Application of a primer pair for detecting sugarcane bacilliform virus in RCA detection

Abstract

The present invention provides the use of a primer pair for detecting sugarcane bacilliform virus (SCBV-F2) in RCA testing, belonging to the field of molecular biological detection technology. The primer pair comprises SCBV-F2 and SCBV-R2. The application includes using sugarcane genomic DNA as a DNA template, performing rolling circle amplification using the primer pair, and then testing the resulting RCA amplification product to determine whether the sugarcane carries free SCBV. Furthermore, PCR and RCA testing can be combined to identify SCBV sequence integration into the sugarcane genome. The present invention establishes a highly sensitive and specific RCA detection method for sugarcane bacilliform virus. Then, RCA and PCR testing are combined to establish a rapid method for identifying sugarcane bacilliform virus integration into the sugarcane genome. This method has the advantages of low cost, simple operation steps, rapidity, and minimal sample material required.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to the application of a primer pair for detecting sugarcane bacilliform virus in RCA detection. Specifically, the present invention relates to a primer pair for RCA detection of sugarcane bacilliform virus, a kit, an RCA detection method for sugarcane bacilliform virus, and a method for identifying SCBV sequence and sugarcane genome integrated material by combining PCR and RCA detection. Background Art

[0002] Sugarcane bacilliform virus ( Sugarcane bacilliform virus SCBV (ScBV) is the causative agent of sugarcane bacilliform virus disease. It belongs to the genus Badnavirus (Badnavirus) of the family Caulimoviridae. Its genome consists of circular double-stranded DNA, 7.3-8.0 kb in size. It is the causative agent of sugarcane bacilliform virus disease and has been shown to integrate its genome or fragments into the host genome. In most cases, SCBV infection of sugarcane results in chlorotic patches, mottled patches, or chlorotic streaks of varying lengths on leaves, leaf shrinkage, cracks in the stem internodes, and dwarfing of the plant's bunch tops. SCBV infection reduces sugarcane stem weight, juice yield, and sucrose content, resulting in poor quality and yield. Severe SCBV infection can cause yield losses of 25-35% in some sensitive sugarcane varieties.

[0003] Currently, SCBV is present in sugarcane-producing areas in over 20 countries and regions. It has become widespread in major sugarcane-producing regions in my country, including Guangdong, Guangxi, Yunnan, and Hainan, with high infection rates. In 2020, the average SCBV infection rate in my country's sugarcane-producing areas was reported to be 68.41%, with some areas experiencing rates as high as 73.08%. SCBV is one of the major viruses that harm sugarcane production and poses a significant threat to the safe production of my country's sugarcane industry.

[0004] DNA viruses can integrate their genomes or fragments into the host genome. SCBV belongs to the genus Baculovirus. Cai Yanqing et al., using Southern blot hybridization, found that SCBV can integrate its genome into the sugarcane genome. Once integrated, DNA viruses can maintain their presence within the host, maintaining long-term infection within host cells. Furthermore, the viral integration sequence can be activated under specific conditions, causing disease in the host plant. Therefore, when propagating sugarcane seedlings, germplasm material with SCBV integrated into the sugarcane genome must be removed. While Southern blot hybridization can identify SCBV integration into the sugarcane genome, it has the disadvantages of requiring large sample sizes, being time-consuming, and being expensive.

[0005] Since the rolling-circle amplification method (RCA) is an isothermal amplification technology that can be performed at room temperature, exponential rolling circle amplification can be achieved using two or more primers. It is characterized by accuracy, speed, and wide applicability. The bacteriophage phi29 DNA polymerase used in this method only recognizes circular DNA templates during amplification, and does not recognize linear DNA templates. SCBV free virus is circular double-stranded DNA, which can be recognized and amplified by the phi29 DNA polymerase target during the RCA process. The integrated sequence is linear DNA and cannot be recognized and amplified by the phi29 DNA polymerase. It can be seen that RCA can specifically detect free circular SCBV virus. In summary, it is necessary to develop a method for detecting SCBV using RCA. Summary of the Invention

[0006] To address the above issues, the present invention provides an application of a primer pair for detecting sugarcane bacilliform virus in RCA detection. Specifically, the present invention provides a primer pair for RCA detection of sugarcane bacilliform virus, a kit, an RCA detection method for sugarcane bacilliform virus, and a method for combining PCR and RCA detection to identify SCBV sequences integrated into the sugarcane genome.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] A primer pair for detecting sugarcane bacilliform virus in RCA detection, wherein the primer pair is used to detect sugarcane bacilliform virus by RCA detection method;

[0009] The nucleotide sequence of the upstream primer SCBV-F2 of the primer pair is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer SCBV-R2 is shown in SEQ ID NO: 2.

[0010] A kit for detecting sugarcane bacilliform virus by RCA, comprising the above primer pair.

[0011] Furthermore, the kit also includes 10× buffer, dNTP, BSA, phi29 DNA polymerase and ddH2O.

[0012] A method for detecting sugarcane bacilliform virus (SCBV) by RCA is disclosed. The method uses sugarcane genomic DNA as a DNA template and the above-mentioned primer pair as amplification primers to perform rolling circle amplification. The resulting RCA amplification product is then subjected to electrophoresis detection. If a target band appears (positive), the sugarcane carries free SCBV virus. If no target band appears (negative), the sugarcane does not carry free SCBV virus, and the sugarcane is either free of SCBV virus or has SCBV sequence integrated into the sugarcane genome.

[0013] Furthermore, the rolling circle amplification system includes 10× buffer, dNTP, the above-mentioned SCBV-F2 and SCBV-R2, a DNA template, BSA, phi29 DNA polymerase and ddH2O.

[0014] Furthermore, the rolling circle amplification program was: 30°C for 18 h, 65°C for 10 min.

[0015] A method for identifying SCBV sequence integrated into sugarcane genome by combining PCR and RCA detection, the method comprising the following steps:

[0016] The sugarcane to be tested is tested using the PCR detection method. If the target band appears (positive), the sugarcane to be tested carries free SCBV virus or SCBV sequence, and the sugarcane is sugarcane carrying free SCBV virus, or sugarcane with SCBV sequence integrated into the sugarcane genome. If the target band does not appear (negative), the sugarcane does not carry free SCBV virus and SCBV sequence, and the sugarcane is sugarcane without free SCBV virus and SCBV sequence.

[0017] The sugarcane to be tested is tested using the above-mentioned RCA detection method. If the target band appears (positive), the sugarcane carries free SCBV virus; if the target band does not appear (negative), the sugarcane to be tested does not carry free SCBV virus. In this case, the sugarcane does not carry free SCBV virus, or the SCBV sequence is integrated into the sugarcane genome.

[0018] Analyze the PCR test results and the RCA test results. If the target band appears in the PCR test but not in the RCA test, the sugarcane to be tested is the material where the SCBV sequence has been integrated with the sugarcane genome.

[0019] Furthermore, the PCR test uses sugarcane genomic DNA as a DNA template and the above-mentioned primer pair as amplification primers to perform PCR amplification, and then the obtained PCR amplification product is subjected to electrophoresis detection. If the target band appears (positive), the sugarcane to be tested carries free SCBV virus or SCBV sequence, and the sugarcane is sugarcane carrying free SCBV virus, or sugarcane in which the SCBV sequence is integrated into the sugarcane genome; if the target band does not appear (negative), the sugarcane does not carry free SCBV virus and SCBV sequence, and in this case the sugarcane is sugarcane that does not carry free SCBV virus and SCBV sequence.

[0020] Furthermore, the PCR amplification reaction system includes 2x Taq plus Master Mix, the above-mentioned SCBV-F2 and SCBV-R2, a DNA template, and ddH2O.

[0021] Furthermore, the PCR amplification reaction program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min, 35 cycles; and finally extension at 72°C for 10 min.

[0022] The beneficial effects of the application of the primers for detecting sugarcane bacilliform virus of the present invention in RCA detection are:

[0023] The present invention successfully established the SCBV virus RCA detection method, which can specifically detect the SCBV virus and has the characteristics of strong specificity and high sensitivity;

[0024] The present invention compares the RCA detection method with the PCR detection method. The RCA detection method only requires a sample amount of 10 pg to specifically detect SCBV. The detection sensitivity of the RCA method is 1000 times higher than that of the PCR detection method. It can be seen that the RCA detection method has the advantages of high sensitivity, small sample amount and good specificity.

[0025] Furthermore, the present invention combines RCA detection with PCR detection to rapidly identify the integration of SCBV into the sugarcane genome. Samples that are positive in PCR and negative in RCA are materials where the SCBV sequence has integrated into the sugarcane genome (a positive PCR result indicates that the sample contains the SCBV nucleic acid sequence, while a negative RCA result indicates that the sample does not contain free SCBV virus). This method has the advantages of low cost, simple and rapid operation steps, and requires less sample material.

[0026] The present invention establishes a highly sensitive and specific RCA detection method for sugarcane bacilliform virus and then combines RCA with PCR detection to establish a rapid identification method for the integration of sugarcane bacilliform virus into the sugarcane genome. Compared with the traditional Southern Blot method for identifying the integration of SCBV into the sugarcane genome, this method has the advantages of low cost, simple operation steps, rapidity, and less sample material required.

[0027] The method of the present invention can greatly improve the screening efficiency of SCBV sequence and sugarcane genome integration materials, and has extremely high economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The results of RCA amplification electrophoresis of the three pairs of primers in Example 1 of the present invention are shown below; wherein, M represents DNA Maker DS15000, 1 represents the primer pair used is SCBV-F1 and SCBV-R1, 2 represents the primer pair used is SCBV-F2 and SCBV-R2, and 3 represents the primer pair used is SCBV-F3 and SCBV-R3;

[0029] Figure 2 is the specificity test result of the RCA detection method in Example 1 of the present invention; wherein, M represents DNA MakerDS15000, 1 represents the detection result of SCBV sugarcane virus, 2 represents the detection result of SCYLV sugarcane virus, 3 represents the detection result of SCSMV sugarcane virus, 4 represents the detection result of SrMV sugarcane virus, and 5 represents the detection result of SCMV sugarcane virus;

[0030] Figure 3 Figure 1 is a comparison of the sensitivity of RCA and PCR in Example 1 of the present invention; Figure A shows the sensitivity of the RCA detection method; Figure B shows the sensitivity of the PCR detection method; M in Figures A and B represents DNA Maker, and 1 to 6 represent DNA templates with concentrations of 1000 ng, 100 ng, 10 ng, 1 ng, 100 pg, and 10 pg, respectively;

[0031] Figure 4 These are the PCR and RCA test results for some sugarcane germplasm materials in Example 2 of the present invention; Figure A shows the PCR test results; in Figure A, M represents DNA Maker DS2000, and 1 to 16 represent sugarcane germplasm materials that test positive using different PCR methods; Figure B shows the RCA test results; in Figure B, M represents DNA Maker DS15000, 1 to 12 and 14 represent sugarcane germplasm materials carrying free SCBV virus particles (i.e., RCA test results are positive); 13, 15, and 16 represent materials with SCBV sequences integrated into the sugarcane genome (i.e., RCA test results are negative); 13, 15, and 16 represent Guitang 05-3256, Guitang 37, and ROC16, respectively;

[0032] Figure 5 This is a Southern hybridization blot of the material in which the SCBV sequence is integrated with the sugarcane genome in Example 2 of the present invention; wherein, M represents a DIG-labeled DNA marker for Southern hybridization, 1 represents a Southern hybridization blot of Guitang 05-3256, 2 represents a Southern hybridization blot of Guitang 37, 3 represents a Southern hybridization blot of ROC16, and 4 represents a Southern hybridization blot of sugarcane without the SCBV sequence; the SCBV genome in 1 to 3 is integrated into the sugarcane genome. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present invention are described clearly and completely below. Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The present invention is further described in detail below in conjunction with specific embodiments to facilitate understanding by those skilled in the art.

[0034] Example 1 Establishment of SCBV rolling circle amplification (RCA) detection technology

[0035] The rolling circle amplification (RCA) assay uses the bacteriophage phi29 DNA polymerase, which recognizes only circular templates during amplification. Free SCBV is a circular DNA virus and can be recognized and amplified by phi29 DNA polymerase during the RCA process. Sequences integrated into the sugarcane genome cannot be recognized and amplified by phi29 DNA polymerase. Therefore, RCA is a method that can specifically detect free circular SCBV.

[0036] 1) Establishment of the RCA assay for SCBV

[0037] 11) Take 0.1 g of sugarcane leaf tissue sample and grind it quickly and thoroughly in liquid nitrogen. Extract total DNA from the sugarcane leaf using the CTAB method to obtain a DNA template.

[0038] 12) Based on the reported SCBV complete genome sequence (NCBI accession number: GenBank: JN377533.1), DNAMAN 8 software was used to align the sequences and analyze the conserved regions. Primer Premier 5.0 software was used to design specific primers in the conserved regions. Three primer pairs were designed:

[0039] The first pair of primers were: SCBV-F1: 5′-CATCCCGAGGTAAACCCTA-3′ and SCBV-R1: 5′-ATGGCATCACTAACCATTCATA -3′;

[0040] The second pair of primers were: SCBV-F2: 5′-CCGGATTCGAGATTACAGAAG-3′ and SCBV-R2: 5′-CACCTAGCCAACCGTCATA-3′;

[0041] The third pair of primers were: SCBV-F3: 5'-CTGGCTAGAAGTCTGGAG-3' and SCBV-R3: 5'-TGTTTCTGGAAGTTGTTC-3'.

[0042] Using bacteriophage phi29 DNA polymerase as the replicase and the above three pairs of primers as specific primers, rolling circle amplification was performed on SCBV-positive samples to obtain RCA amplification products.

[0043] The rolling circle amplification system was as follows: 10× buffer 2.0 μL, dNTP (10 mM / L) 2.0 μL, forward primer and reverse primer (100 μM / L) 0.6 μL each, DNA template 10 ng, BSA 0.2 μL, phi29 DNA polymerase 0.4 μL, and ddH2O was added to make up to 20 μL.

[0044] The rolling circle amplification program was: 30°C for 18 h, 65°C for 10 min.

[0045] 13) The RCA amplified products were then detected by 1% agarose gel electrophoresis at 80V for 40 minutes. The gel imager was used to observe and photograph the amplification results of the three primer pairs. Figure 1 shown.

[0046] pass Figure 1 As can be seen, the primer pair SCBV-F2 and SCBV-R2 exhibited the best amplification effect among the three primer pairs, and thus the primer pair SCBV-F2 and SCBV-R2 was selected in the present invention. Specifically, the nucleotide sequence of the upstream primer SCBV-F2 of the primer pair of the present invention is shown in SEQ ID NO: 1, i.e., 5'-CCGGATTCGAGATTACAGAAG-3'; and the nucleotide sequence of the downstream primer SCBV-R2 is shown in SEQ ID NO: 2, i.e., 5'-CACCTAGCCAACCGTCATA-3'.

[0047] In summary, the present invention extracts genomic DNA from sugarcane leaves as a DNA template, uses primer pairs SCBV-F2 and SCBV-R2, and adopts the above-mentioned rolling circle amplification system and rolling circle amplification procedure to perform rolling circle amplification. The resulting RCA amplification products are then subjected to electrophoresis detection. If the target band appears (the size of the target band is greater than 7.3 kb), it means that the sugarcane carries free SCBV virus; if the target band does not appear, it means that the sugarcane does not carry free SCBV virus. In this case, the sugarcane is sugarcane that does not carry free SCBV virus, or the SCBV sequence is integrated into the sugarcane genome.

[0048] It should be noted that the sugarcane in which the SCBV sequence is integrated with the sugarcane genome in the present invention does not carry free SCBV virus, but carries the SCBV sequence.

[0049] 2) Specificity test of RCA assay

[0050] The established RCA assay was used to simultaneously detect five sugarcane viruses, namely SCBV, SCYLV, SCSMV, SrMV, and SCMV. The amplification results were observed and the specificity of the RCA assay for SCBV was analyzed as follows:

[0051] 21 Take the DNA or cDNA of five sugarcane viruses, SCBV, SCYLV, SCSMV, SrMV and SCMV, as the corresponding amplification templates. Among them, SCBV is a DNA virus and the template is DNA, while SCYLV, SCSMV, SrMV and SCMV are RNA viruses and the template is cDNA;

[0052] 22) Perform RCA assays on each amplified template and compare and analyze the specificity of the RCA assay for SCBV as follows:

[0053] Each amplification template was taken for rolling circle amplification to obtain the corresponding RCA amplification product.

[0054] The rolling circle amplification system was as follows: 10× buffer 2.0 μL, dNTP (10 mM / L) 2.0 μL, SCBV-F2 and SCBV-R2 (100 μM / L) 0.6 μL each, amplification template 10 ng, BSA 0.2 μL, phi29 DNA polymerase 0.4 μL, and ddH2O was added to make up to 20 μL.

[0055] The rolling circle amplification program was: 30°C for 18 h, 65°C for 10 min.

[0056] Each RCA amplification product was then detected by 1% agarose gel electrophoresis at a voltage of 80 V and an electrophoresis time of 40 minutes. The gel imager was used to observe, photograph, and save the results. Figure 2 shown.

[0057] Depend on Figure 2 It can be seen that only SCBV can be amplified to obtain the target band, while the other four sugarcane viruses (SCYLV, SCSMV, SrMV and SCMV) have no target band. This shows that the RCA detection method established in the present invention can specifically amplify SCBV virus and will not induce nonspecific amplification of other sugarcane viruses. The specificity of the RCA detection method is strong.

[0058] 3) Sensitivity test of RCA detection method

[0059] The sensitivity of the established RCA assay for SCBV was tested as follows:

[0060] 31) The total DNA of SCBV positive samples was diluted 10-fold (10 ~ 10 6 ), and the diluted DNA was used as DNA template.

[0061] 32) Perform RCA and PCR tests separately, observe the amount of amplified product, and compare and analyze the sensitivity of the RCA test for SCBV. The specific detection methods are as follows:

[0062] 321) RCA assay

[0063] Each diluted DNA was subjected to rolling circle amplification to obtain RCA amplification products.

[0064] The rolling circle amplification system was as follows: 10× buffer 2.0 μL, dNTP (10 mM / L) 2.0 μL, SCBV-F2 and SCBV-R2 (100 μM / L) 0.6 μL each, DNA template 10 ng, BSA 0.2 μL, phi29 DNA polymerase 0.4 μL, and ddH2O was added to make up to 20 μL.

[0065] The rolling circle amplification program was: 30°C for 18 h, 65°C for 10 min.

[0066] The RCA amplified product was then detected by 1% agarose gel electrophoresis at a voltage of 80 V for 40 minutes. The gel imager was used to observe, photograph, and save the results. Figure 3 Figure A in .

[0067] If the target band appears in the RCA test results (positive), the sugarcane carries free SCBV virus and is sugarcane carrying free SCBV virus; if the target band does not appear (negative), the sugarcane does not carry free SCBV virus and is sugarcane that does not carry free SCBV virus, or the SCBV sequence is integrated into the sugarcane genome.

[0068] 322) PCR detection method

[0069] Each diluted DNA was taken for PCR amplification to obtain PCR amplification products.

[0070] The PCR amplification reaction system was as follows: 12.5 μL of 2x Taq plus Master Mix, 1.0 μL each of SCBV-F2 and SCBV-R2 (10 μM / L), 1.0 μL of DNA template, and ddH O was added to make up to 25 μL;

[0071] PCR amplification reaction program: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, 35 cycles; final extension at 72°C for 10 min.

[0072] The PCR amplified products were then detected by 1% agarose gel electrophoresis at a voltage of 80 V for 40 minutes. The gel imager was used to observe and photograph the products. Figure 3 Figure B in .

[0073] If the target band appears in the PCR test results (positive), the sugarcane to be tested carries free SCBV virus or SCBV sequence, and the sugarcane is sugarcane carrying free SCBV virus, or sugarcane in which the SCBV sequence is integrated into the sugarcane genome; if the target band does not appear (negative), the sugarcane does not carry free SCBV virus and SCBV sequence, and the sugarcane is sugarcane that does not carry free SCBV virus and SCBV sequence.

[0074] Depend on Figure 3 It can be seen that the minimum concentration of DNA template that can be detected by RCA detection is 10pg ( Figure 3 Figure A in the figure); However, when the DNA template is diluted to 10 ng in PCR detection, the target band is darker, and when it is diluted to 1 ng, the target band cannot be detected ( Figure 3 As shown in Figure B, the minimum concentration of DNA template that can be detected by PCR is 10 ng, and the sensitivity of RCA detection is 1000 times higher than that of PCR detection.

[0075] Example 2 Method for identifying SCBV sequence integrated into sugarcane genome by combining PCR and RCA detection

[0076] 1. Identification of sugarcane germplasm materials

[0077] First, 221 sugarcane germplasm materials were tested using the PCR detection method described in step 3) of Example 1. The sugarcane germplasm materials that were screened and tested positive by the PCR detection method were then tested using the RCA detection method described in step 3) of Example 1. The PCR and RCA detection results were statistically analyzed to screen out samples that were PCR-positive (the target band appeared) and RCA-negative (the target band did not appear). In other words, samples that were free of free SCBV virus and had the SCBV sequence integrated into the sugarcane genome were obtained. These samples were materials with the SCBV sequence integrated into the sugarcane genome.

[0078] Among them, 140 of the 221 sugarcane germplasm materials tested by PCR were positive (some test results are as follows Figure 4As shown in Figure A of the figure, these 140 sugarcane germplasm materials contained SCBV nucleic acid sequences (i.e., the PCR test was positive, indicating that the sugarcane germplasm materials included sugarcane carrying free SCBV virus and materials with SCBV sequences integrated into the sugarcane genome). RCA testing was then performed on these 140 PCR-positive sugarcane germplasm materials, and the results showed that 130 sugarcane germplasm materials were able to amplify the target band (partial results are shown in Figure A). Figure 4 (As shown in Figure B), these 130 sugarcane accessions carried free SCBV virions (i.e., RCA-positive). Ten accessions failed to amplify the target band, and their RCA-negative results indicated that the accessions did not contain free SCBV virions. In other words, these 10 accessions tested positive for SCBV nucleic acid, but negative for RCA, indicating that the SCBV sequences in these accessions were not present in the form of free SCBV virions but rather integrated into the sugarcane genome. Ten accessions were screened for SCBV genome-integrated sequences: Guitang 05-3256, Co1149, CP00-1100, Q198, Guitang 03-3005, Guitang 73-167, Guitang 37, Guitang 13-532, ROC16, and Guitang 05-1474.

[0079] 2. Verification of SCBV sequence and sugarcane genome integration

[0080] Southern hybridization was performed on the SCBV sequences and sugarcane genome integration materials obtained by the above method to verify the accuracy of the above method of screening SCBV sequences and sugarcane genome integration materials using a combination of PCR detection and RCA detection, as follows:

[0081] Three SCBV sequences, Guitang 05-3256, Guitang 37, and ROC16, obtained by the above method, were selected for Southern hybridization with the sugarcane genome-integrated materials, specifically as follows: the CTAB method was used to extract a large amount of total DNA from the leaves of the SCBV sequence-integrated materials of the sugarcane genome, and 30 μg of total leaf DNA was digested with the restriction endonuclease BamHI. The enzyme digestion system was: 30 μg of total DNA from sugarcane leaves, 10 μL of 10×M Buffer, 10 μL of BamHI, and sterile ddH2O was added to make up to 100 μL; the enzyme digestion was incubated at 37°C for 16 h.The digested product was subjected to low-voltage electrophoresis on 0.8% agarose gel for 16 hours (voltage: 18V). After electrophoresis, the membrane was transferred to HyboodTM-N+ nylon membrane according to the instructions of the DIGHigh Prime DNA Labeling and Detection Starter Kit I (Roche). The membrane was then cross-linked using a UV cross-linker and hybridized with the prepared probe (probe sequence:). The hybridization temperature was 41°C, the pre-hybridization time was 2 hours, and the hybridization time was 16 hours. After hybridization, the nylon membrane was washed and then chemically developed with BCIP / NBT at room temperature in the dark. The position of the blot band was observed after 16 hours of development. The results are as follows. Figure 5As shown, the Southern hybridization bands for the three samples, Guitang 05-3256, Guitang 37, and ROC16, were located between 9416 bp and 23130 bp, while the control sugarcane plant samples, which did not carry SCBV sequences, showed no bands. Furthermore, the size of the episomal SCBV genome was approximately 7300 bp to 8000 bp, but none of these three samples showed bands corresponding to the episomal SCBV genome. This indicates that the SCBV in these three samples did not exist as an episomal viral genome, but rather was integrated into the sugarcane genome. Therefore, the results of the traditional Southern hybridization method were consistent with the experimental results of the combined PCR and RCA assay developed by the present invention for identifying materials integrated with the SCBV sequence and the sugarcane genome, demonstrating that the combined PCR and RCA assay developed by the present invention for identifying materials integrated with the SCBV sequence and the sugarcane genome is accurate and reliable.

[0082] The method developed in the present invention for identifying SCBV sequences integrated into the sugarcane genome using a combination of PCR and RCA detection is compared to the traditional Southern hybridization method for identifying integrated SCBV sequences. Southern hybridization requires a sample size of 30 μg, and the experimental cycle takes up to 144 hours. This method is complex and consumes a lot of manpower and resources. In contrast, the method developed in the present invention for identifying SCBV sequences integrated into the sugarcane genome using a combination of PCR and RCA detection requires only 10 ng of sample, one-thousandth the sample size required for Southern hybridization, and can be completed in just 20 hours. This method is simple to operate and requires less manpower and resources. Therefore, the method developed in the present invention for identifying SCBV sequences integrated into the sugarcane genome using a combination of PCR and RCA detection has the advantages of low cost, simple operation steps, rapidity, and minimal sample material requirements.

[0083] All other parts not described in detail are prior art. Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Those skilled in the art can also derive other embodiments based on this embodiment without inventiveness, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A primer pair for detecting sugarcane bacilliform virus in RCA detection, characterized in that: The application is to use the primer pair to detect sugarcane bacilliform virus through the RCA detection method; The nucleotide sequence of the upstream primer SCBV-F2 of the primer pair is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer SCBV-R2 is shown in SEQ ID NO:

2.

2. A kit for detecting sugarcane bacilliform virus by RCA, characterized in that: The kit comprises the primer pair according to claim 1.

3. The kit for detecting sugarcane bacilliform virus by RCA according to claim 2, characterized in that: The kit also includes 10× buffer, dNTP, BSA, phi29 DNA polymerase and ddH2O.

4. A RCA detection method for sugarcane bacilliform virus, characterized in that: The RCA detection method uses sugarcane genomic DNA as a DNA template and the primer pair described in claim 1 as an amplification primer to perform rolling circle amplification, and then detects the obtained RCA amplification product. If the target band appears, the sugarcane carries free SCBV virus; if the target band does not appear, the sugarcane does not carry free SCBV virus.

5. The RCA detection method for sugarcane bacilliform virus according to claim 4, characterized in that: The rolling circle amplification system comprises 10× buffer, dNTP, SCBV-F2 and SCBV-R2 according to claim 1, a DNA template, BSA, phi29 DNA polymerase and ddH2O.

6. The RCA detection method for sugarcane bacilliform virus according to claim 4 or 5, characterized in that: The rolling circle amplification program was: 30°C for 18 h, 65°C for 10 min.

7. A method for identifying SCBV sequence integrated into the sugarcane genome by combining PCR and RCA detection, characterized in that: The method comprises the following steps: The sugarcane to be tested is detected using a PCR detection method, wherein the genomic DNA of the sugarcane to be tested is used as a DNA template and the primer pair described in claim 1 is used as an amplification primer to perform PCR amplification, and then the obtained PCR amplification product is detected. If a target band appears, the sugarcane to be tested carries free SCBV virus or SCBV sequence; Detecting the sugarcane to be tested using the RCA detection method according to any one of claims 4 to 6, if no target band appears, the sugarcane to be tested does not carry free SCBV virus; Analyze the PCR test results and the RCA test results. If the target band appears in the PCR test but not in the RCA test, the sugarcane to be tested is the material where the SCBV sequence has been integrated with the sugarcane genome.

8. The method for identifying SCBV sequence integrated into the sugarcane genome by combining PCR and RCA detection according to claim 7, characterized in that: The PCR amplification reaction system includes 2x Taq plus Master Mix, SCBV-F2 and SCBV-R2 according to claim 1, a DNA template, and ddH2O.

9. The method for identifying SCBV sequence and sugarcane genome integrated materials by combining PCR and RCA detection according to claim 7, characterized in that: The PCR amplification reaction program was as follows: pre-denaturation at 95°C for 5 min; 35 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min; and finally extension at 72°C for 10 min.

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