Rapid analysis method of metal corrosive microorganism sulfate reducing bacteria

Abstract

The invention provides a rapid analysis method of metal corrosive microorganism sulfate reducing bacteria, and belongs to the technical field of biology.The rapid analysis method comprises the steps that a to-be-detected microorganism genome DNA sample and a photosensitizer As2MoO6 atNiS are taken to jointly construct a triple asymmetric photon PCR system, asymmetric photon PCR amplification is carried out under the condition of LED light source excitation, after amplification is completed, the sample is detected, and then the sample is detected. The obtained amplification product is mixed with a photo-thermal signal probe prepared from a photosensitizer As2MoO6 coated NiS, and photo-thermal temperature change detection is carried out. The rapid analysis method provided by the invention has the characteristics of high efficiency, convenience and accuracy, and is suitable for rapid detection of sulfate reducing bacteria.

Description

Technical Field

[0001] The invention belongs to the technical field of inspection and detection, and specifically relates to a rapid analysis method for metal-corrosive microorganisms, sulfate-reducing bacteria. Background Art

[0002] Sulfate-reducing bacteria (SRB) are a widely distributed facultative anaerobic bacteria that use organic matter or iron as electron donors and sulfate as electron acceptors. They can reduce sulfate to H2S and induce chemical and electrochemical reactions, thereby corroding metals. In industry, SRB may cause corrosion of facilities and equipment, so the detection of SRB is of great significance to industrial process control.

[0003] At present, the dilution culture counting method (most probable number, MPN), fluorescence sensor method, polymerase chain reaction method, enzyme-linked immunosorbent assay and electrochemical biosensor method have been used for the detection of SRB. These methods complement each other in practical applications, but each has its own advantages and disadvantages. MPN has high reliability, but its operation is cumbersome and time-consuming; the detection method based on characteristic metabolites is simple to operate and has a wide range of applications, but it still has the problem of being time-consuming; the cell-based detection method has a short detection cycle, but the stability and specificity need to be further improved. The detection method based on genetic markers has strong specificity and high sensitivity, but its amplification speed needs to be further improved. At present, there is still a blank in the field rapid detection method based on genetic markers.

[0004] Asymmetric PCR is a method that uses unequal amounts of a pair of primers to produce a large amount of single-stranded DNA (ssDNA). Photon PCR is an innovative polymerase chain reaction (PCR) technology. Photon PCR uses the principle of photon thermal conversion to generate heat at the photosensitizer interface through photon-electron-phonon coupling, thereby achieving rapid heating and cooling of the PCR reaction solution. This process significantly shortens the thermal cycle time of PCR and improves the amplification efficiency to achieve faster, more efficient and more sensitive nucleic acid amplification and detection. Photon PCR can complete the amplification process that traditional PCR takes tens of minutes or even hours in a few minutes; due to its fast amplification speed and high efficiency, photon PCR can detect extremely low concentrations of nucleic acid samples, improving the sensitivity of detection; photon PCR systems usually use low-power light sources (such as LEDs) as heat sources, reducing energy consumption and costs; in addition, due to its small size and simple operation, photon PCR systems are very suitable for on-site detection or instant diagnosis. At present, there is no report on the combination of asymmetric PCR technology and photon PCR technology for SRB detection.

[0005] Lateral flow immunochromatographic test strips (LFIA) are widely used in the design of portable analytical detection sensors due to their fast analytical performance, good selectivity, low cost, small sample volume requirements, ease of large-scale production, and stable long-term storage. The signal of photothermal LFIA is caused by temperature changes due to the photothermal effect, with low background signals and sensitive sensing. It can be performed on various substrates of different colors and has great application potential. At present, new nanomaterials with higher photothermal conversion efficiency are still needed as signal probes to further improve the performance of photothermal LFIA. Summary of the invention

[0006] The existing analysis methods for metal-corrosive microorganisms, sulfate-reducing bacteria, have limitations, including that the traditional PCR method is time-consuming, complicated to operate, and has insufficient detection sensitivity for low-abundance target bacteria; at the same time, there is a lack of efficient, simultaneous amplification and detection technology for sulfate-reducing bacteria-specific genes such as 16SrDNA, DSR genes and APS genes; in addition, the existing detection methods are insufficient in rapid quantification and on-site application, and the present invention provides a rapid analysis method for metal-corrosive microorganisms, sulfate-reducing bacteria.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is: A rapid analysis method for metal-corrosive microorganisms, sulfate-reducing bacteria, comprising the following specific steps: Construction of triple asymmetric photon PCR system: Take the microbial genomic DNA sample to be detected, photosensitizer As2MoO6@NiS, 16SrDNA primer pair, DSR Primer pairs for genes and APS The primer pairs of the genes are used to construct a triple asymmetric photon PCR system; LED light source stimulates asymmetric photon PCR: LED light source is used to irradiate the triple asymmetric photon PCR system to perform asymmetric photon PCR amplification to obtain the amplified product; Photothermal detection: The detection area of ​​the photothermal test paper is fixed with complementary sequences of three amplification products to be tested in sequence, and the quality control area of ​​the test paper body is fixed with a sequence complementary to the signal probe; The amplified product was mixed with the running buffer and the photothermal signal probe, and then dripped onto the test paper sample pad for photothermal temperature change detection to obtain a photothermal temperature change signal that was positively correlated with the content of the amplified product.

[0008] Furthermore, the total volume of the triple asymmetric photon PCR system is 25 mL, including the primer pair for 16S rDNA, DSR Primer pairs for genes and APS1.0~2.0mL of each primer pair of the gene, 1.0~3.0mL of photosensitizer As2MoO6@NiS, 10mL of PCR premix (Taq plus mix), 1.0~2.0mL of the microbial genomic DNA sample to be detected, and ultrapure water; Among them, the primer pairs for 16SrDNA, DSR Primer pairs for genes and APS The volume ratio of the primer pairs of the genes to the genomic DNA samples of the microorganisms to be detected was 1:1; The ratio between any upstream primer (i.e., F) and its corresponding downstream primer (R) in the three primer pairs is 1:0.1~1:10.

[0009] Further, the 16SrDNA sequence of the sulfate-reducing bacteria is shown in SEQ ID NO: 1; The primer pair for 16SrDNA included the upstream primer 16SrDNA-1-F: 5′-GTGCCAGCGCCGCGG-3′, the downstream primer 16SrDNA-1-R: 5′-AAAAAAAAAAGGTTACCTTGTTACGACTT-3′; Sulfate-reducing bacteria DSR The gene sequence is shown in SEQ ID NO: 2; DSR The primer pairs for the gene included upstream primer DSR-F: 5′-ACSCACTGGAAGCACGGCGG-3′, downstream primer DSR-R: 5′-AAAAAAAAAAGTGGMRCCGTGCAKRTTGG-3′; Sulfate-reducing bacteria APS The gene sequence is shown in SEQ ID NO: 3; APS The primer pair for the gene included the upstream primer APS-F: 5′-TGGCAGATMATGATYMACGGG-3′ and the downstream primer APS-R: 5′-AAAAAAAAAAGGGCCGTAACCGTCCTTGAA-3′.

[0010] Furthermore, the preparation process of the photosensitizer As2MoO6@NiS is as follows: The photosensitizer As2MoO6 nanoparticles were synthesized by hydrothermal method; Take 1~2 mg of As2MoO6 nanoparticles and add them to 5~10 mL of deionized water containing 10~20 mg of polyethylene glycol (MW5000-10000), ultrasonicate for 20~30 min, stir overnight to obtain a mixed solution; 1 mL of the mixed solution was added to 1 mL of 1-10 mg / mL NiS aqueous solution and stirred overnight. The resulting solution was directly used as the photosensitizer As2MoO6@NiS solution and applied to the triple asymmetric photon PCR system; or the dark gray powder was collected by centrifugation and dried to obtain the solid photosensitizer As2MoO6@NiS for further surface modification reaction or storage.

[0011] Furthermore, the preparation process of the three photothermal signal probes is as follows: Take 10mL of 0.1M three nucleic acid chains GTGCCAGCG, ACSCACTGGA, and TGGCAGATMA, which are partially complementary to the asymmetric photon PCR amplification product, and mix them with 1mL of 1mg / mL photosensitizer As2MoO6@NiS, respectively. Incubate in 1´PBS buffer in the dark and shake at room temperature overnight to obtain three corresponding photothermal signal probes, namely, the photothermal signal probe of 16SrDNA of sulfate-reducing bacteria, DSR Photothermal signal probes for genes and APS Photothermal signal probe for genes.

[0012] Furthermore, the LED light source irradiation is irradiated by using an 808nm external coaxial reflection LED; During the irradiation process, the excitation power is 4.5~6W and the total excitation time is 5~15min.

[0013] Furthermore, the reaction conditions of asymmetric photon PCR amplification are as follows: the concentration of the photosensitizer As2MoO6@NiS solution is 0.1~1.0 mg / mL, the pre-denaturation excitation power is 6W, 1min; 20-60 denaturation-annealing-extension cycles: 6W, 5s; 4.5W, 2s; 5W, 2s; extension 5W, 1min.

[0014] Furthermore, based on the photothermal temperature change signal, 16SrDNA, DSR Genes and APS The genes of the three gene entries were quantitatively analyzed as follows: After asymmetric photon PCR amplification and photothermal detection of three samples with unknown target gene concentrations, the photothermal temperature change signals of the three genes are substituted into the equation of the quantitative curve, and the corresponding concentrations of the three genes are calculated, thereby achieving quantitative analysis of the target genes; Among them, the quantitative curves of the genes of the three items are as follows: The quantitative curve of 16S rDNA is: y = 5.16x + 0.50, R 2 =0.9961; DSR The quantitative curve of the gene is: y=4.06x+0.78, R 2 =0.9899; APS The quantitative curve of the gene is y=4.91x+0.54, R 2 =0.9987.

[0015] Furthermore, the microbial genomic DNA sample to be detected is prepared by collecting microorganisms on the metal surface and extracting genomic DNA; Methods for collecting microorganisms on metal surfaces include but are not limited to direct scraping, cotton swab wiping or brushing; Methods for extracting genomic DNA include, but are not limited to, thermal lysis method, kit method, and SDS method.

[0016] Furthermore, the detection area (T area) of the photothermal test paper is fixed with complementary sequences of three amplification products to be tested (T probe of 16SrDNA: GTGCCAGCG, T probe of DSR: ACSCACTGGA, T probe of APS: TGGCAGAT) in sequence, and the quality control area (C area) of the test paper body is fixed with a sequence complementary to the signal probe (C probe: TTTTTTTTTT).

[0017] The beneficial effects of the rapid analysis method of metal-corrosive microorganisms sulfate-reducing bacteria of the present invention are: The present invention provides a rapid analysis method for metal-corrosive microorganisms, sulfate-reducing bacteria, which uses the photoactivity of the photosensitizer As2MoO6@NiS to generate photoelectrons and photothermal effects, can quickly control the temperature and directly contact the amplification system at the molecular level, which is significantly more efficient than traditional water baths and metal baths. At the same time, the photosensitizer is used as a signal probe of the photothermal test paper to provide a sensitive and quantifiable signal. The entire method of the present invention can quickly achieve efficient amplification and detection of multiple target DNAs. The rapid analysis method of the present invention is efficient, convenient and accurate, and is suitable for the rapid detection of sulfate-reducing bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of the workflow of the present invention; Figure 2 The photothermal temperature rise of the photothermal material in step S3 of Example 1 of the present invention; Figure 3 The final test paper detection result corresponding to the asymmetric photon PCR system with different upstream and downstream primer ratios in step S6 of Example 1 of the present invention; Figure 4 The comparison results of gel electrophoresis analysis of the corresponding amplification products obtained by the asymmetric photon PCR method and the traditional PCR method in step S7 of Example 1 of the present invention; Figure 5 This is a photothermal test paper photo in step S6 of Example 2 of the present invention; Figure 6 The quantitative curves of the genes of the three entries; Figure 6 The left figure is the quantitative curve of 16SrDNA, and the middle figure is DSR The quantitative curve of the gene is shown on the right. APS Quantification curves of genes; the detection limits (i.e., sensitivity) of the three genes were all 10 copies / mL. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present invention are described clearly and completely below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below. The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0020] In addition, if no specific techniques or conditions are specified in the specific examples disclosed below, the techniques or conditions described in the literature in the field or the product instructions are used. If the manufacturer of the reagents is not specified, they are all conventional products that can be purchased.

[0021] The present invention discloses a rapid analysis method for metal-corrosive microorganisms, sulfate-reducing bacteria, and specifically relates to a method for using a photosensitizer As2MoO6@NiS to perform asymmetric photon PCR and photothermal test paper to detect 16SrDNA, DSR Gene, APS Rapid gene analysis and testing methods, such as Figure 1 As shown, the following steps are included: S1. Primer sequence design According to the PCR principle, the target gene sulfate-reducing bacteria 16SrDNA, DSR Gene, APS A primer set is designed for amplification of the gene sequence, wherein the primer set includes two upstream and downstream primers.

[0022] S2. Sample collection and nucleic acid extraction Microorganisms on metal surfaces were collected by direct scraping, cotton swab wiping or brushing methods; and microbial genomic DNA was extracted by thermal pyrolysis, kit method and SDS method.

[0023] S3. Preparation of photosensitizer As2MoO6@NiS The photosensitizer As2MoO6 nanoparticles were synthesized by hydrothermal method; Take 1~2 mg of As2MoO6 nanoparticles and add them to 5~10 mL of deionized water containing 10~20 mg of polyethylene glycol (MW5000-10000), ultrasonicate for 20~30 min, stir overnight to obtain a mixed solution; Take 1 mL of the mixed solution and add it to 1 mL of NiS aqueous solution with a concentration of 1-10 mg / mL and stir overnight. The resulting solution can be directly used as a photosensitizer As2MoO6@NiS solution and applied to the triple asymmetric photon PCR system; the dark gray powder can also be collected by centrifugation and dried to obtain a solid photosensitizer As2MoO6@NiS for further surface modification reaction or storage.

[0024] S4. Construction of triple asymmetric photon PCR system The 16SrDNA of sulfate-reducing bacteria in the microbial genomic DNA extracted in step S2, DSR Gene, APS The gene is used as a template to construct a triple asymmetric photon PCR system. The triple asymmetric photon PCR system includes the following components: the primer set in step S1, the photosensitizer As2MoO6@NiS prepared in step S3, PCR premix (Taq plus mix), the microbial genomic DNA extracted in step S2 as a template, and ultrapure water.

[0025] S5, LED light source stimulates asymmetric photon PCR The triple asymmetric photon PCR system is irradiated with an 808nm external coaxial reflection LED to excite the photosensitizer in the triple asymmetric photon PCR system to provide the energy required for asymmetric photon PCR. The excitation power is 4.5-6W and the total excitation time is 5-15min.

[0026] Then asymmetric photon PCR amplification was performed, and the reaction conditions were: the concentration of the photosensitizer As2MoO6@NiS solution was 0.1~1.0 mg / mL, the pre-denaturation excitation power was 6W, 1min; 20-60 denaturation-annealing-extension cycles: 6W, 5s; 4.5W, 2s; 5W, 2s; extension 5W, 1min.

[0027] After the triple asymmetric photon PCR system is completed, the amplification product is obtained.

[0028] S6. Photothermal detection The photosensitizer As2MoO6@NiS was modified by nucleic acid chains to obtain three corresponding photothermal signal probes.

[0029] After the amplified product is mixed with three photothermal signal probes, the sample is loaded, and the detection area of ​​the photothermal test paper captures the amplified product and the signal probe in a sandwich form, and gives a photothermal temperature change signal that is positively correlated with the content of the amplified product, based on which the target gene is quantified. The amplified product is detected using the photothermal test paper, and the temperature signal is read by a smartphone for quantification.

[0030] The main body of the photothermal test paper is a nitrocellulose membrane, with a glass fiber sample pad and absorbent paper at both ends. The complementary sequences of the three amplification products to be tested (16SrDNA T probe: GTGCCAGCG, DSR T probe ACSCACTGGA, APS T probe: TGGCAGAT) are fixed in sequence on the detection area (T area) of the photothermal test paper, and the sequence complementary to the signal probe (C probe: TTTTTTTTTT) is fixed on the quality control area (C area) of the test paper.

[0031] At the same time, the three corresponding photothermal signal probes can be mixed with the amplified product as in the above method (i.e., independent of the test paper body), or they can be dripped on the binding pad and dried first, and the binding pad is integrated on the test paper, and the amplified product is directly loaded when used. During the detection process, one end of the amplified product binds to the T probe and the other end binds to the photothermal signal probe, thereby giving a photothermal temperature change signal that is positively correlated with the content of the amplified product, and the target gene is quantified accordingly.

[0032] The rapid analysis method of the present invention is used to detect metal-corrosive microorganisms, sulfate-reducing bacteria. During application, it is necessary to use components including a light source, an asymmetric photon PCR amplification system bracket, the above-mentioned photothermal test paper, a test paper bracket, a device with temperature collection accessories (such as a smart phone), a light-proof and heat-insulating shell, etc., to realize the integrated and convenient operation of the above-mentioned method.

[0033] The present invention can realize the 16SrDNA, DSR Genes and APS Efficient amplification of genes. The following examples use the 16S rDNA of sulfate-reducing bacteria as an example to fully and in detail illustrate the entire amplification method.

[0034] Example 1 A rapid analysis method for 16S rDNA in metal-corrosive microorganisms, sulfate-reducing bacteria This embodiment is a method for rapid analysis of 16SrDNA in metal-corrosive microorganisms, sulfate-reducing bacteria, specifically a method for using the photothermal effect of the photosensitizer As2MoO6@NiS to drive asymmetric photon PCR with 16SrDNA of sulfate-reducing bacteria as a template. The method includes the following specific steps: S1. Primer sequence design: According to the principle of asymmetric PCR, an amplification primer set was designed for two different regions of the 16SrDNA sequence of the target gene sulfate-reducing bacteria. The primer set included upstream and downstream primers. The specific primer set sequence is shown in Table 1; the 16SrDNA sequence of sulfate-reducing bacteria is shown in SEQ ID NO: 1.

[0035] Table 1 Sequences of primer sets for asymmetric photon PCR

[0036] S2. Sample collection and nucleic acid extraction Use the cotton swab wiping method to collect microorganisms on the metal surface. Specifically, dip a sterile cotton swab in normal saline and gently wipe the metal surface to collect the microorganisms on the surface. Place the wiped cotton swab in a sterile container and add 10 mL of sterile water or normal saline to dilute it.

[0037] The microbial genomic DNA was extracted by thermal lysis method, specifically: 0.5 mL of bacterial solution was taken, heated in a 100°C water bath for 10 min, and after standing, 100 μL of the supernatant was taken as the microbial genomic DNA sample to be tested (i.e., 16SrDNA containing sulfate-reducing bacteria).

[0038] At the same time, Nano Drop micro-spectrophotometer was used to measure the DNA content, A260nm / A280nm ratio, and A260nm / A230nm ratio in the microbial genomic DNA samples to ensure the quality of the microbial genomic DNA samples.

[0039] The 16SrDNA gene reference used to prepare the standard curve is a custom-made sequence that is artificially synthesized. The required concentration can be obtained by diluting it step by step according to the copy number concentration provided by the supplier.

[0040] S3. Preparation and characterization of photosensitizer As2MoO6@NiS As2MoO6 nanoparticles were synthesized by hydrothermal method; Take 2 mg of As2MoO6 nanoparticles and add them to 10 mL of deionized water containing 20 mg of polyethylene glycol (MW = 10000), ultrasonicate for 30 min, and stir overnight to obtain a mixed solution; Take 1 mL of the mixed solution and add it to 1 mL of 1 mg / mL NiS aqueous solution and stir overnight. The resulting solution can be directly used as a photosensitizer As2MoO6@NiS solution and applied to an asymmetric photon PCR system. Alternatively, the dark gray powder can be collected by centrifugation and dried to obtain a solid photosensitizer As2MoO6@NiS for further surface modification reaction or storage.

[0041] The photosensitizer As2MoO6@NiS solution (concentration of 0.1 mg / mL) was irradiated with 808 nm laser and compared with the As2MoO6 solution (the concentration of As2MoO6 in the As2MoO6 solution was 0.1 mg / mL) and NiS solution (the solvent of the NiS solution was deionized water and the NiS concentration was 0.1 mg / mL) with the same concentration. The photothermal heating curves of the three are shown in Figure 2. Figure 2 As shown, it can be seen that the heating efficiency of As2MoO6@NiS solution is significantly improved compared with that of a single substance solution, which is beneficial to improving its efficiency as a photosensitizer.

[0042] S4. Construction of asymmetric photon PCR system The microbial genomic DNA sample to be detected is used as a DNA template to construct an asymmetric photon PCR system in a PCR tube. The asymmetric photon PCR system includes the following components: the primer set in step S1, the photosensitizer As2MoO6@NiS prepared in step S3, PCR premix (Taq plus mix), the microbial genomic DNA sample to be detected, and ultrapure water. The specific concentrations of the components and the reaction volumes of the asymmetric photon PCR system are shown in Table 2.

[0043] Table 2 Summary of component concentrations and reaction volumes for asymmetric photon PCR amplification of 16S rDNA of sulfate-reducing bacteria

[0044] S5, LED light source stimulates asymmetric photon PCR An 808nm external coaxial reflection LED was used to illuminate the asymmetric photon PCR system by repeatedly turning it on (10s) and off (5s) at a distance of 3cm from the PCR tube to excite the photosensitizer As2MoO6@NiS in the asymmetric photon PCR system to provide energy for asymmetric photon PCR. The excitation power was 4.5W and the total excitation time was 10 min.

[0045] Then asymmetric photon PCR amplification was performed, and the reaction conditions were as follows: the concentration of the photosensitizer As2MoO6@NiS solution was 0.1~1.0 mg / mL, the pre-denaturation excitation power was 6W, 1min; 20 denaturation-annealing-extension cycles: 6W, 5s; 4.5W, 2s; 5W, 2s; extension 5W, 1min. Asymmetric photon PCR amplification took 5min. According to the gel electrophoresis of the amplified product, the primer pairs 16SrDNA-1-F and 16SrDNA-1-R can give effective amplification, while the primer pairs 16SrDNA-2-F and 16SrDNA-2-R cannot give effective amplification, so the subsequent asymmetric photon PCR amplification and traditional amplification both used primer pairs 16SrDNA-1-F and 16SrDNA-1-R.

[0046] For the reference sample of sulfate-reducing bacteria with 1000 copies of 16SrDNA, asymmetric photon PCR was performed on 16SrDNA at 20 and 40 amplification cycles, respectively, to obtain the corresponding amplification products.

[0047] According to 20 amplification cycles, asymmetric photon PCR was performed on samples with the same 16SrDNA copy number and different upstream and downstream primer ratios to obtain the corresponding amplification products.

[0048] According to 20 amplification cycles, asymmetric photon PCR amplification was performed on a reference series with 16SrDNA copy numbers of 1, 10, 100, 1000, 10000, and 100000 to obtain corresponding asymmetric photon PCR amplification products.

[0049] S6. Photothermal detection The main body of the photothermal test paper is a nitrocellulose membrane, with a glass fiber sample pad and absorbent paper at both ends. The detection area (T area) of the photothermal test paper is fixed with the 16SrDNA T probe: GTGCCAGCG, and the quality control area (C area) of the test paper body is fixed with a sequence complementary to the signal probe (C probe: TTTTTTTTTT).

[0050] Take 10mL of 0.1M nucleic acid chain (the nucleic acid chain is TTACGACTT) and 1mL of 1mg / mL photosensitizer As2MoO6@NiS, in 1´PBS buffer, in the dark and shake at room temperature overnight to obtain the photothermal signal probe of 16SrDNA.

[0051] Take 10mL of asymmetric photon PCR amplification product, 10mL of 16SrDNA photothermal signal probe and 80mL of running buffer (4×SSC, 0.25% Tween-20), mix them, and load them. Capture the amplification product and photothermal signal probe in the detection area of ​​the photothermal test paper in a sandwich form. After loading for 10 minutes, air-dry the test paper and collect the photothermal temperature change signal of the detection area with a smartphone. Then, draw a quantitative curve with the initial target gene copy number as the horizontal axis and the corresponding photothermal temperature change signal as the vertical axis. The results are shown in Figure 6 As shown in the left picture.

[0052] Take 10mL of amplification product corresponding to the ratio of upstream and downstream primers, mix it with 10mL of 16SrDNA photothermal signal probe and 80mL of running buffer (4×SSC, 0.25% Tween-20), load it, and capture the amplification product and photothermal signal probe in the detection area of ​​the photothermal test paper in a sandwich form. After 10 minutes of loading, air-dry the test paper, collect the photothermal temperature change signal of the detection area with a smartphone, and bring the measured photothermal temperature change signal into the quantitative curve, and quantify the target gene according to the quantitative curve.

[0053] from Figure 3 It can be seen that under the conditions of using different upstream and downstream primer ratios, the photothermal test paper detection area gives different photothermal signals, that is, the yield of asymmetric photon PCR amplification products is different. When the upstream and downstream primer ratios F:R are 1:2, 1:4 and 1:6 respectively, there is no significant difference in the photothermal signal. From the perspective of saving primers, the present invention prefers an upstream and downstream primer ratio F:R of 1:2.

[0054] S7. Comparison with traditional PCR The microbial genomic DNA sample obtained in step S2 is used as a DNA template and amplified by conventional PCR, wherein the amplification system of conventional PCR is shown in Table 3: Table 3. List of concentrations and reaction volumes of components for conventional PCR amplification of 16S rDNA of Acidithiobacillus ferrooxidans

[0055] The traditional PCR program was set as follows: pre-denaturation at 94°C for 5 min, followed by 20 cycles of denaturation at 94°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 10 s. It took about 20 min to obtain the traditional PCR amplification product.

[0056] The microbial genomic DNA sample obtained in step S2 is used as a DNA template, the upstream and downstream primer ratio F:R is 1:2, and amplification is performed for 5 minutes according to the method in step S4 to step S5 to obtain an asymmetric photon PCR amplification product.

[0057] The conventional PCR amplification products and asymmetric photon PCR amplification products were analyzed by gel electrophoresis. The results are as follows: Figure 4 As shown. It can be seen that there is a significant difference in the amplification effect between the asymmetric photon PCR method of the present invention and the traditional PCR method. Under the premise of the same initial DNA template concentration, the traditional PCR amplification after 20 minutes of gel electrophoresis did not produce obvious product bands, while the asymmetric photon PCR method of the present invention amplified after 5 minutes of gel electrophoresis, and obvious bands were visible. This shows that the asymmetric photon PCR method of the present invention can effectively improve the amplification efficiency and shorten the amplification time.

[0058] Example 2 A rapid analysis method for metal-corrosive microorganisms, sulfate-reducing bacteria This embodiment is a rapid analysis method for metal corrosive microorganisms, sulfate-reducing bacteria, specifically a method of using photosensitizer As2MoO6@NiS as a photothermal signal probe to detect 16SrDNA, DSR Genes and APS A rapid analysis method for asymmetric photon PCR products of genes, the method comprising the following specific steps: S1. Primer sequence design: According to the principle of asymmetric PCR, the 16SrDNA of the target gene sulfate-reducing bacteria, DSR Gene, APS The primer pairs for amplification of the genes were designed respectively, and the primer pairs included upstream and downstream primers. The specific primer sets and primer pair sequences are shown in Table 4; wherein the 16SrDNA sequence of sulfate-reducing bacteria is shown in SEQ ID NO: 1; DSR The gene sequence is shown in SEQ ID NO: 2; APS The gene sequence is shown in SEQ ID NO: 3; Table 4 List of primer sequences for asymmetric photon PCR

[0059] S2. Sample collection and nucleic acid extraction Sample collection and nucleic acid extraction are performed according to the method in step S2 of Example 1 to obtain a microbial genomic DNA sample to be detected.

[0060] For the 16SrDNA used in the preparation of quantitative curves, DSR Gene, APS Gene reference products are artificially synthesized custom sequences, which can be diluted step by step according to the copy number concentration provided by the supplier to obtain the required concentration.

[0061] S3. Preparation of photosensitizer As2MoO6@NiS The photosensitizer As2MoO6@NiS solution was prepared according to the method in step S3 of Example 1.

[0062] S4. Construction of triple asymmetric photon PCR system Using 16SrDNA, DSR Gene, APS The gene mixture was used as a DNA template, and an amplification system was constructed according to the method in step S4 of Example 1. The concentrations of the components and the reaction volumes in the obtained amplification system are shown in Table 5.

[0063] Table 5 Asymmetric photon PCR amplification of 16SrDNA, DSR Gene, APS Table of concentrations and reaction volumes of each gene component

[0064] S5, LED light source stimulates asymmetric photon PCR Amplification was performed according to the method in step S5 of Example 1. After amplification, 16SrDNA, DSR Genes and APS Amplification products of genes.

[0065] S6. Photothermal detection The main body of the photothermal test paper is a nitrocellulose membrane, with a glass fiber sample pad and absorbent paper at both ends. The complementary sequences of the three amplification products to be tested (16SrDNA T probe: GTGCCAGCG, DSR T probe ACSCACTGGA, APS T probe: TGGCAGAT) are fixed in sequence on the detection area (T area) of the photothermal test paper, and the sequence complementary to the signal probe (C probe: TTTTTTTTTT) is fixed on the quality control area (C area) of the test paper.

[0066] 10 mL of each of the three nucleic acid chains GTGCCAGCG, ACSCACTGGA, and TGGCAGATMA with a concentration of 0.1 M, which are partially complementary to the asymmetric photon PCR amplification product, were taken and mixed with 1 mL of 1 mg / mL photosensitizer As2MoO6@NiS, respectively, and incubated in 1´PBS buffer in the dark and at room temperature overnight to obtain three corresponding photothermal signal probes of 16SrDNA of sulfate-reducing bacteria, DSR Photothermal signal probes for genes and APS Photothermal signal probe for genes.

[0067] Take 10mL of the amplified product from step 5, 10mL of each of the three photothermal signal probes, and 60mL of running buffer (4×SSC, 0.25% Tween-20), mix them, and apply the sample. The amplified product and the signal probe are captured in a sandwich form in the detection area of ​​the photothermal test paper. After 10 minutes of application, the test paper is air-dried, and the photothermal temperature change signal of the detection area is collected with a smartphone. The results are as follows: Figure 5 shown.

[0068] Depend on Figure 5 As can be seen from the photothermal test paper photos, the test paper can detect the amplification products of specific target genes in the triple asymmetric photon PCR system in the photothermal mode.

[0069] S7. Quantitative curve drawing The target gene 16SrDNA with the target gene copy number of 1, 10, 100, 10000, 100000, DSR Genes and APS The mixture of genes is used as a DNA template, and asymmetric photon PCR amplification is performed according to the method in step S4 to step S5 to obtain a corresponding amplification mixture; Take 10mL of the amplification mixture corresponding to the number of copies of each gene, mix it with 10mL of each of the three photothermal signal probes and 60mL of running buffer (4×SSC, 0.25% Tween-20), and then load it. Capture the amplification product and the photothermal signal probe in the detection area of ​​the photothermal test paper in a sandwich form. After loading for 10 minutes, air-dry the test paper and use a smartphone to collect the photothermal temperature change signal of the detection area. Then, draw the quantitative curve of the three genes with the initial number of copies of the target gene as the horizontal axis and the corresponding photothermal temperature change signal as the vertical axis. The results are as follows: Figure 6 shown.

[0070] Among them, the quantitative curves of the genes of the three items are as follows: The quantitative curve of 16S rDNA is: y = 5.16x + 0.50, R 2 =0.9961; DSR The quantitative curve of the gene is: y=4.06x+0.78, R 2 =0.9899; APS The quantitative curve of the gene is y=4.91x+0.54, R 2 =0.9987.

[0071] When performing quantitative analysis on the three genes, samples of three unknown target gene concentrations are subjected to asymmetric photon PCR amplification and photothermal detection, and the photothermal temperature change signals of the three genes are substituted into the equation of the quantitative curve for calculation to obtain the corresponding concentrations of the three genes, thereby achieving quantitative analysis of the target genes.

[0072] At the same time, from Figure 6 It can also be seen that the present invention can successfully amplify and quantitatively detect the 16SrDNA, DSR Gene, APS The detection limits of the three genes were all 10 copies / mL.

[0073] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, rather than all the embodiments. A person skilled in the art can also obtain other embodiments based on the embodiments without inventiveness, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A rapid analysis method for metal corrosive microorganisms sulfate-reducing bacteria, characterized in that: The rapid analysis method comprises the following specific steps: Construction of triple asymmetric photon PCR system: Take the microbial genomic DNA sample to be detected, photosensitizer As2MoO6@NiS, 16SrDNA primer pair, DSR Primer pairs for genes and APS The primer pairs of the genes are used to construct a triple asymmetric photon PCR system; LED light source excites triple asymmetric photon PCR system: LED light source is used to irradiate triple asymmetric photon PCR system to perform asymmetric photon PCR amplification to obtain amplified products; Photothermal detection: The complementary sequences of three amplification products to be tested are fixed in sequence on the detection area of ​​the photothermal test paper, and the sequence complementary to the signal probe is fixed on the quality control area of ​​the test paper body; after the amplification product is mixed with the running buffer and the photothermal signal probe, it is dripped onto the sample pad of the test paper for photothermal temperature change detection to obtain a photothermal temperature change signal that is positively correlated with the content of the amplification product.

2. The rapid analysis method of metal corrosive microorganisms sulfate-reducing bacteria according to claim 1, characterized in that: The triple asymmetric photon PCR system includes a primer pair for 16S rDNA, DSR Primer pairs for genes and APS 1.0~2.0mL of each primer pair of the gene, 1.0~3.0mL of photosensitizer As2MoO6@NiS, 10mL of PCR premix and 1.0~2.0mL of the genomic DNA sample of the microorganism to be detected.

3. The rapid analysis method of metal corrosive microorganism sulfate-reducing bacteria according to claim 1 or 2, characterized in that: The primer pair for 16SrDNA included the upstream primer 16SrDNA-1-F: 5′-GTGCCAGCGCCGCGG-3′, the downstream primer 16SrDNA-1-R: 5′-AAAAAAAAAAGGTTACCTTGTTACGACTT-3′; DSR The primer pairs for the gene included upstream primer DSR-F: 5′-ACSCACTGGAAGCACGGCGG-3′, downstream primer DSR-R: 5′-AAAAAAAAAAGTGGMRCCGTGCAKRTTGG-3′; APS The primer pairs for the gene included the upstream primer APS-F: 5′-TGGCAGATMATGATYMACGGG-3′, the downstream primer APS-R: 5′-AAAAAAAAAAGGGCCGTAACCGTCCTTGAA-3′; The ratio between any upstream primer and its corresponding downstream primer in the three primer pairs is 1:0.1~1:

10.

4. The rapid analysis method of metal corrosive microorganism sulfate-reducing bacteria according to claim 1 or 2, characterized in that: The preparation process of photosensitizer As2MoO6@NiS is as follows: As2MoO6 nanoparticles were added to deionized water containing polyethylene glycol, ultrasonicated, and stirred overnight to obtain a mixed solution; The mixed solution was added to the NiS aqueous solution and stirred overnight, and the obtained solution was directly used as the photosensitizer As2MoO6@NiS solution; or the dark grey powder was collected by centrifugation and dried to obtain the solid photosensitizer As2MoO6@NiS.

5. The rapid analysis method of metal corrosive microorganism sulfate-reducing bacteria according to claim 1 or 2, characterized in that: The preparation process of the three photothermal signal probes is as follows: Three nucleic acid chains GTGCCAGCG, ACSCACTGGA, and TGGCAGATMA, which are partially complementary to the asymmetric photon PCR amplification product, were taken and incubated with the photosensitizer As2MoO6@NiS in 1´PBS buffer in the dark and at room temperature overnight to obtain three corresponding photothermal signal probes of 16SrDNA of sulfate-reducing bacteria, DSR Photothermal signal probes for genes and APS Photothermal signal probe for genes.

6. The rapid analysis method of metal corrosive microorganism sulfate-reducing bacteria according to claim 1 or 2, characterized in that: LED light source irradiation is performed using 808nm external coaxial reflection LED; during the irradiation process, the excitation power is 4.5~6W, and the total excitation time is 5~15min.

7. The rapid analysis method of metal corrosive microorganism sulfate-reducing bacteria according to claim 1 or 2, characterized in that: The reaction conditions of asymmetric photon PCR amplification were as follows: the concentration of photosensitizer As2MoO6@NiS solution was 0.1~1.0 mg / mL, the pre-denaturation excitation power was 6 W, 1 min; 20-60 denaturation-annealing-extension cycles: 6 W, 5 s; 4.5 W, 2 s; 5 W, 2 s; extension 5 W, 1 min.

8. The rapid analysis method of metal corrosive microorganism sulfate-reducing bacteria according to claim 1 or 2, characterized in that: Based on the photothermal temperature change signal, 16SrDNA, DSR Genes and APS The genes of the three gene entries were quantitatively analyzed as follows: After asymmetric photon PCR amplification and photothermal detection of three samples with unknown target gene concentrations, the photothermal temperature change signals of the three genes are substituted into the equation of the quantitative curve, and the corresponding concentrations of the three genes are calculated, thereby achieving quantitative analysis of the target genes; Among them, the quantitative curves of the genes of the three items are as follows: The quantitative curve of 16S rDNA is: y = 5.16x + 0.50, R 2 =0.9961; DSR The quantitative curve of the gene is: y=4.06x+0.78, R 2 =0.9899; APS The quantitative curve of the gene is y=4.91x+0.54, R 2 =0.9987.

9. The rapid analysis method of metal corrosive microorganism sulfate-reducing bacteria according to claim 1 or 2, characterized in that: The microbial genomic DNA sample to be detected is prepared by collecting microorganisms on the metal surface and extracting genomic DNA.

10. The rapid analysis method of metal corrosive microorganism sulfate-reducing bacteria according to claim 1 or 2, characterized in that: The detection area of ​​the photothermal test paper has three complementary sequences of the amplification products to be tested fixed in sequence, and the quality control area of ​​the test paper body has a sequence complementary to the signal probe fixed; Among them, the detection area is area T, and the quality control area is area C; 16S rDNA T probe: GTGCCAGCG, DSR T probe of gene ACSCACTGGA, APS T probe for gene: TGGCAGAT; C probe: TTTTTTTTTT.

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