CN114085930A - SERS detection kit and method for detecting SARS-CoV-2 nucleic acid - Google Patents
SERS detection kit and method for detecting SARS-CoV-2 nucleic acid Download PDFInfo
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Abstract
The invention discloses a SERS detection kit and a method for detecting SARS-CoV-2 nucleic acid, wherein the SERS detection kit comprises a SERS detection chip, a first reagent, a second reagent and a third reagent. The SERS detection kit provided by the invention can realize rapid, specific and high-sensitivity detection of SARS-CoV-2 nucleic acid by detecting the Raman signal of the SERS probe on the SERS detection chip through the mutual cooperation of the SERS detection chip, the first reagent, the second reagent and the third reagent, and can be further applied to rapid, more sensitive, multi-channel and on-site instant virus nucleic acid detection.
Description
Technical Field
The invention belongs to the technical field of functional nano materials and biological detection, and particularly relates to a Surface Enhanced Raman Scattering (SERS) detection kit for detecting SARS-CoV-2 nucleic acid, a preparation method thereof, and a method for detecting SARS-CoV-2 nucleic acid based on SERS.
Background
Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) is a newly discovered infectious coronavirus, which causes a pandemic of novel coronavirus pneumonia (COVID-19), and has posed a great threat to human life and health. Therefore, early diagnosis of COVID-19 remains an important approach to preventing infection at this stage before effective drugs and vaccines are developed to suppress the prevalence of COVID-19. Furthermore, given that COVID-19 infection can spread within a latent period without overt symptoms, rapid and early detection of SARS-CoV-2 plays a crucial role for screening and diagnosis. Compared to serological detection of virus-specific antibodies (IgM/IgG), molecular diagnosis of SARS-CoV-2 RNA wiped from the throat or nasal cavity of a patient has been approved for use, enabling early detection of viral infection.
Currently, common detection methods for SARS-CoV-2 nucleic acid are developed based on enzyme-mediated nucleic acid amplification strategies such as real-time Polymerase Chain Reaction (PCR), loop-mediated isothermal amplification (LAMP), and Recombinase Polymerase Amplification (RPA). Among them, the real-time fluorescence PCR detection is the most widely used SARS-CoV-2 RNA detection technology at present, and it realizes high-sensitivity detection by continuous amplification of target nucleic acid under the action of polymerase.
Although the technology has great success in the aspect of virus nucleic acid detection, the technology still has obvious defects and limitations in the aspects of reaction condition control, experimental operation and the like, so that the technology has the problems of high detection cost, long time consumption and the like. However, because COVID-19 can propagate rapidly in a short time, long backlog tests can produce unpredictable results for the spread of a pandemic. In addition, the general detection limit of the commercial fluorescent PCR technology is higher than 200 copies/mL, so that the development of a novel ultra-fast and ultra-high sensitive detection method has important significance for realizing rapid molecular diagnosis of ultra-low analytes in an early detection window. Surface Enhanced Raman Scattering (SERS) with single molecule level sensitivity is considered to be an effective analytical means for high sensitivity determination of trace species, and can provide a possible solution for rapid and ultrasensitive detection to meet the demanding requirements of virus screening in early detection windows.
Therefore, the PCR technology based on the enzyme-mediated nucleic acid amplification strategy has the defects of detection sensitivity, specificity, timeliness, portable operability and the like, and the SARS-CoV-2 nucleic acid detection method which is quicker, more specific and more sensitive is very necessary to be provided.
Disclosure of Invention
Based on the above, one of the objectives of the present invention is to provide a SERS assay kit for detecting SARS-CoV-2 nucleic acid, which has the advantages of short time, high sensitivity and strong specificity when used for detecting SARS-CoV-2 nucleic acid.
The specific technical scheme for realizing the aim of the invention comprises the following steps:
a SERS detection kit for detecting SARS-CoV-2 nucleic acids, the SERS detection kit comprising:
(1) SERS detection chip
The SERS detection chip is a silver nanorod array substrate with a surface modified with a single-chain capture shown in SEQ ID NO. 4;
(2) a first reagent
The first reagent is obtained by mixing an auxiliary single strand with a sequence shown as SEQ ID NO.1 and a hairpin type DNA single strand H1 with a sequence shown as SEQ ID NO.2 and annealing at 90-95 ℃; the concentration ratio of the auxiliary single strand to the hairpin type DNA single strand H1 is 1: 0.8 to 2;
(3) a second reagent
The second reagent is a hairpin type DNA single-chain H2 with the sequence shown in SEQ ID NO. 3;
(4) a third reagent
The third reagent is gold nanoparticles with the surface modified with a probe single chain with a sequence shown as SEQ ID NO.5 and 5,5 '-dithiobis (2-nitrobenzoic acid), and the concentration ratio of the probe single chain to the 5, 5' -dithiobis (2-nitrobenzoic acid) is 1: 1 to 3.
In some embodiments, the silver nanorod array substrate comprises 3 × 10 array-type small holes, and each small hole has a hole diameter of 3 mm to 5mm and a depth of 0.8 mm to 1.2 mm.
In some of these embodiments, the concentration ratio of the auxiliary single strand to the hairpin DNA single strand H1 is 1: 1 to 1.25.
In some of these embodiments, the working concentration of the first agent is between 5 μ M and 10 μ M; the working concentration of the second reagent is 5-20 mu M; the working concentration of the third reagent is 0.1 nM-10 nM.
In some of these embodiments, the working concentration of the third agent is 3.2 nM to 3.4 nM.
In some of the embodiments, the gold nanoparticles have a particle size of 15nm to 100 nm.
In some of these embodiments, the gold nanoparticles have a particle size of 25 nm to 35 nm.
The invention also provides a preparation method of the SERS detection kit for detecting SARS-CoV-2 nucleic acid.
The specific technical scheme for realizing the aim of the invention comprises the following steps:
a preparation method of SERS detection kit for detecting SARS-CoV-2 nucleic acid comprises the following steps:
(1) preparing SERS detection chip
According to the mol ratio of 1: mixing a capture single chain with the concentration of 500 nM-2000 nM and the sequence shown in SEQ ID NO.4 with a tri-carboxyethyl phosphine solution in a proportion of 100-1000, reacting for 4-12 hours in a mixing instrument at a constant temperature of 25-37 ℃, co-culturing with a silver nanorod array substrate for 3-5 hours, and cleaning with a TM buffer solution to obtain the nano-silver-coated nano-rod array substrate; the co-culture conditions are as follows: the temperature is 25-37 ℃, and the humidity is 60-80%;
(2) preparing the first reagent
The auxiliary single strand with the sequence shown as SEQ ID NO.1 and the hairpin type DNA single strand H1 with the sequence shown as SEQ ID NO.2 are mixed according to the concentration ratio of 1: mixing the raw materials in a ratio of 0.8-2, and annealing at 90-95 ℃ for 4-6 min to obtain the product;
(3) preparing the second reagent
Designing and synthesizing a hairpin DNA single strand H2 with a sequence shown in SEQ ID NO.3 according to the hairpin DNA single strand H1;
(4) preparing a third reagent
According to the mol ratio of 1: mixing a probe single chain with a sequence shown as SEQ ID NO.5 with a tricarboxyethylphosphine solution according to a ratio of 100-1000, reacting in a constant-temperature mixing instrument at 25-37 ℃ for 4-12 hours, and mixing 8-12 mu L of 10 mu M-100 mu M probe single chain and 450-550 mu L of 0.1 nM-10 nM gold nanoparticle solution in a TBE solution for overnight culture; adding NaCl solution in batches until the final concentration of NaCl in the mixture is 160-200 mM, and co-culturing overnight; adding 8-12 mul of 5, 5' -dithiobis (2-nitrobenzoic acid) to react for 2.5-3.5 hours; centrifuging to remove supernatant, dispersing the centrifugal sediment by using a TBE solution and fixing the volume to obtain the product, wherein the concentration ratio of the 5, 5' -dithiobis (2-nitrobenzoic acid) to the single chain of the probe is 1-3: 1.
in some embodiments, the single capture strand concentration in step (1) is 500nM to 1000 nM.
In some embodiments, in the step (1) of preparing the SERS detection chip, after the TM buffer washing, the method further includes: and (3) dropwise adding 15-25 mu L of 0.5-2 mM 6-mercaptohexanol to the surface of the silver nanorod array, and reacting in a constant-temperature mixing instrument at 25-37 ℃ for 8-12 minutes.
The invention also provides a method for detecting SARS-CoV-2 nucleic acid based on surface enhanced Raman scattering for non-diagnosis purpose, which can simply and quickly detect whether the sample to be detected contains SARS-CoV-2 nucleic acid.
A method for detecting SARS-CoV-2 nucleic acid for non-diagnostic purposes based on surface enhanced raman scattering, comprising the steps of:
(1) mixing a sample to be detected with a first reagent, a second reagent and a third reagent, then dropwise adding the mixture to the surface of the SERS detection chip, and culturing at 25-37 ℃ at 200-400 rpm for 40-60 minutes;
(2) cleaning an SERS detection chip by ultrapure water, carrying out SERS detection on the SERS detection chip to obtain an SERS spectrum and a characteristic peak signal intensity value thereof, and calculating the concentration of SARS-CoV-2 nucleic acid in a sample to be detected according to a working curve;
the working curve is drawn by the following steps:
(a) the first reagent, the second reagent, the third reagent and the concentration are respectively 102copies/mL、103copies/mL、104copies/mL、105copies/mL、106Mixing copies/mLSARS-CoV-2 nucleic acid standard solution, dropwise adding the mixture to the surface of an SERS detection chip, and culturing at 25-37 ℃ at 200-400 rpm for 40-60 minutes;
(b) cleaning an SERS detection chip by ultrapure water, and performing SERS detection on the SERS detection chip to obtain SERS spectra corresponding to SARS-CoV-2 nucleic acid standard solutions with different concentrations and characteristic peak signal intensity values thereof;
(c) and drawing a working curve by taking the logarithm of the concentration of the SARS-CoV-2 nucleic acid standard solution as an abscissa and the ratio of the characteristic peak signal intensity of the SARS-CoV-2 nucleic acid standard solution to the blank sample (TM buffer solution) as an ordinate.
Compared with the prior art, the invention has the following beneficial effects:
1. the kit comprises a SERS detection chip, a first reagent, a second reagent and a third reagent; the SERS detection chip is a silver nanorod array (namely a substrate) with a capture single chain C modified on the surface, and the capture single chain C is fixed on the surface of the silver nanorod array (namely the substrate) through a covalent bond formed by sulfydryl and silver; the first reagent is a detection probe and is formed by mixing and annealing an auxiliary single-stranded primer and a hairpin type DNA single-stranded H1; the second reagent is a hairpin DNA single strand H2 synthesized according to H1; the third reagent is an SERS probe which is gold nanoparticles modified with a probe single chain P and Raman signal molecules. Mixing a first reagent, a second reagent, a third reagent and a sample to be detected, then dripping the mixture on the surface of a SERS detection chip, if a SARS-CoV-2 nucleic acid chain exists in the sample to be detected, H1 of the first reagent is triggered by the SARS-CoV-2 nucleic acid chain to open the first reagent partially, the SARS-CoV-2 nucleic acid chain is hybridized with H1, the second reagent H2 can compete with the SARS-CoV-2 nucleic acid chain hybridized with H1 and the auxiliary single-stranded primer for hybridization, the SARS-CoV-2 nucleic acid chain and the H1-H2 duplex are released, the SARS-CoV-2 nucleic acid chain is released for recycling (thereby realizing high-sensitivity detection of SARS-CoV-2 nucleic acid), a plurality of auxiliary single-stranded primers are released from the first reagent, a DNA fragment of the released auxiliary single-stranded primer can be captured by the SERS detection chip through specific hybridization with a capture probe C, the other DNA fragment of the auxiliary single-stranded primer can further capture the SERS probe. Therefore, the SERS detection kit provided by the invention can realize rapid and specific (can effectively distinguish low-concentration SARS-CoV-2 RNA from other virus RNA) and high-sensitivity (the sensitivity can reach 51.38 copies/mL) detection on SARS-CoV-2 nucleic acid by the mutual matching of the SERS detection chip, the first reagent, the second reagent and the third reagent and by detecting the Raman signal of the SERS probe on the SERS detection chip.
2. The method for detecting SARS-CoV-2 nucleic acid by using the kit of the invention has simple operation, does not need amplification, can realize detection within 1.5 hours by using a portable Raman spectrometer, and can be further applied to quicker, more sensitive, multi-channel and on-site instant virus nucleic acid detection.
Drawings
FIG. 1 is a schematic diagram of the surface enhanced Raman scattering detection kit for SARS-CoV-2 nucleic acid detection according to the present invention.
FIG. 2 is an optimization experiment of the first reagent (detection probe) in which the auxiliary single-stranded primer was assembled with the hairpin type DNA single-stranded H1 in test example 1 of the present invention.
FIG. 3 is an optimization experiment of the concentration of single-chain C captured by SERS detection chip surface immobilization in experimental example 2 of the present invention.
FIG. 4 shows SERS intensities obtained by blocking 6-Mercaptohexanol (MCH) on the surface of a SERS detection chip in experimental example 2 of the present invention for different times.
FIG. 5 is a time-optimized assay for detecting SARS-CoV-2 nucleic acid using the SERS assay kit of Experimental example 3 of the present invention.
FIG. 6 is a working curve of SERS detection kit in test example 4 of the present invention for detecting SARS-CoV-2 nucleic acid of different concentrations.
FIG. 7 shows the specificity of SERS detection kit for detecting SARS-CoV-2 nucleic acid in test example 5 of the present invention.
FIG. 8 shows the uniformity of SARS-CoV-2 nucleic acid detected by SERS assay kit in test example 6 of the present invention.
FIG. 9 shows the stability of SERS detection kit for detecting SARS-CoV-2 nucleic acid in test example 7 of the present invention.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
In one aspect of the present invention, there is provided a surface enhanced Raman Scattering detection kit for detecting SARS-CoV-2 nucleic acid, comprising:
1. SERS detection chip
The SERS detection chip isThe method comprises the following steps of taking a silver nanorod array as a substrate, modifying a capturing single chain C on the surface of the substrate, fixing the capturing single chain C on the surface of the substrate through a covalent bond formed by sulfydryl and silver, and then dropwise adding 6-Mercaptohexanol (MCH) on the surface of the substrate for sealing to reduce nonspecific adsorption of the substrate, thereby obtaining the SERS detection chip. In the present invention, the silver nanorod array is prepared by a vacuum electron beam evaporation coating technique (based on documents c.y. Song, j.l. Abell, y.p. He, s.h. Murph, y.p. Cui, y.p. zhao, Gold-modified silver nanoarray: growth dynamics and improved SERS properties, Journal of Materials Chemistry, 2012, 22(3): 1150-1159, the preparation comprises: AgNR array substrates were prepared by Oblique Angle Deposition (OAD), loading a clean glass slide (3 inch × 1 inch) into a vacuum deposition chamber, the substrate being perpendicular to the incident vapor direction first, 20 nm Ti and 200 Ag films were deposited sequentially at 0.2 nm/s and 0.3 nm/s respectively, then the substrate normal was rotated 86 degrees with respect to the incident vapor direction, AgNRs with a thickness of 3000 Ag film deposited at a rate of 0.3 nm/s to make arrays the entire evaporation process is under high vacuum conditions (.<3×10-6Torr)), and encapsulating the surface with a Polydimethylsiloxane (PDMS) film, wherein the PDMS film comprises 3 x 10 array type pores, the aperture of each pore is 3 mm-5 mm, and the depth of each pore is 0.8 mm-1.2 mm.
2. A first reagent (i.e., a detection probe) consisting of a mixture of two probes at a concentration ratio of 1: mixing 0.8-2 of the auxiliary single-stranded primer with the hairpin DNA single-strand H1, and annealing at 90-95 ℃ for 4-6 min to obtain the final product. Preferably, the concentration ratio of the auxiliary single-stranded primer to the hairpin DNA single strand H1 is 1: 1 to 1.25. The working concentration of the first reagent is 5-10 mu M, and the preferable working concentration is 5 mu M.
3. And a second reagent (namely, a hairpin DNA single strand H2), wherein the hairpin DNA single strand H2 is designed and synthesized according to the hairpin DNA single strand H1, and the working concentration of the second reagent is 5-20 mu M, and the preferred working concentration is 10 mu M.
4. Third reagent (i.e. SERS probe)
The third reagent is a SERS probe used in cooperation with the SERS detection chip, and the SERS probe is modified with a probe single-chain P and a Raman molecule 5, 5' -dithiobis (2-nitrobenzoic acid) (DTNB) on the surface of the gold nanoparticle (generally, the enhancement effect of SERS is not only related to the particle size of the gold nanoparticle, but also influenced by other factors (such as particle stability and the like), and the larger the particle size of the gold nanoparticle is, the stronger the enhancement effect of SERS is, the maximum value of the enhancement effect of SERS can be achieved by the gold nanoparticle within a certain size and density range, the particle size is 15 nm-100 nm, and preferably 30 nm). In the process of preparing the third reagent, the concentration of the used probe single strand is 10-100 mu M, and the concentration of the Raman molecule is 1-3 times of the concentration of the probe single strand. The working concentration of the third reagent is 0.1 nM-10 nM, preferably 3.3 nM.
The DNA nucleotide sequence fragments used above were all obtained by artificial synthesis and synthesized by Takara Biotechnology, Inc. (Chinese Dalian). The base sequences of the auxiliary single-stranded primer, the hairpin type DNA single strands H1 and H2, the capture single strand C and the probe single strand P are respectively as follows:
helper single strand primer (SEQ ID NO. 1):
5’-GCAGTAGGCCCGTCTGCCGT-3’
hairpin DNA single strand H1(SEQ ID No. 2):
5’-TTTTTTATACCGCAGACGGTACAGACTGTGCAAAGTCGCTGC ACAGTCTGTAGCCTACTGC-3’
hairpin DNA single strand H2(SEQ ID No. 3):
5’-TACAGACTGTGCAGCGACTTTGCACAGTCTGTACAGTCTGCG GAGACTGTGCAAAGTCGCTG-3’
capture single strand C (SEQ ID No. 4):
5’-SH-(CH2)6-TTTTTTTTTACGGCAGACG-3’
probe single-stranded P (SEQ ID NO. 5):
5’-GGCCTACTGCTTTTTTTTTTTT-SH-(CH2)6-3’。
please refer to FIG. 1, which is a schematic diagram of the surface enhanced Raman scattering detection kit for SARS-CoV-2 nucleic acid detection according to the present invention. Mixing a first reagent (detection probe), a second reagent, a third reagent (SERS probe) and a sample to be detected, and then dropwise adding the mixture on the surface of the SERS detection chip. If the sample to be detected contains SARS-CoV-2 nucleic acid, the H1 of the first reagent is triggered to open by the SARS-CoV-2 nucleic acid chain, so that the first reagent is partially opened, and the SARS-CoV-2 nucleic acid chain is hybridized with H1; the second reagent can compete with the SARS-CoV-2 nucleic acid chain hybridized on H1 and the auxiliary single-stranded primer for hybridization, the SARS-CoV-2 nucleic acid chain and the H1-H2 duplex are released, the released SARS-CoV-2 nucleic acid chain can be recycled, so that a plurality of auxiliary single-stranded primers are released from the detection probe, one piece of DNA of the released auxiliary single-stranded primers can be captured by the SERS detection chip through specific hybridization with the capture probe C, and the other piece of DNA of the primers on the chip can further capture the SERS probe. Therefore, by detecting the Raman signal of the SERS probe on the SERS detection chip, the rapid, specific and high-sensitivity detection of SARS-CoV-2 nucleic acid is realized.
The present invention will be described in further detail with reference to the following specific embodiments and the accompanying drawings.
EXAMPLE 1 preparation of surface enhanced Raman Scattering assay kit for SARS-CoV-2 nucleic acid detection
The SERS detection kit for SARS-CoV-2 nucleic acid detection of this embodiment comprises:
1. SERS detection chip
The method is characterized in that a silver nanorod array is used as a substrate, a capture single chain C with the concentration of 500nM is modified on the surface of the substrate, the silver nanorod array substrate comprises 3 x 10 array type small holes, the aperture of each small hole is 4 mm, and the depth of each small hole is 1 mm.
2. First reagent (i.e., detection probe)
It is prepared from the following components in a concentration ratio of 1: 1 and mixing the auxiliary single-chain primer with the hairpin type DNA single-chain H1, and annealing at 90-95 ℃ for 5min to obtain the first reagent with the working concentration of 5 mu M.
3. Second reagent (i.e., hairpin type DNA single strand H2)
Hairpin DNA Single Strand H1 hairpin DNA Single Strand H2 designed and synthesized, the working concentration of the second reagent was 10. mu.M.
4. Third reagent (i.e. SERS probe)
Namely, 10. mu.L of 50. mu.M probe single-stranded P and 10. mu.L of 100. mu.M Raman molecule 5, 5' -dithiobis (2-nitrobenzoic acid) (DTNB) -modified gold nanoparticles having a particle size of 30nM were used, and the working concentration of the third reagent was 3.3 nM.
The preparation method of the SERS detection kit for SARS-CoV-2 nucleic acid detection of the embodiment comprises the following steps:
1. preparation of SERS detection chip
The SERS detection chip is obtained by using a silver nanorod array as a substrate, modifying the surface of the substrate with a capture single chain C, and then dropwise adding 6-Mercaptohexanol (MCH) on the surface of the substrate for sealing, and specifically comprises the following steps:
(1) preparing a silver nanorod array (the silver nanorod array is prepared by adopting a vacuum electron beam evaporation coating technology and is packaged on the surface of the silver nanorod array by a Polydimethylsiloxane (PDMS) film, the substrate comprises 3 multiplied by 10 array type small holes, the aperture of each small hole is 4 mm, the depth of each small hole is 1 mm), and the silver nanorod array is repeatedly washed by ultrapure water;
(2) taking the trapped single chain C and a tricarboxyethylphosphine solution (TCEP) in a molar ratio of 1: 1000, mixing, and placing in a homothermal mixer at 25 ℃ for reaction for 4 hours;
(3) putting the silver nanorod array and 20 mu L of 500nM solution for capturing the single-chain C into a constant-temperature mixer at 37 ℃ for co-culture (the culture condition is that the mixture is kept still for 3 hours at 37 ℃ in an 80% humidity environment), and fixing the captured single-chain C on the surface of the silver nanorod array through the covalent bond formed by sulfydryl and silver;
(4) using TM buffer (10 mM Tris and 5mM MgCl)2pH 8.0), dropping 20 μ L of 1 mM 6-Mercaptohexanol (MCH) on the surface of the substrate, and reacting in a mixer at 37 deg.C for 10 min;
(5) and cleaning the substrate for multiple times by sequentially using TM buffer solution and ultrapure water to obtain the SERS detection chip.
2. Preparing the first reagent
And (3) mixing the auxiliary single-stranded primer and the hairpin type DNA single strand H1 according to the concentration ratio of 1: 1, annealing at 90-95 ℃ for 5min to obtain a first reagent (namely a detection probe), and diluting by using a TM buffer solution to ensure that the working concentration of the first reagent is 5 mu M.
3. Preparation of the second reagent
Designing and synthesizing a hairpin DNA single strand H2, namely a second reagent according to the sequence of the hairpin DNA single strand H1, and diluting by using a TM buffer solution to ensure that the working concentration of the second reagent is 10 mu M.
4. A third reagent (i.e., SERS probe) is prepared.
(1) And (3) mixing the single-stranded probe with a tricarboxyethylphosphine solution (TCEP) in a molar ratio of 1: 1000, mixing, and placing in a homothermal mixer at 25 ℃ for reaction for 4 hours;
(2) 10 μ L of 50 μ M probe single-stranded P and 500 μ L of 0.33 nM gold nanoparticle (AuNP) solution were mixed in 0.5 XTBE solution and incubated overnight at 25 ℃ at 300 rpm;
(3) 5, 10, 15 and 20. mu.L of 2M NaCl solution were added to the mixture every 30 minutes to give a final concentration of 178mM NaCl, and co-incubated overnight at 25 ℃ at 300 rpm;
(4) adding 10 mu L of 100 mu M Raman molecule 5, 5' -dithiobis (2-nitrobenzoic acid) (DTNB) for reaction for 3 hours;
(5) and finally, removing the supernatant through centrifugation, dispersing the centrifugal sediment by using 0.5 xTBE solution, and fixing the volume to 50 mu L by using 0.5 xTBE solution to obtain a third reagent with the working concentration of 3.3 nM.
Test example 1 optimization experiment of detection Probe (i.e., first reagent) assembled by Assist Single-stranded primer and hairpin-type DNA Single-stranded H1
During the preparation of the first reagent (i.e., the detection probe), the helper single-strand primer and the hairpin DNA single-strand H1 are each mixed in a ratio of 1: 0.8, 1: 1. 1: 1.25, 1: 1.5 and 1: 2 and annealing at 90-95 ℃ for 5min to obtain 5 first reagents.
A first reagent (4. mu.L, 5. mu.M), a second reagent (4. mu.L, 10. mu.M), a third reagent (10. mu.L, 3.3 nM) and 2. mu.L of a blank sample (TM buffer)/10 fM/10pM of SARS-CoV-2 nucleic acid strand (DNA synthesized fragment, synthesized by Takara Biotechnology, Inc. (David Co., Ltd.)) were simultaneously added to the wells of a SERS chip (prepared by the method of example 1), and after incubation in a homomixer at 37 ℃ and 300rpm for 50 minutes, the wells of the SERS chip were washed with TM buffer and ultrapure water in this order. After natural air drying, a portable Raman spectrometer is used for carrying out SERS detection on the SERS detection chip (the detection conditions comprise that the scanning time is 1s, the accumulated times are 1 time, and the excitation light wavelength is 785nm), and an SERS spectrum and a characteristic signal intensity value thereof are obtained.
The results are shown in FIG. 2. FIG. 2 (A) is a SERS spectrum obtained by using detection probes assembled by auxiliary single-stranded primer and hairpin type DNA single-stranded H1 in different concentration ratios for detecting SARS-CoV-2 nucleic acid chains; FIG. 2 (B) is the SERS peak intensity ratio of detected SARS-CoV-2 nucleic acid chain and blank sample (TM buffer) ((B))I T/I blank)。
As can be seen from FIG. 2, when the molar concentration ratio of the auxiliary single-stranded primer to the hairpin DNA single strand H1 is 1: 1, better SERS response than other ratios was obtained. The optimal molar concentration ratio of the auxiliary single-stranded primer and the hairpin type DNA single-stranded H1 for assembling the first reagent is 1: 1.
experimental example 2 optimization experiment of SERS detection chip
Optimization experiment for fixing and capturing concentration of single-chain C on surface of SERS detection chip
The method comprises the following steps:
1. preparing a silver nanorod array (as in example 1) and washing with ultrapure water multiple times;
2. taking the capture single chain C and a tricarboxyethylphosphine solution (TCEP) in a molar ratio of 1: 1000, mixing, and placing in a homothermal mixer at 25 ℃ for reaction for 4 hours;
3. placing the silver nanorod array and 20 mu L of capture single-chain C solutions of 100nM, 200 nM, 300 nM, 500nM, 1000nM and 2000 nM respectively in a constant-temperature mixer for co-culture (culture condition: 37 ℃, standing for 3 hours in 80% humidity environment), wherein the capture single-chain C is immobilized on the surface of the silver nanorod array through covalent bond formed by sulfydryl and silver;
4. TM buffer (10 mM Tris and 5mM MgCl) was used2pH 8.0), dropping 20 μ L of 1 mM 6-Mercaptohexanol (MCH) on the surface of the substrate, and reacting in a mixer at 37 deg.C for 10 min;
5. and cleaning the substrate for multiple times by sequentially using TM buffer solution and ultrapure water to respectively obtain 6 SERS detection chips.
6. A first reagent (prepared by annealing a helper single-strand primer to a hairpin DNA single-strand H1 at a concentration ratio of 1: 1, 4. mu.L of 5. mu.M), a second reagent (4. mu.L of 10. mu.M), a third reagent (prepared by the method of example 1, 10. mu.L of 3.3 nM), and 2. mu.L of a blank sample (TM buffer)/10 pM of SARS-CoV-2 nucleic acid strand (DNA synthetic fragment, synthesized by Takara Biotechnology, Inc. (Chinese Dalian Co., Ltd.) were simultaneously added to each of the wells of 6 SERS detection chips, and after incubation in a 37 ℃ 300rpm homomixer for 50 minutes, the wells of the SERS detection chips were washed with TM buffer and ultrapure water in this order. After natural air drying, a portable Raman spectrometer is used for carrying out SERS detection on the SERS detection chip (the detection conditions comprise scanning time of 1s, cumulative times of 1 time and exciting light wavelength of 785nm), and an SERS spectrum and a characteristic signal intensity value thereof are obtained.
The results are shown in FIG. 3. FIG. 3 (A) is a SERS spectrum obtained by fixing different concentrations of the capture single-chain C on the surface of the SERS detection chip; FIG. 3 (B) is a 1330 cm spectrum of FIG. 3 (A)-1SERS peak intensity corresponding to raman shift.
As can be seen from FIG. 3, when the silver nanorod array is co-cultured with 20 μ L of 100-500 nM capture single-chain C solution, the SERS signal gradually increases and reaches a saturation state as the C concentration increases; when the C concentration is higher than 500nM, the SERS signal no longer changes with the C concentration as the C concentration increases. It was demonstrated that the concentration of the solution for trapping single strands C of 500nM was determined as the optimum concentration for immobilization on the silver nanorod array substrate.
Second, optimization experiment of SERS detection chip surface 6-Mercaptohexanol (MCH) blocking time
In the preparation process of the SERS detection chip, a silver nanorod array and 20 muL of 500nM capture single-chain C solution are placed in a constant-temperature mixer for co-culture (the culture condition is 37 ℃, and the mixture is kept stand for 3 hours in an 80% humidity environment), after a substrate is washed by using TM buffer solution, 20 muL of 1 mM 6-Mercaptohexanol (MCH) is dripped onto the surface of the substrate and placed in the constant-temperature mixer at 37 ℃ for reaction for 0, 5, 10, 20, 40 and 60 minutes respectively, and the substrate is washed by using the TM buffer solution and ultrapure water for multiple times, so that 6 SERS detection chips with different sealing times are obtained.
A first reagent (prepared by annealing a helper single-stranded primer to a hairpin DNA single-stranded H1 at a concentration ratio of 1: 1, 4. mu.L of 5. mu.M), a second reagent (4. mu.L of 10. mu.M), a third reagent (prepared by the method of example 1, 10. mu.L of 3.3 nM), and 2. mu.L of a blank sample (TM buffer)/10 pM of a SARS-CoV-2 nucleic acid strand (DNA synthetic fragment, synthesized by Takara Biotechnology, Inc. (David.) were simultaneously added to the wells of the SERS chip, and after incubation at 37 ℃ for 50 minutes in a 300rpm homomixer, the wells of the SERS chip were washed with TM buffer and ultrapure water in this order. After natural air drying, a portable Raman spectrometer is used for carrying out SERS detection on the SERS detection chip (the detection conditions comprise scanning time of 1s, cumulative times of 1 time and exciting light wavelength of 785nm), and an SERS spectrum and a characteristic signal intensity value thereof are obtained. The results are shown in FIG. 4.
As can be seen from FIG. 4, the background decreased with longer MCH blocking time, while the SERS intensity detected for the 10pM SARS-CoV-2 nucleic acid strand also decreased significantly. Therefore, in order to effectively reduce the background and maintain a specific detected signal, the blocking time of 10min is selected as the optimal condition for subsequent sensing.
Experimental example 3 detection time optimization experiment for detecting SARS-CoV-2 nucleic acid by SERS detection kit
In the preparation process of the SERS detection chip, a silver nanorod array and 20 muL of 500nM capture single-chain C solution are placed in a constant-temperature mixer at 37 ℃ for co-culture (the culture condition is 37 ℃, the mixture is kept still for 3 hours in an 80% humidity environment), after the substrate is washed by using TM buffer solution, 20 muL of 1 mM 6-Mercaptohexanol (MCH) is dripped onto the surface of the substrate, the substrate is placed in the constant-temperature mixer at 37 ℃ for reaction for 10 minutes respectively, and the substrate is washed by using the TM buffer solution and ultrapure water for multiple times, so that the SERS detection chip is obtained.
After 4. mu.L of 5. mu.M first reagent (prepared by annealing auxiliary single-stranded primer to hairpin type DNA single-stranded H1 at a concentration ratio of 1: 1), 4. mu.L of 10. mu.M second reagent, 10. mu.L of 3.3nM third reagent (prepared by the method of example 1), and 2. mu.L of 10pM SARS-CoV-2 nucleic acid strand (DNA synthetic fragment, synthesized by Takara Biotechnology, Inc. (Daizian Co., Ltd.) were put into a 37 ℃ 300rpm homomixer and reacted for 30, 40, 50, 60, 90 and 120 minutes, respectively, cleaning the small holes of the SERS detection chip by using TM buffer solution and ultrapure water in sequence, naturally drying, then using a portable Raman spectrometer, and carrying out SERS detection on the SERS detection chip (under the detection conditions of 1s of scanning time, 1 cumulative time and 785nm of exciting light wavelength) to obtain an SERS spectrum and a characteristic signal intensity value thereof.
The results are shown in FIG. 5. Wherein (A) in FIG. 5 is a SERS spectrum corresponding to different detection times of the SERS detection kit for detecting SARS-CoV-2 nucleic acid chain; FIG. 5 (B) is a graph showing the spectral lines at 1330 cm in FIG. 5 (A)-1SERS peak intensity corresponding to raman shift.
As can be seen from FIG. 5, the SERS intensity gradually increased within 30min to 50min, and the SERS signal was saturated at 50min of co-culture, indicating that 50min is the optimal SARS-CoV-2 nucleic acid detection time.
Experimental example 4 SERS detection kit working curve and detection limit for detecting SARS-CoV-2 nucleic acid
mu.L of 5. mu.M first reagent (prepared by the method of example 1), 4. mu.L of 10. mu.M second reagent, 10. mu.L of 3.3nM third reagent (prepared by the method of example 1), and 2. mu.L of a range of concentrations (10)2copies/mL~106copies/mL) of SARS-CoV-2 nucleic acid standard (purchased from Jingzhen Gene technology, Ltd.) was mixed, added dropwise to the wells of the SERS detection chip (prepared by the method of example 1), incubated at 37 ℃ for 50 minutes in a 300rpm homomixer, and then the wells were washed with TM buffer and ultrapure water several times. After natural air drying, using a portable Raman spectrometer to carry out SERS detection on the SERS detection chip (the detection conditions are that the scanning time is 1s, the accumulated times are 1 time, and the exciting light wavelength is 785nm), and obtaining an SERS spectrum and the intensity ratio of SERS characteristic peaks of a SARS-CoV-2 nucleic acid standard sample and a blank sample (TM buffer solution) ((I T/I blank) (intensity ratio of SERS characteristic peak of SARS-CoV-2 nucleic acid standard sample to blank sample (TM buffer) by taking logarithm of concentration of SARS-CoV-2 nucleic acid standard sample as abscissaI T/I blank) And drawing a working curve for the ordinate, and calculating the detection limit of the SERS detection kit for detecting SARS-CoV-2 nucleic acid according to the working curve.
The results are shown in FIG. 6, wherein (A) in FIG. 6 is SERS detection kit detectionSERS spectrograms corresponding to SARS-CoV-2 nucleic acid standard samples with different concentrations, and (B) in FIG. 6 is the intensity ratio of SERS characteristic peak of SARS-CoV-2 nucleic acid standard sample and blank sample (TM buffer solution) (TM)I T/I blank)。
For the detection of SARS-CoV-2 nucleic acid, a working curve was obtained asI T/I blank=0.17×LogC T+0.75(R2=0.980), the calculation of the detection limit is 51.38copies/mL, which shows that the sensitivity of detecting SARS-CoV-2 nucleic acid by using the SERS detection kit of the invention is far higher than the sensitivity of detecting SARS-CoV-2 nucleic acid by using the existing PCR technology based on the enzyme-mediated nucleic acid amplification strategy.
Experimental example 5 specific characterization of SARS-CoV-2 nucleic acid by SERS assay kit
SARS-CoV-2 nucleic acid standards (purchased from Jinglian Gene technology (Shenzhen) Co., Ltd.) were diluted to 10 with TM buffer solutions respectively6copies/mL and 103copies/mL, TM buffer without any additional biomolecules added was used as a blank and other viral RNA was selected for detection as interfering samples together. Other viral RNAs include influenza a virus nucleic acid standards (purchased from qian gene technology (shenzhen) ltd), influenza b virus nucleic acid standards (purchased from qian gene technology (shenzhen) ltd), rhinovirus inactivated virus standards (purchased from shanghai heng biont ltd), and syncitial virus and adenovirus throat swab samples of unknown concentrations (from guangzhou gold-field medical testing center ltd).
mu.L of 5. mu.M first reagent (prepared by the method of example 1), 4. mu.L of 10. mu.M second reagent, and 10. mu.L of 3.3nM third reagent (prepared by the method of example 1) were combined with 2. mu.L of 106copies/mLSARS-CoV-2 nucleic acid Standard (purchased from Jinglian Gene technology (Shenzhen) Co., Ltd.), 2. mu.L 103 copies/mL SARS-CoV-2 nucleic acid standard, 2. mu.L of other viral RNAs (including influenza A virus nucleic acid standard (purchased from Jinglian Gene technology (Shenzhen) Co., Ltd.), influenza B virus nucleic acid standard (purchased from Jinglian Gene technology (Shenzhen) Co., Ltd.), and rhinovirus inactivated virus standard (purchased from Shanghai province, Ltd.), and other virus RNAYihengshi, Inc.), syncytium virus and adenovirus throat swab samples (from Guangzhou gold-region medical examination center, Inc.) with unknown concentrations, and 2 μ L of blank samples (TM buffer) were mixed, added dropwise to the wells of the SERS detection chip (prepared as in example 1), incubated at 37 ℃ for 50 minutes in a homomixer at 300rpm, and the wells were washed with TM buffer and ultra-pure water several times in sequence. After natural air drying, using a portable Raman spectrometer to carry out SERS detection on the SERS detection chip (detection conditions: scanning time is 1s, cumulative times are 1 time, excitation wavelength is 785nm), obtaining an SERS spectrum and an intensity ratio of SERS characteristic peaks of virus RNA and a blank sample (TM buffer solution) ((I T/I blank)。
The results are shown in FIG. 7, wherein (A) in FIG. 7 is SERS spectra for detecting different virus RNA samples (including novel coronavirus, influenza A virus, influenza B virus, rhinovirus, syncitial disease and adenovirus), and (B) in FIG. 7 is the intensity ratio of SERS characteristic peak for detecting virus RNA and blank sample (TM buffer) ((TM buffer))I T/I blank)。
As can be seen from FIG. 7, the SERS detection kit of the present invention can better distinguish SARS-CoV-2 RNA from other viral RNAs, which indicates that the SERS detection kit has good specificity.
Experimental example 6 characterization of uniformity of SARS-CoV-2 nucleic acid detected by SERS assay kit
mu.L of 5. mu.M first reagent (prepared as described in example 1), 4. mu.L of 10. mu.M second reagent, 10. mu.L of 3.3nM third reagent (prepared as described in example 1), and 2. mu.L of different concentrations (0, 10)3copies/mL and 106copies/mL) of SARS-CoV-2 nucleic acid standard (purchased from Jingzhen gene technology, Inc.), dropwise added into the wells of the SERS detection chip (prepared by the method of example 1), incubated in a 37 ℃ constant temperature mixer at 300rpm for 50 minutes, and then the wells were washed with TM buffer and ultrapure water several times. After natural air drying, using portable Raman spectrometer to carry out SERS detection on 20 random points (small holes) on SERS detection chip of SARS-CoV-2 nucleic acid standard sample with different concentrations (detection condition: scanning time 1s, accumulatedCounting for 1 time, exciting light wavelength 785nm) to obtain 20 random points (pores) at 1330 cm-1The corresponding SERS peak intensity is used for researching the uniformity of the silver nanorod array substrate with the single chain C captured by surface modification.
The results are shown in fig. 8, from which it can be seen that: the Relative Standard Deviation (RSD) of the SERS peak intensity of 20 random points is very small (RSD <7.71%), which indicates that the SERS detection kit of the invention has good uniformity in detecting SARS-CoV-2 nucleic acid.
Experimental example 7 characterization of stability of SERS assay kit for detecting SARS-CoV-2 nucleic acid
mu.L of 5. mu.M first reagent (prepared as described in example 1), 4. mu.L of 10. mu.M second reagent, 10. mu.L of 3.3nM third reagent (prepared as described in example 1), and 2. mu.L of 10. mu.M of each reagent6copies/mLSARS-CoV-2 nucleic acid Standard (purchased from Jinglian Gene technology (Shenzhen) Co., Ltd.), 2. mu.L 103The copies/mL SARS-CoV-2 nucleic acid standard (from Jingzhen gene technology, Inc.), 2 μ L other viral RNA (including influenza A virus nucleic acid standard (purchased from Jingzhen gene technology, Inc.), influenza B virus nucleic acid standard (purchased from Jingzhen gene technology, Inc.), rhinovirus inactivated virus standard (purchased from Shanghai Hensheng, Inc.) and syncytium virus and adenovirus swab samples (from Guangzhou Jinju medical inspection center, Inc.) with unknown concentrations), and 2 μ L blank samples (TM buffer) were mixed to obtain a mixed solution.
The mixed solution of the same sample obtained above was measured using 3 different batches of SERS chips (each prepared by the method of example 1). And (3) dropwise adding the mixed solution into a small hole of the SERS detection chip, culturing in a constant-temperature mixer at 37 ℃ and 300rpm for 50min, and sequentially washing the small hole for multiple times by using TM buffer solution and ultrapure water. After natural air drying, SERS detection is carried out on SERS detection chips of different batches by using a portable Raman spectrometer (the detection conditions are that the scanning time is 1s, the accumulated times are 1 time, and the wavelength of exciting light is 785nm), and the strength of SERS characteristic peaks of SARS-CoV-2 nucleic acid standard samples, other virus nucleic acid standard samples and blank samples (TM buffer solution) is obtainedDegree ratio (I T/I blank) And the stability of the silver nanorod array substrate with the surface modified and captured single-chain C is researched.
FIG. 9 shows the SERS characteristic peak intensity ratio (of different batches of SERS detection chips) between the virus nucleic acid standard and the blank sample (TM buffer solution) ((SERS detection chip))I T/I blank) The results show that: the SERS detection chips in different batches have better consistency on the output SERS signals for SARS-CoV-2 nucleic acid detection, which shows that the SERS detection kit has good stability for detecting SARS-CoV-2 nucleic acid.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
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Claims (10)
1. A SERS detection kit for detecting SARS-CoV-2 nucleic acids, the SERS detection kit comprising:
(1) SERS detection chip
The SERS detection chip is a silver nanorod array substrate with a surface modified with a single-chain capture shown in SEQ ID NO. 4;
(2) a first reagent
The first reagent is obtained by mixing an auxiliary single strand with a sequence shown as SEQ ID NO.1 and a hairpin type DNA single strand H1 with a sequence shown as SEQ ID NO.2 and annealing at 90-95 ℃; the concentration ratio of the auxiliary single strand to the hairpin type DNA single strand H1 is 1: 0.8 to 2;
(3) a second reagent
The second reagent is a hairpin type DNA single-chain H2 with the sequence shown in SEQ ID NO. 3;
(4) a third reagent
The third reagent is gold nanoparticles with the surface modified with a probe single chain with a sequence shown as SEQ ID NO.5 and 5,5 '-dithiobis (2-nitrobenzoic acid), and the concentration ratio of the probe single chain to the 5, 5' -dithiobis (2-nitrobenzoic acid) is 1: 1 to 3.
2. The SERS detection kit for detecting SARS-CoV-2 nucleic acid according to claim 1, wherein the silver nanorod array substrate comprises 3 x 10 array type pores, each pore has a pore diameter of 3 mm to 5mm and a depth of 0.8 mm to 1.2 mm.
3. The SERS assay kit for detecting SARS-CoV-2 nucleic acid according to claim 1, wherein the concentration ratio of the auxiliary single strand to the hairpin DNA single strand H1 is 1: 1 to 1.25.
4. The SERS detection kit for detecting SARS-CoV-2 nucleic acid as claimed in claim 1, wherein the gold nanoparticles have a particle size of 15nm to 100 nm.
5. The SERS detection kit for detecting SARS-CoV-2 nucleic acid according to any one of claims 1 to 4, wherein the working concentration of the first reagent is 5 μ M to 10 μ M; and/or the working concentration of the second reagent is 5-20 mu M; and/or the working concentration of the third reagent is 0.1nM to 10 nM.
6. The SERS detection kit for detecting SARS-CoV-2 nucleic acid as claimed in claim 5, wherein the working concentration of the third reagent is 3.2 nM-3.4 nM.
7. A preparation method of SERS detection kit for detecting SARS-CoV-2 nucleic acid is characterized by comprising the following steps:
(1) preparing SERS detection chip
According to the mol ratio of 1: mixing a capture single chain with the concentration of 500 nM-2000 nM and the sequence shown in SEQ ID NO.4 with a tri-carboxyethyl phosphine solution in a proportion of 100-1000, reacting for 4-12 hours in a mixing instrument at a constant temperature of 25-37 ℃, co-culturing with a silver nanorod array substrate for 3-5 hours, and cleaning with a TM buffer solution to obtain the nano-silver-coated nano-rod array substrate; the co-culture conditions are as follows: the temperature is 25-37 ℃, and the humidity is 60-80%;
(2) preparing the first reagent
The auxiliary single strand with the sequence shown as SEQ ID NO.1 and the hairpin type DNA single strand H1 with the sequence shown as SEQ ID NO.2 are mixed according to the concentration ratio of 1: mixing the raw materials in a ratio of 0.8-2, and annealing at 90-95 ℃ for 4-6 min to obtain the product;
(3) preparing the second reagent
Designing and synthesizing a hairpin DNA single strand H2 with a sequence shown in SEQ ID NO.3 according to the hairpin DNA single strand H1;
(4) preparing a third reagent
According to the mol ratio of 1: mixing a probe single chain with a sequence shown as SEQ ID NO.5 with a tricarboxyethylphosphine solution according to a ratio of 100-1000, reacting in a constant-temperature mixing instrument at 25-37 ℃ for 4-12 hours, and mixing 8-12 mu L of 10 mu M-100 mu M probe single chain and 450-550 mu L of 0.1 nM-10 nM gold nanoparticle solution in a TBE solution for overnight culture; adding NaCl solution in batches until the final concentration of NaCl in the mixture is 160-200 mM, and co-culturing overnight; adding 8-12 mu L of 5, 5' -dithiobis (2-nitrobenzoic acid) to react for 2.5-3.5 hours; centrifuging to remove supernatant, dispersing the centrifugal sediment by using a TBE solution and fixing the volume to obtain the product, wherein the concentration ratio of the 5, 5' -dithiobis (2-nitrobenzoic acid) to the single chain of the probe is 1-3: 1.
8. the method for preparing an SERS assay kit for detecting SARS-CoV-2 nucleic acid as claimed in claim 7, wherein the step (1) of preparing the SERS assay chip further comprises the following steps after washing the TM buffer solution: and (3) dropwise adding 15-25 mu L of 0.5-2 mM 6-mercaptohexanol to the surface of the silver nanorod array, and reacting in a constant-temperature mixing instrument at 25-37 ℃ for 8-12 minutes.
9. The method for preparing an SERS detection kit for detecting SARS-CoV-2 nucleic acid as claimed in claim 7, wherein in step (1), the concentration of the capturing single strand is 500nM to 1000 nM.
10. A method for detecting SARS-CoV-2 nucleic acid for non-diagnostic purpose based on SERS, comprising the steps of using the SERS detection kit for detecting SARS-CoV-2 nucleic acid according to any one of claims 1 to 6 to perform detection, wherein the method comprises:
(1) mixing a sample to be detected with a first reagent, a second reagent and a third reagent, then dropwise adding the mixture to the surface of the SERS detection chip, and culturing at 25-37 ℃ at 200-400 rpm for 40-60 minutes;
(2) cleaning an SERS detection chip by ultrapure water, carrying out SERS detection on the SERS detection chip to obtain an SERS spectrum and a characteristic peak signal intensity value thereof, and calculating the concentration of SARS-CoV-2 nucleic acid in a sample to be detected according to a working curve;
the working curve is drawn by the following steps:
(a) the first reagent, the second reagent, the third reagent and the concentration are respectively 102 copies/mL、103 copies/mL、104 copies/mL、105 copies/mL、106 Mixing copies/mL of SARS-CoV-2 nucleic acid standard solution, dropwise adding the mixture to the surface of an SERS detection chip, culturing at 25-37 ℃ at 200-400 rpm for 40-60 minutes;
(b) cleaning an SERS detection chip by ultrapure water, and performing SERS detection on the SERS detection chip to obtain SERS spectra corresponding to SARS-CoV-2 nucleic acid standard solutions with different concentrations and characteristic peak signal intensity values thereof;
(c) and drawing a working curve by taking the logarithm of the concentration of the SARS-CoV-2 nucleic acid standard solution as the abscissa and the ratio of the SARS-CoV-2 nucleic acid standard solution to the characteristic peak signal intensity of the blank sample as the ordinate.
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