Phalaenopsis virus detection method based on multiplex RT-PCR and application thereof

Abstract

The invention provides a phalaenopsis virus detection method based on multiple RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and application thereof. Specific primers aiming at capsid protein genes of an odontoglossus ringspot virus (ORSV), a tobacco mosaic virus (TMV), a tomato mottle mosaic virus (ToMMV), a pepper mild mottle virus (PMMoV), a plum pox virus (PPV) and a tobacco etch virus (TEV) are designed, and efficient, sensitive and specific detection on a plurality of viruses is realized by adopting multiple RT-PCR (Reverse Transcription-Polymerase Chain Reaction). The invention provides an important technical means for early diagnosis and control of virus diseases in phalaenopsis production and management, can effectively reduce the transmission risk of viruses, improves the effect of quarantine measures, and is suitable for further popularization and application.

Description

Technical Field

[0001] The invention belongs to the technical field of plant disease quarantine, and specifically relates to a Phalaenopsis virus detection method based on multiple RT-PCR and an application thereof. Background Art

[0002] Phalaenopsis is a popular ornamental plant, but it is susceptible to a variety of viral infections, which seriously affects its ornamental value and market value. Current methods for detecting viruses in Phalaenopsis mainly include indicator plant methods, electron microscopy observations, and serological tests, but these methods have the disadvantages of low detection efficiency, complex operations, or high costs. In particular, with the expansion of planting scale and the frequent international trade, viral diseases are posing an increasingly serious threat to the Phalaenopsis industry.

[0003] In recent years, the development of molecular biology technology has provided new means for plant virus detection. High-throughput sequencing technology can comprehensively analyze known and unknown viruses in samples, while multiplex RT-PCR technology can achieve simultaneous detection of multiple viruses, thereby improving detection efficiency and accuracy. However, the application of these technologies in Phalaenopsis virus detection is still limited. Summary of the invention

[0004] The purpose of the present invention is to provide a Phalaenopsis virus detection method based on multiple RT-PCR, which is used for early identification of multiple virus infections in Phalaenopsis and improvement of the efficiency of Phalaenopsis disease management and quarantine.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] The invention provides a set of primer pairs for identifying Phalaenopsis viruses based on multiple RT-PCR, which consists of a specific primer pair A for identifying Odontoglossum wheel spot virus, a specific primer pair B for identifying tobacco mosaic virus, a specific primer pair C for identifying tomato mottle mosaic virus, a specific primer pair D for identifying pepper mild mottle virus, a specific primer pair E for identifying plum pox virus and a specific primer pair F for identifying tobacco etch virus;

[0007] The specific primer pair A consists of primer ORSV-F and primer ORSV-R; wherein the sequence of ORSV-F is shown in SEQ ID NO.1, and the sequence of ORSV-R is shown in SEQ ID NO.2;

[0008] The specific primer pair B consists of primer TMV-F and primer TMV-R; wherein the sequence of TMV-F is shown in SEQ ID NO.3, and the sequence of TMV-R is shown in SEQ ID NO.4;

[0009] The specific primer pair C consists of primer ToMMV-F and primer ToMMV-R; wherein the sequence of ToMMV-F is shown in SEQ ID NO.5, and the sequence of ToMMV-R is shown in SEQ ID NO.6;

[0010] The specific primer pair D consists of primer PMMoV-F and primer PMMoV-R; wherein the sequence of PMMoV-F is shown in SEQ ID NO.7, and the sequence of PMMoV-R is shown in SEQ ID NO.8;

[0011] The specific primer pair E consists of primer PPV-F and primer PPV-R; wherein the sequence of PPV-F is shown in SEQ ID NO.9, and the sequence of PPV-R is shown in SEQ ID NO.10;

[0012] The specific primer pair F consists of primer TEV-F and primer TEV-R; wherein the sequence of TEV-F is shown in SEQ ID NO.11, and the sequence of TEV-R is shown in SEQ ID NO.12.

[0013] Based on the above, the present invention also provides a kit containing the above set of primer pairs.

[0014] As a preferred embodiment, preferably, the concentration ratio of primers ORSV-F / R, TMV-F / R, ToMMV-F / R, PMMoV-F / R, PPV-F / R, and TEV-F / R is 2:5:5:5:5:5, among which the F / R ratio of primers for the same virus is 1:1.

[0015] The kit provided by the present invention can be used in any one of the following I) to IV);

[0016] 1) Identification of 1-6 of the six Phalaenopsis viruses;

[0017] II) Identify whether the virus to be tested is one of the six Phalaenopsis viruses;

[0018] III) Identification of six Phalaenopsis viruses;

[0019] IV) detecting whether the sample to be tested contains six types of Phalaenopsis viruses;

[0020] The six Phalaenopsis viruses are Odontoglossum wheel spot virus, tobacco mosaic virus, tomato mottle mosaic virus, pepper mild mottle virus, plum pox virus and tobacco etch virus.

[0021] Based on the above, the present invention also provides a Phalaenopsis virus detection method based on multiple RT-PCR, comprising the following steps:

[0022] 1) Preparing Phalaenopsis samples and extracting RNA from the samples;

[0023] 2) performing multiple RT-PCR amplification using the primer set as claimed in claim 1 to obtain RT-PCR amplification products;

[0024] If the RT-PCR amplification product contains a band with a length of 477 bp, the Phalaenopsis sample contains Odontoglossum wheel spot virus;

[0025] If the RT-PCR amplification product contains a band with a length of 135 bp, the Phalaenopsis sample contains tobacco mosaic virus;

[0026] If the RT-PCR amplification product contains a band with a length of 277 bp, the Phalaenopsis sample contains Tomato Mottle Mosaic Virus;

[0027] If the RT-PCR amplification product contains a band with a length of 312 bp, the Phalaenopsis sample contains pepper mild mottle virus;

[0028] If the RT-PCR amplification product contains a band with a length of 680 bp, the Phalaenopsis sample contains plum pox virus;

[0029] If the RT-PCR amplification product contains a band with a length of 770 bp, the Phalaenopsis sample contains tobacco etch virus.

[0030] As a possible implementation, further, the method for preparing the Phalaenopsis sample in step 1) comprises the following steps:

[0031] 1.1) Collect Phalaenopsis leaf samples, quickly freeze them in liquid nitrogen and store them at -80°C;

[0032] 1.2) Grind leaf tissue under liquid nitrogen environment to obtain Phalaenopsis samples.

[0033] As a possible implementation, the Phalaenopsis virus detection method based on multiplex RT-PCR further includes: quantitatively analyzing the virus load through multiplex RT-PCR, and counting the relative abundance of the virus in the sample to evaluate the severity of the virus infection in the sample.

[0034] The beneficial effects of the invention are:

[0035] The present invention develops an efficient and sensitive method for detecting Phalaenopsis viruses based on multiple RT-PCR technology. The method can simultaneously detect a variety of common Phalaenopsis viruses, providing a reliable technical means for early diagnosis, effective prevention and control, and quarantine management of viral diseases, helping to reduce the risk of virus transmission during planting and circulation, and improving the production and management level of Phalaenopsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Figure AF shows the alignment depth of six viruses in Phalaenopsis leaf samples. Figure A shows the alignment depth of different viral genome positions, among which Figure A is Odontoglossum wheel spot virus (ORSV), with an alignment depth of more than 1.5 million reads; Figure B is tobacco mosaic virus (TMV), with an alignment depth of about 500 reads; Figure C is tomato mottle mosaic virus (ToMMV); Figure D is pepper mild mottle virus (PMMoV); Figure E is plum pox virus (PPV); and Figure F is tobacco etch virus (TEV). The alignment depths of the latter three viruses are all less than 100 reads. The figure shows the relative abundance of each virus in the sample.

[0037] Figure 2 The results of multiplex RT-PCR detection of six Phalaenopsis viruses based on specific primers;

[0038] Figure A is a gel electrophoresis of single RT-PCR products of 6 viruses; M in the figure represents DNA molecular weight standards, 1-6 represent Odontoglossum wheel spot virus (ORSV), tobacco mosaic virus (TMV), tomato mottle mosaic virus (ToMMV), pepper mild mottle virus (PMMoV), plum pox virus (PPV) and tobacco etch virus (TEV), respectively;

[0039] Figure B shows the concentration optimization results of different primers for detecting 6 viruses; M in the figure represents a DNA molecular weight standard; 1-3 are the detection results of three different primer concentration optimizations.

[0040] Figure C shows the results of multiple one-step RT-PCR applied to newly collected Phalaenopsis samples; M in the figure represents the DNA molecular weight standard; 1-10 are the test results of 10 different samples. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] The invention provides a set of primer pairs for identifying Phalaenopsis viruses based on multiple RT-PCR, which consists of a specific primer pair A for identifying Odontoglossum wheel spot virus, a specific primer pair B for identifying tobacco mosaic virus, a specific primer pair C for identifying tomato mottle mosaic virus, a specific primer pair D for identifying pepper mild mottle virus, a specific primer pair E for identifying plum pox virus and a specific primer pair F for identifying tobacco etch virus;

[0043] The specific primer pair A consists of primer ORSV-F and primer ORSV-R; wherein the sequence of ORSV-F is shown in SEQ ID NO.1, and the sequence of ORSV-R is shown in SEQ ID NO.2;

[0044] The specific primer pair B consists of primer TMV-F and primer TMV-R; wherein the sequence of TMV-F is shown in SEQ ID NO.3, and the sequence of TMV-R is shown in SEQ ID NO.4;

[0045] The specific primer pair C consists of primer ToMMV-F and primer ToMMV-R; wherein the sequence of ToMMV-F is shown in SEQ ID NO.5, and the sequence of ToMMV-R is shown in SEQ ID NO.6;

[0046] The specific primer pair D consists of primer PMMoV-F and primer PMMoV-R; wherein the sequence of PMMoV-F is shown in SEQ ID NO.7, and the sequence of PMMoV-R is shown in SEQ ID NO.8;

[0047] The specific primer pair E consists of primer PPV-F and primer PPV-R; wherein the sequence of PPV-F is shown in SEQ ID NO.9, and the sequence of PPV-R is shown in SEQ ID NO.10;

[0048] The specific primer pair F consists of primer TEV-F and primer TEV-R; wherein the sequence of TEV-F is shown in SEQ ID NO.11, and the sequence of TEV-R is shown in SEQ ID NO.12.

[0049] Based on the above, the present invention also provides a kit containing the above-mentioned set of primer pairs, wherein the concentration ratio of primers ORSV-F / R, TMV-F / R, ToMMV-F / R, PMMoV-F / R, PPV-F / R, and TEV-F / R in the kit is 2:5:5:5:5:5, wherein the F / R ratio of the primers for the same virus is 1:1. The kit can be used in any one of the following I) to IV);

[0050] I) Identify 1-6 of the six Phalaenopsis viruses; II) Identify whether the virus to be tested is one of the six Phalaenopsis viruses; III) Identify the six Phalaenopsis viruses; IV) Detect whether the sample to be tested contains the six Phalaenopsis viruses; Among them, the six Phalaenopsis viruses are Odontoglossum wheel spot virus, tobacco mosaic virus, tomato mottle mosaic virus, pepper mild mottle virus, plum pox virus and tobacco etch virus.

[0051] Based on the above, the present invention also provides a Phalaenopsis virus detection method based on multiple RT-PCR, comprising the following steps:

[0052] 1) Collect Phalaenopsis leaf samples, quickly freeze them in liquid nitrogen and store them at -80°C; grind the leaf tissues in liquid nitrogen to obtain Phalaenopsis samples, and then extract RNA from the samples;

[0053] 2) performing multiple RT-PCR amplification using the primer set as claimed in claim 1 to obtain RT-PCR amplification products;

[0054] If the RT-PCR amplification product contains a band with a length of 477bp, the Phalaenopsis sample contains Odontoglossum wheel spot virus; if the RT-PCR amplification product contains a band with a length of 135bp, the Phalaenopsis sample contains tobacco mosaic virus; if the RT-PCR amplification product contains a band with a length of 277bp, the Phalaenopsis sample contains tomato mottle mosaic virus; if the RT-PCR amplification product contains a band with a length of 312bp, the Phalaenopsis sample contains pepper mild mottle virus; if the RT-PCR amplification product contains a band with a length of 680bp, the Phalaenopsis sample contains plum pox virus; if the RT-PCR amplification product contains a band with a length of 770bp, the Phalaenopsis sample contains tobacco etch virus.

[0055] 3) Quantitative analysis of viral load was performed by multiplex RT-PCR to count the relative abundance of viruses in the samples in order to assess the severity of viral infection in the samples.

[0056] Example 1: High-throughput sequencing screening for Phalaenopsis virus

[0057] 1. Sample collection and preparation

[0058] 82 Phalaenopsis leaf samples were collected, quickly frozen in liquid nitrogen and stored at -80°C. Leaf tissue was ground in liquid nitrogen environment, 0.2g of ground powder was weighed for each sample, mixed and ground again, and the ground samples were divided into 4 portions, each of 1.2g was stored at -80°C to be sent to the sequencing company, and the remaining samples were also stored at -80°C for standby use.

[0059] 2. High-throughput sequencing

[0060] The four samples were sent to Biomark Biotech, and the samples were sequenced using a high-throughput sequencing platform using a ribosomal RNA-free library construction process. The resulting sequence data were preprocessed, and low-quality reads were removed before assembly and alignment.

[0061] 3. Virus screening and identification

[0062] The sequence data were aligned to the NCBI virus database using BLAST alignment software to screen for possible viruses in the samples. The types of viruses were determined based on the alignment results, including six viruses: Odontoglossum ring spot virus (ORSV), tobacco mosaic virus (TMV), tomato mottle mosaic virus (ToMMV), pepper mild mottle virus (PMMoV), plum pox virus (PPV), and tobacco etch virus (TEV).

[0063] Example 2: Multiplex RT-PCR detection of Phalaenopsis virus

[0064] 1. Specific primer design

[0065] Specific primers were designed for the capsid protein gene region of each target virus to ensure the specificity and sensitivity of multiplex RT-PCR. The sequences of each primer and the product length are shown in Table 1 below.

[0066] Table 1. Primer Appendix

[0067]

[0068] 2. Single virus RT-PCR detection

[0069] To verify the specificity of each primer pair, a single RT-PCR test was performed for each virus. The test product was subjected to gel electrophoresis to ensure that a clear single band was produced at a specific primer concentration; the test results were as follows: Figure 2 As shown in A.

[0070] 3. Multiplex RT-PCR system optimization

[0071] According to the results of single virus RT-PCR, different mixed concentrations of virus primers with high accumulation of three viruses, namely, Orchid wheel spot virus (ORSV), Plum pox virus (PPV) and Tobacco etch virus (TEV), were optimized and tested to reduce the interference of these viruses on adjacent virus detection bands. Ultimately, it was ensured that the specific bands of six target viruses could be detected simultaneously. During the optimization test, the concentrations of multiple RT-PCR mixed primers are shown in Table 2 below:

[0072] Table 2. Multiplex RT-PCR mixed primer concentrations

[0073]

[0074] During the test, the primer concentrations of the three viruses, tobacco mosaic virus (TMV), tomato mottle mosaic virus (ToMMV), and pepper mild mottle virus (PMMoV), were all 0.5 μM; the primer concentrations of the orchid wheel spot virus (ORSV) were 0.4 μM, 0.2 μM, and 0.1 μM, respectively; the primer concentrations of the plum pox virus (PPV) were 0.3 μM, 0.5 μM, and 0.7 μM, respectively; and the primer concentrations of the tobacco etch virus (TEV) were 0.3 μM, 0.5 μM, and 0.7 μM, respectively.

[0075] Test results such as Figure 2 As shown in Figure B, it can be seen that the second primer concentration combination in Table 2 above is the optimal primer concentration combination, in which the ORSV primer concentration is 0.2 μM, and the primer concentrations of TMV, ToMMV, PMMoV, PPV and TEV are all 0.5 μM. Under this primer concentration combination, it can be ensured that the specific bands of the six target viruses can be detected simultaneously.

[0076] Then, the second primer concentration combination in Table 2 above was applied to the newly collected 10 groups of Phalaenopsis samples for multiplex RT-PCR detection; the test results are as follows Figure 2 C and shown in Table 3 below.

[0077] Table 3. Results of multiplex RT-PCR detection of 6 viruses in 10 Phalaenopsis samples

[0078]

[0079] It can be seen from the test results that this primer concentration combination can achieve simultaneous detection of multiple viruses.

[0080] Example 3: Quantitative detection and analysis of viruses

[0081] 1. Quantification of viral load

[0082] In samples where the virus was detected, the viral load was quantified by multiplex RT-PCR to assess the severity of viral infection in different samples.

[0083] 2. Data Analysis

[0084] The relative abundance of viruses in different samples is counted, and a comparison depth map of each virus is drawn to intuitively display the distribution of viruses in the samples.

[0085] The depth of comparison of the CP genes of six viruses in the high-throughput sequencing results of 82 Phalaenopsis mixed samples (see attached Figure 1Figure 1 shows the sequencing depth of CP genes of different viruses. The depth of the sequencing of CP genes of Figure A, Odontoglossum wheel spot virus (ORSV), is more than 1.5 million reads; Figure B, Tobacco mosaic virus (TMV), is about 500 reads; Figure C, Tomato mottle mosaic virus (ToMMV); Figure D, Pepper mild mottle virus (PMMoV); Figure E, Plum pox virus (PPV); Figure F, Tobacco etch virus (TEV), is less than 100 reads for the latter three viruses.

[0086] The results can intuitively show the relative abundance of the six viruses in 82 mixed samples. The differences in virus accumulation and genome coverage are mainly attributed to the biological characteristics of the virus. For example, ORSV is known for its high stability and efficient replication in host plants, which may explain its high accumulation in this Phalaenopsis high-throughput sequencing. Another virus, TMV, with similar biological characteristics, also showed a relatively high abundance. In addition, host-virus compatibility plays a key role in viral replication. ORSV has been widely reported as a major pathogen of orchids and has a strong adaptability to Phalaenopsis. TEV and PPV mainly infect plants of other families, such as Solanaceae, and have a low affinity with Phalaenopsis, thereby reducing replication efficiency. Finally, sampling bias may also have affected the results, because 82 samples were mixed for testing, further diluting the comparison depth of low-abundance viruses (such as TEV and PPV).

[0087] 3. Result verification

[0088] To verify the reliability of the method, additional samples were collected from different varieties of Phalaenopsis and the above test method was repeated to ensure the applicability and stability of the method in different samples. The final test results were consistent with the above results.

[0089] The embodiments of the present invention demonstrate the effectiveness of multiplex RT-PCR technology in the detection of Phalaenopsis viruses. Multiplex RT-PCR can achieve the simultaneous detection of multiple viruses, and the detection results are sensitive and specific, and are suitable for disease management during Phalaenopsis planting and quarantine. The detection method of the present invention provides an efficient and convenient technical means for the early diagnosis and control of Phalaenopsis virus diseases.

[0090] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A set of primer pairs for identifying Phalaenopsis orchid viruses based on multiplex RT-PCR, characterized in that: The method comprises a specific primer pair A for identifying Odontoglossum wheel spot virus, a specific primer pair B for identifying tobacco mosaic virus, a specific primer pair C for identifying tomato mottle mosaic virus, a specific primer pair D for identifying pepper mild mottle virus, a specific primer pair E for identifying plum pox virus and a specific primer pair F for identifying tobacco etch virus; The specific primer pair A consists of primer ORSV-F and primer ORSV-R; wherein the sequence of ORSV-F is shown in SEQ ID NO.1, and the sequence of ORSV-R is shown in SEQ ID NO.2; The specific primer pair B consists of primer TMV-F and primer TMV-R; wherein the sequence of TMV-F is shown in SEQ ID NO.3, and the sequence of TMV-R is shown in SEQ ID NO.4; The specific primer pair C consists of primer ToMMV-F and primer ToMMV-R; wherein the sequence of ToMMV-F is shown in SEQ ID NO.5, and the sequence of ToMMV-R is shown in SEQ ID NO.6; The specific primer pair D consists of primer PMMoV-F and primer PMMoV-R; wherein the sequence of PMMoV-F is shown in SEQ ID NO.7, and the sequence of PMMoV-R is shown in SEQ ID NO.8; The specific primer pair E consists of primer PPV-F and primer PPV-R; wherein the sequence of PPV-F is shown in SEQ ID NO.9, and the sequence of PPV-R is shown in SEQ ID NO.10; The specific primer pair F consists of primer TEV-F and primer TEV-R; wherein the sequence of TEV-F is shown in SEQ ID NO.11, and the sequence of TEV-R is shown in SEQ ID NO.

12.

2. A kit comprising the primer set according to claim 1.

3. The kit according to claim 2, characterized in that The concentration ratio of primers ORSV-F / R, TMV-F / R, ToMMV-F / R, PMMoV-F / R, PPV-F / R, and TEV-F / R was 2:5:5:5:5:5, and the F / R ratio of primers for the same virus was 1:

1.

4. A use of the kit according to claim 2 or 3, characterized in that: The use of the kit in any one of the following I) to IV); 1) Identification of 1-6 of the six Phalaenopsis viruses; II) Identify whether the virus to be tested is one of the six Phalaenopsis viruses; III) Identification of six Phalaenopsis viruses; IV) detecting whether the sample to be tested contains six types of Phalaenopsis viruses; The six Phalaenopsis viruses are Odontoglossum wheel spot virus, tobacco mosaic virus, tomato mottle mosaic virus, pepper mild mottle virus, plum pox virus and tobacco etch virus.

5. A method for detecting Phalaenopsis virus based on multiplex RT-PCR, characterized in that: The steps include: 1) Preparing Phalaenopsis samples and extracting RNA from the samples; 2) performing multiple RT-PCR amplification using the primer set as claimed in claim 1 to obtain RT-PCR amplification products; If the RT-PCR amplification product contains a band with a length of 477 bp, the Phalaenopsis sample contains Odontoglossum wheel spot virus; If the RT-PCR amplification product contains a band with a length of 135 bp, the Phalaenopsis sample contains tobacco mosaic virus; If the RT-PCR amplification product contains a band with a length of 277 bp, the Phalaenopsis sample contains Tomato Mottle Mosaic Virus; If the RT-PCR amplification product contains a band with a length of 312 bp, the Phalaenopsis sample contains pepper mild mottle virus; If the RT-PCR amplification product contains a band with a length of 680 bp, the Phalaenopsis sample contains plum pox virus; If the RT-PCR amplification product contains a band with a length of 770 bp, the Phalaenopsis sample contains tobacco etch virus.

6. The method for detecting Phalaenopsis orchid virus based on multiple RT-PCR according to claim 5, characterized in that: The method for preparing the Phalaenopsis sample in step 1) comprises the following steps: 1.1) Collect Phalaenopsis leaf samples, quickly freeze them in liquid nitrogen and store them at -80°C; 1.2) Grind leaf tissue under liquid nitrogen environment to obtain Phalaenopsis samples.

7. The method for detecting Phalaenopsis orchid viruses based on multiplex RT-PCR according to claim 5, characterized in that: Also includes: The viral load was quantitatively analyzed by multiplex RT-PCR, and the relative abundance of the virus in the sample was counted to assess the severity of viral infection in the sample.

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