Princtoria parviflora detection method based on RPA-CRISPR-LFD technology

Abstract

The invention relates to a Princidium parvum detection method based on an RPA-CRISPR / Cas12a-LFD technology. The Princidium parvum detection method comprises the following steps: firstly, carrying out The method has the advantages that the sensitivity is high, the lowest detection limit of the genome can reach 1.5 * 10 <-2 > pg / mu L, and the lowest detection limit of a simulation sample can reach 1.12 * 10 <-2 > cells / mL; the specificity is good, and crRNA and RPA primers are both designed according to the ITS target sequence of Prlinella parvum; the detection speed is high, and detection can be completed only through two-step reaction; the requirements on instruments and equipment are low, a PCR instrument, an electrophoresis instrument and a gel imaging system are not needed, and amplification and detection can be completed by only one constant-temperature heater; the method has low requirements on professional quality of operators, does not need professional algal experts, and can complete all detection processes of the Princiinella parvata by only having a little molecular biology basis.

Description

Technical Field

[0001] The present invention belongs to the field of water environment protection, and more particularly, to a method for detecting Pleurotus eryngii based on RPA-CRISPR / Cas12a-LFD technology. Background Art

[0002] Prymnesium parvum belongs to the phylum Haptophyta, class Coccolithophyceae, orders Prymnesiales, family Prymnesiaceae, and genus Prymnesium. It was first discovered and recorded in Danish waters in 1930 and is a globally widespread species. Prymnesium parvum is the most influential algae species among non-cyanobacterial inland harmful algal blooms in the United States, affecting at least 23 states and causing tens of millions of dollars in economic losses. In China, although there have been no reports of coastal Prymnesium blooms in recent years, high densities of Prymnesium parvum are often found in aquaculture ponds, with cell densities reaching 10 2 -10 7 cells / ml. It is worth noting that even when no large-scale reproduction is observed, low densities of Purlina can affect the ecosystem. Therefore, monitoring the presence of Purlina in water environments is of great significance for ecological protection and the development of aquaculture.

[0003] At present, the methods used for the detection of P. microphylla are mainly divided into traditional microscopic examination and molecular biological methods. Traditional microscopic examination is time-consuming and laborious, and requires experienced experts to make more accurate identifications. It has no advantages among various detection methods. Although molecular detection technologies such as PCR and molecular probes have the advantages of being fast and sensitive, they have too high requirements for experimental equipment and require professional personnel to conduct detection, which cannot achieve the goal of rapid detection of harmful algae in the field.

[0004] Our team has used recombinase polymerase amplification (RPA) technology to achieve rapid detection in the field, but we still hope to further improve its detection sensitivity and specificity. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a method for detecting Pleurotus eryngii in a water sample, comprising the following steps:

[0006] S1: extracting DNA from the water sample;

[0007] S2: amplifying the characteristic site by recombinase polymerase amplification method to obtain an RPA amplification product, wherein the sequence of the characteristic site is shown in SEQ ID NO: 2;

[0008] S3: preparing a CRISPR-12a reaction system using the RPA amplification product, performing a CRISPR reaction, and obtaining a CRISPR reaction product;

[0009] S4: Detecting the labeled molecules in the CRISPR reaction product.

[0010] In a specific embodiment, in S2, the primer sequences used to amplify the characteristic sites are shown in SEQ ID NOs: 8 and 9.

[0011] In a specific embodiment, in S3, the CRISPR-12a reaction system further includes the following components: Cas12a protein, ssDNA with a labeled molecule, and crRNA.

[0012] In a specific embodiment, the concentration of the ssDNA carrying the labeled molecule is 500 nmol / L.

[0013] In a specific embodiment, the CRISPR reaction time is 40 min.

[0014] In a specific embodiment, in S4, the labeled molecule is detected using a lateral flow test strip on which a substance capable of capturing the labeled molecule is immobilized.

[0015] In a specific embodiment, in S4, PEG is added to the reaction product before lateral flow test strip detection, and the working concentration of PEG is 4.6%.

[0016] In a specific embodiment, the lateral flow test strip uses gold-labeled nanoparticles as a color developer, and the content of the gold-labeled nanoparticles is 1.5×10 2 pg / μL.

[0017] The RPA-CRISPR / Cas12a-LFD detection method of Pleurotus eryngii provided by the present invention has the following advantages:

[0018] 1. High sensitivity, the minimum detection limit of genome can reach 1.5×10 -2 pg / μL, the minimum detection limit of the simulated sample can reach 1.12×10 -2 cells / mL.

[0019] 2. Good specificity, crRNA and RPA primers were designed based on the ITS target sequence of P. microphylla.

[0020] 3. The detection speed is fast, and only two steps of reaction are required to complete the detection.

[0021] 4. Low requirements for instruments and equipment. No PCR instrument, electrophoresis instrument or gel imaging system is required. Only a constant temperature heater is needed to complete amplification and detection.

[0022] 5. The requirements for the professional quality of the operators are relatively low. No professional algae experts are required. Only a little knowledge of molecular biology is needed to complete the entire detection process of the small purinol algae. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 :RPA-CRISPR-LFD detection flowchart.

[0024] Figure 2 : Screening of crRNA and RPA primers for P. microphylla. Among them, (a) shows the optimal crRNA screening; (b) shows the optimal crRNA; (c) shows the RPA primer amplification results, NC is the blank control with ddH2O as the template, M: DL2000 DNA marker; (d) shows the optimal RPA primer screening.

[0025] Figure 3 :Optimization of RPA-CRISPR / Cas12a-LFD system. Among them, (a) is the ssDNA concentration screening; (b) is the PEG concentration primary screening (negative); (c) is the PEG concentration primary screening (positive); (d) is the PEG concentration rescreening (negative).

[0026] Figure 4 : Optimization of gold-labeled nanoparticle concentration in test strips. (a) shows the screening of test strip length; (b) shows the screening of CRISPR reaction time.

[0027] Figure 5:Method evaluation. Among them, Figures (a, c) and (b, d) respectively show the specificity and sensitivity of two different detection methods: RPA-CRISPR / Cas12a-LFD (conventional) and RPA-CRISPR / Cas12a-LFD (trimmed). NC is a blank control with ddH2O as a template, M: DL2000 DNA marker; specific algae species used: Prymnesium parvum, Isochrysis galbana, Chrysotila dentata, Karlodinium veneficum, Trichodesmium erythraeum, Kareniamikimotoi, Skeletonema costatum, Chaetoceros debilis, Thalassiosira pseudonana, Phaeodactylum tricornutum, Pseudo-nitzschia multiseries, Prorocentrumdonghaiense, Chaetoceros curvisetus), Thalassiosirarotula;

[0028] Figure 6 : Simulated field sample verification results.

[0029] Figure 7 :Real field sample verification results. Among them, stations B1-B14 represent water samples from Bohai Sea, stations L1-L14 represent water samples from Lianjiang River, stations XS1-XS2 represent water samples from Xiangshan Bay, stations SM1-SM6 represent water samples from Sanmen Bay, and stations SS1-SS15 represent water samples from South China Sea. DETAILED DESCRIPTION

[0030] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0031] 1. Principles of RPA-CRISPR-LFD technology

[0032] RPA-CRISPR-LFD technology is a new technology that combines recombinase polymerase amplification technology (RPA), clustered regularly interspaced short palindromic repeats (CRISPR) and lateral flow dipstick (LFD).

[0033] Its working principle is as follows Figure 1 As shown, RPA, as a pre-amplification step, only needs to react for 10 to 20 minutes in a constant temperature environment of 35 to 39°C to achieve exponential high-specificity DNA amplification. CRISPR / Cas12a is activated for trans-cleavage activity, and the cleavage probe produces the target molecule. LFD is a detection device that can be combined with a variety of detection methods to greatly shorten the time it takes for the test results to appear. The fundamental principle of the LFD test strip is the combination of antibodies and antigens. The validity of the test strip can be judged by the control line, and the test results can be judged by the test line in the detection area, and visualized with the naked eye.

[0034] RPA-CRISPR-LFD technology is fast and efficient, highly sensitive, and specific, and has no equipment or technical limitations. It has obvious advantages over traditional detection methods.

[0035] To achieve the above detection, it is necessary to design RPA primers based on the genome sequence of P. microphylla to amplify and produce target fragments. After the target fragment binds to crRNA, it activates the cutting activity of cas12a, cuts the probe, produces target molecules, and optimizes other detection systems on this basis.

[0036] The probe used in the present invention is ssDNA-FB: 5'-FAM-TTATT-Biotin-3', or ssDNA-FT: 5'-FAM-TTATT-TAMRA-3'.

[0037] 2. Microalgae used in the experiment

[0038] The 14 algae strains used in the present invention include: Prymnesium parvum (NMBjih029), Chaetoceros curvisetus (NMBguh003-10), Thalassiosirapseudonana (NMBguh006), Skeletonema costatum (NMBguh0042), Isochrysis galbana (NMBjih021-2), Pseudo-nitzschia multiseries (NMBguh002-1-1), Karenia mikimotoi (NMBjah052), Karlodinium veneficum (NMBjah047-1), Prorocentrum donghaiense (NMBguh006-2), and Pseudo-nitzschia multiseries (NMBguh002-1-1). donghaiense (NMBjah045), and Chrysotila dentata (NMBjih026-1), were provided by the Microalgae Germplasm Bank of Ningbo University; Thalassiosira rotula (MMDL50319) and Chaetoceros debilis (MMDL50116), were provided by Xiamen University; Phaeodactylum tricornutum (CCMP2561), was provided by Westlake University; and Trichodesmium erythraeum (IMS101), was provided by the University of Southern California.

[0039] The culture medium used was f / 2 medium. The algae were cultured in a 100 ml conical flask under fluorescent light with a light intensity of 15 to 20 μmol / (m 2 ·s), light-dark cycle of 12:12, and culture temperature of 16°C.

[0040] 3. Design and screening of crRNA and RPA primers

[0041] Based on the ITS sequence in the genome of P. purinus, we identified the PAM site (TTTN) and designed multiple crRNAs. We found that three of them had good effects, namely crRNA-1, crRNA-2 and crRNA-3 (sequences are SEQ ID NO: 1-3 respectively). The ITS sequence of P. purinus amplified by universal primers was used as a substrate, and the best crRNA was screened from these three crRNAs by fluorescence value. The results are as follows Figure 2 As shown in a, crRNA-2 has the best fluorescence value performance. The schematic diagram of crRNA-2 binding to the target is as shown in Figure 2As shown in b.

[0042] According to crRNA-2, multiple pairs of RPA primers were designed, and the three pairs of RPA primers with the best amplification effect (RPA-1-F / R, sequences as shown in SEQ ID NOs: 4 and 5; RPA-2-F / R, sequences as shown in SEQ ID NOs: 4 and 7; RPA-3-F / R, sequences as shown in SEQ ID NOs: 8 and 9) were selected for the next step of screening. The electrophoresis photos of the RPA amplification products are shown in Figure 2. Figure 2 c, the fluorescence value is shown as Figure 2 As shown in d.

[0043] The above results show that the best crRNA is crRNA-2; the best RPA primer pair is RPA-3-F / R.

[0044] 4. Optimization of RPA-CRISPR-LFD system

[0045] 4.1 Optimization of ssDNA concentration

[0046] Reaction system: ssDNA-FB 1μL, ddH2O 19μL. 8 concentration gradients of ssDNA-FB were set, namely 5000nmol / L, 500nmol / L, 100nmol / L, 50nmol / L, 25nmol / L, 5nmol / L, 0nmol / L. The reaction results are shown in Figure 3 As shown in a.

[0047] In the LFD reaction, if the concentration of ssDNA is too high, the gold-labeled nanoparticles bound to ssDNA will escape from the control line and enter the detection line. If the concentration of ssDNA is too low, the gold-labeled nanoparticles will not be able to completely bind to ssDNA, ultimately leading to false positive results. In order to minimize this effect, concentration screening was performed and 500nmol / L, which had the lightest detection line when negative, was selected as the optimal ssDNA concentration for subsequent experiments.

[0048] 4.2 LFD buffer optimization

[0049] In the LFD reaction, the appropriate addition of PEG can reduce the fluidity of the buffer, thereby allowing the gold-labeled nanoparticles to combine more fully with the control line, and to some extent reduce the impact of false positives. This study used PEG 6000 to optimize the buffer.

[0050] The concentration gradient of PEG 6000 was set as follows: 0%, 2%, 4%, 4.2%, 4.4%, 4.6%, 4.8%, 5.0%, 5.2%, 5.4%, 5.6%, 5.8%, 6%, 8%. The reaction results are shown in Figure 3As shown in Figures b and c, when the PEG 6000 concentration is 6%, the detection line band in the negative result of the test strip basically disappears, but the color of the band in the positive result is also greatly reduced. Therefore, in order to increase the concentration of the positive band as much as possible on the basis of the disappearance of the negative band, optimization was performed again between 4% and 6%, and finally 4.6% was selected as the optimal PEG 6000 concentration for LFD buffer optimization ( Figure 3 d).

[0051] 4.3 CRISPR reaction time optimization

[0052] The CRISPR reaction time was optimized based on the optimal ssDNA concentration and PEG 6000 concentration. The time gradient was set to 40min, 30min, 20min, 15min, and 10min. When the reaction time was 40min, the positive band of the test strip was significantly higher than that of other reaction groups, so 40min was selected as the optimal reaction time for subsequent experiments.

[0053] 5. Optimization of the number of gold-labeled nanoparticles in the test strip

[0054] The gold-labeled nanoparticles of the test strip are distributed in its sample loading area. Therefore, this study achieves the purpose of changing the number of gold-labeled nanoparticles by trimming the length of the sample loading area of ​​the test strip. The trimming length is set to three gradients: 1.5±0.1cm, 1.0±0.1cm, and 0.5±0.1cm. Subsequently, by setting 5 gradients of 0min, 40min, 30min, 15min, and 10min, the CRISPR reaction time was screened with the optimal values ​​of ssDNA concentration and LFD band length. The reaction results are shown in Figure 4 Finally, the optimal reaction conditions were selected as the trimming length of 1.5±0.1 cm and the reaction time of 40 min.

[0055] The RPA-CRISPR-LFD system optimization and the gold-labeled nanoparticle quantity optimization were used as two parallel optimization schemes for subsequent specificity experiments and sensitivity experiments. Their specificity and sensitivity were comprehensively compared to select the best optimization scheme.

[0056] 6. Detection of Pleurotus erythrocytes using RPA-CRISPR-LFD technology

[0057] The above 14 common harmful algae were used to verify the specificity of the RPA-CRISPR-LFD detection system established in the present invention, and ddH2O was selected as a template as a negative control. After extracting the algae genomic DNA, amplification was performed, and the amplification results were as follows: Figure 5 As shown in a and c, it is shown that the primers and probe sequences provided by the present invention have good specificity and do not produce positive amplification reactions with other algae.

[0058] The sensitivity of the RPA-CRISPR-LFD detection system established in the present invention was verified. The extracted genomic DNA template of P. microphylla (original concentration 1.5×10 4 pg / μL) was diluted 10 times as a reaction template. After the complete RPA-CRISPR-LFD reaction, two parallel optimization schemes, RPA-CRISPR-LFD system optimization and gold-labeled nanoparticle quantity optimization, were used for sensitivity determination, and 2% agarose gel electrophoresis was used for conventional RPA sensitivity detection.

[0059] The amplification results are as follows Figure 5 b and d. The sensitivity experiment of the genomic DNA of Micropurinola showed that the sensitivity of the optimized RPA-CRISPR-LFD system was 1.5×10 2 pg / μL, and the sensitivity of the optimized solution for the number of gold-labeled nanoparticles was 1.5×10 - 2 pg / μL. By comparison, the sensitivity of the gold-labeled nanoparticle quantity optimization scheme was 10,000 times higher than that of the RPA-CRISPR-LFD system optimization scheme, so the gold-labeled nanoparticle quantity optimization scheme was selected as the final optimization scheme.

[0060] 7. Field validation of RPA-CRISPR-LFD technology for detecting P. microphylla

[0061] The simulated field samples were water samples from the Xianbei Sea mountainous area of ​​the South China Sea. After confirming that there was no Plinia microphylla in the water samples by microscopic examination, a certain amount of Plinia microphylla cells (cell number 1.12×10 5 cells / ml), and then the genomic DNA was extracted and the sensitivity of RPA-CRISPR-LFD was tested. Figure 6 The results show that the primers provided by the present invention can detect P. microphylla in simulated water samples with a detection limit of 1.12×10 -2 cells / mL.

[0062] Real field samples were collected from Bohai Sea (14 samples), Xiangshan Bay (2 samples), Sanmen Bay (6 samples), Lianjiang (14 samples) and South China Sea (15 samples). All water samples were filtered through a 0.22 μm membrane. The sample stations almost covered China's oceans and offshore aquaculture areas, representing the distribution of P. microphylla from north to south. The test results are as follows: Figure 7 As shown in the figure, a total of 37 samples tested positive, but the overall positive results were weak and the bands were lighter. Among them, the 6 positive samples from the South China Sea showed relatively brighter bands. This shows that although P. microphylla is widely present in Chinese waters, its cell concentration is extremely low.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting Pleurotus eryngii in a water sample, characterized in that: The following steps are involved: S1: extracting DNA from the water sample; S2: amplifying the characteristic site by recombinase polymerase amplification method to obtain an RPA amplification product, wherein the sequence of the characteristic site is shown in SEQ ID NO: 2; S3: preparing a CRISPR-12a reaction system using the RPA amplification product, performing a CRISPR reaction, and obtaining a CRISPR reaction product; S4: Detecting the labeled molecules in the CRISPR reaction product.

2. The method according to claim 1, characterized in that In S2, the primer sequences used to amplify the characteristic sites are shown in SEQ ID NOs: 8 and 9.

3. The method according to claim 1, characterized in that In S3, the CRISPR-12a reaction system also includes the following components: Cas12a protein, ssDNA with labeled molecules, and crRNA.

4. The method according to claim 3, characterized in that The concentration of the ssDNA with the labeled molecule is 500 nmol / L.

5. The method according to claim 3, characterized in that: The CRISPR reaction time is 40 minutes.

6. The method according to claim 1, characterized in that In S4, the marker molecule is detected using a lateral flow test strip on which a substance capable of capturing the marker molecule is fixed.

7. The method according to claim 6, characterized in that In S4, PEG is added to the reaction product and then tested by lateral flow test strip, and the working concentration of PEG is 4.6%.

8. The method according to claim 6, characterized in that The lateral flow test strip uses gold-labeled nanoparticles as a color developer, and the content of the gold-labeled nanoparticles is 1.5×10 -2 pg / μL.

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