Primer probe combination for detecting fusarium laminatum, RCA visual kit and detection method

Abstract

The invention belongs to the field of biological detection, and particularly relates to a primer probe combination for detecting fusarium laminatum, an RCA visual kit and a detection method. Comprising the following sequences: a lock-type probe PLP, an RCA amplification upstream primer, an RCA amplification downstream primer and a ZIP sequence. The sensitivity is 100 times higher than that of the conventional PCR.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to a primer and probe combination, an RCA visualization kit, and a detection method for detecting Fusarium solani. Background Technology

[0002] Fusarium moniliforme ( Fusarium proliferatum Diseases reduce the yield and quality of various crops. The most common method for detecting Fusarium solani remains traditional morphological identification. However, this method suffers from drawbacks such as low efficiency, poor timeliness, and high subjectivity.

[0003] Polymerase chain reaction (PCR) is one of the techniques for detecting various pathogenic microorganisms. However, in PCR, primers are consumed in each cycle. Therefore, starting from a template DNA molecule, theoretically, a maximum of 2... n With extremely low concentrations of starting template, the amount of target product is minimal in early cycles and may be masked by background noise, non-specific enzyme activity, or primer dimers. Even if detection is eventually achieved, more cycles are required, and the reaction may have already entered an inefficient phase before then. There is a theoretical upper limit to the signal amplification factor produced by each template molecule. Furthermore, PCR primers, especially when the sequence is not completely specific or the annealing conditions are not optimized, may bind to non-target sequence portions, producing primer dimers or non-specific products. PCR primers / probes are typically very short, around 20 nt, and mutations in any key base in their binding region can severely affect annealing efficiency and extension, leading to a sharp drop in amplification efficiency or even complete failure.

[0004] Therefore, although PCR primers are highly efficient, timely, and objective in PCR detection, they have low sensitivity. Summary of the Invention

[0005] To address the technical problem of low detection sensitivity in existing technologies, this invention provides a primer-probe combination, an RCA visualization kit, and a detection method for detecting Fusarium solani.

[0006] A primer-probe combination for detecting pathogenic Fusarium moniliformes, comprising the sequences: SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.

[0007] An RCA visualization kit for detecting Fusarium latum includes the aforementioned primer-probe combination, as well as a buffer, a ligation sequence, and an LFD lateral flow immunoassay strip.

[0008] Furthermore, the connection sequence is a ZIP sequence, as follows: 5'-FAM-CTTCAACGCTCCCGCTAT-3', SEQ ID NO.4.

[0009] Furthermore, the buffer solution includes a ligation buffer and an amplification buffer.

[0010] The application of the primer-probe combination or the RCA visualization kit in the detection of Fusarium latum.

[0011] Furthermore, the method for detecting Fusarium laminaria using the RCA visualization kit is as follows: Using the primer-probe combination described above, the nucleic acid of the sample to be tested was subjected to an RCA reaction in a buffer environment, and then the reaction product was detected using an LFD lateral flow immunoassay strip.

[0012] Furthermore, the RCA reaction includes a ligation reaction and an amplification reaction.

[0013] Furthermore, the temperature of the ligation reaction is 55℃~65℃, and the number of cycles is 6~15; the temperature of the amplification reaction is 62℃~68℃, and the reaction time is 30min~90min.

[0014] Furthermore, the detection temperature using the LFD lateral flow immunoassay strip is room temperature, and the time is 5 min to 15 min.

[0015] The principle of this invention: Rolling circle amplification (RCA) is an isothermal amplification technique that mimics the replication process of viral nucleic acids and plasmids. The core principle of RCA can be summarized as follows: using a special DNA polymerase, a circular DNA template is used as the core, and the polymerase continuously replicates and synthesizes a very long single-stranded DNA molecule composed of hundreds to thousands of repeating units, much like "traveling in a circle." This ultra-long DNA strand can be easily detected, thus indirectly proving the presence of the initial target molecule. This technique mainly relies on a locking probe, PLP. The PLP consists of approximately 100 bases, with complementary target sequence regions at both ends, a ligation sequence in the middle, primer binding sites for amplification, and a zipcode sequence for hybridization. Only when the PLP and the corresponding target DNA are both present in the ligation system and are completely complementary can the linear locking probe be effectively ligated into a circular form under the action of the ligase. During the amplification reaction, the circular PLP is amplified under the action of the polymerase and primers.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides a primer-probe combination for detecting Fusarium latum based on rolling circle amplification (RoBA) technology, including a lock-type probe PLP, an upstream primer for RCA amplification, and a downstream primer for RCA amplification. The detection sensitivity is 100 times higher than that of conventional PCR.

[0017] 2. In this invention, nucleic acid amplification is first performed using rolling circle amplification (RCA) technology, and then the amplification results are visualized using LFD lateral flow immunoassay strips.

[0018] 3. The reagent kit of the present invention has high specificity, high accuracy and reliability, and is simple and quick to operate, and has a wide range of applications. Attached Figure Description

[0019] Figure 1 For the establishment and detection of the reaction system; (A) is the 2.0% agarose gel electrophoresis image of PLP concentration optimization, lane M is the electrophoresis of DL 1000 marker, and the PLP concentrations corresponding to lanes 1-6 are 10pM, 100pM, 1000pM, 1000pM, 10000pM and 10000pM respectively; (B) is the 2.0% agarose gel electrophoresis image of RCA reaction system verification, lane M is the electrophoresis of DL1000 marker; lane 1 is the positive control group, lane 2 is the PLP-free group, lane 3 is the template-free group and lane 4 is the Taq DNA ligase-free group.

[0020] Figure 2 In the image, (A) shows 2.0% agarose gel electrophoresis of Fusarium moniliforme PLP at different circularization ligation temperatures, with lane M representing the electrophoresis using a DL 1000 marker; lanes 1-6 represent electrophoresis images after amplification at ligation temperatures of 55℃, 57℃, 59℃, 61℃, 63℃, and 65℃, respectively; (B) shows 2.0% agarose gel electrophoresis of Fusarium moniliforme PLP at different circularization ligation cycles, with lane M representing the electrophoresis using a DL 1000 marker; lanes 1-6 represent electrophoresis images after amplification at ligation cycles of 0, 3, 6, 9, 12, and 15, respectively; (C) shows 2.0% agarose gel electrophoresis of Fusarium moniliforme PLP at different amplification temperatures, with lane M representing the electrophoresis using a DL 1000 marker. Electrophoresis with 1000 marker; lanes 1-6 represent electrophoresis after amplification at temperatures of 55℃, 58℃, 61℃, 64℃, 67℃ and 70℃, respectively; (D) is an agarose gel electrophoresis image of Fusarium moniliforme PLP at different amplification times, lane M is electrophoresis with DL 1000 marker; lanes 1-6 represent electrophoresis after amplification at times of 15 min, 30 min, 45 min, 60 min, 75 min and 90 min, respectively.

[0021] Figure 3In the image, (A) shows an agarose gel electrophoresis image of PCR reaction of total DNA from various Fusarium species, with lane M representing the DL1000 marker; (B) shows an agarose gel electrophoresis image of RCA reaction of genomic DNA from various Fusarium species, with lane M representing the DL1000 marker. (C) Electrophoresis of 2000 markers; RCA-LFD test of total DNA of various Fusarium species after RCA reaction; In (A), 1-18 represent agarose gel electrophoresis of PCR reactions of Fusarium flocculation, Fusarium graminearum, Fusarium oxysporum, Fusarium solani, Fusarium pseudograminearum, Fusarium asiaticum, Fusarium temperateum, Fusarium verticillatum, Fusarium brückii, Fusarium fusiformis, Fusarium niger, Fusarium oatum, Fusarium australis, Fusarium xanthophylloides, Fusarium trifidum, Fusarium rosenbergii, Fusarium moniliforme, and blank control; In (B), 1-18 represent agarose gel electrophoresis of PCR reactions of Fusarium flocculation, Fusarium graminearum, Fusarium oxysporum, Fusarium solani, Fusarium pseudograminearum, Fusarium asiaticum, and blank control. Agarose gel electrophoresis of RCA reactions for *Fusarium*, *Fusarium* var. *temperate*, *Fusarium* verticillatum, *Fusarium brevicornum*, *Fusarium fusiforme*, *Fusarium oxysporum*, *Fusarium solani*, *Fusarium oxysporum*, *Fusarium lancifolium*, *Fusarium rosenbergii*, *Fusarium moniliforme*, and a blank control; (C) 1-18 represent the LFD test strip detection results of RCA reactions for *Fusarium* var. *temperate*, *Fusarium* verticillatum, *Fusarium brevicornum*, *Fusarium fusiforme*, *Fusarium solani*, *Fusarium moniliforme*, *Fusarium anaphyllum*, and a blank control, respectively.

[0022] Figure 4 In the images, (A) is an agarose gel electrophoresis (2.0%) image of Fusarium moniliforme PCR sensitivity test. Lane M is used for DL ​​1000 marker electrophoresis; (B) is an agarose gel electrophoresis (2.0%) image of Fusarium moniliforme RCA sensitivity test. Lane M is used for DL ​​2000 marker electrophoresis; (C) is a Fusarium moniliforme RCA-LFD sensitivity test. Lanes 1-7: Genomic DNA concentrations are 4.3 ng / μL and 4.3 × 10⁻⁶, respectively. −1 ng / μL, 4.3×10 −2 ng / μL, 4.3×10 −3 ng / μL, 4.3×10 −4 ng / μL, 4.3×10 −5 The ng / μL and blank control were water.

[0023] Figure 5 In the diagram, (A) is the RCA test; (B) is the RCA-LFD test; M is the DL 1000 marker; lanes 1 and 2 are PLP and negative PLP, respectively. Figure 6In the diagram, (A) represents the PCR test; (B) represents the RCA test; (C) represents the RCA-LFD test; M represents the DL 2000 marker; lanes 1-6 were 2.1×10 ng / µL, 2.1 ng / µL, and 2.1×10 ng / µL, respectively. −1 ng / µL, 2.1×10 −2 ng / µL, 2.1×10 −3 The blank control group consisted of ng / µL and genomic DNA replaced with water. Detailed Implementation

[0024] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0025] Key terms and abbreviations are as follows: Padlock probes (abbreviated as PLP).

[0026] Rolling Circle Amplification (RCA)

[0027] Lateral flow dipsticks (LFD) are a technique used in lateral flow chromatography.

[0028] The following examples further illustrate the technical solution of the present invention. The buffer solution is from Xiamen Donglin Jieshi Biotechnology Co., Ltd., catalog number DF001; the nucleic acid test strip is an LFD lateral flow immunoassay strip from Xiamen Donglin Jieshi Biotechnology Co., Ltd., catalog number DF001.

[0029] Lock-on probe PLP: 5'P-CAAGATGTACCCCGCCAGATCTTGGTCGGATTAGACGACTGCTGGGAGCAGCAACTTCTTCGTCTTCAACGCTCCCGCTATCAGCATGTTGTCTTC-3', SEQ ID NO.1; P is a phosphorylation label; RCA amplification upstream primer: 5'-Bio-CCAGCAGTCGTCTAATCC-3', Bio represents biotin label, SEQ ID NO.2; RCA amplification downstream primer: 5'-GAGCAGCAACTTCTTCGT-3', SEQ ID NO.3; Linked sequence, ZIP sequence: 5'-FAM-CTTCAACGCTCCCGCTAT-3', SEQ ID NO.4; The upstream primer for TEF-1α sequence amplification is any one of the following: TEF-1α sequence amplification upstream primer 1: 5'-ATGGGTAAGGAAGACAAGAC-3', SEQ ID NO.5; TEF-1α sequence amplification upstream primer 2: 5'-ATGGGTAAGGAGGACAAGAC-3', SEQ ID NO.6; The downstream primer for TEF-1α sequence amplification is any one of the following: TEF-1α sequence amplification downstream primer 1: 5'-GGAGGTACCAGTGATCATGTT-3', SEQ ID NO.7; TEF-1α sequence amplification downstream primer 2: 5'-GGAGGTACCAGTCATCATGTT-3', SEQ ID NO.8; TEF-1α sequence amplification downstream primer 3: 5'-GGAAGTACCAGTGATCATGTT-3', SEQ ID NO.9; TEF-1α sequence amplification downstream primer 4: 5'-GGAAGTACCAGTCATCATGTT-3', SEQ ID NO.10; TEF-1α sequence of Fusarium moniliforme: 5'--3', SEQ ID NO.11; T1 sequence of the 5' end arm of the locking probe: 5'-CAAGATGTACCCCGCCAGATCTTGGTC-3', SEQ ID NO.12; T2 sequence of the 3' end arm of the locking probe: 5'-CAGCATGTTGTCTTC-3', SEQ ID NO.13; Specific PCR amplification primers for Fusarium moniliforme: Fp-F 5'-ACAGACCGGTCACTTGATCT-3', SEQ ID NO.14; Specific PCR amplification primers for Fusarium moniliforme: Fp-R 5'-CAGGAAAGGGCAAAAACGC-3', SEQ ID NO.15; All were synthesized at Shanghai Sangon Biotech Co., Ltd.

[0030] Example 1: Lock-on Probe PLP Design S1. Obtain total DNA Filter 20 mL of culture medium containing Fusarium chrysogenum and control strains in the logarithmic growth phase. Collect the filtered bacterial pellets, freeze them in liquid nitrogen, and grind them. Then, extract genomic DNA using a fungal genomic DNA extraction kit (Sangon Biotech, Shanghai, China) according to the manufacturer's instructions. Determine the DNA concentration using an NSA-99 microviolet spectrophotometer (Thermo Fisher Scientific, MA, USA). Calculate the ratio (A260 / A280) of the absorbance at 260 nm (A260) and 280 nm (A280) to measure DNA purity. Store the obtained DNA sample at -20°C for later use. The target bacterium is Fusarium chrysogenum (…). Fusarium proliferatum ) and control bacteria including Fusarium graminearum ( Fusarium graminearum Fusarium oxysporum ( Fusarium oxysporum Fusarium solani ( ), Fusarium solani Fusarium pseudograss () Fusarium pseudograminearum ), Fusarium tumefaciens ( Fusarium asiaticum ), temperate Fusarium ( Fusarium temperatum ), Fusarium verticillata ( Fusarium verticillioides Fusarium brucellosis ( Fusarium boothii ), Fusarium oxysporum ( Fusarium fujikuroi Fusarium oxysporum ( ), Fusarium and iyazi ), Fusarium oxysporum ( Fusarium avenaceum Fusarium moniliforme ( ), Fusarium meridionale ), Fusarium oxysporum ( Fusarium culmorum Fusarium trifidum ( Fusarium tricinctum Fusarium rosenbergii ( ), Fusarium incarnatum ) and Fusarium moniliforme ( Fusarium moniliforme Total DNA was obtained from *Fusarium moniliforme*, *Fusarium graminearum*, *Fusarium oxysporum*, *Fusarium solani*, *Fusarium pseudograminearum*, *Fusarium asiaticum*, *Fusarium tempera*, *Fusarium verticillatum*, *Fusarium brevicornum*, *Fusarium fusiforme*, *Fusarium niger*, *Fusarium oatum*, *Fusarium septemlobum*, *Fusarium trifidum*, *Fusarium rosenbergii*, and *Fusarium moniliforme*.

[0031] S2. Obtain the TEF-1α sequence of Fusarium effusum. Using total DNA from *Fusarium effusum* as a template, the TEF-1α sequence of *Fusarium effusum* was amplified. The PCR reaction was performed in a DNA thermal cycler (Bioer Technology, Hangzhou, China), with a reaction volume of 12.5 µL, as detailed below:

[0032] 10×PCR buffer, 2.5mM MgCl2, 2.5mM dNTP mixture, 10mM TEF-1α sequence amplification upstream primer, 10mM TEF-1α sequence amplification downstream primer, 1.25U Taq polymerase (Sangon Biotech, Shanghai, China) and 1µL genomic DNA.

[0033] The amplification program was as follows: pre-denaturation at 94℃ for 5 min; 29 cycles of denaturation at 94℃ for 45 s, annealing at 55℃ for 30 s, extension at 72℃ for 50 s; and a final extension at 72℃ for 7 min. The PCR products were purified using the Sanprep Type DNA Gel Extraction Kit from Shanghai Sangon Biotech Co., Ltd. The purified products were ligated into the pMDTM18-T vector from TaKaRa Biotechnology Co., Ltd. (Dalian, China) to prepare recombinant plasmids, which were then transformed into competent *E. coli* cells. Positive clones were identified by colony PCR and then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0034] S3, Obtain the lock probe PLP After removing the primers at both ends of the sequences obtained from sequencing positive clone strains, the sequences were uploaded to NCBI. https: / / www.ncbi.nlm.nih.gov / ; Analysis using the online search engine BLASTn confirmed the correctness of the target sequence. TEF-1α sequences of closely related species were found in Genbank, and compared with the TEF-1α sequence of *Fusarium solani* using BioEdit software.

[0035] https: / / bioedit.software.informer.com / ; ClustalX alignment analysis was performed to identify hypervariable regions as the 5' end arm T1 and 3' end arm T2 of the lock-type probe. The tiger prawn (Litopenaeus monodon), which is distantly related to Fusarium moniliforme, was used as a reference. Penaeus monodon The actin gene (Accession No. JQ241179) is used as the basic sequence. Primers were designed using PrimerPremier 5.0 software.

[0036] https: / / www.premierbiosoft.com / primerdesign / overview.html; T1, T2, ZIP sequences, upstream primers for RCA amplification, and downstream primers for RCA amplification were ligated using ligase to obtain linear PLPs. Furthermore, based on the ClustalX alignment results mentioned above, hypervariable regions of the target sequence were visually identified, and specific PCR amplification primers Fp-F and Fp-R for Fusarium effusum were designed using Primer Premier 5.0.

[0037] Example 2 RCA reaction The RCA ligation reaction system is shown in Table 1, and the RCA amplification reaction system is shown in Table 2. The advanced ligation reaction yields the reaction products, and the feasibility of RCA detection is then tested by amplification reaction.

[0038] Table 1 RCA Linkage Reaction System Note: 10×Taq DNA Ligase Buffer and 40.0 U / µL Taq DNA ligase are used as ligation buffer.

[0039] The ligation procedure was as follows: pre-denaturation at 94℃ for 4 min; 15 cycles, 94℃ for 30 s, ligation temperature at 60℃ for 5 min; after the reaction, inactivation of the ligase at 95℃ for 15 min, followed by ice bath for 5 min to obtain the ligation product.

[0040] Table 2 RCA amplification reaction system Note: 10×Bst DNA Polymerase Buffer, 10mM dNTP Mixture, 3.50M Betaine and 8U / µL Bst DNA Polymerase are amplification buffers.

[0041] Amplification program: react at 60℃ for 90 min.

[0042] Without adding total DNA from Fusarium effusus, the concentration of PLP was optimized by performing the RCA reaction according to Tables 1 and 2, and the results are as follows: Figure 1 As shown in (A), the RCA product appeared with increasing PLP concentration, indicating that the PLP probe underwent self-ligation, resulting in false positives. To ensure amplification efficiency, the PLP concentration used in subsequent experiments was 100 pM.

[0043] Groups were set up separately: the PLP-free group (with PLP removed) in Table 1, the positive control group as set in Table 1, the Taq DNA ligase-free group (with Taq DNA ligase removed) in Table 1, and the template-free group (with total Fusarium effusum DNA removed) in Table 1. The results are as follows: Figure 1 As shown in (B), only the positive control ligation system showed amplification. Conversely, without any of the components PLP, DNA template, and Taq DNA ligase, RCA could not proceed normally, indicating that the established RCA system was normal.

[0044] Example 3 RCA reaction The RCA ligation reaction system is shown in Table 1 of Example 1, and the RCA amplification reaction system is shown in Table 2 of Example 1. Following the systems in Tables 1 and 2, the ligation temperature was varied, with temperature gradients of 55℃, 57℃, 59℃, 61℃, 63℃, and 65℃. The results are as follows: Figure 2 As shown in (A), the probes can effectively form rings and initiate the RCA reaction within the temperature range of 55°C to 65°C. The RCA yield gradually increases as the temperature rises from 55°C to 61°C; however, the yield decreases above 63°C. Therefore, the optimal cyclization temperature is 61°C.

[0045] In addition to the 15 loops, five more gradient loops with intervals of 0, 3, 6, 9, and 12 were set. The result is as follows... Figure 2 As shown in (B), no RCA products were detected when the number of cycles was less than 3; products began to be detected after more than 6 cycles; when the number of cycles increased from 9 to 15, the amount of product showed an increasing trend, but the difference was not significant, and the condition of 9 cycles was optimal.

[0046] The RCA amplification temperatures for the system in Table 2 were set at six gradients: 58℃, 60℃, 62℃, 64℃, 66℃, and 68℃. Figure 2 As shown in (C), no RCA product is generated below 60℃; the amplification reaction can occur in the range of 62℃ to 68℃, and the product amount shows a trend of first increasing and then decreasing with increasing temperature. Therefore, 64℃ is the optimal amplification temperature.

[0047] Different RCA amplification times were set, with six gradients: 15 min, 30 min, 45 min, 60 min, 75 min, and 90 min. The results are as follows: Figure 2 As shown in (D), RCA products can be detected after 30 minutes of amplification; when the amplification time is extended from 30 minutes to 60 minutes, the product amount gradually increases; while in the range of 45 minutes to 90 minutes, no significant difference in product amount is observed, and 45 minutes is the optimal amplification time.

[0048] Example 4 The RCA reaction system was identical to Tables 1 and 2 in Example 1, except for the total DNA. The ligation reaction procedure was as follows: pre-denaturation at 94°C for 4 min; 15 cycles of 94°C for 30 s and 61°C for 5 min; after the reaction, the ligase was inactivated at 95°C for 15 min, followed by an ice bath for 5 min to obtain the ligation product; the amplification reaction procedure was 64°C for 90 min.

[0049] Various bacteria were subjected to routine PCR reactions. The primers were upstream primers for TEF-1α sequence amplification and downstream primers for TEF-1α sequence amplification. The reaction system consisted of 10× PCR buffer, a mixture of 2.5 mM MgCl2 and 2.5 mM dNTPs, 10 mM upstream primers for TEF-1α sequence amplification, 10 mM downstream primers for TEF-1α sequence amplification, 1.25 U Taq polymerase from Sangon Biotech in Shanghai, China, and 1 µL of genomic DNA.

[0050] The RCA amplification products of various bacteria were detected by LFD lateral flow immunoassay strips and agarose gel electrophoresis. The results are shown in [Figure number missing]. Figure 3 The RCA-LFD analysis results were the same as those of RCA. RCA-LFD analysis of the target bacterial species produced two pink indicator lines on the test strip: the detection line and the control line. However, RCA-LFD analysis of non-Fusarium species and the blank control produced only one pink indicator line on the test strip: the control line. In summary, both RCA and RCA-LFD analysis results indicate that the established detection method is specific to the target bacteria.

[0051] Comparative Example 1: PCR Detection Using the total DNA sample of *Fusarium solani* from Example 1, conventional PCR was performed using primers Fp-F and Fp-R with genomic DNA solutions diluted 10-fold as templates. Figure 4 As shown in (A), the detectable genomic DNA concentration range is 4.3 ng / µL–4.3 × 10⁻⁶. −2 ng / µL.

[0052] The ligation procedure using the RCA reaction system in Example 4 was as follows: pre-denaturation at 94°C for 4 min; 15 cycles of 94°C for 30 s, 61°C for 5 min; after the reaction, the ligase was inactivated at 95°C for 15 min, followed by an ice bath for 5 min to obtain the ligation product; the amplification reaction procedure was 64°C for 90 min; the genomic DNA testing results are as follows. Figure 4 As shown in (B) and (C) of the sensitivity detection, the detection concentration range for both positive RCA and RCA-LFD of genomic DNA is 4.3~4.3×10⁻⁶. −4 ng / µL. RCA and RCA-LFD are 100 times more sensitive than conventional PCR.

[0053] Comparative Example 2 Based on Example 1, T1 and T2 in the PLP probe were replaced to design a PLP probe as a negative control. The specific method is as follows:

[0054] According to the African clawed frog ( Xenopus laevis PLP probes were designed based on the gene sequence of elongation factor 1 alpha (Accession No: NM_001101761.2), as shown in the following sequence: 5'P-CAAGGGAACAAACCGGTAAAGACCTGTGGATTAGACGACTGCTGGGAGCAGCAACTTCTTCGTCTTCAACGCTCCCGCTATTCCGGTCTTCTCGCA-3', SEQ ID NO.16; P is a phosphorylation label; Lock-on probe 5' end arm T1 sequence: 5'-CAAGGGAACAAACCGGTAAAGACCTGT-3', SEQ ID NO.17; T2 sequence of the 3' end arm of the locking probe: 5'-TCCGGTCTTCTCGCA-3', SEQ ID NO.18; The negative PLP was verified using the Fusarium moniliforme total DNA sample from Example 1. The RCA ligation and amplification systems were performed according to Example 2; the RCA reaction procedure was performed according to Example 4. The ligation procedure was: 94°C pre-denaturation for 4 min; 15 cycles: 94°C, 30 s; 61°C, 5 min; after the reaction, the ligase was inactivated at 95°C for 15 min, followed by an ice bath for 5 min to obtain the ligation product; the amplification reaction procedure was 64°C for 90 min. The experimental results are as follows: Figure 5 As shown, PLP produces an amplification band, with two pink indicator lines on the test strip, namely the detection line and the control line; while the negative control does not produce an amplification band, and only one pink indicator line, namely the control line, is produced on the test strip; this further indicates that the probe, negative PLP, cannot be used to detect Fusarium latum.

[0055] Application Example 1: Detection of bacteria-laden soil Fusarium moniliformes were artificially added to sterilized soil. After 7 days, total DNA was extracted from the samples. The extracted total DNA was then diluted 10-fold and used as templates for PCR, RCA, and RCA-LFD detection. PCR was a standard PCR procedure, and the RCA system was consistent with that in Example 4.

[0056] The results are as follows Figure 6As shown in (A) of the detection of contaminated soil samples, the concentration of genomic DNA detectable by PCR in the diluted solution of artificially inoculated soil samples ranges from 2.1 × 10 ng / µL to 2.1 × 10 −1 ng / µL. Results of genomic DNA testing experiments using RCA and RCA-LFD systems are as follows: Figure 6 As shown in (B) and (C), the detection concentration range of positive RCA and RCA-LFD for total DNA is 2.1 × 10⁻⁶. 1 –2.1×10 −2 ng / µL. In the detection of spiked samples, RCA and RCA-LFD are 10 times more sensitive than conventional PCR.

[0057] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention also intends to include these modifications and variations.

Claims

1. A primer-probe combination for detecting Fusarium laurylae based on rolling circle amplification technology, characterized in that, Includes the following sequences: Lock-on probe PLP: 5'P-CAAGATGTACCCCGCCAGATCTTGGTCGGATTAGACGACTGCTGGGAGCAGCAACTTCTTCGTCTTCAACGCTCCCGCTATCAGCATGTTGTCTTC-3', SEQ ID NO.1; RCA amplification upstream primer: 5'-Bio-CCAGCAGTCGTCTAATCC-3', SEQ ID NO.2; RCA amplification downstream primer: 5'-GAGCAGCAACTTCTTCGT-3', SEQ ID NO.

3.

2. An RCA visualization kit for detecting Fusarium laurylates, characterized in that, The kit includes the primer-probe combination of claim 1, and further includes buffer, ligation sequence, and LFD lateral flow immunoassay strip.

3. The RCA visualization kit for detecting Fusarium sp. according to claim 2, characterized in that, The connection sequence is a ZIP sequence, as follows: 5'-FAM-CTTCAACGCTCCCGCTAT-3', SEQ ID NO.

4.

4. The RCA visualization kit for detecting Fusarium sp. according to claim 2, characterized in that, The buffer solution includes a ligation buffer and an amplification buffer.

5. A method for detecting Fusarium laurylates using the RCA visualization kit of claim 2, characterized in that, include: Using the primer-probe combination described above, the nucleic acid of the sample to be tested was subjected to an RCA reaction in a buffer environment, and then the reaction product was detected using an LFD lateral flow immunoassay strip.

6. The method for detecting Fusarium laminaria using the RCA visualization kit according to claim 5, characterized in that, The RCA reaction includes a ligation reaction and an amplification reaction.

7. The method for detecting Fusarium laminaria using the RCA visualization kit according to claim 6, wherein the ligation reaction temperature is 55℃~65℃ and the number of cycles is 6~15; the amplification reaction temperature is 62℃~68℃ and the reaction time is 30min~90min.

8. The method according to claim 5, characterized in that, The detection temperature using the LFD lateral flow immunoassay strip is room temperature, and the time is 5 min to 15 min.

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