PCR primers, kit for simultaneously detecting multiple free banana streak virus and application thereof

Abstract

The application discloses a PCR primer, a kit for simultaneously detecting multiple free-state banana streak virus and application thereof, and belongs to the technical field of banana virus detection. The application optimizes the primer concentration and annealing temperature conditions in the PCR system, determines that the upstream primer concentration and the downstream primer concentration of BSOLV, BSGFV and BSIMV are respectively 0.25 muM / 0.25 muM, 0.1 muM / 0.1 muM and 0.25 muM / 0.25 muM, and the amplification effect is best when the annealing temperature is 56.5 DEG C. The multiple PCR system has a detection sensitivity of 1 x 10 3 copies per muL, has good specificity, and can accurately and sensitively detect the three free-state different BSVs in banana samples. Therefore, the multiple PCR system is suitable for rapidly detecting the three free-state viruses in field banana samples.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of banana virus detection, and particularly relates to a PCR primer and kit for simultaneously detecting multiple free banana streak viruses and application thereof. BACKGROUND

[0002] Banana streak virus (BSV) belongs to Caulimoviridae, Badnavirus, and is a double-stranded circular DNA virus with a size of 7-9 kb (Harper et al, 2005; Fei Jifeng et al, 2001). So far, 11 types of BSVs that can cause banana streak have been reported, and banana streak OL virus (BSOLV), banana streak GF virus (BSGFV) and banana streak IM virus (BSIMV) are three major BSVs that harm bananas. The three BSVs can be integrated into the banana genome and can be released from the banana genome to infect bananas under environmental stress (Chabannes et al, 2013), causing banana streak. Free BSVs include BSVs released from the banana genome and BSVs from external infection, and can infect bananas alone or in combination to cause banana streak, but the symptoms produced on banana plants are often different due to differences in banana varieties, virus types and natural environment (Fan Wubuo et al, 2007). Some bananas infected by BSVs are symptomless; some have broken or continuous yellowing stripes on the leaves, and some have necrotic spots on the banana leaves in the later stage (Dahal et al, 1998). The initial symptoms of banana streak are often difficult to distinguish from the leaf symptoms caused by cucumber mosaic virus (CMV), but BSVs will cause necrosis on bananas while CMV will not (He Yunwei et al, 2009).

[0003] Banana is an important economic crop in China, and BSV is one of the important viruses that restricts the production of banana and affects the development of banana industry in China. Banana streak disease caused by BSV has the characteristics of serious damage, difficult prevention and control, and easy to occur. Banana plants infected with banana streak disease grow slowly, yield and quality decrease, and there is no effective pesticide to prevent and control banana streak disease at present. Because most of the bananas planted in China are derived from banana test-tube seedlings produced by tissue culture, the virus in banana suckers or seedlings is easy to be transmitted to the offspring; and in the process of tissue culture, BSV integrated in the banana genome can be released to infect banana. Therefore, establishing an effective virus detection method can eliminate virus-carrying banana seedlings in time, which is an important prevention and control measure to prevent the occurrence and spread of the disease.

[0004] At present, the main virus detection methods are serological and molecular biological methods. Molecular biological methods are more sensitive than serological methods, and polymerase chain reaction (PCR) as a representative of molecular biological methods is widely used in the detection of different viruses (Li Y et al., 2011). Single PCR can only detect one virus, while multiplex PCR can detect multiple viruses at the same time, reducing the detection time and saving the detection cost. This method has the characteristics of sensitivity, rapidness, high efficiency, etc., and is suitable for rapid detection of banana samples. Therefore, it has broad prospects to study and design a PCR primer, kit for simultaneous detection of multiple free-state banana streak viruses and its application.

[0005] References:

[0006] [1] Chabannes M, Baurens FC, Duroy PO, et al. Three infectious viral species lying in wait in the banana genome. J Virol. 2013, 87(15): 8624-37.

[0007] [2] Dahal G, Hughes JD, Thottappilly G, et al. Effect of temperature on symptom expression and reliability of banana streak badnavirus detection in naturally infected plantain and banana (Musa spp.). Plant Dis. 1998, 82(1): 16-21.

[0008] [3] Harper G, Hart D, Moult S, et al. The diversity of banana streak virus isolates in Uganda Archives of Virology, 2005, 150(12): 2407-2420.

[0009] [4] Fan W B, Wu D Q, Wang J H, et al. Research progress of banana streak virus and its disease. Tropical Agricultural Science, 2007, 27(5): 58-63.

[0010] [5] Fei J F, Xiao H G, Li H P, et al. Research progress of banana streak virus disease. Virus Journal, 2001, (4): 381-385.

[0011] [6] He Y W, Chen X, Ruan X L, et al. Prokaryotic expression of ORF II gene of banana streak virus and preparation of antibody. Plant Disease Report, 2009, 39(1): 100-103.

[0012] [7] Li Y Y, Wu Z Q, Li Y, et al. Multiplex PCR detection of three major viruses of banana. Plant Quarantine, 2011, 25(4): 39-41. SUMMARY

[0013] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present application is to provide a PCR primer for simultaneously detecting multiple free banana streak virus.

[0014] Another purpose of the present application is to provide a kit for simultaneously detecting multiple free banana streak virus.

[0015] Another purpose of the present application is to provide the application of the above-mentioned PCR primer or kit.

[0016] Still another purpose of the present application is to provide a method for simultaneously detecting multiple free banana streak virus.

[0017] The purpose of the present application is achieved by the following technical solutions:

[0018] A PCR primer for simultaneously detecting multiple free banana streak virus, comprising at least one of a BSOLV primer group, a BSGFV primer group and a BSIMV primer group; the BSOLV primer group comprises primers BSOLV-F / R;

[0019] BSOLV-F: 5'-GAAGAGCATGGGCCTCAGAA-3';

[0020] BSOLV-R; 5'-CGCATGCCTTGCAGATAGTC-3';

[0021] The BSGFV primer set comprises BSGFV-F / R;

[0022] BSGFV-F: 5'-GCCTTCATGACCTGGCGAAT-3';

[0023] BSGFV-R; 5'-ATGTACGGGTGTATTGCCGC-3';

[0024] The BSIMV primer set comprises BSIMV-F / R;

[0025] BSIMV-F: 5'-TCTTAGACACAGGAGCTGCCA-3';

[0026] BSIMV-R: 5'-ATTCCTCCTTCCATTGCACG-3';

[0027] The PCR primer is used for preparing a kit for simultaneously detecting multiple free banana streak viruses.

[0028] A kit for simultaneously detecting multiple free banana streak viruses comprises the PCR primer.

[0029] The kit further comprises reagents required for PCR reaction; further 2x Surper Taq PCR StarMix.

[0030] The kit further comprises positive control, negative control, and blank control; wherein the positive control is Brazilian banana DNA infected by three viruses of BSOLV, BSGFV, and BSIMV; the negative control is healthy Brazilian banana DNA; and the blank control is sterile double distilled water.

[0031] The PCR primer or the kit is used for detecting multiple free banana streak viruses in bananas.

[0032] The banana streak virus comprises at least one of BSOLV, BSGFV, and BSIMV.

[0033] A method for simultaneously detecting multiple free banana streak viruses comprises the following steps:

[0034] (1) Preparation of virus particles in a sample to be tested;

[0035] (2) Using the virus particles in step (1) as a template, the PCR primer is used for reaction to obtain a PCR amplification product;

[0036] (3) According to the amplification result, it is determined whether the free state banana line virus is contained in the sample to be tested, wherein, each batch of experiment must simultaneously make positive control, negative control and blank control, and the three controls must be completely consistent, and the result is confirmed to be reliable, and the positive plasmid is used as the template.

[0037] In step (1), the preparation method of virus particles in the sample to be tested comprises the following steps:

[0038] The sample to be tested is taken, and the grinding buffer is added according to the ratio of 100 mg:1 mL, and then incubated at room temperature, washed for the first time, and then treated with DNase mix to remove residual banana genome DNA; after DNase inactivation, washed for the second time to obtain virus particles.

[0039] Preferably, the grinding buffer is 2% PVP, 0.2% BSA and 0.2% Na2SO3 added in PBST buffer.

[0040] Preferably, the incubation time is 2h; the first washing is PBST washing for 3 times and ddH2O washing for 1 time; the treatment condition of DNase mix is 37℃ for 1 hour; the inactivation condition is 95℃ for 10min; the second washing is ddH2O washing for 1 time.

[0041] In step (2), the molar ratio of BSOLV primer group, BSGFV primer group and BSIMV primer group in the reaction system is (2-5):(2-5):(2-5); preferably 5:2:5.

[0042] Further, the final concentration of each primer of BSOLV primer group in the reaction system is 0.1-0.25μM, the final concentration of each primer of BSGFV primer group is 0.1-0.25μM, and the final concentration of each primer of BSIMV primer group is 0.1-0.25μM; preferably, the final concentration of each primer of BSOLV primer group in the reaction system is 0.25μM, the final concentration of each primer of BSGFV primer group is 0.1μM, and the final concentration of each primer of BSIMV primer group is 0.25μM.

[0043] Further, the reaction system is 20μL, 2×Surper Taq PCR StarMix 10μL, the final concentration of each primer of BSOLV-F / R is 0.1-0.25μM, the final concentration of each primer of BSGFV-F / R is 0.1-0.25μM, the final concentration of each primer of BSIMV-F / R is 0.1-0.25μM, and the virus particles are used as the template, and ddH2O is supplemented to 20μL.

[0044] Preferably, the reaction system is 20 μL, 2×Surper Taq PCR StarMix is 10 μL, the final concentration of primers BSOLV-F / R is 0.25 μM respectively, the final concentration of primers BSGFV-F / R is 0.1 μM respectively, the final concentration of primers BSIMV-F / R is 0.25 μM respectively, the virus particle is used as a template, and ddH2O is supplemented to 20 μL.

[0045] In step (2), the reaction conditions are as follows: pre-denaturation at 94℃ for 2 min, denaturation at 94℃ for 15 s, annealing at 55-65℃ for 15 s, extension at 72℃ for 15 s, and the cycle number is 40; and the final extension is at 72℃ for 5 min.

[0046] Further, in step (2), the reaction conditions are as follows: pre-denaturation at 94℃ for 2 min, denaturation at 94℃ for 15 s, annealing at 55-61℃ for 15 s, extension at 72℃ for 15 s, and the cycle number is 40; and the final extension is at 72℃ for 5 min.

[0047] Still further, in step (2), the reaction conditions are as follows: pre-denaturation at 94℃ for 2 min, denaturation at 94℃ for 15 s, annealing at 55-58.4℃ for 15 s, extension at 72℃ for 15 s, and the cycle number is 40; and the final extension is at 72℃ for 5 min.

[0048] Still further, in step (2), the reaction conditions are as follows: pre-denaturation at 94℃ for 2 min, denaturation at 94℃ for 15 s, annealing at 56.5℃ for 15 s, extension at 72℃ for 15 s, and the cycle number is 40; and the final extension is at 72℃ for 5 min.

[0049] In step (3), the banana streak virus includes at least one of BSOLV, BSGFV and BSIMV.

[0050] In step (3), the method for determining whether the free banana streak virus is contained in the sample according to the amplification result is an electrophoresis detection technology; the PCR amplification product of step (2) is observed by electrophoresis, if at least one of specific bands of 744 bp, 480 bp and 256 bp is amplified, it is a positive reaction, indicating that the sample contains at least one of BSOLV, BSGFV and BSIMV; if no specific bands of 744 bp, 480 bp and 256 bp are amplified, it is a negative reaction, indicating that the sample does not contain BSOLV, BSGFV and BSIMV.

[0051] The present application has the following advantages and effects relative to the prior art:

[0052] The application optimizes the primer concentration and annealing temperature conditions in the PCR system, and establishes a simple and accurate multiplex PCR method for detecting three different BSVs in banana. 3 The detection sensitivity of the multiplex PCR system is 1x10 3 The multiplex PCR is suitable for rapid detection of the three different BSVs in banana samples in the field. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 is the total DNA agarose gel electrophoresis detection result of banana leaves;

[0054] Figure 2 is the single PCR detection result; Note: M: molecular weight standard DL 2000; Lanes 1-3: BSOLV, BSGFV, BSIMV; N: negative control; CK: blank control.

[0055] Figure 3 is the multiplex PCR primer concentration optimization; Note: M: molecular weight standard DL 2000; Lanes 1-5 correspond to treatment 1-treatment 5; N: negative control; CK: blank control.

[0056] Figure 4 is the multiplex PCR annealing temperature optimization; Note: M: molecular weight standard DL 2000; Lanes 1-7: 65℃, 64℃, 62.9℃, 60.9℃, 58.4℃, 56.5℃, 55℃; N: negative control; CK: blank control.

[0057] Figure 5 is the multiplex PCR specificity detection of three BSV plasmid DNAs; Note: M: molecular weight standard DL 2000; Lane 1: BSOLV+BSGFV; Lane 2: BSGFV+BSIMV; Lane 3: BSOLV+BSIMV; Lane 4: BSOLV+BSGFV+BSIMV; Lane 5: BSOLV; Lane 6: BSGFV; Lane 7: BSIMV; Lane 8: CMV-CP plasmid; N: negative control; CK: blank control.

[0058] Figure 6RT-PCR detection of CMV in Brazilian banana; Note: M: molecular weight marker DL 2000; Lanes 1-5: CMV-infected Brazilian banana; P: CMV positive control; N: negative control; CK: blank control; Lane 6: BSOLV, BSGFV and BSIMV complex-infected Brazilian banana.

[0059] Figure 7 Specific amplification of three BSV multiplex PCR in CMV-infected Brazilian banana; Note: M: molecular weight marker DL Maker 5000; Lane 1: CMV positive control; Lanes 2-6: CMV-infected Brazilian banana; P: BSOLV, BSGFV and BSIMV complex-infected Brazilian banana; N: negative control; CK: blank control.

[0060] Figure 8 Sensitivity of multiplex PCR; Note: M: molecular weight marker DL 2000; Lanes 1-10: 1 x 10 10 1 copies / μL; N: negative control; CK: blank control.

[0061] Figure 9 Application of multiplex PCR; Note: M: molecular weight marker DL 2000; Lanes 1-9: field banana samples; P: positive control; N: negative control; CK: blank control. DETAILED DESCRIPTION

[0062] The application will be further described in conjunction with the examples and drawings, but the embodiments of the application are not limited thereto.

[0063] The test methods in the following examples, unless otherwise specified, are generally in accordance with conventional test procedures or as suggested by the manufacturer. The materials, reagents and the like used are those available commercially unless otherwise specified.

[0064] BSOLV, BSGFV and BSIMV used in the examples are disclosed in the literature “Rao X, Wu Z, Wang W, Zhou L, Sun J, Li H*. Genetic diversity analysis reveals new badnaviruses infecting banana in South China. J Plant Pathol, 2020, 102: 1065-1075”.

[0065] CMV used in the examples is disclosed in the patent document “201610826246.3, A method for detecting banana cucumber mosaic virus by immunocapture-reverse transcription loop-mediated isothermal amplification reaction”. ​

[0066] Example 1

[0067] I. Materials and Methods

[0068] 1.1 Materials

[0069] The test plants were banana samples collected from fields with obvious symptoms of banana streak and suspected symptoms of banana streak.

[0070] Reagents: New plant total DNA extraction kit, DNase mix without RNAase, purchased from Tiangen Biosciences Co., Ltd. (Beijing); 2x Surper Taq PCR StarMix, PMD18T vector, purchased from Beijing Genstar Biotech Co., Ltd. (Genstar). One-step reverse transcription kit PrimeScript R One Step RT-PCR Kit Ver.2, purchased from Dalian Baobioengineering Biotechnology Co., Ltd.

[0071] 1.2 Methods

[0072] 1.2.1 Primer design and synthesis

[0073] According to the conserved sequences of coat protein (CP) and RNase H (RH) of BSOLV, BSGFV and BSIMV in GenBank, primers were designed. According to the conserved sequence of coat protein (CP) of CMV in GenBank, primers were designed. Primer synthesis was performed by Shanghai Biomed Engineering Co., Ltd. The primer sequences are shown in Table 1.

[0074] Table 1 Specific primers for three banana streak viruses and CMV

[0075]

[0076] 1.2.2 Extraction of nucleic acids

[0077] DNA extraction was performed using the new plant total DNA extraction kit from Tiangen, and the specific operation was referred to the instruction manual. The extracted DNA was determined for concentration and purity, and then stored at -20℃ for later use.

[0078] RNA extraction was performed using the plant tissue RNA extraction kit from Tiangen, and the specific operation was referred to the instruction manual. The extracted RNA was determined for concentration and purity, and then stored at -20℃ for later use.

[0079] 1.2.3 Single PCR and RT-PCR

[0080] The healthy Brazilian banana was used as negative control (N), and sterile double distilled water was used as blank control (CK). The virus particles prepared from the Brazilian banana samples infected with free BSV (BSOLV, BSGFV and BSIMV) were used as templates, and the specific primers of 1.2.1 were used for single PCR amplification. The reaction system was 20 μL, including 2×SurperTaq PCR StarMix 10 μL, 0.5 μL of each of the upstream and downstream primers (10 μM) (0.25 μM final concentration), virus particles or plasmid DNA as template, and ddH2O to make up to 20 μL. The reaction conditions were 94°C pre-denaturation for 2 min, 94°C denaturation for 15 s, 58°C annealing for 15 s, 72°C extension for 15 s, 40 cycles, and 72°C final extension for 5 min. After the PCR amplification reaction was completed, 10 μL of the amplification reaction product was subjected to agarose gel electrophoresis detection.

[0081] The RNA extracted from the CMV-infected Brazilian banana sample was used as a template for RT-PCR amplification. The 10 μL RT-PCR reaction system was as follows: 2×1Step Buffer 5 μL; 0.4 μL of each of the upstream and downstream primers (10 μmol / L) (0.4 μM final concentration); RNA template 1 μL; PrimeScript 1Step Enzyme Mix (Dalian Baobio) 0.2 μL; and RNase-free dH2O to make up to 10 μL. The RT-PCR reaction program was as follows: 50°C reverse transcription for 30 min, 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; and 72°C final extension for 5 min. After the reaction was completed, agarose gel electrophoresis detection was performed. The CMV-infected Brazilian banana was used as positive control; the healthy Brazilian banana was used as negative control; and sterile double distilled water was used as blank control.

[0082] 1.2.4 Construction of recombinant plasmid

[0083] The amplified products from electrophoresis were recovered and connected to PMD-18T cloning vector, the connection system: target fragment 5 μL, vector 1 μL, solution 4 μL. After mixing the system gently, it was connected at 16℃ overnight. The connection product was transformed into E. coli Trans 5a for culture, and the positive clone product was screened by blue-white spot, and the white colonies on the screening plate were inoculated into LB liquid medium for expansion. The bacterial liquid PCR was identified by specific primers of BSV (BSOLV-F / R, BSGFV-F / R, BSIMV-F / R) and specific primers of CMV (CMV-F / R), and the plasmid was extracted by using the plasmid extraction kit and sent to the Bioengineering Co., Ltd. for sequencing. The fragment amplified by CMV-F / R was named as CMV-CP plasmid.

[0084] 1.2.5 Optimization and establishment of multiplex PCR detection reaction conditions

[0085] Healthy Brazilian banana was used as negative control (N), and sterile double distilled water was used as blank control (CK); 1 μL BSOLV, BSGFV and BSIMV mixed plasmid DNA was used as template, and the multiplex PCR reaction was carried out by adding specific primers of the above three viruses according to the single PCR reaction system. Several main conditions (primer addition amount, annealing temperature) that could affect multiplex PCR were optimized in turn, and other reaction conditions should be kept unchanged when optimizing a certain condition.

[0086] The use amount ratio of primers BSOLV-F / R, BSGFV-F / R, BSIMV-F / R was set as five treatments: treatment one: 0.5 μL / 0.5 μL (0.25 μM / 0.25 μM): 0.5 μL / 0.5 μL (0.25 μM / 0.25 μM): 0.5 μL / 0.5 μL (0.25 μM / 0.25 μM); treatment two: 0.2 μL / 0.2 μL (0.1 μM / 0.1 μM): 0.5 μL / 0.5 μL (0.25 μM / 0.25 μM): 0.5 μL / 0.5 μL (0.25 μM / 0.25 μM); treatment three: 0.5 μL / 0.5 μL (0.25 μM / 0.25 μM): 0.2 μL / 0.2 μL (0.1 μM / 0.1 μM): 0.5 μL / 0.5 μL (0.25 μM / 0.25 μM); treatment four: 0.5 μL / 0.5 μL (0.25 μM / 0.25 μM): 0.5 μL / 0.5 μL (0.25 μM / 0.25 μM): 0.2 μL / 0.2 μL (0.1 μM / 0.1 μM); treatment five: 0.2 μL / 0.2 μL (0.1 μM / 0.1 μM): 0.2 μL / 0.2 μL (0.1 μM / 0.1 μM): 0.2 μL / 0.2 μL (0.1 μM / 0.1 μM). The annealing temperature was set as 55-65 °C, respectively, and the PCR products were detected by 1.5% gel electrophoresis.

[0087] 1.2.6 Specificity detection of multiplex PCR

[0088] To detect the specificity of the multiplex PCR reaction system to positive plasmids, single, two and three mixed plasmids and CMV-CP plasmid were used as templates, and the optimized system was used for multiplex PCR reaction. Healthy Brazilian banana was used as negative control (N), and sterilized double distilled water was used as blank control (CK).

[0089] To detect the specificity of the multiplex PCR reaction system to different BSV (BSOLV, BSGFV and BSIMV) complex infection and CMV infection of Brazilian banana samples, virus particles prepared from CMV infected Brazilian banana were used as templates, and BSOLV, BSGFV and BSIMV specific primers were used for multiplex PCR amplification. Meanwhile, Brazilian banana infected with BSOLV, BSGFV and BSIMV was used as positive control (P), healthy Brazilian banana was used as negative control (N), and sterilized double distilled water was used as blank control (CK).

[0090] 1.2.7 Sensitivity of multiplex PCR

[0091] Healthy Brazilian banana as negative control (N) and sterile double distilled water as blank control (CK); the plasmids of BSOLV, BSGFV and BSIMV were mixed (the initial concentration of each plasmid was 1 x 10 10 The template was diluted by 10 times gradient dilution, and 10 gradients were selected for optimization of the multiplex PCR system reaction.

[0092] 1.2.8 Multiplex PCR application

[0093] The banana leaves suspected to be infected by banana streak virus (100 mg) were ground in 1 mL grinding buffer (2% PVP, 0.2% BSA, 0.2% Na2SO3 in PBST buffer), and 25 μL supernatant was taken into eight tubes. The blank control (CK) was added with sterile double distilled water, and incubated at room temperature for 2 h. After washing with PBST for 3 times and ddH2O for 1 time, the residual banana genomic DNA was removed by DNase mix (3 μL 10 x buffer RDD, 3 μL DNase I, and water to 30 μL) at 37 °C for 1 h. The DNase I was inactivated at 95 °C for 10 min. After washing with ddH2O for 1 time, the virus particles adsorbed on the tube were used as templates, and the established multiplex PCR method was used to detect BSOLV, BSGFV and BSIMV simultaneously. Meanwhile, the Brazilian banana infected by BSOLV, BSGFV and BSIMV was used as positive control (P), and healthy Brazilian banana was used as negative control (N).

[0094] II. Results and analysis

[0095] 2.1 Extraction of plant total DNA and quality detection

[0096] The high-quality nucleic acid was extracted by using the plant new DNA extraction kit of Tiangen. The DNA gel electrophoresis graph showed that the genomic band was single and bright, and there was no diffuse band, indicating that the DNA quality was good, and the subsequent experiment could be carried out. Figure 1

[0097] 2.2 Optimization and determination of multiplex PCR reaction system

[0098] The DNA of Brazilian banana sample with free virus BSV (infected by BSOLV, BSGFV and BSIMV) was used as template, and the specific primers of BSOLV, BSGFV and BSIMV were used for single PCR amplification, respectively, and 744 bp, 480 bp and 256 bp specific bands were amplified, respectively, which were consistent with the expected band size Figure 2 ​). The PCR products were cloned and sequenced. The correct BSOLV, BSGFV and BSIMV positive DNA plasmids were sequenced and used as templates for the multiplex PCR reaction system. The multiplex PCR reaction system was optimized based on single-PCR, and the primer amount and annealing temperature were optimized.

[0099] The results of the specific primer optimization experiment showed that the different primer addition amount combinations had little effect on the amplification products, and the amplification effect of the three target bands was good, and the amplification bands were the most uniform. After comprehensive comparison of the amplification brightness of the three target bands under different addition amounts, the selected primer ratio combination of the multiplex PCR detection system was treatment three: 0.5 μL / 0.5 μL (0.25 μM / 0.25 μM): 0.2 μL / 0.2 μL (0.1 μM / 0.1 μM): 0.5 μL / 0.5 μL (0.25 μM / 0.25 μM). The three virus amplification target bands were relatively bright Figure 3 ). The annealing temperature optimization experiment showed that when the annealing temperature was 65℃, 64℃, 62.9℃, 60.9℃, 58.4℃, 56.5℃, and 55℃, the target bands could be amplified. When the annealing temperature was 56.5℃, the amplification effectiveness of each target band was close and the band shape was relatively uniform, so 56.5℃ was selected as the optimal annealing temperature of the multiplex PCR Figure 4 ).

[0100] 2.3 Specificity of the multiplex PCR system

[0101] The specificity experiment showed that the established optimized system could amplify the corresponding bands in single plasmid, two mixed plasmids, and three mixed plasmids, but no target bands were amplified from the CMV-CP plasmid, healthy Brazilian banana DNA, and blank control Figure 5 ), indicating that the established multiplex PCR system had good specificity.

[0102] RT-PCR was performed on CMV-infected Brazilian bananas, and the corresponding CMV target bands were amplified Figure 6 ). PCR amplification was performed on CMV-infected Brazilian bananas using BSOLV, BSGFV, and BSIMV specific primers. Except for the BSV positive control, no bands were amplified, indicating that the BSOLV, BSGFV, and BSIMV primers had specificity for BSV amplification in banana samples Figure 7 ).

[0103] 2.4 Sensitivity of the multiplex PCR

[0104] The sensitivity experiment showed that the established multiplex PCR method could still amplify the target bands when the plasmid was diluted to 1×10 3 copies / μL.Figure 8 ), indicating that the system can detect 1 x 10 3 copies of virus per μL.

[0105] 2.5 Application of the multiplex PCR detection method

[0106] Banana samples were detected by using the optimized multiplex PCR system Figure 9 Suspected symptom banana leaves were picked from the field and detected by multiplex PCR. The banana samples in the field were mostly infected by different viruses. From the results of the multiplex PCR, it can be seen that sample 3 and 4 amplified 3 bands, which were 3 viruses of BSOLV, BSGFV and BSIMV. Sample 9 amplified 2 bands, which were BSGFV and BSIMV. Sample 2 and 6 amplified a single band of BSGFV, and sample 7 and 8 amplified a single band of BSIMV. Samples 1 and 5 did not detect the three viruses. Figure 9

[0107] III. Conclusion

[0108] In this study, the primer concentration and annealing temperature in the PCR system were optimized, and a simple and accurate multiplex PCR method was established for detecting 3 different free BSV in banana. Through the exploration of primer concentration, the concentrations of the upstream and downstream primers of BSOLV, BSGFV and BSIMV were determined as 0.25 μM / 0.25 μM, 0.1 μM / 0.1 μM and 0.25 μM / 0.25 μM, respectively. The amplification effect was the best when the annealing temperature was 56.5°C. The detection sensitivity of the multiplex PCR system was 1 x 10 3 copies of virus per μL, and it had good specificity, and could accurately and sensitively detect different free BSV in the composite infection of banana samples. Therefore, the multiplex PCR is suitable for rapid detection of the three free viruses in banana samples in the field.

[0109] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are included in the protection scope of the present application.​

Claims

1. A PCR primer set for simultaneous detection of multiple free banana streak viruses, characterized in that: Including the BSOLV primer set, BSGFV primer set, and BSIMV primer set; The BSOLV primer set includes primers BSOLV-F / R; BSOLV-F: 5'-GAAGAGCATGGGCCTCAGAA-3'; BSOLV-R;5'-CGCATGCCTTGCAGATAGTC-3'; The BSGFV primer set includes BSGFV-F / R; BSGFV-F: 5'-GCCTTCATGACCTGGCGAAT-3'; BSGFV-R;5'-ATGTACGGGTGTATTGCCGC-3'; The BSIMV primer set includes BSIMV-F / R; BSIMV-F: 5'-TCTTAGACACAGGAGCTGCCA-3'; BSIMV-R: 5'-ATTCCTCCTTCCATTGCACG-3'.

2. The application of the PCR primer set described in claim 1 in the preparation of a kit for simultaneous detection of multiple free banana stripe viruses, characterized in that: The banana-striped viruses mentioned include BSOLV, BSGFV, and BSIMV.

3. A kit for simultaneously detecting multiple free-state banana stripe viruses, characterized in that, Includes the PCR primer set as described in claim 1.

4. The reagent kit according to claim 3, characterized in that: The kit also includes reagents required for PCR reaction, a positive control, a negative control, and a blank control; the positive control is the DNA of a banana plantation infected with three viruses: BSOLV, BSGFV, and BSIMV; the negative control is the DNA of a healthy banana plantation; and the blank control is sterile double-distilled water.

5. The application of the PCR primer set according to claim 1 or the kit according to any one of claims 3-4 in the detection of multiple free banana striae viruses in bananas, characterized in that: The banana-striped viruses mentioned include BSOLV, BSGFV, and BSIMV.

6. A method for simultaneously detecting multiple free banana streak viruses using the PCR primer set described in claim 1, characterized in that: The banana-striped viruses mentioned include BSOLV, BSGFV, and BSIMV.

7. The method for simultaneously detecting multiple free-state banana stripe viruses according to claim 6, characterized in that: Includes the following steps: (1) Preparation of viral particles in the sample to be tested; (2) Using the viral particles from step (1) as a template, the PCR primer set described in claim 1 is used to carry out the reaction to obtain the PCR amplification product; the molar ratio of BSOLV primer set, BSGFV primer set and BSIMV primer set in the reaction system is (2~5):(2~5):(2~5). (3) Determine whether the sample to be tested contains free banana line virus based on the amplification results. For each batch of experiments, a positive control, a negative control and a blank control must be performed simultaneously. Only when the three controls are completely consistent can the reliability of the results be confirmed. The banana-striped viruses mentioned include BSOLV, BSGFV, and BSIMV; In step (1), the method for preparing viral particles in the sample to be tested includes the following steps: Take the sample to be tested, add the grinding buffer at a ratio of 100 mg: 1 mL, grind, incubate at room temperature, wash for the first time, and then treat with DNase mix to remove residual banana genomic DNA; wash a second time after DNase inactivation to obtain virus particles; In step (3), the method for determining whether the sample to be tested contains free banana line virus based on the amplification results is electrophoresis detection technology; The electrophoresis detection technique is as follows: the PCR amplification product of step (2) is observed by electrophoresis. If at least one specific band of 744bp, 480bp and 256bp is amplified, it is a positive reaction, indicating that the sample contains at least one of BSOLV, BSGFV and BSIMV; if no specific band of 744bp, 480bp and 256bp is amplified, it is a negative reaction, indicating that the sample does not contain BSOLV, BSGFV and BSIMV.

8. The method for simultaneously detecting multiple free-state banana stripe viruses according to claim 7, characterized in that: In step (2), the molar ratio of BSOLV primer set, BSGFV primer set and BSIMV primer set in the reaction system is 5:2:

5.

9. The method for simultaneously detecting multiple free-state banana stripe viruses according to claim 7, characterized in that: In step (2), the final concentration of each primer in the BSOLV primer set is 0.1–0.25 μM, the final concentration of each primer in the BSGFV primer set is 0.1–0.25 μM, and the final concentration of each primer in the BSIMV primer set is 0.1–0.25 μM.

10. The method for simultaneously detecting multiple free-state banana stripe viruses according to claim 9, characterized in that: In step (2), the final concentration of each primer in the BSOLV primer set is 0.25 μM, the final concentration of each primer in the BSGFV primer set is 0.1 μM, and the final concentration of each primer in the BSIMV primer set is 0.25 μM.

11. The method for simultaneously detecting multiple free-state banana stripe viruses according to claim 7, characterized in that: In step (2), the reaction system is 20 μL, 2×Surper Taq PCR StarMix is ​​10 μL, the final concentrations of primers BSOLV-F / R are 0.1-0.25 μM, the final concentrations of BSGFV-F / R are 0.1-0.25 μM, the final concentrations of BSIMV-F / R are 0.1-0.25 μM, and ddH2O is added to make up to 20 μL using viral particles as templates. In step (2), the reaction conditions are: 94 °C pre-denaturation for 2 min, 94 °C denaturation for 15 s, 55-65 °C annealing for 15 s, 72 °C extension for 15 s, with a cycle number of 40; and 72 °C final extension for 5 min.

12. The method for simultaneously detecting multiple free-state banana stripe viruses according to claim 11, characterized in that: In step (2), the reaction system is 20 μL, 2×Surper Taq PCR StarMix is ​​10 μL, the final concentrations of primers BSOLV-F / R are 0.25 μM each, the final concentrations of BSGFV-F / R are 0.1 μM each, and the final concentrations of BSIMV-F / R are 0.25 μM each. Viral particles are used as templates, and ddH2O is added to make up to 20 μL. In step (2), the reaction conditions are: 94 °C pre-denaturation for 2 min, 94 °C denaturation for 15 s, 55-61 °C annealing for 15 s, 72 °C extension for 15 s, with a cycle number of 40; and 72 °C final extension for 5 min.

13. The method for simultaneously detecting multiple free-state banana stripe viruses according to claim 12, characterized in that: In step (2), the reaction conditions are: 94 °C pre-denaturation for 2 min, 94 °C denaturation for 15 s, 55-58.4 °C annealing for 15 s, 72 °C extension for 15 s, with a cycle number of 40; and 72 °C final extension for 5 min.

14. The method for simultaneously detecting multiple free-state banana stripe viruses according to claim 13, characterized in that: In step (2), the reaction conditions are: 94 °C pre-denaturation for 2 min, 94 °C denaturation for 15 s, 56.5 °C annealing for 15 s, 72 °C extension for 15 s, with a cycle number of 40; and 72 °C final extension for 5 min.

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