Primer probe combination, kit and method for identifying banana wilt subtype TR4 by fluorescent RPA (recombinase polymerase amplification)

Abstract

The invention relates to the field of biological detection, and discloses a primer probe combination, a kit and a method for identifying banana fusarium wilt subtype TR4 through fluorescent RPA (recombinase polymerase amplification). The primer probe combination provided by the invention is used for detecting the banana wilt subtype TR4 by a PRA fluorescence method, and has the advantages of good specificity, high accuracy, high sensitivity and the like. According to the nucleic acid extraction method, purification is not needed after the sample is ground and split, and the banana wilt pathogenic bacterium nucleic acid can be extracted within 10-15 min. Meanwhile, aiming at the current RPA method, freeze-dried powder containing buffer and an enzyme system is creatively used, the RPA system can be completed only by adding a nucleic acid extracting solution and a reaction reagent into the freeze-dried powder, the operation is simple and convenient, meanwhile, an amplification product is detected by using a fluorescence detector, and the defect that aerosol pollution is caused due to the fact that a cover of the amplification product needs to be opened when a test paper card is used for detection is avoided.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological detection, in particular to a primer probe combination, kit and method for identifying banana wilt subtype TR4 by fluorescence RPA. BACKGROUND

[0002] Banana wilt is an international plant quarantine object, which is divided into FOC1, FOC2, TR4 and STR4 subtypes. At present, TR4 is the most widely prevalent and harmful subtype, which has caused devastating damage to the global banana industry. At present, due to the lack of effective drugs and disease-resistant varieties, it is extremely difficult to control in production.

[0003] Therefore, it is crucial to quickly and accurately detect the pathogen of the disease in order to take timely measures to destroy and isolate to prevent the spread and spread of the disease.

[0004] At present, accurate detection of the disease is mainly through molecular means, such as ordinary PCR and constant temperature amplification RPA and LAMP. PCR or QPCR is a basic means of molecular detection and is the most widely used molecular detection technology. PCR and QPCR are generally combined with traditional nucleic acid extraction methods to form PCR detection technology. Traditional nucleic acid extraction methods such as column extraction or magnetic bead method are time-consuming and complex to operate. PCR or QPCR requires a complex reaction system, which is complicated and complex to operate, has many steps, takes a long time to detect, and requires complex and precise temperature changing equipment and professional operation, so that PCR detection technology cannot achieve rapid and instant detection, and cannot be completed in the field, which limits its use.

[0005] Recombinase polymerase amplification (RPA) is a new type of constant temperature nucleic acid amplification technology that has developed rapidly in recent years. It is sensitive and can rapidly amplify DNA at a lower constant temperature (40±2℃), which shortens the constant temperature amplification reaction time to 10-15 min. By designing and synthesizing specific fluorescent RPA primers and probes, combined with fluorescent detection instruments for detecting RPA amplification products, the fluorescent RPA technology not only takes advantage of the rapid and simple amplification of RPA, but also avoids the disadvantages of aerosol pollution caused by test strip card detection. SUMMARY

[0006] In view of the shortcomings of the prior art, the present application aims to provide a primer probe combination, kit and method for identifying banana wilt subtype TR4 by fluorescence RPA, in order to accelerate the rapid and accurate identification of banana wilt subtype TR4 in the field.

[0007] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions: A primer-probe combination for identifying the banana wilt subtype TR4 using fluorescent RPA includes an F1 primer, an R1 primer, and a P1 probe. The nucleotide sequence of the F1 primer is as follows: 5'-TGAGGGATTGGAGCGACTTACCGTGAAATGG-3'; The nucleotide sequence of the R1 primer is as follows: 5'-TCAGATAGCTGATATCTCCCTGCTCCATCA-3'; The nucleotide sequence of the P1 probe is as follows: 5'-AGGATGATACTATTGCTTTATTTTCTGTGGTTATTTGGAGTCACTCTA-3'; The thymine at a position 28 bp from the 5' end of the P1 probe is modified with the fluorescent group FAM. The G base at a position 30 bp from the 5' end of the P1 probe is replaced by dSpacer; The thymine at a position 31 bp from the 5' end of the P1 probe is modified with the quenching group BHQ1. The P1 probe is connected to ddC at its 3' end. A kit for identifying the banana wilt subtype TR4 using fluorescent RPA, wherein the kit comprises the primer-probe combination described above.

[0008] The kit for identifying the banana wilt subtype TR4 using fluorescent RPA further includes lysis buffer, nucleic acid dilution buffer, amplification lyophilized powder, and activator.

[0009] The kit for identifying the banana wilt subtype TR4 using fluorescent RPA further includes a lysis buffer, a nucleic acid dilution buffer, a mixed enzyme solution, a buffer, and an activator.

[0010] The kit for identifying the banana wilt subtype TR4 using fluorescent RPA, wherein the mixed enzyme solution comprises the following components: BSU polymerase at a concentration of 10–30 ng / μL, GB32 recombinase at a concentration of 300–500 ng / μL, UvsX recombinase at a concentration of 50–80 ng / μL, UvsY protein at a concentration of 5–20 ng / μL, creatine kinase at a concentration of 20–50 ng / μL, and EXO enzyme at a concentration of 1–4 U / μL.

[0011] The kit for identifying the banana wilt subtype TR4 using fluorescent RPA comprises a lysis buffer containing 0.2–0.3 M NaOH, 0.1–0.3 M Tris at pH 12.0–12.5, and 1–5 mM DTT; and a nucleic acid dilution buffer containing 10–50 mM Tris-H at pH 7.0–8.0.

[0012] A method for identifying the banana wilt subtype TR4 using fluorescent RPA, employing the aforementioned kit, includes the following steps: S1. Sampling and processing of the samples to be tested; S2. Obtain the nucleic acid from the sample to be tested; S3. Mix the nucleic acid, lyophilized amplification powder, activator, and primer probe combination of the sample to be tested, and then place it in a fluorescence detector for reaction to obtain the detection results; or mix the nucleic acid, mixed enzyme solution, buffer, activator, and primer probe combination of the sample to be tested, and then place it in a fluorescence detector for reaction to obtain the detection results.

[0013] The method for identifying the banana wilt subtype TR4 using fluorescent RPA, wherein in step S1, the sample to be tested is a banana pseudostem suspected of being infected with banana wilt and located close to the ground; and the banana pseudostem is processed to have a length and width of 1.0–2.0 cm and a surface area of ​​2–2.5 cm². 2 Samples with a thickness of 2-3 mm.

[0014] The method for identifying the banana wilt subtype TR4 using fluorescent RPA, wherein step S2 specifically involves: placing the processed sample into a container, adding lysis buffer to the container, and crushing the sample; transferring the liquid from the container into a centrifuge tube, and placing the centrifuge tube in a fluorescence detector for incubation; after the sample incubation is complete, transferring the sample into a centrifuge tube containing nucleic acid diluent, mixing thoroughly, and obtaining a nucleic acid extract.

[0015] The method for identifying the banana wilt subtype TR4 using fluorescent RPA includes the following steps in step S3: The primer-probe combination and activator are mixed and diluted to obtain a reaction agent; nucleic acid extract is added to a reaction tube containing lyophilized amplification powder, followed by the addition of the reaction agent, and the mixture is thoroughly mixed to obtain a homogeneous solution; the homogeneous solution is transferred to a detection tube, which is then placed in a fluorescence detector. After the fluorescence detector completes its reaction, the detection result is read. Alternatively, the primer-probe combination, activator, nucleic acid extract, mixed enzyme solution, buffer, and denuclease-free water are added to a detection tube, mixed thoroughly, and then the detection tube is placed in a fluorescence detector. After the fluorescence detector completes its reaction, the detection result is read.

[0016] Beneficial effects: This invention provides a primer-probe combination, kit, and method for identifying the banana wilt subtype TR4 using fluorescent RPA. The primer-probe combination, used for PRA fluorescence detection of banana wilt subtype TR4, exhibits advantages such as high specificity, high accuracy, and high sensitivity. This invention optimizes the nucleic acid extraction method, requiring only two steps. After sample grinding and lysis, purification is unnecessary, and the nucleic acid of the banana wilt pathogen can be extracted within 10-15 minutes, which can be directly used as a template. Furthermore, current RPA methods generally require complex reaction systems and incubation equipment. This invention optimizes the process, eliminating the need for complex reaction systems. It creatively uses lyophilized powder containing buffer and enzyme system; simply adding the nucleic acid extract and reaction reagents to the lyophilized powder completes the RPA system, making the operation simple and convenient. Simultaneously, the amplification product is detected using a fluorescence detector, avoiding the aerosol contamination caused by opening the test strip. Overall, this invention simplifies the nucleic acid extraction method and RPA process, offering simple, time-saving, and safe operation, enabling real-time and rapid field detection. Detailed Implementation

[0017] This invention provides a primer-probe combination, kit, and method for identifying the TR4 subtype of banana wilt disease using fluorescent RPA. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the following examples further illustrate the invention in detail. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the scope of protection of this invention.

[0018] This invention provides a primer-probe combination for identifying the banana wilt subtype TR4 using fluorescent RPA, including an F1 primer, an R1 primer, and a P1 probe; The nucleotide sequence of the F1 primer is as follows: 5'-TGAGGGATTGGAGCGACTTACCGTGAAATGG-3', whose nucleotide sequence is SEQ ID NO: 1; The nucleotide sequence of the R1 primer is as follows: 5'-TCAGATAGCTGATATCTCCCTGCTCCATCA-3', whose nucleotide sequence is SEQ ID NO: 2; The nucleotide sequence of the P1 probe is as follows: 5'-AGGATGATACTATTGCTTTATTTTCTGTGGTTATTTGGAGTCACTCTA-3', whose nucleotide sequence is SEQ ID NO: 3; The thymine at the P1 probe, located 28 bp from the 5' end, is modified with the fluorescent group FAM. FAM is a fluorescent group that emits fluorescence at a specific wavelength under excitation light. FAM is chemically linked to the thymine (T) residue in the oligonucleotide chain.

[0019] The C base at the position 30 bp from the 5' end of the P1 probe is replaced by dSpacer; dSpacer is a chemically modified sequence or linker that does not participate in base pairing. Its function is to separate the fluorescent group (FAM) and the quencher group (BHQ1) to avoid abnormal fluorescence quenching efficiency caused by the two being too close. It is also the specific recognition and cleavage site of EXO enzyme in the system.

[0020] The thymine at a position 31 bp from the 5' end of the P1 probe is modified with the quencher group BHQ1. BHQ1 is a highly efficient quencher that absorbs the fluorescence emitted by FAM and does not emit fluorescence itself. The two form a reporter-quencher pair through the structure "FAM-dT-dSpacer-BHQ1-dT". When the P1 probe is not bound to the target sequence, the probe is in a free state. The spatial distance between FAM and BHQ1 is relatively small, and the fluorescence of FAM is effectively quenched by BHQ1, resulting in no fluorescence signal release. When the P1 probe specifically binds to the target sequence (RPA amplification product) of the banana wilt subtype TR4 to form a double-stranded complex, the dSpacer site on the probe is exposed. The EXO enzyme (exonuclease) in the system recognizes this site and cuts it along the 5'→3' direction, causing the probe to break into a 5' end fragment containing FAM and a 3' end fragment containing BHQ1. As the cleavage reaction occurs, FAM and BHQ1 completely separate, significantly increasing their spatial distance. The quenching effect of BHQ1 on FAM completely disappears, and FAM begins to emit a fluorescent signal, which can be captured in real time by a fluorescence detector. Since the more target sequence amplification products there are, the more probes bind to the P1 probe and are cleaved by the EXO enzyme, resulting in a stronger emitted fluorescence signal, monitoring changes in fluorescence intensity with a fluorescence detector provides a direct indication of the TR4 target sequence amplification, thus enabling the identification of the banana wilt subtype TR4.

[0021] The P1 probe is attached to ddC at its 3' end. ddC is an abbreviation for dideoxycytidine, used to terminate chain extension.

[0022] The present invention also provides a kit for identifying the banana wilt subtype TR4 using fluorescent RPA, the kit comprising the primer-probe combination as described in claim 1.

[0023] In one embodiment, the kit for identifying the banana wilt subtype TR4 using fluorescent RPA further includes a lysis buffer, a nucleic acid dilution buffer, an amplification lyophilized powder, and an activator. Specifically, the amplification lyophilized powder includes amplification-related enzymes and a buffer.

[0024] In another embodiment, the kit for identifying the banana wilt subtype TR4 using fluorescent RPA further includes lysis buffer, nucleic acid dilution buffer, mixed enzyme solution, buffer, and activator.

[0025] Specifically, the lysis buffer in both embodiments includes NaOH at a concentration of 0.2–0.3 M, Tris at a concentration of 0.1–0.3 M and a pH of 12.0–12.5, and DTT at a concentration of 1–5 mM; the nucleic acid dilution buffer is Tris-H at a concentration of 10–50 mM and a pH of 7.0–8.0.

[0026] Specifically, the mixed enzyme solution comprises the following components: BSU polymerase at a concentration of 10–30 ng / μL, wherein the BSU polymerase is a large fragment of Bst DNA polymerase with strand displacement function, which, after the primer binds to the template, extends the primer along the 5'→3' direction using dNTPs as raw materials to synthesize a new DNA strand; GB32 recombinase at a concentration of 300–500 ng / μL, wherein the GB32 recombinase is an E. coli RecA family recombinase that specifically binds to the primer to form a "recombinase-primer complex," which actively searches for and recognizes homologous sequences in the template DNA, opening local regions of the double-stranded DNA through ATP energy, allowing the primer to bind to the template single strand and providing an initiation site for subsequent extension; and UvsX recombinase at a concentration of 50–80 ng / μL, wherein the UvsX recombinase is a recombinase derived from bacteriophage T4, whose function is synergistically complementary to GB32, used to enhance the binding efficiency of the recombinase-primer complex to the template DNA and assist in opening the template double strand. It maintains the stability of the single-stranded template, reduces non-specific binding, further improves the efficiency and specificity of strand replacement, and ensures that the primers accurately bind to the target sequence; UvsY protein at a concentration of 5-20 ng / μL, wherein the UvsY protein is a UvsX helper protein derived from phage T4, which can bind to UvsX and activate its recombinase activity, while promoting the assembly and stability of UvsX on the DNA strand, enhancing the formation efficiency of the "UvsX-primer-template" complex, and indirectly improving the rate of strand replacement and primer binding; creatine kinase at a concentration of 20-50 ng / μL, wherein the creatine kinase has ATP regeneration function, used to continuously provide ATP (energy source for the strand replacement process) for the recombinase (GB32, UvsX); and EXO enzyme at a concentration of 1-4 U / μL, wherein the EXO enzyme is an exonuclease derived from phage T7. Once the fluorescent probe in the system binds to the amplification product to form a double strand, the EXO enzyme recognizes the specific cleavage site (dSpacer) on the probe and cleaves the probe along the 5'→3' direction, separating the fluorescent group (FAM) from the quencher group (such as BHQ1) on the probe and releasing a fluorescent signal. By monitoring changes in fluorescence intensity, the formation of the amplification product can be reflected in real time, enabling quantitative or qualitative detection of the target sequence. Furthermore, by limiting the concentration range of the EXO enzyme, a balance can be struck between probe cleavage efficiency and excessive degradation of the amplification product. Too low an EXO enzyme concentration will result in a weak fluorescence signal and insufficient detection sensitivity; too high a concentration may degrade the target amplification product and inhibit the reaction.

[0027] This invention also provides a method for identifying the banana wilt subtype TR4 using fluorescent RPA, comprising the following steps: S1. Sampling and processing of the samples to be tested.

[0028] Collect banana pseudostem tubers suspected of being infected with banana wilt subtype TR4 and located close to the ground; then process the banana pseudostem tubers to a length and width of 1.0–2.0 cm and a surface area of ​​2–2.5 cm². 2 Samples with a thickness of 2-3 mm.

[0029] S2. Obtain the nucleic acid from the sample to be tested.

[0030] The specific steps are as follows: (1) Place the prepared sample in the bottom corner of the grinding bag, open the grinding bag, and add 800-1000 μL of lysis buffer into the grinding bag in 4-5 portions using a 200 μL pipette. Remove the air from the grinding bag, seal the grinding bag, and then fold the grinding bag diagonally. Lay the sample and lysis buffer flat on a horizontal surface, ensuring that the sample is immersed in the lysis buffer. Gently and quickly tap the sample with a small claw hammer or other suitable tool (be careful not to tap too hard to avoid breaking the grinding bag) for about 1-2 minutes until the sample becomes a paste. Then, use a 200 μL pipette to draw 100 μL of liquid from the grinding bag and add the liquid to a 1.5 mL centrifuge tube. Place the centrifuge tube in a fluorescence detector and keep it warm for 10 minutes.

[0031] (2) Prepare a new 1.5 mL centrifuge tube, add 380 μL of nucleic acid diluent to the centrifuge tube in advance, and after the sample is incubated, use a 200 μL pipette to aspirate 20 μL of the sample supernatant into the 1.5 mL centrifuge tube containing the nucleic acid diluent, mix well, and obtain the nucleic acid extract.

[0032] S3.PRA reaction and detection In one embodiment, primer-probe combination and activator are mixed and diluted to obtain a reaction agent; nucleic acid extract is added dropwise to a reaction tube containing amplification lyophilized powder (containing mixed enzyme solution, buffer, etc. required for amplification), and then the reaction agent is added dropwise and mixed evenly to obtain a mixture; the mixture is transferred to a detection tube, and then the detection tube is placed in a fluorescence detector. After the fluorescence detector finishes its reaction, the instrument will automatically read the negative or positive result of the sample within 15 minutes.

[0033] In another embodiment, the primer probe combination, activator, nucleic acid extract, mixed enzyme solution, buffer and nuclease-free water are added to the detection tube, mixed evenly, and then the detection tube is placed in the fluorescence detector. After the fluorescence detector finishes its reaction, the instrument will automatically read the negative or positive result of the sample within 15 minutes.

[0034] To further illustrate the primer-probe combination, kit, and method for identifying the banana wilt subtype TR4 using fluorescent RPA, the following examples and comparative examples are provided.

[0035] Example 1 A primer-probe combination for identifying the banana wilt subtype TR4 using fluorescent RPA includes an F1 primer: 5'-TGAGGGATTGGAGCGACTTACCGTGAAATGG-3', the nucleotide sequence of which is SEQ ID NO: 1; R1 primer: 5'-TCAGATAGCTGATATCTCCCTGCTCCATCA-3', whose nucleotide sequence is SEQ ID NO: 2; P1 probe: 5'-AGGATGATACTATTGCTTTATTTTCTGTGGTTATTTGGAGTCACTCTA-3', its nucleotide sequence is SEQ ID NO: 3; the thymine at the 28 bp position from the 5' end of the P1 probe is modified with the fluorescent group FAM; the C base at the 30 bp position from the 5' end of the P1 probe is replaced with dSpacer; the thymine at the 31 bp position from the 5' end of the P1 probe is modified with the quencher group BHQ1; the P1 probe is connected to ddC at the 3' end.

[0036] A method for identifying the banana wilt subtype TR4 using fluorescent RPA includes the following steps: S1. Sampling and processing of the samples to be tested: Take banana pseudostems suspected of being infected with banana wilt subtype TR4 and located close to the ground; process the banana pseudostems to a length and width of 1.0–2.0 cm and a surface area of ​​2–2.5 cm². 2 Samples with a thickness of 2-3 mm.

[0037] S2. Obtain the nucleic acid from the sample to be tested. The specific steps are as follows: (1) Place the prepared sample in the bottom corner of the grinding bag, open the grinding bag, and add 800-1000 μL of lysis buffer into the grinding bag in 4-5 portions using a 200 μL pipette. Remove the air from the grinding bag, seal the grinding bag, and then fold the grinding bag diagonally. Lay the sample and lysis buffer flat on a horizontal surface, ensuring that the sample is immersed in the lysis buffer. Gently and quickly tap the sample with a small claw hammer or other suitable tool (be careful not to tap too hard to avoid breaking the grinding bag) for about 1-2 minutes until the sample becomes a paste. Then, use a 200 μL pipette to draw 100 μL of liquid from the grinding bag and add the liquid to a 1.5 mL centrifuge tube. Place the centrifuge tube in a fluorescence detector and keep it warm for 10 minutes.

[0038] (2) Prepare a new 1.5 mL centrifuge tube, add 380 μL of nucleic acid diluent to the centrifuge tube in advance, and after the sample is incubated, use a 200 μL pipette to aspirate 20 μL of the sample supernatant into the 1.5 mL centrifuge tube containing the nucleic acid diluent, mix well, and obtain the nucleic acid extract.

[0039] S3.PRA reaction and detection (1) Commercially available RPA reagents were used as raw materials. The reagent kit included lyophilized amplification powder (containing amplification-related enzymes and buffer) and activator. The activator in the reagent kit was dissolved at room temperature and then mixed evenly with a 200 μL pipette. 60-65 μL of activator, 40-45 μL each of F1 and R1 primers, and 10-15 μL of P1 probe were pipetted into a 1.5 mL centrifuge tube. Then, 530-550 μL of nucleic acid dilution buffer was added to the 1.5 mL centrifuge tube, bringing the total liquid volume to 700 μL. The mixture was shaken until homogeneous to obtain the reaction reagent.

[0040] (2) Take out the reaction tube containing the amplification lyophilized powder, open the cap, and use a 200μL pipette to add 25μL of nucleic acid extraction solution to the amplification lyophilized powder.

[0041] (3) Take out a 1.5 mL centrifuge tube containing the reagent, use a 200 μL pipette to draw 25 μL of the reagent and add it to the amplification lyophilized powder. Use a 200 μL pipette to carefully blow the liquid formed after the amplification lyophilized powder melts, blow 15 to 20 times until completely uniform, and then transfer all the liquid to the detection tube matched with the fluorescence detector and cover it with the rubber cap.

[0042] (4) Open the cover of the fluorescence detector, put the detection tubes into the wells of the fluorescence detector in sequence, close the cover of the instrument, start the instrument, and the instrument will automatically read the positive and negative results of the sample within 15 minutes. The results are displayed directly on the instrument screen. The instrument displays "+" for positive and "-" for negative.

[0043] In the RPA reaction, the commercially available RPA reagent is specifically the fluorescent RPA (Exo probe method) (in situ lyophilized powder) produced by Yisheng Biotechnology (Shanghai) Co., Ltd.

[0044] Performance Test 1: Specificity Detection Nucleic acids extracted from the hyphae of pure cultures of 11 different Fusarium wilt diseases were used to test the specificity of the primer-probe combination provided in Example 1. Numbers 1-8 belong to four different subspecies of banana Fusarium wilt: FOC1, FOC2, TR4, and STR4. Three other species of Fusarium wilt diseases were also included: cucumber Fusarium wilt, tomato Fusarium wilt, and tobacco Fusarium wilt. A negative control was also included, for a total of 12 groups for specificity testing. Sample information and fluorescence detector results are shown in Table 1.

[0045] Table 1 Specific Detection Results

[0046] Note 1: FOC1-1, FOC1-2, and FOC1-3 belong to different FOC1 subtypes; STR4-1 and STR4-2 belong to different STR4 subtypes; cucumber FO, tomato FO, and tobacco FO belong to different species of wilt disease.

[0047] Note 2: The time to positive result refers to the time when a positive result is first detected in the sample. The shorter the time, the higher the degree of positivity of the sample.

[0048] Results and Conclusions: Of the 11 Fusarium wilt diseases tested, 8 were different subspecies of banana Fusarium wilt, belonging to FOC1, FOC2, TR4, and STR4 respectively, and 3 were Fusarium wilt diseases of other species. The test results (Table 1) showed that only TR4 showed a positive result, and the positive result appeared in 4 minutes and 23 seconds, indicating that the detection efficiency was high. All other samples and negative controls showed negative results, indicating that the method has good specificity and can accurately detect banana Fusarium wilt TR4 from different subspecies and different species of Fusarium wilt, with high specificity.

[0049] Performance Test 2: Sensitivity Test A suitable sample was taken from the pseudostem near the ground of a banana plant infected with banana wilt disease TR4. Nucleic acid was extracted according to the nucleic acid extraction method in Example 1. Then, using this as the stock solution, a 2-fold dilution method was used to obtain a total of 11 different concentrations of DNA template, including the stock solution and stock solution diluted 2, 4, 8, 16, 32, 64, 128, 256, 512, and 1024 times, as well as 1 negative control. The RPA reaction and detection method in Example 1 were then used for detection. The results are shown in Table 2.

[0050] Table 2 Sensitivity Test Results Table 1

[0051] Results and Conclusions: It can be seen that even after the original DNA template was diluted 256 times, a positive result could still be detected, although the time to positive results was close to the instrument's threshold (15 minutes). A negative result was obtained after a 512-fold dilution. The dilution calculations theoretically indicate that if the amount of pathogen infecting a banana plant infected with Fusarium wilt TR4 is reduced by approximately 250 times, it can still be detected in the early stages of infection, even when the banana is asymptomatic. This demonstrates the extremely high sensitivity of this method. The extracted original DNA, measured by a micro-spectrophotometer, had a concentration of 4.32 nanograms per microliter. Even after a 250-fold dilution, it was still detectable, resulting in a detection sensitivity of 1.73 × 10⁻⁶. -2 nanograms per microliter.

[0052] Example 2 A method for identifying the banana wilt subtype TR4 using fluorescent RPA is basically the same as that in Example 1, except that the reagents used in the RPA reaction are different.

[0053] In S01, 450 μL of nucleic acid diluent was added to a 1.5 mL centrifuge tube; 50 μL of the intermediate liquid was added to the 1.5 mL centrifuge tube containing 450 μL of nucleic acid diluent, the cap was tightened, and the tube was shaken upside down to mix well to obtain nucleic acid extract (template DNA). In S02, instead of using amplified lyophilized powder, a mixed enzyme solution and its buffer are used. The mixed enzyme solution contains the following components: The concentrations of BSU polymerase (20 ng / μL), GB32 recombinase (400 ng / μL), UvsX recombinase (60 ng / μL), UvsY protein (15 ng / μL), creatine kinase (30 ng / μL), and EXO enzyme (2 U / μL) were included.

[0054] The specific steps of the RPA reaction are as follows: (1) 10 minutes in advance, place the buffer (reaction buffer) and activator (350mM magnesium acetate) at room temperature to dissolve completely. After the reagents are dissolved, gently shake to mix for 3-5 seconds, centrifuge for 2-3 seconds, and add the reagents to the test tube in the order shown in Table 2.

[0055] Table 2. Addition of Components in RPA Reaction

[0056] The buffer (2×) consists of: 100 mM Tris-HCl solution at pH 7.9, 200 mM potassium acetate, 400 μM dNTPs, 4 mM dithiothreitol, 100 mM creatine phosphate, and 6 mM ATP.

[0057] (2) After adding the above components, add 2 μL of activator to the cap of the detection tube, carefully put the cap on, and then centrifuge for 2-3 seconds to allow the activator to enter the reaction mixture. Immediately invert the tube 8-10 times or shake it to mix for 3-5 seconds, and then immediately centrifuge for 2-3 seconds to allow all reagents to sink to the bottom of the tube. (3) Open the cover of the fluorescence detector, put the detection tubes into the holes of the fluorescence detector in sequence, close the cover of the instrument, start the instrument, and the instrument will automatically read the positive and negative results of the sample within 15 minutes. The results are displayed directly on the instrument screen. The instrument displays "+" for positive and "-" for negative.

[0058] Sensitivity test Nucleic acid was extracted according to the nucleic acid extraction method in Example 2. Then, using this as the stock solution, a 2-fold dilution method was adopted to obtain a total of 11 different concentrations of DNA template, including the stock solution and the stock solution diluted 2, 4, 8, 16, 32, 64, 128, 256, 512, and 1024 times, as well as 1 negative control. Then, the RPA reaction and detection method in Example 2 were used for detection. The results are shown in Table 3.

[0059] Table 3 Sensitivity Test Results Table 2

[0060] Results analysis: As can be seen from the duration of positive results for each sample in Table 3, the RPA reaction system in Example 2 also has extremely high sensitivity.

[0061] Comparative Example 1 The main difference between the method of Comparative Example 1 for identifying the banana wilt subtype TR4 using fluorescent RPA and Example 2 is that the concentration of BSU polymerase in the mixed enzyme solution is 5 ng / μL.

[0062] Sensitivity test Nucleic acid was extracted according to the nucleic acid extraction method in Example 2. Then, using this as the stock solution, a 2-fold dilution method was used to obtain a total of 9 different concentrations of DNA template, including the stock solution and the stock solution diluted 2, 4, 8, 16, 32, 64, and 128 times, as well as 1 negative control. The RPA reaction and detection method in Example 2 were then used for detection. The results are shown in Table 4.

[0063] Table 4 Sensitivity Test Results Table 3

[0064] Results Analysis: BSU polymerase, as a strand displacement DNA polymerase, is the core enzyme for primer extension in the RPA reaction. When its concentration decreased to 5 ng / μL, insufficient enzyme quantity led to a slower primer extension rate. The time to positive result in the original solution (8 min 30 s) was delayed by 1 min 10 s compared to Example 2 (7 min 20 s), and the delay time gradually increased with increasing dilution factor. In Example 2, samples diluted 64 times, 128 times, and 256 times could all be detected positive within 15 min, while the detection limit of Comparative Example 1 decreased from 256 times to 32 times, indicating a significant decrease in sensitivity. Therefore, when the BSU polymerase concentration is below 10 ng / μL, it cannot meet the requirements for efficient extension of medium and low concentration templates, resulting in detection sensitivity and efficiency that do not reach the optimized system level, and failing to achieve accurate detection of low bacterial count samples in the early stage of banana wilt TR4 infection.

[0065] Comparative Example 2 The main difference between Comparative Example 2 and Example 2 is that the concentration of UvsX recombinant enzyme in the mixed enzyme solution is 46 ng / μL.

[0066] Sensitivity test Nucleic acid was extracted according to the nucleic acid extraction method in Example 2. Then, using this as the stock solution, a 2-fold dilution method was used to obtain a total of 9 different concentrations of DNA template, including the stock solution and the stock solution diluted 2, 4, 8, 16, 32, 64, and 128 times, as well as 1 negative control. The RPA reaction and detection method in Example 2 were then used for detection. The results are shown in Table 5.

[0067] Table 5 Sensitivity Test Results Table 4

[0068] Results Analysis: The core function of UvsX recombinase is to bind primers and open the template DNA double strand. When the concentration drops to 46 ng / μL, its mediated strand displacement efficiency decreases, leading to a slower primer-template binding speed. The time to positive results for a 16-fold diluted sample (14 min 38 s) is close to the 15 min threshold. Therefore, the UvsX recombinase concentration needs to be maintained at 50-80 ng / μL. Below 50 ng / μL, the strand displacement efficiency cannot support detection within 15 min for medium-concentration templates (e.g., 32-fold dilution), resulting in reduced detection sensitivity and failing to meet the detection requirements for different bacterial loads in actual samples.

[0069] Comparative Example 3 The main difference between Comparative Example 3 and Example 2 is that the concentration of EXO enzyme in the mixed enzyme solution is 0.5 U / μL.

[0070] Sensitivity test Nucleic acid was extracted according to the nucleic acid extraction method in Example 2. Then, using this as the stock solution, a 2-fold dilution method was adopted to obtain a total of 11 different concentrations of DNA template, including the stock solution and the stock solution diluted 2, 4, 8, 16, 32, 64, 128, 256, 512, and 1024 times, as well as 1 negative control. Then, the RPA reaction and detection method in Example 2 were used for detection. The results are shown in Table 6.

[0071] Table 6 Sensitivity Test Results (Table 5)

[0072] Results Analysis: EXO enzyme, as a fluorescent probe cleavage enzyme, needs to specifically cleave the P1 probe (releasing the FAM fluorescent group) bound to the TR4 amplification product. When the concentration drops to 0.5 U / μL, the probe cleavage efficiency decreases significantly. Therefore, when the EXO enzyme concentration is below 1 U / μL, the fluorescence signal release efficiency cannot meet the requirements for detection within 15 minutes of low-concentration templates, directly leading to a significant reduction in detection sensitivity. This makes it impossible to achieve rapid and accurate identification of the banana wilt subtype TR4, especially in samples with low bacterial loads in the early stages of infection.

[0073] Comparative Example 4 A primer-probe combination for identifying the banana wilt subtype TR4 using fluorescent RPA, comprising: F2 primer: 5'-GACAAGAAATTGGCGTAGCGTTTCGGGGCAT-3', whose nucleotide sequence is SEQ ID NO: 4; R2 primer: 5'-CGCGCCAAATATTACGAAATAACAGCATTCAT-3', whose nucleotide sequence is SEQ ID NO: 5; P2 probe: 5'-CGACTTACCGTGAAATGGAGAAAAGGATGATACTATTGCTTTATTTTCT-3'ddC, its nucleotide sequence is SEQ ID NO: 6; the thymine at the 28 bp position from the 5' end of the P2 probe is modified with the fluorescent group FAM; the G base at the 29 bp position from the 5' end of the P2 probe is replaced by dSpacer; the thymine at the 31 bp position from the 5' end of the P2 probe is modified with the quenching group BHQ1; the P2 probe is connected to ddC at the 3' end.

[0074] The primer-probe combination in Example 1 and the primer-probe combination in Comparative Example 4 were compared and tested using the method described in Example 1. Two replicates were set for each primer-probe combination. The grouping and test results are shown in Table 7.

[0075] Table 7. Fluorescence test results of the two primer-probe combinations.

[0076] Results analysis: As can be seen from the results in Table 7, neither of the two replicate experiments with the RPA2 primer-probe combination could detect TR4, while both replicate experiments with the RPA1 primer-probe combination could detect TR4 of banana wilt disease normally, indicating that the RPA1 primer-probe combination was more effective.

[0077] In summary, this invention enables rapid nucleic acid extraction, is time-efficient, and simple to operate, with extraction time limited to only 10-15 minutes. Furthermore, the specifically designed fluorescent RPA primer-probe combination exhibits high specificity and sensitivity, theoretically allowing detection of banana samples even in the early stages of infection when asymptomatic. The entire detection method and fluorescence detector are characterized by their short processing time and ease of operation; the time from nucleic acid extraction to detection for two samples is controlled within 22-30 minutes. No special facilities or professional personnel are required, enabling portable and rapid field testing.

[0078] It is understood that those skilled in the art can make equivalent substitutions or changes to the technical solution and inventive concept of the present invention, and all such changes or substitutions should fall within the protection scope of the present invention.

Claims

1. A primer-probe combination for identifying the banana wilt subtype TR4 using fluorescent RPA, characterized in that, The primer-probe combination includes an F1 primer, an R1 primer, and a P1 probe; The nucleotide sequence of the F1 primer is as follows: 5'-TGAGGGATTGGAGCGACTTACCGTGAAATGG-3'; The nucleotide sequence of the R1 primer is as follows: 5'-TCAGATAGCTGATATCTCCCTGCTCCATCA-3'; The nucleotide sequence of the P1 probe is as follows: 5'-AGGATGATACTATTGCTTTATTTTCTGTGGTTATTTGGAGTCACTCTA-3'; The thymine at a position 28 bp from the 5' end of the P1 probe is modified with the fluorescent group FAM. The G base at a position 30 bp from the 5' end of the P1 probe is replaced by dSpacer; The thymine at a position 31 bp from the 5' end of the P1 probe is modified with the quenching group BHQ1. The P1 probe is connected to ddC at its 3' end.

2. A kit for identifying the banana wilt subtype TR4 using fluorescent RPA, characterized in that, The kit includes the primer-probe combination as described in claim 1.

3. The kit for identifying banana wilt subtype TR4 using fluorescent RPA according to claim 2, characterized in that, The kit also includes lysis buffer, nucleic acid dilution buffer, amplification lyophilized powder, and activator.

4. The kit for identifying banana wilt subtype TR4 using fluorescent RPA according to claim 2, characterized in that, The kit also includes lysis buffer, nucleic acid dilution buffer, mixed enzyme solution, buffer, and activator.

5. The kit for identifying banana wilt subtype TR4 using fluorescent RPA according to claim 4, characterized in that, The mixed enzyme solution comprises the following components: BSU polymerase at a concentration of 10–30 ng / μL, GB32 recombinase at a concentration of 300–500 ng / μL, UvsX recombinase at a concentration of 50–80 ng / μL, UvsY protein at a concentration of 5–20 ng / μL, creatine kinase at a concentration of 20–50 ng / μL, and EXO enzyme at a concentration of 1–4 U / μL.

6. The kit for identifying banana wilt subtype TR4 using fluorescent RPA according to claim 3 or 4, characterized in that, The lysis buffer includes 0.2–0.3 M NaOH, 0.1–0.3 M Tris with a pH of 12.0–12.5, and 1–5 mM DTT; the nucleic acid dilution buffer is 10–50 mM Tris-H with a pH of 7.0–8.

0.

7. A method for identifying the banana wilt subtype TR4 using fluorescent RPA, characterized in that, The detection using the kit described in claim 3 or 4 includes the following steps: S1. Sampling and processing of the samples to be tested; S2. Obtain the nucleic acid from the sample to be tested; S3. Mix the nucleic acid, lyophilized amplification powder, activator, and primer probe combination of the sample to be tested, and then place it in a fluorescence detector for reaction to obtain the detection results; or mix the nucleic acid, mixed enzyme solution, buffer, activator, and primer probe combination of the sample to be tested, and then place it in a fluorescence detector for reaction to obtain the detection results.

8. The method for identifying banana wilt subtype TR4 using fluorescent RPA according to claim 7, characterized in that, In step S1, the sample to be tested is a banana pseudostem tuber suspected of being infected with banana wilt disease and located close to the ground; the banana pseudostem tuber is then processed to have a length and width of 1.0–2.0 cm and a surface area of ​​2–2.5 cm². 2 Samples with a thickness of 2-3 mm.

9. The method for identifying banana wilt subtype TR4 using fluorescent RPA according to claim 8, characterized in that, The specific operation of step S2 is as follows: put the processed sample into a container, add lysis buffer to the container, crush the sample; aspirate the liquid in the container into a centrifuge tube, and place the centrifuge tube in a fluorescence detector for incubation; After the sample is incubated, it is transferred to a centrifuge tube containing nucleic acid diluent, mixed thoroughly, and the nucleic acid extract is obtained.

10. The method for identifying banana wilt subtype TR4 using fluorescent RPA according to claim 9, characterized in that, The specific operation of step S3 is as follows: mix and dilute the primer probe combination and activator to obtain the reaction agent; add nucleic acid extraction solution to the reaction tube containing amplification lyophilized powder, then add the reaction agent and mix evenly to obtain a mixture; transfer the mixture to the detection tube, then place the detection tube into the fluorescence detector, and read the detection result of the fluorescence detector after the reaction is completed; or add the primer probe combination, activator, nucleic acid extraction solution, mixed enzyme solution, buffer and nuclease-free water to the detection tube, mix evenly, place the detection tube into the fluorescence detector, and read the detection result of the fluorescence detector after the reaction is completed.

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