Fomitopsis pinicola real-time fluorescence RAA detection primer, probe and detection method

Abstract

The invention discloses a real-time fluorescent RAA detection primer, a probe and a detection method for Fomitopsis pinicola. The sequences of the real-time fluorescent RAA detection primer and the probe disclosed by the invention are shown as SEQ ID NO: 1-3. The invention further provides an RAA (recombinase-assisted amplification) fluorescence detection method of fomitopsis pinicola, the method has the characteristics of simplicity and convenience in operation, rapidness, high efficiency and high sensitivity, the fir heart rot can be rapidly and accurately diagnosed under the field condition, and a technical support is provided for timely prevention and control of diseases.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to primers and probes for real-time fluorescence RAA detection of phyllotoxinia rubrum and a detection method. Background Art

[0002] As an important forest resource in Linzhi, Tibet, fir is of great significance to maintaining ecological balance and local economic development. However, in recent years, a large number of fir heart rots have been found in Linzhi, resulting in the death of a large number of fir trees and serious damage to local forest resources. In an in-depth study of fallen fir wood, it was found that red-margined phyllotoxin (Fomyces erythroxysporum) frequently appeared in trees with fir heart rot and in the surrounding areas. Fomitopsis pinicola ), this phenomenon has attracted great attention of researchers.

[0003] Fomitopsis rubripes is a fungus widely distributed in various forests and has a strong ability to decompose lignin. The fungus can invade the body of trees and degrade the xylem tissue by secreting enzymes, causing the heartwood of the tree to decay and eventually lead to the death of the tree. Combining the symptoms of fir heart rot and the biological characteristics of Fomitopsis rubripes, it is reasonable to infer that Fomitopsis rubripes is the pathogen of fir heart rot.

[0004] Although it is preliminarily inferred that F. rubrum is the pathogen of fir heart rot, the current detection methods for this disease still have many shortcomings. Traditional detection methods such as morphological identification and molecular biological identification, although highly accurate, are complex and time-consuming to operate, and require professional experimental equipment and personnel, which are difficult to meet the needs of rapid on-site detection. Especially in remote areas such as Linzhi, Tibet, the lack of advanced experimental conditions and professional technicians has led to a lag in disease monitoring and prevention and control. Summary of the invention

[0005] The invention aims to provide primers and probes for real-time fluorescence RAA detection of Fomitopsis rubrum and a detection method.

[0006] The invention designs RAA detection primers according to the TEF gene (NCBI number: MK236361.1) of the red margined phyllotoxin. The primer design principle is as follows: the primer length is 30-35 bp, the optimum amplification product size is 100-200 bp, the minimum is 70-80 bp, the GC content is 30%-70%, the secondary structure can be avoided, the Tm value is 50-80°C, a plurality of primer pairs are designed, primer screening is performed, and then according to the result of the primer screening, a real-time fluorescent RAA probe is designed for the screened primer pairs with good amplification performance and no obvious dimer.

[0007] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a red margin phyllotoxin ( Fomitopsis pinicola) Real-time fluorescence RAA detection primers and probes, the sequences of real-time fluorescence RAA detection primers FPKF3 and FPKR2 are shown in SEQ ID NO:1-2, and the sequence of the probe is shown in SEQ ID NO:3.

[0008] Upstream primer FPKF3: 5'-CCATCAATTTTCATGTTGCTGATGCGCTTTGC-3' (SEQ ID NO: 1) Downstream primer FPKR2: 5'-TGCCGCTGACCATCTGCAACACTTACTTGACA-3' (SEQ ID NO: 2) Probe: 5'-TCAAGAAGGTCGGATACAACCCGAAGGCTGTCTCCTTCGTCCCCATCT-3' (SEQ IDNO: 3) In a second aspect, the present invention provides a real-time fluorescent RAA detection kit for F. rubrum, comprising real-time fluorescent RAA detection primers FPKF3 and FPKR2 as shown in SEQ ID NO: 1-2 and a probe RAA-KP; The probe RAA-KP is: 5'-TCAAGAAGGTCGGATACAACCCGAAGGCTG / i6FAMdT / / idSp / / iBHQ1 dT / CCTTCGTCCCCATCT-SpC3-3'; Among them, i6FAM dT is the base T labeled with a fluorescent group, iBHQ1 dT is the base T labeled with a quenching group, and SpC3 is the blocking group C3-Spacer. idSp is internal D-spacer, also known as / thf / or abasic. As a base modification, it represents the addition of a tetrahydrofuran (THF) spacer inside the oligonucleotide.

[0009] Furthermore, the real-time fluorescent RAA detection kit also includes RAA reaction buffer, purified water (nuclease-free water), lyophilized powder, magnesium acetate, etc.

[0010] In a third aspect, the present invention provides a real-time fluorescent RAA detection method for F. rubrum, comprising the following steps: (1) Extracting genomic DNA from the sample to be tested as a template; (2) Performing real-time fluorescent RAA detection using the real-time fluorescent RAA detection kit: adding 12.5 μL RAA reaction buffer, 10 μM FPKF3 and 10 μM FPKR2, 1.05 μL each, 10 μM RAA-KP 0.3 μL, 6.6 μL purified water, and 1 μL DNA template to a PCR tube containing lyophilized powder (one tube is two portions), and finally adding 2.5 μL magnesium acetate. After standing at 39° C. for 4 min, take out, flick the PCR tube by hand, mix the system thoroughly, and centrifuge briefly; (3) After centrifugation, place the PCR tube in a fluorescence detector and set the program to a constant temperature of 39°C. Collect the fluorescence signal every 30 seconds, repeat 50 times, and the reaction time is 25 minutes. After the reaction is completed, analyze the real-time fluorescence RAA detection results.

[0011] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: (I) The present invention provides a RAA (recombinase-assisted amplification) fluorescence detection method for Fomitopsis rubrum, which is simple to operate, rapid and efficient, and highly sensitive. It can achieve rapid and accurate diagnosis of fir heart rot under field conditions, providing technical support for timely prevention and control of the disease.

[0012] (ii) The primer set for detecting F. rubrum based on real-time fluorescence RAA provided by the present invention has no false positives, good amplification effect and high specificity. The detection sensitivity is ten times that of the ordinary PCR method.

[0013] (III) The real-time fluorescent RAA based on the primer set of the present invention can be used to perform rapid real-time detection of F. rubrum on site. The operation is simple and does not require complex temperature control equipment. It overcomes the dependence on laboratory equipment and can be used for on-site detection in areas where equipment conditions are insufficient. This saves time on sample transportation and avoids the impact of transportation on the test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Figure 1 is the result of basic RAA amplification using RAA primer set 001 in a preferred embodiment of the present invention. Fomitopsis pinicola; 2: Melanoporia tropica ; 3: Fomitopsis cajander;; 4: Fomitopsis dickinsii; ; 5: Fomitopsis marianii; 6: Rhodofomes roseus ; 7: Candolleomyces eurysporus ; n: negative control; M: 100bp Marker; p: positive control.

[0015] Figure 2 Figure 1 is the result of PCR specific detection in a preferred embodiment of the present invention. Fomitopsis pinicola 2.Porodaedalea alpicola ; 3. Rhodofomes roseus ; 4. Heterobasidion linzhiense ; n: negative control; M: 100bp Marker.

[0016] Figure 3 This is a graph showing the real-time fluorescence RAA specific detection results in a preferred embodiment of the present invention.

[0017] Figure 4 Figure 1 is the result of PCR sensitivity test in a preferred embodiment of the present invention. 1: 9.97×10 3 copies / μL; 2: 9.97×10 2 copies / μL; 3: 9.97×10 1 copies / μL; 4: 9.97×10 0 copies / μL; 5: 9.97×10 - 1 copies / μL; 6: 9.97×10 -2 copies / μL; 7: 9.97×10 -3 copies / μL; 8: 9.97×10 -4 copies / μL; 9: 9.97×10 -5 copies / μL; n: negative control; M: 100bp Marker.

[0018] Figure 5 This is a graph showing the results of real-time fluorescence RAA sensitivity detection in a preferred embodiment of the present invention.

[0019] Figure 6 The figure is a graph of the actual sample detection results of PCR in a preferred embodiment of the present invention. 1: N-7-2; 2: N-11-3; 3: N-12-1; 4: N-12-2; 5: N-16-1; 6: N-17-3; 7: N-19-3; 8: N-20-1; 9: F-12-1; 10: F-12-2; 11: F-13-1; 12: F-14-3; 13: F-16-2; 14: F-17-3; 15: F-19-3; 16: F-20-2; n: negative control; M: 100 bp Marker.

[0020] Figure 7 This is a graph showing the real-time fluorescence RAA specific detection results in a preferred embodiment of the present invention.

[0021] Figure 8 This is the appearance of a portable fluorescence instrument in a preferred embodiment of the present invention.

[0022] Figure 9The portable fluorescence instrument in the preferred embodiment of the present invention observes the detection results. 1: N-7-2; 2: N-11-3; 3: N-12-1; 4: N-12-2; 5: N-16-1; 6: N-17-3; 7: N-19-3; 8: n(1); 9: N-20-1; 10: F-12-1 11: F-12-2; 12: F-13-1; 13: F-14-3; 14: F-16-2; 15: F-17-3; 16: n(2); 17: F-19-3; 18: F-20-2; 19: n(3). DETAILED DESCRIPTION

[0023] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0024] Example 1 Primer design and screening for RAA detection Primers for RAA detection were designed based on the TEF gene of F. rubrum (NCBI No.: MK236361.1). The primer design principles are as follows: primer length is 30-35 bp, the optimal amplification product size is 100-200 bp, the GC content is 30%-70%, and the Tm value is 50-80. Multiple pairs of primers were designed and synthesized by Sangon Biotech Co., Ltd. Melanoporia tropica , Fomitopsis cajander, Fomitopsis dickinsii, Fomitopsis marianii, Rhodofomes roseus, Candolleomyces eurysporus The genomic DNA of was used as a template for RAA amplification. The RAA amplification reaction was carried out according to the RAA nucleic acid amplification kit (fluorescence method). The amplified product was mixed with chloroform and centrifuged for 8 minutes in a handheld centrifuge. The supernatant was taken for 1% agarose gel electrophoresis. The primer pairs with good amplification effect, no obvious dimer and good specificity were screened out ( Figure 1 ).

[0025] After screening of the basic RAA amplification method, FPKF3 and FPKR2 were selected as primers for RAA detection of F. rubrum.

[0026] RAA Primer Set 001: FPKF3: 5'-CCATCAATTTTCATGTTGCTGATGCGCTTTGC-3' (SEQ ID NO: 1) FPKR2: 5'-TGCCGCTGACCATCTGCAACACTTACTTGACA-3' (SEQ ID NO: 2) from Figure 1It can be seen that RAA primer set 001 has good specificity, but there are negative bands. It is inferred that it may be contaminated or affected by the primers, but the introduction of the probe can solve this problem well.

[0027] This embodiment provides a primer and probe for real-time fluorescence RAA of the pathogen of fir heart rot, Fomitopsis rubrum: Upstream primer FPKF3: 5'-CCATCAATTTTCATGTTGCTGATGCGCTTTGC-3' (SEQ ID NO: 1) Downstream primer FPKR2: 5'-TGCCGCTGACCATCTGCAACACTTACTTGACA-3' (SEQ ID NO: 2) (Unsubstituted probe sequence used in fluorescence method): 5'-TCAAGAAGGTCGGATACAACCCGAAGGCTGTCTCCTTCGTCCCCATCT-3' (SEQ ID NO: 3) Example 2 Primer set probe and detection method for real-time fluorescent RAA of the pathogenic fungus Fomitopsis rubrum 1. Design of fluorescent RAA probe Primer set for detecting F. rubrum based on real-time fluorescence RAA (SEQ ID NO: 1-2), the fluorophore and quencher of the probe are labeled on thymine (T), the distance between the fluorophore and the quencher is 1-5nt, and a larger distance will reduce the quenching efficiency; dSpacer is a base replaced between the fluorophore and the quencher, and the number of base intervals between the dT-quencher or the dT-fluorophore and the dSpacer is 0, 1 or 2; the blocking group C3-Spacer needs to be added to the 3' end of the probe. The probe sequence after replacement is as follows: RAA-KP: 5'-TCAAGAAGGTCGGATACAACCCGAAGGCTG / i6FAMdT / / idSp / / iBHQ1 dT / CCTTCGTCCCCATCT-SpC3-3' The fluorescent RAA kit also includes RAA reaction buffer, magnesium acetate (MgAc), purified water, and lyophilized powder. Among them, the RAA reaction buffer comes from the nucleic acid amplification kit (F00001) of Jiangsu Qitian Gene Biotechnology Co., Ltd., and the kit used in the basic RAA reaction process is the nucleic acid amplification kit (B00000) of Jiangsu Qitian Gene Biotechnology Co., Ltd.

[0028] 2. Detection of F. rubrum based on real-time fluorescence RAA The steps include: (1) Extract genomic DNA from the sample to be tested.

[0029] (2) Using the above-mentioned fluorescent RAA fluorescent primer set FPKF3 / FPKR2 as the upstream and downstream primers for amplification, add 12.5 μL RRAA reaction buffer, 1.05 μL each of 10 μM FPKF3 and 10 μM FPKR2, 0.3 μL of 10 μM RAA-KP, 6.6 μL purified water, and 1 μL template to a PCR tube containing lyophilized powder (one tube is the amount of two portions). Finally, add 2.5 μL magnesium acetate and let it stand at 39°C for 4 min. After 4 min, take out the tube and flick the PCR tube with a little force to mix the reagent and sample thoroughly, then place it in a handheld centrifuge for a short centrifuge.

[0030] (3) Immediately after centrifugation, place the PCR tube in a fluorescence detector and set the program to a constant temperature of 39°C. Collect the fluorescence signal every 30 seconds, cycle 50 times, and the reaction time is 25 minutes. Set up a set of NTC controls (no template negative control) for each set of primers and probes. The NTC controls do not contain template DNA to determine whether false positives occur.

[0031] 3. Real-time fluorescence RAA-specific detection Since researchers have found a relatively large number of fruiting bodies of Pseudomonas erythrorhizon, Pseudomonas alpina, Pseudomonas rosae, and Heterobasidiomycetes in Linzhi, Tibet, PCR primer specificity screening of Pseudomonas erythrorhizon was performed using Pseudomonas alpina, Pseudomonas rosae, and Heterobasidiomycetes. The above RAA primers FPKF3 and FPKR2 were used for PCR specificity experiments, and the results are shown in Table 1. Figure 2 From the figure, it can be seen that only the channel with the DNA of F. rubrum has the target band, which is 197 bp in size, indicating that the primers have reference value for PCR detection.

[0032] The primer sets FPKF3, FPKR2, and RAA-KP designed by screening were validated for specificity. Melanoporia tropica , Fomitopsis cajander, Fomitopsis dickinsii, Fomitopsis marianii, Rhodofomes roseus, Candolleomyces eurysporusThe genomic DNA of the sample was used as a template for RAA reaction. A group of no-template controls (NTC) and a group of positive controls (PTC) were set up. The RAA nucleic acid amplification kit (fluorescence method) was used for amplification. The RAA reaction buffer, purified water, and magnesium acetate were taken out 30 minutes in advance, melted at room temperature, and oscillated to mix. The total volume of the amplification reaction was 25 μL. 12.5 μL of RAA reaction buffer, 1.05 μL of 10 μM FPKF3 and 10 μM FPKR2, 0.3 μL of 10 μM RAA-KP, 6.6 μL of purified water, and 1 μL of template were added to the PCR tube containing lyophilized powder (one tube is the amount of two copies). Finally, 2.5 μL of magnesium acetate I was added, mixed, and centrifuged quickly. After pre-incubation for 4 minutes, the sample was taken out, and the PCR tube was flicked with a little force to mix the reagents and samples thoroughly, and then placed in a handheld centrifuge for short centrifugation. Immediately place the PCR in a fluorescence detection instrument, set the program to a constant temperature of 39°C, collect fluorescence signals every 30 seconds, cycle 50 times, and the reaction time is 25 minutes. The results obtained are as follows Figure 3 As shown, the real-time fluorescence RAA results using the genomic DNA of Pseudomonas erythrorhizium as template and the positive control (PTC) were positive, while the results of the real-time fluorescence RAA reaction system using other genomic DNA as templates and no template DNA were negative, indicating that the detection system can specifically detect Pseudomonas erythrorhizium with good detection specificity.

[0033] 4. Real-time fluorescence RAA minimum detection limit determination The concentration of the constructed plasmid was measured with an ultra-micro spectrophotometer and converted to the copy number according to the formula: copy number = (plasmid concentration × 10 -9 ×6.02×10 23 ) / (660×base bp size) copies / μL. The calculated plasmid copy number of F. rubrum was 9.97×10 10 The plasmid was diluted to 9.97×10 3 9.97×10 2 9.97×10 1 , 9.97×10 0 , 9.97×10 -1 , 9.97×10 -2 , 9.97×10 -3 9.97×10 -4 , 9.97×10 -5copies / μL, take 1μL as template for RAA reaction to evaluate the minimum detection limit of real-time fluorescent RAA, use RAA nucleic acid amplification kit (fluorescence method) for amplification, take out RAA reaction buffer, purified water, and magnesium acetate 30 minutes in advance, melt at room temperature, and shake to mix. The total volume of the amplification reaction was 25 μL. 12.5 μL RAA reaction buffer, 10 μM FPKF3 and 10 μM FPKR2, 1.05 μL each, 10 μM RAA-KP 0.3 μL, 6.6 μL purified water, and 1 μL template were added to the PCR tube containing lyophilized powder (one tube is the amount of two copies). Finally, 2.5 μL magnesium acetate was added, mixed, and centrifuged quickly. After pre-incubation for 4 minutes, the PCR tube was taken out and flicked with a little force. After the reagents and samples were fully mixed, they were placed in a handheld centrifuge for a short centrifugation. A set of no-template controls (NTC) was set up, and the PCR tube was immediately placed in the fluorescence detection instrument. The program was set to a constant temperature of 39°C, and the fluorescence signal was collected every 30 seconds. The cycle was repeated 50 times, and the reaction time was 25 minutes. The results are as follows: Figure 4 As shown in the figure, it can be observed that the target band appears in channel 1, while no target band appears in the other channels. Therefore, the sensitivity of the primer set FPKF3 and FPKR2 in the PCR reaction is 9.97×10 3 copies / μL.

[0034] Depend on Figure 5 It can be observed that under the condition of 37℃ for 25min, the minimum detection limit of F. rubrum based on real-time fluorescence RAA is 9.97×10 2 copies / μL (it is normal for the fluorescence value to fluctuate around 100), and the sensitivity increases with time.

[0035] 5. Use real-time fluorescence RAA method to detect red-margined phyllotoxin in samples Abies were randomly selected from the sample plots in Tibet, and the cores of the fir trees were drilled for detection. The collected samples were detected using the real-time fluorescence RAA detection system established by the present invention, and compared with the PCR method.

[0036] PCR method to detect actual samples: The PCR used a 25 μL reaction system, as follows:

[0037] Primer F is primer FPKF3, and primer R is primer FPKR2.

[0038] PCR reaction procedure:

[0039] The PCR amplification products were verified by 1.0% agarose gel electrophoresis detection conditions were 130V, 25min. Figure 6 It can be seen that there are 7 positive samples (the target bands appear in channels 5, 10, 11, 12, 13, 14, and 15) and 9 negative samples.

[0040] Comparison of PCR and real-time fluorescence RAA detection results is shown in Table 1: Table 1 Comparison of PCR and real-time fluorescence RAA test results (+ indicates positive, - indicates negative)

[0041] Real-time fluorescence RAA specific detection results are shown in Figure 7 .

[0042] In this study, the sensitivity of PCR and real-time fluorescence RAA for detecting F. rubrum was 9.97×10 3 copies / μL and 9.97×10 2 The results of the two methods for detecting samples were consistent and met the sensitivity requirements. Therefore, the results of the real-time fluorescence RAA detection method established in this experiment were correct.

[0043] Furthermore, a portable fluorescence instrument (from the Forest Engineering and Forest Protection Team of Beijing Forestry University, Figure 8 ) Observe the real-time fluorescence RAA actual sample detection results, the results are shown in Figure 9 The results show that positive samples are all green, negative samples are blue, and only sample No. 14 has a low fluorescence signal, so the signal is not obvious on the portable fluorescence meter, which is consistent with the results of the real-time fluorescence meter (Bio-rad).

[0044] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Phomitopsis rubrum Fomitopsis pinicola ) Real-time fluorescent RAA detection primers and probes, characterized in that, The sequences of the real-time fluorescent RAA detection primers FPKF3 and FPKR2 are shown in SEQ ID NO:1-2, and the sequence of the probe is shown in SEQ ID NO:

3.

2. A real-time fluorescence RAA detection kit for F. rubrum, characterized in that: It includes real-time fluorescence RAA detection primers FPKF3 and FPKR2 and probe RAA-KP as shown in SEQ ID NO: 1-2; The probe RAA-KP is: 5'-TCAAGAAGGTCGGATACAACCCGAAGGCTG / i6FAMdT / / idSp / / iBHQ1 dT / CCTTCGTCCCCATCT-SpC3-3'; Among them, i6FAM dT is base T labeled with a fluorescent group, iBHQ1 dT is base T labeled with a quenching group, SpC3 is a blocking group C3-Spacer, and idSp is an internal D-spacer.

3. The real-time fluorescent RAA detection kit according to claim 2, characterized in that: The kit also includes RAA reaction buffer, purified water, lyophilized powder, and magnesium acetate.

4. A real-time fluorescence RAA detection method for F. rubrum, characterized in that: The following steps are involved: (1) Extracting genomic DNA from the sample to be tested as a template; (2) Using the kit described in claim 3 to perform real-time fluorescent RAA detection: add 12.5 μL RAA reaction buffer, 1.05 μL each of 10 μM FPKF3 and 10 μM FPKR2, 0.3 μL 10 μM RAA-KP, 6.6 μL purified water, and 1 μL DNA template to a PCR tube containing lyophilized powder, and finally add 2.5 μL magnesium acetate. After standing at 39°C for 4 minutes, take out, flick the PCR tube by hand, mix the system thoroughly, and centrifuge briefly; (3) After centrifugation, place the PCR tube in a fluorescence detector and set the program to a constant temperature of 39°C. Collect the fluorescence signal every 30 seconds, repeat 50 times, and the reaction time is 25 minutes. After the reaction is completed, analyze the real-time fluorescence RAA detection results.

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