Rapid and high-sensitivity mycobacterium tuberculosis drug-resistant strain identification and Raman quantitative analysis method

Abstract

The invention discloses a rapid and high-sensitivity mycobacterium tuberculosis drug-resistant strain identification and Raman quantitative analysis method, which comprises the following steps of: firstly, constructing a semiconductor surface enhanced Raman substrate with high stability and high enhancement factors by adopting a self-assembly method to amplify a Raman signal of a signal molecule to be detected, and providing high sensitivity for analysis and detection; secondly, designing and synthesizing a plurality of mycobacterium tuberculosis nucleic acid recognition units with single base recognition capability according to mutation sites of key drug-resistant genes of the binding bacilli; appropriate Raman signal molecules and internal reference analysis are selected, a high-selectivity and high-sensitivity enzyme-free nucleic acid amplification process-free rapid mycobacterium tuberculosis drug-resistant gene Raman analysis probe is constructed, and a brand new mycobacterium tuberculosis drug-resistant strain identification and Raman quantitative analysis chip and method are constructed through spatial arrangement design. And finally, normal-temperature long-term preservation of the analysis reagent, low-cost rapid typing of common binding bacterium drug-resistant strains, and high-sensitivity and high-selectivity quantitative analysis detection are realized.

Description

Technical Field

[0001] The invention discloses a rapid and highly sensitive identification and Raman quantitative analysis method for drug-resistant Bacillus strains, and belongs to the technical field of material synthesis and biosensor technology. Background Art

[0002] According to the "2022 Global Tuberculosis Report", the estimated number of new tuberculosis patients in the world in 2021 is 10.6 million, with an incidence rate of 134 / 100,000. The estimated number of new tuberculosis patients in China in 2021 is 780,000, and my country ranks third in the estimated number of tuberculosis cases among the 30 countries with a high burden of tuberculosis. There are many carriers, many patients, and many deaths in China. Therefore, in order to more effectively curb the spread of multiple tuberculosis bacteria, rapid and sensitive detection of multiple pathogens is imminent. At present, the common traditional detection methods for pathogens such as tuberculosis infection are mainly: ① asking medical history and symptoms, physical examination; ② chest X-ray, CT and other imaging examinations; ③ sputum smear acid-fast staining to check acid-fast bacilli; ④ mycobacterium isolation and culture and bacterial species identification; ⑤ molecular biological detection: sputum smear examination is a simple, rapid, easy and reliable method, but it is not sensitive. A positive sputum smear test can only indicate that the sputum contains acid-fast bacilli, and cannot distinguish whether it is Mycobacterium tuberculosis or non-tuberculous mycobacteria. Mycobacterium isolation and culture is the gold standard for diagnosing tuberculosis, but the in vitro culture cycle is long, generally 2-8 weeks, and the positive detection rate is not high, which brings certain difficulties to clinical early diagnosis and determination of drug treatment. Polymerase chain reaction (PCR) technology for detecting Mycobacterium tuberculosis can greatly improve the detection rate and specificity of Mycobacterium tuberculosis, but the traditional PCR method is prone to false positives and false negatives due to low specificity or inhibitory factors, specimen pretreatment, DNA extraction and improper operation methods.

[0003] More importantly, with the large-scale use of drugs to treat tuberculosis, Mycobacterium tuberculosis has gradually evolved into a variety of drug-resistant strains of varying degrees, such as the common: multidrug-resistant tuberculosis (MDR-TB) that is resistant to two major anti-tuberculosis drugs, isoniazid and rifampicin; delayed drug-resistant tuberculosis (DDR-TB) that is resistant to anti-tuberculosis drugs such as isoniazid, rifampicin and / or pyrazinamide; extremely drug-resistant tuberculosis (XDR-TB) that is resistant to two or more second-line anti-tuberculosis drugs (such as amikacin, moxifloxacin, ciprofloxacin, kanamycin, etc.) in addition to isoniazid and rifampicin; and all drug-resistant tuberculosis (TDR-TB) that is resistant to all first-line and second-line anti-tuberculosis drugs. The emergence of these drug-resistant strains poses a major challenge to the treatment of tuberculosis. Studies have shown that the main reason for the drug resistance of Mycobacterium tuberculosis is the mutation of its specific gene sequence. It has been found that the mutation of multiple Mycobacterium tuberculosis gene sites, including rpoB, katG, gyrA, gyrB and inhA, is significantly associated with drug resistance. Therefore, the development of new methods for specific gene detection targeting specific mutation sites of Mycobacterium tuberculosis is of great significance for the identification and quantitative analysis of drug-resistant strains of Mycobacterium tuberculosis.

[0004] Raman spectroscopy is a method of quantitative spectral analysis based on the Raman spectral signal of the probe molecule and the signal intensity and position information. Its fast analysis response speed and narrow half-peak width make it very suitable for constructing multi-channel and rapid analysis probes to achieve high-throughput rapid analysis of specific biomarkers. More importantly, surface enhanced Raman spectroscopy (SERS) is an ultra-sensitive analysis and detection technology that uses the interaction between nanomaterials and molecules on the surface of materials to enhance the Raman signal of molecules. Its enhancement factor can reach up to 10 10 Above. Therefore, SERS-based analysis techniques usually have extremely high sensitivity and rapid response, and can realize the analysis and detection of trace substances in a variety of environments, while effectively avoiding the nucleic acid amplification process in the RT-PCR analysis method, greatly saving economic costs and time costs. In addition, in terms of the selection of SERS substrate materials, compared with traditional noble metal nanomaterials such as Au, Ag, Cu, etc., semiconductor nanomaterial substrates have greater advantages in analysis selectivity, signal stability and cost. The construction of SERS substrate materials and Mycobacterium tuberculosis Raman analysis probes based on semiconductor nanomaterials plays an important role in realizing rapid, accurate, sensitive identification and analysis of Mycobacterium tuberculosis and reducing costs. Summary of the invention

[0005] The purpose of the present invention is to invent a multi-channel Mycobacterium tuberculosis resistance gene Raman analysis chip using semiconductor nanomaterial-Cu2O, which has the advantages of fast analysis speed, high molecular selectivity, sensitive detection, low price, easy storage and the like.

[0006] Compared with the prior art, the core invention of the present invention is that a semiconductor nanoarray is first prepared as a surface enhanced Raman spectroscopy substrate material, and based on this, a new multi-channel Mycobacterium tuberculosis resistance gene Raman analysis chip with high selectivity, high sensitivity, low price and easy storage is further designed and prepared, which realizes the simultaneous, sensitive and rapid analysis and detection of multiple Mycobacterium tuberculosis resistant strains.

[0007] The present invention first provides a method for preparing a novel surface enhanced Raman spectroscopy (SERS) substrate material, comprising the following steps:

[0008] (1) using copper acetate (CuAc2), ultrapure water, and o-anisidine mixed solution as reactants to prepare Cu2O nanowires through a hydrothermal reaction;

[0009] (2) Based on the Cu2O nanowires obtained by centrifugation in step (1), a SERS substrate material is constructed on the surface of a solid substrate through surface modification, oil / water interface self-assembly, heating and drying, and the like.

[0010] In step (1), the dosage ratio of CuAc2, ultrapure water and o-anisidine is 0.05g-0.2g:30-50mL:0.05-0.2mol / L; preferably, 0.1g:50mL:0.1mol / L.

[0011] In step (1), the solution is mixed for 15 min to 45 min; preferably, for 30 min.

[0012] In step (1), the volume of the hydrothermal reaction kettle is 50 mL or 100 mL; preferably, 50 mL.

[0013] In step (1), the temperature of the hydrothermal reaction is 160°C-200°C; preferably, 180°C.

[0014] In step (1), the hydrothermal reaction time is 8h-15h; preferably, 10h.

[0015] In step (2), the centrifugal rotation speed is 3000rpm-8000rpm; preferably, it is 5000rpm.

[0016] In step (2), the centrifugation time is 3 min-10 min; preferably, 5 min.

[0017] In step (2), the surface modification agent of the Cu2O nanowires is one of polyvinyl pyrrolidone (PVP), polyvinyl chloride (PVC), polystyrene (PS), etc.; preferably, it is polystyrene (PS).

[0018] Wherein, the modification group of the surface modification active agent is thiol (-SH), amino (-NH2), carboxyl (-COOH); preferably, it is amino (-NH2).

[0019] The concentration of the surface modification active agent is 1 mM-50 mM; preferably, 10 mM.

[0020] In step (2), the molecular weight of the surface modification active agent used for assembling the Cu2O nanowires is one of 100-1000000; preferably, it is 100000.

[0021] In step (2), the amount of Cu2O nanowires used is 10-100 μg; preferably, 20 μg.

[0022] In step (2), the solvent used for surface modification of the Cu2O nanowires is ultrapure water or anhydrous ethanol, etc.; preferably, it is ultrapure water.

[0023] In step (2), the time used for the surface modification process of the Cu2O nanowires is 8-15 hours; preferably, 12 hours.

[0024] In step (2), the temperature used in the Cu2O nanowire surface modification process is 20-40°C; preferably, 30°C.

[0025] In step (2), the oil / water interface is one of water / chloroform, water / cyclohexane, water / dichloromethane, etc.; preferably, water / cyclohexane.

[0026] In step (2), the oil / water ratio at the oil / water interface is 1:0.5-1:3; preferably, 1:1.

[0027] In step (2), the total volume of the oil / water dispersion solvent is 0.5-2 mL; preferably, 1 mL.

[0028] In step (2), the drying temperature is 40°C-80°C; preferably, 60°C.

[0029] In step (2), the drying time is 10 min-40 min; preferably, 20 min.

[0030] In one specific embodiment, 0.1g CuAc2 is dissolved in 50mL ultrapure water, and o-anisidine is added thereto at a concentration of 0.1mol / L. After the mixture is fully stirred for 30min, it is transferred to a 50mL hydrothermal reactor and kept in an oven at 180°C for 10h. Then, the hydrothermal reactor is naturally cooled to room temperature, the dispersion is transferred to a centrifuge tube, and then centrifuged at 5000rpm to obtain a precipitate. Further, amino-modified polystyrene (NH2-PS) with a molecular weight of 100,000 is dissolved in 5mL ultrapure water to prepare a 10mM NH2-PS solution, to which 30μg Cu2O nanowires are added as a precipitate, and stirring is continued at 30°C for 12h. Subsequently, the resulting nanowires are fully dispersed in 500μL cyclohexane and transferred to a 2cm 2 Then, 500 μL of ultrapure water was dripped onto the surface of the single crystal silicon substrate, and after the interface was stabilized, it was placed in a dry environment and slowly evaporated in a 60°C oven to obtain a Cu2O nano-SERS substrate.

[0031] The present invention also provides a semiconductor SERS substrate material prepared by the above method.

[0032] The present invention also provides the use of the semiconductor SERS substrate material in constructing a SERS analysis probe and an analysis chip for Mycobacterium tuberculosis drug-resistant genes, and in the analysis of Mycobacterium tuberculosis drug-resistant genes.

[0033] The present invention also provides a method for preparing a tuberculosis resistance gene SERS analysis probe, the core of which is to design a tuberculosis resistance gene nucleic acid sequence as a recognition unit based on the tuberculosis resistance gene nucleic acid sequence, use the previously constructed semiconductor SERS substrate material as a signal amplification unit, use the corresponding nucleic acid signal sequence as a signal unit, modify the Raman signal molecule and the reference molecule respectively, use a coupling agent to achieve signal sequence-molecule modification and substrate-recognition sequence modification, and establish a tuberculosis resistance gene SERS analysis probe. The specific steps are as follows:

[0034] Based on the above-mentioned semiconductor SERS substrate material, a nucleic acid recognition probe is designed for a specific Mycobacterium tuberculosis resistance gene sequence, and a Mycobacterium tuberculosis resistance gene SERS analysis probe is constructed by modifying the signal molecule and the reference molecule.

[0035] The tuberculosis resistance gene nucleic acid gene sequence is one of the tuberculosis gyrA, gyrB, rpoB, intA and katG sequences; preferably, it is the rpoB gene.

[0036] Wherein, the nucleic acid recognition sequence and the nucleic acid signal sequence are DNA molecules or RNA molecules; preferably, they are DNA molecules.

[0037] Wherein, the number of bases of the nucleic acid recognition sequence is 35-40 nt; preferably, 37 nt.

[0038] Wherein, the number of bases of the nucleic acid signal sequence is 6-10 nt; preferably, 8 nt.

[0039] Wherein, the modification group at the 5' end of the nucleic acid recognition sequence is one of an amino group, a thiol group, a hydroxyl group, a carboxylic acid group, etc.; preferably, it is a thiol group.

[0040] The modification group at the 3' end of the nucleic acid signal sequence is one of an amino group, a thiol group, a hydroxyl group, a carboxylic acid group, etc.; preferably, it is an amino group.

[0041] Wherein, the base sequence of the nucleic acid recognition sequence is one of the following four types:

[0042] Sequence 1: 5'-SH-GCC AAC TGGAGG CCC CAG CGC CAACAG TCG GCG CTT T-3' (SEQ IDNO.1)

[0043] Sequence 2: 5'-SH-GCC GTCATG TAC CTC GAT GCC GGT GGT GAT CGC GTC T-3' (SEQID NO. 2)

[0044] Sequence 3: 5'-SH-GCC ACCAAT GAT GCG CAC CAG GGT GTC GAT GAT CGAT-3' (SEQ IDNO.3)

[0045] Sequence 4: 5'-SH-GCCACT GGTAGC CTGAAC TTC GGG GTT CTT TAG CAC T-3' (SEQ IDNO.4)

[0046] Preferably, it is sequence 1 (SEQ ID NO.1);

[0047] Wherein, the base sequence of the nucleic acid signal sequence is one of the following four types:

[0048] 5′-CCAGTT GG-NH2-3′;

[0049] 5'-CAT GAC GG-NH2-3'

[0050] 5'-ATT GGT GG-NH2-3'

[0051] 5'-ACCAGT GG-NH2-3'

[0052] Preferably, it is 5'-CCAGTT GG-NH2-3';

[0053] Among them, the Raman signal molecule is one of perylenetetracarboxylic dianhydride (PTCDA), rhodamine 6G (R6G), rhodamine B (RhB), cyanine and its derivatives, tetracyanoquinodimethane (TCNQ) and its derivatives, bathocuproine (BCP) and its derivatives, phenylacetylene and its derivatives, etc.; preferably, it is perylenetetracarboxylic dianhydride (PTCDA).

[0054] Wherein, the Raman reference molecule is one of perylenetetracarboxylic dianhydride (PTCDA), rhodamine 6G (R6G), rhodamine B (RhB), cyanine and its derivatives, tetracyanoquinodimethane (TCNQ) and its derivatives, bathocuproine (BCP) and its derivatives, phenylacetylene and its derivatives, etc.; preferably, it is tetracyanoquinodimethane (TCNQ).

[0055] Wherein, the concentration of the reference molecule solution is 100 μM-2 mM; preferably, 1 mM.

[0056] Wherein, the volume of the reference molecule solution is 100 μL-1000 μL; preferably, 500 μL.

[0057] Wherein, the modification time of the reference molecule is 30 min-60 min; preferably, 30 min.

[0058] Wherein, the concentration of the Raman signal molecule solution used for the signal sequence-molecule modification is 100 μM-1000 μM; preferably, it is 500 μM.

[0059] Wherein, the total volume of the Raman signal molecule system used for the sequence-molecule modification is 1 mL-20 mL; preferably, 10 mL.

[0060] The coupling agent in the Raman signal molecule system used for the sequence-molecule modification is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and the ratio is 2:1.

[0061] Wherein, in the Raman signal molecule system used for the sequence-molecule modification, the dosage of the nucleic acid signal sequence is 10 μM-100 μM; preferably, 20 μM.

[0062] The reaction time for the sequence-molecule modification is 10 h-20 h; preferably, 16 h.

[0063] Wherein, the reaction temperature used for the sequence-molecule modification is 60°C-90°C; preferably, 85°C.

[0064] Wherein, the concentration of the nucleic acid recognition sequence used for the substrate-recognition sequence modification is 10-50 μM; preferably, 20 μM.

[0065] The volume of the solution of the nucleic acid recognition sequence used for the substrate-recognition sequence modification is 100 μL-1 mL; preferably, 500 μL.

[0066] Wherein, the reaction temperature of the nucleic acid recognition sequence used for the substrate-recognition sequence modification is 20°C-50°C; preferably, 37°C.

[0067] The reaction time of the nucleic acid recognition sequence used for the substrate-recognition sequence modification is 3h-5h; preferably, 3h.

[0068] In a specific embodiment, the preparation step of the tuberculosis resistance gene Raman probe includes: adding 500 μL of a 20 μM nucleic acid recognition sequence solution to the prepared SERS substrate surface, and incubating at 37°C for 3 hours. Then, adding 500 μL of 1 mM reference small molecule TCNQ to the nanoarray surface, further incubating at 37°C for 30 minutes, and washing the surface with ultrapure water 3 times to obtain the tuberculosis resistance gene Raman probe. At the same time, 10 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 5 mg of N-hydroxysuccinimide (NHS) were added to 1 mL of an aqueous solution of a nucleic acid signal sequence having a concentration of 20 μM, and perylenetetracarboxylic dianhydride (PTCDA) was added thereto to make its concentration 500 μM. The above reaction system was continued to react at a constant temperature of 85° C. for 16 hours, and the product was further repeatedly washed three times with 100 mL of 1 M hydrochloric acid, and the precipitate was filtered and retained for later use.

[0069] The present invention also provides a multi-channel Raman analysis chip for Mycobacterium tuberculosis drug-resistant gene constructed based on the Mycobacterium tuberculosis drug-resistant gene Raman probe.

[0070] The present invention also provides the application of the tuberculosis resistance gene Raman analysis chip for performing high-specificity analysis on the tuberculosis resistance gene in artificial simulated sputum.

[0071] The present invention also provides a method for preparing a multi-channel nucleic acid Raman analysis chip based on the Raman probe of the tuberculosis resistance gene, comprising the following steps:

[0072] (1) Taking the nucleic acid sequences of different tuberculosis resistance genes as monitoring targets, Raman probes of tuberculosis resistance genes were designed for different tuberculosis resistance strains according to the preparation method of Raman probes of tuberculosis resistance genes.

[0073] (2) The series of Mycobacterium tuberculosis resistance gene Raman probes prepared in step (1) are uniformly fixed to the surface of a large-size substrate to construct a multi-channel Mycobacterium tuberculosis resistance gene Raman analysis chip.

[0074] (3) Using different tuberculosis resistance gene nucleic acid sequences as targets, simulated sputum samples of tuberculosis resistance genes were prepared and dripped onto the chip surface. Subsequently, a probe solution containing a nucleic acid signal sequence was added. After incubation, a Raman spectrometer was used for spectral acquisition and data analysis.

[0075] In step (1), the tuberculosis resistance gene is one or more of rpoB, gyrA, gyrB and katG, etc.; preferably, it is rpoB.

[0076] In step (1), the nucleic acid recognition sequences of the specific recognition Raman probes are:

[0077] rpoB: SEQ ID NO.1

[0078] katG: SEQ ID NO.2

[0079] gyrA: SEQ ID NO.3

[0080] gyrB: SEQ ID NO.4

[0081] In step (1), the nucleic acid signal sequences in the specific recognition Raman probe are:

[0082] rpoB: 5′-CCAGTT GG-NH2-3′;

[0083] katG: 5'-CAT GAC GG-NH2-3'

[0084] gyrA: 5′-ATT GGT GG-NH2-3′

[0085] gyrB: 5′-ACCAGT GG-NH2-3′

[0086] In step (2), the substrate sheet is one of a glass sheet, a quartz sheet, a mica sheet, etc.; preferably, it is a quartz sheet.

[0087] In step (3), the volume of the simulated sputum sample of the Mycobacterium tuberculosis variant strain is 200 μL-800 μL; preferably, 500 μL.

[0088] In step (3), the temperature of the co-incubation is 30°C-80°C; preferably, 60°C.

[0089] In step (3), the co-incubation time is 1-10 min; preferably, 5 min.

[0090] In step (3), the volume of the solution containing the nucleic acid signal sequence probe is 100 μL-500 μL; preferably, 200 μL.

[0091] In step (3), the concentration of the solution containing the nucleic acid signal sequence probe is 0.5-2 μM; preferably, 1 μM.

[0092] In step (3), the laser wavelength used in the Raman spectrum test is one of 532 nm, 633 nm and 785 nm; preferably, 633 nm.

[0093] In step (3), the laser power used in the Raman spectroscopy test is 0.5-5 mW; preferably, 1 mW.

[0094] In step (3), the test exposure time used in the Raman spectrum test is 0.5-2s; preferably, 1s.

[0095] In a specific embodiment, the preparation of the multi-channel Mycobacterium tuberculosis resistance gene Raman analysis chip and the Mycobacterium tuberculosis resistance gene detection method include: first, according to the Mycobacterium tuberculosis resistance gene nucleic acid sequence, correspondingly design different nucleic acid recognition sequences and nucleic acid signal sequences, and prepare Mycobacterium tuberculosis resistance gene Raman probes, and then use a quartz plate as a substrate to evenly fix different Mycobacterium tuberculosis resistance gene Raman probes on the surface of the quartz substrate, take a 4-channel Mycobacterium tuberculosis resistance gene Raman analysis chip as an example, adopt a 1-channel control group and a 3-channel Mycobacterium tuberculosis resistance gene detection group to prepare a multi-channel Raman analysis chip, and drip 500 μL of Mycobacterium tuberculosis sputum simulation samples containing different concentrations and different nucleic acid sequences, and 200 μL of a mixed solution containing 1 μM modified nucleic acid signal sequence onto the surface of the Raman analysis chip, and incubate at 60°C for 5 minutes. Detection is performed using a Raman spectrometer, wherein the laser wavelength used by the Raman spectrometer is 633nm, the laser power is 1mW, and the exposure time is 1s.

[0096] In the present invention, the specific analysis and detection of the tuberculosis resistance gene in the sample can be achieved by measuring the different degrees of Raman enhancement effects of the signal molecule PTCDA and the reference molecule TCNQ on the SERS substrate chip and taking the ratio of the PTCDA Raman signal intensity to the TCNQ Raman signal intensity.

[0097] In a specific embodiment of the present invention, the multi-channel Raman analysis chip construction method for Mycobacterium tuberculosis drug resistance gene and the detection method for Mycobacterium tuberculosis drug resistance gene nucleic acid include the following steps:

[0098] (1) Preparation of SERS substrate materials

[0099] Dissolve 0.1g CuAc2 in 50mL ultrapure water, and add o-anisidine at a concentration of 0.1mol / L. After stirring the mixture for 30min, transfer it to a 50mL hydrothermal reactor and keep it in an oven at 180°C for 10h. Then, cool the hydrothermal reactor naturally to room temperature, transfer the dispersion to a centrifuge tube, and centrifuge at 5000rpm to obtain a precipitate. Further, dissolve amino-modified polystyrene (NH2-PS) with a molecular weight of 100,000 in 5mL ultrapure water to prepare a 10mM NH2-PS solution, add 30μg Cu2O nanowires to precipitate, and continue stirring at 30°C for 12h. Subsequently, the resulting nanowires are fully dispersed in 500μL cyclohexane and transferred to a 2cm 2 The surface of the single crystal silicon substrate. Then 500 μL of ultrapure water was added to the surface of the single crystal silicon substrate, and after the interface was stabilized, it was placed in a dry environment and slowly evaporated in a 60°C oven to obtain a Cu2O nano-SERS substrate material.

[0100] (2) Construction of Raman analysis probe

[0101] The preparation steps of the Raman analysis probe for tuberculosis resistance gene include: dropping 500 μL of a 20 μM nucleic acid recognition sequence solution onto the surface of the SERS substrate material prepared above, and incubating at 37°C for 3 hours. Then, dropping 500 μL of a 1 mM reference small molecule TCNQ onto the surface of the nanoarray, further incubating at 37°C for 30 minutes, and washing the surface with ultrapure water for 3 times to obtain the tuberculosis resistance gene Raman probe. At the same time, 10 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 5 mg of N-hydroxysuccinimide (NHS) were added to 1 mL of an aqueous solution of a nucleic acid signal sequence having a concentration of 20 μM, and perylenetetracarboxylic dianhydride (PTCDA) was added thereto to make its concentration 500 μM. The above reaction system was continued to react at a constant temperature of 85° C. for 16 hours, and the product was further repeatedly washed three times with 100 mL of 1 M hydrochloric acid, and the precipitate was filtered and retained for later use.

[0102] (3) Construction of a multi-channel Raman analysis chip and analysis of sputum simulation samples of drug-resistant strains of Mycobacterium tuberculosis

[0103] The preparation method of the multi-channel Mycobacterium tuberculosis resistance gene Raman analysis chip and the Mycobacterium tuberculosis resistance strain detection method include: first, according to the Mycobacterium tuberculosis resistance gene nucleic acid sequence, correspondingly designing different nucleic acid recognition sequences and nucleic acid signal sequences, and preparing Mycobacterium tuberculosis resistance gene probes, and then using a quartz plate as a substrate, evenly fixing different Mycobacterium tuberculosis resistance gene probes on the surface of the quartz substrate, taking a 4-channel Mycobacterium tuberculosis resistance gene Raman analysis chip as an example, using a 1-channel control group and a 3-channel (the 4 channels are: 1. negative experimental control group detection channel; 2. Mycobacterium tuberculosis rpoB gene; 3. Mycobacterium tuberculosis katG gene; 4. Mycobacterium tuberculosis gyrB gene;) Mycobacterium tuberculosis resistance gene detection group design to prepare a multi-channel Raman analysis chip, 500 μL of sputum simulation samples of Mycobacterium tuberculosis resistance strains with different concentrations and different nucleic acid sequences, and 200 μL of a mixed solution containing 1 μM modified nucleic acid signal sequence are dripped onto the surface of the Raman analysis chip, and incubated at 60°C for 5 minutes. The detection was performed using a Raman spectrometer, wherein the laser wavelength used by the Raman spectrometer was 633 nm, the laser power was 1 mW, and the exposure time was 1 s.

[0104] The beneficial effect of the present invention is that, firstly, based on Cu2O nanowires, the present invention constructs a surface enhanced Raman spectroscopy substrate material with a high enhancement effect through material self-assembly. And on this basis, for the tuberculosis resistance gene nucleic acid sequence, a series of multiple Raman analysis probes with the ability to identify and distinguish tuberculosis resistance strains are specifically designed, and further through spatial combination, a multi-channel tuberculosis resistance gene Raman analysis chip is designed and manufactured, which realizes the simultaneous analysis and detection of multiple tuberculosis resistance gene nucleic acid sequences. Based on the ultra-high sensitivity and high selectivity Raman enhancement effect of the SERS substrate material on the signal molecules PTCDA and TCNQ, the Raman peak intensity ratio of the PTCDA molecule to the TCNQ molecule is used as the quantitative basis, and without using biological enzymes and nucleic acid amplification technology, multi-channel simultaneous sensitive detection of different tuberculosis resistance gene nucleic acid sequences is achieved, and the detection limit reaches 48 copies / mL, and the average detection time is only 5 minutes. The chip can not only realize the rapid detection and screening of tuberculosis resistant strains of nucleic acid, but also the cost of a single test is only 1 yuan. The relevant detection reagents can be stored at room temperature for more than 6 months, which greatly saves economic and time costs. It provides a highly sensitive, highly selective and highly stable biological analysis platform for the rapid screening and analysis of tuberculosis resistant genes, which plays a significant positive role in pathogen detection and prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1 This is an analytical selectivity diagram of the nucleic acid Raman probe prepared in Example 3 of the present invention.

[0106] Figure 2It is a Raman spectrum linear diagram of the multi-channel Mycobacterium tuberculosis resistance gene Raman analysis chip in Example 4 of the present invention responding to three different Mycobacterium tuberculosis resistance gene nucleic acids, wherein A is the rpoB gene, B is the katG gene, and C is the gyrB gene.

[0107] Figure 3 This is the response time curve of the multi-channel Mycobacterium tuberculosis resistance gene Raman analysis chip in Example 4 of the present invention to three different Mycobacterium tuberculosis resistance gene nucleic acids.

[0108] Figure 4 This is a graph of long-term analysis stability test data of the multi-channel Mycobacterium tuberculosis resistance gene Raman analysis chip prepared in Example 4 of the present invention. DETAILED DESCRIPTION

[0109] The present invention is further described in detail with reference to the following specific examples and drawings. The process, conditions, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0110] Example 1: Preparation of SERS substrate material

[0111] Dissolve 0.1g CuAc2 in 50mL ultrapure water, and add o-anisidine at a concentration of 0.1mol / L. After stirring the mixture for 30min, transfer it to a 50mL hydrothermal reactor and keep it in an oven at 180°C for 10h. Then, cool the hydrothermal reactor naturally to room temperature, transfer the dispersion to a centrifuge tube, and centrifuge at 5000rpm to obtain a precipitate. Further, dissolve amino-modified polystyrene (NH2-PS) with a molecular weight of 100,000 in 5mL ultrapure water to prepare a 10mM NH2-PS solution, add 30μg Cu2O nanowires to precipitate, and continue stirring at 30°C for 12h. Subsequently, the resulting nanowires are fully dispersed in 500μL cyclohexane and transferred to a 2cm 2 The surface of the single crystal silicon substrate. Then 500 μL of ultrapure water was added to the surface of the single crystal silicon substrate, and after the interface was stabilized, it was placed in a dry environment and slowly evaporated in a 60°C oven to obtain a Cu2O nano-SERS substrate material.

[0112] Example 2: Preparation of SERS substrate material

[0113] Dissolve 0.1g CuAc2 in 50mL ultrapure water, and add o-anisidine at a concentration of 0.1mol / L. After stirring the mixture for 30min, transfer it to a 50mL hydrothermal reactor and keep it in an oven at 180°C for 10h. Then, cool the hydrothermal reactor naturally to room temperature, transfer the dispersion to a centrifuge tube, and centrifuge at 5000rpm to obtain a precipitate. Further, dissolve polyvinylpyrrolidone (PVP) with an amino-modified molecular weight of 30,000 in 5mL ultrapure water to prepare a 10mM NH2-PS solution, add 30μg Cu2O nanowires to precipitate, and continue stirring at 30°C for 12h. Subsequently, the resulting nanowires are fully dispersed in 500μL cyclohexane and transferred to a 2cm 2 The surface of the single crystal silicon substrate. Then 500 μL of ultrapure water was added to the surface of the single crystal silicon substrate, and after the interface was stabilized, it was placed in a dry environment and slowly evaporated in a 60°C oven to obtain a Cu2O nano-SERS substrate material.

[0114] Example 3 Construction of Raman Analysis Probe

[0115] The preparation steps of the nucleic acid Raman probe for the drug resistance gene of Mycobacterium tuberculosis include: dropping 500 μL of a 20 μM nucleic acid recognition sequence solution onto the surface of the SERS substrate material prepared in Example 1 of the present invention, and incubating at 37°C for 3 hours. Then, 500 μL of a 1 mM reference small molecule TCNQ is dropped onto the surface of the nanoarray, and further incubated at 37°C for 30 minutes, and the surface is washed with ultrapure water 3 times to obtain a nucleic acid Raman probe for mutations in the drug resistance gene rpoB, katG, gyrA or gyrB of Mycobacterium tuberculosis. At the same time, 10 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 5 mg of N-hydroxysuccinimide (NHS) were added to 1 mL of an aqueous solution of a nucleic acid signal sequence having a concentration of 20 μM, and perylenetetracarboxylic dianhydride (PTCDA) was added thereto to make its concentration 500 μM. The above reaction system was continued to react at a constant temperature of 85° C. for 16 hours, and the product was further repeatedly washed three times with 100 mL of 1 M hydrochloric acid, and the precipitate was filtered and retained for later use.

[0116] Figure 1 3 is a summary diagram of the analysis selectivity data of the Raman analysis probe for the rpoB, katG, gyrA and gyrB gene sequences of Mycobacterium tuberculosis. It can be seen from the figure that the constructed Raman analysis probe does not respond to other nucleic acid sequences or proteins except rpoB, katG, gyrA and gyrB genes, which proves that the SERS analysis probe constructed in the embodiment of the present invention has high selectivity.

[0117] Example 4 Construction of a multi-channel Raman analysis chip and analysis of simulated tuberculosis sputum samples

[0118] The preparation of a multi-channel Mycobacterium tuberculosis resistance gene Raman analysis chip and a method for detecting Mycobacterium tuberculosis resistance strains include: first, according to the Mycobacterium tuberculosis resistance gene nucleic acid sequence, different nucleic acid recognition sequences and nucleic acid signal sequences are correspondingly designed, and Mycobacterium tuberculosis resistance gene probes are prepared, and then, with a quartz plate as a substrate, different Mycobacterium tuberculosis resistance gene probes are evenly fixed on the surface of the quartz substrate. Taking a 4-channel Mycobacterium tuberculosis resistance gene Raman analysis chip as an example (wherein the 4 channels are: 1. negative experimental control group detection channel; 2. Mycobacterium tuberculosis rpoB gene; 3. Mycobacterium tuberculosis katG gene; 4. Mycobacterium tuberculosis gyrB gene;), a multi-channel Raman analysis chip is prepared by using a 1-channel control group and a 3-channel Mycobacterium tuberculosis resistance gene detection group design, 500 μL of Mycobacterium tuberculosis resistance gene sputum simulation samples containing different concentrations and different nucleic acid sequences and 200 μL of a mixed solution containing 1 μM modified nucleic acid signal sequence are dripped onto the surface of the Raman analysis chip and incubated at 60°C for 5 minutes. The detection was performed using a Raman spectrometer, wherein the laser wavelength used by the Raman spectrometer was 633 nm, the laser power was 1 mW, and the exposure time was 1 s.

[0119] like Figure 2 As shown, this embodiment takes a 4-channel Mycobacterium tuberculosis resistance gene nucleic acid Raman analysis chip as an example, and prepares a multi-channel Mycobacterium tuberculosis resistance gene Raman analysis chip. The 4 channels include a negative control analysis group and Raman analysis probes developed for 3 different Mycobacterium tuberculosis resistance genes, which can simultaneously realize high-sensitivity analysis and detection of three different Mycobacterium tuberculosis resistance genes. Figure 2 The Raman test response linear curves of the three different Raman analysis probes in the chip to the changes in nucleic acid sequence concentration were shown. The minimum detection limit of the chip reached 48 copies / mL. Figure 3 The figure shows the response time statistics of the three Raman analysis probes in the Raman chip to their corresponding nucleic acid sequences. It can be found that the average response time of the Raman analysis chip is about 5 minutes. Figure 4 The long-term stability of the constructed Raman analysis chip is shown in the figure. It can be seen from the figure that after the constructed Raman analysis chip is stored at room temperature for 6 months, its Raman analysis performance has not decreased significantly, proving that it has long-term storage and analysis and detection capabilities for more than 6 months. Table 1 calculates the cost of the Raman analysis chip for the analysis of tuberculosis resistance gene nucleic acid. Through calculation, it can be seen that the single production measurement cost of the chip for tuberculosis resistance gene nucleic acid is only about 1 yuan.

[0120] Table 1

[0121]

[0122] The beneficial effect of the present invention is that, firstly, based on Cu2O nanowires, the present invention constructs a surface enhanced Raman spectroscopy substrate material with a high enhancement effect through material self-assembly. And on this basis, for the tuberculosis resistance gene nucleic acid sequence, a series of multiple Raman analysis probes with the ability to identify and distinguish tuberculosis resistance strains are specifically designed, and further through spatial combination, a multi-channel tuberculosis resistance strain Raman analysis chip is designed and manufactured, which realizes the simultaneous analysis and detection of multiple tuberculosis resistance gene nucleic acid sequences. Based on the ultra-high sensitivity and high selectivity Raman enhancement effect of the SERS substrate material on the signal molecules PTCDA and TCNQ, the Raman peak intensity ratio of the PTCDA molecule to the TCNQ molecule is used as the quantitative basis, and without using biological enzymes and nucleic acid amplification technology, multi-channel simultaneous sensitive detection of different tuberculosis resistance gene nucleic acid sequences is achieved, and the detection limit reaches 48 copies / mL, and the average detection time is only 5 minutes. The chip can not only realize the rapid detection and screening of tuberculosis resistance gene nucleic acid, but also the cost of a single test is only 1 yuan. The relevant detection reagents can be stored at room temperature for more than 6 months, which greatly saves economic and time costs. It provides a highly sensitive, highly selective and highly stable biological analysis platform for the rapid screening and variant analysis of tuberculosis resistance strains, and plays a significant positive role in pathogen detection and prevention and control.

[0123] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A method for preparing a surface enhanced Raman SERS substrate material, characterized in that: The following steps are involved: (1) using copper acetate CuAc2, ultrapure water, and o-anisidine mixed solution as reactants to prepare Cu2O nanowires through a hydrothermal reaction; (2) Based on the Cu2O nanowires obtained by centrifugation in step (1), a SERS substrate material is constructed on the surface of a solid substrate through surface modification, oil / water interface self-assembly, and heating and drying.

2. The preparation method according to claim 1, characterized in that: In step (1), the dosage ratio of CuAc2, ultrapure water and o-anisidine is 0.05g-0.2g:30-50mL:0.05-0.2mol / L; and / or the temperature of the hydrothermal reaction is 160℃-200℃; and / or the time of the hydrothermal reaction is 8h-15h; and / or the time of mixing the solution is 15min-45min.

3. The preparation method according to claim 1, characterized in that: In step (2), the centrifugal speed is 3000rpm-8000rpm; and / or, the centrifugal time is 3min-10min; and / or, the surface modifier of the Cu2O nanowire is one of polyvinyl pyrrolidone, polyvinyl chloride, and polystyrene; and / or, the molecular weight of the surface modifier used for the assembly of the Cu2O nanowire is one of 100-1000000; and / or, the amount of the Cu2O nanowire is 10-100μg; and / or, the surface modifier of the Cu2O nanowire is 100-100μg. The solvent used for the surface modification of the Cu2O nanowires is ultrapure water or anhydrous ethanol; and / or, the time used for the surface modification process of the Cu2O nanowires is 8-15 hours; and / or, the temperature used for the surface modification process of the Cu2O nanowires is 20-40°C; and / or, the oil / water interface is one of water / chloroform, water / cyclohexane, and water / dichloromethane; and / or, the oil / water ratio of the oil / water interface is 1:0.5-1:3; and / or, the total volume of the oil / water dispersion solvent is 0.5-2 mL; and / or, the drying temperature is 40°C-80°C.

4. A surface enhanced Raman spectroscopy (SERS) substrate material prepared according to the method of any one of claims 1 to 3.

5. Use of the surface enhanced Raman SERS substrate material according to claim 4 in constructing a SERS analysis probe and an analysis chip for Mycobacterium tuberculosis drug resistance gene, and in the analysis of Mycobacterium tuberculosis drug resistance gene.

6. A method for preparing a SERS analysis probe for Mycobacterium tuberculosis drug resistance gene, characterized in that: Based on the surface enhanced Raman SERS substrate material described in claim 4, a nucleic acid recognition probe is designed for a specific tuberculosis resistance gene, and a tuberculosis resistance gene SERS analysis probe is constructed by modifying a signal molecule and a reference molecule.

7. The preparation method according to claim 6, characterized in that: The nucleic acid sequence of the tuberculosis resistance gene is one of the tuberculosis resistance gene fragments katG, gyrB, rpoB, and gyrA; and / or the nucleic acid recognition sequence and the nucleic acid signal sequence are DNA molecules or RNA molecules; and / or the number of bases of the nucleic acid recognition sequence is 35-40nt; and / or the number of bases of the nucleic acid signal sequence is 6-10nt; and / or the 5' end modification group of the nucleic acid recognition sequence is one of amino, thiol, hydroxyl, and carboxylic acid; and / or the 3' end modification group of the nucleic acid signal sequence is one of amino, thiol, hydroxyl, and carboxylic acid.

8. The preparation method according to claim 6, characterized in that: The base sequence of the nucleic acid recognition sequence is one of those shown in SEQ ID NO.1 to 6; and / or, the base sequence of the nucleic acid signal sequence is one of 5'-CCAGTTGG-NH2-3', 5'-CATGACGG-NH2-3', 5'-ATTGGTGG-NH2-3', and 5'-ACCAGTGG-NH2-3'; and / or, the Raman signal molecule is perylenetetracarboxylic dianhydride, rhodamine 6G, rhodamine B, cyanine and its derivatives, tetracyanoquinodimethane and its derivatives, bathocuproin and its derivatives, phenylacetylene and its derivatives and / or, the Raman reference molecule is one of perylenetetracarboxylic dianhydride, rhodamine 6G, rhodamine B, cyanine and its derivatives, tetracyanoquinodimethane and its derivatives, bathocuproin and its derivatives, phenylacetylene and its derivatives; and / or, the concentration of the reference molecule solution is 100 μM-2 mM; and / or, the volume of the reference molecule solution is 100 μL-1000 μL; and / or, the modification time of the reference molecule is 30 min-60 min; and / or, the signal sequence-molecule modification The concentration of the Raman signal molecule solution used is 100 μM-1000 μM; and / or, the total volume of the Raman signal molecule system used for the sequence-molecule modification is 1 mL-20 mL; and / or, the coupling agent in the Raman signal molecule system used for the sequence-molecule modification is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and the ratio is 2:1; and / or, in the Raman signal molecule system used for the sequence-molecule modification, the amount of the nucleic acid signal sequence is 10 μM-10 0μM; and / or, the reaction time used for the sequence-molecule modification is 10h-20h; and / or, the reaction temperature used for the sequence-molecule modification is 60℃-90℃; and / or, the concentration of the nucleic acid recognition sequence used for the substrate-recognition sequence modification is 10-50μM; and / or, the volume of the solution of the nucleic acid recognition sequence used for the substrate-recognition sequence modification is 100μL-1mL; and / or, the reaction temperature of the nucleic acid recognition sequence used for the substrate-recognition sequence modification is 20℃-50℃.

9. A method for preparing a SERS analysis chip for Mycobacterium tuberculosis drug resistance gene, characterized in that: The method comprises the following steps: based on the Mycobacterium tuberculosis resistance gene SERS analysis probe prepared by the method as claimed in claim 6, a series of SERS analysis probes with different recognition functions are fixed on the surface of a quartz substrate for a variety of different Mycobacterium tuberculosis resistance gene sequences, and then a Mycobacterium tuberculosis resistance gene SERS analysis chip is constructed to perform Raman spectroscopy testing.

10. The preparation method according to claim 9, characterized in that: The substrate sheet is one of a glass sheet, a quartz sheet, and a mica sheet; and / or the volume of the bacillus nucleic acid sputum simulation sample is 200 μL-800 μL; and / or the temperature of the co-incubation is 30°C-80°C; and / or the time of the co-incubation is 1-10 min; and / or the volume of the nucleic acid signal sequence probe solution is 100 μL-500 μL; and / or the concentration of the nucleic acid signal sequence probe solution is 0.5-2 μM; and / or the laser wavelength used in the Raman spectroscopy test is one of 532 nm, 633 nm and 785 nm; and / or the laser power used in the Raman spectroscopy test is 0.5-5 mW; and / or the test exposure time used in the Raman spectroscopy test is 0.5-2 s.

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