Primer group for synchronously detecting seven soybean viruses based on multiple RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and detection method

Abstract

The invention discloses a primer group for synchronously detecting seven soybean viruses based on multiple RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and a detection method. The viruses comprise a common kidney bean mosaic virus, a soybean mosaic virus, a light cowpea mottle virus, a soybean green disease virus, a soybean chlorotic leaf roll virus and a pueraria lobata mosaic virus) and a citrus related rhabdovirus. According to the detection method, seven pairs of specific primers (SEQ ID NO.1-14) are included, the cross reaction between the primers is effectively overcome, seven soybean virus fragments can be synchronously amplified in a PCR system, stripes are clear and uniform in distribution, and the detection sensitivity reaches the capacity of independent PCR detection. And after a multiplex PCR amplification system is further optimized, the detection can be economically, quickly and effectively carried out within 2 hours. The method disclosed by the invention has very important significance in monitoring the occurrence condition of soybean virus diseases in the field and predicting disease epidemic.

Description

Technical Field

[0001] This invention relates to the field of plant pathogen detection technology, and specifically to primer sets and detection methods for simultaneous detection of seven soybean viruses based on multiplex RT-PCR. Background Technology

[0002] Soybean (Glycine max (L.) Merr) is a globally important oilseed and protein crop, and its production stability is directly related to food security and the agricultural economy. However, soybeans are often threatened by various pathogens during their growth and development, among which soybean viral diseases are the most damaging. Research reports that more than 110 plant viruses can infect soybeans globally, with nearly 50 viruses capable of infecting soybeans under natural conditions. Among these, soybean mosaic virus (SMV), bean common mosaic virus (BCMV), and cowpea mild mottle virus (CPMMV) can cause soybean diseases in many countries and regions worldwide.

[0003] SMV is a single-stranded, positive-sense RNA virus belonging to the genus Pottyvirus in the family Pottyviridae. It is primarily transmitted through seed and non-persistent transmission via aphids, and is the most prevalent and destructive virus in the field. SMV infection can affect the roots, stems, leaves, and seeds of soybean. BCMV belongs to the same genus as SMV, and both are currently the two main Potyviruses causing soybean mosaic disease in China. Both can be transmitted through seeds and aphids. Although BCMV primarily hosts common beans, some strains can cross-host and infect soybeans, causing systemic yellowing and seed-borne infection. In 2021, co-infection of BCMV and SMV was detected in Shandong, China, with a co-infection rate of 24.3%. Currently, BCMV has occurred in soybean-growing areas in Northeast, East, and South China, showing a trend of localized spread. CPMMV is a positive-sense RNA virus belonging to the genus Carlavirus in the family Betaflexiviridae. It is transmitted non-persistently by whiteflies. Since its initial discovery in Ghana in 1973, CPMMV has spread worldwide. This virus can cause stem necrosis in soybeans, significantly reducing plant height, pod number, and thousand-seed weight, resulting in economic losses in soybean production. The virus was first discovered in cowpeas in my country in 2019, and subsequently occurred in other legumes such as soybeans and broad beans. It can co-infect with SMV, leading to more severe symptoms.

[0004] In addition to the known harm caused by viruses to soybeans, in recent years, the inventors have identified a novel recombinant geminivirus derived from the genera *Mastrevirus* and *Begomovirus*—soybean stay-green associated geminivirus (SoSGV). This virus is a single-stranded DNA virus and is currently the main cause of soybean stay-green syndrome (SGS) in the Huang-Huai-Hai region of China. Infected soybeans remain green and cannot be harvested at normal maturity, accompanied by empty pods or shriveled seeds.

[0005] In 2023, a naturally occurring soybean viral disease occurred in the soybean-producing area of ​​Sichuan, China. The disease symptoms were complex, and three newly emerging soybean viruses were identified through deep small RNA sequencing and PCR: kudzu mosaic virus (KuMV), citrus-associated rhabdovirus (CiaRV), and soybean chlorotic leafroll virus (SbCLRV). KuMV is a two-component DNA virus belonging to the genus *Begomovirus* in the family Geminiviridae. It was first reported in 2009 on kudzu plants exhibiting typical leaf vein yellowing symptoms in Fuzhou, Fujian, China. CiaRV is a negative-sense RNA virus belonging to the genus *Cytorhabdovirus* in the family Rhabdoviridae. Discovered and identified in citrus in Chongqing, China in 2018, it also infected passion fruit (Passiflora edulis) and daphne (Edgeworthia chrysantha) nearby. The research team confirmed for the first time that these two viruses can infect soybeans under natural conditions and cause large-scale co-infection with CPMMV. SbCLRV, the first Polerovirus virus identified in Guizhou, China in 2022 capable of infecting soybeans, was detected in a few samples. Infected soybeans often experience co-infection, exhibiting different symptoms depending on the specific virus involved. Some show mosaic and wrinkled leaves, others develop pustular spots along leaf veins, and still others show downward curling and yellowing of leaf edges, affecting soybean yield and quality. Therefore, developing an efficient and accurate multiplex detection technology to achieve simultaneous identification of multiple soybean viruses is of significant practical value for early diagnosis, integrated control, and monitoring of emerging soybean viral diseases.

[0006] Currently, the detection of soybean viruses mainly relies on molecular biology techniques, such as PCR and RT-PCR, and serological methods, such as enzyme-linked immunosorbent assay (ELISA). However, these methods can only detect one virus at a time. For cases of co-infection with multiple viruses, especially in the field where DNA and RNA viruses often co-infect, multiple independent tests are required, leading to low efficiency, increased reagent and consumable costs, and increased sample volume requirements. Furthermore, these detection methods are highly dependent on known viral genome information and cannot effectively identify emerging viruses. Although small RNA deep sequencing technology is widely used for the detection of known viruses and the identification of unknown viruses, this technology has strict requirements for sample quality and a lengthy sequencing cycle, making it difficult to meet the rapid detection needs of large-scale field samples. Therefore, developing a method capable of detecting multiple emerging and common soybean viruses in parallel is crucial.

[0007] Multiplex PCR (mPCR) enables the simultaneous amplification of multiple target genes by adding multiple pairs of specific primers to a single reaction system. Typically, multiplex PCR is mainly used for the detection of fewer than five pathogens. When more than five pathogens are detected, cross-reactivity of the added primers is likely, and differences in the concentration of different pathogens lead to decreased detection sensitivity. Therefore, designing efficient primers and detection techniques for more than five pathogens presents certain challenges. Based on this principle, this patent optimizes primer combinations, reaction conditions, and primer concentrations for seven viruses, establishing for the first time a detection method for seven major prevalent soybean viruses in my country—BCMV, SMV, CPMMV, SbCLRV, KuMV, SoSGV, and CiaRV—using a single RT-PCR reaction. This method has significant practical value for the accurate diagnosis and integrated control of common and emerging soybean viral diseases in the field. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a method for the simultaneous detection of seven major soybean viruses in my country: BCMV, SMV, CPMMV, SbCLRV, KuMV, SoSGV, and CiaRV. This method effectively overcomes cross-reactions between primers and can simultaneously amplify fragments of these seven prevalent soybean viruses in a single PCR system. These include two DNA viruses and five RNA viruses (ranging from 195 to 1127 bp). The bands are clear and uniformly distributed, and the detection sensitivity reaches the capability of standalone PCR detection. The established simultaneous detection method can economically, accurately, efficiently, and sensitively detect major prevalent and emerging soybean viruses in my country within two hours.

[0009] In a first aspect, the present invention provides primers for the simultaneous detection of seven soybean viruses: BCMV, SMV, CPMMV, SoSGV, SbCLRV, KuMV, and CiaRV. The primer pairs used include:

[0010] The nucleotide sequences of the forward and reverse primers of the specific primer pair for amplifying BCMV are shown in SEQ ID NO.6 and SEQ ID NO.7.

[0011] The nucleotide sequences of the forward and reverse primers of the specific primer pair for amplifying SMV are shown in SEQ ID NO.17 and SEQ ID NO.19, respectively.

[0012] The nucleotide sequences of the forward and reverse primers of the specific primer pair for amplifying CPMMV are shown in SEQ ID NO.22 and SEQ ID NO.23.

[0013] The nucleotide sequences of the forward and reverse primers of the specific primer pair for amplifying SoSGV are shown in SEQ ID NO.12 and SEQ ID NO.13.

[0014] The nucleotide sequences of the forward and reverse primers of the specific primer pair for amplifying SbCLRV are shown in SEQ ID NO.14 and SEQ ID NO.16.

[0015] The nucleotide sequences of the forward and reverse primers of the specific primer pair for amplifying KuMV are shown in SEQ ID NO.20 and SEQ ID NO.21.

[0016] The nucleotide sequences of the forward and reverse primers of the specific primer pair for amplifying CiaRV are shown in SEQ ID NO.3 and SEQ ID NO.2.

[0017] Secondly, the present invention provides a reagent kit for the simultaneous detection of seven soybean viruses: BCMV, SMV, CPMMV, SoSGV, SbCLRV, KuMV, and CiaRV, comprising the primers described in the first aspect.

[0018] Thirdly, this invention provides a method for simultaneously detecting seven soybean viruses—BCMV, SMV, CPMMV, SoSGV, SbCLRV, KuMV, and CiaRV—based on multiplex PCR technology, comprising the following steps:

[0019] S1: Extract total RNA from the sample;

[0020] S2: Using the total RNA extracted in step S1 as a template, reverse transcription was performed to obtain cDNA;

[0021] S3: Using the cDNA obtained in step S2 as a template, perform PCR amplification using the primers described in claim 1 to obtain the amplification product;

[0022] S4: Agarose gel electrophoresis was used to detect the amplification products. The target bands of different sizes represent SoSGV 195 bp, SbCLRV 268 bp, KuMV 347 bp, CPMMV 481 bp, SMV 625 bp, BCMV 849 bp, and CiaRV 1127 bp, respectively.

[0023] Furthermore, the method for extracting total nucleic acid from the sample in step S1 is the TRIzol method.

[0024] Furthermore, the system and conditions for reverse transcription in step S2 are as follows: 1 µL of 5 Primer, 2 µg of nucleic acid template, with the sum of their volumes not exceeding 15.75 µL; heat treatment at 70°C for 5 min, followed by cooling on ice for 2 min; add 1.25 µL of dNTP (10 mM), 5 µL of M-MLV RT 5× Buffer, 1 µL of Recombinant Rnase Inhibitor (RRI), 1 µL of M-MLV Reverse Transcriptase to the product, supplement with ddH2O to 25 µL, heat treatment at 42°C for 1 h, and store the obtained cDNA product at -20°C.

[0025] Furthermore, the total volume of the PCR system described in step S3 is 20 µL, containing 10× PCR Buffer (with Mg). 2+ ) 2 μL, dNTP (10 mM) 0.4 μL, 10 μmol·L -1 0.15 µL of upstream and downstream primers for BCMV, SMV, CPMMV, SoSGV, SbCLRV, KuMV and CiaRV, 0.2 µL of Taq plus DNA polymerase, 0.5 µL of cDNA template, and 14.7 µL of ddH2O.

[0026] Furthermore, the PCR amplification reaction conditions described in step S3 are as follows: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 50 s, for a total of 33 cycles; 72℃ overall extension for 10 min.

[0027] Furthermore, the conditions for agarose gel electrophoresis of the PCR reaction products described in step S4 are as follows: 2% agarose gel electrophoresis at a constant voltage of 180 V for 25 min in a 0.5×TBE buffer environment.

[0028] Fourthly, the present invention provides the application of the primers described in the first aspect, the kits described in the second aspect, or the methods described in the third aspect in detecting whether biological samples contain seven soybean viruses: BCMV, SMV, CPMMV, SoSGV, SbCLRV, KuMV, and CiaRV.

[0029] Beneficial effects:

[0030] 1. This invention is the first to propose a multiplex RT-PCR system that can simultaneously detect 7 common and emerging soybean viruses from 6 genera and 5 families in soybeans, covering the major soybean viruses circulating in my country. It can significantly improve diagnostic efficiency and save time and costs to a great extent in the face of complex multi-infection of viruses in the field.

[0031] 2. All primers designed and screened in this invention exhibit good specificity, stability, and compatibility. By comparing conserved regions of the genomic sequences of seven viruses, a 21-25 bp sequence with a CG content of 40%-50% and similar annealing temperatures was selected as primers, avoiding hairpin structures or self-complementarity and cross-complementarity. In particular, by screening and comparing different primer pair combinations, cross-reactivity between primers was effectively overcome, enabling the simultaneous amplification of fragments of seven major soybean viruses circulating in my country within a single PCR system.

[0032] 3. This invention further optimizes the multiplex PCR amplification system and conditions, significantly shortening the detection time (total detection time can be reduced to 2 hours) while ensuring reliable detection results. The reaction system of this invention has good sensitivity, and the lowest effective dilution factor for the 7-fold RT-PCR detection system is 10⁻⁶. -4 times (5×10 -3 (ng / µL).

[0033] 4. The simultaneous rapid detection technology established in this invention uses the extracted total RNA of the sample as a template to achieve simultaneous detection of DNA viruses and RNA viruses.

[0034] Therefore, the seven viruses targeted in this invention represent the major viral diseases currently affecting soybeans in my country, originating from five families and six genera, including Common Bean Mosaic Virus (BCMV), Soybean Mosaic Virus (SMV), Cowpea Light Mottle Virus (CPMMV), Soybean Green Leaf Curl Virus (SoSGV), Soybean Chlorotic Leaf Roll Virus (SbCLRV), Kudzu Mosaic Virus (KuMV), and Citrus-Associated Rumble Virus (CiaRV). This detection method contains seven pairs of specific primers (SEQ ID NO. 1-14), effectively overcoming cross-reactivity between primers. It can simultaneously amplify fragments of seven major prevalent soybean viruses in my country within a single PCR system, including two DNA viruses and five RNA viruses (ranging from 195 to 1127 bp). The bands are clear, uniformly distributed, and the detection sensitivity reaches the reproducibility of single PCR detection. Optimized multiplex PCR amplification systems demonstrate that the optimized multiplex RT-PCR reaction system can economically, rapidly, and effectively detect viral infection in soybean field samples in my country within two hours. This invention is of great significance for monitoring the occurrence of soybean viral diseases in the field and predicting disease outbreaks. Attached Figure Description

[0035] Figure 1 In Example 2, singlet RT-PCR was used to verify the specificity and amplification effect of three sets of viral primers. Lanes A-7 represent: Soybean Green Leaf Roll Virus (SoSGV) 274 bp, Kudzu Mosaic Virus (KuMV) 347 bp, Cowpea Light Mottle Virus (CPMMV) 481 bp, Soybean Mosaic Virus (SMV) 576 bp, Soybean Chlorotic Leaf Roll Virus (SbCLRV) 668 bp, Common Bean Mosaic Virus (BCMV) 887 bp, and Citrus-Associated Rumble Virus (CiaRV) 1062 bp. Lanes B-7 represent: Soybean Chlorotic Leaf Roll Virus (SbCLRV) 268 bp, Kudzu Mosaic Virus (KuMV) 347 bp, Cowpea Light Mottle Virus (CPMMV) 481 bp, Soybean Mosaic Virus (SMV) 576 bp, and Soybean Green Leaf Roll Virus (SoSGV) 666 bp. bp, Common Bean Mosaic Virus (BCMV) 887 bp, Citrus-associated Rumble Virus (CiaRV) 1062 bp; C: Lanes 1-7 are SoSGV 195 bp, SbCLRV 268 bp, KuMV 347 bp, CPMMV 481 bp, SMV 625 bp, BCMV 849 bp, CiaRV 1127 bp respectively; H: Healthy soybean plant; M: DNA marker.

[0036] Figure 2In Example 3, a multiplex RT-PCR detection system was used to screen for the optimal primer combination. cDNA from mixed samples infected with BCMV, SMV, CPMMV, SoSGV, SbCLRV, KuMV, and CiaRV was used as a template for amplification. A, B, and C are the simultaneous amplification results of the three primer combinations in Table 2. Lanes 1 and 2 represent two replicates; M: DNA marker.

[0037] Figure 3 The following is a graph showing the sensitivity determination results of the multiplex RT-PCR system in Example 4 of this disclosure. Lanes 1-8 contain mixed plasmids with amplification fragments of 7 viruses, at concentrations of 5 × 10⁻⁶. 1 ng / µL, 5×10 0 ng / µL, 5×10 -1 ng / µL, 5×10 -2 ng / µL, 5×10 -3 ng / µL, 5×10 -4 ng / µL, 5×10 -5 ng / µL, 5×10 -6 ng / µL; N: negative control; M: DNA marker.

[0038] Figure 4 Example 5 of this disclosure shows the detection results of soybean-related viruses in soybean leaf samples from different regions using multiplex RT-PCR. Lanes 1-5 are samples from soybean producing areas in Sichuan; lane 6 is a sample from Fenhu area in Suzhou; lanes 7 and 8 are laboratory-transmitted insect-borne soybean samples; lane 9 is a healthy soybean plant; lane 10 is a positive control with simultaneous amplification of 7 viruses; M: Trans2K plus DNA marker (Takara, China). Detailed Implementation

[0039] The technical solution of the present invention will be clearly and completely described below with reference to embodiments of the present invention. These are merely some of the specific implementation methods that can be adopted by the present invention, not all of the embodiments, and should not be construed as limiting the innovative solution of the present invention. Any solution that adopts the same inventive concept as the present invention should be included within the protection scope of the present invention.

[0040] Example 1

[0041] This embodiment describes the design of specific detection primers for multiplex RT-PCR. To obtain optimal amplification efficiency and detection sensitivity, 13 specific primers were designed for 7 viruses and divided into 3 groups based on band size. Primer design was performed using Primer 5.0 software. A 21-25 bp sequence with a CG content of 40%-50% and similar annealing temperatures, which does not form hairpin structures or exhibits self-complementarity or cross-complementarity, was selected as the primer. The reference sequences for the primer pairs for the 7 viruses were downloaded from the NCBI database: CiaRV accession number MT302543.1; BCMV accession number KF919300.1; SMV accession number MN539670.1; CPMMV accession number KF554108.1; KuMV accession number DQ641690.1; SbCLRV accession number OM507197.1; and SoSGV accession number OQ079703.1. All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., with a concentration of 10 μmol·L⁻¹. -1 Store at -20℃ for later use. Primer sequences and amplification lengths are shown in Table 1.

[0042] Table 1. Information on multiplex RT-PCR primers for 7 soybean viruses

[0043]

[0044] Example 2

[0045] This embodiment performed singlet RT-PCR validation to test the specificity and amplification of the primers in Table 1. RT-PCR amplification was performed on the corresponding positive soybean samples using virus-specific primers. cDNA from healthy samples was used as a negative control. The specific steps are as follows:

[0046] 1. Total RNA was extracted from the samples using the TRIzol method.

[0047] Weigh 0.08-0.1 g of soybean leaves and place them in a 2.0 mL centrifuge tube. Quickly freeze the tube in liquid nitrogen. Add 3-4 small steel balls and grind the leaf tissue into a fine powder using a grinder at 50 Hz for 30 s, repeating 2-3 times. Add 1 mL of Trizol and vortex for 15 s until no obvious clumps remain. Let the mixture stand at room temperature for 5 min. Add 200 µL of chloroform (1 / 5 volume of the lysis buffer) to the lysis buffer and vortex for 30 s until an emulsion forms. Let the mixture stand at room temperature for 5 min. Centrifuge the mixture at 12000 rpm for 15 min at 4°C. The mixture will separate into three layers, with the upper aqueous phase containing RNA. Carefully pipette 500 µL of the aqueous phase and add an equal volume of isopropanol. Invert the tube repeatedly to mix the solution thoroughly and let it stand at room temperature for 10 min (a faint white flocculent substance may be visible). Centrifuge at 12000 rpm for 10 min at 4°C. A white precipitate of varying sizes is usually visible at the bottom of the tube. Collect the precipitate and discard the supernatant. Add 1 mL of 75% ethanol, shake and tap or gently pipette to suspend the precipitate, inverting several times. Centrifuge at 12000 rpm for 5 min at 4°C, discard the supernatant, cap the tube, centrifuge at 4°C for 30 s to collect the ethanol at the bottom of the tube, aspirate the remaining liquid with a pipette, and retain the precipitate. Dry the precipitate in a clean environment at room temperature for 2-5 min, add 60-100 µL of RNase-free ddH2O to dissolve the precipitate, and store the obtained total RNA product at -80°C.

[0048] 2. Obtaining cDNA template via RNA reverse transcription

[0049] cDNA was synthesized using M-MLV reverse transcriptase. Reaction system and conditions: Primer 1 µL, nucleic acid template 2 µg, the total volume of both not exceeding 15.75 µL; heat at 70℃ for 5 min, then cool on ice for 2 min; add 1.25 µL of dNTP (10 mM), 5 µL of M-MLV RT 5× Buffer, 1 µL of Recombinant Rnase Inhibitor, 1 µL of M-MLV Reverse Transcriptase to the product, add ddH2O to a final volume of 25 µL, heat at 42℃ for 1 h, and store the resulting cDNA product at -20℃.

[0050] 3. PCR amplification

[0051] Using the cDNA obtained in step 2 as a template, PCR amplification was performed using the primers described in Table 1. The system was as follows: 10×PCR Buffer (with Mg) 2+ 1 μL of dNTPs (10 mM), 0.2 μL of dNTPs (10 mM), 0.1 μL of Taq plus DNA polymerase, and 10 μmol·L⁻¹ virus-specific primers.-1 Add 0.2 μL of each of the following: cDNA template and 0.5 μL of ddH2O to a final volume of 10 μL.

[0052] Amplification program: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 50 s, for a total of 33 cycles; 72℃ extension for 10 min. After completion, the PCR products were electrophoresed on a 1.5% agarose gel at 180 V for 20 min in 0.5×TBE buffer, and the results were observed.

[0053] The results are as follows Figure 1 As shown, all primer pairs were able to amplify the target fragment of the corresponding size in the tested samples, with no nonspecific background bands. No amplification products were obtained in healthy plants. However, the band spacing of different primer combinations varied, and subsequent multiplex RT-PCR tests were used to screen primer combinations.

[0054] 4. Verification of the viral sequence of the sample to be tested.

[0055] After the target fragment amplified by RT-PCR was excised and recovered from the gel, it was ligated into the pMDTM19-T Vector cloning vector and transformed into DH5α. Escherichia coli competent cells. Positive clones were identified and screened by colony PCR, and plasmids were extracted and sequenced. The sequences were verified by BLAST search of the NCBI nucleotide database (https: / / blast.ncbi.nlm.nih.go-v / Blast.cgi). The results showed that the target fragment amplified by singleton PCR was a relevant viral sequence, and the above primer pairs can be used for RT-PCR detection of relevant viruses.

[0056] Example 3

[0057] This embodiment screened for the optimal primer combination by simultaneously amplifying seven soybean viruses. The specific combinations are shown in Table 2.

[0058] Table 2. Primer combinations for multiplex RT-PCR

[0059]

[0060] To identify soybean samples infected with seven viruses (CiaRV, SbCLRV, KuMV, SoSGV, SMV, BCMV, and CPMMV), total nucleic acids were mixed in equal proportions (m=2 µg) and reverse transcribed to obtain cDNA products. Using these cDNA products as templates, the three sets of virus-specific primers listed in Table 2 were thoroughly mixed and added to a multiplex RT-PCR system, with each set amplified in duplicate. The specific steps are as follows:

[0061] 1. Total RNA was extracted from the samples using the TRIzol method.

[0062] Same as step 1 in Example 2.

[0063] 2. Obtaining cDNA template via RNA reverse transcription

[0064] Same as step 2 in Example 2.

[0065] 3. Multiplex RT-PCR

[0066] The multiplex RT-PCR detection system is as follows: 20 µL Taq Plus system: 10× PCR Buffer (with Mg) 2+ ) 2 μL, dNTP (10 mM) 0.4 μL, 10 μmol·L -1 0.15 µL of upstream and downstream primers for BCMV, SMV, CPMMV, SoSGV, SbCLRV, KuMV and CiaRV, 0.2 µL of Taq plus DNA polymerase, 0.5 µL of cDNA template, and 14.7 µL of ddH2O.

[0067] The multiplex RT-PCR amplification conditions were as follows: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 50 s, for a total of 33 cycles; and 72℃ final extension for 10 min. After completion, the PCR products were electrophoresed on a 2% agarose gel at 180 V for 25 min in 0.5×TBE buffer, and the results were observed.

[0068] The results are as follows Figure 2 As shown. When the primer addition amount was 0.15 µL, SbCLRV and SoSGV were not detected by the specific primers in group A. Figure 2 (A) After shortening the amplified sequence of SbCLRV from 668 bp to 268 bp, an effective amplification was obtained. Figure 2 Group B). The Rep gene of SoSGV was not effectively detected in the specific primers of Group B, and the amplification effect of CiaRV was poor. Figure 2 (B) The C group primers changed the amplification target of SoSGV from the Rep gene to the CP gene and shortened the target fragment length, increasing the spacing between the CiaRV and BCMV amplification bands. This achieved, for the first time, simultaneous amplification of seven viruses, with uniform spacing, clear distribution, and reproducibility, meeting the expected results. Figure 2 (C). This embodiment screened the optimal primer combination for simultaneous amplification using multiplex RT-PCR, and clarified the amplification system and reaction conditions for multiplex detection.

[0069] Example 4

[0070] This example analyzes the sensitivity of the multiplex RT-PCR system.

[0071] The recombinant plasmid containing the target sequences of seven soybean viruses from step 4 of Example 2 was subjected to concentration determination (50 ng / µL) and then serially diluted to 10 ng / µL. 0 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 There are a total of 8 gradients. Diluents of the same gradient are mixed in equal proportions, and the recombinant plasmids of these 8 gradients are amplified using the multiplex RT-PCR detection system and conditions in step 3 of Example 3.

[0072] The results are as follows Figure 3 As shown, in the established multiplex RT-PCR detection system, the effective detection dilution factor excluding SMV is 10. -4 times (5×10 -3 (ng / µL), the other 6 viruses at a dilution factor of 10 -5 times (5×10 -4 Even at concentrations of ng / µL, effective bands were still detected, with CiaRV, BCMV, CPMMV, and KuMV being relatively clear. The lowest effective dilution for the 7-fold RT-PCR assay was 10⁻⁶. -4 times (5×10 -3 The established multiplex RT-PCR system (ng / µL) has sufficient sensitivity and is suitable for virus diagnosis of routine samples.

[0073] Example 5

[0074] This embodiment provides an application example of multiplex RT-PCR detection of soybean-related viruses.

[0075] 5. We used the multiplex RT-PCR system established in Example 3 to clean and disinfect soybean samples collected from soybean-producing areas in Sichuan, Fenhu in Suzhou, and those infected with insect-borne viruses in the laboratory. Three leaves from each region and each insect-borne sample were used as replicates, and 0.1g of the mixture was weighed for subsequent RNA extraction. The total RNA extraction method, reverse transcription, and PCR methods and procedures are described in Example 2.

[0076] The results are as follows Figure 4As shown, samples from soybean producing areas in Sichuan Province were mostly found to contain co-infections of CPMMV and CiaRV, with different combinations of viruses involved. Samples from Suzhou Province were found to contain BCMV alone. In laboratory-transmitted soybeans, KuMV and SoSGV were detected, consistent with the observed viral phenotypes. These virus detection results are consistent with previous singleton PCR detection results, indicating that the multiplex RT-PCR detection system established in this invention can be used for the detection of field standards, and the results are reliable.

[0077] In summary, the primer set and multiplex RT-PCR detection system disclosed herein can be used to simultaneously detect seven major soybean viruses prevalent in my country. This method is more efficient, economical, and convenient than conventional PCR, and can meet the requirements of simultaneous detection of multiple pathogens in production.

[0078] It should be understood that the various forms of processes shown above can be used to rearrange, add, or delete steps. For example, the steps described in this disclosure can be executed in parallel, sequentially, or in different orders, as long as the desired result of the technical solution of this disclosure can be achieved, and this is not limited herein.

Claims

1. A primer combination for multiplex RT-PCR for simultaneous detection of seven soybean viruses of BCMV, SMV, CPMMV, SoSGV, SbCLRV, KuMV and CiaRV, characterized by: The primer pair comprises: The nucleotide sequence of the forward and reverse primers of the specific primer pair for amplifying BCMV is shown in SEQ ID NO. 6 and SEQ ID NO. 7; The nucleotide sequence of the forward and reverse primers of the specific primer pair for amplifying SMV is shown in SEQ ID NO. 17 and SEQ ID NO. 19; The nucleotide sequence of the forward and reverse primers of the specific primer pair for amplifying CPMMV is shown in SEQ ID NO. 22 and SEQ ID NO. 23; The nucleotide sequence of the forward and reverse primers of the specific primer pair for amplifying SoSGV is shown in SEQ ID NO. 12 and SEQ ID NO. 13; The nucleotide sequence of the forward and reverse primers of the specific primer pair for amplifying SbCLRV is shown in SEQ ID NO. 14 and SEQ ID NO. 16; The nucleotide sequence of the forward and reverse primers of the specific primer pair for amplifying KuMV is shown in SEQ ID NO. 20 and SEQ ID NO. 21; The nucleotide sequence of the forward and reverse primers of the specific primer pair for amplifying CiaRV is shown in SEQ ID NO. 3 and SEQ ID NO.

2.

2. A kit for multiplex RT-PCR for simultaneous detection of seven soybean viruses of BCMV, SMV, CPMMV, SoSGV, SbCLRV, KuMV and CiaRV, characterized in that, The kit comprises the primer combination of claim 1, and optionally, detection reagents such as total RNA extraction reagents, reverse transcription reagents.

3. A method for simultaneous detection of seven soybean viruses of BCMV, SMV, CPMMV, SoSGV, SbCLRV, KuMV and CiaRV based on multiplex PCR technology, characterized in that: The primer combination of claim 1 is used to detect the soybean sample to be detected in the same PCR reaction.

4. The method of claim 3, wherein, The kit comprises the following steps: The extracted total RNA of the soybean sample to be detected is used as a template for reverse transcription to obtain cDNA, and the primer of claim 1 is used for PCR amplification to obtain an amplification product; The amplification product is detected for judgment.

5. The method of claim 4, wherein, The method for detecting the amplification product is sequencing or agarose gel electrophoresis; Specifically, if the 195 bp target amplification product exists, it indicates that the sample contains SoSGV; If the 268 bp target amplification product exists, it indicates that the sample contains SbCLRV; If the 347 bp target amplification product exists, it indicates that the sample contains KuMV; If the 481 bp target amplification product exists, it indicates that the sample contains CPMMV; If the 625 bp target amplification product exists, it indicates that the sample contains SMV; If the 849 bp target amplification product exists, it indicates that the sample contains BCMV; If the 1127 bp target amplification product exists, it indicates that the sample contains CiaRV.

6. The method of claim 4, wherein, The system and conditions for reverse transcription are as follows: 5 Primer 1 μL, nucleic acid template 2 μg, the sum of the volumes of the two is not more than 15.75 μL, 70℃ heating treatment for 5 min, cooling on ice for 2 min; adding dNTP (10 mM) 1.25 μL, M-MLV RT 5 × Buffer 5 μL, Recombinant RnaseInhibitor (RRI) 1 μL, M-MLV Reverse Transcriptase 1 μL, and supplementing ddH2O to 25 μL, 42℃ heating treatment for 1 h, and storing the obtained cDNA product at -20℃; In PCR amplification, the total volume of the amplification system is 20 µL, containing 10× Mg2+. 2+ PCR Buffer2 μL, 10 mMdNTP0.4 μL, 10 μmol·L -1 0.15 µL of upstream and downstream primers for BCMV, SMV, CPMMV, SoSGV, SbCLRV, KuMV and CiaRV, 0.2 µL of Taq plus DNA polymerase, 0.5 µL of cDNA template, and 14.7 µL of ddH2O.

7. The method of claim 4, wherein: The reaction conditions for PCR amplification are as follows: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 50 s, a total of 33 cycles; and 72℃ overall extension for 10 min.

8. The method of claim 5, wherein: The conditions for agarose gel electrophoresis are as follows: 2% agarose gel in 0.5 × TBE buffer environment, 180 V constant voltage electrophoresis for 25 min.

9. The method according to any one of claims 3 to 8, wherein, The soybean samples are soybean leaves, pods and seeds, which are collected from fields or imported soybeans.

10. Use of the primer combination according to claim 1 or the kit according to claim 2 or the method according to any one of claims 3-9 for simultaneously detecting whether a biological sample contains seven viruses of BCMV, SMV, CPMMV, SoSGV, SbCLRV, KuMV and CiaRV.

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