Method, primer and probe combination and kit for detecting apple tree pathogenic bacteria

Abstract

The invention belongs to the technical field of pathogen detection, and discloses a method for detecting apple tree pathogens, a primer and probe combination and a kit. The invention designs a combination of a specific primer and a probe aiming at the apple tree pathogenic bacteria Botryosphaeria dothidea, and establishes a detection method of the apple ring spot pathogenic bacteria on the basis of a TaqMan qPCR (Quantitative Polymerase Chain Reaction) technology. Through verification, the detection method provided by the invention is high in sensitivity and good in specificity, has no cross reaction with pathogenic bacteria of other genus, and has important significance on early diagnosis of apple tree diseases, determination of the optimal prevention and treatment period and establishment of an effective disease prevention strategy.

Description

Technical Field

[0001] The invention belongs to the technical field of pathogen detection and relates to a method for detecting apple tree pathogens, a combination of primers and probes, and a kit. Background Art

[0002] Apple has become a major consumer fruit due to its strong ecological adaptability, high nutritional value and long supply cycle. However, apple ring rot caused by Botryosphaeria is a major fungal disease threatening the apple industry. This disease not only reduces apple yield and quality, but also causes a large number of fruit trees to die and even destroy the orchard in severe cases. Therefore, it is particularly important to monitor apple tree diseases for orchard management and disease prevention.

[0003] At present, a variety of methods for pathogen detection have been developed, such as PCR, qRT-PCR, etc. Compared with conventional PCR, the advantages of qRT-PCR technology are higher sensitivity and specificity, good stability, short time consumption, and low pollution. At the same time, the detection results can be qualitative as well as quantitative. The principle is to add fluorescent substances to the PCR reaction system. As the reaction proceeds, the cumulative amount of the reaction product is positively correlated with the intensity of the fluorescent signal generated by the fluorescent substance. Therefore, the change in fluorescence intensity can reflect the change in the amount of amplified products in real time. After counting all fluorescent signals, an amplification curve can be obtained. The amplification curve is S-shaped and can be divided into three stages: background signal stage, exponential amplification stage, and plateau stage. In the amplification curve, the number of cycles when the fluorescence intensity reaches a certain set fluorescence threshold (Threshold) is called the Ct value (Threshold cycle). The Ct value is directly affected by the starting copy number of the template DNA. The smaller the starting copy number of the template DNA, the larger the Ct value, and vice versa.

[0004] However, these conventional methods have poor sensitivity and specificity, resulting in inaccurate test results. This requires an apple pathogen detection method that is easy to operate, highly specific, and highly sensitive to solve the above problems. TaqMan qPCR technology combines TaqMan probes with qRT-PCR. This technology can greatly improve the specificity of detection and can also achieve quantitative detection of pathogens. TaqMan qPCR technology has been used for plant pathogen detection, but it is difficult to screen probes with strong specificity using TaqMan qPCR technology. Therefore, it has only been used in the detection of a few plant pathogens and has not yet been implemented in the detection of apple ring rot. Therefore, there is an urgent need for a detection method for apple ring rot pathogens with strong specificity and sensitivity. Summary of the invention

[0005] In view of the above problems existing in the prior art, the present invention provides a combination of primers and probes for detecting apple tree pathogens based on TaqMan qPCR technology, and establishes a method for detecting apple tree pathogens with strong specificity and high sensitivity. Another object of the present invention is to provide a kit containing a specific detection agent for apple ring rot pathogens, so as to achieve rapid detection of apple ring rot. To this end, the present invention provides the following technical solutions.

[0006] In one aspect, the present invention provides a combination of primers and probes for detecting apple tree pathogens based on TaqMan qPCR technology, wherein the forward sequence of the primer is shown in SEQ ID NO.1, and the reverse sequence of the primer is shown in SEQ ID NO.2. The sequence of the probe is shown in SEQ ID NO.3. The apple tree pathogen is Botryosphaeria berengeriana.

[0007] On the other hand, the present invention seeks to protect the use of the above combination in detecting apple tree pathogens.

[0008] On the other hand, the present invention seeks to protect the use of the above combination in the preparation of a detection reagent or a kit for apple tree pathogens.

[0009] On the other hand, the present invention also provides an apple tree pathogen detection kit based on TaqMan qPCR technology, the kit comprising a detection reagent, the detection reagent comprising at least one amount of the above combination. In the combination, the primer is used to initiate the extension of the DNA chain; the probe specifically binds to the target DNA sequence and is cut by Taq DNA polymerase during PCR amplification, thereby releasing a fluorescent signal.

[0010] Furthermore, in the above-mentioned apple tree pathogen detection kit, the kit also includes: 2×TaqMan FastqPCRMasterMix and deionized water.

[0011] Finally, the present invention provides a method for detecting apple tree pathogens based on TaqMan qPCR technology, the method comprising the following steps:

[0012] 1) Extracting DNA from the sample to be tested;

[0013] 2) using DNA as a template and performing qPCR amplification using the above primer and probe combination;

[0014] 3) Determine whether the sample is positive based on the Ct value that reaches the specified fluorescence threshold during qPCR amplification.

[0015] Specifically, in the above method, the apple tree pathogen is Botryosphaeria berengeriana.

[0016] Specifically, in the above method, when the Ct value is less than 40, the result is positive; when the Ct value is greater than 40, the result is negative.

[0017] Compared with the prior art, the present invention "a method for detecting apple tree pathogens, a combination of primers and probes, and a kit" has the following beneficial effects:

[0018] 1) The TaqMan qPCR detection method provided by the present invention is highly specific. The combination of primers and probes is obtained through a large number of experimental verifications and screenings, and can specifically detect the pathogen of apple ring rot, without cross-reaction with other pathogens.

[0019] 2) The TaqMan qPCR detection method provided by the present invention has extremely high detection sensitivity. The method can detect the genomic DNA of the apple ring rot pathogen at a minimum of 1 pg / μL.

[0020] 3) The TaqMan qPCR detection method provided by the present invention can quantitatively detect pathogens, while conventional PCR can only perform qualitative detection.

[0021] 4) The TaqMan qPCR detection system used in the present invention can directly interpret the detection results through the Ct value, which is convenient and fast.

[0022] 5) The present invention is suitable for quantitative detection of apple ring rot pathogens, and can be further used to study the relationship between the occurrence of apple ring rot and the amount of fungus sources. Apple ring rot develops rapidly in the field and is difficult to prevent and control once it occurs. The method provided by the present invention can be used for early prediction and forecast of apple ring rot, which is of great significance for early diagnosis of the disease, determination of the best prevention and control period, and establishment of effective prevention and control strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the result of TaqMan qPCR specific detection of apple ring rot pathogen. Figure 1In the table, 1: apple brown rot pathogen (Moniliniafructicola); 2: apple wilt pathogen (Fusarium oxysporum); 3: apple rot pathogen (Alternaria spp); 4: apple anthracnose pathogen (Colletotrichum gloeosporioides); 5: apple anthracnose leaf blight pathogen (Colletotrichum fructicola); 6: apple dry rot pathogen (Botryosphaeria ribis); 7: apple ring rot pathogen CK+ (Botryosphaeria berengeriana); NC: double distilled water.

[0024] Figure 2 For the standard curve. Figure 2 In the table, 1 to 5 represent a series of concentrations of apple ring rot pathogens, 1: 10 ng / μL; 2: 1 ng / μL; 3: 100 pg / μL; 4: 10 pg / μL; 5: 1 pg / μL. DETAILED DESCRIPTION

[0025] The present invention is explained below in conjunction with the embodiments, and the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0027] The test strains and test reagents involved in the examples are as follows.

[0028] 1. Test strains

[0029] The test strains were isolated and preserved by the laboratory of the fruit tree disease pathogen biology and integrated prevention and control research team of Northwest Agriculture and Forestry University (see Table 1). The 7 test strains in Table 1 were all cultured using PDA solid medium. First, the bacterial block stored in 25% glycerol (frozen at -80°C) was inoculated on the PDA solid medium, and the plate was placed at 25°C for 48 hours. The cultured mycelium was used to extract genomic DNA.

[0030] Table 1. Test strain information

[0031] Serial number Bacteria Strain name Host describe 1 Monilinia fructicola HF apple Apple brown rot pathogen 2 Fusariumoxysporum LD apple Apple wilt pathogen 3 Alternariaspp LG apple Apple core rot pathogen 4 Colletotrichum gloeosporioides TJ apple Apple Anthracnose Pathogen 5 Colletotrichum fructicola TJK apple Apple Anthracnose Leaf Blight Pathogen 6 Botryosphaeriaribis GF apple Apple stem rot pathogen 7 Botryosphaeriadothidea (CK+) LW apple Apple ring rot pathogen

[0032] 2. Test Reagents

[0033] The fungal genomic DNA extraction kit was purchased from Sangon Biotech (Shanghai) Co., Ltd.; the primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0034] Example 1

[0035] This example describes the extraction of genomic DNA from the test strains.

[0036] Use the fungal genomic DNA extraction kit to extract the strain genomic DNA according to the instructions. The specific steps are as follows:

[0037] (1) Take 50-100 mg of fresh fungal mycelium, grind it into powder with liquid nitrogen, add it to a 1.5 mL centrifuge tube, add 400 μL of Buffer Digestion and 4 μL of β-mercaptoethanol, and oscillate to mix. Incubate in a 65°C water bath for 1 h until the cells are completely lysed.

[0038] (2) Add 200 μL of Buffer PF, mix thoroughly by inversion, and place in a -20°C refrigerator for 5 min.

[0039] (3) Centrifuge at 10,000 rpm for 5 min at room temperature and transfer the supernatant (about 500-550 μL) to a new 1.5 mL centrifuge tube.

[0040] (4) Add an equal volume of isopropanol, invert 5 to 8 times to mix thoroughly, and leave at room temperature for 2 to 3 minutes. Centrifuge at 10,000 rpm for 5 minutes at room temperature and discard the supernatant.

[0041] (5) Add 1 mL of 75% ethanol, invert and rinse for 1 to 3 minutes, centrifuge at 10,000 rpm for 2 minutes, and discard the supernatant.

[0042] (6) Repeat step (5) once.

[0043] (7) Open the lid and invert at room temperature for 5 to 10 minutes until the remaining ethanol is completely evaporated.

[0044] (8) Dissolve the extracted DNA in 100 μL TE Buffer. The extracted DNA can be used immediately for the next experiment or stored at -20°C.

[0045] Example 2

[0046] This example describes the design of TaqMan qPCR primers and probes for detecting the pathogen of apple ring rot and the establishment of a TaqMan qPCR detection method.

[0047] The elongation factor gene (EF-1α gene) of the apple ring rot pathogen was used as the probe binding sequence of TaqMan qPCR, and then Primer Express 3.0 was used to design the primers and probes of TaqMan probe method fluorescence quantitative PCR. The 5' end of the probe was labeled with the fluorescent group HEX, and the 3' end was labeled with the quenching group BHQ1. The primers and the probe cooperate with each other. Specifically, the primers are used to initiate the extension of the DNA chain; the probe specifically binds to the target DNA sequence and is cut by Taq DNA polymerase during the PCR amplification process, thereby releasing a fluorescent signal. As the amplification proceeds, the fluorescent signal gradually increases, and an amplification curve is obtained.

[0048] The designed primer sequences are as follows:

[0049] LW-taq-F: 5'-CCTCGTTTGGTCTTCGGCAA-3' (SEQ ID NO. 1);

[0050] LW-taq-R: 5'-CCTGTTGTGGCGGTGGTTAG-3' (SEQ ID NO. 2).

[0051] The probe sequences are as follows:

[0052] LW-taq-Probe:5'-HEX-ACGCGAACAACCCCTCACCAACGCTT-BHQ1-3'(S

[0053] EQ ID NO.3).

[0054] The extracted genomic DNA was used as a template and the above primer and probe combination was used to perform TaqMan qPCR reaction. The reaction system was as follows:

[0055] 10μM primer LW-taq-F 0.4μL, 10μM primer LW-taq-R 0.4μL, 10μM probe LW-taq-Probe0.2μL, 1μL DNA template and 10μL 2×TaqMan Fast qPCR MasterMix, and deionized water was added to the total reaction system of 20μL.

[0056] Furthermore, referring to "Real-time fluorescence quantitative PCR molecular detection technology for apple brown spot pathogens" (Zhang Lixia et al., Acta Phytopathologica Sinica, 2023.12.22), the Ct value was used as the basis for judgment. When the Ct value is less than 40, the result is positive, indicating that the sample contains the pathogen of apple ring rot; when the Ct value is greater than 40, the result is negative, indicating that the sample does not contain the pathogen of apple ring rot.

[0057] Example 3

[0058] This example describes the analysis of TaqMan qPCR assay specificity.

[0059] In order to verify the specificity of TaqMan qPCR detection, one apple ring rot pathogen and six apple endophytic fungi (see Table 1 for details) were selected and genomic DNA was used as a template to verify the specificity of the TaqMan qPCR system. Figure 1 As shown by Figure 1 It can be seen that only the apple ring rot pathogen was positive, that is, the Ct value was less than 40. The DNA test results of the other 6 strains were all negative, that is, the Ct value was greater than 40. This shows that the designed TaqMan qPCR reaction system is highly specific.

[0060] Example 4

[0061] This example describes the sensitivity analysis of TaqMan qPCR detection and the establishment of a standard curve.

[0062] In order to verify the sensitivity of TaqMan qPCR detection, the genomic DNA of the apple ring rot pathogen was extracted as a template, and the extracted genomic DNA concentration was diluted 10 times in sequence to obtain 8 gradients with mass concentrations of 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL, and 1 fg / μL, respectively. The template DNA was replaced with an equal amount of ultrapure water as a blank control (CK).

[0063] The results are as follows Figure 2 As shown, when the initial concentration of genomic DNA of apple ring rot pathogen was 10ng / μL, 1ng / μL, 100pg / μL, 10pg / μL, and 1pg / μL, the Ct value of TaqMan qPCR detection was less than 40, and the result was positive; when the initial concentration was 100fg / μL, 10fg / μL, and 1fg / μL, the Ct value of TaqMan qPCR detection was greater than 40, and the result was negative. This result shows that the sensitivity of the TaqMan qPCR detection system is 1pg / μL.

[0064] The standard curve was drawn with the logarithm of the copy number of the apple ring rot pathogen DNA concentration as the horizontal axis and the Ct value as the vertical axis. Figure 2 As shown in the figure, with the decrease of DNA template concentration of apple ring rot pathogen strain, Ct value gradually increased. When the DNA template concentration of standard strain was in the range of 10 ng / μL to 1 pg / μL, it had a good linear relationship with Ct value. 2 =0.9998, the slope is -3.4893, and the linear equation of the standard curve is y=-3.4893x+25.895.

[0065] In summary, the present invention designs a combination of specific primers and probes for the apple tree pathogen Botryosphaeria dothidea, and establishes a detection method for the apple ring rot pathogen based on TaqMan qPCR technology. It has been verified that the detection method provided by the present invention has high sensitivity and good specificity, and has no cross-reaction with pathogens of other genera. It is of great significance for the early diagnosis of apple tree diseases, determining the best prevention and control period, and establishing effective disease prevention strategies.

[0066] The embodiments described above are only some embodiments of the present invention, not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but only represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. A combination of primers and probes for detecting apple tree pathogens based on TaqMan qPCR technology, characterized in that: In the combination, the forward primer sequence of the primer is shown as SEQ ID NO.1, the reverse primer sequence of the primer is shown as SEQ ID NO.2; the sequence of the probe is shown as SEQ ID NO.3; The apple tree pathogen is Botryosphaeria berengeriana.

2. Use of the combination of primers and probes described in claim 1 in detecting apple tree pathogens.

3. Use of the combination of primers and probes according to claim 1 in the preparation of a detection reagent or kit for apple tree pathogens.

4. A detection kit for apple tree pathogens based on TaqMan qPCR technology, characterized in that: The kit comprises a detection reagent, wherein the detection reagent comprises at least one amount of the combination of the primer and the probe according to claim 1; In the combination, the primer is used to initiate extension of the DNA chain; The probe specifically binds to the target DNA sequence and is cleaved by Taq DNA polymerase during PCR amplification, thereby releasing a fluorescent signal.

5. The apple tree pathogen detection kit according to claim 4, characterized in that: The kit also includes: 2×TaqMan Fast qPCR MasterMix and deionized water.

6. A method for detecting apple tree pathogens based on TaqMan qPCR technology, characterized in that: The method comprises the following steps: 1) Extracting DNA from the sample to be tested; 2) using DNA as a template and performing qPCR amplification using the combination of primers and probes described in claim 1; 3) Determine whether the sample is positive based on the Ct value that reaches the specified fluorescence threshold during qPCR amplification.

7. The method according to claim 6, characterized in that The apple tree pathogen is Botryosphaeria berengeriana.

8. The method according to claim 7, characterized in that When the Ct value is less than 40, the result is positive; when the Ct value is greater than 40, the result is negative.

Images