Primer probe combination, kit and RPA-LFD method for detecting citrus huanglongbing

Abstract

The invention relates to the field of biological detection, and discloses a primer probe combination, a kit and an RPA-LFD method for detecting Candidatus Liberobacter asiaticum. The primer probe combination provided by the invention can be used for RPA-LFD detection of Citrus Huanglongbing, and has the advantages of wide sampling range, good specificity, high accuracy and high sensitivity. According to the method, a sample does not need to be purified after being ground and split and can be directly used as a template, meanwhile, a specific primer probe aiming at the citrus huanglongbing is designed by utilizing an RPA-LFD technology, an RPA reaction is carried out by utilizing palm heat preservation, and tools and operation are simplified by combining the characteristics of rapidness, simplicity, result visualization and the like of an LFD detection test paper card, so that the detection efficiency is improved, and the detection cost is reduced. Therefore, the method has the characteristics of simplicity in operation, no need of equipment, short time consumption, high detection sensitivity and the like, has huge advantages on the traditional PCR and QPCR technologies, and provides a new method and path for early-stage rapid and mobile diagnosis of the citrus huanglongbing.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and in particular to a primer-probe combination, kit, and RPA-LFD method for detecting Huanglongbing (HLB) in citrus. Background Technology

[0002] Citrus Huanglongbing, also known as yellow shoot disease or yellow blight, is caused by infection with Bacillus thuringiensis. The disease is primarily transmitted through psyllids and spreads extremely rapidly. Currently, control measures mainly focus on removing diseased trees, controlling citrus psyllids, and using disease-free seedlings. Therefore, early diagnosis of citrus Huanglongbing is crucial for controlling the disease and preventing large-scale infection of citrus orchards.

[0003] Research on citrus Huanglongbing (HLB) has been ongoing for many years, and various detection methods exist, such as conventional PCR, nested PCR, and real-time fluorescent PCR. However, conventional PCR methods suffer from low sensitivity, inability to quantify, complex operation, and long processing times. While fluorescent PCR and nested PCR offer high sensitivity, they also suffer from numerous operational steps, long processing times, the need for specialized facilities and equipment, and the inability to enable mobile field testing. Currently, recombinase polymerase amplification (RPA) technology offers advantages such as simple operation, short processing time (20-30 minutes), no need for complex equipment, mobile real-time detection, and high sensitivity. As a novel technology, it has developed rapidly in recent years and is particularly well-suited for the early detection of plant quarantine diseases. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide a primer-probe combination, kit and RPA-LFD method for detecting citrus Huanglongbing, aiming to solve the problems of long nucleic acid extraction time, complicated steps, long detection time, and the need for precision instruments and professional operators in traditional molecular detection methods.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A primer-probe combination for detecting citrus Huanglongbing (HLB), the primer-probe combination comprising an F1 primer, an R1 primer, and a P1 probe; The nucleotide sequence of the F1 primer is as follows: 5'-Biotin-TATATTGGTTGACCTATGCAGTATCATCCA-3'; The nucleotide sequence of the R1 primer is as follows: 5'-TATTAACATATCTAGATATCGAAGTACTTG-3'; The nucleotide sequence of probe P1 is as follows: 5'-CTCTACAATGAAGAACGCATAGGAATCCTCATTAGGAATGGACACA-3'; The C base of the P1 probe is replaced by dSpacer at a position 30 bp from the 5' end; the P1 probe is attached to a fluorescent group at the 5' end and to ddC at the 3' end.

[0006] The primer-probe combination for detecting citrus Huanglongbing is described above, wherein the fluorescent group is FAM.

[0007] An RPA-LFD kit for detecting citrus Huanglongbing (HLB) includes the primer-probe combination described above.

[0008] The RPA-LFD kit for detecting citrus Huanglongbing further includes lysis buffer, nucleic acid dilution buffer, amplification lyophilized powder, activator, LFD test strip, and product dilution buffer.

[0009] The RPA-LFD kit for detecting citrus Huanglongbing further includes lysis buffer, nucleic acid diluent, mixed enzyme solution, buffer, activator, LFD test strip, and product diluent.

[0010] The RPA-LFD kit for detecting citrus Huanglongbing (HLB) comprises the following components in the mixed enzyme solution: BSU polymerase at a concentration of 10–30 ng / μL, GB32 recombinase at a concentration of 200–400 ng / μL, UvsX recombinase at a concentration of 30–60 ng / μL, UvsY protein at a concentration of 5–20 ng / μL, creatine kinase at a concentration of 20–50 ng / μL, and NFO enzyme at a concentration of 0.5–1 U / μL.

[0011] The RPA-LFD kit for detecting citrus Huanglongbing (HLB) comprises the following: the lysis buffer includes 0.15–0.3 M NaOH, 0.05–0.1 M Tris at pH 12.0–12.5, and 1–5 mM EDTA; the nucleic acid dilution buffer is 10–50 mM Tris-H at pH 7.0–8.0; and the product dilution buffer is 20–50 mM Tris-H at pH 7.0–8.0.

[0012] An RPA-LFD method for detecting citrus Huanglongbing (HLB) using the aforementioned RPA-LFD 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. Perform an RPA reaction on the nucleic acid of the sample to be tested and the primer and probe combination to obtain the amplification product; S4. Dilute the amplification product and add it dropwise to the LFD test strip for detection to obtain the test results.

[0013] The RPA-LFD method for detecting citrus Huanglongbing (HLB) includes, in step S1, samples to be tested including tender branch samples suspected of being infected with HLB, citrus leaf samples, fruit samples suspected of being infected with HLB, and psyllid samples.

[0014] The RPA-LFD method for detecting citrus Huanglongbing (HLB) includes the following steps in step S2: Samples of suspected HLB-infected tender branches, citrus leaves, and fruits are collected and placed in a container. A lysis buffer is added to the container, and the samples are crushed and allowed to stand for 2-3 minutes. Alternatively, psyllid samples are placed in a container, a lysis buffer is added, and the psyllid samples are ground for 2-3 minutes and allowed to stand. The supernatant from the crushed or ground samples is extracted and added to a container containing nucleic acid diluent. The mixture is then thoroughly mixed to obtain the nucleic acid extract.

[0015] The RPA-LFD method for detecting citrus Huanglongbing (HLB) includes the following steps in step S3: The primer-probe combination and activator are mixed and diluted to obtain a reaction agent; nucleic acid extraction solution is added to a reaction tube containing lyophilized amplification powder, followed by the addition of the reaction agent and thorough mixing; or the primer-probe combination, activator, nucleic acid extraction solution, mixed enzyme solution, buffer, and denuclease-free water are added to the reaction tube and thoroughly mixed; the reaction tube is incubated at 37–42°C for 12–15 min to obtain the amplification product.

[0016] The RPA-LFD method for detecting citrus Huanglongbing, wherein step S4 specifically involves: taking aspirate the amplification product, adding it to a container containing product diluent, mixing thoroughly to obtain the amplification product diluent; and adding the amplification product diluent to the LFD test strip to obtain the detection result.

[0017] Beneficial effects: This invention provides a primer-probe combination, kit, and RPA-LFD method for detecting citrus Huanglongbing (HLB). The primer-probe combination, used in the RPA-LFD method for detecting HLB, offers advantages such as wide sampling range, high specificity, high accuracy, and high sensitivity. This invention creatively provides a rapid nucleic acid extraction technology; samples can be directly used as templates after grinding and lysis without purification. Utilizing RPA-LFD technology (recombinase polymerase isothermal amplification and immunoassay strip technology), specific primers and probes targeting HLB were designed. The RPA reaction is performed using hand warming, combined with the rapid, simple, and visually appealing characteristics of LFD test strips. Furthermore, the invention creatively uses pipettes and droppers to simplify tools and operations, making the entire solution simple to operate, requiring no equipment, time-efficient, and highly sensitive. This offers significant advantages over traditional PCR and qPCR technologies, providing a new method and pathway for early, rapid, and mobile diagnosis of HLB, and is of great significance for controlling HLB and promptly blocking its rapid spread and infection. Attached Figure Description

[0018] Figure 1 Electrophoresis images of three pairs of RPA primers for citrus Huanglongbing (HLB).

[0019] Figure 2 The results are for RPA-LFD testing of the RPA primer-probe combination of Example 1 and Comparative Example 1.

[0020] Figure 3 This is the specific detection result of Example 1.

[0021] Figure 4 The results are the sensitivity test results for Example 1. Detailed Implementation

[0022] This invention provides a primer-probe combination, kit, and RPA-LFD method for detecting citrus Huanglongbing (HLB). To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the following detailed description, with reference to the accompanying drawings and embodiments, further illustrates the invention. It should be understood that the specific embodiments described herein are merely illustrative and are not intended to limit the scope of protection of this invention.

[0023] This invention provides a primer-probe combination for detecting citrus Huanglongbing (HLB), the primer-probe combination comprising an F1 primer, an R1 primer, and a P1 probe; The nucleotide sequence of the F1 primer is as follows: The nucleotide sequence of the 5'-Biotin-TATATTGGTTGACCTATGCAGTATCATCCA-3' primer is shown in SEQ ID NO: 1. The forward primer is labeled with biotin at the 5' end for easy detection of the subsequent product.

[0024] The nucleotide sequence of the R1 primer is as follows: 5'-TATTAACATATCTAGATATCGAAGTACTTG-3', whose nucleotide sequence is shown in SEQ ID NO: 2.

[0025] The nucleotide sequence of probe P1 is as follows: 5'-CTCTACAATGAAGAACGCATAGGAATCCTCATTAGGAATGGACACA-3', the nucleotide sequence of which is shown in SEQ ID NO: 3; the C base of the P1 probe is replaced by dSpacer at a position 30 bp from the 5' end; the P1 probe is attached to a fluorescent group at the 5' end and to ddC at the 3' end.

[0026] Specifically, the fluorescent group is FAM.

[0027] The present invention also provides an RPA-LFD kit for detecting citrus Huanglongbing, comprising the above-mentioned primer and probe combination.

[0028] In one embodiment, the RPA-LFD kit further includes lysis buffer, nucleic acid diluent, amplification lyophilized powder, activator, LFD test strip, and product diluent.

[0029] In another embodiment, the RPA-LFD kit further includes lysis buffer, nucleic acid diluent, mixed enzyme solution, buffer, activator, LFD test strip, and product diluent.

[0030] 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 substitution function; GB32 recombinase at a concentration of 200–400 ng / μL, wherein the GB32 recombinase is an Escherichia coli RecA family recombinase; UvsX recombinase at a concentration of 30–60 ng / μL, wherein the UvsX recombinase is a recombinase derived from bacteriophage T4; UvsY protein at a concentration of 5–20 ng / μL, wherein the UvsY protein is a UvsX accessory protein derived from bacteriophage T4; creatine kinase at a concentration of 20–50 ng / μL, wherein the creatine kinase has ATP regeneration function; and NFO enzyme at a concentration of 0.5–1 U / μL, wherein the NFO enzyme is a nuclease capable of cleaving uracil-containing DNA strands.

[0031] More specifically, the lysis buffer comprises 0.15–0.3 M NaOH, 0.05–0.1 M Tris at pH 12.0–12.5, and 1–5 mM EDTA. The nucleic acid diluent is 10–50 mM Tris-H at pH 7.0–8.0. The product diluent is 20–50 mM Tris-H at pH 7.0–8.0.

[0032] This invention also provides an RPA-LFD method for detecting citrus Huanglongbing (HLB), using the aforementioned RPA-LFD kit for identification, comprising the following steps: S1. Sampling and processing of the samples to be tested.

[0033] The specific steps are as follows: (1) Citrus branch samples: Take tender branches suspected of being infected with citrus Huanglongbing, remove the leaves from the branches with scissors, cut the branches into 0.2-0.3cm lengths, and put 5-7 branch pieces into a grinding bag.

[0034] (2) Citrus leaf samples: For trees suspected of being diseased, prioritize collecting leaves showing signs of mottled yellowing. If no typical symptoms are observed, collect leaves from the four cardinal directions of the tree canopy, taking 1–1.5 cm samples. 2 Place the citrus leaves of different sizes into a grinding bag.

[0035] (3) Citrus fruit samples: Select fruits suspected of being infected with citrus Huanglongbing, cut off the fruit stalk, fruit stem, and orange pith, and put 2-3g into a grinding bag.

[0036] (4) Samples of the disease vector, the citrus psyllid: During the period of new shoot emergence in citrus trees, a certain number of adult insects or larvae of the 3rd instar or above are collected from each tree. Take 4-5 psyllid samples and place them in a 1.5ml centrifuge tube.

[0037] S2. Obtain the nucleic acid from the sample to be tested.

[0038] The specific steps are as follows: (1) Place the prepared sample (twigs, leaves, fruit) in the bottom corner of the grinding bag (tweezers can be used), open the grinding bag, draw up the lysis solution and add it to the grinding bag, remove the air from the grinding bag, and seal the grinding bag. Fold the grinding bag diagonally, lay the sample and lysis solution flat on a horizontal surface, ensuring that the sample is immersed in the lysis solution, and gently and quickly hammer the sample with a small claw hammer or other suitable tool (be careful not to hammer too hard to avoid breaking the grinding bag), hammer for about 1 minute until the sample is crushed, and let it stand for 2-3 minutes. Psyllid sample: Take 4-5 psyllid samples, put them into a 1.5 mL centrifuge tube, draw up the lysis solution and add it to the 1.5 mL centrifuge tube, grind with a pestle for about 2-3 minutes, and let it stand for 1 minute.

[0039] (2) After the sample is crushed or ground, squeeze the sample and lysis buffer into one corner of the grinding bag, take 20 μL of the supernatant and add it to a container containing 380 μL of nucleic acid diluent. Dilute the supernatant 20 times and mix well to obtain the nucleic acid extract.

[0040] S3. Perform an RPA reaction on the nucleic acid of the sample to be tested and the primer and probe combination to obtain the amplification product.

[0041] The specific steps are as follows: In one embodiment, the specific steps for performing an RPA reaction on the nucleic acid of the sample to be tested and the primer-probe combination to obtain the amplification product are as follows: The 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 the amplification lyophilized powder, followed by the addition of the reaction agent and thorough mixing; the mixture is incubated at 37–42°C for 12–15 minutes to obtain the amplification product. In outdoor locations such as fields, the reaction temperature can be simulated by holding the reaction tube in hand, further reducing the difficulty of detecting Huanglongbing (HLB). It should be noted that the amplification lyophilized powder contains enzymes and buffers related to amplification.

[0042] In another embodiment, the specific operation of performing an RPA reaction on the nucleic acid of the sample to be tested and the primer-probe combination to obtain the amplification product is as follows: the primer-probe combination, activator, nucleic acid extraction solution, mixed enzyme solution, buffer and nuclease-free water are added to the reaction tube and mixed evenly; the mixture is incubated in a metal bath at 37-42℃ for 12-15 min to obtain the amplification product.

[0043] S4. Dilute the amplification product and add it dropwise to the LFD test strip for detection to obtain the test results.

[0044] The specific steps are as follows: Aspirate the amplification product and add it to a container containing the product diluent. Mix well to obtain the amplification product diluent. Add the amplification product diluent to the LFD test strip to obtain the test results.

[0045] To further illustrate the primer-probe combination, kit, and RPA-LFD method for detecting citrus Huanglongbing provided by the present invention, the following examples are provided.

[0046] Example 1 PCR verification and screening of 3 pairs of RPA primers for citrus Huanglongbing (1) RPA1~RPA3 primer pair RPA1 primer pair: The nucleotide sequence of the F1 primer is as follows: 5'-TATATTGGTTGACCTATGCAGTATCATCCA-3'; The nucleotide sequence of the R1 primer is as follows: 5'-TATTAACATATCTAGATATCGAAGTACTTG-3'.

[0047] RPA2 primer pair: The nucleotide sequence of the F2 primer is: 5'-TGCTATTTTCTAATCGGAGACCTAAACCAT-3'; The nucleotide sequence of the R2 primer is as follows: 5'-AACCTATACTTGATGCCTTCTAGAGCGTC-3'.

[0048] RPA3 primer pair: The nucleotide sequence of the F3 primer is as follows: 5'-TCGGATAGTCCTGTTATTGCTCCTAAA-3'; The nucleotide sequence of the R3 primer is as follows: 5'-AAATAGCACGAACAACTTGAGTCTGTG-3'.

[0049] (2) RPA1 and RPA2 primer-probe combination RPA1 primer-probe combination: The nucleotide sequence of the F1 primer is as follows: 5'-Biotin-TATATTGGTTGACCTATGCAGTATCATCCA-3', the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0050] The nucleotide sequence of the R1 primer is as follows: 5'-TATTAACATATCTAGATATCGAAGTACTTG-3', whose nucleotide sequence is shown in SEQ ID NO: 2.

[0051] The nucleotide sequence of probe P1 is as follows: 5'-CTCTACAATGAAGAACGCATAGGAATCCTCATTAGGAATGGACACA-3', the nucleotide sequence of which is shown in SEQ ID NO: 3; the C base of the P1 probe is replaced by dSpacer at a position 30 bp from the 5' end; the P1 probe is connected to a FAM fluorescent group at the 5' end and to ddC at the 3' end.

[0052] RPA2 primer-probe combination: The nucleotide sequence of the F2 primer is: 5'-Biotin-TGCTATTTTCTAATCGGAGACCTAAACCAT-3', the nucleotide sequence of which is shown in SEQ ID NO: 4.

[0053] The nucleotide sequence of the R2 primer is as follows: 5'-AACCTATACTTGATGCCTTCTAGAGCGTC-3', whose nucleotide sequence is shown in SEQ ID NO: 5.

[0054] The nucleotide sequence of the P2 probe is as follows: 5'-TAGCCTCGAAGCGCCACTTAACGGAAACTGAGTAGCTGCTGAGCACAG-3', the nucleotide sequence of which is shown in SEQ ID NO: 6; the T base of the P2 probe is replaced by dSpacer at a position 33 bp from the 5' end; the P2 probe is connected to a FAM fluorescent group at the 5' end and to ddC at the 3' end.

[0055] Three pairs of primers for citrus Huanglongbing (HLB) RPA were designed and synthesized (the specific nucleotide sequences are shown above). Four different HLB-positive citrus samples (citrus fruit, citrus psyllids, citrus branches, and citrus leaves) were used for PCR and electrophoresis to screen the primer pairs. Results are as follows: Figure 1 As shown, where Figure 1 Numbers 1-4 represent electrophoresis images after PCR of fruit, psyllid, branch, and leaf samples from the same positive citrus Huanglongbing plant using primer pair RPA1; numbers 5-8 represent electrophoresis images after PCR of fruit, psyllid, branch, and leaf samples from the same positive citrus Huanglongbing plant using primer pair RPA2; numbers 9-12 represent electrophoresis images after PCR of fruit, psyllid, branch, and leaf samples from the same positive citrus Huanglongbing plant using primer pair RPA3.

[0056] Analysis and conclusions: From Figure 1The results show that primer pairs RPA1 and RPA2 detected bands in all four samples, indicating good specificity. Primer pair RPA3 only showed a band in sample number 11 (branch sample), and the band was blurry. Therefore, RPA1 and RPA2 were selected as candidate primers. Based on this, corresponding probes were designed and two pairs of RPA primer probes were synthesized. The corresponding nucleotide sequences are shown above.

[0057] Example 2 An RPA-LFD method for detecting citrus Huanglongbing (HLB) includes the following steps: S1. Sample Sampling and Sample Processing (1) Citrus branch samples: Take tender branches suspected of being infected with citrus Huanglongbing, remove the leaves from the branches with scissors, cut the branches into 0.2-0.3cm lengths, and put 5-7 branch pieces into a grinding bag.

[0058] (2) Citrus leaf samples: For trees suspected of being diseased, prioritize collecting leaves showing signs of mottled yellowing. If no typical symptoms are observed, collect leaves from the four cardinal directions of the tree canopy, taking 1–1.5 cm samples. 2 Place the citrus leaves of different sizes into a grinding bag.

[0059] (3) Citrus fruit samples: Select fruits suspected of being infected with citrus Huanglongbing, cut off the fruit stalk, fruit stem, and orange pith, and put 2-3g into a grinding bag.

[0060] (4) Samples of the disease vector, the citrus psyllid: During the period of new shoot emergence in citrus trees, a certain number of adult insects or larvae of the 3rd instar or above are collected from each tree. Take 4-5 psyllid samples and place them in a 1.5ml centrifuge tube.

[0061] S2. Nucleic acid extraction Place the prepared samples (twigs, leaves, fruits) in the bottom corner of the grinding bag (tweezers can be used as a tool). Open the grinding bag and add the lysis buffer to the bag in 5 portions using a 200μL pipette. Expel the air from the grinding bag and seal it. Fold the grinding bag diagonally, laying the sample and lysis buffer flat on a horizontal surface, ensuring 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 minute until the sample is crushed. Let it stand for 2-3 minutes. For psyllid samples: Take 4-5 psyllid samples and place them in a 1.5mL centrifuge tube. Add the lysis buffer to the 1.5mL centrifuge tube in 2 portions using a 200μL pipette. Grind with a pestle for about 2-3 minutes and let it stand for 1 minute. Specifically, the lysis buffer includes NaOH at a concentration of 0.15–0.3 M, Tris at a concentration of 0.05–0.1 M and a pH of 12.0–12.5, and EDTA at a concentration of 1–5 mM.

[0062] After crushing or grinding the sample, squeeze the sample and lysis buffer into a corner of the grinding bag. Use a 20μL pipette to draw 20μL of the supernatant. Unscrew the cap of dropper #1 (containing 380μL of nucleic acid diluent), dilute the supernatant 20-fold, add it to dropper #1, tighten the cap, and shake several times to mix the liquid thoroughly, thus obtaining the nucleic acid extract. Specifically, the nucleic acid diluent is Tris-H with a concentration of 10–50 mM and a pH of 7.0–8.0.

[0063] S3.RPA reaction Using commercially available RPA reagents as raw materials, the reagent kit includes lyophilized amplification powder (containing amplification-related enzymes and buffer) and activator. Dissolve the activator in the reagent kit at room temperature, then pipette it evenly with a 200 μL pipette. Pipette 60–65 μL of activator, 40–45 μL each of F1 and R1 primers, and 10–15 μL of P1 probe into an empty dropper bottle No. 2. Then, pipette 530–550 μL of nucleic acid diluent into dropper bottle No. 2, bringing the total liquid volume to 700 μL. Cap dropper bottle No. 2 and shake it until the liquid is homogeneous to obtain the reaction reagent.

[0064] Take out the reaction tube containing the amplification lyophilized powder, open the cap, and use the No. 1 dropper bottle containing nucleic acid extraction solution. After opening the cap, first drop 2 drops into the waste liquid container to ensure the accuracy of the liquid volume, and then drop 1 drop into the amplification lyophilized powder.

[0065] Take out dropper bottle No. 2, open the cap and drop 2 drops into the waste liquid container, then drop 1 drop of the reaction agent into the amplification lyophilized powder. Use a pipette to carefully blow and stir the liquid formed after the amplification lyophilized powder melts, blowing and stirring 15 to 20 times until completely uniform.

[0066] The amplification product was obtained by incubating at 37–42℃ for 12–15 min.

[0067] In the RPA reaction, the commercially available RPA reagent is specifically the chromatographic RPA (NFO probe) (in situ lyophilized powder) produced by Yisheng Biotechnology (Shanghai) Co., Ltd.

[0068] S4.LFD test strip detection After RPA incubation, carefully open the cap and use a 20μL double-ended pipette to draw 20μL of amplification product. Add this product to a dropper bottle containing 1180μL of product diluent, diluting 60 times. Tighten the cap and shake the bottle upside down 5–7 times to ensure thorough mixing. Specifically, the product diluent is Tris-H with a concentration of 20–50 mM and a pH of 7.0–8.0.

[0069] After shaking well, open the cap, drop 2 drops into the waste liquid container, and then drop 3 drops into the sample loading area (S) of the LFD test strip. Wait 2-3 minutes and observe the results. The results displayed within 1-5 minutes shall be taken as the standard. Results displayed after 5 minutes shall not be used as the basis for judging positive or negative.

[0070] Result interpretation: If there are lines at both C (control line) and T (test line) on the reagent card (even if the line is lighter in color, it is considered positive), the result is positive; if there is only a line at C, the result is negative; if there is no line at C (regardless of whether there is a line at T), the reagent card is invalid and needs to be replaced for testing.

[0071] Example 3 Comparative verification results of two pairs of citrus Huanglongbing RPA primer probes on LFD test strips Two pairs of RPA primers and probes for citrus Huanglongbing (nucleotide sequences will be as described in Example 1) were designed. Using citrus Huanglongbing branch samples, nucleic acid extraction and RPA reaction were performed as described in Example 2, with three replicates. RPA-LFD testing was conducted to screen and select the most suitable RPA primer and probe pairs. The results are as follows: Figure 2 As shown, where Figure 2 In the numbers 1 to 3, the RPA1 primer probe is represented by three replicates; in the numbers 4 to 6, the RPA2 primer probe is represented by three replicates.

[0072] Analysis and conclusions: From Figure 2 The results showed that among the two pairs of citrus Huanglongbing RPA primer-probe pairs, all three replicates of the RPA1 primer-probe showed positive results, while the results of the RPA2 primer-probe were unstable, with one strong positive result, one weak positive result, and one negative result. Therefore, the RPA1 primer-probe was initially selected as the primer-probe of this invention, and subsequent specificity and sensitivity tests were carried out.

[0073] Example 4 Specific detection Using RPA1 primers and probes F1, R1, and P1, DNA from citrus Huanglongbing (HLB), citrus canker, citrus anthracnose, tomato wilt, citrus scab, citrus scab, citrus sooty mold, and citrus yellow spot was used as a template, with nuclease-free water as a negative control to verify specificity. Specific sample information is shown in Table 1, and the comparison results of the LFD test strips are as follows. Figure 3 As shown.

[0074] Table 1 Sample Information

[0075] Analysis and Conclusions: A total of 8 samples and one negative control (nuclease-free water) were tested. The 8 samples, from left to right, were: citrus Huanglongbing (HLB), citrus canker, citrus anthracnose, tomato bacterial wilt, citrus scab, citrus scab, citrus sooty mold, and citrus yellow spot. Figure 3 It can be seen that only sample number 1 (citrus Huanglongbing sample) tested positive out of the eight samples, indicating that the primer-probe combination provided in Example 1 has good specificity. The negative control result was negative, indicating that there were no false positive results.

[0076] Example 5 Sensitivity test Nucleic acid extracted from positive samples of citrus Huanglongbing (HLB) branches was serially diluted eight times (2, 4, 8, 16, 32, 64, 128, and 256 times) using a two-fold serial dilution method. RPA-LFD assays were then performed to test the sensitivity of this method. The results are as follows: Figure 4 As shown, where Figure 4 The numbers 1 to 8 represent positive samples diluted 2, 4, 8, 16, 32, 64, 128, and 256 times with the nucleic acid extract, respectively.

[0077] Analysis and conclusions: From Figure 4 The results showed that when the nucleic acid extracted from the positive sample of citrus Huanglongbing was diluted to 128 times, a blurry band could still be seen. However, when diluted to 256 times, no band was observed. This indicates that the method can still detect the virus even when the viral infection content is very low, which demonstrates the high sensitivity of the method.

[0078] Example 6 The primer-probe combination in Example 6 is the RPA1 primer-probe combination.

[0079] An RPA-LFD method for detecting citrus Huanglongbing (HLB) includes the following steps: S1. Sample sampling and sample processing: Same as in Example 2.

[0080] S2. Nucleic acid extraction Place the prepared samples (twigs, leaves, fruits) in the bottom corner of the grinding bag (tweezers can be used). Open the grinding bag and use a 200μL pipette to add the lysis buffer to the grinding bag in 5 portions. Remove the air from the grinding bag and seal it. Fold the grinding bag diagonally, laying the sample and lysis buffer flat on a horizontal surface, ensuring 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 minute until the sample is crushed. Let it stand for 2-3 minutes. For psyllid samples: Take 4-5 psyllid samples and place them in a 1.5mL centrifuge tube. Use a 200μL pipette to add the lysis buffer to the 1.5mL centrifuge tube in 2 portions. Grind with a pestle for about 2-3 minutes and let it stand for 1 minute. Specifically, the lysis buffer includes NaOH at a concentration of 0.15–0.3 M, Tris at a concentration of 0.05–0.1 M and a pH of 12.0–12.5, and EDTA at a concentration of 1–5 mM.

[0081] After crushing or grinding the sample, squeeze the sample and lysis buffer into a corner of the grinding bag. Use a 20 μL pipette to draw 20 μL of the supernatant and add it to a 1.5 mL centrifuge tube containing 380 μL of nucleic acid diluent. Dilute the supernatant 20 times, cap the tube, and pipette several times to mix the liquid thoroughly, obtaining the nucleic acid extract. Specifically, the nucleic acid diluent is Tris-H with a concentration of 10–50 mM and a pH of 7.0–8.0.

[0082] S3.RPA reaction Using commercially available RPA reagents as raw materials, the reagent kit includes lyophilized amplification powder (containing amplification-related enzymes and buffer) and activator. Dissolve the activator in the kit at room temperature, then pipette it thoroughly with a 200 μL pipette. Pipette 60–65 μL of activator, 40–45 μL each of F1 and R1 primers, 10–15 μL of P1 probe, and 530–550 μL of nucleic acid diluent into a new 1.5 mL centrifuge tube, bringing the total liquid volume to 700 μL. Cap the tube and pipette it several times until homogeneous to obtain the reaction mixture.

[0083] Take out the reaction tube containing the amplification lyophilized powder, open the cap, and use a 200μL pipette to add 25μL of the nucleic acid extraction solution obtained in step S2 and 25μL of the reaction reagent obtained in step S3 to the amplification lyophilized powder. Then use a pipette to blow and mix 15 to 20 times until completely homogeneous.

[0084] Wearing gloves, hold the reaction tube (PCR tube) and keep it warm for 12-15 minutes to obtain the amplification product. It should be noted that when holding the reaction tube, the body temperature is close to 37°C, and the amplification reaction can also be carried out under this condition.

[0085] In the RPA reaction, the commercially available RPA reagent is specifically the chromatographic RPA (NFO probe) (in situ lyophilized powder) produced by Yisheng Biotechnology (Shanghai) Co., Ltd.

[0086] S4.LFD test strip detection After RPA incubation, carefully open the cap and use a 200 μL pipette to aspirate 10 μL of the amplification product. Add this product to a 1.5 mL centrifuge tube containing 590 μL of product diluent. Mix the product thoroughly by pipetting several times. Specifically, the product diluent is Tris-H with a concentration of 20–50 mM and a pH of 7.0–8.0.

[0087] Then open the cap and use a 200μL pipette to draw 80μL of the diluted amplification product into the sample loading area (S) of the LFD test strip. Wait 2-3 minutes and observe the results. The results displayed within 1-5 minutes are considered the standard. Results displayed after 5 minutes are not considered as the basis for judging positive or negative.

[0088] Example 6 uses a pipette and centrifuge tubes instead of the dropper bottle and double-capsule pipette used in Example 2.

[0089] Example 7 An RPA-LFD method for detecting citrus Huanglongbing (HLB) differs from Example 6 mainly in that: In the S3.RPA reaction, instead of using amplification lyophilized powder, a mixed enzyme solution and buffer are used. The mixed enzyme solution includes the following components: BSU polymerase at a concentration of 20 ng / μL, GB32 recombinase at a concentration of 300 ng / μL, UvsX recombinase at a concentration of 50 ng / μL, UvsY protein at a concentration of 15 ng / μL, creatine kinase at a concentration of 30 ng / μL, and NFO enzyme at a concentration of 0.5 U / μL.

[0090] 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 reaction tube in the order shown in Table 2.

[0091] Table 2. Addition of Components in RPA Reaction

[0092] 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.

[0093] (2) After adding the above components, add 2 μL of activator to the cap of the reaction 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) Immediately add the reaction tube to a metal bath (40±2℃) and incubate for 15 min.

[0094] Sensitivity test Nucleic acid extracted from positive samples of citrus Huanglongbing branches was subjected to RPA reaction according to the method in Example 7 to obtain amplification products. Positive samples diluted 2, 4, 8, 16, 32, 64, 128, and 256 times were obtained according to the method in Example 5. RPA-LFD tests were performed on each sample, and the results are shown in Table 3.

[0095] Table 3 Sensitivity Test Results of Example 7

[0096] Table 3 shows that when the positive samples of citrus Huanglongbing branches were diluted 128 times, blurry bands could still be seen, but when diluted 256 times, no bands were visible. This indicates that this method can still detect the virus even when the viral infection content is very low, which means that this method has high sensitivity.

[0097] Comparative Example 1 The main difference between the RPA-LFD method for detecting citrus Huanglongbing in Comparative Example 1 and Example 7 is that the concentration of GB32 recombinant enzyme in the mixed enzyme solution is 150 ng / μL.

[0098] Sensitivity test Nucleic acid extracted from positive samples of citrus Huanglongbing branches in Example 7 was subjected to RPA reaction according to the method in Example 7 to obtain amplification products. Positive samples diluted 2, 4, 8, 16, 32, 64, 128, and 256 times were obtained according to the method in Example 5. RPA-LFD tests were performed on each sample, and the results are shown in Table 4.

[0099] Table 4: Sensitivity Test Results of Comparative Example 1

[0100] Table 4 shows that when the concentration of GB32 recombinase is below the lower limit of the range, the amplification effect on the product is poor. When the positive sample is diluted 32 times, the band becomes blurred. When the positive sample is diluted 64 times, there is no band on the LFD test strip.

[0101] Comparative Example 2 The main difference between the RPA-LFD method for detecting citrus Huanglongbing in Comparative Example 2 and Example 7 is that the concentration of UvsX recombinase in the mixed enzyme solution is 20 ng / μL.

[0102] Sensitivity test Nucleic acid extracted from positive samples of citrus Huanglongbing branches in Example 7 was subjected to RPA reaction according to the method in Example 7 to obtain amplification products. Positive samples diluted 2, 4, 8, 16, 32, 64, 128, and 256 times were obtained according to the method in Example 5. RPA-LFD tests were performed on each sample, and the results are shown in Table 5.

[0103] Table 5. Sensitivity Test Results for Comparative Example 2

[0104] Table 5 shows that when the concentration of UvsX recombinase is below the lower limit of the range, it also affects the amplification effect of the product. When the positive sample is diluted 64 times, the band becomes blurred. When the positive sample is diluted 128 times, there is no band on the LFD test strip. The effect of UvsX recombinase concentration is less than the effect of GB32 recombinase concentration on RPA reaction.

[0105] In summary, this invention enables rapid nucleic acid extraction, is time-efficient, and simple to operate, reducing the extraction time to 5 minutes. Furthermore, the specifically designed RPA primer-probe combination for citrus Huanglongbing (HLB) exhibits high specificity and sensitivity, allowing detection even in the early stages of plant disease when symptoms are not obvious. Moreover, the provided RPA-LFD detection method is time-efficient, completing the detection of a single sample within 30 minutes. It boasts high specificity, sensitivity, and simple operation; using dropper bottles and pipettes, it requires no complex equipment, and even non-professionals can perform portable detection in fields and outdoors after simple training.

[0106] 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 detecting citrus Huanglongbing (HLB), 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'-Biotin-TATATTGGTTGACCTATGCAGTATCATCCA-3'; The nucleotide sequence of the R1 primer is as follows: 5'-TATTAACATATCTAGATATCGAAGTACTTG-3'; The nucleotide sequence of the P1 probe is as follows: 5'-CTCTACAATGAAGAACGCATAGGAATCCTCATTAGGAATGGACACA-3'; The C base of the P1 probe is replaced by dSpacer at a position 30 bp from the 5' end; the P1 probe is attached to a fluorescent group at the 5' end and to ddC at the 3' end.

2. The primer-probe combination for detecting citrus Huanglongbing according to claim 1, characterized in that, The fluorescent group is FAM.

3. An RPA-LFD kit for detecting citrus Huanglongbing (HLB), characterized in that, The RPA-LFD kit includes the primer-probe combination as described in claim 1 or 2.

4. The RPA-LFD kit for detecting citrus Huanglongbing according to claim 3, characterized in that, The RPA-LFD kit also includes lysis buffer, nucleic acid dilution buffer, amplification lyophilized powder, activator, LFD test strip, and product dilution buffer.

5. The RPA-LFD kit for detecting citrus Huanglongbing according to claim 3, characterized in that, The RPA-LFD kit also includes lysis buffer, nucleic acid diluent, mixed enzyme solution, buffer, activator, LFD test strip, and product diluent.

6. The RPA-LFD kit for detecting citrus Huanglongbing according to claim 5, 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 200–400 ng / μL, UvsX recombinase at a concentration of 30–60 ng / μL, UvsY protein at a concentration of 5–20 ng / μL, creatine kinase at a concentration of 20–50 ng / μL, and NFO enzyme at a concentration of 0.5–1 U / μL.

7. The RPA-LFD kit for detecting citrus Huanglongbing according to claim 4 or 5, characterized in that, The lysis buffer comprises 0.15–0.3 M NaOH, 0.05–0.1 M Tris at pH 12.0–12.5, and 1–5 mM EDTA; the nucleic acid diluent is 10–50 mM Tris-H at pH 7.0–8.0; and the product diluent is 20–50 mM Tris-H at pH 7.0–8.

0.

8. An RPA-LFD method for detecting citrus Huanglongbing (HLB), characterized in that, The detection using the RPA-LFD kit as described in claim 4 or 5 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. Perform an RPA reaction on the nucleic acid of the sample to be tested and the primer and probe combination to obtain the amplification product; S4. Dilute the amplification product and add it dropwise to the LFD test strip for detection to obtain the test results.

9. The RPA-LFD method for detecting citrus Huanglongbing according to claim 8, characterized in that, The samples to be tested in step S1 are tender branch samples suspected of being infected with citrus Huanglongbing, citrus leaf samples, fruit samples suspected of being infected with citrus Huanglongbing, and psyllid samples.

10. The RPA-LFD method for detecting citrus Huanglongbing according to claim 9, characterized in that, Step S2 is performed as follows: Samples of tender branches, leaves, and fruits suspected of being infected with citrus Huanglongbing (HLB) were collected and placed in a container. Lysis buffer was added to the container, and the samples were crushed and allowed to stand for 2–3 minutes. Alternatively, psyllid samples were placed in a container, lysis buffer was added, and the psyllid samples were ground for 2–3 minutes and then allowed to stand. Absorb the supernatant of the crushed or ground sample, add the supernatant to a container containing nucleic acid diluent, mix well, and obtain nucleic acid extract.

11. The RPA-LFD method for detecting citrus Huanglongbing according to claim 10, 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 the amplification lyophilized powder, and then add the reaction agent and mix well; or add the primer probe combination, activator, nucleic acid extraction solution, mixed enzyme solution, buffer and nuclease-free water to the reaction tube and mix well; incubate the reaction tube at 37-42℃ for 12-15 min to obtain the amplification product.

12. The RPA-LFD method for detecting citrus Huanglongbing according to claim 11, characterized in that, The specific operation of step S4 is as follows: aspirate the amplification product, add it to a container containing the product diluent, mix well to obtain the amplification product diluent; add the amplification product diluent to the LFD test strip to obtain the test result.

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