Probe primer set, kit and detection method for detecting larch dieback pathogen based on hyperbranching rolling circle amplification

Abstract

The present invention discloses a probe primer set, a kit and a detection method for detecting larch dieback pathogen based on hyperbranching rolling circle amplification, wherein the probe primer set includes: a padlock probe NL-FD, a first rolling circle amplification primer NL-F1, a second rolling circle amplification primer NL-F2, and a detection probe cZip. The present invention designs a padlock probe based on the conservative sequence of the ITS gene of larch dieback pathogen as the target gene, establishes a complete rolling circle amplification system, and combines it with a nucleic acid test strip to achieve visual detection. The present invention uses hyperbranching rolling circle amplification technology combined with lateral flow test strips to detect larch dieback pathogen, which can make the HRCA amplification detection results more intuitive and rapid, and at the same time has high specificity and sensitivity, with a minimum detection limit concentration of 1pg / μL, showing its potential for on-site detection and meeting the accurate identification application of larch dieback pathogen at the grassroots level.

Description

Technical Field

[0001] The invention belongs to the field of molecular diagnosis and relates to the field of forest disease detection, and particularly to a probe primer set, a kit and a detection method for detecting larch dieback pathogen based on hyperbranching rolling circle amplification. Background Art

[0002] Larch is one of the important forest resources in my country, with significant ecological, economic and social benefits. However, various diseases that have occurred in larch in recent years have caused great harm to the growth, development, and maturity of larch. Among them, larch dieback ( Neofusicoccum laricinum ) is particularly harmful. When larch is infected, new shoots die, and in severe cases, the tree crown becomes bald or even dies, causing huge losses to forestry production and ecological environment construction.

[0003] When the larch dieback pathogen infects larch, the initial symptoms are not obvious and difficult to detect. In addition, there is a lack of rapid detection technology for larch dieback. Once the disease spreads, its prevention and control is too difficult, the effect is not ideal, and it is difficult to eradicate. Therefore, if the pathogen can be detected efficiently and quickly before the disease spreads on a large scale, it can be blocked to the maximum extent. Scientific and effective outposts provide a basis for efficient and rapid detection of the pathogen in the early stage of the disease. At present, the domestic quarantine for this disease is mainly carried out according to LY / T 2215-2013 "Technical Regulations for Quarantine of Larch Dieback Disease", and its inspection and identification methods mainly include direct inspection method, moisturizing culture method and separation culture method. These identification methods are all based on typical symptoms, and require tissue isolation and culture of pathogens. They are interfered by human factors and environmental conditions to a certain extent, and are time-consuming. It is difficult to carry out early and rapid detection of suspected diseased larch.

[0004] At present, the detection of larch dieback pathogen in my country is based on the design of primers based on the ITS sequence of larch branch-dwelling fungi. The primers were used to separate the dead branches of larch and 9 other branch-dwelling fungi, and a basic PCR amplification method was initially established, which provided an experimental basis for the rapid diagnosis of larch dieback. However, this primer is easy to form a hairpin structure, which is difficult to design, and the PCR test is prone to false positive results, with low sensitivity, and cannot be used in production practice. At the same time, due to the long sample pretreatment time, long test time, low detection efficiency, and the test site is limited to laboratories with conditions, it is difficult to adapt to the rapid detection of suspected diseased branches, and it is also difficult to meet the monitoring and detection technology of key plant quarantine targets at ports, which must be accurate, fast, and simple.

[0005] Rolling circle amplification (RCA) based on the unique design of padlock probes not only has the advantages of high specificity and high sensitivity of PCR (polymerase chain reaction) technology, but also can realize parallel detection of multiple targets. This feature makes RCA show great potential and growing practical value in the theoretical research and practical application of molecular biology. Its reaction conditions do not require complex high-end equipment, and only common constant temperature equipment such as water bath or constant temperature incubator is needed to smoothly carry out the amplification reaction. However, this technology is prone to aerosol phenomenon, causing false positives and affecting the interpretation of results. In order to accurately interpret the sample, the lateral flow dipstick (LFD) technology is combined. LFD combines immunochromatography technology and molecular biology methods to anchor specific fluorescent antibodies on the test strips, react with fluorescent labeled on the RCA product, and form a colored detection line on the test strips, thereby detecting the correct target product, greatly reducing the probability of false positives. However, there are no reports on its application in the detection of larch dieback pathogens. Summary of the invention

[0006] In view of the key technical problems in the background technology, the purpose of the present invention is to provide a probe primer set, a kit and a detection method for detecting larch dieback pathogen based on hyperbranching rolling circle amplification, which solves the defects of the prior art in detecting larch fungal diseases, such as long detection cycle, low specificity, and easy occurrence of false positives.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides a probe primer set for detecting larch dieback pathogen based on hyperbranching rolling circle amplification, comprising: a padlock probe NL-FD, a detection probe cZip, and a first rolling circle amplification primer NL-F1 and a second rolling circle amplification primer NL-F2, wherein:

[0009] The nucleotide sequence of the padlock probe NL-FD is shown in SEQ ID NO.1, the nucleotide sequence of the primer detection probe cZip is shown in SEQ ID NO.2, the nucleotide sequence of the first amplification primer NL-F1 is shown in SEQ ID NO.3, and the nucleotide sequence of the second amplification primer NL-F2 is shown in SEQ ID NO.4.

[0010] Preferably, the 5 ′ The second rolling circle amplification primer NL-F2 is labeled with 6-carboxyfluorescein (6-FAM). ′ End-labeled with biotin.

[0011] The second aspect of the present invention provides a kit for detecting larch dieback pathogen based on hyperbranching rolling circle amplification, comprising the above-mentioned probe primer set.

[0012] The third aspect of the present invention provides a method for detecting larch dieback pathogen based on hyperbranching rolling circle amplification, comprising the following steps:

[0013] S1. Extract DNA from the sample to be tested;

[0014] S2, using the DNA extracted from the sample to be tested as a template and using the probe primer set to perform HRCA amplification; specifically comprising the following steps:

[0015] S21, performing a ligation and cyclization reaction on the extracted DNA and the padlock probe NL-FD using a thermal cycle ligation method;

[0016] S22, adding enzyme digestion premix to the reaction product obtained in step S21 to perform exonuclease digestion reaction;

[0017] S23, using the exonuclease digestion reaction product obtained in step S22 as a template, using the first rolling circle amplification primer NL-F1 and the second rolling circle amplification primer NL-F2 to perform HRCA amplification reaction to obtain an HRCA amplification product;

[0018] S3, performing hybridization reaction between the obtained HRCA amplification product and the detection probe cZip;

[0019] S4. Use lateral flow test strips (LFD) to visually detect the hybrid reaction products and determine whether the sample to be tested contains larch dieback pathogen based on the test results.

[0020] Preferably, in step S21, the ligation cyclization reaction system is 10 μL, and is composed of: 2-5 μL DNA template, 1-3 μL 40U / μL Taq DNA ligase buffer, 0.1-0.5 μL 40U / μL Taq DNA ligase, 0.2-0.5 μL 20pmol / L padlock probe NL-FD, 1-3 μL DNA template, and Sterilized ddH2O to make up to 10 μL.

[0021] The thermocycling ligation method is used for the ligation circularization reaction program: 94°C, 4 min; 94°C, 30s; 65°C, 5 min, 15 cycles; 95°C, 15 min, inactivation of Taq DNA ligase.

[0022] Preferably, in step S22, the enzyme digestion premix is ​​10 μL, and is composed of: 1-2 μL of 10× exonuclease I buffer; 2-3 μL of 5U / μL exonuclease I; and Sterilized ddH2O to make up to 10 μL.

[0023] Preferably, the exonuclease digestion reaction procedure is: 37°C, water bath for 2.5 h; 80°C, water bath for 20 min.

[0024] Preferably, in step S23, the HRCA amplification reaction system is 25 μL, and is composed of: 2-4 μL of 10×Bst DNA polymerase buffer, 0.5-1 μL of 8U / μL Bst DNA polymerase, 1-2 μL of 10μmol / L NL-F1, 1-2 μL of 10μmol / L NL-F2, 0.5-1 μL of 10mmol / L dNTPs, 4 μL of ligation circularization digestion product, and SterilizedddH2O to make up to 25 μL.

[0025] Preferably, the HRCA amplification reaction procedure is: 55-62°C, 1.0h.

[0026] Preferably, in step S3, the hybridization reaction is carried out at a temperature of 55-60° C. and for a time of 5-8 min.

[0027] Preferably, the detection results are divided into the following three situations:

[0028] (1) Positive (+), that is, a blue band appears on the test strip at the quality control line (C line) and a red band appears on the test line (T line), indicating that the sample to be tested contains the nucleic acid fragment of pine blight pathogen;

[0029] (2) Negative (-), that is, a blue band appears on the test strip quality control line (C line) and no band appears on the test line (T line), indicating that the sample to be tested does not contain the nucleic acid fragment of pine blight pathogen, or its content is lower than the minimum detection amount of the test strip;

[0030] (3) Invalid, that is, no bands appear on the test strip quality control line (C line) and the test line (T line), indicating that the test strip or amplification reagent used may be damaged, ineffective or operated incorrectly. Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention firstly designs a specific padlock probe NL-FD, a pair of primers (a first rolling circle amplification primer NL-F1 and a second rolling circle amplification primer NL-F2) and a detection probe cZip based on the ITS gene sequence of larch dieback pathogen by selecting conservative sequence fragments with large differences from similar species and major fungal groups on larch, and then performs circularization connection, exonuclease digestion of the uncircularized linear probe, and then uses the first rolling circle amplification primer and the second rolling circle amplification primer to perform super-branched rolling circle amplification and verify through electrophoresis, then hybridizes the super-branched rolling circle amplification product with the detection probe, and finally uses a lateral flow test strip (LFD) to visually detect the hybrid reaction product, and determines whether the sample to be tested contains larch dieback pathogen according to the detection result. The present invention uses hyperbranched rolling circle amplification technology and lateral flow test strips to detect larch dieback pathogen. The combination of the two makes the HRCA amplification detection results more intuitive and rapid, while having high specificity and sensitivity. The minimum detection concentration is 1pg / μL, showing its potential for on-site detection and meeting the requirements for accurate identification of larch dieback pathogen at the grassroots level. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1 This is the HRCA-LFD schematic diagram of larch dieback pathogen;

[0034] Figure 2 This is the gel electrophoresis diagram for the specific detection of larch dieback pathogen: Lane M represents 100 bp DNA Marker; Lane 1 represents larch dieback pathogen [ Neofusicoccum laricinum ]; 2 means Epicoccum Fungus; 3 means Diaporthe Fungus; 4 means Alternaria Fungus; 5 means Didymella Fungus; 6 means Pestalotiopsis genus fungi; 7 represents sterile water control;

[0035] Figure 3 The test strip diagram for specific detection of larch dieback pathogen: Among them, the test strip results 1-7 are Figure 2 The middle lanes correspond to 1-7;

[0036] Figure 4The gel electrophoresis diagram of the sensitivity detection of larch dieback pathogen: wherein, lane M represents 100 bp DNA Marker, lanes 1-6 represent the DNA concentrations of larch dieback pathogen, which are 10 ng / μl, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, respectively; lane 7 represents the sterile water control;

[0037] Figure 5 This is a test strip diagram for the sensitivity test of larch dieback pathogen: Among them, test strip results 1-7 and Figure 4 The middle lanes correspond to 1-7;

[0038] Figure 6 This is the gel electrophoresis diagram of the actual sample test of the diseased branches of larch dieback disease: wherein, lane M represents the 100bp DNA marker, 1-3 represent the raised bark parts with suspected symptoms, 4-6 represent the diseased branches, and 7 represents the healthy branches;

[0039] Figure 7 This is a test strip diagram of the actual sample of larch dieback disease branches: Among them, the test strip results 1-7 are Figure 6 The middle lanes correspond to 1-7. DETAILED DESCRIPTION

[0040] In the following description, for the purpose of illustration rather than limitation, specific details such as specific system structures and technologies are provided to provide a thorough understanding of the embodiments of the present invention. However, it should be clear to those skilled in the art that the present invention may also be implemented in other embodiments without these specific details.

[0041] Example 1

[0042] 1. Experimental Materials and Methods

[0043] 1.1 Experimental Materials and Reagents

[0044] Larix gmelinii dieback pathogen Neofusicoccum laricinum ], Taq DNA ligase, ExonucleaseⅠ, Bst DNA polymerase, dNTPs, and Sterilized ddH2O were all purchased from New England Biolabs (NEB), 100 bp DNA ladder was purchased from Promega, and disposable nucleic acid detection strips were purchased from Tiosbio.

[0045] 1.2 Experimental methods

[0046] 1.2.1 Design of padlock probes and primers:

[0047] In this embodiment, the padlock probe is designed based on the specific fragment of ITS of larch dieback pathogen, and is compared with similar species by software such as Blast and Mega to obtain a sequence with a large base difference with similar species, which is used as the specific detection arm (T1, T2) of the padlock probe. Then, according to the design principle of the padlock probe, the padlock probe is designed using software Oligo and premier5. The detection arms T1 and T2 at both ends of the designed padlock probe are complementary to the base sequences of the specific fragment (see Table 1, the uppercase letter sequence in the padlock probe), and another Zipcode sequence randomly designed from a protein gene with no homology to the template sequence is added to the connecting region in the middle of the padlock probe. The detection probe cZip is designed based on the reverse complementarity of the Zipcode sequence for binding to the LFD test strip. Mfold (http: / / www.bioinfo.rpi.edu / applications / mfold / ) was used to predict the secondary structure of the designed padlock probe to ensure that the secondary structure energy value was minimized and to prevent the generation of hairpin structure. At the same time, the 5' end of the padlock probe was phosphorylated; then the designed padlock probe sequence and a pair of universal amplification primers P1 and P2 were homologously compared in the NCBI database to screen out padlock probes with strong specificity and amplification primers with stable performance and easy amplification. Among them, the 5' end of the detection probe cZip is 6-carboxyfluorescein (6-FAM); the 5' end of NL-F2 is biotin labeled. The padlock probe designed in this embodiment is a single-stranded DNA with a length of about 100-120 bp, mainly composed of five parts: 5' end specific detection arm T1, universal amplification primer regions P1 and P2, Zipcode region specific for hybridization, and 3' end specific detection arm T2. Aiming at the specific region of ITS of larch dieback pathogen, the present invention designs a padlock probe NL-FD and a detection probe cZip, as well as a first primer NL-F1 and a second primer NL-F2 for rolling circle amplification targeting the pathogen.

[0048] Table 1. Padlock probe and rolling circle amplification primer pair information

[0049]

[0050] 1.2.2 DNA extraction

[0051] The operation was performed according to the instructions of the genomic DNA extraction kit.

[0052] 1.2.3 Ligation cyclization reaction

[0053] The padlock probe NL-FD was connected and cyclized with the DNA templates of larch blight pathogen and other control strains by thermal cycle ligation. The ligation cyclization reaction system was 10 μL, consisting of: DNA template 2-5 μL, 40U / μLTaq DNA ligase buffer 1-3 μL, 40U / μL Taq DNA ligase 0.1-0.5 μL, 20 pmol / L padlock probe NL-FD 0.2-0.5 μL, DNA template 1-3 μL, Sterilized ddH2O supplemented to 10 μL, and the conditions for thermal cycle ligation were set as follows: 94℃, 4min; 94℃, 30s; 65℃, 5min, 15 cycles; 95℃, 15min, inactivate TaqDNA ligase. After the reaction, the PCR reaction tube was immediately placed in an ice bath for 5min and then stored in a -20℃ refrigerator for later use.

[0054] 1.2.4 Exonuclease digestion

[0055] Since there are still many double-stranded and single-stranded DNAs in the ligation reaction system, background signals will be generated during the amplification process, which may lead to false positive results. In this embodiment, exonuclease I is used to digest the product to remove the uncircularized linear padlock probe and DNA template. The specific steps are: add 10 μL of enzyme digestion mixture to the PCR reaction tube after ice bath, react at 37°C for 2.5h, completely digest the uncircularized linear padlock probe, and then react at 80°C for 20min to inactivate the remaining exonuclease I; wherein, the enzyme digestion premix is ​​10 µL, composed of: 10× exonuclease I buffer 1-2 µL; 5U / µL exonuclease I 2-3 µL; Sterilized ddH2O is added to 10 µL.

[0056] 1.2.5 HRCA amplification reaction

[0057] The exonuclease digested product was used as a template, and the first primer NL-F1 and the second primer NL-F2 were used for HRCA amplification reaction. The HRCA amplification reaction system was 25 μL, and the composition was: 10×Bst DNA polymerase buffer 2-4 μL, 8U / μL Bst DNA polymerase 0.5-1 μL, 10 μmol / L NL-F1 1-2 μL, 10 μmol / L NL-F2 1-2 μL, 10mmol / L dNTPs 0.5-1 μL, exonuclease digested product 4 μL, Sterilized ddH2O was added to 25 μL. The amplification reaction was carried out at 60.0℃ for 1 hour.

[0058] 1.2.6 Gel electrophoresis of amplified products

[0059] 7 μL of HRCA amplification product was electrophoresed on 2% (mass fraction) agarose gel for verification, stained, and photographed and analyzed using an ultraviolet gel imaging system.

[0060] 1.2.7 HRCA-LFD color reaction

[0061] Visualize test results using lateral flow test strips.

[0062] Add the detection probe cZip labeled with 6-carboxyfluorescein (6-FAM) to the rolling circle amplification reaction product and react at 55-60℃ for 5-8 minutes. After the reaction, add Washing Buffer and mix well. Insert the detection end of the lateral flow test strip vertically into the test liquid, let it stand for 5 minutes, and observe the results with the naked eye.

[0063] 2. Specificity test

[0064] The pathogen of larch dieback disease Neofusicoccum laricinum ] as standard strains, and other fungi attached to the surface of larch branches ( Epicoccum , Diaporthe , Alternaria , Didymella , Pestalotiopsis ) were used as reference strains, and their DNA was extracted. 2 μL DNA samples were taken from each strain. Sterilized ddH2O was used as a blank control. The above steps 1.2.3-1.2.7 were followed. The results are shown in the figure. Figure 2-3 .

[0065] Depend on Figure 2 and Figure 3 The results showed that for the DNA of larch dieback pathogen, HRCA-LFD, only larch dieback pathogen [ Neofusicoccum laricinum ] can be detected, and the red detection line and blue quality control line can also be observed on the test strip in the gel electrophoresis band.

[0066] 3. Sensitivity test

[0067] The DNA samples of larch dieback pathogen were diluted with ultrapure water in a gradient manner, in the order of 10 ng / μl, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, and 100 fg / μL. 2 μL of the DNA samples with different dilutions were taken as templates for HRCA-LFD detection to determine the sensitivity of the detection. The results are shown in Figure 4-5 .

[0068] Depend on Figure 4 and Figure 5The results showed that the minimum detection concentration of HRCA-LFD for the DNA of larch dieback pathogen was 1 pg / μL. At this concentration, gel electrophoresis bands could be detected and red detection lines and blue quality control lines could be observed on the test strips.

[0069] 4. Actual sample testing

[0070] Take the seriously diseased tips of suspected larch dieback samples and observe them under a stereo microscope. Use a blade to cut 3 suspected symptom raised bark parts and 3 diseased branches and put them into centrifuge tubes as test samples. Use healthy branches as controls. Use small steel beads to grind the samples and use a kit to extract DNA from each sample. Figure 1 According to the detection principle and established system, each sample was tested using the rolling circle amplification test strip method. The results are shown in Figure 6 and Figure 7 .

[0071] Depend on Figure 6 and Figure 7 The results showed that gel electrophoresis bands could be detected in the raised part of the diseased part of larch branches and the suspected diseased branches, and red detection lines and blue quality control lines could also be observed on the test strips. Compared with the existing technology, the detection method of the present invention greatly shortens the time and is more suitable for grassroots personnel.

[0072] The present invention is not limited to the above-mentioned specific implementation modes. Various changes made by ordinary technicians in this field based on the above-mentioned concepts without creative work are all within the protection scope of the present invention.

Claims

1. A probe primer set for detecting larch dieback pathogen based on hyperbranching rolling circle amplification, characterized in that: include: Padlock probe NL-FD, detection probe cZip, first rolling circle amplification primer NL-F1, second rolling circle amplification primer NL-F2, wherein, The nucleotide sequence of the padlock probe NL-FD is shown in SEQ ID NO.1, the nucleotide sequence of the detection probe cZip is shown in SEQ ID NO.2, the nucleotide sequence of the first rolling circle amplification primer NL-F1 is shown in SEQ ID NO.3, and the nucleotide sequence of the second rolling circle amplification primer NL-F2 is shown in SEQ ID NO.4; The detection probe cZip 5 ′ The second rolling circle amplification primer NL-F2 is labeled with 6-carboxyfluorescein at the 5 ′ End-labeled with biotin.

2. A kit for detecting larch dieback pathogen based on hyperbranching rolling circle amplification, characterized in that: Comprising the probe primer set according to claim 1.

3. A method for detecting larch dieback pathogen based on hyperbranching rolling circle amplification, characterized in that: The following steps are involved: S1. Extract DNA from the sample to be tested; S2, using the DNA extracted from the sample to be tested as a template, and using the probe primer set described in claim 1 or 2 to perform HRCA amplification; specifically comprising the following steps: S21, performing a ligation and cyclization reaction on the extracted DNA and the padlock probe NL-FD using a thermal cycle ligation method; S22, adding enzyme digestion premix to the reaction product obtained in step S21 to perform exonuclease digestion reaction; S23, using the exonuclease digestion reaction product obtained in step S22 as a template, using the first rolling circle amplification primer NL-F1 and the second rolling circle amplification primer NL-F2 to perform HRCA amplification reaction to obtain an HRCA amplification product; S3, performing hybridization reaction between the obtained HRCA amplification product and the detection probe cZip; S4. Use lateral flow test strips to visually detect the hybrid reaction product, and determine whether the sample to be tested contains larch dieback pathogen based on the test results.

4. The method for detecting larch dieback pathogen based on hyperbranching rolling circle amplification according to claim 3, characterized in that: In step S21, the ligation cyclization reaction system is 10 μL, and is composed of: 2-5 μL DNA template, 1-3 μL 40U / μL Taq DNA ligase buffer, 0.1-0.5 μL 40U / μL Taq DNA ligase, 0.2-0.5 μL 20 pmol / L padlock probe NL-FD, 1-3 μL DNA template, and Sterilized ddH2O to 10 μL; The ligation and cyclization reaction program using the thermal cycle ligation method is: 94°C, 4 min; 94°C, 30s; 65°C, 5 min, 15 cycles; 95°C, 15 min, inactivation of Taq DNA ligase.

5. The method for detecting larch dieback pathogen based on hyperbranching rolling circle amplification according to claim 3, characterized in that: In step S22, the enzyme digestion premix in the exonuclease digestion reaction is 10 μL, and the composition is: 1-2 μL of 10× exonuclease I buffer; 2-3 μL of 5U / μL exonuclease I; Sterilized ddH2O is added to make up to 10 μL; The exonuclease digestion reaction procedures are: 37°C, water bath for 2.5 h; 80°C, water bath for 20 min.

6. The method for detecting larch dieback pathogen based on hyperbranching rolling circle amplification according to claim 3, characterized in that: In step S23, the HRCA amplification reaction system is 25 μL, and is composed of: 2-4 μL of 10×Bst DNA polymerase buffer, 0.5-1 μL of 8U / μL Bst DNA polymerase, 1-2 μL of 10μmol / L NL-F1, 1-2 μL of 10μmol / L NL-F2, 0.5-1 μL of 10mmol / L dNTPs, 4 μL of ligation circularization digestion product, and Sterilized ddH2O to make up to 25 μL; The HRCA amplification reaction procedure is: 55-62° C., 1.0 h.

7. The method for detecting larch dieback pathogen based on hyperbranching rolling circle amplification according to claim 3, characterized in that: In step S3, the hybridization reaction is carried out at a temperature of 55-60° C. for 5-8 min.

8. The method for detecting larch dieback pathogen based on hyperbranching rolling circle amplification according to claim 3, characterized in that: The test results are divided into the following three situations: (1) Positive (+), that is, a blue band appears on the test strip at the quality control line (C line) and a red band appears on the test line (T line), indicating that the sample to be tested contains the nucleic acid fragment of pine blight pathogen; (2) Negative (-), that is, a blue band appears on the test strip quality control line (C line) and no band appears on the test line (T line), indicating that the sample to be tested does not contain the nucleic acid fragment of pine blight pathogen, or its content is lower than the minimum detection amount of the test strip; (3) Invalid, that is, no bands appear on the test strip quality control line (C line) and the detection line (T line), indicating that the test strip or amplification reagent used may be damaged, ineffective or operated incorrectly.

Images