WO2022109833A1 - Method for constructing model for detecting novel coronavirus nucleic acids on basis of surface-enhanced infrared spectroscopy and principal component analysis - Google Patents

Method for constructing model for detecting novel coronavirus nucleic acids on basis of surface-enhanced infrared spectroscopy and principal component analysis Download PDF

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WO2022109833A1
WO2022109833A1 PCT/CN2020/131334 CN2020131334W WO2022109833A1 WO 2022109833 A1 WO2022109833 A1 WO 2022109833A1 CN 2020131334 W CN2020131334 W CN 2020131334W WO 2022109833 A1 WO2022109833 A1 WO 2022109833A1
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nucleic acid
sample
new coronavirus
infrared spectroscopy
acid single
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喻学锋
姚智奇
张琼珶
周文华
李佳
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深圳先进技术研究院
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    • C—CHEMISTRY; METALLURGY
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light

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  • the invention belongs to the technical field of analytical chemistry, and in particular relates to a method for constructing a new coronavirus nucleic acid detection model based on surface-enhanced infrared spectroscopy and principal component analysis.
  • Reverse transcription-polymerase chain reaction (RT-PCR) technology has high detection sensitivity and specificity, and is currently the main method for the detection of new coronaviruses.
  • This technology first extracts the viral RNA in the sample and reverses it into cDNA using reverse transcription technology, and then uses PCR technology to continuously replicate the cDNA.
  • the fluorescent probes in the kit are also working at the same time. Each time the cDNA is amplified, the fluorescent signal will increase, and the new coronavirus can be detected according to the intensity curve of the fluorescent signal.
  • this technology also has obvious shortcomings.
  • this method requires a lot of manpower and material resources, and in the stage of large-scale outbreaks, it may not be able to provide enough resources to carry out comprehensive testing;
  • RT-PCR testing takes a long time to complete The entire detection technology usually takes 4-6 hours, which may delay the timing of epidemic prevention and control;
  • the detection cost of RT-PCR technology is relatively high, which is not conducive to the long-term use of this method as the main detection method (that is, it has a high demand for human and material resources. , long detection time, high detection cost, etc.). Therefore, it is necessary to develop a rapid and low-cost detection method as a complement to RT-PCR detection technology.
  • the purpose of the present invention is to provide a method for constructing a new coronavirus nucleic acid detection model based on surface-enhanced infrared spectroscopy and principal component analysis.
  • the present invention provides a method for constructing a new coronavirus nucleic acid detection model based on surface-enhanced infrared spectroscopy and principal component analysis, comprising the following steps:
  • the nucleic acid single strand of the new coronavirus sample is a new coronavirus complementary nucleic acid single strand that is complementary to the DNA probe
  • the nucleic acid single strand of the healthy sample is a single strand that does not pair with the DNA probe and does not belong to the new coronavirus sequence
  • the surface-enhanced infrared spectroscopy substrate provided with DNA probes on the surface includes an infrared transparent substrate, the surface of the infrared transparent substrate is provided with a gold film, and the surface of the gold film is provided with DNA probes.
  • the infrared transparent substrate is selected from calcium fluoride, silicon wafer, sapphire and the like.
  • the thickness of the gold film is 10-20 nm.
  • the preparation method of the surface-enhanced infrared spectroscopy substrate with DNA probes on the surface comprises the following steps:
  • step 2) placing the substrate obtained in step 1) in a DNA probe solution modified by sulfhydryl groups, combining the DNA probe with gold to form a gold-sulfur covalent bond, and forming a monomolecular self-assembly layer on the gold surface to obtain the Surface-enhanced infrared spectroscopy substrate with DNA probes on the surface.
  • the pretreatment of the infrared transparent substrate is also included;
  • the pretreatment of the infrared transparent substrate is to wash the infrared transparent substrate in acetone, absolute ethanol and ultrapure water in sequence, and then blow dry with a N 2 gun.
  • nucleotide sequence of the nucleic acid single strand of the new coronavirus sample is shown in SEQ ID NO: 2.
  • nucleotide sequence of the nucleic acid single strand of the healthy sample is shown in SEQ ID NO: 3.
  • the method that the model is used to detect the nucleic acid of the new coronavirus includes the following steps:
  • the present invention provides a novel coronavirus nucleic acid detection kit based on surface-enhanced infrared spectroscopy and principal component analysis, comprising a surface-enhanced infrared spectroscopy substrate with DNA probes on the surface, the DNA probes having nucleotides The sequence is shown in SEQ ID NO: 1;
  • the nucleic acid single-stranded solution of the healthy sample and the nucleic acid single-stranded solution of the new coronavirus sample are also included.
  • the nucleic acid single strand of the virus sample is the new coronavirus complementary nucleic acid single strand that is complementary to the DNA probe;
  • nucleotide sequence of the nucleic acid single strand of the new coronavirus sample is shown in SEQ ID NO: 2;
  • nucleotide sequence of the nucleic acid single strand of the healthy sample is shown in SEQ ID NO: 3.
  • the beneficial effects of the present invention are as follows: the present invention organically combines surface-enhanced infrared spectroscopy and principal component analysis, distinguishes healthy samples from virus samples according to nucleic acid pairing and complementation, and provides a novel high-sensitivity, low-cost and fast-testing method. New coronavirus detection methods;
  • the detection method of the invention is very fast, and the test result can be analyzed within 5 minutes of collecting the infrared spectrum;
  • the detection method of the present invention has relatively low cost, only needs an infrared spectrometer, and does not need other special conditions or chemicals.
  • Figure 1 shows the infrared transmission spectra of 1 ⁇ M thiol-modified DNA probe solution deposited on 10 nm Au and 50 nm Au, respectively;
  • Figure 2 is the infrared transmission spectrum of various samples of t-nCoV-N, t-non-nCoV, t-nCoV-ORF, p-nCoV-N, none SH;
  • Figure 3a is the scatter plot of the PCA analysis of t-nCoV-N and p-nCoV-N data
  • Figure 3b is the original infrared spectrum of t-nCoV-N, the second differential spectrum, and the loading plot of PC1 from top to bottom and the loading plot of PC2;
  • Figure 4a is the scatter plot of PCA analysis of t-nCoV-ORF and p-nCoV-N group data
  • Figure 4b is the scatter plot of t-nCoV-ORF and t-nCoV-N group data PCA analysis
  • Figure 4c is t - Scatter plot of PCA analysis of nCoV-ORF, p-nCoV-N and t-nCoV-N group data
  • Figure 5a is the scatter plot of PCA analysis of t-non-nCoV and p-nCoV-N group data
  • Figure 5b is the scatter plot of t-non-nCoV and t-nCoV-N group data PCA analysis
  • Figure 5c is t - Scatter plot of PCA analysis of non-nCoV, p-nCoV-N and t-nCoV-N group data.
  • a calcium fluoride window was used as the raw material, and a 10nm Au film was evaporated on it as a surface-enhanced infrared substrate (10nm Au); compared with a 50nm flat gold film (50nm Au).
  • 1 ⁇ M of thiol-modified DNA probe solution was deposited on 10 nm Au and 50 nm Au, respectively, and the infrared transmission spectrum was measured. The results are shown in Figure 1. It can be seen from Fig. 1 that 10 nm Au significantly enhanced the infrared spectral signal of 1 ⁇ M thiol-modified DNA probe.
  • test group
  • the preparation process is as follows: the calcium fluoride window substrate is sequentially placed in acetone, absolute ethanol and ultrapure water for cleaning for 10min, and dried with a N2 gun; A 10nm gold film was evaporated on a clean substrate to form a randomly distributed gold nanoparticle structure; the substrate was placed in a DNA probe solution modified with thiol groups, and the DNA probe was combined with gold to form an Au-S covalent bond. A single-molecule self-assembly layer was formed on the surface of Au, and the nucleotide sequence of the DNA probe was shown in SEQ ID NO: 1 (p-nCoV-N).
  • Nucleic acid single-stranded solution of the new coronavirus sample The nucleotide sequence of the nucleic acid single-stranded of the new coronavirus sample is shown in SEQ ID NO: 2 (t-nCoV-N).
  • Nucleic acid single strand solution of healthy sample The nucleotide sequence of nucleic acid single strand of healthy sample is shown in SEQ ID NO: 3 (t-non-nCoV).
  • the preparation process is as follows: the calcium fluoride window substrate is placed in acetone, anhydrous ethanol and ultrapure water in sequence for 10 min, and then blown dry with a N 2 gun; A 10nm gold film was evaporated on the substrate to form a randomly distributed gold nanoparticle structure; the substrate was placed in a DNA probe solution that was not modified by thiol groups, and the DNA probe could not combine with gold to form Au-S covalent bonds.
  • the nucleotide sequence of the DNA probe is shown in SEQ ID NO: 1 (none SH).
  • Nucleic acid single-stranded solution that is not paired with the DNA probe but is the same as the new coronavirus The nucleoside sequence of the nucleic acid single-stranded that is not paired with the DNA probe but is the same as the new coronavirus is shown in SEQ ID NO: 4 (t-nCoV-ORF ).
  • nucleic acid single-stranded solution of the new coronavirus sample Drop the nucleic acid single-stranded solution of the new coronavirus sample, the nucleic acid single-stranded solution of the healthy sample, and the nucleic acid single-stranded solution of the new coronavirus that is not paired with the DNA probe, respectively, on the surface-enhanced infrared ray with the DNA probe on the surface.
  • Spectral substrate surface (among them, nucleic acid single strands of healthy samples and nucleic acid single strands that are not paired with DNA probes but are both new coronaviruses cannot be complementary to existing DNA probes, while nucleic acid single strands of new coronavirus samples can be compatible with existing DNA probes.
  • the DNA probes are complementary to each other), after the solvent is naturally volatilized, it is rinsed repeatedly with ultrapure water to remove the nucleic acid single strands physically adsorbed on the surface, and it is blown dry with a N
  • the signal of none SH was used to compare with the thiol-modified DNA probe, which proved that the obtained infrared signal came from the DNA bound to the substrate.
  • the signal of p-nCoV-N is compared with the signals of t-nCoV-N, t-non-nCoV, and t-nCoV-ORF to see whether the surface-enhanced infrared spectroscopy substrate with DNA probes on the surface is post-treated. There will be differences.
  • the nucleic acid single-stranded solution that is not paired with the DNA probe but is also a new coronavirus is used as a control experiment. Although it belongs to the nucleic acid sequence of the new coronavirus, it cannot be successfully paired if it is not in the pairing range, which further shows the specificity of the DNA probe.
  • Figure 3a is a scatter plot of the PCA analysis of the t-nCoV-N and p-nCoV-N group data, PC1 and PC2 represent the two principal components with the largest overall difference, and the ellipses represent the 95% confidence interval for each group of samples.
  • Figure 3a a distinction can be made between the t-nCoV-N and p-nCoV-N groups.
  • Figure 3b shows, from top to bottom, the original infrared spectrum of t-nCoV-N, the second differential spectrum, the loading plot of PC1 and the loading plot of PC2. It can be seen from Fig. 3b that the second differential spectrum can reveal all the peaks covered by the broad peaks in the original spectrum, which can make it easier to distinguish the position of each peak. The farther away from 0 in the Loading plot, the greater the contribution of the infrared spectral peaks to distinguishing the difference between the two groups of data.
  • Figure 4a is a scatter plot of the PCA analysis of the t-nCoV-ORF and p-nCoV-N group data, PC1 and PC2 represent the two principal components with the largest overall difference, and the ellipses represent the 95% confidence interval for each group of samples. From Figure 4a, it can be seen that the t-nCoV-ORF and p-nCoV-N groups cannot be distinguished.
  • Figure 4b is a scatter plot of the PCA analysis of the t-nCoV-ORF and t-nCoV-N group data, PC1 and PC2 represent the two principal components with the largest overall difference, and the ellipses represent the 95% confidence interval for each group of samples. From Figure 4b it can be seen that the t-nCoV-ORF and t-nCoV-N groups can be distinguished.
  • Figure 4c is a scatter plot of the PCA analysis of the t-nCoV-ORF, p-nCoV-N and t-nCoV-N group data, PC1 and PC2 represent the two principal components with the largest overall difference, and the ellipses represent 95% of the samples in each group. confidence interval.
  • Figure 5a is a scatter plot of the PCA analysis of the t-non-nCoV and p-nCoV-N group data, PC1 and PC2 represent the two principal components with the largest overall difference, and the ellipses represent the 95% confidence interval of each group of samples. From Figure 5a, it can be seen that no distinction can be made between the t-non-nCoV and p-nCoV-N groups.
  • Figure 5b is a scatter plot of the PCA analysis of the t-non-nCoV and t-nCoV-N group data, PC1 and PC2 represent the two principal components with the largest overall difference, and the ellipses represent the 95% confidence interval of each group of samples. From Figure 5b, it can be seen that the t-non-nCoV and t-nCoV-N groups can be distinguished.
  • Figure 5c is the scatter plot of the PCA analysis of the t-non-nCoV, p-nCoV-N and t-nCoV-N group data, PC1 and PC2 represent the two principal components with the largest overall difference, and the ellipses represent 95% of the samples in each group. confidence interval.
  • the method of the present invention can successfully distinguish virus samples from healthy samples.

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Abstract

A method for constructing a model for detecting novel coronavirus nucleic acids on the basis of surface-enhanced infrared spectroscopy and principal component analysis. The method comprises the following steps: (1) preparing a surface-enhanced infrared spectroscopy substrate with a DNA probe on the surface, a single-stranded nucleic acid solution from a healthy sample and a single-stranded nucleic acid solution from a novel coronavirus sample, wherein the nucleotide sequence of the DNA probe is as shown in SEQ ID NO: 1; (2) respectively dropping the single-stranded nucleic acid solution from the healthy sample and the single-stranded nucleic acid solution from the novel coronavirus sample on the surface of the surface-enhanced infrared spectroscopy substrate with the DNA probe on the surface, volatilizing the solvent naturally, and then rinsing the residue with ultrapure water repeatedly to remove the single-stranded nucleic acids which are physically adsorbed on the surface thereof; (3) collecting the infrared transmission spectrum; (4) performing spectrum preprocessing; and (5) processing the sample data in step (4) by means of using the PCA Spectroscopy program to obtain an analysis model of the principal components from the health sample and the novel coronavirus. The detection method is very fast and relatively inexpensive in cost, requiring only an infrared spectrometer.

Description

基于表面增强红外光谱和主成分分析的新冠病毒核酸检测模型的构建方法Construction method of new coronavirus nucleic acid detection model based on surface-enhanced infrared spectroscopy and principal component analysis 技术领域technical field
本发明属于分析化学技术领域，具体涉及一种基于表面增强红外光谱和主成分分析的新冠病毒核酸检测模型的构建方法。The invention belongs to the technical field of analytical chemistry, and in particular relates to a method for constructing a new coronavirus nucleic acid detection model based on surface-enhanced infrared spectroscopy and principal component analysis.
背景技术Background technique
反转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)技术检测灵敏度高，特异性强，是目前对于新冠病毒检测的主要手段。此技术首先将样本中的病毒RNA提取出来并利用反转录技术反转成cDNA，之后利用PCR技术使cDNA不断复制，在cDNA扩增的同时，试剂盒中的荧光探针也在同时工作，cDNA每完成扩增，荧光信号便会增强，根据荧光信号的强度曲线从而检测出新冠病毒。然而，此技术也存在明显的缺陷，首先，此方法需要大量的人力和物力，在疫情大规模爆发阶段，可能无法提供足够的资源来实行全面检测；其次，RT-PCR检测时间较长，完成整个检测技术通常需要4-6个小时，可能会延误疫情防控的时机；最后，RT-PCR技术检测成本较为高昂，不利于长期使用此方法作为主要的检测手段(即具有人力物力资源需求高、检测时间长、检测成本高等缺陷)。因此有必要开发一种快速、成本低廉的检测方法作为RT-PCR检测技术的补充。Reverse transcription-polymerase chain reaction (RT-PCR) technology has high detection sensitivity and specificity, and is currently the main method for the detection of new coronaviruses. This technology first extracts the viral RNA in the sample and reverses it into cDNA using reverse transcription technology, and then uses PCR technology to continuously replicate the cDNA. At the same time as the cDNA is amplified, the fluorescent probes in the kit are also working at the same time. Each time the cDNA is amplified, the fluorescent signal will increase, and the new coronavirus can be detected according to the intensity curve of the fluorescent signal. However, this technology also has obvious shortcomings. First, this method requires a lot of manpower and material resources, and in the stage of large-scale outbreaks, it may not be able to provide enough resources to carry out comprehensive testing; secondly, RT-PCR testing takes a long time to complete The entire detection technology usually takes 4-6 hours, which may delay the timing of epidemic prevention and control; finally, the detection cost of RT-PCR technology is relatively high, which is not conducive to the long-term use of this method as the main detection method (that is, it has a high demand for human and material resources. , long detection time, high detection cost, etc.). Therefore, it is necessary to develop a rapid and low-cost detection method as a complement to RT-PCR detection technology.
发明内容SUMMARY OF THE INVENTION
为了解决上述背景技术中所提出的问题，本发明的目的在于提供一种基于表面增强红外光谱和主成分分析的新冠病毒核酸检测模型的构建方法。In order to solve the problems raised in the above background technology, the purpose of the present invention is to provide a method for constructing a new coronavirus nucleic acid detection model based on surface-enhanced infrared spectroscopy and principal component analysis.
为了达到上述目的，本发明所采用的技术方案为：一方面，本发明提供了一种基于表面增强红外光谱和主成分分析的新冠病毒核酸检测模型的构建方法，包括以下步骤：In order to achieve the above purpose, the technical solution adopted in the present invention is as follows: On the one hand, the present invention provides a method for constructing a new coronavirus nucleic acid detection model based on surface-enhanced infrared spectroscopy and principal component analysis, comprising the following steps:
(1)材料的准备：表面设有DNA探针的表面增强红外光谱基底、健康样本的核酸单链溶液和新冠病毒样本的核酸单链溶液，所述DNA探针的核苷酸序列如SEQ ID NO：1所示；(1) Preparation of materials: surface-enhanced infrared spectroscopy substrates with DNA probes on the surface, nucleic acid single-stranded solutions of healthy samples and nucleic acid single-stranded solutions of new coronavirus samples, the nucleotide sequences of the DNA probes are as shown in SEQ ID NO: shown in 1;
其中，所述新冠病毒样本的核酸单链为与DNA探针互补配对的新冠病毒互补核酸单链，所述健康样本的核酸单链为与DNA探针不配对且不属于新冠病毒序列的单链；Wherein, the nucleic acid single strand of the new coronavirus sample is a new coronavirus complementary nucleic acid single strand that is complementary to the DNA probe, and the nucleic acid single strand of the healthy sample is a single strand that does not pair with the DNA probe and does not belong to the new coronavirus sequence ;
(2)将健康样本的核酸单链溶液与新冠病毒样本的核酸单链溶液分别滴在表面设有DNA探针的表面增强红外光谱基底表面，待其溶剂自然挥发后，用超纯水反复润洗，除去表面物理吸附的核酸单链；(2) Drop the nucleic acid single-stranded solution of the healthy sample and the nucleic acid single-stranded solution of the new coronavirus sample respectively on the surface of the surface-enhanced infrared spectroscopy substrate with the DNA probe on the surface. After the solvent is naturally volatilized, it is repeatedly moistened with ultrapure water. Washing to remove nucleic acid single strands physically adsorbed on the surface;
(3)红外透射光谱的采集：采集光谱波长范围为1100-1800cm -1的红外指纹区； (3) Collection of infrared transmission spectrum: the infrared fingerprint region with the wavelength range of 1100-1800 cm -1 is collected;
(4)光谱预处理：将所采集的红外透射光谱进行平滑、基线校准、曲线均一化、二次微分处理；(4) Spectral preprocessing: smoothing, baseline calibration, curve normalization, and quadratic differential processing of the collected infrared transmission spectrum;
(5)判别模型的建立：利用PCA Spectroscopy程序处理(4)中的样本数据，得到健康、新冠病毒的主成分分析模型。(5) Establishment of the discriminant model: The PCA Spectroscopy program was used to process the sample data in (4) to obtain the principal component analysis model of health and new coronavirus.
进一步地，所述表面设有DNA探针的表面增强红外光谱基底包括红外透明的衬底，所述红外透明的衬底表面设有金膜，所述金膜的表面设有DNA探针。Further, the surface-enhanced infrared spectroscopy substrate provided with DNA probes on the surface includes an infrared transparent substrate, the surface of the infrared transparent substrate is provided with a gold film, and the surface of the gold film is provided with DNA probes.
进一步地，所述红外透明的衬底选自氟化钙、硅片、蓝宝石等。Further, the infrared transparent substrate is selected from calcium fluoride, silicon wafer, sapphire and the like.
进一步地，所述金膜的厚度为10-20nm。Further, the thickness of the gold film is 10-20 nm.
进一步地，所述表面设有DNA探针的表面增强红外光谱基底的制备方法包括以下步骤：Further, the preparation method of the surface-enhanced infrared spectroscopy substrate with DNA probes on the surface comprises the following steps:
1)在红外透明的衬底上蒸镀金膜，形成随机分布的金纳米颗粒结构；1) Evaporating a gold film on an infrared transparent substrate to form a randomly distributed gold nanoparticle structure;
2)将步骤1)所得衬底置于被巯基修饰的DNA探针溶液中，使DNA探针与金结合形成金-硫共价键，在金表面生层单分子自组装层，得到所述表面设有DNA探针的表面增强红外光谱基底。2) placing the substrate obtained in step 1) in a DNA probe solution modified by sulfhydryl groups, combining the DNA probe with gold to form a gold-sulfur covalent bond, and forming a monomolecular self-assembly layer on the gold surface to obtain the Surface-enhanced infrared spectroscopy substrate with DNA probes on the surface.
进一步地，所述步骤1)前还包括红外透明的衬底的预处理；Further, before the step 1), the pretreatment of the infrared transparent substrate is also included;
优选地，所述红外透明的衬底的预处理为将红外透明的衬底依次置于丙酮、无水乙醇和超纯水之中清洗，然后用N 2枪吹干。 Preferably, the pretreatment of the infrared transparent substrate is to wash the infrared transparent substrate in acetone, absolute ethanol and ultrapure water in sequence, and then blow dry with a N 2 gun.
进一步地，所述新冠病毒样本的核酸单链的核苷酸序列如SEQ ID NO：2所示。Further, the nucleotide sequence of the nucleic acid single strand of the new coronavirus sample is shown in SEQ ID NO: 2.
进一步地，所述健康样本的核酸单链的核苷酸序列如SEQ ID NO：3所示。Further, the nucleotide sequence of the nucleic acid single strand of the healthy sample is shown in SEQ ID NO: 3.
进一步地，所述模型用于检测新冠病毒核酸的方法包括以下步骤：Further, the method that the model is used to detect the nucleic acid of the new coronavirus includes the following steps:
(1)将待测样品滴在表面设有DNA探针的表面增强红外光谱基底表面，待其溶剂自然挥发后，用超纯水反复润洗；(1) drop the sample to be tested on the surface of the surface-enhanced infrared spectroscopy substrate provided with the DNA probe on the surface, and after the solvent is naturally volatilized, rinse it repeatedly with ultrapure water;
(2)采集红外透射光谱：采集光谱波长范围为1100-1800cm -1的红外指纹区； (2) Collecting infrared transmission spectrum: collecting the infrared fingerprint region with a spectral wavelength range of 1100-1800 cm -1 ;
(3)光谱预处理：将所采集的红外透射光谱进行平滑、基线校准、曲线均一化、二次微分处理；(3) Spectral preprocessing: smoothing, baseline calibration, curve normalization, and quadratic differential processing of the collected infrared transmission spectrum;
(4)利用PCA Spectroscopy程序一起处理(3)中待测样品、上述所述的新冠病毒样本和健康样本的数据，从PCA分析图中判断待测样品落在新冠病毒样本的置信区间内还是健康样本的置信区间内，进而得到待测样品健康、新冠病毒的类别。(4) Use the PCA Spectroscopy program to process the data of the sample to be tested, the above-mentioned new coronavirus sample and the healthy sample in (3), and determine whether the sample to be tested falls within the confidence interval of the new coronavirus sample or is healthy from the PCA analysis chart Within the confidence interval of the sample, the health of the sample to be tested and the category of the new coronavirus can be obtained.
另一方面，本发明提供了一种基于表面增强红外光谱和主成分分析的新冠病毒核酸检测试剂盒，包括表面设有DNA探针的表面增强红外光谱基底，所述DNA探针的核苷酸序列如SEQ ID NO：1所示；In another aspect, the present invention provides a novel coronavirus nucleic acid detection kit based on surface-enhanced infrared spectroscopy and principal component analysis, comprising a surface-enhanced infrared spectroscopy substrate with DNA probes on the surface, the DNA probes having nucleotides The sequence is shown in SEQ ID NO: 1;
优选地，还包括健康样本的核酸单链溶液和新冠病毒样本的核酸单链溶液，所述健康样本的核酸单链为与DNA探针不配对且不属于新冠病毒序列的单链，所述新冠病毒样本的核酸单链为与DNA探针互补配对的新冠病毒互补核酸单链；Preferably, the nucleic acid single-stranded solution of the healthy sample and the nucleic acid single-stranded solution of the new coronavirus sample are also included. The nucleic acid single strand of the virus sample is the new coronavirus complementary nucleic acid single strand that is complementary to the DNA probe;
优选地，所述新冠病毒样本的核酸单链的核苷酸序列如SEQ ID NO：2所示；Preferably, the nucleotide sequence of the nucleic acid single strand of the new coronavirus sample is shown in SEQ ID NO: 2;
优选地，所述健康样本的核酸单链的核苷酸序列如SEQ ID NO：3所示。Preferably, the nucleotide sequence of the nucleic acid single strand of the healthy sample is shown in SEQ ID NO: 3.
本发明的有益效果是：本发明将表面增强红外光谱与主成分分析法有机结合，根据核酸配对互补情况以区分健康样本与病毒样本，提供了一种新型的灵敏度高、成本低廉、测试快速的新冠病毒检测方法；The beneficial effects of the present invention are as follows: the present invention organically combines surface-enhanced infrared spectroscopy and principal component analysis, distinguishes healthy samples from virus samples according to nucleic acid pairing and complementation, and provides a novel high-sensitivity, low-cost and fast-testing method. New coronavirus detection methods;
本发明的检测方法非常快速，在采集红外光谱的5min内便可分析出测试结果；The detection method of the invention is very fast, and the test result can be analyzed within 5 minutes of collecting the infrared spectrum;
本发明的检测方法成本相对低廉，只需要红外光谱仪，不需要其他特殊条件或化学药品。The detection method of the present invention has relatively low cost, only needs an infrared spectrometer, and does not need other special conditions or chemicals.
附图说明Description of drawings
图1是1μM被巯基修饰的DNA探针溶液分别沉积在10nm Au和50nm Au上的红外透射光谱；Figure 1 shows the infrared transmission spectra of 1 μM thiol-modified DNA probe solution deposited on 10 nm Au and 50 nm Au, respectively;
图2是t-nCoV-N、t-non-nCoV、t-nCoV-ORF、p-nCoV-N、none SH各种样本的红外透射光谱；Figure 2 is the infrared transmission spectrum of various samples of t-nCoV-N, t-non-nCoV, t-nCoV-ORF, p-nCoV-N, none SH;
图3a为t-nCoV-N和p-nCoV-N组数据PCA分析的散点图；图3b从上到下依次为t-nCoV-N的原始红外光谱、二次微分光谱、PC1的loading plot和PC2的loading plot；Figure 3a is the scatter plot of the PCA analysis of t-nCoV-N and p-nCoV-N data; Figure 3b is the original infrared spectrum of t-nCoV-N, the second differential spectrum, and the loading plot of PC1 from top to bottom and the loading plot of PC2;
图4a为t-nCoV-ORF和p-nCoV-N组数据PCA分析的散点图；图4b为t-nCoV-ORF和t-nCoV-N组数据PCA分析的散点图；图4c为t-nCoV-ORF、p-nCoV-N和t-nCoV-N组数据PCA分析的散点图；Figure 4a is the scatter plot of PCA analysis of t-nCoV-ORF and p-nCoV-N group data; Figure 4b is the scatter plot of t-nCoV-ORF and t-nCoV-N group data PCA analysis; Figure 4c is t - Scatter plot of PCA analysis of nCoV-ORF, p-nCoV-N and t-nCoV-N group data;
图5a为t-non-nCoV和p-nCoV-N组数据PCA分析的散点图；图5b为t-non-nCoV和t-nCoV-N组数据PCA分析的散点图；图5c为t-non-nCoV、p-nCoV-N和t-nCoV-N组数据PCA分析的散点图。Figure 5a is the scatter plot of PCA analysis of t-non-nCoV and p-nCoV-N group data; Figure 5b is the scatter plot of t-non-nCoV and t-nCoV-N group data PCA analysis; Figure 5c is t - Scatter plot of PCA analysis of non-nCoV, p-nCoV-N and t-nCoV-N group data.
具体实施方式Detailed ways
为了更好地理解本发明的内容，下面结合具体实施方法对本发明内容作进一步说明，但本发明的保护内容不局限以下实施例。In order to better understand the content of the present invention, the content of the present invention will be further described below in conjunction with specific implementation methods, but the protection content of the present invention is not limited to the following examples.
实施例1Example 1
采用氟化钙窗片为原材料，在其上蒸镀10nm Au膜，作为表面增强红外衬底(10nm Au)；对比50nm的平整金膜(50nm Au)。将1μM被巯基修饰的DNA探针溶液分别沉积在10nm Au 和50nm Au上，进行红外透射光谱的测定，其结果如图1所示。从图1可以看出，10nm Au显著增强1μM被巯基修饰的DNA探针的红外光谱信号。A calcium fluoride window was used as the raw material, and a 10nm Au film was evaporated on it as a surface-enhanced infrared substrate (10nm Au); compared with a 50nm flat gold film (50nm Au). 1 μM of thiol-modified DNA probe solution was deposited on 10 nm Au and 50 nm Au, respectively, and the infrared transmission spectrum was measured. The results are shown in Figure 1. It can be seen from Fig. 1 that 10 nm Au significantly enhanced the infrared spectral signal of 1 μM thiol-modified DNA probe.
实施例2Example 2
(1)材料的准备：(1) Preparation of materials:
实验组：test group:
表面设有DNA探针的表面增强红外光谱基底：制备过程为，将氟化钙窗片衬底依次置于丙酮、无水乙醇和超纯水之中清洗10min，用N 2枪吹干；在干净的衬底上蒸镀10nm金膜，形成随机分布的金纳米颗粒结构；将衬底置于被巯基修饰的DNA探针溶液中，使DNA探针与金结合形成Au-S共价键，在Au表面生层单分子自组装层，DNA探针的核苷酸序列如SEQ ID NO：1所示(p-nCoV-N)。 Surface-enhanced infrared spectroscopy substrate with DNA probes on the surface: the preparation process is as follows: the calcium fluoride window substrate is sequentially placed in acetone, absolute ethanol and ultrapure water for cleaning for 10min, and dried with a N2 gun; A 10nm gold film was evaporated on a clean substrate to form a randomly distributed gold nanoparticle structure; the substrate was placed in a DNA probe solution modified with thiol groups, and the DNA probe was combined with gold to form an Au-S covalent bond. A single-molecule self-assembly layer was formed on the surface of Au, and the nucleotide sequence of the DNA probe was shown in SEQ ID NO: 1 (p-nCoV-N).
新冠病毒样本的核酸单链溶液：新冠病毒样本的核酸单链的核苷酸序列如SEQ ID NO：2所示(t-nCoV-N)。Nucleic acid single-stranded solution of the new coronavirus sample: The nucleotide sequence of the nucleic acid single-stranded of the new coronavirus sample is shown in SEQ ID NO: 2 (t-nCoV-N).
健康样本的核酸单链溶液：健康样本的核酸单链的核苷酸序列如SEQ ID NO：3所示(t-non-nCoV)。Nucleic acid single strand solution of healthy sample: The nucleotide sequence of nucleic acid single strand of healthy sample is shown in SEQ ID NO: 3 (t-non-nCoV).
对照组：Control group:
表面没有DNA探针的表面增强红外光谱基底：制备过程为，将氟化钙窗片衬底依次置于丙酮、无水乙醇和超纯水之中清洗10min，用N 2枪吹干；在干净的衬底上蒸镀10nm金膜，形成随机分布的金纳米颗粒结构；将衬底置于没有被巯基修饰的DNA探针溶液中，DNA探针不能与金结合形成Au-S共价键，DNA探针的核苷酸序列如SEQ ID NO：1所示(none SH)。 Surface-enhanced infrared spectroscopy substrate without DNA probes on the surface: the preparation process is as follows: the calcium fluoride window substrate is placed in acetone, anhydrous ethanol and ultrapure water in sequence for 10 min, and then blown dry with a N 2 gun; A 10nm gold film was evaporated on the substrate to form a randomly distributed gold nanoparticle structure; the substrate was placed in a DNA probe solution that was not modified by thiol groups, and the DNA probe could not combine with gold to form Au-S covalent bonds. The nucleotide sequence of the DNA probe is shown in SEQ ID NO: 1 (none SH).
与DNA探针不配对但同为新冠病毒的核酸单链溶液：与DNA探针不配对但同为新冠病毒的核酸单链的核苷序列如SEQ ID NO：4所示(t-nCoV-ORF)。Nucleic acid single-stranded solution that is not paired with the DNA probe but is the same as the new coronavirus: The nucleoside sequence of the nucleic acid single-stranded that is not paired with the DNA probe but is the same as the new coronavirus is shown in SEQ ID NO: 4 (t-nCoV-ORF ).
(2)将新冠病毒样本的核酸单链溶液、健康样本的核酸单链溶液、与DNA探针不配对但同为新冠病毒的核酸单链溶液分别滴在表面设有DNA探针的表面增强红外光谱基底表面(其中健康样本的核酸单链、与DNA探针不配对但同为新冠病毒的核酸单链无法与已有的DNA探针形成互补，新冠病毒样本的核酸单链则可以与已有的DNA探针形成互补)，待其溶剂自然挥发后，用超纯水反复润洗，除去表面物理吸附的核酸单链，用N 2枪吹干。 (2) Drop the nucleic acid single-stranded solution of the new coronavirus sample, the nucleic acid single-stranded solution of the healthy sample, and the nucleic acid single-stranded solution of the new coronavirus that is not paired with the DNA probe, respectively, on the surface-enhanced infrared ray with the DNA probe on the surface. Spectral substrate surface (among them, nucleic acid single strands of healthy samples and nucleic acid single strands that are not paired with DNA probes but are both new coronaviruses cannot be complementary to existing DNA probes, while nucleic acid single strands of new coronavirus samples can be compatible with existing DNA probes. The DNA probes are complementary to each other), after the solvent is naturally volatilized, it is rinsed repeatedly with ultrapure water to remove the nucleic acid single strands physically adsorbed on the surface, and it is blown dry with a N2 gun.
(3)将(2)中处理好的健康样本基底、新冠病毒样本基底、与DNA探针不配对但同为新冠病毒样本的基底、表面设有DNA探针的表面增强红外光谱基底、表面没有DNA探针 的表面增强红外光谱基底分别置于傅立叶红外光谱仪的红外显微镜之下，采集各自的红外透射光谱，采集光谱波长范围为1100-1800cm -1的红外指纹区，采集面积50μm*50μm。结果如图2所示，从图2可以看出各组(t-nCoV-N、t-non-nCoV、t-nCoV-ORF、p-nCoV-N)红外光谱之间无明显差异，肉眼无法分辨其差异。 (3) The healthy sample substrate processed in (2), the new coronavirus sample substrate, the substrate that is not paired with the DNA probe but is also the new coronavirus sample, the surface-enhanced infrared spectroscopy substrate with the DNA probe on the surface, and the surface without The surface - enhanced infrared spectroscopy substrates of the DNA probes were placed under the infrared microscope of the Fourier transform infrared spectrometer, respectively, and their respective infrared transmission spectra were collected. The results are shown in Figure 2. From Figure 2, it can be seen that there is no significant difference between the infrared spectra of each group (t-nCoV-N, t-non-nCoV, t-nCoV-ORF, p-nCoV-N), and the naked eye cannot distinguish the difference.
其中，none SH的信号用来与被巯基修饰的DNA探针做比较，证明所得到的红外信号来自与衬底相结合的DNA。Among them, the signal of none SH was used to compare with the thiol-modified DNA probe, which proved that the obtained infrared signal came from the DNA bound to the substrate.
p-nCoV-N的信号与t-nCoV-N、t-non-nCoV、t-nCoV-ORF的信号做比较，看表面设有DNA探针的表面增强红外光谱基底经过后处理之后红外光谱是否会有差异。The signal of p-nCoV-N is compared with the signals of t-nCoV-N, t-non-nCoV, and t-nCoV-ORF to see whether the surface-enhanced infrared spectroscopy substrate with DNA probes on the surface is post-treated. There will be differences.
以与DNA探针不配对但同为新冠病毒的核酸单链溶液为对照实验，虽然同属于新冠病毒核酸序列，但是不在配对区间就无法配对成功，更加说明DNA探针的特异性。The nucleic acid single-stranded solution that is not paired with the DNA probe but is also a new coronavirus is used as a control experiment. Although it belongs to the nucleic acid sequence of the new coronavirus, it cannot be successfully paired if it is not in the pairing range, which further shows the specificity of the DNA probe.
实施例3Example 3
将实施例2中采集的t-nCoV-N和p-nCoV-N的红外光谱数据进行平滑、基线校准、曲线均一化、二次微分处理，之后利用Origin中的PCASpectroscopy程序处理，得到如图3所示的结果。图3a为t-nCoV-N和p-nCoV-N组数据PCA分析的散点图，PC1和PC2代表总体差异最大的两个主成分，椭圆代表每组样品95％的置信区间。从图3a可以看出，t-nCoV-N和p-nCoV-N组之间可以区别开。图3b从上到下依次为t-nCoV-N的原始红外光谱、二次微分光谱、PC1的loading plot和PC2的loading plot。从图3b可以看出，二次微分光谱可以将原光谱中宽峰所掩盖的峰都显现出来，可以更容易分辨各个峰的位置。而在Loading plot中偏离0越远的位置代表此处红外光谱峰对于分辨两组数据差异的贡献越大。The infrared spectral data of t-nCoV-N and p-nCoV-N collected in Example 2 were processed by smoothing, baseline calibration, curve normalization, and quadratic differentiation, and then processed by the PCA Spectroscopy program in Origin, as shown in Figure 3 results shown. Figure 3a is a scatter plot of the PCA analysis of the t-nCoV-N and p-nCoV-N group data, PC1 and PC2 represent the two principal components with the largest overall difference, and the ellipses represent the 95% confidence interval for each group of samples. As can be seen from Figure 3a, a distinction can be made between the t-nCoV-N and p-nCoV-N groups. Figure 3b shows, from top to bottom, the original infrared spectrum of t-nCoV-N, the second differential spectrum, the loading plot of PC1 and the loading plot of PC2. It can be seen from Fig. 3b that the second differential spectrum can reveal all the peaks covered by the broad peaks in the original spectrum, which can make it easier to distinguish the position of each peak. The farther away from 0 in the Loading plot, the greater the contribution of the infrared spectral peaks to distinguishing the difference between the two groups of data.
将实施例2中采集的t-nCoV-ORF和p-nCoV-N的红外光谱数据进行平滑、基线校准、曲线均一化、二次微分处理，之后利用Origin中的PCASpectroscopy程序处理，得到如图4a所示的结果。图4a为t-nCoV-ORF和p-nCoV-N组数据PCA分析的散点图，PC1和PC2代表总体差异最大的两个主成分，椭圆代表每组样品95％的置信区间。从图4a可以看出t-nCoV-ORF和p-nCoV-N组之间不能区别开。将实施例2中采集的t-nCoV-ORF和t-nCoV-N的红外光谱数据进行平滑、基线校准、曲线均一化、二次微分处理，之后利用Origin中的PCASpectroscopy程序处理，得到如图4b所示的结果。图4b为t-nCoV-ORF和t-nCoV-N组数据PCA分析的散点图，PC1和PC2代表总体差异最大的两个主成分，椭圆代表每组样品95％的置信区间。从图4b可以看出t-nCoV-ORF和t-nCoV-N组之间可以区别开。将实施例2中采集的t-nCoV-ORF、p-nCoV-N和t-nCoV-N的红外光谱数据进行平滑、基线校准、曲线均一化、二次微分处理， 之后利用Origin中的PCASpectroscopy程序处理，得到如图4c所示的结果。图4c为t-nCoV-ORF、p-nCoV-N和t-nCoV-N组数据PCA分析的散点图，PC1和PC2代表总体差异最大的两个主成分，椭圆代表每组样品95％的置信区间。从图4c可以看出t-nCoV-N和t-nCoV-ORF、p-nCoV-N组之间可以区别开，t-nCoV-ORF和p-nCoV-N组之间不能区别开。The infrared spectral data of t-nCoV-ORF and p-nCoV-N collected in Example 2 were processed by smoothing, baseline calibration, curve normalization, and quadratic differentiation, and then processed by the PCA Spectroscopy program in Origin, as shown in Figure 4a results shown. Figure 4a is a scatter plot of the PCA analysis of the t-nCoV-ORF and p-nCoV-N group data, PC1 and PC2 represent the two principal components with the largest overall difference, and the ellipses represent the 95% confidence interval for each group of samples. From Figure 4a, it can be seen that the t-nCoV-ORF and p-nCoV-N groups cannot be distinguished. The infrared spectral data of t-nCoV-ORF and t-nCoV-N collected in Example 2 were processed by smoothing, baseline calibration, curve normalization, and quadratic differentiation, and then processed by the PCA Spectroscopy program in Origin, as shown in Figure 4b results shown. Figure 4b is a scatter plot of the PCA analysis of the t-nCoV-ORF and t-nCoV-N group data, PC1 and PC2 represent the two principal components with the largest overall difference, and the ellipses represent the 95% confidence interval for each group of samples. From Figure 4b it can be seen that the t-nCoV-ORF and t-nCoV-N groups can be distinguished. The infrared spectral data of t-nCoV-ORF, p-nCoV-N and t-nCoV-N collected in Example 2 were processed for smoothing, baseline calibration, curve normalization, and quadratic differentiation, and then the PCASpectroscopy program in Origin was used processing, and the result shown in Figure 4c is obtained. Figure 4c is a scatter plot of the PCA analysis of the t-nCoV-ORF, p-nCoV-N and t-nCoV-N group data, PC1 and PC2 represent the two principal components with the largest overall difference, and the ellipses represent 95% of the samples in each group. confidence interval. It can be seen from Figure 4c that the t-nCoV-N and t-nCoV-ORF and p-nCoV-N groups can be distinguished, but the t-nCoV-ORF and p-nCoV-N groups cannot be distinguished.
将实施例2中采集的t-non-nCoV和p-nCoV-N的红外光谱数据进行平滑、基线校准、曲线均一化、二次微分处理，之后利用Origin中的PCASpectroscopy程序处理，得到如图5a所示的结果。图5a为t-non-nCoV和p-nCoV-N组数据PCA分析的散点图，PC1和PC2代表总体差异最大的两个主成分，椭圆代表每组样品95％的置信区间。从图5a可以看出t-non-nCoV和p-nCoV-N组之间不能区别开。将实施例2中采集的t-non-nCoV和t-nCoV-N的红外光谱数据进行平滑、基线校准、曲线均一化、二次微分处理，之后利用Origin中的PCASpectroscopy程序处理，得到如图5b所示的结果。图5b为t-non-nCoV和t-nCoV-N组数据PCA分析的散点图，PC1和PC2代表总体差异最大的两个主成分，椭圆代表每组样品95％的置信区间。从图5b可以看出t-non-nCoV和t-nCoV-N组之间可以区别开。将实施例2中采集的t-non-nCoV、p-nCoV-N和t-nCoV-N的红外光谱数据进行平滑、基线校准、曲线均一化、二次微分处理，之后利用Origin中的PCASpectroscopy程序处理，得到如图5c所示的结果。图5c为t-non-nCoV、p-nCoV-N和t-nCoV-N组数据PCA分析的散点图，PC1和PC2代表总体差异最大的两个主成分，椭圆代表每组样品95％的置信区间。从图4c可以看出t-nCoV-N和t-non-nCoV、p-nCoV-N组之间可以区别开，t-non-nCoV和p-nCoV-N组之间不能区别开。The infrared spectral data of t-non-nCoV and p-nCoV-N collected in Example 2 were processed by smoothing, baseline calibration, curve normalization, and quadratic differentiation, and then processed by the PCA Spectroscopy program in Origin, as shown in Figure 5a. results shown. Figure 5a is a scatter plot of the PCA analysis of the t-non-nCoV and p-nCoV-N group data, PC1 and PC2 represent the two principal components with the largest overall difference, and the ellipses represent the 95% confidence interval of each group of samples. From Figure 5a, it can be seen that no distinction can be made between the t-non-nCoV and p-nCoV-N groups. The infrared spectral data of t-non-nCoV and t-nCoV-N collected in Example 2 were processed by smoothing, baseline calibration, curve normalization, and quadratic differentiation, and then processed by the PCA Spectroscopy program in Origin, as shown in Figure 5b results shown. Figure 5b is a scatter plot of the PCA analysis of the t-non-nCoV and t-nCoV-N group data, PC1 and PC2 represent the two principal components with the largest overall difference, and the ellipses represent the 95% confidence interval of each group of samples. From Figure 5b, it can be seen that the t-non-nCoV and t-nCoV-N groups can be distinguished. The infrared spectral data of t-non-nCoV, p-nCoV-N, and t-nCoV-N collected in Example 2 were smoothed, baseline calibrated, curve normalized, and second-differentiated, and then processed using the PCASpectroscopy program in Origin processing, and the result shown in Figure 5c is obtained. Figure 5c is the scatter plot of the PCA analysis of the t-non-nCoV, p-nCoV-N and t-nCoV-N group data, PC1 and PC2 represent the two principal components with the largest overall difference, and the ellipses represent 95% of the samples in each group. confidence interval. It can be seen from Figure 4c that the t-nCoV-N and t-non-nCoV and p-nCoV-N groups can be distinguished, but the t-non-nCoV and p-nCoV-N groups cannot be distinguished.
综上，本发明的方法可以成功区分病毒样本与健康样本。In conclusion, the method of the present invention can successfully distinguish virus samples from healthy samples.
以上所述仅为本发明的具体实施方式，不是全部的实施方式，本领域普通技术人员通过阅读本发明说明书而对本发明技术方案采取的任何等效的变换，均为本发明的权利要求所涵盖。The above descriptions are only specific embodiments of the present invention, not all of the embodiments. Any equivalent transformations to the technical solutions of the present invention that are taken by those of ordinary skill in the art by reading the description of the present invention are covered by the claims of the present invention. .

Claims (10)

  1. 一种基于表面增强红外光谱和主成分分析的新冠病毒核酸检测模型的构建方法，其特征在于，包括以下步骤：A method for constructing a new coronavirus nucleic acid detection model based on surface-enhanced infrared spectroscopy and principal component analysis, comprising the following steps:
    (1)材料的准备：表面设有DNA探针的表面增强红外光谱基底、健康样本的核酸单链溶液和新冠病毒样本的核酸单链溶液，所述DNA探针的核苷酸序列如SEQ ID NO：1所示；(1) Preparation of materials: surface-enhanced infrared spectroscopy substrates with DNA probes on the surface, nucleic acid single-stranded solutions of healthy samples and nucleic acid single-stranded solutions of new coronavirus samples, the nucleotide sequences of the DNA probes are as shown in SEQ ID NO: shown in 1;
    其中，所述新冠病毒样本的核酸单链为与DNA探针互补配对的新冠病毒互补核酸单链，所述健康样本的核酸单链为与DNA探针不配对且不属于新冠病毒序列的单链；Wherein, the nucleic acid single strand of the new coronavirus sample is a new coronavirus complementary nucleic acid single strand that is complementary to the DNA probe, and the nucleic acid single strand of the healthy sample is a single strand that does not pair with the DNA probe and does not belong to the new coronavirus sequence ;
    (2)将健康样本的核酸单链溶液与新冠病毒样本的核酸单链溶液分别滴在表面设有DNA探针的表面增强红外光谱基底表面，待其溶剂自然挥发后，用超纯水反复润洗，除去表面物理吸附的核酸单链；(2) Drop the nucleic acid single-stranded solution of the healthy sample and the nucleic acid single-stranded solution of the new coronavirus sample respectively on the surface of the surface-enhanced infrared spectroscopy substrate with the DNA probe on the surface. After the solvent is naturally volatilized, it is repeatedly moistened with ultrapure water. Washing to remove nucleic acid single strands physically adsorbed on the surface;
    (3)红外透射光谱的采集：采集光谱波长范围为1100-1800cm -1的红外指纹区； (3) Collection of infrared transmission spectrum: the infrared fingerprint region with the wavelength range of 1100-1800 cm -1 is collected;
    (4)光谱预处理：将所采集的红外透射光谱进行平滑、基线校准、曲线均一化、二次微分处理；(4) Spectral preprocessing: smoothing, baseline calibration, curve normalization, and quadratic differential processing of the collected infrared transmission spectrum;
    (5)判别模型的建立：利用PCA Spectroscopy程序处理(4)中的样本数据，得到健康、新冠病毒的主成分分析模型。(5) Establishment of the discriminant model: The PCA Spectroscopy program was used to process the sample data in (4) to obtain the principal component analysis model of health and new coronavirus.
  2. 根据权利要求1所述的基于表面增强红外光谱和主成分分析的新冠病毒核酸检测模型的构建方法，其特征在于，所述表面设有DNA探针的表面增强红外光谱基底包括红外透明的衬底，所述红外透明的衬底表面设有金膜，所述金膜的表面设有DNA探针。The method for constructing a new coronavirus nucleic acid detection model based on surface-enhanced infrared spectroscopy and principal component analysis according to claim 1, wherein the surface-enhanced infrared spectroscopy substrate with DNA probes on the surface comprises an infrared transparent substrate , the surface of the infrared transparent substrate is provided with a gold film, and the surface of the gold film is provided with a DNA probe.
  3. 根据权利要求2所述的基于表面增强红外光谱和主成分分析的新冠病毒核酸检测模型的构建方法，其特征在于，所述红外透明的衬底选自氟化钙、硅片、蓝宝石。The method for constructing a new coronavirus nucleic acid detection model based on surface-enhanced infrared spectroscopy and principal component analysis according to claim 2, wherein the infrared transparent substrate is selected from calcium fluoride, silicon wafer, and sapphire.
  4. 根据权利要求2所述的基于表面增强红外光谱和主成分分析的新冠病毒核酸检测模型的构建方法，其特征在于，所述金膜的厚度为10-20nm。The method for constructing a new coronavirus nucleic acid detection model based on surface-enhanced infrared spectroscopy and principal component analysis according to claim 2, wherein the thickness of the gold film is 10-20 nm.
  5. 根据权利要求2-4任一项所述的基于表面增强红外光谱和主成分分析的新冠病毒核酸检测模型的构建方法，其特征在于，所述表面设有DNA探针的表面增强红外光谱基底的制备方法包括以下步骤：The method for constructing a new coronavirus nucleic acid detection model based on surface-enhanced infrared spectroscopy and principal component analysis according to any one of claims 2-4, wherein the surface of the surface-enhanced infrared spectroscopy substrate with DNA probes is provided on the surface. The preparation method includes the following steps:
    1)在红外透明的衬底上蒸镀金膜，形成随机分布的金纳米颗粒结构；1) Evaporating a gold film on an infrared transparent substrate to form a randomly distributed gold nanoparticle structure;
    2)将步骤1)所得衬底置于被巯基修饰的DNA探针溶液中，使DNA探针与金结合形成金-硫共价键，在金表面生层单分子自组装层，得到所述表面设有DNA探针的表面增强红外光谱基底。2) placing the substrate obtained in step 1) in a DNA probe solution modified by sulfhydryl groups, combining the DNA probe with gold to form a gold-sulfur covalent bond, and forming a monomolecular self-assembly layer on the gold surface to obtain the Surface-enhanced infrared spectroscopy substrate with DNA probes on the surface.
  6. 根据权利要求5所述的基于表面增强红外光谱和主成分分析的新冠病毒核酸检测模型的构建方法，其特征在于，所述步骤1)前还包括红外透明的衬底的预处理；The method for constructing a new coronavirus nucleic acid detection model based on surface-enhanced infrared spectroscopy and principal component analysis according to claim 5, characterized in that, before the step 1), preprocessing of the infrared transparent substrate is further included;
    优选地，所述红外透明的衬底的预处理为将红外透明的衬底依次置于丙酮、无水乙醇和超纯水之中清洗，然后用N 2枪吹干。 Preferably, the pretreatment of the infrared transparent substrate is to wash the infrared transparent substrate in acetone, absolute ethanol and ultrapure water in sequence, and then blow dry with a N 2 gun.
  7. 根据权利要求1所述的基于表面增强红外光谱和主成分分析的新冠病毒核酸检测模型的构建方法，其特征在于，所述新冠病毒样本的核酸单链的核苷酸序列如SEQ ID NO：2所示。The method for constructing a new coronavirus nucleic acid detection model based on surface-enhanced infrared spectroscopy and principal component analysis according to claim 1, wherein the nucleotide sequence of the nucleic acid single strand of the new coronavirus sample is as shown in SEQ ID NO: 2 shown.
  8. 根据权利要求1所述的基于表面增强红外光谱和主成分分析的新冠病毒核酸检测模型的构建方法，其特征在于，所述健康样本的核酸单链的核苷酸序列如SEQ ID NO：3所示。The method for constructing a new coronavirus nucleic acid detection model based on surface-enhanced infrared spectroscopy and principal component analysis according to claim 1, wherein the nucleotide sequence of the nucleic acid single strand of the healthy sample is as shown in SEQ ID NO: 3 Show.
  9. 根据权利要求1所述的基于表面增强红外光谱和主成分分析的新冠病毒核酸检测模型的构建方法，其特征在于，所述模型用于检测新冠病毒核酸的方法包括以下步骤：The method for constructing a new coronavirus nucleic acid detection model based on surface-enhanced infrared spectroscopy and principal component analysis according to claim 1, wherein the method for detecting the new coronavirus nucleic acid by the model comprises the following steps:
    (1)将待测样品、健康样本的核酸单链溶液与新冠病毒样本的核酸单链溶液分别滴在表面设有DNA探针的表面增强红外光谱基底表面，待其溶剂自然挥发后，用超纯水反复润洗；(1) Drop the nucleic acid single-stranded solution of the sample to be tested, the healthy sample and the nucleic acid single-stranded solution of the new coronavirus sample respectively on the surface of the surface-enhanced infrared spectroscopy substrate with the DNA probe on the surface. Repeated rinsing with pure water;
    (2)采集红外透射光谱：采集光谱波长范围为1100-1800cm-1的红外指纹区；(2) Collect infrared transmission spectrum: collect the infrared fingerprint region with the wavelength range of 1100-1800cm-1;
    (3)光谱预处理：将所采集的红外透射光谱进行平滑、基线校准、曲线均一化、二次微分处理；(3) Spectral preprocessing: smoothing, baseline calibration, curve normalization, and quadratic differential processing of the collected infrared transmission spectrum;
    (4)利用PCA Spectroscopy程序一起处理(3)中待测样品、权利要求1中新冠病毒样本和健康样本的数据，从PCA分析图中判断待测样品落在新冠病毒样本的置信区间内还是健康样本的置信区间内，进而得到待测样品健康、新冠病毒的类别。(4) Use the PCA Spectroscopy program to process the data of the sample to be tested in (3), the new coronavirus sample in claim 1 and the healthy sample, and determine whether the sample to be tested falls within the confidence interval of the new coronavirus sample or is healthy from the PCA analysis chart Within the confidence interval of the sample, the health of the sample to be tested and the category of the new coronavirus can be obtained.
  10. 一种基于表面增强红外光谱和主成分分析的新冠病毒核酸检测试剂盒，其特征在于，包括表面设有DNA探针的表面增强红外光谱基底，所述DNA探针的核苷酸序列如SEQ ID NO：1所示；A new coronavirus nucleic acid detection kit based on surface-enhanced infrared spectroscopy and principal component analysis, characterized in that it comprises a surface-enhanced infrared spectroscopy substrate with a DNA probe on the surface, and the nucleotide sequence of the DNA probe is such as SEQ ID NO: shown in 1;
    优选地，还包括健康样本的核酸单链溶液和新冠病毒样本的核酸单链溶液，所述健康样本的核酸单链为与DNA探针不配对且不属于新冠病毒序列的单链，所述新冠病毒样本的核酸单链为与DNA探针互补配对的新冠病毒互补核酸单链；Preferably, the nucleic acid single-stranded solution of the healthy sample and the nucleic acid single-stranded solution of the new coronavirus sample are also included. The nucleic acid single strand of the virus sample is the new coronavirus complementary nucleic acid single strand that is complementary to the DNA probe;
    优选地，所述新冠病毒样本的核酸单链的核苷酸序列如SEQ ID NO：2所示；Preferably, the nucleotide sequence of the nucleic acid single strand of the new coronavirus sample is shown in SEQ ID NO: 2;
    优选地，所述健康样本的核酸单链的核苷酸序列如SEQ ID NO：3所示。Preferably, the nucleotide sequence of the nucleic acid single strand of the healthy sample is shown in SEQ ID NO: 3.
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