloses a corn necrotic stripe virus TaqMan real-time fluorescent quantitative RT-PCR detection fluorescent probe, a kit and application, and relates to the technical field of virus detection.The nucleotide sequence of the fluorescent probe is 5'-FAM-CGCTGGTACACTAGGATGGCAAC-TAMRA-3'; the nucleotide sequence of the primer is: an upstream primer: 5'-CCTGCTCCCTCTGCTATTCG-3'; and a downstream primer: 5'-CAGACTCTCCCTTGGCAGTGT-3'. The kit at least comprises the above-mentioned fluorescent probe and the above-mentioned primer.The TaqMan real-time fluorescent quantitative RT-PCR detection method established by the application is simple in operation, high in specificity, high in sensitivity and good in repeatability, can be used for qualitative identification and can also realize accurate quantitative analysis, and is at least 100 times higher in sensitivity than a common RT-PCR detection method.The application can solve the problem that there is no TaqMan real-time fluorescent quantitative RT-PCR detection method for detecting corn necrotic stripe virus in the prior art.

Description

Technical Field

[0001] This invention relates to the field of virus detection technology, specifically to TaqMan real-time quantitative RT-PCR detection fluorescent probes, kits, and applications for maize necrosis stripe virus. Background Technology

[0002] Corn is a major food crop in my country. Viral diseases affecting corn are widespread, and their severity is second only to diseases caused by fungi and bacteria. Compared to other types of diseases, viral diseases are characterized by their insidious and long-term nature, making control measures very complex and difficult to implement.

[0003] Maize Necrotic Streak Virus (MNeSV) is a single-stranded, positive-sense RNA virus, a member of the family Tombusvirus and the genus Zeavirus. This virus contains five open reading frames encoding the p89, p30, capsid, p21, and p19 proteins. Infection causes noticeable symptoms on maize leaves, including pale green or yellow spots and streaks. These symptoms worsen as the plant grows, severely impacting photosynthetic capacity and overall health. This virus not only reduces crop yield but can also lead to plant death, causing significant economic losses for farmers.

[0004] Therefore, there is an urgent need to provide a convenient, rapid, and highly sensitive probe and kit for detecting maize necrosis stripe virus. Summary of the Invention

[0005] The purpose of this invention is to propose a TaqMan real-time quantitative RT-PCR detection fluorescent probe, kit, and application for maize necrosis stripe virus, thereby solving the technical problem of the lack of a fluorescent RT-PCR detection method for detecting maize necrosis stripe virus nucleic acid in the prior art.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] This invention provides a fluorescent probe for TaqMan real-time quantitative RT-PCR detection of maize necrosis stripe virus, the nucleotide sequence of which is:

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

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

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

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

[0012] This invention provides a TaqMan real-time quantitative RT-PCR detection kit for maize necrosis stripe virus, comprising at least the fluorescent probe and primers described above.

[0013] This invention provides the application of the above-described fluorescent probes and primers or the above-described kits in the preparation of reagents for detecting maize necrosis stripe virus.

[0014] Preferably, the TaqMan real-time quantitative RT-PCR detection method for maize necrosis stripe virus is as follows: real-time fluorescent PCR amplification is performed using the above-mentioned fluorescent probes and primers or the above-mentioned kit, including the following steps:

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

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

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

[0018] Preferably, the amplification reaction system in step 2 is as follows: 12.5 μL of TaqMan real-time fluorescent PCR premix, 0.5 μL each of 10 μM upstream and downstream primers, 0.2 μL of 10 μM fluorescent probe, 3.0 μL of cDNA template, and ddH2O to make up to a total volume of 25 μL.

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

[0020] Preferably, the rule for judgment in step 3 is as follows:

[0021] When the Ct value is ≤35 and a standard S-type amplification curve appears, the sample result is judged to be positive for corn necrosis stripe virus.

[0022] When there is no Ct value, a Ct value > 40, or no standard S-shaped amplification curve, the sample result is judged to be negative for corn necrosis stripe virus.

[0023] When 35 < Ct value ≤ 40 and a standard S-shaped curve appears, the sample result is considered suspicious and requires retesting; 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 necrosis stripe virus; otherwise, it is negative for corn necrosis stripe virus.

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

[0025] This invention provides a fluorescent probe or kit for TaqMan real-time quantitative RT-PCR detection of maize necrosis stripe virus. It is suitable for quantitative PCR amplification, exhibits good specificity and high sensitivity, and is suitable for widespread application in the field of plant disease diagnosis. The real-time fluorescent RT-PCR detection method based on the fluorescent probe or kit of this invention can detect up to 10... 3 The fg / μL concentration level is at least 100 times more sensitive than the ordinary RT-PCR method; and experimental verification shows that this detection method can accurately identify MNesv from maize necrosis stripe virus, maize chlorotic mottle virus, potato virus Y, and wheat mosaic virus WSMV. Attached Figure Description

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

[0027] Figure 2 This is a specific experimental result diagram of the real-time fluorescence quantitative RT-PCR detection method established in this invention (Mnesv is maize necrosis stripe virus, MCMV is maize chlorotic mottle virus, MDM is potato virus Y, WSMV is wheat mosaic virus, and NC is negative control).

[0028] Figure 3 This is a graph showing the sensitivity test results of the RT-PCR detection method (M represents DNA Marker 2000, total RNA from maize leaves infected with Mnesv, concentration 10). 8 10 7 10 6 10 5 10 4 10 3 fg / uL and 10 2 fg / uL, NC is a negative control);

[0029] Figure 4This is a sensitivity test result graph of the real-time quantitative RT-PCR detection method established in this invention (2~8 are the logarithms of RNA concentration, corresponding to concentrations of 10). 8 10 7 10 6 10 5 10 4 10 3 fg / uL and 10 2 fg / uL; NC was a negative control).

[0030] Figure 5 This is the standard curve of the real-time fluorescence quantitative RT-PCR detection method established in this invention;

[0031] Figure 6 The results are obtained by detecting maize leaf samples suspected of being infected with MNESV in different regions using the real-time fluorescence quantitative RT-PCR detection method established in this invention (LX is Linxia, ​​LZ 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 positive control, and NC is negative control). Detailed Implementation

[0032] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be described in further detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0033] Example 1

[0034] This embodiment illustrates the process of using the present invention for TaqMan real-time quantitative RT-PCR detection of maize necrosis stripe virus.

[0035] I. Test reagents:

[0036] The positive sample was collected from Yuzhong County, Gansu Province, where the disease was relatively severe, and Mnesv virus was detected in the early stages through high-throughput sequencing. Examples 2-4 and the positive control (CK) in the experimental cases all used cDNA from this sample as a template.

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

[0038] Fluorescent probes and primers were designed using Primer Express 3.0 software, and RT-PCR primers were designed using Primer 3 input.

[0039] II. A TaqMan real-time quantitative RT-PCR method for detecting maize necrosis stripe virus, comprising the following steps:

[0040] 1. RNA extraction

[0041] RNA was extracted from plant tissues suspected of being infected with MNESV virus using a universal RNA extraction kit (RE611, Coolaber). The specific procedures are as follows:

[0042] (1) Sample preparation: 100 mg of plant tissue was placed in a mortar and ground quickly into a fine powder after adding an appropriate amount of liquid nitrogen. Then, 1 mL of lysis buffer CZ was added and the mixture was ground until a homogeneous slurry was formed. The homogeneous slurry was transferred to a 1.5 mL centrifuge tube and allowed to stand at room temperature for 5 minutes.

[0043] (2) Separation and purification:

[0044] Add 200 μL of chloroform to the mixture above, shake vigorously for about 15 seconds to mix thoroughly, and incubate at room temperature for another 3 minutes.

[0045] After centrifuging at 12,000 rpm for 10 minutes at 4°C, carefully aspirate the supernatant (approximately 500 μL) and transfer it to a new 1.5 mL centrifuge tube.

[0046] Add 250 μL of anhydrous ethanol to the supernatant, gently invert several times to mix, and then transfer all liquid and any precipitate that may have formed into the RNA adsorption column. Centrifuge again at 12,000 rpm for 1 minute at 4°C, discard the permeate, and return the adsorption column to the original collection tube.

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

[0048] (3) Washing and storage:

[0049] Remove the old collection tube and replace it with a new 1.5 mL centrifuge tube free of RNase enzyme contamination 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 again at 4°C and 12,000 rpm for 1 minute to elute the RNA. The resulting RNA solution should be stored immediately at -80°C for later use.

[0050] 2. cDNA synthesis:

[0051] RNA was reverse transcribed into cDNA using a kit (6210A, Takara). The specific steps are as follows:

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

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

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

[0055] 3. Real-time quantitative PCR detection

[0056] MNESV was detected by real-time quantitative PCR using a kit (RR390A, Takara). The optimal reaction mixture in a 25 μL reaction system consisted of: 12.5 μL Primix Ex Taq Cprobe qPCR (2×), 0.5 μL upstream primer (10 μM), 0.5 μL downstream primer (10 μM), 0.2 μL TaqMan probe (10 μM), 2 μL template cDNA, and 9.3 μL RNase-free H2O. The specific reaction procedure was as follows: pre-denaturation at 95°C for 30 seconds, followed by 40 cycles, each cycle consisting of 5 seconds of denaturation at 95°C, followed by 30 seconds of annealing and extension at 60°C.

[0057] Example 2

[0058] This embodiment describes the primers and fluorescent probes used in the TaqMan real-time quantitative RT-PCR detection of maize necrosis stripe virus in this invention.

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

[0060] Three sets of primers and probes were designed using Primer Express 3.0 software targeting the replication protease and capsid protein sequences of the MNESV virus (see Table 1). The TaqMan probes were labeled with a FAM fluorescent reporter group at the 5' end and a TAMRA fluorescent quencher group at the 3' end. After design, all primers and probes were sent to Qingke Biotechnology Co., Ltd. for synthesis.

[0061] Table 1 Primers and probes used for three sets of real-time fluorescent primers for quantitative RT-PCR detection

[0062]

[0063] Referring to Example 1, real-time quantitative PCR was performed on three sets of probes and primers. Each experiment included two replicates and one negative control (NC), where the negative control used RNase-free H2O as a template. Real-time quantitative PCR was performed on MNESV-positive samples using the three sets of primers and probes described above. The results showed that the Ct values ​​of the negative controls for all three sets of primers and probes were higher than 40, indicating no nonspecific amplification (see Example 1). Figure 1 Since the probes and primers of group P1 have the lowest Ct values ​​and are located on the replicase-related protein sequence of MNesv, the fluorescent probes and primers of group P1 were selected for subsequent experiments in this invention.

[0064] Example 3

[0065] This embodiment illustrates the specificity of the TaqMan real-time quantitative RT-PCR detection method for maize necrosis stripe virus of the present invention.

[0066] In this embodiment, the fluorescent probes and primers of group P1 from Example 2 were used to perform real-time quantitative PCR detection on different virus samples to evaluate the specificity of the primers, probes, and the established TaqMan real-time quantitative RT-PCR detection method for maize necrosis stripe virus designed in this invention.

[0067] The replicase-associated protein sequences of the following viruses were synthesized: MCMV (maize chlorotic mottle virus), MDMV (potato virus Y), and WSMV (wheat mosaic virus). Using these synthesized sequences as templates, quantitative real-time PCR detection was performed according to the detection method in Example 1, with a negative control (NC) also included.

[0068] The results showed that, except for MNESV, the Ct values ​​of other viruses were all greater than 35, and no standard "S-shaped" amplification curve was observed, indicating that the primers and fluorescent probes designed in this declaration for MNESV have high specificity. This also verified the accuracy and reliability of the primers and fluorescent probes designed in this declaration for MNESV in detecting different viruses (see...). Figure 2 ).

[0069] Example 4

[0070] This embodiment illustrates the sensitivity of the TaqMan real-time quantitative RT-PCR detection method for maize necrosis stripe virus of the present invention.

[0071] By comparing the sensitivity of RT-PCR and real-time quantitative PCR, the application potential of the two methods in practical detection is comprehensively evaluated.

[0072] (1) RT-PCR sensitivity assessment

[0073] Total RNA from MNESV-infected maize leaves was diluted to different concentrations: 10 8 10 7 10 6 10 5 10 4 10 3 fg / μL and 10 2 fg / μL was used, with a negative control (NC) also included. RT-PCR amplification primers were designed based on the primers designed in Example 2 (see Table 2). RT-PCR detection was performed using a kit (CW2849L, Kangwei Reagent Co., Ltd.). The sensitivity of this method was evaluated.

[0074]

[0075] The results showed that at an RNA concentration of 10 8 fg / μL, 10 7 fg / μL, and 10 6 At fg / μL, clear amplification bands were observed, while when the RNA concentration was reduced to 10... 5 At a concentration of fg / μL, only a weak amplification band could be observed. Therefore, the minimum RNA concentration at which RT-PCR can detect MNesv is 10. 5 fg / μL, which also demonstrates that RT-PCR has a certain detection capability in high-concentration samples (see fg / μL). Figure 3 ).

[0076] (2) Sensitivity assessment and standard curve construction of real-time quantitative PCR

[0077] Total RNA from MNESV-infected maize leaves was diluted to different concentrations: 10 8 10 7 10 6 10 5 10 4 10 3 fg / μL and 10 2 fg / μL, with a negative control (NC) also set up. Real-time quantitative PCR was performed on templates of different concentrations according to the detection method in Example 1 to evaluate the sensitivity of the primers, probes, and the established TaqMan real-time quantitative RT-PCR detection method against maize necrosis stripe virus designed in this invention, and a standard curve for quantitative PCR amplification was constructed.

[0078] The results showed that when the RNA concentration was greater than 10... 3 At a concentration of fg / μL, the corresponding Ct value is less than 35; while when the RNA concentration is 10... 2 When fg / μL or lower, the corresponding Ct value is greater than 35, and there is no standard "S-shaped" amplification curve; at this point, MNesv virus is considered undetectable. Therefore, the lowest RNA concentration at which quantitative real-time PCR can detect MNesv is 10. 3 fg / μL indicates that the sensitivity of the TaqMan real-time quantitative RT-PCR detection method against maize necrosis stripe virus established in this invention is 100 times that of the RT-PCR method (see fg / μL). Figure 4 ).

[0079] A standard curve was constructed using the logarithm of RNA concentration and its corresponding Ct value, yielding an R² = 0.9816 standard curve with the standard equation y = -2.136*X + 42.45. The above experiments validated the high sensitivity and accuracy of the TaqMan real-time quantitative RT-PCR detection method for maize necrosis stripe virus established in this invention in low-concentration samples (see...). Figure 5 ).

[0080] Experimental example:

[0081] Samples of suspected MNESV from different regions were collected for quantitative real-time PCR detection.

[0082] Maize leaf samples suspected of being infected with MNesv were collected from Lanzhou, Tianshui, Wuwei, Zhangye, Pingliang, Jiuquan, Qingyang, Dingxi, Longnan, Linxia, ​​Gannan, and Jinchang in Gansu Province. After preliminary screening and processing, these samples were tested using real-time quantitative PCR. These experiments aimed to assess the MNesv infection status in each region and analyze the distribution characteristics of the virus in different areas.

[0083] The results showed that the highest levels of MNesv virus were found in samples from Gannan, followed by Dingxi; in addition, MNesv virus was also detected in samples from Longnan (see...). Figure 6 These results indicate that the distribution of MNesv varies significantly across different regions, suggesting the need for tailored prevention and control measures based on the specific circumstances of each region. This finding is of great significance for understanding the transmission mechanism of MNesv and developing effective prevention and control strategies.

[0084] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. The application of fluorescent probes and primers for TaqMan real-time quantitative RT-PCR detection of maize necrosis stripe virus in the preparation of reagents for detecting maize necrosis stripe virus, characterized in that, The nucleotide sequence of the fluorescent probe is as follows: MNeSP1: 5'-FAM-CGCTGGTACACTAGGATGGCAAC-TAMRA-3'; The nucleotide sequence of the primer is as follows: Upstream primer MNeSF1: 5'-CCTGCTCCCTCTGCTATTCG-3'; Downstream primer MNeSR1: 5'-CAGACTCTCCCTTGGCAGTGT-3'.

2. Use according to claim 1, wherein The method for detecting maize necrosis stripe virus using TaqMan real-time quantitative RT-PCR is as follows: Real-time fluorescence PCR amplification is performed using the fluorescent probe and primers described in claim 1, comprising the following steps: Step 1: Extract RNA from the sample to be tested and reverse transcribe it into a cDNA template; Step 2: Prepare the amplification reaction system and perform real-time fluorescence PCR amplification using the amplification reaction program to obtain the amplification curve. The amplification reaction system includes at least the cDNA template from Step 1, the fluorescent probe and primers as described in claim 1. Step 3: Analyze the amplification curve and make a judgment.

3. Use according to claim 2, wherein the compound is ###0002### The amplification reaction system described in step 2 is as follows: 12.5 μL of TaqMan real-time fluorescent PCR premix, 0.5 μL each of 10 μM upstream and downstream primers, 0.2 μL of 10 μM fluorescent probe, 3.0 μL of cDNA template, and ddH2O to make up to a total volume of 25 μL.

4. The use according to claim 2, wherein The amplification reaction program described in step 2 is as follows: 95℃ for 30s, 95℃ for 5s, 60℃ for 30s, for 40 cycles.

5. The application as described in any one of claims 2-4, characterized in that, The specific rules for judgment in step 3 are as follows: When the Ct value is ≤35 and a standard S-type amplification curve appears, the sample result is judged to be positive for corn necrosis stripe virus. When there is no Ct value, a Ct value > 40, or no standard S-shaped amplification curve, the sample result is judged to be negative for corn necrosis stripe virus. When 35 < Ct value ≤ 40 and a standard S-shaped curve appears, the sample result is considered suspicious and requires retesting; 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 necrosis stripe virus; otherwise, it is negative for corn necrosis stripe virus.

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