CEEA-exo kit and detection method for rapidly detecting larix gmelinii

Abstract

The invention discloses a CEEA-exo kit and a detection method for rapidly detecting dahurian larch blight. The kit comprises a fluorescent probe NYT-1, a first constant-temperature amplification primer NYF-4 and a second constant-temperature amplification primer NYR-1, wherein nucleotide sequences of the fluorescent probe NYT-1, the first constant-temperature amplification primer NYF-4 and the second constant-temperature amplification primer NYR-1 are sequentially shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3. A fluorescent probe NYT-1 is designed on the basis of a specific fragment of a larix gmelinii genome, the fluorescent probe NYT-1, a first constant-temperature amplification primer NYF-4, a second constant-temperature amplification primer NYR-1 and an enzyme are prepared into freeze-dried powder, A Buffer and B Buffer are prepared into microspheres, and the CEEA-exo kit is obtained through assembling. Based on the kit, the invention establishes a rapid detection method for the larix gmelinii in combination with a digital isothermal amplification technology, the method can rapidly and accurately detect target pathogenic bacteria from diseased branches, and has the advantages of strong specificity, high sensitivity, good applicability and the like; therefore, the method is of great significance to early warning of the larch shoot blight and monitoring of pathogens in quarantine areas.

Description

Technical Field

[0001] This invention belongs to the field of rapid detection of fungal diseases of forest trees, specifically relating to a CEEA-exo kit and detection method for rapid detection of larch dieback fungus. Background Technology

[0002] Currently, the target is *Clostridium neocarpa* (…). Neofusicoccum laricinum Quarantine of twig blight caused by larch is mainly conducted according to LY / T 2215-2013 "Technical Regulations for Quarantine of Larch Twig Blight". The main identification methods include direct testing, moist culture, and isolation culture. However, these identification methods are all based on the presence of typical symptoms and require tissue isolation and culture of the pathogen. They are susceptible to interference from human factors and environmental conditions, and are time-consuming, making it difficult to conduct early and rapid detection of suspected diseased larch.

[0003] To improve detection efficiency, molecular detection techniques are gradually being applied to the identification of this disease. For example, researchers have established a nested PCR method based on ITS sequences, which can distinguish between pathogens and some arboreal fungi, laying the foundation for molecular detection. However, this type of PCR technology relies on precise temperature control equipment and laboratory conditions, is cumbersome and time-consuming to operate, and is mainly for isolated and cultured live strains, making it unsuitable for rapid on-site detection at the grassroots level.

[0004] Chinese patent CN116162731B discloses a locking probe-based rolling circle amplification primer set and its detection method. Specifically, it discloses a locking probe-based rolling circle amplification (RCA) technique that amplifies the signal through specific circularization of the probe and target sequence followed by isothermal rolling circle extension. This detection method, through the design of specific locking probes, involves circularization, exonuclease digestion, and superbranched rolling circle amplification, with the results finally verified by agarose gel electrophoresis, achieving high detection specificity and sensitivity. However, this detection method involves multiple enzymatic reactions, making the process complex and time-consuming, and it requires electrophoresis equipment for result interpretation.

[0005] To further simplify operation and visualize results, Chinese patent CN118995995A discloses an optimization of high-branch rolling circle amplification (HRCA) technology based on RCA, combined with lateral flow detection strips (LFD), forming an HRCA-LFD detection system. The detection method based on this system introduces a zipcode sequence and corresponding labeled probes into the lock-type probe, allowing the amplification products to be directly read via the test strip, improving the convenience and on-site applicability of the detection. However, this detection method suffers from problems such as complex probe RCA operation and long overall time consumption.

[0006] In addition, isothermal amplification systems represented by recombinant enzyme-mediated complex enzyme exponential amplification technology (CEEA) have shown potential in rapid on-site detection of pathogens because they can achieve rapid accumulation of target sequences under constant temperature conditions and are easy to operate. However, such technologies still face some challenges in practical application, such as the difficulty of real-time, quantitative fluorescence monitoring of the amplification process, and the risk of false positives due to aerosol pollution.

[0007] In view of this, the present application provides a CEEA-exo kit and a detection method for rapid detection of the pathogen of larch canker. SUMMARY

[0008] In view of the technical problems in the background art, the present application aims to provide a CEEA-exo kit and a detection method for rapid detection of the pathogen of larch canker, which solves the defects of the prior art such as complex RCA operation, long time consumption, limited sensitivity and speed of HRCA-LFD, and limitations of existing isothermal amplification techniques in real-time monitoring, pollution prevention, and ultra-high sensitivity.

[0009] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: The first aspect of the present application provides a CEEA-exo kit for rapid detection of the pathogen of larch canker, comprising a probe primer set, wherein the probe primer set comprises a fluorescent probe NYT-1, a first isothermal amplification primer NYF-4, and a second isothermal amplification primer NYR-1, wherein The nucleotide sequence of the fluorescent probe NYT-1 is shown in SEQ ID NO. 1, the nucleotide sequence of the first isothermal amplification primer NYF-4 is shown in SEQ ID NO. 2, and the nucleotide sequence of the first isothermal amplification primer NYR-1 is shown in SEQ ID NO. 3.

[0010] The second aspect of the present application provides a preparation method of the above-mentioned CEEA-exo kit for detection of the pathogen of larch canker, comprising: freeze-drying the fluorescent probe NYT-1, the first isothermal amplification primer NYF-4, the second isothermal amplification primer NYR-1, and the enzyme to obtain a freeze-dried powder, preparing microparticle balls from A Buffer and B Buffer, and then assembling the freeze-dried powder and the microparticle balls in the same reagent tube to obtain the CEEA-exo kit.

[0011] The third aspect of the present application provides a rapid detection method for the pathogen of larch canker based on the above-mentioned CEEA-exo kit combined with digital isothermal amplification technology, comprising the following steps: S1, take out the freeze-dried powder reagent tube in the CEEA-exo kit and the liquid components as negative and positive controls, place the liquid components at room temperature for equilibrium to complete melting for standby; S2, DNA extraction in the sample to be tested: cut the diseased tissue section of the diseased larch branch and add the nucleic acid rapid release agent, shake slightly and then stand still to allow the DNA to be fully released, obtaining the DNA extraction solution; S3, dilute the DNA extraction solution obtained in step S2 to a certain concentration with ddH2O; S4, add the DNA dilution solution of a certain concentration obtained in step S3 to the freeze-dried powder reagent tube, tightly cover the tube cap, invert up and down to mix, then put it into the digital constant temperature amplifier, stand still for amplification reaction at 39-42 ℃ for 20 minutes, and directly read the result value; S5, result interpretation: if the read value is greater than or equal to 5, it is determined to be positive for larch canker disease bacteria; if the read value is less than 5, it is determined to be negative.

[0012] Preferably, the total volume of the amplification reaction system is 50 μL, including: A Buffer 29.4 µL, first constant temperature amplification primer NYF-4 (upstream primer) 2 μL, second constant temperature amplification primer NYR-1 (downstream primer) 2 μL, ddH2O and DNA solution 13.5 μL, B Buffer 2.5 μL, fluorescent probe NYT-1 0.6 μL.

[0013] The present application has the following beneficial effects: The present application designs a high-sensitivity and strong-specificity fluorescent probe based on the specific fragment of the larch canker disease bacteria genome, and respectively makes the fluorescent probe, specific primer and enzyme into freeze-dried powder, makes A Buffer and B Buffer into microparticles, assembles them in the same PC cryopreservation tube, and obtains the CEEA-exo kit. Based on the kit, the present application establishes a rapid detection method of larch canker disease bacteria combined with digital constant temperature amplification technology, which can quickly and accurately detect the target pathogenic bacteria from the diseased branches, significantly shortens the traditional detection time, improves the detection efficiency, has the advantages of simple operation, visual amplification process and result, and is helpful to realize the early diagnosis of diseases and provide technical support for the prevention and control of larch canker disease. The detection system constructed by the method has the advantages of strong specificity, high sensitivity and good applicability, and has important significance for early warning and quarantine area pathogen monitoring of larch canker disease. BRIEF DESCRIPTION OF DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only constitute some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0015] Figure 1 CEEA-exo primer probe screening results for the larch canker disease fungus; Figure 2 CEEA-exo specific detection results for the larch canker disease fungus; Figure 3 CEEA-exo sensitivity detection results for the larch canker disease fungus; Figure 4 CEEA-exo applicability detection results for the larch canker disease fungus; Figure 5 CEEA-exo in-situ sample test results for the larch canker disease fungus; Figure 6 CEEA-exo kit combined with digital isothermal amplification system for detecting the larch canker disease fungus; Figure 7 In-situ detection results for the larch canker disease fungus. DETAILED DESCRIPTION

[0016] In the following description, specific details are set forth in order to provide a thorough understanding of embodiments of the application. However, persons of ordinary skill in the art will readily recognize that the application can be practiced without these specific details, which are presented merely to provide that understanding, and are not intended to limit the present application.

[0017] The present application is based on a specific fragment of the genome of the larch canker disease fungus, and uses a probe to complementarily combine with an amplified fragment, and then a specific site of the probe is cut by an exonuclease, so that a fluorescent group and a quenching group are separated and a fluorescent signal is released, and real-time monitoring of the amplification process is realized by combining with a fluorescence detection device.

[0018] Embodiment 1 1. Experimental materials and methods 1.1 Experimental materials and reagents Positive control larch canker disease fungus (Gremmeniella abietina) Neofusicoccum laricinum Plasmid, negative control (ddH2O), healthy larch branches, CEEA-exo rapid detection kit, nucleic acid rapid release agent.

[0019] 1.2 Experimental methods 1.2.1 Designing fluorescent probes and primers: The present application is based on a specific fragment of the genome of the larch canker disease fungus (Gremmeniella abietina) Neofusicoccum laricinum) The specific fragment of the genome is aligned with the similar species sequence of Botryosphaeria dothidea by Blast, Mega and other software, and the region with significant base difference is selected as the target gene for designing the fluorescent probe. The fluorescent probe is designed between the upstream and downstream primers, with a length of 46-52 nt, complementary to the target fragment and not covering the primer recognition site; the sequence needs to avoid palindromic structure, internal secondary structure and continuous repeated base. The fluorescent probe designed in this embodiment is a single-stranded DNA about 51 bp in length, mainly composed of four modified sites: exonuclease recognition site, fluorescent labeling group, quenching labeling group and modification at the end. At the same time, the primer design principles are followed, covering the length, GC content and other aspects. If there are degenerate bases, they should not appear continuously, and the number of degenerate bases in a single primer should be controlled within a lower range, generally not more than five, and not placed at the two ends of the primer.

[0020] Under constant temperature conditions, the recombinase forms a complex with the primer, invades the double-stranded DNA template under the cooperation of the helper protein and single-stranded binding protein, binds the primer to the complementary region to form a D-loop structure, and then the recombinase dissociates; the DNA polymerase binds to the 3' end of the primer to start synthesis, realizing the exponential amplification of the target fragment. During the amplification process, the specific molecular probe binds to the complementary region, the exonuclease cuts the probe tetrahydrofuran (THF) site, separates the fluorescent group and the quenching group, and releases the fluorescent signal, which can be monitored in real time by a fluorescence detection device. Based on the above principle, the present application designs 5 upstream primers, 4 downstream primers and 1 fluorescent probe for the specific region of the genome of the larch canker disease fungus, and 20 combinations are obtained by matching the upstream and downstream primers with the probe. According to the reaction system (see Table 1) and reaction conditions (39-42 ℃, 20 minutes) of CEEA-exo for detecting the larch canker disease fungus, negative screening is carried out to exclude false positive primer pairs with non-specific amplification, and positive screening is carried out to select primer pairs with early amplification start and low CT value, so as to determine the optimal primer probe combination NYF4+NYR1, NYT1 (see Figure 1 ), and finally the optimal fluorescent probe NYT-1 and isothermal amplification primer pair NYF-4 and NYR-1 (see Table 2) are screened and determined.

[0021] Table 1. Reaction system for detecting larch canker disease fungus based on CEEA-exo

[0022] Table 2 Sequence information of fluorescent probe and isothermal amplification primer pair

[0023] 1.2.2 Preparation of CEEA-exo kit According to the reaction system ratio confirmed by screening, the fluorescence probe NYT-1, isothermal amplification primer pair NYF-4 and NYR-1, and enzyme (a multi-enzyme system composed of exonuclease and other auxiliary enzymes) were made into freeze-dried powder, A Buffer and B Buffer were made into microparticle balls, and were assembled in the same PC tube to form the CEEA-exo kit.

[0024] 1.2.3 Reagent preparation According to the number of samples to be tested, the freeze-dried powder reagent tube in the CEEA-exo kit was taken out and labeled with the sample number for standby, and 1 tube of positive control (plasmid) and negative control (ddH2O) was taken out and placed at room temperature for equilibrium until completely melted for standby. Neofusicoccum laricinum

[0025] 1.2.4 DNA extraction According to the number of samples to be tested, the corresponding centrifuge tube was taken and labeled with the sample number, and 1-2 sections of each of the diseased tissue larch branches with a length of 0.8-1.2 cm were added to each tube, and 450-550 µL of nucleic acid rapid release agent was added, and after slight shaking, it was placed for 5 min to allow the DNA to be fully released, obtaining the DNA solution.

[0026] 1.2.5 DNA concentration dilution 8-12 µL of the above obtained DNA solution was taken into a new centrifuge tube, and ddH2O was added and mixed to 50 µL.

[0027] 1.2.6 Constant temperature amplification digital display 50 µL of the DNA dilution liquid of step (1.2.5) was added to the freeze-dried powder reagent tube prepared in step (1.2.3), the tube cap was tightly covered, and it was inverted and mixed for 8-10 times, then it was placed in the CEEA-exo mini digital constant temperature amplifier, and after 20 min of amplification reaction at 39-42 ℃, the result value was directly read.

[0028] 1.2.7 Result interpretation If the instrument reads a value ≥5, it is determined to be positive for the larch canker disease fungus, and if the instrument reads a value <5, it is determined to be negative for the larch canker disease fungus.

[0029] 2. Specificity test The DNA of the preserved K7 strain of Ophiostoma novo-ulmi (Buisman) Nannfeldt was used as the positive control, ddH2O was used as the negative control, and the DNA of other fungi (including Neofusicoccum laricinum Allantophomopsis sp., Aplosporella sp., Botryosphaeria sp., Cytospora sp., Diaporthe sp., Diplodia sp., Tympanis sp., Phaeobotryon sp. etc.) isolated from the larch branch canker was used as the sample to be tested, and the CEEA-exo kit was used for detection with the fluorescence PCR instrument, and the results are shown in Figure 2 ​​.

[0030] Figure 2 The results showed that only strain K7 was positive among all tested samples, and its fluorescence PCR showed a significant amplification curve. No fluorescence signal was detected in the other tested strains and the negative control, and the results were consistent across three replicates. This indicates that the CEEA-exo detection kit developed in this invention has good specificity for *Larix chinensis*, the causal agent of larch blight, and the results can be visually interpreted through fluorescence curves, making it suitable for the accurate detection of this pathogen.

[0031] 3. Sensitivity Test Using ddH2O as a negative control, preserved *Clostridium neoformans* (a type of blight pathogen) was collected. Neofusicoccum laricinum DNA samples from strain K7 were serially diluted 10-fold with ultrapure water to concentrations of 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL, 1 fg / μL, and 0.1 fg / μL. The CEEA-exo detection kit was used in conjunction with a fluorescence PCR instrument to determine the detection sensitivity. Results are shown below. Figure 3 . Depend on Figure 3 The results showed that, except for concentrations of 10 fg / μL, 1 fg / μL and 0.1 fg / μL which did not show amplification curves, all other concentrations showed clear amplification curves. The lowest detectable concentration for larch blight pathogens was 100 fg / μL, and repeated experiments verified that this concentration could be detected stably.

[0032] 4. Applicability Testing The preserved larch shoot blight pathogen, *Clostridium neoformans* (… Neofusicoccum laricinum DNA from strain K7 was used as a positive control, while ddH2O and healthy larch branches were used as negative controls. DNA from larch blight pathogen strains (H030-1, Y042-3, HC-2, JX-21, 37-7) from different regions was detected using a CEEA-exo detection kit in conjunction with a fluorescence PCR instrument. Results are shown below. Figure 4 .

[0033] Figure 4 The results showed that positive amplification curves were detected in all strains of larch dieback pathogen from all sources, while no positive amplification was detected in the negative controls (ddH2O and healthy larch branches), indicating that the system has good detection applicability.

[0034] 5. In-situ sample testing To verify the performance of the fluorescent probes, specific primers, and the constructed technology system, the assembly, preparation, and stability testing of the CEEA-exo kit combined with the digital isothermal amplification system were first completed. (Refer to...) Figure 6A rapid in-situ detection method for *Neocytospora larvae*, the causal agent of larch blight, using the CEEA-exo kit combined with digital isothermal amplification technology, is described. The specific procedure is as follows: DNA is extracted from the sample (suspected in-situ sample of larch blight) using a rapid nucleic acid release agent, diluted with ddH2O, and 50 μL of the diluted solution is added to a lyophilized powder test tube from the CEEA-exo kit. After mixing, the sample is placed in a digital isothermal rapid detection instrument and incubated at 42°C for 20 minutes to read the results. Following this procedure, *Neocytospora larvae*, the causal agent of larch blight, is detected in preserved samples. Neofusicoccum laricinum K7 strain DNA served as a positive control, while ddH2O and healthy larch branches served as negative controls. Results are shown below. Figure 5 .

[0035] Figure 5 The results showed that both the in situ sample and the positive control were positive, while the negative control showed no detection. The CT value (cycle threshold) refers to the number of cycles required for the fluorescence signal to reach a set threshold during PCR amplification.

[0036] 6. Practical Applications of the CEEA-exo Reagent Kit Detection of preserved *Clostridium neoformans* var. *negeri* (larch blight pathogen) Neofusicoccum laricinum K7 strain DNA served as a positive control, while ddH2O and healthy larch branches served as negative controls. Eighty-four larch dieback samples (collected in three batches) from Maoershan Forest Farm, Heilongjiang Province, were tested. After excluding atypical samples, 1 cm tissue was taken from the dieback area of ​​the remaining 39 samples. DNA was lysed using a rapid nucleic acid release agent, diluted with ddH2O, and mixed thoroughly. The mixture was then added to PC tubes of the CEEA-exo kit, inverted 8-10 times, rapidly centrifuged, and immediately placed in a digital thermostat for 20 minutes. Some results are shown below. Figure 7 The results showed that 35 samples from the three batches tested positive, while 4 samples did not. Both the tested samples and the positive control were positive, and the negative control showed no signal, indicating that the method of the present invention has good reliability.

[0037] In summary, this invention, by designing specific primers and fluorescent probes, introduces an exonuclease-mediated signal release mechanism into the CEEA amplification system, enabling real-time fluorescence monitoring during the amplification process. Combined with a digital isothermal amplification instrument, the fluorescence signal is directly converted into numerical values, thus completing the process from in-situ sample processing to direct result interpretation in approximately 20 minutes. The detection sensitivity is increased to 100 fg / μL, with good specificity and applicability. This invention provides a rapid, accurate, and easy-to-operate technical means for early warning, on-site quarantine, and disease monitoring of larch twig blight.

[0038] The present application is not limited to the above-mentioned specific embodiments, and various modifications made by those skilled in the art based on the above concept without creative labor are within the scope of the present application.

Claims

1. A rapid detection kit for larch blight pathogens, CEEA-exo, characterized in that, include: The fluorescent probe NYT-1, the first isothermal amplification primer NYF-4, and the second isothermal amplification primer NYR-1, among which, The nucleotide sequence of the fluorescent probe NYT-1 is shown in SEQ ID NO.1, the nucleotide sequence of the first isothermal amplification primer NYF-4 is shown in SEQ ID NO.2, and the nucleotide sequence of the second isothermal amplification primer NYR-1 is shown in SEQ ID NO.

3.

2. The preparation method of the CEEA-exo reagent kit as described in claim 1, characterized in that, include: The fluorescent probe NYT-1, the first isothermal amplification primer NYF-4, the second isothermal amplification primer NYR-1, and the enzyme were prepared into lyophilized powder. Buffer A and Buffer B were prepared into microspheres. The lyophilized powder and microspheres were then assembled into the same reagent tube to obtain the CEEA-exo kit.

3. A rapid detection method for larch blight pathogens based on the CEEA-exo kit of claim 1 combined with digital isothermal amplification technology, characterized in that, Includes the following steps: S1. Take out the lyophilized powder test tube and the liquid components used as negative and positive controls from the CEEA-exo kit, and place the liquid components at room temperature until they are completely thawed for later use. S2. DNA extraction from the sample to be tested: Add the diseased larch branches to be tested to the nucleic acid rapid release agent, shake gently and let stand to allow the DNA to be fully released, and obtain the DNA extract. S3. Dilute the DNA extract obtained in step S2 to a certain concentration using ddH2O; S4. Add the DNA diluent of a certain concentration from step S3 to the lyophilized powder test tube in step S1, tighten the cap, invert and mix well, then place it together with the positive and negative control test tubes into a digital isothermal amplification instrument, and let it stand at 39-42 ℃ for 20 minutes for amplification reaction. After the reaction is complete, read the result value directly. S5. Result Interpretation: If the read value is ≥5, it is determined to be positive for larch blight pathogen; if the read value is <5, it is determined to be negative.

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