CN-122012673-A - ICP-MS analysis method for simultaneously detecting multiple respiratory pathogens by combining CRISPR/Cas12a with DNA nanomachines

Abstract

The invention discloses a detection method for simultaneously measuring multiple respiratory pathogens. The invention completes the simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (H1N 1) and Mycoplasma Pneumoniae (MP) in respiratory pathogens by means of a recognized excellent signal amplification tool, namely a CRISPR/Cas12a system and a DNA nano machine, an auxiliary metal element mark and an ICP-MS detection technology, and is beneficial to maintaining the respiratory system safety of whole human beings. When the complex formed by Cas12a/crRNA specifically binds with the corresponding target DNA, the trans-shearing activity of the CRISPR/Cas12a system is activated, so that the corresponding DNAzyme is sheared, the DNA nanomachines cannot operate, lanthanide metal elements on the DNA nanomachines cannot be released, and therefore ICP-MS outputs a low signal, and quantitative analysis of multiple DNAs can be realized. The method for detecting respiratory pathogens can realize multi-component simultaneous detection, reduces detection cost and sample consumption, and has the advantages of high sensitivity and good specificity.

Inventors

• LIU RUI
• WANG SIYI
• Lv ge
• HU YUELI
• CHEN CHENG
• YAN SHUGUANG
• HUANG ZILI
• YAN XUETING

Assignees

• 四川大学

Dates

Publication Date

20260512

Application Date

20241108

Claims (2)

1. 1. An ICP-MS analysis method for simultaneously detecting multiple respiratory pathogens by combining CRISPR/Cas12a with a DNA nanomachine, characterized in that: The following operation steps are carried out: (1) Preparation of lanthanide metal element 159 Tb、 165 Ho、 169 Tm labeled DNA substrate strand: a. Dissolving 1,4,7,10 tetraazacyclododecane-1, 4, 7-triacetic acid-10-maleimidoethyl acetamide (MMA-DOTA) and terbium chloride hexahydrate (TbCl 3 ·6H 2 O), holmium chloride hexahydrate (HoCl 3 ·6H 2 O) and thulium chloride hexahydrate (TmCl 3 ·6H 2 O) respectively in an ammonium acetate (NH 4 Ac) buffer solution (0.5M NH 4 Ac, pH 5.8), MMA-DOTA to a concentration of 5mM, tbCl 3 ·6H 2 O、HoCl 3 ·6H 2 O and TmCl 3 ·6H 2 O to a concentration of 10mM, storing at 4 ℃, dissolving 3 disulfide-modified respiratory pathogens corresponding DNA substrate chains (SARS-CoV-2-substrate, MP-substrate and H1N 1-substrate) in the NH 4 Ac buffer solution, respectively, and storing at 4 ℃; b. Mixing 20 mu L of 5 mu M MMA-DOTA solution with 20 mu L of 10mM Tb 3+ 、Ho 3+ 、Tm 3+ solution respectively, diluting to 200 mu L with NH 4 Ac buffer solution, and incubating at 37 ℃ for 1 hour after shaking to obtain three chelates of DOTA-Tb, DOTA-Ho and DOTA-Tm; c. 3 100. Mu.M DNA substrate strands were mixed with 10mM tris (2-carboxyethyl) phosphine (TCEP) solution, respectively, in a 1:1 volume ratio, and incubated at 37℃for 30min to reduce disulfide bonds; d. The resulting solution was purified 2 times with Amicon-3K ultrafiltration tube using wash buffer (20 mM Tris-HCl,150mM NaCl,pH7.4), then 1 time with NH 4 Ac buffer, and the remaining TCEP was removed; e. Adding 150 mu L b of the prepared chelate compound into the purified solution obtained in the step d respectively, and vibrating and incubating for 2 hours at 37 ℃ to obtain 159 Tb、 165 Ho、 169 Tm marked DNA substrate chain conjugate; f. Purifying the obtained conjugate with Amicon-3K ultrafiltration tube washing buffer for 3 times, removing excessive Tb 3+ 、Ho 3+ 、Tm 3+ metal ions, purifying with Tris-HCl buffer (20 mM Tris-HCl,150mM NaCl,pH 7.4) for 1 time, diluting the final product to 1220 mu L, and preserving at 4deg.C for later use; (2) Preparation of DNA nanomachines: a. 100. Mu.L of streptavidin-modified magnetic beads (SA-MBs) were dispersed in a binding and washing (B & W) buffer solution (5 mM Tris-HCl,1M NaCl,0.5mM EDTA,pH 7.4), and after 3 washes 500. Mu.L of the prepared 159 Tb、 165 Ho、 169 Tm-labeled DNA substrate strand with biotin modification and 500. Mu.L of 2 XB & W buffer solution (10 mM Tris-HCl,2M NaCl,1mM EDTA,pH 7.4) were added, respectively, and incubated with shaking at 25℃for 2 hours; b. The prepared DNA nanometer machine is washed for 4 times by Tris-HCl buffer solution to remove redundant substrate chains, reduce experimental background, finally diluted to 750 mu L by Tris-HCl buffer solution and preserved at 4 ℃ for standby.
2. Analytical procedure for icp-MS detection of various respiratory pathogens: a. Three respiratory pathogen DNAs (SARS-CoV-2-cDNA, MP-DNA, H1N 1-DNA) and corresponding DNAzyme (SARS-CoV-2-DNAzyme, MP-DNAzyme, H1N 1-DNAzyme) were formulated into solutions of the desired concentrations in a super clean bench with DEPC treatment water and NEBuffer r2.1 (10 mM Tris-HCl,50mM NaCl,10mM MgCl 2 , 100. Mu.g/mL Recombinant Albumin, pH 7.9); b. mu.L of three respiratory pathogen DNA at a concentration and corresponding 10. Mu.L of 20nM Cas12a/crRNA (1:3 ratio), 10. Mu.L of 100nM DNAzyme were mixed and incubated for 1 hour at 37 ℃; c. Then, the reaction is carried out for 5min at 85 ℃ by a PCR instrument, so that the activity of the Cas12a protease is lost; d. Mixing 30 mu L of inactivated solution uniformly in a new PCR tube, adding 45 mu L of Mg 2+ and 45 mu L of DNA nano machine mixed solution which is successfully prepared, and carrying out oscillation reaction for 40min at 25 ℃ to release Tb 3+ 、Ho 3+ 、Tm 3+ metal ions on the magnetic beads; e. The supernatant was separated by magnetic attraction, dispersed in 1mL of 1% HNO 3 , mixed well and sent to inductively coupled plasma mass spectrometry (ICP-MS) for analysis.

Description

ICP-MS analysis method for simultaneously detecting multiple respiratory pathogens by combining CRISPR/Cas12a with DNA nanomachines Technical Field The invention belongs to the field of detection of analytical chemistry, in particular to a nucleic acid detection technology, and particularly relates to an ICP-MS analysis method for simultaneously detecting multiple respiratory pathogens by combining CRISPR/Cas12a with a DNA nano machine. Background Respiratory tract viruses (including novel coronaviruses and influenza a viruses), bacteria, mycoplasma pneumoniae, fungi and other pathogens may cause respiratory tract infections, even pneumonia, and may cause extensive infections through contact or air transmission, severely jeopardizing public health. Respiratory tract infections caused by a variety of related variants and other pathogens have occurred since the outbreak of the new coronavirus infection epidemic, exhibiting enhanced transmissibility and pathogenicity. Early diagnosis is the best way to control pandemic due to the extremely spread of respiratory pathogens and the potential for positive asymptomatic patients. In addition, the clinical symptoms of respiratory tract infections are relatively similar, and are generally manifested as sore throat, cough, headache, etc., and it is difficult to judge the infectious pathogens by symptoms at the early stage of infection. Under the current situation, multiple and accurate respiratory pathogen detection can be developed to provide assistance in early diagnosis phases, and simultaneously reduce drug abuse and pain of patients. Real-time reverse transcription polymerase chain reaction (RT-PCR) is a gold standard diagnostic method for detecting respiratory virus infection, and although the detection method has high accuracy and specificity, the processing time, complicated equipment and complicated procedures are required. In recent years, analytical methods for simultaneous detection of multiple disease markers are gradually developed, so that the analytical time can be shortened, the operation steps can be simplified, more information can be provided, and the analytical methods are always hot spots in the fields of life sciences and nucleic acid detection. The invention can detect multiple respiratory pathogens simultaneously, thus achieving good analysis effect. Inductively coupled plasma mass spectrometry (ICP-MS) has the advantages of high resolution, low matrix effect, high sensitivity and the like, and interference of band overlapping does not exist when multiple metal elements are simultaneously analyzed, so that the inductively coupled plasma mass spectrometry (ICP-MS) is an ideal detection tool in multiple analysis research, and has been widely applied to multiple detection of biomolecules. The DNA nanomachines form double-chain starting reaction through base complementation pairing of DNAzyme and DNA substrate chains, and the DNAzyme continuously and circularly shears rA sites on the substrate chains with the aid of specific metal ions, so that signal amplification is completed. CRISPR/Cas12a can be sheared rapidly by means of trans-lytic activity up to 1250 times per second, is an effective means of generating signal amplification, and has unique sequence programmability, which has been widely used in quantitative analytical studies of biomolecules. Disclosure of Invention The invention develops a high-sensitivity analysis method for simultaneously detecting various respiratory tract pathogen related DNAs based on metal element labeling and inherent multiple detection capability of ICP-MS and combined with cascade signal amplification of a CRISPR/Cas12a system and a DNA nano machine. The principle of the invention is shown in figure 1, wherein the complex formed by the designed 3 crRNAs which are completely complementary with target DNA and Cas12a protease does not have trans-cleavage activity, and after the complex is combined with different target DNA to form a ternary complex structure, the trans-cleavage activity of a corresponding CRISPR/Cas12a system can be triggered, and the DNAzyme which is pre-introduced into the system is subjected to disordered cleavage. After the cleavage is completed, the cut DNAzyme cannot travel on the surface of the corresponding nanomachine according to a predetermined track, so that the DNA nanomachine cannot operate, and therefore the labeled lanthanide metal probe remains stably combined with the magnetic bead, and finally a lower IC-PMS signal is output. The nucleic acid sequence information used in the present invention is set forth in FIG. 2. When target DNA does not exist in the system, the CRISPR/Cas12a complex cannot show cleavage activity, DNAzyme in the system keeps complete, binds with a DNA substrate chain on the surface of a corresponding nano machine, cuts a specific site with the assistance of Mg 2+, and continues to move after releasing the DNA fragment. Therefore, the lanthanide metal element is