A Multiplex RT-PCR Method for Simultaneous Detection of Three Viruses and Two Viral Species in Chrysanthemum

Abstract

The present invention discloses a method for simultaneously detecting three viruses and two viroids in chrysanthemum by multiplex RT-PCR, using the following primer pair combination: CSVd-F and CSVd-R, whose nucleotide sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2; CChMVd-F and CChMVd-R, whose nucleotide sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4; CVB-F and CVB-R, whose nucleotide sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6; TAV-F and TAV-R, whose nucleotide sequences are shown in SEQ ID NO: 7 and SEQ ID NO: 8; TSWV-F and TSWV-R, whose nucleotide sequences are shown in SEQ ID NO: 9 and SEQ ID NO: 10. The present invention can effectively detect plant viruses.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology of garden plants, and in particular to a method for simultaneously detecting three viruses and two viroids in chrysanthemums using multiplex RT-PCR. Background Art

[0002] There are over 20 viruses that infect chrysanthemums worldwide, with 14 currently reported in my country. These viruses are numerous and spread through a wide range of pathways, acting as a kind of "cancer" for plants. Ideal prevention and control measures are lacking. Early quarantine, prevention of spread, and suppression of transmission vectors are the most effective means of prevention and control. Rapid virus detection is a prerequisite for virus prevention and control.

[0003] Currently, the main technologies for chrysanthemum virus detection include biological detection, enzyme-linked immunosorbent assay (ELISA), nucleic acid hybridization (NAM), and PCR. Biological detection involves infecting a virus indicator plant with the virus by rubbing or grafting it, and then identifying the virus species by observing its symptoms. While simple to perform and intuitive, the detection process is long and susceptible to environmental influences. ELISA is one of the most commonly used methods for virus detection. It involves linking an antigen or antibody to an enzyme to form an enzyme conjugate. The color reaction of the substrate determines the presence and type of virus. While rapid, intuitive, and inexpensive, it is cumbersome, lacks specificity and sensitivity, and cannot detect viroids or viruses without coat proteins. Nucleic acid hybridization (NAM) utilizes nucleic acids hybridized with labeled, specific probes to detect viruses. However, with the increasing adoption of PCR, its application in virus detection has declined. PCR can rapidly amplify viral genetic material in large quantities, offering advantages such as speed, accuracy, and high sensitivity. However, it can only detect one virus at a time. In chrysanthemum cultivation, multiple viruses often infect the plant, necessitating repeated PCR tests to identify the pathogen. Summary of the Invention

[0004] The technical problem addressed by this invention is to provide a method for simultaneously detecting three viruses and two viroids in chrysanthemums using multiplex RT-PCR. The multiplex RT-PCR technology designed in this invention is simple to operate, has a short detection time, and is highly sensitive. It can simultaneously detect five viruses and viroids, significantly reducing detection time and costs.

[0005] The present invention discloses a method for simultaneously detecting three viruses and two viroids in chrysanthemums by multiplex RT-PCR, including Chrysanthemum virus B (CVB), Tomato aspermy virus (TAV), Tomato spotted wilt virus (TSWV), Chrysanthemum stunt viroid (CSVd), and Chrysanthemum chlorotic mottleviroid (CChMVd). The method uses the following primer pair combination:

[0006] CSVd-F and CSVd-R, whose nucleotide sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2;

[0007] CChMVd-F and CChMVd-R, whose nucleotide sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4;

[0008] CVB-F and CVB-R, whose nucleotide sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6;

[0009] TAV-F and TAV-R, whose nucleotide sequences are shown in SEQ ID NO: 7 and SEQ ID NO: 8;

[0010] The nucleotide sequences of TSWV-F and TSWV-R are shown in SEQ ID NO:9 and SEQ ID NO:10.

[0011] The method of the present invention for simultaneously detecting three viruses and two viroids in chrysanthemum by multiplex RT-PCR has the following multiplex PCR reaction system:

[0012]

[0013] Reaction procedure: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 55.3°C for 30 s, and 72°C for 30 s, for 35 cycles; extension at 72°C for 10 min; and storage at 4°C.

[0014] The method of the present invention for simultaneously detecting three viruses and two viroids in chrysanthemum by multiple RT-PCR differs from the prior art in that:

[0015] PCR technology is widely used in virus detection. Conventional PCR technology can only detect one virus or viroid at a time, while the multiplex RT-PCR detection technology of the present invention can detect five chrysanthemum viruses and viroids at a time. Its sensitivity is slightly lower than that of single PCR, and it can effectively detect plant viruses, greatly saving detection time and consumables such as reagents. It is suitable for large-scale virus surveys and provides a method for rapid and efficient virus detection.

[0016] The method of the present invention for simultaneously detecting three viruses and two viroids in chrysanthemum by multiplex RT-PCR is further described below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the results of a single PCR test in the present invention; wherein M is a 500 marker and N is a negative control;

[0018] Figure 2 This is a graph showing the detection results of the primer concentration combination optimization in the present invention;

[0019] Figure 3 This is a graph showing the detection results of the annealing temperature optimization in the present invention;

[0020] Figure 4 This is another test result diagram of annealing temperature optimization in the present invention;

[0021] Figure 5 This is a sensitivity test result diagram in the present invention. DETAILED DESCRIPTION

[0022] 1. Experimental Materials

[0023] Chrysanthemum virus disease samples were collected from chrysanthemum growing areas in suburban counties such as Shunyi, Pinggu, and Yanqing. 1 g of fresh leaves were quick-frozen in liquid nitrogen and stored in a -80°C refrigerator.

[0024] 2. Primer design

[0025] The gene sequences of five viruses and viroids (Chrysanthemum B virus, Tomato Sterility Virus, Tomato Spotted Wilt Virus, Chrysanthemum Dwarf Viroid, and Chrysanthemum Chlorotic Mottle Viroid) were downloaded from the NCBI database. Sequence alignment was performed using ClustalX software. Primers were then selected from conserved regions and designed using Primer 5.0 software. The design process required that the annealing temperature of each primer be between 50 and 60°C, the GC content be between 40 and 60%, and that dimers and hairpin loops be avoided between primers. To ensure effective electrophoretic differentiation, the amplified sequences of different viruses and viroids differed by at least 50 bp. Specific primer sequences, annealing temperatures, and product sizes are shown in Table 1.

[0026] Table 1 Primer design for simultaneous detection of three viruses and two viroids in chrysanthemum by multiplex RT-PCR

[0027]

[0028] a Y=C / T;R=G / A;H=A / T / C

[0029] 3. RNA Extraction and Reverse Transcription

[0030] Total RNA was extracted using the MiniBEST Plant RNA Extraction Kit from Takara, and 1 μg of RNA was reverse transcribed using the Reverse Transcription System from Promega in a final volume of 100 μl.

[0031] 4. Single PCR detection

[0032] Reaction system:

[0033]

[0034] Reaction procedure: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing for 30 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for another 10 min; storage at 4°C.

[0035] The PCR amplification products were detected by 2% agarose gel electrophoresis. Figure 1 The purpose of the single PCR was to screen for specific primers for five viruses and viroids. The results showed that the designed primers had good specificity, with no nonspecific bands, and could be used for subsequent multiplex PCR experiments.

[0036] 5. Multiplex PCR detection

[0037] Based on single PCR, the primer concentration combination (Table 2) and annealing temperatures (50, 52, 54, 56, 58, 60), (53, 53.4, 54.3, 55.3, 56.8, 57.8, 58.5, 59) were optimized.

[0038]

[0039] The concentration combination optimization results are as follows Figure 2As shown in the figure, when the primer dosages for the five viruses and viroids were the same, only TAV could be detected. When the primer dosages for combination 4 were used, all five viruses and viroids could be detected. However, the other combinations failed to detect all five viruses and viroids simultaneously. Therefore, the final primer ratio was determined to be TSWV:TAV:CVB:CChMVd:CSVd = 0.6:0.1:0.4:1.4:1.2.

[0040] The annealing temperature optimization results are as follows Figure 3 and Figure 4 As shown in the figure, when the annealing temperature is higher than 56°C, the bands begin to darken; when the annealing temperature is lower than 54.2°C, nonspecific bands are generated; and when the annealing temperature is 55.3°C, the bands are clear and there are no nonspecific bands. Therefore, 55.3°C is selected as the optimal annealing temperature.

[0041] 6. Multiplex PCR optimization results

[0042] Reaction system:

[0043]

[0044] Reaction procedure: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 55.3°C for 30 s, and 72°C for 30 s, for 35 cycles; extension at 72°C for 10 min; and storage at 4°C.

[0045] 7. Sensitivity test

[0046] The total cDNA of five viruses (viroids) including CVB, TAV, TSWV, CSVd and CChMVd were sequentially subjected to 10 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 Single PCR and multiplex PCR were performed respectively, and the PCR amplification products were detected by 2% agarose gel electrophoresis.

[0047] like Figure 5 As shown in the results, the sensitivity of TSWV was 10 when using a single PCR test. 2 , the sensitivity of TAV is 10 5 , the sensitivity of CVB is 10 2 , the sensitivity of CChMVd is 10 0 , the sensitivity of CSVd is 10 1 ; When using multiplex PCR detection, the sensitivity of TSWV is 10 1 , the sensitivity of TAV is 10 3, the sensitivity of CVB is 10 3 , the sensitivity of CChMVd is 10 2 , the sensitivity of CSVd is 10 2 In general, the sensitivity of multiplex PCR is lower than that of single PCR.

[0048] Although the sensitivity of the present invention is slightly lower than that of single PCR, it is completely sufficient for virus detection. It can effectively detect plant viruses, greatly saving detection time and consumables such as reagents. It is suitable for large-scale virus surveys and provides a method for rapid and efficient virus detection.

[0049] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention. Sequence Listing <110> Beijing Agricultural Biotechnology Research Center <120> A Multiplex RT-PCR Method for Simultaneous Detection of Three Viruses and Two Viral Species in Chrysanthemum <130> 2021-5 <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <400> 1 ggaggaagtc cgacgagat 19 <210> 2 <211> twenty one <212> DNA <213> Artificial Sequence <400> 2 tccaggagag gaaggaaact a 21 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <400> 3 gctctctcca cagcctcat 19 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <400> 4 agcgaaccaa ctgggact 18 <210> 5 <211> twenty one <212> DNA <213> Artificial Sequence <400> 5 catcygagga tatggtgaag a 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 atrgchgacc aatcagaagg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 atcccttcaa catccgactt 20 <210> 8 <211> twenty one <212> DNA <213> Artificial Sequence <400> 8 ctcagcaccg acaytagaca t 21 <210> 9 <211> twenty three <212> DNA <213> Artificial Sequence <400> 9 aatctggtag cattcaactt caa 23 <210> 10 <211> twenty two <212> DNA <213> Artificial Sequence <400> 10 gcttccctgg tgtcatactt ct 22

Claims

1. A method for simultaneous detection of three viruses and two viroids in chrysanthemum by multiplex RT-PCR, characterized by: The following primer pair combinations were used: CSVd-F and CSVd-R, whose nucleotide sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2; CChMVd-F and CChMVd-R, whose nucleotide sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4; CVB-F and CVB-R, whose nucleotide sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6; TAV-F and TAV-R, whose nucleotide sequences are shown in SEQ ID NO: 7 and SEQ ID NO: 8; TSWV-F and TSWV-R, whose nucleotide sequences are shown in SEQ ID NO:9 and SEQ ID NO:10; The three viruses are chrysanthemum B virus, tomato sterility virus and tomato spotted wilt virus, and the two viroids are chrysanthemum stunt viroid and chrysanthemum chlorotic mottle viroid; In the method for simultaneously detecting three viruses and two viroids in chrysanthemum using multiplex RT-PCR, the PCR reaction system is as follows: Reaction procedure: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 55.3°C for 30 s, and 72°C for 30 s, for 35 cycles; extension at 72°C for 10 min; and storage at 4°C.

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