A kind of RT-qPCR primer for detecting mulberry leaf roll associated virus and application thereof
By designing specific RT-qPCR primers and kits, and combining them with real-time quantitative PCR technology, the problems of low sensitivity and insufficient standards in traditional detection methods have been solved, achieving high sensitivity and specificity in the detection of mulberry leaf curl-related viruses and improving the accuracy of detection results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGSU UNIV OF SCI & TECH
- Filing Date
- 2026-03-05
- Publication Date
- 2026-06-02
AI Technical Summary
In existing technologies, traditional detection methods such as agarose gel electrophoresis have low sensitivity for detecting mulberry leaf curl-associated virus (MMLRaV), making it difficult to meet the needs for rapid and accurate detection. Furthermore, the lack of a stable RT-qPCR detection standard system leads to insufficient accuracy in the detection results.
Design and apply specific RT-qPCR primers and kits, including reagents for RNA extraction, RNA reverse transcription reagents, and qPCR reagents. Use reverse transcriptase, DNA polymerase, etc., in conjunction with NovoSTAR® SYBR Qpcr SuperMix for real-time quantitative PCR to establish an efficient and reliable detection system.
It achieves high sensitivity and high specificity in the detection of mulberry leaf curl-related viruses, accurately screening for symptomatic and asymptomatic cases, and improving the reliability of test results.
Smart Images
Figure CN122128471A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to an RT-qPCR primer for detecting mulberry leaf curl-related viruses and its application, belonging to the field of molecular biology and plant virus detection technology. Background Technology
[0002] Mulberry mosaic leaf roll associated virus (MMLRaV) occupies a unique position in the field of plant virology. It can cause typical symptoms of mulberry leaves such as mosaic, leaf rolling, wrinkling, and yellowing. On the other hand, there is a complex interaction between plants (including mulberry trees) and pathogens (MMLRaV). The viral load in mulberry trees is an important indicator and sign of pathogenicity, mulberry tree resistance, and mulberry tree physiological status. It is an important parameter for conducting research on MMLRaV.
[0003] Traditional methods for detecting plant viruses, such as agarose gel electrophoresis, suffer from low sensitivity and high false negative rates, failing to meet the demands for rapid and accurate detection. Currently, real-time quantitative PCR (RT-qPCR) for plant virus detection has become the preferred method due to its high sensitivity, specificity, and quantification capabilities. Primers containing viral nucleic acids are crucial for the effective application of this technology. However, RT-qPCR detection of MMLRaV still faces a technical bottleneck: the lack of a stable standard system, leading to insufficient accuracy in the results.
[0004] Therefore, developing an RT-qPCR detection method based on the MMLRaV coat protein CP gene recombinant plasmid standard and establishing an efficient and reliable detection system is of great significance for achieving accurate quantitative detection of MMLRaV and disease control. Summary of the Invention
[0005] Purpose of the invention: The purpose of this invention is to provide a highly sensitive detection primer and its application.
[0006] Technical solution: The nucleotide sequences of the RT-qPCR primers for detecting mulberry leaf curl-related viruses described in this invention are shown in SEQ ID No: 3~4.
[0007] The application of the RT-qPCR primers for detecting mulberry leaf curl-related viruses described in this invention in the preparation of a reagent kit.
[0008] The kit for mulberry leaf curl-related virus of the present invention includes primers shown in SEQ ID No: 3-4.
[0009] Furthermore, the kit also includes reagents for RNA extraction, RNA reverse transcription reagents, and qPCR reagents.
[0010] Furthermore, the kit also includes reverse transcriptase, DNA polymerase, negative control, positive control, reverse transcription buffer, PCR buffer, dNTP mixture, enzyme-free purified water, RNA extraction reagent, RNase inhibitor, and fluorescent dye.
[0011] Furthermore, the positive control is a recombinant plasmid containing the CP gene of the coat protein MMLRaV.
[0012] Furthermore, the nucleotide sequence of the CP gene, the outer coat protein of MMLRaV, is shown in SEQ ID No: 5.
[0013] The application of the kit for detecting mulberry leaf curl-related virus described in this invention in the detection of mulberry leaf curl-related virus.
[0014] Furthermore, the real-time quantitative PCR reaction system consisted of: 10 μL NovoSTAR® SYBR Qpcr SuperMixPlus, 7.6 µL ddH2O, 2 μL cDNA, and 0.2 μL each of forward and reverse primers.
[0015] Furthermore, the real-time quantitative PCR reaction conditions were as follows: pre-denaturation temperature 95℃, time 3 minutes; denaturation temperature 95℃, time 20 seconds; annealing temperature 60℃, time 30 seconds; extension temperature 72℃, time 30 seconds; 40 cycles.
[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The primer sequences of this application have high sensitivity and specificity when used for mulberry leaf curl-related virus, and the detection results are reliable, making the method of the present invention an effective means of screening for symptomatic and latent mulberry leaf curl-related virus. Attached Figure Description
[0017] Figure 1 Agarose gel electrophoresis image of cDNA amplified from the MMLRaV capsid protein CP gene using the primers of this patent; lane M: DNA Maker, lane NC: negative control, lane 1: MMLRaV-3F / MMLRaV-3R amplification product.
[0018] Figure 2 This is an agarose gel electrophoresis image of the MMLRaV capsid protein CP gene plasmid diluted 10 times in this patent; Lane M: DNA Marker, Lane NC: Negative control, Lane 1: MMLRaV capsid protein CP gene plasmid diluted 10 times. -1 Lane 2: MMLRaV coat protein CP gene plasmid diluted 10 times. -2Lane 3: MMLRaV coat protein CP gene plasmid diluted 10 times. -3 Lane 4: MMLRaV capsid protein CP gene plasmid diluted 10 times. -4 Lane 5: MMLRaV capsid protein CP gene plasmid diluted 10 times. -5 times.
[0019] Figure 3 A bar chart showing the Ct values of plasmids used in real-time quantitative PCR for gradient dilution of the MMLRaV coat protein CP gene.
[0020] Figure 4 The regression curve is shown as the copy number log of the plasmid template of the MMLRaV coat protein CP gene versus the CT value of RT-qPCR amplification.
[0021] Figure 5 The image shows the agarose gel electrophoresis results of the actual samples; lane M: DNA Marker, lane NC: negative control, lane PC: positive control, lane 1: S23-1, lane 2: S13-2, lane 3: N31-10, lane 4: S13-1, lane 5: S1-1, lane 6: N31-7, lane 7: S2-5.
[0022] Figure 6 This is a graph showing the results of real-time quantitative PCR for actual samples. Detailed Implementation
[0023] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, all technical means of the present invention are conventional means in the art, and the methods used can be referred to classic experimental manuals such as "Molecular Cloning: A Laboratory Manual (4th Edition)" and "A Concise Laboratory Manual for Molecular Biology"; all reagents and consumables used can be obtained commercially.
[0024] Example 1 Primer Design Based on the MMLRaV sequence located at SEQ ID No: 5 (458nt) of the CP gene coat protein in the NCBI database, primers for agarose gel electrophoresis and real-time quantitative PCR were designed using Primer Premier 6 software according to primer design principles. The sequences are as follows: MMLRaV-3F (SEQ ID No: 1): 5'-TTTTTGGCTCTTTTTGTTCCC-3' MMLRaV-3R (SEQ ID No: 2): 5'-GGAATCTAGACTCCAAGTG-3' MMLRaV-qF1 (SEQ ID No: 3):5'-GGGTGAGAGTTTGAGTCGTCGAGTG-3' MMLRaV-qR2 (SEQ ID No: 4):5'-GAAAGTGGCGTCCTTGGTGGAGAAA-3'.
[0025] Example 2 Primer Specificity Determination The primers of this patent were used to perform PCR on the cDNA of the MMLRaV capsid protein CP gene. The amplification products were detected by agarose gel electrophoresis to verify the amplification specificity of the primers of this patent for the MMLRaV capsid protein CP gene cDNA.
[0026] The PCR reaction system for detection by agarose gel electrophoresis was: 10 × LA PCR Buffer Mg 2+ 2.5 μL of dNTP Mixture, 1 μL of cDNA, 0.5 μL of LA Taq Polymerase, 0.25 μL of forward primer (MMLRaV-qF1), 0.25 μL of reverse primer (MMLRaV-qR2), and 0.25 μL of ddH2O were used. The reaction conditions were: pre-denaturation temperature 95℃ for 3 minutes; denaturation temperature 94℃ for 20 seconds; annealing temperature 53℃ for 30 seconds; extension temperature 72℃ for 30 seconds; 35 cycles; and extension at 72℃ for 7 minutes.
[0027] The results are as follows Figure 1 As shown, this primer pair can effectively amplify the cDNA of the MMLRaV capsid protein CP gene.
[0028] Example 3 Primer sensitivity determination The standard plasmids of the MMLRaV coat protein CP gene of this patent were serially diluted for detection by agarose gel electrophoresis and real-time quantitative PCR. Water was used as a negative control (NC).
[0029] Preparation of standard quality plasmids: MMLRaV genome was extracted and cDNA was prepared by reverse transcription. RNA of mulberry leaf curl-associated virus (MMLRaV) was extracted from mulberry samples using the Trizol method. The reverse transcription reaction system was as follows: The first stage included 1 µL of RNA, 1 µL of Random Primer (Takara), 0.5 µL of dNTP Mixture (Takara), and 3.5 µL of ddH2O, with the reaction conditions being 60℃ for 5 minutes, followed by rapid incubation on ice for 2 minutes; The second stage included 2 µL of 5×ReverseTranscriptase M-MLV Buffer (Takara), 0.25 µL of PRI (Takara), 0.25 µL of ReverseTranscriptase M-MLV (Takara), and 1.5 µL of ddH2O, with the reaction conditions being 30℃ for 10 minutes and 42℃ for 60 minutes. The obtained cDNA was stored at -20℃. The method for extracting and reverse transcribing the MMLRaV genome to prepare cDNA is also applicable to the pretreatment of samples to be tested.
[0030] The MMLRaV fragment located in the CP gene of the coat protein obtained using the primers of this patent (SEQ ID No: 5) was ligated to the pMD19-T vector using T4 DNA ligase. The specific ligation system (total volume 10 μL) included: 1.0 μL 10×T4 DNA ligase buffer, 0.5 μL T4 DNA ligase, 0.25 μL pMD19-T vector, 3.0 μL gel-purified target DNA fragment, and 5.25 μL ddH2O. After ligation in a 16℃ metal bath for 4-6 hours, the fragment was transformed into *E. coli* (…). Eschericherichia coli In DH5α, a standby plasmid system was obtained to preserve this cDNA for long-term use in the detection of MMLRaV.
[0031] The initial concentration of the plasmid was determined using a Nanodrop spectrophotometer, and the formula copies / µL = (plasmid concentration ng / µL × 10⁻⁶) was applied. -9 The logarithm of the plasmid copy number was calculated as (6.02 × 10²³) / (DNA length × 660). The extracted plasmid was then subjected to six consecutive 10-fold serial dilutions, resulting in a concentration range of 3.86 × 10⁻⁶. 9 -3.86×10 3 A standard system of copies / µL.
[0032] The reaction system for detection by agarose gel electrophoresis was: 10 × LA PCR Buffer Mg2+ 2.0 μL of dNTP Mixture, 1 μL of cDNA, 0.2 µL of TaKaRa LA Taq, 0.2 μL of forward primer (MMLRaV-3F), 0.2 μL of reverse primer (MMLRaV-3R), and 15.4 µL of ddH2O were used. The reaction conditions were as follows: pre-denaturation temperature 95℃ for 3 minutes; denaturation temperature 94℃ for 20 seconds; annealing temperature 53℃ for 30 seconds; extension temperature 72℃ for 30 seconds; 35 cycles; and extension at 72℃ for 7 minutes. Figure 2 This is an agarose gel electrophoresis image of the MMLRaV coat protein CP gene concentration gradient dilution plasmid of this patent, showing that the gradient dilution plasmid only has the first three concentration gradients (10). -1 ~10 -3 The amplified band was observed, indicating that the detected concentration range was 3.86 × 10⁻⁶. 9 -3.86×10 7 copies / μL.
[0033] The reaction system for real-time quantitative PCR was as follows: 10 μL NovoSTAR® SYBR Qpcr SuperMix Plus, 7.6 µL ddH2O, 2 μL cDNA, 0.2 μL forward primer (MMLRaV-qF1), and 0.2 μL reverse primer (MMLRaV-qR2). The reaction conditions were as follows: pre-denaturation temperature 95℃ for 3 minutes; denaturation temperature 95℃ for 20 seconds; annealing temperature 60℃ for 30 seconds; extension temperature 72℃ for 30 seconds; and 40 cycles. Figure 3 The bar chart shows the Ct values of the plasmid after 10... 6 Even after multiple dilutions, it can still be detected, thus the sensitivity of the qPCR detection method is determined to be 3.86 × 10⁻⁶. 3 The number of copies / µL indicates a detection range of 3.86 × 10⁻⁶. 9 -3.86×10 3 copies / μL.
[0034] RT-qPCR showed significantly better detection sensitivity, therefore it was selected as the optimal detection method, and a corresponding standard curve was established. Three technical replicates were performed for each concentration. A standard curve was constructed using Excel software, with the logarithm of cDNA copy number as the X-axis and Ct value as the Y-axis, and the linearity of the standard curve was determined. The results are as follows: Figure 4 As shown, the standard curve y = -3.296x + 41.176 displays the correlation coefficient R. 2= 0.998, the amplification efficiency of this quantitative primer pair for amplifying gradient dilution plasmids is 99.526%, close to the ideal value of 100%, showing its excellent amplification performance.
[0035] Example 4: Determination of actual detection sensitivity Seven samples were selected from a mulberry orchard in Zhenjiang based on the plant virus infection statistics chart. Four of the samples showed MMLRaV infection symptoms, while three samples were asymptomatic. Water was used as a negative control. The total RNA of the virus was first extracted and then reverse transcribed into cDNA, following the same process as in Example 3.
[0036] The reaction system for detection by agarose gel electrophoresis was: 10 × LA PCR Buffer Mg 2+ 2.0 μL of dNTPMixture, 1 μL of cDNA, 0.5 μL of TaKaRa LA Taq, 0.2 μL of forward primer (MMLRaV-3F), 0.2 μL of reverse primer (MMLRaV-3R), and 15.9 μL of ddH2O were used. The reaction conditions were: pre-denaturation temperature 95℃ for 3 minutes; denaturation temperature 94℃ for 20 seconds; annealing temperature 53℃ for 30 seconds; extension temperature 72℃ for 30 seconds; 35 cycles; and extension at 72℃ for 7 minutes.
[0037] The real-time fluorescence quantitative PCR reaction system and procedure are the same as in Example 3.
[0038] Agarose gel electrophoresis results are as follows Figure 5 As shown, three positive bands were found in four samples with symptoms of MMLLRaV infection, while no positive band was detected in sample S13-1.
[0039] Real-time quantitative PCR results are as follows Figure 6 As shown, all four samples with MMLLRaV infection symptoms tested positive for the virus. Using the standard curve equation described above, the viral load of S13-1 was calculated to be 1.95 × 10⁻⁶. 6 copies / μL.
Claims
1. An RT-qPCR primer for detecting mulberry leaf curl-related virus, characterized in that, The nucleotide sequences of the primers are shown in SEQ ID No: 3~4.
2. The use of the RT-qPCR primers for detecting mulberry leaf curl-related viruses as described in claim 1 in the preparation of the kit.
3. A kit for detecting mulberry leaf curl-related viruses, characterized in that, Includes the primers shown in SEQ ID No: 3~4.
4. The kit for detecting mulberry leaf curl-related viruses according to claim 3, characterized in that, The kit also includes reagents for RNA extraction, RNA reverse transcription reagents, and qPCR reagents.
5. The kit for detecting mulberry leaf curl-related viruses according to claim 3, characterized in that, The kit also includes reverse transcriptase, DNA polymerase, negative control, positive control, reverse transcription buffer, PCR buffer, dNTP mixture, enzyme-free purified water, RNA extraction reagent, RNase inhibitor, and fluorescent dye.
6. The kit for detecting mulberry leaf curl-related viruses according to claim 5, characterized in that, The positive control is a recombinant plasmid containing the CP gene of the outer coat protein of MMLRaV.
7. The kit for detecting mulberry leaf curl-related viruses according to claim 6, characterized in that, The nucleotide sequence of the CP gene, the outer shell protein of MMLRaV, is shown in SEQ ID No:
5.
8. The use of the kit for detecting mulberry leaf curl-associated virus according to any one of claims 3 to 7 in the detection of mulberry leaf curl-associated virus.
9. The application according to claim 8, characterized in that, The real-time quantitative PCR reaction system consisted of: 10 μL NovoSTAR®SYBR Qpcr SuperMix Plus, 7.6 µL ddH2O, 2 μL cDNA, and 0.2 μL each of forward and reverse primers.
10. The application according to claim 8, characterized in that, The real-time quantitative PCR reaction conditions were as follows: pre-denaturation temperature 95℃, time 3 minutes; denaturation temperature 95℃, time 20 seconds; annealing temperature 60℃, time 30 seconds; extension temperature 72℃, time 30 seconds; 40 cycles.