Primer-probe combination and detection method for rapid identification of *Candidatus Liberibacter asiaticus* based on MIRA-LFD

Abstract

The present invention provides a primer-probe combination and a detection method for rapidly identifying *Candidatus Liberibacter asiaticus* based on MIRA-LFD. The primer-probe combination includes a primer pair and a probe. The sequences of the primer pair are shown in SEQ ID NO.1-2, and the probe sequence is shown in SEQ ID NO.3 or 4. This primer-probe combination has high sensitivity and specificity. Using this primer-probe combination, the present invention provides a rapid detection method for *Candidatus Liberibacter asiaticus* by combining MIRA with LFD technology, realizing rapid, sensitive, specific, and visual diagnosis of citrus huanglongbing. Moreover, this method is simple to operate, time-saving, efficient, and the results are reliable, solving the problems of long time consumption and cumbersome steps of traditional detection techniques. The detection process does not require special instruments and equipment, the detection cost is low, and it can be applied to special environments such as field sites, which is of great significance for the early detection and diagnosis of *Candidatus Liberibacter asiaticus* in the field.

Description

Technical Field

[0001] The present invention belongs to the detection technology of citrus huanglongbing bacterium, and particularly relates to a primer-probe combination and a detection method for rapidly identifying citrus huanglongbing bacterium based on MIRA-LFD Background Art

[0002] Citrus huanglongbing (HLB) is the most devastating disease in citrus production worldwide and is also an important quarantine pest at home and abroad, causing huge losses to citrus production and seriously threatening the healthy development of the citrus industry. Citrus huanglongbing is a bacterial disease caused by Candidatus Liberibacter. So far, this bacterium has not been able to be obtained in pure culture in vitro. The spread of huanglongbing mainly occurs through the feeding and diffusion of Diaphorina citri, and in addition, the transportation of diseased plants and scions will also cause the long-distance spread of the disease. The latest research shows that citrus huanglongbing bacterium can also infect flower organs and can be spread through the pollination process, greatly increasing the risk of citrus huanglongbing

[0003] At present, the main diagnostic methods for citrus huanglongbing bacterium include field symptom diagnosis, serological diagnosis, electron microscopy diagnosis, PCR and its derivative technologies (such as nested PCR, real-time fluorescence quantitative PCR, etc.). Especially the molecular detection technology based on PCR is widely used. However, the above detection methods are mostly limited to laboratory detection, highly dependent on instruments and equipment, with relatively cumbersome operation processes and long operation times, seriously restricting their popularization and application at the grass-roots level

[0004] Therefore, there is still an urgent need for a detection method for citrus huanglongbing bacterium that is simple in the detection process, low in cost and does not rely on instruments and equipment Summary of the Invention

[0005] Aiming at the problems in the prior art, the present invention uses MIRA (Multienzyme isothermal rapid amplification)-LFD (Lateral flow dipstick) technology to construct a detection method for rapidly identifying citrus huanglongbing bacterium. This method does not require DNA extraction or special instruments and equipment, can be directly used for sample detection in the field, and the detection results can be directly judged by the naked eye, which is conducive to grass-roots promotion and application

[0006] The technical solution of the present invention is specifically as follows

[0007] In the first aspect of the present invention, a primer-probe combination for rapidly identifying citrus huanglongbing bacterium based on MIRA-LFD is provided, including a primer pair and a probe

[0008] Among them, the sequences of the primer pairs are as follows:

[0009] Forward primer RPnrdB-F1: 5’-CATCATGCGAGATGAATCACTGCATCTCAA-3’,

[0010] Reverse primer RPnrdB-R1: 5’-Biotin-TGATATTCTGTGACCCGAGTTTCGAAGAAG-3’;

[0011] The probe is probe P1 or probe P2. Specifically, probe P1 is:

[0012] 5’-6-FAM-CGCACCATGCTCCATGAAGCTACCCTCCTCGA(THF)ATCGCCTATGCACA TG-C3-spacer,

[0013] Specifically, probe P2 is:

[0014] 5’-6-FAM-ATGCTCCCTCATGTGAACAATATATGCAGT(THF)TCATCGCCAATCGTC-C3-spacer.

[0015] In the above primer-probe combination, the primers and probes are designed based on the nrdB gene of Huanglongbing bacterium and obtained through a large number of experiments. They are not only applicable to the detection of different Huanglongbing bacteria, but also have good specificity and will not have non-specific reactions with other common citrus pathogens.

[0016] In the above primer-probe combination, the 5’ end of the reverse primer is labeled with biotin, the 5’ end of the probe is labeled with carboxyfluorescein (FAM), and a tetrahydrofuran (THF) recognition site for the nfo enzyme replaces one base of the probe at about 30 nt from the 5’ end of the probe. It should be noted that the THF site should be ≥15 nt from the 3’ end of the probe; the 3’ end of the probe is modified with a C3-Spacer group that blocks polymerase amplification.

[0017] In the above primer-probe combination, both probe P1 and probe P2 can achieve good detection effects, and probe P1 is slightly better than probe P2.

[0018] The second aspect of the present invention provides a detection method for rapidly identifying Huanglongbing bacterium in citrus based on MIRA-LFD. This method utilizes the primer-probe combination provided in the first aspect of the present invention. The specific steps are as follows:

[0019] S1. Take the midrib of citrus leaves, cut it into thin slices, add it to NaOH solution and grind, and dilute the obtained juice to be used as a template;

[0020] S2. Add a template, forward and reverse primers, and a probe to the MIRA multi-enzyme system, and perform a reaction on the resulting reaction system;

[0021] S3. Drop the reaction product obtained in S2 onto a colloidal gold test strip, and the result can be observed within 5 min; the colloidal gold test strip contains a test line and a quality control line, the test line contains a biotin ligand, and the quality control line contains an anti-FAM antibody.

[0022] In the above detection method, the thickness of the thin slice in step S1 is preferably 0.1 - 0.15 mm, and the number of thin slices is preferably 6 - 24. Experiments show that when the number of thin slices is at least 6, the detection can be achieved by the method of the present invention. However, in actual detection, to avoid missed detection, it is recommended to cut as many thin slices as possible.

[0023] In the above detection method, according to the amount of the midrib of the leaf cut, an appropriate amount of 10 - 20 μL of 0.5 mol / L NaOH solution is added for sufficient grinding; after grinding, the obtained juice is diluted with TE Buffer and can be used as a template for the MIRA reaction.

[0024] In the above detection method, in addition to the template, forward and reverse primers, and a probe, the reaction system further includes enzymes necessary for the MIRA amplification reaction. It can be known from the prior art that the multi-enzyme isothermal rapid amplification technology is an isothermal nucleic acid rapid amplification technology, which relies on the synergistic action of multiple functional proteins (helicase, recombinase, single-stranded binding protein, DNA polymerase, etc.) to achieve rapid nucleic acid amplification. Therefore, the MIRA multi-enzyme system in step S2 should at least include various functional proteins necessary to support the reaction.

[0025] In an embodiment of the present invention, a commercially available freeze-dried mixed enzyme powder is directly used to prepare the reaction system. The specific process is as follows: Add A Buffer (dissolution buffer) to the freeze-dried enzyme powder tube to completely melt and mix evenly, then add upstream and downstream primers and ddH2O and mix evenly, dispense into centrifuge tubes, add a template and a probe, and finally add B Buffer and invert up and down 8 - 10 times to mix evenly, and then forcefully flick (or instantaneously centrifuge) the reaction solution to the bottom of the tube for reaction.

[0026] In the above detection method, the reaction system is preferably: 0.5 μL of each of the 10 μmol / L forward and reverse primers, 2 μL of the template, 1.2 μL of the 1 μmol / L probe, the mixed enzyme and buffer required for the MIRA reaction, and ddH2O is added to make up to 12.5 μL.

[0027] In the above detection method, the reaction temperature in step S2 is 36-37 °C, and the reaction time is 11-12 min. It should be noted that in the detection system provided by the present invention, the reaction time cannot exceed 12 min, otherwise it will affect the results.

[0028] In the above detection method, in step S2, the reaction can be directly carried out using the hand temperature with PE gloves, that is, the bottom of the reaction tube (where the reaction solution is located) is held in the palm of the hand. After ensuring contact between the palm and the reaction tube, a disposable PE glove is worn, and the reaction is directly completed using the hand temperature. The role of the PE glove is to prevent heat dissipation. This method can carry out the reaction without a constant temperature device such as a water bath, which is conducive to the direct detection of field samples and grass-roots promotion and application. In addition, it should be noted that the hand temperature when bare hands is not sufficient to make the reaction of the present invention proceed smoothly. Therefore, wearing a PE glove mentioned in the present invention is a necessary step to ensure the smooth progress of the reaction.

[0029] In the above detection method, before detecting with the colloidal gold test strip in step S3, the reaction product obtained in S2 can be diluted with ddH2O, and then the diluted solution is taken and dropped onto the sample injection end of the colloidal gold test strip.

[0030] The principle of the detection method for citrus huanglongbing bacteria established by the present invention is specifically as follows: Specific primers labeled with biotin and specific probes labeled with fluorescein (FAM) amplify double-labeled amplification products after matching with the template. After dropping the reactants onto the test strip, they bind to the colloidal gold-labeled anti-FAM antibody to form a ternary complex. When it flows through the test line, the biotin ligand captures the complex and shows color, forming a test line, which is positive; when the sample to be detected does not contain huanglongbing bacteria, neither the primer nor the probe can produce effective amplification, so it cannot bind to the biotin ligand on the test line of the test strip, and only the quality control line shows color, which is negative. Therefore, the method for reading the results in step S3 is specifically as follows: One quality control line (C) appears as negative, and one quality control line (C) and one test line (T) appear as positive; if the T line is lighter, it is weakly positive, indicating that the content of huanglongbing bacteria in the citrus sample is low; if the T line is darker, it is strongly positive, indicating that the content of huanglongbing bacteria in the citrus sample is high.

[0031] In addition, it should be noted that in the detection method for citrus huanglongbing bacteria established by the present invention, the template for the reaction in step S2 can be obtained not only by the preparation in step S1, but also by extracting the total DNA of citrus samples and other methods.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] (1) Simple and easy to implement: The operation is simple and easy to carry out. It does not require the extraction of sample DNA and does not rely on various instrument devices, and can be directly applied to the detection of field samples.

[0034] (2) Fast speed and short time: It can be completed within 15 minutes from sample processing to obtaining reliable test results, greatly saving time and improving the detection efficiency.

[0035] (3) Low cost: No need for various reagents for DNA extraction, and at the same time, the reaction does not require special instrument equipment, saving the test cost.

[0036] (4) Wide application: This detection method is not limited to the laboratory, but can also be used for field detection, and the operation is simple, easy to understand, and conducive to popularization and application.

[0037] (5) High throughput: Suitable for large-scale sample analysis.

[0038] (6) Material saving: It can detect trace citrus samples, that is, it is suitable for the detection of precious materials with less sample materials, and also suitable for the rapid detection of a large number of sample materials. Description of the Drawings

[0039] Figure 1 It is the detection gel diagram of simultaneously amplifying 3 different Huanglongbing positive templates with different primer pairs in Example 1;

[0040] Figure 2 It is the schematic diagram of the best sampling part for detecting Huanglongbing bacterium in citrus in Example 2;

[0041] Figure 3 It is the specific detection result of MIRA-LFD in Example 3;

[0042] Figure 4 It is the sensitivity detection result of MIRA-LFD in Example 3;

[0043] Figure 5 It is the PCR detection result (A) and MIRA-LFD (B) detection result of some citrus samples in Example 4. Detailed Embodiments

[0044] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments. It should be understood that the specific embodiments described here are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0045] In the following embodiments, unless otherwise specified, they are all conventional methods; the reagents and materials, unless otherwise specified, can be obtained from commercial channels.

[0046] Example 1

[0047] This example provides a primer-probe combination for the detection of *Candidatus Liberibacter asiaticus*, including a primer pair and a probe. Among them, the primer pair is designed according to the *nrdB* gene of *Candidatus Liberibacter asiaticus*, and the sequences are specifically as follows:

[0048] Forward primer RPnrdB-F1: 5’-CATCATGCGAGATGAATCACTGCATCTCAA-3’ (SEQ ID NO.1);

[0049] Reverse primer RPnrdB-R1: 5’-TGATATTCTGTGACCCGAGTTTCGAAGAAG-3’ (SEQ ID NO.2).

[0050] Moreover, experiments show that the primer pair provided in this example has excellent amplification effects compared with other primer pairs (such as those shown in SEQ ID NO.5-10, also designed according to the conserved genes of *Candidatus Liberibacter asiaticus*) (see Figure 1 ). As can be seen from Figure 1 , when using the following 4 primer pairs to simultaneously amplify 3 different positive templates of huanglongbing, only RPnrdB-F1 / RPnrdB-R1 can stably amplify the target band.

[0051] Forward primer 5315-F: 5’-TCTTCTGGCTATGCTTTATCTGGCAGTAGT-3’ (SEQ ID NO.5),

[0052] Reverse primer 5315-R: 5’-TCTTCTGGCTATGCTTTATCTGGCAGTAGT-3’ (SEQ ID NO.6);

[0053] Forward primer 4560-F1: 5’-CGTCGTGCTAGCAGGCTATCTTTGGAAGAG-3’ (SEQ ID NO.7),

[0054] Reverse primer 4560-R1: 5’-ATGCTACGTCCCATAGCTTTAACGACGAGT-3’ (SEQ ID NO.8);

[0055] Forward primer 4560-F2: 5’-CTATTAGTAGTTGTGACCTCGGTGATTCCA-3’ (SEQ ID NO.9),

[0056] Reverse primer 4560-R2: 5’-CAAGCACCTCTGAGGTTGCTGAGGGTATGA-3’ (SEQ ID NO.10).

[0057] On the basis of obtaining the RPnrdB-F1 / RPnrdB-R1 primers with good amplification effect, in order to smoothly carry out the next test strip test, a specific fluorescent probe containing 3 modification sites was designed and synthesized according to the characteristics of the nrdB gene sequence. And a modification group was added to the 5' end of the reverse primer. The specific sequence characteristics are as follows:

[0058] RPnrdB-F1: 5'-CATCATGCGAGATGAATCACTGCATCTCAA-3',

[0059] RPnrdB-R1: 5'-Biotin-TGATATTCTGTGACCCGAGTTTCGAAGAAG-3';

[0060] Probe P1:

[0061] 5'-6-FAM-CGCACCATGCTCCATGAAGCTACCCTCCTCGA(THF)ATCGCCTATGCACA TG-C3-Spacer(SEQ ID NO.3),

[0062] Or probe P2:

[0063] 5'-6-FAM-ATGCTCCCTCATGTGAACAATATATGCAGT(THF)TCATCGCCAATCGTC-C3-spacer(SEQ ID NO.4).

[0064] It can be seen that the 5' end of the reverse primer was labeled with biotin (Biotin), the 5' end of the probe was labeled with carboxyfluorescein (FAM), and at the same time, a base of the probe was replaced with the nfo enzyme recognition site tetrahydrofuran (THF) at a position about 30 nt from the 5' end of the probe, and the 3' end of the probe was modified with a C3-Spacer group that blocks polymerase amplification.

[0065] Example 2

[0066] Based on the primer-probe combination provided in Example 1, in this example, a detection method for rapidly identifying *Candidatus Liberibacter asiaticus* based on MIRA-LFD was constructed. The specific process is as follows:

[0067] (1) Preparation of template

[0068] Take out the leaf. As Figure 2 shown, cut off the outermost end of the petiole, and then cut 6 - 24 pieces of the midrib of the leaf (the sampling site is based on Figure 2The parts from a to b and from c to d are the best. Avoid sampling parts including the left end of a, the area between b and c, and the right end of d. Put the midrib slices of the leaf into a 1.5 ml centrifuge tube. Depending on the number of midrib slices of the leaf, add an appropriate amount of 10 - 20 μL of 0.5 mol / L NaOH. After thorough grinding, take 2 μL of the juice and put it into a 0.2 ml PCR tube. Add 98 μL of TE Buffer to dilute it 50 times and use it as a template.

[0069] (2) Reaction system

[0070] First, prepare a 12.5 μL reaction system and then carry out the reaction. The steps are as follows:

[0071] ① Wear disposable PE gloves. On ice, add 29.4 μL of A Buffer to the freeze - dried enzyme powder tube to completely melt and mix evenly. Then add 2.0 μL each of the 10 μmol / L upstream and downstream primers and 1.2 μL of ddH2O, and mix well. Aliquot equal volumes into 4 0.2 mL centrifuge tubes (about 8.65 μL per tube).

[0072] ② Add 2.0 μL of the crude extract template prepared in (1) and 1.2 μL of the 1.0 μmol / L probe to the centrifuge tube. Finally, add 0.65 μL of B Buffer. Invert the tube 8 - 10 times to mix well, and centrifuge (or perform a short - term centrifugation) the reaction solution to the bottom of the tube. Then immediately hold it in the palm of your hand (about 36 - 37 °C) for 12 min (note that the reaction time should not exceed 12 min, otherwise it will affect the results. 11 - 12 min is appropriate).

[0073] (3) Detection using a colloidal gold test strip.

[0074] After the reaction in step ② is completed, take 5 μL of the reaction product and put it into a 0.5 ml centrifuge tube. Add 95 μL of ddH2O to dilute it 20 times. Then pipette 50 μL of the diluted product and drop it onto the sample application end of the colloidal gold test strip. The result can be observed within 5 minutes.

[0075] One C line appears for negative, and one C line and one T line appear for positive. If the T line is lighter, it is weakly positive, indicating a lower content of the pathogen in the sample; if the T line is darker, it is strongly positive, indicating a higher content of the pathogen in the sample.

[0076] Example 3

[0077] In this example, the specificity and sensitivity of the detection method constructed in Example 2 were detected, as follows:

[0078] (1) Specificity detection

[0079] Using the crude body fluids of positive samples of Huanglongbing positive, Huanglongbing negative, Citrus tristeza virus (CTV), Citrus yellow vein virus (CYVCV), Citrus tatter leaf virus (CTLV), Citrus exocortis viroid (CEVd), and Citrus canker bacteria (Canker) as templates, the crude extracts were all prepared according to step (1) in Example 2, and the reaction system was formulated according to step (2) in Example 2, with a reaction time of 9 - 12 min.

[0080] The results were as Figure 3 shown. Except that the detection result of the Huanglongbing positive crude extract was positive with two lines, the rest were all negative, indicating that the MIRA-LFD detection system had good specificity.

[0081] (2) Sensitivity detection

[0082] The initial concentration of the positive plasmid containing the nrdB gene of Huanglongbing bacteria was adjusted to 10 ng / μL, and then gradient dilution was carried out in a 10-fold manner. The dilution multiples were set as 10 -5 、10 -8 、10 -10 、10 -11 、10 -12 、10 -15 respectively. Using the plasmids with the above different concentrations as templates, and setting a negative control, the detection was carried out according to step (2) in Example 2, with a reaction time of 12 min. The copy number was calculated according to the formula: copy number concentration (copies / μL) = [6.02×1023×concentration (ng / μL)×10 -9 / [DNA length×660]. The original copy number corresponding to the initial concentration of 10 ng / μL was 2.58×10 10 copies / μL.

[0083] The detection results were as Figure 4 shown. When the template concentration was 10 ng / μL, the detection line band was the strongest. When the template concentration was diluted 10 -10 times, the detection line band was the weakest. When the template concentration was between 10 -11 and 10 -15 times dilution, there was no obvious detection strip, and the result showed negative. Therefore, the sensitivity of MIRA-LFD could detect a 10 -10 times dilution, that is, 1.0×10 -9 ng / μL. After calculation, the lowest detectable amount was 2.58 copies / μL. It can be seen that the method of the present invention has extremely high detection sensitivity.

[0084] Example 4

[0085] In this example, 50 suspected Huanglongbing samples with different symptoms from different citrus producing areas in China were randomly selected for detection and analysis. For each sample, 24 midrib thin slices of leaves were cut to prepare a crude extract as a template, and the detection was carried out according to the detection method in Example 2; at the same time, DNA templates were extracted by the conventional method, and PCR technology was used for comparative analysis.

[0086] After detection and analysis, the positive detection rate of the 50 suspected Huanglongbing samples by the MIRA-LFD technology was 100%, and the positive detection rate by PCR was 96%. Among them, the PCR detection results of 2 samples were negative, while the MIRA-LFD detection was positive. Further combined with qPCR identification, the 2 samples with negative PCR detection were positive by qPCR detection. It can be seen that the detection sensitivity of MIRA-LFD is higher than that of conventional PCR and is consistent with the qPCR detection results. The PCR and MIRA-LFD detection results of some samples are as Figure 5 shown.

[0087] To sum up, the detection method for citrus Huanglongbing bacteria provided by the present invention has the characteristics of simple, rapid and efficient operation. The detection of a single sample can be realized in about 15 minutes, and no professional equipment is required, so the efficiency is greatly improved. It can be operated without special technical training, which is conducive to grass-roots popularization and application. Moreover, this method also has good specificity and can effectively distinguish citrus Huanglongbing bacteria from other common citrus pathogens. And its sensitivity is higher than that of the conventional PCR method, and the lowest detectable amount is 2.58 copies / μL. In addition, this method can not only qualitatively analyze whether citrus is infected with Huanglongbing bacteria, but also distinguish the high and low bacterial content. It can be seen that the present invention provides a reliable new practical detection technology for the rapid field detection of citrus Huanglongbing bacteria.

[0088] The above is the preferred embodiment of the present invention, and the scope of the rights of the present invention cannot be limited thereby. It should be pointed out that for those of ordinary skill in the art, any modifications, equivalent replacements and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A detection method for rapid identification of *Candidatus Liberibacter asiaticus* based on MIRA-LFD, characterized in that, It includes the following steps: S1. Take the midrib of citrus leaves, cut it into thin slices, add it to the NaOH solution and grind it. Dilute the obtained juice and use it as a template; S2. Add the template, primers and probes to the MIRA multi-enzyme system, and react the obtained reaction system; S3. Drop the reaction product obtained in S2 onto the colloidal gold test strip and observe the result; the colloidal gold test strip contains a detection line and a quality control line. The detection line contains a biotin ligand, and the quality control line contains an anti-FAM antibody; The primers include a forward primer RPnrdB-F1 and a reverse primer RPnrdB-R1, and their sequences are respectively: RPnrdB-F1: 5’-CATCATGCGAGATGAATCACTGCATCTCAA-3’, RPnrdB-R1: 5’-Biotin-TGATATTCTGTGACCCGAGTTTCGAAGAAG-3’; The probe is probe P1 or probe P2, and the probe P1 is: 5’-6-FAM-CGCACCATGCTCCATGAAGCTACCCTCCTCGA(THF)ATCGCCTATGCACATG-C3-spacer; The probe P2 is: 5’-6-FAM-ATGCTCCCTCATGTGAACAATATATGCAGT(THF)TCATCGCCAATCGTC-C3-spacer; The juice is diluted with TE Buffer; The temperature of the reaction in step S2 is 36-37 °C, and the reaction time is 11-12 min; in step S2, the reaction is carried out using the hand temperature with PE gloves; In step S1, the thickness of the thin slices is 0.1-0.15 mm, and the number of the thin slices is 6-24; the concentration of the NaOH solution is 0.5 mol / L, and the addition amount of the NaOH solution is 10-20 μL; The reaction system is: 0.5 μL of each of the 10 μmol / L forward and reverse primers, 2 μL of the template, 1.2 μL of the 1 μmol / L probe, the mixed enzymes and buffer required for the MIRA reaction, and then ddH2O is added to make up to 12.5 μL.

2. The detection method for rapid identification of *Candidatus Liberibacter asiaticus* based on MIRA-LFD according to claim 1, characterized in that, In step S3, the reaction product obtained in S2 is diluted with ddH2O and then dropped onto the colloidal gold test strip for observation.

3. The detection method for rapid identification of *Candidatus Liberibacter asiaticus* based on MIRA-LFD according to claim 1, characterized in that, The method for reading the result is: one quality control line appears for negative, one quality control line and one detection line appear for positive, and the depth of the detection line is linearly related to the bacterial content in the template.

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