LAMP (loop-mediated isothermal amplification) primer for detecting fomitopsis pinicola and detection method

Abstract

The invention discloses an LAMP (Loop-Mediated Isothermal Amplification) primer for detecting Fomitopsis pinicola and a detection method. The sequences of the LAMP primers disclosed by the invention are as shown in SEQ ID NO: 1-6. The LAMP primer for detecting fomitopsis pinicola, the fomitopsis pinicola early detection method established based on the primer and the application of the fomitopsis pinicola in prevention and treatment of fir heart rot have remarkable technical advantages and practical application value, and have important significance in guaranteeing the ecological safety of fir forests in high-altitude areas such as Tibetan ganoderma lucidum and the like. Early warning: pathogenic bacteria can be rapidly detected in the early stage of disease occurrence, and a precious time window is provided for disease prevention and control.

Description

Technical Field

[0001] The invention relates to molecular biology detection technology, in particular to a LAMP primer and a detection method for detecting Famophilus rubrum. Background Art

[0002] In high-altitude ecological areas such as Linzhi, Tibet, fir, as an important forest vegetation, plays an irreplaceable role in maintaining ecological balance and biodiversity. Through in-depth investigation, researchers frequently found red-margined phyllotoxins ( Fomitopsis pinicola ). F. rubrum is a fungus widely distributed in various forest trees, with strong lignin decomposition ability. The fungus can invade the tree body, degrade the xylem tissue by secreting enzymes, cause the heartwood of the tree to decay, and eventually cause the tree to die. Combining the symptoms of fir heart rot and the biological characteristics of F. rubrum, we infer that F. rubrum is the pathogen of fir heart rot.

[0003] At present, the identification and prevention of the pathogens of fir heart rot mainly rely on traditional morphological observations, physiological and biochemical tests, and pathogenicity experiments. Although these methods can confirm the pathogens to a certain extent, they mainly have the following problems: 1) Poor detection timeliness: Traditional methods usually take a long time to obtain test results and cannot guide disease prevention and control in a timely manner. 2) High operation complexity: Professional laboratory equipment and technicians are required, which is not suitable for rapid on-site detection. 3) High cost: The high cost of equipment and reagents limits the popularization and application of detection technology. Existing methods are difficult to meet the needs of early warning and rapid prevention and control of diseases. Summary of the invention

[0004] The invention aims to provide a LAMP primer and a detection method for detecting Famophilus rubrum.

[0005] The present invention selects a sequence of TEF gene (NCBI number: MK236361.1) with large difference between F. rubrum and its similar species and inputs it into primerexplorer v5 for design. The LAMP detection of the present invention is mainly based on the change of pH of the reaction system before and after the reaction to achieve detection.

[0006] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a method for detecting the red margined phytomycetes ( Fomitopsis pinicola ) of LAMP primers, including: Outer forward primer F3: 5'-CATGTTGCTGATGCGCTTTG -3' (SEQ ID NO: 1); Outer reverse primer B3: 5'-ACGGAGCGCATATAATGAGG -3' (SEQ ID NO: 2); Internal forward primer FIP: 5'-GCCTTCGGGTTGTATCCGACCTGGAGCGAGGACCGTTTC -3' (SEQ ID NO: 3); Internal reverse primer BIP: 5′-TGTCTCCTTCGTCCCCATCTCTGCTGACCATCTGCAACACTT -3′ (SEQ ID NO: 4); Loop primer LF: 5′-TTCTTGATGAAGGTGGACGTCTC -3′ (SEQ ID NO: 5); Loop primer LB: 5'-ACAACATGTTGGAGGAGTCTGTC -3' (SEQ ID NO: 6).

[0007] The LAMP primers of the present invention need to be obtained by HPLC purification.

[0008] In a second aspect, the present invention provides a detection reagent or a kit containing the LAMP primers.

[0009] In a third aspect, the present invention provides use of the LAMP primer or the detection reagent or kit in the detection of Fomitopsis rubrum.

[0010] In a fourth aspect, the present invention provides a LAMP detection method for Fomitopsis rubrum, comprising the following steps: 1) Extract DNA from the sample to be tested; 2) using the DNA extracted in step 1) as a template, and performing a LAMP amplification reaction using the LAMP primers; 3) Determination of amplification results.

[0011] Furthermore, the reaction system used in step 2) was: 2× Master Mix with UDG 12.5 μL, 10× LAMP Primer Mix 2.5 μL, and ddH2O 9 μL.

[0012] Among them, the concentrations of primers F3, B3, FIP, BIP, LF and LB in 10× LAMP Primer Mix are 2μM, 2μM, 16μM, 16μM, 4μM and 4μM, respectively.

[0013] 2× Master Mix with UDG is WarmStart® Colorimetric LAMP 2X Master Mix with UDG (M1804S) from New England Biolabs.

[0014] Furthermore, the reaction conditions used in step 2) are: 65° C., 35 min.

[0015] Furthermore, step 3) includes: after the amplification reaction is completed, determining the amplification status of the target gene according to the color change of the reaction system before and after the reaction.

[0016] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The LAMP primers for detecting F. rubrum and the early detection method of F. rubrum established based on the primers provided by the present invention and their application in the prevention and treatment of fir heart rot have significant technical advantages and practical application value, and are of great significance for ensuring the ecological security of fir forests in high-altitude areas such as Linzhi, Tibet. The specific advantages are as follows: (I) The LAMP reaction primers and detection method for fir heart rot provided by the present invention can detect F. rubrum without false positives, and have good amplification effect and high specificity.

[0017] (ii) The fir heart rot LAMP reaction primers and detection method provided by the present invention can be used for on-site rapid real-time detection of fir. The operation is simple, and no complicated temperature control equipment is required. This overcomes the dependence on laboratory equipment and allows 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.

[0018] (III) Early warning: It can quickly detect pathogens in the early stages of a disease, providing a valuable time window for disease prevention and control.

[0019] (IV) Visual detection: realize intuitive visualization of detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the detection result of LAMP primer set 001 in Example 1 of the present invention. 1: Fomitopsis pinicola; 2: Melanoporia tropica ; 3: Fomitopsis cajander; 4: Fomitopsis dickinsii; 5: Fomitopsis marianii; 6: Rhodofomes roseus ; 7: Candolleomyces eurysporus ; n: negative control.

[0021] Figure 2This is the detection result of LAMP primer set 002 in Example 1 of the present invention. 1: Fomitopsis pinicola; 2: Melanoporia tropica ; 3: Fomitopsis cajander; 4: Fomitopsis dickinsii; 5: Fomitopsis marianii; 6: Rhodofomes roseus ; 7: Candolleomyces eurysporus ; n: negative control.

[0022] Figure 3 The results of the test in Example 1 of the present invention are shown in Figure 1, where ddH2O is replaced with DEPC water. 1: Fomitopsis pinicola; 2: Melanoporia tropica ; 3: Fomitopsis cajander; 4: Fomitopsis dickinsii; 5: Fomitopsis marianii; 6: Rhodofomes roseus ; 7: Candolleomyces eurysporus ; n: negative control.

[0023] Figure 4 This is the result of LAMP sensitivity detection in Example 1 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.

[0024] Figure 5 This is the PCR specific detection result in Example 1 of the present invention. 1. Fomitopsispinicola; 2. Porodaedalea alpicola; 3. Rhodofomes roseus; 4. Heterobasidion linzhiense; n: negative control; M: 100 bp Marker.

[0025] Figure 6 This is the PCR sensitivity test result in Example 1 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: 9.97×100 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.

[0026] Figure 7 The actual sample detection results of PCR in Example 1 of the present invention are shown in Figure 1. 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.

[0027] Figure 8 The results of detecting actual samples using the fir heart rot LAMP reaction primers and detection method of the present invention in Example 1 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. DETAILED DESCRIPTION

[0028] 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.

[0029] Example 1 Design of LAMP primers and detection method for F. rubrum This embodiment provides an early detection method for F. rubrum based on LAMP (loop-mediated isothermal amplification) technology, which can quickly and accurately detect pathogens in the early stage of the disease, providing strong support for early warning and timely prevention and control of fir heart rot.

[0030] 1. Primer design and screening based on LAMP detection Based on the TEF gene of F. rubrum (NCBI number: MK236361.1), the authors designed primers based on LAMP detection. The authors selected a sequence that was quite different between F. rubrum and its closely related species and designed it using primerexplorer v5. There are usually three pairs of LAMP primers, namely F3 / B3, FIP / BIP, and LF / LB. The primer design is relatively complicated. When performing LAMP detection, sometimes LF and LB primers are not added. Adding LF and LB can improve amplification efficiency and speed up the reaction time.

[0031] The following two pairs of primers were selected for the experiment: LAMP primer 001 (final selection): F3: 5'-CATGTTGCTGATGCGCTTTG-3' (SEQ ID NO: 1) B3: 5'-ACGGAGCGCATATAATGAGG-3' (SEQ ID NO: 2) FIP: 5'-GCCTTCGGGTTGTATCCGACCTGGAGCGAGGACCGTTTC-3' (SEQ ID NO:3) BIP: 5'-TGTCTCCTTCGTCCCCATCTCTGCTGACCATCTGCAACACTT-3' (SEQ ID NO:4) LF: 5'-TTCTTGATGAAGGTTGGACGTCTC-3' (SEQ ID NO:5) LB: 5'-ACAACATGTTGGAGGAGTCTGTC-3' (SEQ ID NO:6) LAMP Primer 002: F3: 5'-TTGTACAGTTGCTCGAACCA-3' B3: 5'-GCTGACCATCTGCAACACTT-3' FIP: 5'-AGGTGGACGTCTCCTTGACGATCATGTTGCTGATGCGCTTTG-3' BIP: 5'-GTCGGATACAACCCGAAGGCTGAACATGTTGTCGCCGTGC-3' LF: 5'-AACGGTCCTCGCTCCACTG-3' The detection of F. rubrum based on LAMP includes the following steps: 1) Extract genomic DNA from the sample to be tested; 2) First, prepare the primer premix (LAMP Primer Mix) according to the system in Table 1, and then prepare the LAMP reaction solution according to the system in Table 2; Table 1 Primer premix

[0032] Note: For the primer premix of primer set 002, loop primer LB is not included, and the other concentrations are the same as above.

[0033] Table 2 LAMP reaction system

[0034] Note: 2× Master Mix with UDG is WarmStart® Colorimetric LAMP 2X Master Mix with UDG (M1804S) from New England Biolabs.

[0035] 3) Mix the reaction solution and centrifuge to collect the liquid. The reaction solution should be bright pink. Place the prepared sample in a 65℃ environment and react for 35 minutes. You can also take out the prepared sample during the reaction and observe the result with the naked eye. If there is no change, continue the reaction for 35 minutes. Within 35 minutes, the sensitivity increases with the extension of the reaction time.

[0036] 4) The results can be observed with the naked eye. If the reaction sample is yellow, it indicates that the sample contains F. rubrum. If the reaction sample is still pink, it indicates that the sample does not contain F. rubrum, or the content of F. rubrum in the test sample is very small, below the detection limit of LAMP.

[0037] 2. LAMP specific detection by Melanoporia tropica, Fomitopsis cajander, Fomitopsis dickinsii, Fomitopsis marianii, Rhodofomes roseus, Candolleomyces eurysporus The genomic DNA of UDG was used as a template, and the DNA concentration was 1ng / μL. LAMP amplification was performed, and a set of no-template controls (NTC) was set up. 2× MasterMix with UDG, ddH2O, LAMP primer set, and DNA were melted at room temperature and mixed by oscillation. The total volume of the amplification reaction was 25μL. First, 10× LAMP primer premix was prepared according to the system in Table 1 above. 2× Master Mix with UDG 12.5μL, 10× LAMP Primer Mix 2.5μL, ddH2O 9μL were added to each reaction tube, and finally 1μL DNA was added. The DNA in the NTC was replaced with ddH2O. After fully mixing the reagents and samples, centrifuge and collect the bottom of the tube.

[0038] The prepared samples were placed in a 65°C environment and reacted for 30 minutes. The results of the two sets of primers are as follows Figure 1 (LAMP Primer Set 001) and Figure 2 (LAMP primer set 002) as shown.

[0039] After reacting at 65℃ for 30min, only the target species in group 001 turned yellow, and the rest turned pink, indicating that the primer group had good specificity and sensitive reaction. The reaction solution of group 002 showed no obvious change, so LAMP primer group 001 was selected for LAMP detection of F. rubrum.

[0040] In addition, in order to test the effect of different water on LAMP reaction, ddH2O was replaced with DEPC water for detection, and the reaction was carried out at 65℃ for 30min. The results are shown in Figure 3 It can be seen that the reaction tube with the DNA of F. rubrum turned yellow, and the reaction tube with the DNA of a closely related species of F. rubrum remained pink, but the NTC turned orange, indicating a false positive phenomenon, indicating that DEPC water cannot be used in this reaction system, which will lead to abnormal test results.

[0041] 3. LAMP minimum sensitivity 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 -5 copies / μL, and 1 μL was used as a template for LAMP reaction to evaluate the minimum detection limit of LAMP.

[0042] Take out 2× Master Mix with UDG, ddH2O, LAMP primer set, and DNA in advance, melt at room temperature, and shake to mix. The total volume of the amplification reaction is 25μL. First, prepare 10× LAMP primer premix according to the system in Table 1 above. Add 12.5μL UDG Mix, 2.5μL Primer Mix, 9μL ddH2O to each reaction tube, and finally add 1μL DNA. Replace the DNA in the NTC with ddH2O. After fully mixing the reagents and samples, centrifuge and collect the bottom of the tube.

[0043] Place the prepared samples in a 65°C environment. Figure 4 When the reaction was carried out at 65℃ for 35min, it was observed that sample 1 began to turn yellow. There was no obvious color change before 35min. The authors continued to observe at 40min and found no obvious color change compared with 35min. Therefore, 65℃ and 35min were selected as the LAMP reaction conditions for F. rubrum. Under these conditions, the detection limit of F. rubrum based on LAMP method was 9.97×10 3 copies / μL.

[0044] 4. Actual sample testing First, PCR testing was performed on samples collected in Tibet.

[0045] The PCR used a 25 μL reaction system, as follows:

[0046] Vazyme's 2 × Rapid Taq Master Mix was used for the PCR reaction.

[0047] PCR reaction procedure:

[0048] The PCR amplification products were verified by 1.0% agarose gel electrophoresis detection conditions were 130V, 25min.

[0049] At present, a large number of fruiting bodies have been found on trees with fir heart rot in Linzhi, Tibet, including red-margined pseudo-Phosphodia, alpine rusty porphyra, rose red porphyra, and Linzhi Heterobasidiomycetes. Therefore, alpine rusty porphyra, rose red porphyra, and Linzhi Heterobasidiomycetes were used to screen the specificity of PCR primers for red-margined pseudo-Phosphodia. PCR primers FPKF3 and FPKR2 (i.e. primers F and R) were used for PCR specificity experiments, and the results are shown in Figure 5 .

[0050] FPKF3 (primer F): 5'-CCATCAATTTTCATGTTGCTGATGCGCTTTGC-3' FPKR2 (Primer R): 5'-TGCCGCTGACCATCTGCAACACTTACTTGACA-3' It can be seen from the figure that only the channel with the DNA of F. rubrum has the target band, which is 197bp in size, indicating that the primers are of reference value for PCR detection.

[0051] The results of PCR sensitivity test are shown in Figure 6 It can be observed that the target band appears in channel 1, but no target band appears in the other channels. Therefore, the sensitivity of the PCR primers FPKF3 and FPKR2 in the PCR reaction is 9.97×10 3 copies / μL.

[0052] The actual sample test results of PCR are shown in Figure 7 From the figure, we can see that there are 7 positive samples and 9 negative samples.

[0053] The actual samples were tested using the LAMP reaction primers and detection method for fir heart rot established in this experiment ( Figure 8 ).

[0054] Comparison of PCR and LAMP test results is shown in Table 3: Table 3 Comparison of PCR and LAMP test results (+ indicates positive, - indicates negative)

[0055] In this experiment, the sensitivity of PCR and LAMP methods in detecting F. rubrum was the same, and the results of detecting actual samples were consistent.

[0056] In this study, the sensitivity of PCR and LAMP methods for detecting F. rubrum was 9.97×10 3 The results of the two methods for detecting actual samples were consistent and met the sensitivity requirements. The LAMP detection results established in this experiment were correct.

[0057] The fir heart rot LAMP reaction primers and the detection method provided by the invention can detect Fomitopsis rubrum without false positive, and have good amplification effect and high specificity.

[0058] 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. Used to detect the red margin of the fungus ( Fomitopsis pinicola ) is characterized in that, include: Outer forward primer F3: 5′-CATGTTGCTGATGCGCTTTG -3′; Outer reverse primer B3: 5′-ACGGAGCGCATATAATGAGG -3′; Internal forward primer FIP: 5′-GCCTTCGGGTTGTATCCGACCTGGAGCGAGGACCGTTTC -3′; Internal reverse primer BIP: 5′-TGTCTCCTTCGTCCCCATCTCTGCTGACCATCTGCAACACTT -3′; Loop primer LF: 5′-TTCTTGATGAAGGTGGACGTCTC -3′; Loop primer LB: 5′-ACAACATGTTGGAGGAGTCTGTC -3′.

2. A detection reagent or kit containing the LAMP primer according to claim 1.

3. Use of the LAMP primers according to claim 1 or the detection reagent or kit according to claim 2 in the detection of Fomitopsis rubrum.

4. The LAMP detection method of Fomitopsis rubrum is characterized in that: The following steps are involved: 1) Extract DNA from the sample to be tested; 2) using the DNA extracted in step 1) as a template, and performing a LAMP amplification reaction using the LAMP primers described in claim 1; 3) Determination of amplification results.

5. The method according to claim 4, characterized in that Step 2) The reaction system used was: 2× Master Mix with UDG 12.5 μL, 10× LAMP Primer Mix 2.5 μL, ddH2O 9 μL; Among them, the concentrations of primers F3, B3, FIP, BIP, LF and LB in 10× LAMP Primer Mix are 2μM, 2μM, 16μM, 16μM, 4μM and 4μM, respectively.

6. The method according to claim 5, characterized in that Step 2) The reaction conditions used were: 65°C, 35 min.

7. The method according to any one of claims 4 to 6, characterized in that: Step 3) includes: after the amplification reaction is completed, determining the amplification status of the target gene according to the change in the color of the reaction system before and after the reaction.

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