ICP-MS (Inductively Coupled Plasma Mass Spectrometry) analysis method for simultaneously detecting multiple respiratory pathogens by combining CRISPR / Cas12a and DNA (Deoxyribonucleic Acid) nano machine

Abstract

The invention discloses a detection method for simultaneously determining multiple respiratory pathogens. According to the present invention, with the help of the recognized excellent signal amplification tool-CRISPR / Cas12a system and the DNA nanometer machine, the metal element labeling and the ICP-MS detection technology are assisted, such that the simultaneous detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the influenza A virus (H1N1) and the mycoplasma pneumoniae (MP) in the respiratory pathogens is completed so as to easily maintain the respiratory system safety of the whole human. When a compound formed by Cas12a / crRNA is specifically combined with a corresponding target DNA, the trans-shear activity of a CRISPR / Cas12a system can be activated, so that corresponding DNAzyme can be sheared, a DNA nano machine cannot run, lanthanide series metal elements on the DNA nano machine cannot be released, ICP-MS outputs a low signal, and quantitative analysis of multiple DNAs can be realized. The method for detecting the respiratory pathogens can realize simultaneous detection of multiple components, reduces the detection cost and the sample consumption, and has the advantages of high sensitivity and good specificity.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry, specifically relating to nucleic acid detection technology, and particularly to an ICP-MS analytical method for simultaneously detecting multiple respiratory pathogens by combining CRISPR / Cas12a with DNA nanomachines. Background Technology

[0002] Respiratory viruses (including the novel coronavirus and influenza A virus), bacteria, Mycoplasma pneumoniae, and fungi can all cause respiratory infections, even pneumonia, leading to widespread infection through contact or airborne transmission and seriously endangering public health. Since the outbreak of the novel coronavirus, respiratory infections caused by various related variants and other pathogens have occurred frequently, exhibiting enhanced transmissibility and pathogenicity. Because respiratory pathogens are highly contagious and there is a possibility of asymptomatic positive cases, early diagnosis is the best way to control large-scale epidemics. Furthermore, the clinical symptoms of respiratory infections are quite similar, generally manifesting as sore throat, cough, and headache, making it difficult to identify the infecting pathogen based on symptoms alone in the early stages of infection. In the current situation, developing multiplex and accurate respiratory pathogen tests can help in the early diagnostic stage, while also reducing drug overuse and alleviating patient suffering.

[0003] Real-time reverse transcription polymerase chain reaction (RT-PCR) is the gold standard diagnostic method for detecting respiratory viral infections. Although this method has high accuracy and specificity, it is time-consuming, requires complex equipment, and involves cumbersome procedures. In recent years, analytical methods for the simultaneous detection of multiple disease biomarkers have gradually developed, which can shorten analysis time, simplify operation steps, and provide more information. This has always been a hot topic in the life sciences and nucleic acid detection fields. This invention enables the simultaneous detection of multiple respiratory pathogens, achieving good analytical results. Inductively coupled plasma mass spectrometry (ICP-MS) has advantages such as high resolution, low matrix effect, and high sensitivity. When analyzing multiple metal elements simultaneously, there is no interference from overlapping bands, making it an ideal detection tool for multiplex analysis and widely used in the multiplex detection of biomolecules. DNA nanomachines form a double-stranded initiation reaction through complementary base pairing between DNAzymes and DNA substrate chains. With the assistance of specific metal ions, the DNAzyme continuously cleaves rA sites on the substrate chain along the substrate chain, completing signal amplification. CRISPR / Cas12a can perform rapid cleavage with up to 1250 trans-cleavage activities per second, making it an effective means of generating signal amplification. It also has unique sequence programmability and has been widely used in quantitative analysis research of biomolecules. Summary of the Invention

[0004] Based on the metal element labeling and the inherent multiplex detection capability of ICP-MS, this invention combines the cascaded signal amplification of the CRISPR / Cas12a system and DNA nanomachines to develop a highly sensitive analytical method for the simultaneous detection of DNA associated with multiple respiratory pathogens.

[0005] The principle of this invention is as follows Figure 1 As shown, the complexes formed by the three designed crRNAs, which are completely complementary to the target DNA, and the Cas12a protease do not possess trans-cleavage activity. However, after binding to different target DNAs to form ternary complexes, they can trigger the corresponding trans-cleavage activity of the CRISPR / Cas12a system, resulting in disordered cleavage of the pre-introduced DNAzyme in the system. After cleavage, the cleaved DNAzyme cannot travel along the predetermined trajectory on the surface of the corresponding nanomachine, causing the DNA nanomachine to malfunction. Therefore, the labeled lanthanide metal probes remain stably bound to the magnetic beads, ultimately outputting a low IC-PMS signal.

[0006] The nucleic acid sequence information used in this invention is Figure 2 Listed in.

[0007] In the absence of target DNA in the system, the CRISPR / Cas12a complex cannot exhibit cleavage activity; the DNAzyme in the system remains intact, binds to the DNA substrate strand on the surface of the corresponding nanomachine, and is contained within Mg. 2+ With the aid of magnetic beads, specific sites are cleaved, releasing DNA fragments which then continue their movement. Therefore, lanthanide metal elements continuously detach from the surface of the magnetic beads and enter the solution, ultimately producing a high ICP-MS signal. The signal strength of the lanthanide metal elements is directly related to the concentration of the target DNA in the system; therefore, the output ICP-MS signal can be used for quantitative analysis of the target analyte.

[0008] In this invention, the practical application capability of the constructed multiplex DNA analysis method is verified by taking respiratory pathogen-associated severe acute respiratory syndrome coronavirus type 2 cDNA (SARS-CoV-2-cDNA), influenza A H1N1 virus DNA (H1N1-DNA), and mycoplasma pneumoniae DNA (MP-DNA) as examples.

[0009] To achieve the above objectives, the present invention provides the following technical solution: The principle of the lanthanide metal element-labeled DNA substrate chain prepared in this invention is as follows: Figure 3As shown, it mainly consists of two parts: a lanthanide metal chelate and a DNA substrate chain (DNA-substrate) modified with a thiol group at the 5' end and biotin at the 3' end. To bind the lanthanide metal chelate and the DNA substrate chain, the bifunctional chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid-10-maleimide ethylacetamide (MMA-DOTA) is used to bind with the lanthanide metal ions. The resulting lanthanide metal chelate DOTA-Tb / Ho / Tm can form covalent bonds with the thiol-modified DNA substrate chains (SARS-CoV-2-substrate, MP-substrate, H1N1-substrate), respectively, thus preparing lanthanide metal-DNA conjugates. The biotin modified at the 3' end of the DNA substrate chain is used to connect streptavidin-modified magnetic beads (SA-MBs) to construct DNA nanomachines.

[0010] The DNA nanomachines used in this invention are prepared by mixing a lanthanide metal element-labeled and biotin-modified DNA substrate chain (DNA-Tb / Ho / Tm) with streptavidin-modified magnetic beads (SA-MBs), shaking the mixture at 25°C, and then washing and purifying it to obtain the assembled DNA nanomachines.

[0011] The method for ICP-MS analysis based on the CRISPR / Cas12a system and DNA nanomachines is as follows: 20 μL of DNA from three respiratory pathogens at specific concentrations and 10 μL of 20 nM Cas12a / crRNA (ratio 1:3) were respectively used to form a ternary complex to activate the trans-cleavage activity of the CRISPR / Cas12a system. Then, 10 μL of 100 nM DNAzyme was added and the mixture was incubated at 37°C for 1 hour. Next, a PCR reaction was performed at 85°C for 5 minutes to deactivate the Cas12a protease activity. 30 μL of the inactivated solution was then mixed thoroughly in a new PCR tube, and 45 μL of MgSO₄ was added. 2+ The mixture of the prepared DNA nanomachines and 45 μL was agitated at 25 °C for 40 min to release Tb from the magnetic beads. 3+ Ho 3+ Tm 3+ Metal ions were removed; the supernatant was separated by magnetic adsorption, dispersed in 1 mL of 1% HNO3, mixed thoroughly, and then sent to ICP-MS for analysis. By establishing linear curves for ICP-MS signal values ​​of different concentrations of target DNA, quantitative analysis of respiratory pathogen-related DNA could be performed.

[0012] This invention develops an ICP-MS analytical method for the simultaneous detection of multiple respiratory pathogens based on the CRISPR / Cas12a system and DNA nanomachines. Researchers in the field can refer to this text and appropriately modify the process parameters to achieve the same result. Unless otherwise specified, the experimental methods used in the following implementation steps are conventional methods. Unless otherwise specified, the materials and reagents used in the following implementation schemes are commercially available. The water used in the following implementation schemes is high-purity deionized water (18.2 MΩ·cm). -1 In the examples below, none of the samples were purified before use; This invention is carried out according to the following specific steps: a. Lanthanide metals 159 Tb, 165 Ho、 169 Preparation of Tm-labeled DNA substrate strands a1 This invention employs the bifunctional chelating agent MMA-DOTA and lanthanide metal elements. 159 Tb, 165 Ho、 169 Tm was chelated. 20 μL of 5 μM MMA-DOTA solution was chelated with 20 μL of 10 mM Tb. 3+ Ho 3+ Tm 3+ The solutions were mixed and then diluted to 200 μL with NH4Ac buffer solution. After shaking, the mixture was incubated at 37°C for 1 hour to obtain three chelates: DOTA-Tb, DOTA-Ho, and DOTA-Tm. Before crosslinking with lanthanide metal chelates, the α2-thiol-modified DNA substrate chains require the reduction of disulfide bonds. Three 100 μM DNA substrate chains were mixed with 10 mM tris(2-carboxyethyl)phosphine (TCEP) solution at a 1:1 volume ratio and incubated at 37°C with shaking for 30 min to break the disulfide bonds between DNA sequences. The resulting solutions were then purified by washing twice with washing buffer and once with NH4Ac buffer solution through an Amicon-3K ultrafiltration tube at 14000 rpm for 15 min to remove excess TCEP. a3: Mix the solutions obtained from a1 and a2, and incubate with shaking at 37°C for 2 hours. After the reaction is complete, the resulting solution is subjected to a reaction at 14000 rpm for 15 min / cycle. 159 Tb, 165 Ho、 169 The Tm-labeled DNA substrate strand conjugate was purified three times by washing with washing buffer using an Amicon-3K ultrafiltration tube to remove excess Tb. 3+ Ho 3+ Tm 3+The metal ions were purified once more with Tris-HCl buffer, and the final product was diluted to a final volume of 1220 μL and stored at 4°C for later use. b. Fabrication of DNA nanomachines b1 Lanthanide metals 159 Tb, 165 Ho、 169 DNA nanomachines were constructed by loading Tm-labeled DNA substrate strands onto SA-MBs. 100 μL of streptavidin-modified magnetic beads (SA-MBs) were dispersed in a binding and washing (B&W) buffer solution. After washing three times, 500 μL of the prepared biotin-modified... 159 Tb, 165 Ho、 169 Tm-labeled DNA orbital strands and 500 μL of 2×B&W buffer solution were incubated with shaking at 25°C for 2 hours. b2 The DNA nanomachines prepared under the condition of 14000 rpm / 15 min / b2 were washed and purified 4 times with Tris-HCl buffer in Amicon-3K ultrafiltration tube to remove excess DNA substrate chains and reduce experimental background. Finally, they were diluted with Tris-HCl buffer to 750 μL and stored at 4℃ for later use. c. Analytical procedures for respiratory pathogen-associated DNA in actual samples Human serum samples and pharyngeal swabs were selected as actual samples for testing. The actual samples were centrifuged at 8000 rpm for 5 min, then the supernatant was collected and diluted 10-fold. The target concentration of DNA was added and mixed, followed by the corresponding Cas12a / crRNA and DNAzyme. The mixture was incubated at 37°C for 1 hour, then reacted at 85°C for 5 min. Mg was then added. 2+ The solution and the pre-prepared DNA nanomachine mixture were reacted with shaking at 25°C for 40 min. After the reaction was complete, the solution was placed on a magnetic rack and allowed to clarify. Then, 160 μL of the supernatant was aspirated and dispersed in 1 mL of 1% HNO3. After thorough mixing, the solution was analyzed by ICP-MS.

[0013] The present invention has the following beneficial effects: This invention develops a method for detecting various respiratory pathogens. It utilizes crRNA to specifically recognize and bind to target DNA, forming a ternary complex with the Cas12a protease. This complex activates the trans-cleavage activity of the CRISPR / Cas12a system, efficiently cleaving pre-introduced DNAzymes. The fragmented DNAzymes cannot travel on the surface of the DNA nanomachines, ultimately presenting the target DNA concentration information as a low metal ion signal on ICP-MS. In the absence of target DNA, the DNAzymes remain intact and travel along tracks designed on the nanomachine surface, releasing a large number of metal tags. The concentration information of various DNA targets is thus converted into lanthanide metal signal intensity on ICP-MS through a metal element labeling strategy, enabling quantitative analysis of multi-component DNA.

[0014] In this invention, the binding arm of the DNAzyme hybridizes with the DNA substrate strand on the surface of the nanomachine without any mutual interference.

[0015] In this invention, ICP-MS can simultaneously detect... 159 Tb, 165 Ho and 169 The three lanthanide metals Tm do not exhibit spectral band overlap.

[0016] This invention validates the analytical capabilities of the construction strategy using DNA associated with various respiratory pathogens as examples. The cascade amplification analysis in this invention improves detection sensitivity, and the multiplex detection strategy reduces sample consumption, shortens analysis time, and provides more information, making it promising for clinical diagnosis of various disease biomarkers. Attached Figure Description

[0017] Figure 1 This is a schematic diagram illustrating the principle of the analytical method of the present invention.

[0018] Figure 2 This refers to the nucleic acid sequence information used in the analytical method of this invention.

[0019] Figure 3 This is a schematic diagram illustrating the synthesis principle of DOTA-Tb / Ho / Tm in the analytical method of this invention.

[0020] Figure 4 This is a feasibility analysis diagram for the analytical method of this invention.

[0021] Figure 5 This is a linear analysis graph showing the detection of respiratory pathogen-related DNA using the analytical method of this invention.

[0022] Figure 6 This is a cross-reactivity analysis diagram of the analytical method of the present invention.

[0023] Figure 7 This is a selective analysis diagram of the analytical method of the present invention. Detailed Implementation

[0024] The following description, in conjunction with the accompanying drawings, further illustrates the present invention, but the analytical method is not limited to the following embodiments: Embodiment 1: Feasibility Study of the Analytical Method of the Present Invention a. To verify the feasibility of the method, the shearing ability of the DNAzyme was verified by 12% non-denaturing polyacrylamide gel electrophoresis (PAGE), using the SARS-CoV-2-cDNA fraction as an example. The PAGE experiment was performed in TBE buffer at a constant voltage of 80V for 2 hours, and the gel was imaged using Fluorchem M. Figure 4 As shown, lanes 1-3 contain DNA standards, DNA zymes, and corresponding DNA substrate strands, respectively; lane 4 contains DNA zymes and substrate strands; and lane 5 contains DNA zymes, DNA substrate strands, and Mg. 2+ Lane 4 exhibits DNAzyme and DNA substrate strand bands corresponding to those in lanes 2 and 3, with the addition of Mg 2+ The DNAzyme cleaves the orbital strand into smaller DNA fragments, resulting in the disappearance of the DNA substrate band in lane 5 and the appearance of smaller DNA fragments, while the DNAzyme band remains. PAGE experiments fully demonstrate the cleavage capability of the DNAzyme.

[0025] Example 2: Analytical Performance of the Method for Detecting Respiratory Pathogen-Related DNA of the Invention In the analytical method of the present invention, respiratory pathogen-related DNA of different concentrations are added to the reaction system and mixed, and reacted at 25°C for 40 min. After the reaction, the solution is placed on a magnetic rack for magnetic separation, and the supernatant is dispersed in 1 mL of 1% HNO3, mixed thoroughly, and then sent to ICP-MS for analysis. Figure 5 As can be seen, taking the logarithm of a portion of the concentration signal reveals a good logarithmic linear relationship between DNA concentration and the ICP-MS signal intensity of lanthanide metals. 159 The ICP-MS signal of Tb was fitted to the concentration of SARS-CoV-2-cDNA (e.g., Figure 5 A) The linear equation in the concentration range of 10 pM to 200 pM is Y = -13684lgX + 32342 (where Y is... 159 The ICPMS signal intensity of Tb (where X is the concentration of SARS-CoV-2-cDNA), and the square of the correlation coefficient R. 2 The value was 0.98, and the limit of detection (LOD, 3σ / k) was 1.4 pM. Meanwhile, it was also found that... 165 The ICP-MS signal of Ho was fitted with the concentration of MP-DNA (e.g., Figure 5 B), the linear equation in the concentration range of 10 pM to 200 pM is Y = -7111lgX + 17288 (where Y is... 165 Ho's ICP-MS signal intensity (where X is the concentration of MP-DNA), and the square of the correlation coefficient R. 2 The value was 0.98, and the detection limit was 1.9 pM. For 169 The ICP-MS signal of Tm was also fitted to the H1N1-DNA concentration (e.g. Figure 5 C), the linear equation in the concentration range of 10 pM to 100 pM is Y = -19370lgX + 39321 (where Y is... 169 The ICP-MS signal intensity of Tm (where X is the concentration of H1N1-DNA), and the square of the correlation coefficient R. 2 The sensitivity was 0.99, and the detection limit was 1.5 pM. This indicates that the multi-component detection mass spectrometry analysis method using the CRISPR / Cas12a system coupled with DNA nanomachines in this invention theoretically has good sensitivity and better analytical performance.

[0026] Example 3: Investigation of the cross-reactivity of the analytical method of the present invention In the analytical method of this invention, samples containing different target DNAs were used to conduct experiments to further explore the cross-reactivity performance of the constructed biosensor. The test samples were: blank sample (Ⅰ), SARS-CoV-2 cDNA (Ⅱ), MPDNA (Ⅲ), H1N1 DNA (Ⅳ), SARS-CoV-2 cDNA and MP DNA (Ⅴ), SARS-CoV-2 cDNA and H1N1 DNA (Ⅵ), MP DNA and H1N1 DNA (Ⅶ), and SARS-CoV-2 cDNA, MP DNA, and H1N1 DNA (Ⅷ). The test results are as follows: Figure 6 As shown, this demonstrates that there is no cross-reaction in the biosensor we constructed for the simultaneous detection of multiple DNA components, and the target DNA can correspond to the ICPMS signal, thus possessing the ability to detect multiple DNA components simultaneously.

[0027] Example 4: Selectivity Exploration of the Analytical Method of the Invention This invention's analytical method mixes several different disease DNAs with target DNA to simulate a complex real-world environment, performing simultaneous detection to evaluate the specificity of the strategy. The concentrations of the target DNA (SARS-CoV-2-cDNA, MP-DNA, and H1N1-DNA) are 150 pM, 150 pM, and 80 pM, respectively, while the concentrations of the interfering agents (influenza B virus, human immunodeficiency virus, hepatitis A virus, and respiratory syncytial virus) are 10 times the concentration of the target DNA. Experimental results are as follows: Figure 7 As shown, the signals of influenza B, HIV, HAV, and RSV were not significantly different from those of the blank sample, while the signal intensity of the target DNA was significantly reduced. These results indicate that the target DNA can be effectively analyzed even in the presence of other high concentrations of disease DNA, demonstrating that the analytical method of this invention has high selectivity for the target DNA.

Claims

1. An ICP-MS analytical method for the simultaneous detection of multiple respiratory pathogens using CRISPR / Cas12a coupled with DNA nanomachines, characterized in that: The following steps should be followed: (1) Lanthanide metals 159 Tb, 165 Ho、 169 Preparation of Tm-labeled DNA substrate strands: a. Dissolve 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid-10-maleimide ethyl acetamide (MMA-DOTA) and terbium chloride hexahydrate (TbCl3·6H2O), holmium chloride hexahydrate (HoCl3·6H2O), and thulium chloride hexahydrate (TmCl3·6H2O) respectively in ammonium acetate (NH4Ac) buffer solution (0.5M NH4Ac, pH). In 5.8), MMA-DOTA was prepared at a concentration of 5 mM, and TbCl3·6H2O, HoCl3·6H2O, and TmCl3·6H2O were prepared at a concentration of 10 mM and stored at 4 °C. The corresponding DNA substrate chains of the three disulfide-modified respiratory pathogens (SARS-CoV-2-substrate, MP-substrate, and H1N1-substrate) were dissolved in NH4Ac buffer solution, and the concentrations were all prepared at 100 μM and stored at 4 °C. b. Take 20 μL of 5 μM MMA-DOTA solution and react it with 20 μL of 10 mM Tb solution. 3+ Ho 3+ Tm 3+ The solutions were mixed and then diluted to 200 μL with NH4Ac buffer solution. After shaking, the mixture was incubated at 37°C for 1 hour to obtain three chelates: DOTA-Tb, DOTA-Ho, and DOTA-Tm. c. Mix the three 100 μM DNA substrate chains with 10 mM tris(2-carboxyethyl)phosphine (TCEP) solution at a volume ratio of 1:1 and incubate at 37 °C for 30 min to reduce disulfide bonds. d. The resulting solution was purified twice with washing buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.4) using an Amicon-3K ultrafiltration tube, and then purified once with NH4Ac buffer to remove the remaining TCEP. e. Add 150 μL of the chelate prepared by b to the purified solution obtained by d, and incubate with shaking at 37°C for 2 hours to obtain... 159 Tb, 165 Ho、 169 Tm-labeled DNA substrate strand conjugates; f. The obtained conjugate was purified three times with washing buffer using an Amicon-3K ultrafiltration tube to remove excess Tb. 3+ Ho 3+ Tm 3+ The metal ions were purified once again with Tris-HCl buffer (20mM Tris-HCl, 150mM NaCl, pH 7.4), and the final product was diluted to 1220μL and stored at 4℃ for later use. (2) Fabrication of DNA nanomachines: a. Disperse 100 μL of streptavidin-modified magnetic beads (SA-MBs) in a binding and washing (B&W) buffer solution (5 mM Tris-HCl, 1 M NaCl, 0.5 mM EDTA, pH 7.4), wash three times, and then add 500 μL of the prepared biotin-modified... 159 Tb, 165 Ho、 169 Tm-labeled DNA substrate strands and 500 μL of 2×B&W buffer solution (10 mM Tris-HCl, 2 M NaCl, 1 mM EDTA, pH 7.4) were incubated at 25 °C with shaking for 2 hours. b. The successfully prepared DNA nanomachines were washed four times with Tris-HCl buffer to remove excess substrate strands and reduce experimental background; finally, they were diluted to 750 μL with Tris-HCl buffer and stored at 4°C for later use.

2. ICP-MS analysis procedure for detecting multiple respiratory pathogens: a. In a clean bench, use DEPC-treated water and NEBuffer r2.1 (10mM Tris-HCl, 50mM NaCl, 10mM MgCl2, 100μg / mL Recombinant Albumin, pH 7.9) to prepare solutions of three respiratory pathogen DNAs (SARS-CoV-2-cDNA, MP-DNA, H1N1-DNA) and their corresponding DNAzymes (SARS-CoV-2-DNAzyme, MP-DNAzyme, H1N1-DNAzyme) and crRNAs (SARS-CoV-2-crRNA, MP-crRNA, H1N1-crRNA) to the required concentrations. b. Mix 20 μL of DNA from three respiratory pathogens at a certain concentration with 10 μL of 20 nM Cas12a / crRNA (ratio 1:3) and 10 μL of 100 nM DNAzyme respectively, and incubate at 37°C for 1 hour. c. Then, use a PCR instrument to react at 85℃ for 5 minutes to deactivate the Cas12a protease activity; d. Take 30 μL of the inactivated solution from each tube, mix thoroughly, and add 45 μL of Mg. 2+ The mixture of the prepared DNA nanomachines and 45 μL was agitated at 25 °C for 40 min to release Tb from the magnetic beads. 3+ Ho 3+ Tm 3+ Metal ions; e. The supernatant was separated by magnetic attraction, dispersed in 1 mL of 1% HNO3, mixed evenly, and then sent to inductively coupled plasma mass spectrometry (ICP-MS) for analysis.

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