A primer set and kit for simultaneously detecting multiple Phalaenopsis viruses and its application

Abstract

The present invention provides a primer set, a kit and an application thereof for simultaneously detecting a plurality of Phalaenopsis viruses. The present invention belongs to the field of gene detection technology. The present invention is directed to the nucleotide sequences of small RNA sequencing of five viruses, ORSV, CyMV, WCMV, YaVX and PoVX, and five pairs of corresponding specific primers are designed respectively, and the five pairs of specific primers are used to simultaneously detect ORSV, CyMV, WCMV, YaVX and PoVX in Phalaenopsis leaves, and the sequence homology of the detected WCMV, YaVX and PoVX with the existing viruses is as low as 52-76%, which is very different from the prior art sequence. In this case, the primer set of the present invention can still achieve the goal of simultaneously detecting 5 kinds of Phalaenopsis viruses in one reaction, which is fast and convenient, with high detection efficiency, and the reagents used in the present invention are commonly used reagents in molecular biology, with low cost, and even if the RNA content of Phalaenopsis virus in the sample is very low, it can still be detected, with high detection sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and more specifically to a primer set and a kit for simultaneously detecting multiple Phalaenopsis viruses and applications thereof. Background Art

[0002] Phalaenopsis plants have important ornamental and economic value, but with the increase in Phalaenopsis plant trade in recent years, the occurrence and spread of viral diseases have accelerated. Cymbidium mosaic virus (CyMV), Odontoglossumringspotvirus (ORSV), White clover mosaic virus (WCMV), Yammosaic virus (YaVX) and Potato virus X (PoVX) are the five main viruses that harm Phalaenopsis. Viral diseases can cause discoloration, necrosis, mosaic and other symptoms on leaves, resulting in poor growth of Phalaenopsis plants, dull leaves, reduced flower quality, and even plant death, reducing the ornamental and economic value of Phalaenopsis and causing serious economic losses to Phalaenopsis production companies.

[0003] At present, there is no effective drug for the prevention and treatment of Phalaenopsis virus disease in the world. Therefore, in the prevention and treatment process of Phalaenopsis virus disease, early detection and early isolation are often used. However, Phalaenopsis diseased plants usually only have slight symptoms in the early stage, or do not show symptoms at all. It is impossible to effectively prevent and treat Phalaenopsis virus disease by simple symptom observation. At present, plant virus detection mostly adopts enzyme-linked immunosorbent assay (i.e. ELISA method) based on antiserum, but antiserum price is high, and one antiserum can only detect one virus, and the detection procedure is cumbersome. There are also problems of false positive reaction and low detection sensitivity to a certain extent. In recent years, in order to improve virus detection efficiency, virus detection technologies based on PCR technology such as RT-PCR and mRT-PCR are developing rapidly. Compared with the single reverse transcription PCR detection method, the multiple reverse transcription PCR detection technology has the advantages of being able to detect multiple viruses at the same time, simple operation, short detection time and low cost, and therefore attracts much attention.

[0004] CyMV, ORSV, WCMV, YaVX and PoVX are all single-stranded RNA viruses, which are prone to mutation, and there are many mutation sites between different mutants. The homology between the WCMV, YaVX and PoVX to be detected by the present invention and the corresponding known virus sequences in the prior art is as low as 80%, and there is a large variation. Therefore, the difficulty of using multiple real-time fluorescence quantitative PCR technology for detection lies in the selection of conservative sequences, the design of primer combinations and the optimization of the detection system. However, there is currently only a document number: CN 104372108 B, the invention name is: A kind of Oncidium virus detection primer and method of the prior art, which only involves the detection of CyMV and ORSV, and the sample is diluted 10 2 Can amplify specific bands, and dilute 10 3 The post-multiplication bands are very weak, and the detection sensitivity is low. In addition, there is no report on the multi-gene synchronous detection technology of five viruses, CyMV, ORSV, WCMV, YaVX and PoVX, in Phalaenopsis.

[0005] Therefore, how to provide an mRT-PCR specific primer set and a detection method with high detection sensitivity and simultaneous detection of CyMV, ORSV, WCMV, YaVX and PoVX is a technical problem that technicians in this field urgently need to solve. Summary of the invention

[0006] In view of this, the present invention provides a primer set, a kit and an application thereof for simultaneously detecting multiple Phalaenopsis viruses. The primer set can simultaneously detect the main pathogenic viruses in five Phalaenopsis orchids, namely CyMV, ORSV, WCMV, YaVX and PoVX, and has the advantages of high detection efficiency, low cost and high detection sensitivity.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A primer set for simultaneously detecting multiple Phalaenopsis viruses, comprising the following 5 primer pairs:

[0009] Forward primer F1 of Jianlan orchid leaf virus: 5'-AGTCTCAACCTGACGTGGAG-3', SEQ ID NO.1,

[0010] Reverse primer F2 of Jianlan orchid leaf virus: 5'-GGGGAAGCGGTGAATATTA-3', SEQ ID NO.2;

[0011] Forward primer F3 of tooth blue ring spot virus: 5'-TATGACAAATCCCAGAACGA-3', SEQ ID NO.3,

[0012] Reverse primer F4 for tooth blue ring spot virus: 5'-GCAAATAGAAACAAACTCCA-3', SEQ ID NO.4;

[0013] White clover mosaic virus forward primer F5: 5'-GTTGTGCTCCCTGTTGAA-3', SEQ ID NO.5,

[0014] White clover mosaic virus reverse primer F6: 5′-GTCCCAAATGGATAAGGATAG-3′, SEQ ID NO.6;

[0015] Forward primer F7 of Chinese yam X virus: 5'-GGCAGAATCTATTGGAAAAA-3', SEQ ID NO.7,

[0016] Reverse primer F8 for Chinese yam X virus: 5'-CCATGAATGACTAAGGGAAG-3', SEQ ID NO.8;

[0017] Potato virus X forward primer F9: 5'-GAGGGTCCCACTTTTGATGC-3', SEQ ID NO.9,

[0018] Potato virus X reverse primer F10: 5'-CAAGCGGTTGGTGATTTTAT-3', SEQ ID NO.10.

[0019] Another object of the present invention is to provide the use of the above primer set in the preparation and identification of Phalaenopsis virus gene products.

[0020] Another object of the present invention is to provide a detection kit for simultaneously detecting multiple Phalaenopsis viruses, comprising the above-mentioned primer set.

[0021] Preferably, the kit further comprises Taq enzyme, Taq enzyme buffer, and dNTPs.

[0022] Another object of the present invention is to provide a detection method for simultaneously detecting multiple Phalaenopsis viruses, using a primer set for detecting multiple Phalaenopsis viruses or a kit for detecting multiple Phalaenopsis viruses, performing mRT-PCR amplification on the viruses to be detected, performing agarose gel electrophoresis analysis on the amplified products, and judging the presence or absence of the virus based on the size of the band.

[0023] Preferably, the mRT-PCR reaction system is composed of the following:

[0024] The total volume of the reaction system was 50 μL, containing 0.3 μL 5U / μL Taq enzyme, 5 μL 10× Taq enzyme buffer, 4 μL 2.5mmol / L dNTPs, 1 μL 20ng / μL template, 0.2-0.4μL Jianlan orchid leaf virus forward primer, 0.2-0.4μL Jianlan orchid leaf virus reverse primer, 1-2.5μL Tooth orchid ringspot virus forward primer, 1-2.5μL Tooth orchid ringspot virus reverse primer, 0.5-2μL White clover mosaic virus forward primer, 0.5-2μL White clover mosaic virus reverse primer, 1.5-3.5μL Huai Yam X virus forward primer, 1.5-3.5μL Huai Yam X virus reverse primer, 1-3μL Potato X virus forward primer, 1-3μL Potato X virus reverse primer, and the remaining ingredients are sterile double distilled water.

[0025] Preferably, the initial concentration of all forward primers and reverse primers in the mRT-PCR reaction system before being added to the reaction system is 10 μmol / L, and the concentrations of the forward primer and reverse primer in each pair of primers in the reaction system are equal.

[0026] Preferably, in the mRT-PCR reaction system, the final concentrations of the forward primer and reverse primer of the Cymbidium orchid leaf virus are 0.04-0.08 μmol / L; the final concentrations of the forward primer and reverse primer of the Cymbidium orchid ringspot virus are 0.2-0.5 μmol / L; the final concentrations of the forward primer and reverse primer of the white clover mosaic virus are 0.1-0.4 μmol / L; the final concentrations of the forward primer and reverse primer of the Chinese yam X virus are

[0027] 0.3-0.7μmol / L; the final concentrations of the forward primer and reverse primer of potato virus X were

[0028] 0.2-0.6μmol / L.

[0029] Preferably, the mRT-PCR reaction procedure is as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 48-56°C for 30 s, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 10 min; and termination of the reaction at 4°C.

[0030] Preferably, when an electrophoretic band of 908 bp appears, it indicates that the sample contains Cymbidium orchid leaf virus; when an electrophoretic band of 1156 bp appears, it indicates that the sample contains Cymbidium orchid ringspot virus; when an electrophoretic band of 561 bp appears, it indicates that the sample contains white clover mosaic virus; when an electrophoretic band of 292 bp appears, it indicates that the sample contains Chinese yam X virus; when an electrophoretic band of 162 bp appears, it indicates that the sample contains potato X virus.

[0031] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The present invention designs and synthesizes five pairs of corresponding specific primers for the small RNA sequencing sequences of five viruses, CyMV, ORSV, WCMV, YaVX and PoVX, respectively, and uses the five pairs of primers to simultaneously perform mRT-PCR reactions on CyMV, ORSV, WCMV, YaVX and PoVX in Phalaenopsis leaves, establishes a new PCR reaction system and reaction procedure, and obtains five amplified fragment sequences of CyMV, ORSV, WCMV, YaVX and PoVX with obvious size differences and lengths of 908 bp, 1156 bp, 561 bp, 292 bp and 162 bp, respectively, thereby achieving the goal of simultaneously detecting five Phalaenopsis viruses in one reaction, and has the advantages of high detection efficiency, low cost and high detection sensitivity.

[0033] (2) The WCMV, YaVX and PoVX detected by the present invention have a sequence homology as low as 52-76% with the corresponding sequences in the prior art, and are new variant strains that do not exist in the prior art. However, the primer set provided by the present invention can effectively identify the above variant strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0035] Figure 1 : 1% agarose gel electrophoresis diagram in Example 2 of the present invention, wherein M: DNA standard DL2000; 1: ORSV; 2: CyMV; 3: WCMV; 4: YaVX; 5: PoVX; 6: negative control; 7: ORSV, CyMV, WCMV, YaVX and PoVX;

[0036] Figure 2 : 1% agarose gel electrophoresis diagram in Example 4 of the present invention. DETAILED DESCRIPTION

[0037] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only 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.

[0038] Example 1

[0039] A primer set for simultaneously detecting multiple Phalaenopsis viruses, comprising the following 5 primer pairs:

[0040] Forward primer F1 of Jianlan orchid leaf virus: 5'-AGTCTCAACCTGACGTGGAG-3', SEQ ID NO.1,

[0041] Reverse primer F2 of Jianlan orchid leaf virus: 5'-GGGGAAGCGGTGAATATTA-3', SEQ ID NO.2;

[0042] Forward primer F3 of tooth blue ring spot virus: 5'-TATGACAAATCCCAGAACGA-3', SEQ ID NO.3,

[0043] Reverse primer F4 for tooth blue ring spot virus: 5'-GCAAATAGAAACAAACTCCA-3', SEQ ID NO.4;

[0044] White clover mosaic virus forward primer F5: 5'-GTTGTGCTCCCTGTTGAA-3', SEQ ID NO.5,

[0045] White clover mosaic virus reverse primer F6: 5′-GTCCCAAATGGATAAGGATAG-3′, SEQ ID NO.6;

[0046] Forward primer F7 of Chinese yam X virus: 5'-GGCAGAATCTATTGGAAAAA-3', SEQ ID NO.7,

[0047] Reverse primer F8 for Chinese yam X virus: 5'-CCATGAATGACTAAGGGAAG-3', SEQ ID NO.8;

[0048] Potato virus X forward primer F9: 5'-GAGGGTCCCACTTTTGATGC-3', SEQ ID NO.9,

[0049] Potato virus X reverse primer F10: 5'-CAAGCGGTTGGTGATTTTAT-3', SEQ ID NO.10.

[0050] Example 2

[0051] A detection method for simultaneously detecting multiple Phalaenopsis viruses

[0052] (1) Small RNA sequencing sequences of Jianlan orchid leaf virus, tooth orchid ringspot virus, white clover mosaic virus, yam X virus and potato X virus were used to design five pairs of specific primers. The designed five pairs of specific primers were calibrated to confirm that there was no self-matching and competitive amplification between the five pairs of primers to ensure that the above five viruses could amplify specific fragments with obvious differences. The above five pairs of specific primers were synthesized by Shanghai Shenggong Biotechnology Co., Ltd.

[0053] (2) 0.2 g of Phalaenopsis leaves infected with CyMV, ORSV, WCMV, YaVX and PoVX and 0.2 g of healthy leaves were taken for RNA extraction, and healthy leaves were used as negative control.

[0054] The RNA extraction process is as follows: using the Trizol method, 0.2g of Phalaenopsis virus-infected leaves or healthy leaves are placed in a mortar, liquid nitrogen is added to grind into powder, then the powdered leaves are placed in the first centrifuge tube, 1mL Trizol is added and violently shaken for 20-30S, then the first centrifuge tube is placed at room temperature for 5min to allow the leaves to fully lyse, then the first centrifuge tube is centrifuged at 10000rpm for 5min; the supernatant is transferred to the second centrifuge tube, 200uL chloroform is added to the second centrifuge tube, and oscillated to mix, and placed at room temperature for 3min, then centrifuged at 4°C and 10000rpm for 15min, then the upper aqueous phase is aspirated to the third centrifuge tube, 0.5mL isopropanol is added to the third centrifuge tube to mix, and placed at room temperature for 10min, then the third centrifuge tube is centrifuged at 4°C and 1000rpm for 10min, and the supernatant is discarded, and the RNA sinks to the bottom of the tube; 1mL is added 75% ethanol was added to the third centrifuge tube and the third centrifuge tube was gently shaken to obtain a suspension, and then centrifuged at 4°C and 10,000 rpm for 5 min, and the supernatant was discarded. The third centrifuge tube was then placed on ice to dry for 10 min, and finally 30 uL of DEPC-treated water was used to dissolve the RNA sample.

[0055] (3) Reverse transcription of the RNA obtained in step (2) into cDNA

[0056] The specific reverse transcription process is as follows: place the reaction tube on ice, add 1uL Primer Oligo (dt), 1uL dNTP mixture (10mmol / L), 4uL DEPC-treated water, and 4uL RNA sample to the reaction tube, mix gently and centrifuge slightly, then heat the reaction tube in a 65°C water bath for 5 minutes, immediately place the reaction tube on ice for 5 minutes, then centrifuge the reaction tube slightly and place the reaction tube on ice again, add 4uL 5× reverse transcriptase buffer, 0.5uL 20U / uL RNAase inhibitor, 1uL200U / uL reverse transcriptase, and 4.5uL DEPC-treated water to the reaction tube, then place the reaction tube in a 42°C water bath and heat for 60 minutes, then place the reaction tube at 70°C for 10 minutes, and finally terminate the reaction.

[0057] (4) Using the cDNA obtained in step (3) as a template and the five pairs of primers obtained in step (1) to simultaneously perform a PCR amplification reaction to obtain a PCR amplification product.

[0058] This embodiment performs a single-plex PCR amplification reaction and a multiplex PCR amplification reaction, so the reaction system of the PCR amplification reaction includes a single-plex PCR reaction system and a multiplex PCR reaction system, wherein the reaction procedures of the single-plex PCR amplification reaction and the multiplex PCR amplification reaction are the same, but the reaction systems are different.

[0059] The specific contents of the reaction system and reaction procedure of the PCR amplification reaction are as follows:

[0060] Single-plex PCR reaction system: The total volume of the reaction system is 50 μL, including 0.3 μL 5 U / μL Taq enzyme, 5 μL 10× Taq enzyme buffer, 4 μL 2.5 mmol / L dNTPs, 1 μL template, 1 μL 10 μmol / L forward primer of CyMV or ORSV or WCMV or YaVX or PoVX, and 1 μL 10 μmol / L reverse primer of CyMV or ORSV or WCMV or YaVX or PoVX.

[0061] Multiplex PCR reaction system: The total volume of the reaction system is 50 μL, containing 0.3 μL 5U / μL Taq enzyme, 5 μL 10× Taq enzyme buffer, 4 μL 2.5 mmol / L dNTPs, 1 μL 20 ng / μL template, 0.3 μL CyMV forward primer, 0.3 μL CyMV reverse primer, 1.5 μL ORSV forward primer, 1.5 μL ORSV reverse primer, 1 μL WCMV forward primer, 1 μL WCMV reverse primer, 2.5 μL YaVX forward primer, 2.5 μL YaVX reverse primer, 2 μL PoVX forward primer, 2 μL PoVX reverse primer, and the remaining components are sterile double distilled water (wherein the concentrations of the forward primer and reverse primer in each primer pair are equal).

[0062] PCR reaction program: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 54°C annealing for 30 s, 72°C extension for 1 min, 35 cycles; 72°C extension for 10 min; 4°C termination reaction.

[0063] (5) Take 5 μL of PCR product and use 1% agarose gel as the electrophoresis support medium. Perform electrophoresis for 30 min at 1×TAE and 120 V. Observe with a UV transilluminator. The experimental results are as follows: Figure 1 shown.

[0064] (6) The PCR amplification products obtained from the multiplex PCR reaction system in step (4) were cut and recovered, and then in vitro ligated with pMD18-T simple vector (TaKaRa Biotechnology (Beijing) Co., Ltd., TaKaRa), and then positive clones were picked. Finally, the plasmids were extracted and sequenced to check the amplification results of the five viruses, CyMV, ORSV, WCMV, YaVX and PoVX.

[0065] Result analysis:

[0066] Depend on Figure 1It can be seen that the single-plex PCR amplification reaction of the ORSV primer pair obtained an amplified fragment with a length of 1156 bp, the single-plex PCR amplification reaction of the CyMV primer pair obtained an amplified fragment with a length of 908 bp, the single-plex PCR amplification reaction of the WCMV primer pair obtained an amplified fragment with a length of 561 bp, the single-plex PCR amplification reaction of the YaVX primer pair obtained an amplified fragment with a length of 292 bp, and the single-plex PCR amplification reaction of the WCMV primer pair obtained an amplified fragment with a length of 162 b. The multiple PCR amplification reaction involving five pairs of primers, namely, ORSV primer pair, CyMV primer pair, WCMV primer pair, YaVX primer pair, and WCMV primer pair, can simultaneously obtain five bright and clear bands with amplified fragment lengths of 1156 bp, 908 bp, 561 bp, 292 bp, and 162 bp. It can be seen that the present invention can simultaneously detect five viruses, namely, CyMV, ORSV, WCMV, YaVX, and PoVX, of Phalaenopsis at one time, with good specificity and high detection efficiency.

[0067] Example 3

[0068] The difference from Example 2 is that:

[0069] Step (4): The reaction system and reaction procedure of the PCR amplification reaction are as follows:

[0070] Multiplex PCR reaction system: the total volume of the reaction system is 50 μL, containing 0.3 μL 5U / μL Taq enzyme, 5 μL 10× Taq enzyme buffer, 4 μL 2.5 mmol / L dNTPs, 1 μL template, 0.25 μL CyMV forward primer, 0.25 μL CyMV reverse primer, 1.5 μL ORSV forward primer, 1.5 μL ORSV reverse primer, 1 μL WCMV forward primer, 1 μL WCMV reverse primer, 2 μL YaVX forward primer, 2 μL YaVX reverse primer, 2 μL PoVX forward primer, 2 μL PoVX reverse primer, and the remaining components are sterile double distilled water;

[0071] PCR reaction program: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 1 min, 35 cycles, 72°C extension for 10 min, and 4°C termination.

[0072] The remaining steps are the same as in Example 2.

[0073] Result analysis:

[0074] The sequencing results of Example 3 are as follows: the amplified products of ORSV, CyMV, WCMV, YaVX and PoVX are sequences consisting of 1156 bp, 908 bp, 561 bp, 292 bp and 162 bp nucleotides respectively:

[0075] The sequence of the ORSV amplification product is shown in SEQ ID NO: 11;

[0076] The sequence of the CyMV amplified product is shown in SEQ ID NO: 12;

[0077] The sequence of the WCMV amplification product is shown in SEQ ID NO: 13;

[0078] The sequence of the YaVX amplification product is shown in SEQ ID NO: 14;

[0079] The sequence of the PoVX amplification product is shown in SEQ ID NO:15.

[0080] The homology analysis results using NCBI showed that:

[0081] The homology rate of the obtained amplified product sequence SEQ ID NO: 11 and the reference sequence, i.e., the sequence of ORSV, reached 99%;

[0082] The sequence homology of SEQ ID NO: 12 and the reference sequence CyMV reached 98%;

[0083] The sequence homology of SEQ ID NO: 13 and the reference sequence WCMV reached 61%;

[0084] The sequence homology of SEQ ID NO: 14 and the reference sequence YaVX reached 52%;

[0085] The sequence homology of SEQ ID NO: 15 and the reference sequence PoVX reaches 76%.

[0086] It can be seen that the obtained amplification product SEQ ID NO: 11-15 sequences are partial sequences of ORSV, CyMV, WCMV, YaVX and PoVX, respectively, which proves the reliability of the detection results of the present invention; and the three sequences of WCMV, YaVX and PoVX obtained by small RNA sequencing in this study have a homology of less than 80% with those in NCBI, which are presumed to be new viruses. The present invention designs new virus primers and successfully establishes a multiplex RT-PCR detection system.

[0087] Example 4

[0088] Sensitivity analysis of the detection method of Phalaenopsis virus of the present invention

[0089] The RNA samples in Example 3 were subjected to 10 -1 (2ng), 10 -2 (0.2ng), 10 -3 (0.02ng), 10 -4 (2*10 - 3 ng), 10 -5 (2*10 -4 ng), 10 -6 (2*10 -5 ng), 10 -7 (2*10 -6 ng), 10 -8 (2*10 -7 ng), a series of gradient dilutions, and the steps are the same as those in Example 3, after multiple RT-PCR amplification reactions, and then 1% agarose gel electrophoresis experiments. The experimental results are as follows Figure 2 shown.

[0090] Result analysis: Figure 2 It can be seen that ORSV, CyMV, WCMV, YaVX and PoVX viruses were diluted 10 4 After dilution of 10 5 The band was very faint after fold increase. ORSV was diluted 10 5 The specific bands can still be amplified after the sample is diluted 10 times. 5 The specific band can still be amplified after the sample is diluted 10 5 The specific band can still be amplified after the sample is diluted 10 times. 8 The specific band can still be amplified after the sample is diluted 10 times. 8 The specific band can still be amplified after multiple times. This shows that the present invention has a high sensitivity.

[0091] The invention designs and synthesizes five pairs of corresponding specific primers respectively for the nucleotide sequences of small RNA sequencing of three viruses, namely, ORSV, CyMV, WCMV, YaVX and PoVX, and uses the five pairs of specific primers to simultaneously perform mRT-PCR reaction on ORSV, CyMV, WCMV, YaVX and PoVX in moth orchid leaves, establishes a new PCR reaction system and reaction procedure, enables the mRT-PCR reaction to obtain five amplified fragment sequences of ORSV, CyMV, WCMV, YaVX and PoVX with obvious size differences and lengths of 1156 bp, 908 bp, 561 bp, 292 bp and 162 bp respectively, realizes the goal of simultaneously detecting five moth orchid viruses, namely, ORSV, CyMV, WCMV, YaVX and PoVX, in one reaction, has high detection efficiency, and the reagents used in the invention are commonly used reagents in molecular biology, with low required cost, and even if the RNA contents of the five moth orchid viruses in the sample are very low, the invention can detect them, so the invention has high detection sensitivity.

[0092] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0093] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A primer set for simultaneously detecting multiple Phalaenopsis viruses, characterized in that: Includes the following 5 sets of primer pairs: Forward primer F1 of Jianlan orchid leaf virus: 5'-AGTCTCAACCTGACGTGGAG-3', SEQ ID NO.1, Reverse primer F2 of Jianlan orchid leaf virus: 5'-GGGGAAGCGGTGAATATTA-3', SEQ ID NO.2; Forward primer F3 of tooth blue ring spot virus: 5'-TATGACAAATCCCAGAACGA-3', SEQ ID NO.3, Reverse primer F4 for tooth blue ring spot virus: 5'-GCAAATAGAAACAAACTCCA-3', SEQ ID NO.4; White clover mosaic virus forward primer F5: 5'-GTTGTGCTCCCTGTTGAA-3', SEQ ID NO.5, White clover mosaic virus reverse primer F6: 5′-GTCCCAAATGGATAAGGATAG-3′, SEQ ID NO.6; Forward primer F7 of Chinese yam X virus: 5'-GGCAGAATCTATTGGAAAAA-3', SEQ ID NO.7, Reverse primer F8 for Chinese yam X virus: 5'-CCATGAATGACTAAGGGAAG-3', SEQ ID NO.8; Potato virus X forward primer F9: 5'-GAGGGTCCCACTTTTGATGC-3', SEQ ID NO.9, Potato virus X reverse primer F10: 5'-CAAGCGGTTGGTGATTTTAT-3', SEQ ID NO.

10.

2. Use of the primer set described in claim 1 in the preparation and identification of Phalaenopsis virus gene products.

3. A detection kit for simultaneously detecting multiple Phalaenopsis viruses, characterized in that: Comprising the primer set of claim 1.

4. The kit according to claim 3, characterized in that It also includes Taq enzyme, Taq enzyme buffer, and dNTPs.

5. A method for simultaneously detecting multiple Phalaenopsis viruses, characterized in that: The primer set described in claim 1 or the kit described in any one of claims 3-4 is used to perform mRT-PCR amplification on the virus to be detected, and the amplified product is analyzed by agarose gel electrophoresis to determine the presence or absence of the virus based on the size of the band.

6. The detection method according to claim 5, characterized in that: The mRT-PCR reaction system is composed of the following: The total volume of the reaction system was 50 μL, containing 0.3 μL 5U / μL Taq enzyme, 5 μL 10× Taq enzyme buffer, 4 μL 2.5mmol / L dNTPs, 1 μL 20ng / μL template, 0.2-0.4μL Jianlan orchid leaf virus forward primer, 0.2-0.4μL Jianlan orchid leaf virus reverse primer, 1-2.5μL Tooth orchid ringspot virus forward primer, 1-2.5μL Tooth orchid ringspot virus reverse primer, 0.5-2μL White clover mosaic virus forward primer, 0.5-2μL White clover mosaic virus reverse primer, 1.5-3.5μL Huai Yam X virus forward primer, 1.5-3.5μL Huai Yam X virus reverse primer, 1-3μL Potato X virus forward primer, 1-3μL Potato X virus reverse primer, and the remaining ingredients are sterile double distilled water.

7. The detection method according to claim 6, characterized in that: The initial concentration of all forward primers and reverse primers in the mRT-PCR reaction system before being added to the reaction system is 10 μmol / L, and the concentrations of the forward primer and the reverse primer in each pair of primers in the reaction system are equal.

8. The detection method according to claim 6, characterized in that: In the mRT-PCR reaction system, the final concentrations of the forward primer and the reverse primer of the Cymbidium orchid leaf virus are both 0.04-0.08 μmol / L; the final concentrations of the forward primer and the reverse primer of the Cymbidium orchid ringspot virus are both 0.2-0.5 μmol / L; the final concentrations of the forward primer and the reverse primer of the white clover mosaic virus are both 0.1-0.4 μmol / L; the final concentrations of the forward primer and the reverse primer of the Chinese yam X virus are both 0.3-0.7 μmol / L; and the final concentrations of the forward primer and the reverse primer of the potato X virus are both 0.2-0.6 μmol / L.

9. The detection method according to claim 5, characterized in that: The mRT-PCR reaction procedure is as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 48-56°C for 30 s, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 10 min; and termination of the reaction at 4°C.

10. The detection method according to claim 9, characterized in that: When an electrophoretic band of 908 bp appears, it indicates that the sample contains Cymbidium orchid leaf virus; when an electrophoretic band of 1156 bp appears, it indicates that the sample contains Cymbidium orchid ringspot virus; when an electrophoretic band of 561 bp appears, it indicates that the sample contains white clover mosaic virus; when an electrophoretic band of 292 bp appears, it indicates that the sample contains Chinese yam X virus; when an electrophoretic band of 162 bp appears, it indicates that the sample contains potato X virus.

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