Rapid analysis method for metal corrosive microorganism acidithiobacillus ferrooxidans

Abstract

The invention provides a rapid analysis method of metal corrosive microorganism acidithiobacillus ferrooxidans, and belongs to the technical field of biology.The rapid analysis method comprises the steps that a microorganism genome DNA sample to be detected and a photosensitizer As2MoO6 at-NiS 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, and the amplification result is obtained; after amplification is completed, the obtained amplification product is mixed with a photo-thermal signal probe prepared from a photosensitizer As2MoO6 at NiS, and photo-thermal temperature change detection is carried out. The rapid analysis method has the characteristics of high efficiency, convenience and accuracy, and is suitable for rapid detection of acidithiobacillus ferrooxidans.

Description

Technical Field

[0001] The invention belongs to the technical field of inspection and detection, and particularly relates to a rapid analysis method for metal corrosive microorganism Acidithiobacillus ferrooxidans. Background Art

[0002] Acidithiobacillus ferrooxidans (AF) is a chemoautotrophic bacterium that uses ferrous ions and low-valent sulfur as energy sources. ²+ ) is the trivalent iron ion (Fe³ + ). This oxidation process not only provides the bacteria with the energy they need to grow, but also changes the chemical environment on the metal surface. Fe³ + Ions have strong oxidizing properties and can react with atoms on the metal surface, causing the metal to be oxidized and corroded. In industry, AF may cause corrosion of facilities and equipment, so the detection of AF is of great significance for 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 AF. 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 analytical methods for the metal-corrosive microorganism Acidithiobacillus ferrooxidans have limitations, including the time-consuming and complicated operation of the traditional PCR method, and the insufficient sensitivity for detecting low-abundance target bacteria; at the same time, there is a lack of specific genes for Acidithiobacillus ferrooxidans, such as aL Gene, cyc2 Gene, rus Efficient, simultaneous gene amplification and detection technology; in addition, existing detection methods have deficiencies in rapid quantification and on-site application. The present invention provides a rapid analysis method for metal-corrosive microorganisms Acidithiobacillus ferrooxidans.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is: A rapid analysis method for metal corrosive microorganisms Acidithiobacillus ferrooxidans, the rapid analysis method comprising the following specific steps: Construction of triple asymmetric photon PCR system: Take the microbial genomic DNA sample to be detected, the photosensitizer As2MoO6@NiS, aL Primer pairs for genes cyc2 Primer pairs for genes and rus 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 photosensitizer is modified with three nucleic acid chains that are partially complementary to the amplified products to prepare three corresponding photothermal signal probes; the complementary sequences of the three amplified 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; the amplified product is 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 is 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 aL Primer pairs for genes cyc2 Primer pairs for genes and rus 1.0~2.0mL 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; in, aL Gene, cyc2 Gene, rus 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 (i.e., R) in the three primer pairs is 1:0.1~1:10.

[0009] Furthermore, Acidithiobacillus ferrooxidans Acidithiobacillus ferrooxidans The genetic information includes aL Gene, cyc2 Gene, rus The gene is included in NCBI GenBank, Accession Number AJ006456.2 (accessed October 19, 2024); Acidithiobacillus ferrooxidans aL The gene sequence is shown in SEQ ID NO: 1; aL The primer pair for the gene includes the upstream primer aL-F: 5'-GTGCCTTTCCCGAACTCACG-3', downstream primer aL -R: 5'-AAAAAAAAAATCCTCCAGCAGACTGAGTCC-3'; Acidithiobacillus ferrooxidans cyc2 The gene sequence is shown in SEQ ID NO: 2; cyc2 The primer pair for the gene includes the upstream primer cyc2 -F: 5'-TGTTGGTGTCTATGGGGCGG-3', downstream primer cyc2 -R: 5'-AAAAAAAAAAAACCCCAATCGTGCTGATAGGC-3'; Acidithiobacillus ferrooxidans rus The gene sequence is shown in SEQ ID NO: 3; rus The primer pair for the gene includes the upstream primer rus -F: 5'-GGCACGCTGGATTCCACATGGAAAGAGGCG-3', downstream primer rus -R: 5'-AAAAAAAAAACTTCAAAGCTCGGGAACGG-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 GTGCCTTTCC, TGTTGGTGTC, and GGCACGCTGG, 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 dark and shake at room temperature overnight to obtain three corresponding photothermal signal probes, namely, Acidithiobacillus ferrooxidans aL Photothermal signal probes for genes, cyc2 Photothermal signal probes for genes and rus 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 10~30min.

[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 6 W, 1 min; 20-60 denaturation-annealing-extension cycles: 6 W, 10 s; 4.5 W, 5 s; 5 W, 5 s; extension 5 W, 3 min.

[0014] Furthermore, based on the light-thermal temperature change signal aL Gene, cyc2 Gene, rus 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: aL The quantitative curve of the gene is: y=5.50x+0.78, R 2 =0.9929; cyc2 The quantitative curve of the gene is: y=4.55x+0.82, R 2 =0.9935; rus The quantitative curve of the gene is y=4.36x+0.34, R 2 =0.9897.

[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 three complementary sequences of the amplification products to be tested ( aL T probe of gene aL :TCCTCCAGCA, cyc2 T probe of gene cyc2 :CCCCAATCGT, rus T probe of gene rus : CTTCAAAGCT), a sequence complementary to the signal probe (C probe: TTTTTTTTTT) is fixed on the quality control area (C area) of the test strip body.

[0017] The beneficial effects of the rapid analysis method of the metal corrosive microorganism Acidithiobacillus ferrooxidans of the present invention are: The rapid analysis method of the present invention uses the photoactivity of the photosensitizer As2MoO6@NiS to generate photoelectrons and photothermal effects, can quickly control 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 a photothermal signal probe for photothermal test paper, providing sensitive and quantifiable signals. Compared with traditional PCR amplification, which takes 40 minutes, and subsequent detection using methods such as gel electrophoresis is also time-consuming, and fluorescent quantitative PCR amplification and detection takes 40 minutes, and its detection limit is about 100 copies, the rapid analysis method of the present invention shortens the entire amplification and detection time to about 20 minutes, and reduces the detection limit to 10 copies, which is 10 times more sensitive than traditional PCR. The rapid analysis method of the present invention can quickly realize the efficient amplification and detection of multiple target DNAs, has the characteristics of high efficiency, convenience and accuracy, and is suitable for the rapid detection of Acidithiobacillus ferrooxidans. 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 with three entries; Figure 6 The left picture in aL The quantitative curve of the gene, the middle figure is cyc2 The quantitative curve of the gene is shown on the right. rus 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 invention discloses a rapid analysis method for metal corrosive microorganisms Acidithiobacillus ferrooxidans, and specifically relates to a method for using a photosensitizer As2MoO6@NiS to perform asymmetric photon PCR and photothermal test paper to detect Acidithiobacillus ferrooxidans. aL Gene, cyc2 Gene, rus 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 Acidithiobacillus ferrooxidans was aL Gene, cyc2 Gene, rus 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 Acidithiobacillus ferrooxidans in the microbial genomic DNA extracted in step S2 aL Gene, cyc2 Gene, rus 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 10-30min.

[0026] Asymmetric photon PCR amplification was then 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 6 W, 1 min; 20-60 denaturation-annealing-extension cycles: 6 W, 10 s; 4.5 W, 5 s; 5 W, 5 s; extension 5 W, 3 min.

[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 detection area (T area) of the photothermal test paper is fixed with three complementary sequences of the amplification products to be tested (T aL :TCCTCCAGCA,T cyc2 :CCCCAATCGT,T rus : CTTCAAAGCT), a sequence complementary to the signal probe (C probe: TTTTTTTTTT) is fixed on the quality control area (C area) of the test strip body.

[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 the metal-corrosive microorganism Acidithiobacillus ferrooxidans. During the application process, 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 aL Gene, cyc2 Gene, rus The following examples are based on the high efficiency amplification of Acidithiobacillus ferrooxidans aL The entire amplification method is described in full detail using genes as an example.

[0034] Example 1 A metal-corrosive microorganism Acidithiobacillus ferrooxidans aL Rapid gene analysis method This example is a metal corrosive microorganism Acidithiobacillus ferrooxidans aL A rapid gene analysis method is specifically a method that uses the photothermal effect of the photosensitizer As2MoO6@NiS to drive the growth of Acidithiobacillus ferrooxidans aLA method for asymmetric photon PCR using a gene as a template, the method comprising the following specific steps: S1. Primer sequence design: According to the principle of asymmetric PCR, the target gene Acidithiobacillus ferrooxidans was aL A set of amplification primer pairs were designed for the gene sequence, the primer pairs included upstream and downstream primers, and the specific primer set sequences are shown in Table 1; aL The gene sequence 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., containing Acidithiobacillus ferrooxidans). aL Gene).

[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] For making standard curves, etc. aL 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.

[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 5 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 0.5 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 pair 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 Asymmetric photon PCR amplification of Acidithiobacillus ferrooxidans aL Table of concentrations and reaction volumes of each gene component

[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 the asymmetric photon PCR. The excitation power was 4.5W and the total excitation time was 10min.

[0045] Then asymmetric photon PCR amplification was performed, and the reaction conditions were: the reaction conditions of asymmetric photon PCR amplification were: the concentration of photosensitizer As2MoO6@NiS solution was 0.5mg / mL, the pre-denaturation excitation power was 6W, 1min; denaturation-annealing-extension cycle: 6W, 10s; 4.5W, 5s; 5W, 5s; extension 5W, 3min. The total time for 20 cycles of asymmetric photon PCR amplification was 10 min, and the total time for 40 cycles of asymmetric photon PCR amplification was 20 min.

[0046] right aL The reference sample with a gene copy number of 1000 was amplified for 20 and 40 cycles respectively. aL The gene was subjected to asymmetric photon PCR to obtain the corresponding amplification product.

[0047] According to 20 amplification cycles, the same aL Asymmetric photon PCR was performed on samples with different gene copy numbers and upstream and downstream primer ratios to obtain corresponding asymmetric photon PCR amplification samples.

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

[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 T-zone probe of alaS is fixed on the detection zone (T zone) of the photothermal test paper: T alaS : TCCTCCAGCA, 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 (ACTGAGTCC) and 1mL of 1mg / mL photosensitizer As2MoO6@NiS, and shake overnight at room temperature in 1´PBS buffer in the dark. aL Photothermal signal probe for genes.

[0051] Take 10mL of asymmetric photon PCR amplification sample and mix with 10mL of aLAfter mixing the photothermal signal probe of the gene with 80mL running buffer (4×SSC, 0.25% Tween-20), the sample was loaded, and the amplified product and the photothermal signal probe were captured in the detection area of ​​the photothermal test paper in a sandwich form. After loading for 10 minutes, the test paper was air-dried and the photothermal temperature change signal of the detection area was collected by a smartphone. Then, the quantitative curve was drawn 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 the amplified product corresponding to the ratio of upstream and downstream primers and mix with 10mL of aL The photothermal signal probe of the gene was mixed with 80 mL of running buffer (4×SSC, 0.25% Tween-20) and loaded. The amplified product and the photothermal signal probe were captured in a sandwich form in the detection area of ​​the photothermal test paper. After 10 minutes of loading, the test paper was air-dried, and the photothermal temperature change signal of the detection area was collected by a smartphone. The measured photothermal temperature change signal was brought into the quantitative curve, and the target gene was quantified based on the quantitative curve.

[0053] from Figure 3 It can be seen that under the conditions of using different upstream and downstream primer ratios, the detection area of ​​the photothermal test paper 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 and 1:5 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. Amplification of Acidithiobacillus ferrooxidans by conventional PCR aL Table of concentrations and reaction volumes of each gene component

[0055] The traditional PCR program was set as follows: pre-denaturation at 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 10 s, and amplification for 30 min. The total time was about 40 min, and the traditional PCR amplification product was obtained.

[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 and 10 minutes respectively according to the method in step S4 to step S5 to obtain asymmetric photon PCR amplification products corresponding to different amplification times.

[0057] The conventional PCR amplification products and the asymmetric photon PCR amplification products corresponding to different amplification times 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 40 minutes of gel electrophoresis shows a weak product band, while the asymmetric photon PCR method of the present invention can show a band after 10 minutes of gel electrophoresis, and the band after 20 minutes of amplification is significantly deeper than the traditional PCR amplification for 40 minutes, that is, the amplification product of the asymmetric photon PCR amplification for 20 minutes is significantly more than the traditional PCR amplification for 40 minutes. 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 Acidithiobacillus ferrooxidans This embodiment is a rapid analysis method for metal corrosive microorganisms Acidithiobacillus ferrooxidans, specifically a method of using photosensitizer As2MoO6@NiS as a photothermal signal probe to detect Acidithiobacillus ferrooxidans. aL Gene, cyc2 Gene, rus 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 target gene Acidithiobacillus ferrooxidans was aL Gene, cyc2 Gene, rus The primer pairs were designed for amplification of the genes, and the primer pairs included upstream and downstream primers. The specific primer sets and primer pair sequences are shown in Table 4. Among them, the aL The gene sequence is shown in SEQ ID NO: 1; cyc2 The gene sequence is shown in SEQ ID NO: 2; rus 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 making standard curves, etc. aL Gene, cyc2Gene, rus 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 use aL Gene, cyc2 Gene, rus 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 Acidithiobacillus ferrooxidans aL Gene, cyc2 Gene, rus 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, aL Gene, cyc2 Gene, rus 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 (T aL :TCCTCCAGCA,T cyc2 :CCCCAATCGT,T rus : CTTCAAAGCT), a sequence complementary to the signal probe (C probe: TTTTTTTTTT) is fixed on the quality control area (C area) of the test strip body.

[0066] 10 mL of each of the three nucleic acid chains GTGCCTTTCC, TGTTGGTGTC, and GGCACGCTGG with a concentration of 0.1 M that are partially complementary to the asymmetric photon PCR amplification product were taken and mixed with 1 mL of 1 mg / mL photosensitizer As2MoO6@NiS in 1´PBS buffer in the dark and shaken at room temperature overnight to obtain the three corresponding Acidithiobacillus ferrooxidans aL Photothermal signal probes for genes, cyc2 Photothermal signal probes for genes and rus 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 Take the target gene with the number of copies of 1, 10, 100, 1000, 10000, and 100000 respectively aL Gene, cyc2 Gene, rus 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), load it, and 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, use the initial target gene copy number as the horizontal axis and the corresponding photothermal temperature change signal as the vertical axis to draw the quantitative curve of the three genes. The results are as follows: Figure 6 shown.

[0070] Among them, the quantitative curves of the genes of the three items are as follows: aL The quantitative curve of the gene is: y=5.50x+0.78, R 2 =0.9929; cyc2 The quantitative curve of the gene is: y=4.55x+0.82, R 2 =0.9935; rus The quantitative curve of the gene is y=4.36x+0.34, R 2 =0.9897.

[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 Acidithiobacillus ferrooxidans aL Gene, cyc2 Gene, rus 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 Acidithiobacillus ferrooxidans, 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, the photosensitizer As2MoO6@NiS, aL Primer pairs for genes cyc2 Primer pairs for genes and rus 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 photosensitizer is modified with three nucleic acid chains that are partially complementary to the amplified products to prepare three corresponding photothermal signal probes; the complementary sequences of the three amplified 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; the amplified product is 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 is positively correlated with the content of the amplified product.

2. The rapid analysis method of metal corrosive microorganism Acidithiobacillus ferrooxidans according to claim 1, characterized in that: The triple asymmetric photon PCR system includes aL Primer pairs for genes cyc2 Primer pairs for genes and rus 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 Acidithiobacillus ferrooxidans according to claim 1 or 2, characterized in that: aL The primer pair for the gene includes the upstream primer aL -F: 5'-GTGCCTTTCCCGAACTCACG-3', downstream primer aL -R: 5'-AAAAAAAAAATCCTCCAGCAGACTGAGTCC-3'; cyc2 The primer pair for the gene includes the upstream primer cyc2 -F: 5'-TGTTGGTGTCTATGGGGCGG-3', downstream primer cyc2 -R: 5'-AAAAAAAAAAAACCCCAATCGTGCTGATAGGC-3'; rus The primer pair for the gene includes the upstream primer rus -F: 5'-GGCACGCTGGATTCCACATGGAAAGAGGCG-3', downstream primer rus -R: 5'-AAAAAAAAAAACTTCAAAGCTCGGGAACGG-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 Acidithiobacillus ferrooxidans 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 Acidithiobacillus ferrooxidans 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 partially complementary to the asymmetric photon PCR amplification product, GTGCCTTTCC, TGTTGGTGTC, and GGCACGCTGG, were taken and reacted with the photosensitizer As2MoO6@NiS, respectively, in 1´PBS buffer, protected from light, and shaken at room temperature overnight to obtain three corresponding Acidithiobacillus ferrooxidans. aL Photothermal signal probes for genes, cyc2 Photothermal signal probes for genes and rus Photothermal signal probe for genes.

6. The rapid analysis method of metal corrosive microorganism Acidithiobacillus ferrooxidans 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 10~30min.

7. The rapid analysis method of metal corrosive microorganism Acidithiobacillus ferrooxidans according to claim 1 or 2, characterized in that: The reaction conditions of asymmetric photon PCR amplification are as follows: the concentration of photosensitizer As2MoO6@NiS solution is 0.1-1.0 mg / mL, the pre-denaturation excitation power is 6 W, 1 min; 20-60 denaturation-annealing-extension cycles: 6 W, 10 s; 4.5 W, 5 s; 5 W, 5 s; extension 5 W, 3 min.

8. The rapid analysis method of metal corrosive microorganism Acidithiobacillus ferrooxidans according to claim 1 or 2, characterized in that: Based on the light-heat-temperature signal aL Gene, cyc2 Gene, rus 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: aL The quantitative curve of the gene is: y=5.50x+0.78, R 2 =0.9929; cyc2 The quantitative curve of the gene is: y=4.55x+0.82, R 2 =0.9935; rus The quantitative curve of the gene is y=4.36x+0.34, R 2 =0.9897.

9. The rapid analysis method of metal corrosive microorganism Acidithiobacillus ferrooxidans 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; aL T probe for gene: TCCTCCAGCA, cyc2 T probe of gene: CCCCAATCGT, rus T probe for gene: CTTCAAAGCT; C probe: TTTTTTTTTT.

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