A method for detecting Alternaria alternata using a thermometer based on CRISPR / Cas12a

Abstract

The present invention discloses a method for detecting Alternaria using a thermometer based on CRISPR / Cas12a. The present invention utilizes a dual signal amplification strategy of high specific surface area of ​​AuNPs and efficient shearing ability of LbCas12a to achieve effective amplification of detection signals, and has very high sensitivity to the detection of Alternaria; at the same time, the present invention utilizes CrRNA and target DNA for specific identification, and has very good specificity for the detection of Alternaria, and can achieve rapid, accurate and sensitive detection of Alternaria in the sample to be tested; in the present invention, there is no need to use large-scale detection instruments and toxic reagents. In the detection method of the present invention, portable on-site detection of Alternaria can be achieved using only a thermometer.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for detecting Alternaria alternata by using a thermometer based on CRISPR / Cas12a. Background Art

[0002] Alternaria is a common plant pathogenic fungus with strong adaptability in natural environments and substrates. Therefore, it can be widely distributed in nature, and more than 95% of its species are facultative parasites on plants, with a wide variety of species and a wide host range. It can infect plants and cause a variety of plant diseases, such as black spot of pears, red dates, and tomatoes, brown spot of lemons and citrus, gray spot of chestnuts, and red spot of tobacco, causing huge economic losses in the field and post-harvest globally, seriously hindering the development of such cash crop industries. Studies have shown that these plant diseases are mainly caused by toxins produced by their secondary metabolites, and most of these toxins have chronic toxicity such as teratogenicity, carcinogenicity, and mutagenicity.

[0003] In addition to causing plant diseases, some species of Alternaria can also infect the skin of humans and animals, causing certain chronic skin diseases and other zoonotic diseases. Although Alternaria can harm the health of plants and animals, it can also be used as an important fungal resource for humans because some biologically active secondary metabolites of Alternaria can kill insects. Biological control agents developed based on the characteristics of certain strains of Alternaria longifolia can prevent and control tobacco brown spot disease with a control effect of more than 90%; Alternaria zinniae can prevent Canada thistle; anticancer drugs such as vinblastine and paclitaxel can be produced by the Alternaria spores of some plant endophytes. In this way, the correct detection of Alternaria has important theoretical and practical significance.

[0004] At present, the detection of Alternaria mainly includes morphological observation, nucleotide sequence analysis, protein gene sequence analysis, isozyme, soluble protein analysis technology, etc. These methods can achieve sensitive and accurate detection of Alternaria, and are widely used in the detection of Alternaria. However, these technologies require cumbersome sample pretreatment, large precision instruments, toxic reagents and professional technicians, which greatly limits their application in the rapid detection of Alternaria. Therefore, it is very urgent to develop other efficient, sensitive and rapid detection methods for Alternaria. Summary of the invention

[0005] The purpose of the present invention is to solve the problems in the prior art that the detection method for Alternaria is complicated, requires large precision instruments or toxic reagents, leads to low detection efficiency and high detection cost, and provides a method for detecting Alternaria with a thermometer based on CRISPR / Cas12a.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: A method for detecting Alternaria alternata with a thermometer based on CRISPR / Cas12a, comprising the following steps:

[0007] S1, prepare gold nanoparticles (AuNPs), Capture ssDNA-MBs capture probes and DetectorssDNA-HRP-AuNPs probes respectively;

[0008] S2, 2 μL 1×Tolo buffer, 2 μM LbCas12a, 5 μM CrRNA, PCR amplification product of the sample DNA to be tested, 1 μM single-stranded DNA substrate S and 10 μL DNase / RNase free water were mixed, the mixture was incubated at 37°C for 30 min, and LbCas12a was inactivated at 80°C;

[0009] S3, cooling the mixture after inactivating LbCas12a in S2 to room temperature, incubating it with capture ssDNA-MBs capture probe, Detector ssDNA-HRP-AuNPs probe and TMB-H2O2 solution in sequence, separating the suspension after incubation, irradiating the suspension with laser for 40s, and monitoring the temperature change of the suspension with a thermometer;

[0010] S4. Result determination: After the suspension is irradiated with laser in S3, if the temperature rises, the DNA of the sample to be tested does not contain Alternaria DNA; if the temperature does not rise, the sample contains Alternaria DNA.

[0011] Preferably, the preparation method of the Detector ssDNA-HRP-AuNPs probe in S1 is as follows: 10 μL of 100 μM detector ssDNA is mixed with 100 μL AuNPs solution, 2 μL of horseradish peroxidase (HRP, 2.5 mg / mL) is added and mixed, and the mixture is frozen at -80°C for 30 min; after thawing, the mixture is washed twice with 0.1% Tween solution, and centrifuged at 12000 r / min at 4°C for 30 min; the resulting solution is resuspended in 100 μL coupling buffer to obtain the Detector ssDNA-HRP-AuNPs probe; and the mixture is stored at 4°C for future use.

[0012] Preferably, the nucleotide sequence of the single-stranded DNA substrate S in S2 is as shown in SEQ ID No.1

[0013] Preferably, the incubation method of the capture ssDNA-MBs capture probe in S3 is: adding 15 μL of capture ssDNA-MBs capture probe and 5 μL of coupling buffer, incubating at room temperature for 20 minutes, and then washing 3 times with coupling buffer.

[0014] Further preferably, the incubation method of the Detector ssDNA-HRP-AuNPs probe in S3 is: adding 10 μL of Detector ssDNA-HRP-AuNPs probe, incubating at room temperature for 10 min, then washing 3 times with coupling buffer, and resuspending in 50 μL of acetate buffer.

[0015] More preferably, the incubation method of the TMB-H2O2 solution in S3 is: adding 50 μL of TMB-H2O2 solution and incubating at 37°C for 10 min.

[0016] The present invention simultaneously modifies the surface of AuNPs (gold nanoparticles) with detector ssDNA (detection DNA) and HRP (horseradish peroxidase) with the ability to catalyze TMB (3,3',5,5'-tetramethylbenzidine) oxidation, and modifies biotin (biotin)-capture ssDNA (capture DNA) on SA-MBs (streptavidin-magnetic beads), and then through a single-stranded DNA substrate S, the three are hybridized to form a sandwich-like structure by using DNA hybridization, thereby connecting TMB-H2O2 and CRISPR Cas12a systems. Since the charge transfer complex of the single-electron oxidation product of TMB (3,3',5,5'-tetramethylbenzidine) has a strong near-infrared laser-driven photothermal effect, the oxidized TMB can be used as a photothermal probe to convert the target signal into heat, and a simple photothermal analysis is performed using a thermometer to achieve portable detection of Alternaria alternata.

[0017] The detection principle of Alternaria alternata is as follows Figure 1 As shown: When the Alternaria gene exists, the Alternaria gene will complementally hybridize with part of the base sequence of CrRNA in the CRISPR-Cas12a system to form a double strand. At this time, the CRISPR-Cas12a system is activated, and the single-stranded DNA substrate S is added. The DNA is cut into several short DNA fragments. When the MBs-capturessDNA and AuNPs-detector ssDNA-HRP probes are added, the three will not hybridize, and almost no hybridization complex will remain on the magnetic beads. When TMB-H2O2 is added, TMB will not be oxidized to TMB +When irradiated with 808nm excitation light, the temperature of the solution will not rise; when the Alternaria gene does not exist, the complex of CrRNA and Cas12a is added, and no DNA is complementary to the partial base sequence of CrRNA in the CISPR-Cas12a system to form a double strand. At this time, the CISPR-Cas12a system cannot be activated, and the single-stranded DNA substrate S is added. The DNA will not be cut by Cas12a, and then MBs-capture ssDNA and AuNPs-detectorssDNA-HRP probes are added. The three are hybridized, and the hybridized complex is connected to the magnetic beads. At this time, TMB-H2O2 is added, and TMB is catalyzed into TMB with photothermal conversion effect. + When irradiated with 808nm excitation light, the temperature of the solution will rise. Therefore, based on this detection principle, the present invention can realize portable detection of Alternaria alternata with only a thermometer.

[0018] The beneficial effects of the present invention are:

[0019] 1) The present invention utilizes the dual signal amplification strategy of the high specific surface area of ​​AuNPs and the efficient shearing ability of LbCas12a to achieve effective amplification of the detection signal, and has a very high sensitivity for the detection of Alternaria; at the same time, the present invention utilizes CrRNA and target DNA for specific identification, and has very good specificity for the detection of Alternaria, which can achieve rapid, accurate and sensitive detection of Alternaria in the sample to be tested;

[0020] 2) The present invention does not require the use of large-scale detection instruments and toxic reagents. In the detection method of the present invention, portable on-site detection of Alternaria can be achieved using only a thermometer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The schematic diagram of the present invention's method for detecting the gene of Alternaria alternata;

[0022] Figure 2 Schematic diagram of the preparation of the Capture ssDNA-MBs probe and the detector ssDNA-HRP-AuNPs probe in Example 1;

[0023] Figure 3 This is a diagram showing the verification results of the method for detecting the gene of Alternaria citri using a thermometer based on CRISPR / Cas12a in Example 2;

[0024] Figure 4 This is a result diagram of detecting genes of other species of Alternaria using a thermometer based on CRISPR / Cas12a in Example 3;

[0025] Figure 5This is a graph showing the results of detecting and analyzing the citrus Alternaria gene using a thermometer based on CRISPR / Cas12a in Example 4. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0027] Example 1

[0028] 1. Synthesis of AuNPs

[0029] Soak the required glassware in aqua regia (HCl:HNO3=3:1) for 15~20min to remove impurities, then wash with ultrapure water and dry for use. Under magnetic stirring, heat 100mL of 1.0mM chloroauric acid (HAuCl4) solution to boiling, and quickly add 1mL of 400mM sodium citrate solution prepared in advance; continue to heat the above mixed solution under magnetic stirring, keep it boiling for 8~10min, when the color of the solution gradually turns purple-red, stop heating and let it cool naturally to room temperature, and a stable AuNPs solution can be obtained, and finally store it in a 4℃ refrigerator for use.

[0030] 2. Preparation of Capture ssDNA-MBs Probe

[0031] As attached Figure 2 -A, first, the surface of the streptavidin-modified magnetic beads (SA-MBs) was thoroughly washed with coupling buffer; then, 100 μL SA-MBs (4 g / L) was reacted with 3 μL 100 μM biotinylated capture ssDNA at 25°C for 30 minutes. Then, the MBs were adsorbed using a magnetic stand and the excess unreacted biotinylated capture ssDNA was removed. After washing three times with coupling buffer, the obtained capture ssDNA-MBs probe was suspended in 100 μL coupling buffer and stored at 4°C for later use.

[0032] Streptavidin-modified magnetic beads (SA-MBs) were purchased from New England Biolabs, Inc. (England).

[0033] Biotinylated capture ssDNA was purchased from Sangon Biotechnology Co., Ltd. The specific sequence is: 5'-biotin-AAAAAAAAAAAAACCTTATGATCTCCCT-3'.

[0034] 3. Preparation of Detector ssDNA-HRP-AuNPs probe

[0035] like Figure 2 -B, in a 1.5mL centrifuge tube, 10μL of 100μM detector ssDNA was mixed with 100μL AuNPs (prepared in step 1 of this embodiment) solution. Then 2μL HRP (2.5mg / mL) was added. After gently mixing, the centrifuge tube was frozen at -80℃ for 30min. After thawing, it was washed twice with 0.1% Tween solution and centrifuged at 12000r / min at 4℃ for 30 minutes. Finally, the resulting solution was resuspended in 100μL coupling buffer and stored at 4℃ for later use.

[0036] detector ssDNA was purchased from Shanghai Bioengineering, with the specific sequence: 5'-AAAAAAAAAAAATGTCCGATGCTC-3'.

[0037] Example 2 Verification of the method for detecting the citrus Alternaria gene using a thermometer based on CRISPR / Cas12a

[0038] In this example, different concentrations of artificially synthesized Alternaria citri DNA (target dsDNA) were detected, and five groups a, b, c, d, and e were set, a was a blank control group, and the target dsDNA concentrations of groups b, c, d, and e were 1000 pM, 200 pM, 40 pM, and 8 pM, respectively.

[0039] The specific detection methods are as follows:

[0040] 2 μL 1×Tolo buffer, 2 μM LbCas12a, 5 μM CrRNA, PCR amplification product of the sample DNA to be tested (synthesized citrus Alternaria DNA), 1 μM single-stranded DNA substrate S and 10 μL DNase / RNase free water were mixed, and the mixture was incubated at 37°C for 30 min, and LbCas12a was inactivated at 80°C. Then 15 μL capture ssDNA-MBs capture probe (prepared in Example 1) and 5 μL coupling buffer were added, incubated at room temperature for 20 min, and then washed 3 times with coupling buffer; 10 μL Detector ssDNA-HRP-AuNPs probe (prepared in Example 1) was added, incubated at room temperature for 10 min, washed 3 times with coupling buffer, and resuspended in 50 μL acetate buffer; 50 μL TMB-H2O2 solution was added and incubated at 37°C for 10 min. After incubation, the suspension was separated from the complex with a magnetic stand and then added to a microplate. The color of the solution was recorded, and the absorption spectrum was measured using a Varioskan LUX microplate reader. Finally, the solution was irradiated with an 808 laser for 40 seconds, and the temperature change of the solution was monitored using a thermometer.

[0041] LbCas12a was purchased from Tolo Biotech Co., Ltd. (Shanghai, China).

[0042] CrRNA was purchased from Shanghai Bioengineering, with the specific sequence: 5′-UAAUUUCUACUAAGUGUAGAUUCCUAGUGGUGGGCGAACCC-3′.

[0043] The nucleotide sequence of the single-stranded DNA substrate S is shown in SEQ ID No.1.

[0044] The test results are as attached Figure 3 As shown in the figure, when the concentration of target dsDNA increases from 8pM to 1000pM, the color of the solution changes from bright blue to light blue. This phenomenon is due to the fact that the PCR product of the synthetic target dsDNA recognized by crRNA triggers the trans-cleavage activity of CRISPR / Cas12a, resulting in complete degradation of S. Therefore, the detection probe cannot be pulled to the surface. After adding TMB and H2O2, the color depth of the solution decreases as the concentration of synthetic target dsDNA increases ( Figure 3 C). Therefore, the characteristic absorption peak of oxTMB at 650 nm decreases with the increase of the concentration of the synthetic target dsDNA ( Figure 3 A). After the detection solution is irradiated by near-infrared laser, the temperature also decreases accordingly. Therefore, as the concentration of synthetic target dsDNA increases, the temperature change increases sharply ( Figure 3 B), which can be easily recorded with a handheld thermometer. The calculation of 3δ, where δ represents the standard deviation of the control group (n=11), shows that the detection limit of the proposed photothermal strategy for synthesizing target dsDNA is 1.5pM. The above verification shows that the detection method of the present invention can indeed detect the citrus Alternaria gene with high sensitivity, and the citrus Alternaria gene can be semi-quantitated by color depth changes.

[0045] Example 3 Detection of other species of Alternaria genes using a thermometer based on CRISPR / Cas12a

[0046] Four groups, a, b, c, and d, were set up, with a being a blank control group, and b, c, and d being apple, tomato, and citrus, respectively. The genetic DNA of the cultured cells of Alternaria alternata of apple, tomato, and citrus were extracted using the classic CTAB method and amplified by PCR. The obtained PCR amplification products were detected and analyzed by agarose gel electrophoresis and the detection method in Example 2, respectively. The experimental results are shown in FIG. Figure 4 shown.

[0047] from Figure 4As can be seen in D, the color depth of the other three groups of solutions is significantly reduced compared to the blank group a. The color of the apple group b is darker than that of the citrus group d and the tomato group c. This is because less genetic DNA is extracted from the cultured Alternaria cells isolated from apples. Through the above-mentioned visual detection, the genetic DNA of the cultured Alternaria isolated from citrus, apples and tomatoes can be semi-quantitatively detected. At the same time, in the presence of the genetic DNA of the cultured Alternaria, the absorption intensity of oxTMB is correspondingly reduced ( Figure 4 A). Therefore, the temperature changes significantly after NIR laser irradiation ( Figure 4 B). This result is consistent with the gel electrophoresis analysis result ( Figure 4 C). The above data indicate that the detection method of the present invention is also very suitable for detecting Alternaria isolated and cultured from different fruit and vegetable products.

[0048] Example 4 Detection of Alternaria citri gene using a thermometer based on CRISPR / Cas12a

[0049] In this example, 9 citrus fruit samples with and without typical brown spot symptoms were collected from Wanzhou and Jiangjin Districts of Chongqing, China, and the corresponding DNA was extracted from them. The appearance of the 9 citrus fruit samples with and without typical brown spot symptoms is shown in the attached figure. Figure 5 As shown in D, these 9 samples are numbered S1-S9.

[0050] The PCR amplification products of the 9 sample DNAs were detected by the detection method in Example 2, and agarose gel electrophoresis was used to further analyze and verify the PCR amplification products of the 9 sample DNAs. The results are shown in the attached figure. Figure 5 As shown. Figure 5 From the data of A, 5B, and 5C, it can be seen that only two samples S5 and S6 were found to be infected with Alternaria among the nine samples; Figure 5 This experimental result can also be verified in the electrophoresis diagram of E. Only S5 and S6 showed two bands in their lanes. After sequencing, it was shown that the band of about 600 bp belonged to Alternaria and the band of about 750 bp belonged to Citrus. Figure 5 D, it is difficult to judge whether citrus is infected by Alternaria only by symptoms on the surface of citrus, because symptomatic samples may also be misleading. Therefore, these results show that the method of the present invention can accurately and effectively identify whether citrus fruit samples are infected with Alternaria, and is suitable for application in actual production.

[0051] In summary, the present invention combines the CRISPR / Cas12a system with TMB-H2O2 to construct a photothermal analysis platform, which can not only quickly detect Alternaria alternata by temperature changes; the Alternaria alternata genes in the test sample can also be semi-quantitatively detected by observing the color depth changes of the solution with the naked eye. At the same time, the detection method in the present invention does not require the use of large-scale detection instruments and toxic reagents. In the detection method of the present invention, portable on-site detection of Alternaria alternata can be achieved using only a thermometer, which is very suitable for application in actual production.

[0052] The specification and drawings of the present invention are considered to be illustrative rather than restrictive. On the basis of the present invention, those skilled in the art can make some substitutions and modifications to some of the technical features according to the disclosed technical content without creative labor, all of which are within the protection scope of the present invention.

Claims

1. A method for detecting Alternaria alternata using a thermometer based on CRISPR / Cas12a, characterized in that: The following steps are involved: S1, prepare gold nanoparticles AuNPs, Capture ssDNA-MBs capture probes and Detector ssDNA-HRP-AuNPs probes respectively; The nucleotide sequence of the Capture ssDNA-MBs probe is: 5'-biotin-AAAAAAAAAAAAACCTTATGATCTCCCT-3'; the nucleotide sequence of the Detector ssDNA-HRP-AuNPs probe is: 5'-AAAAAAAAAAAATGTCCGATGCTC-3'; S2, 2 μL 1×Tolo buffer, 2 μM LbCas12a, 5 μM CrRNA, PCR amplification product of the sample DNA to be tested, 1 μM single-stranded DNA substrate S and 10 μL DNase / RNase free water were mixed, the mixture was incubated at 37°C for 30 min, and LbCas12a was inactivated at 80°C; the nucleotide sequence of the single-stranded DNA substrate S is shown in SEQ ID No.1; S3, cooling the mixture after inactivating LbCas12a in S2 to room temperature, incubating it with capture ssDNA-MBs capture probe, Detector ssDNA-HRP-AuNPs probe and TMB-H2O2 solution in sequence, separating the suspension after incubation, irradiating the suspension with laser for 40s, and monitoring the temperature change of the suspension with a thermometer; S4. Result determination: If the DNA of the sample to be tested does not contain Alternaria DNA, the capture ssDNA-MBs capture probe, the Detector ssDNA-HRP-AuNPs probe and the single-stranded DNA substrate S hybridize to form a complex, and TMB is catalyzed to oxidized TMB with a photothermal conversion effect. At this time, after the suspension is irradiated with a laser in S3, the temperature of the suspension rises; if the sample contains Alternaria DNA, the capture ssDNA-MBs capture probe, the Detector ssDNA-HRP-AuNPs probe and the single-stranded DNA substrate S cannot hybridize to form a complex, and TMB is not catalyzed. At this time, after the suspension is irradiated with a laser in S3, the temperature of the suspension does not rise; the sample is a plant sample.

2. The method according to claim 1, characterized in that The preparation method of the Detector ssDNA-HRP-AuNPs probe in S1 is as follows: 10 μL of 100 μM detector ssDNA is mixed with 100 μL AuNPs solution, 2 μL of 2.5 mg / mL horseradish peroxidase HRP is added and mixed, and the mixture is frozen at -80°C for 30 min; after thawing, the mixture is washed twice with 0.1% Tween solution, and centrifuged at 12000 r / min at 4°C for 30 min; the resulting solution is resuspended in 100 μL coupling buffer to obtain the Detector ssDNA-HRP-AuNPs probe; and the mixture is stored at 4°C for future use.

3. The method according to claim 1, characterized in that The incubation method of the capture ssDNA-MBs capture probe in S3 is: adding 15 μL of capture ssDNA-MBs capture probe and 5 μL of coupling buffer, incubating at room temperature for 20 minutes, and then washing 3 times with coupling buffer.

4. The method according to claim 1, characterized in that: The incubation method of the Detector ssDNA-HRP-AuNPs probe in S3 is as follows: add 10 μL of Detector ssDNA-HRP-AuNPs probe, incubate at room temperature for 10 min, wash three times with coupling buffer, and resuspend in 50 μL of acetate buffer.

5. The method according to claim 1, characterized in that The incubation method of the TMB-H2O2 solution in S3 is: add 50 μL of TMB-H2O2 solution and incubate at 37° C. for 10 minutes.

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