A method for controlling the formation of red tide using natural sphalerite and synergistically reducing the abundance of resistance genes and virulence factors

Abstract

The present application belongs to the technical field of environmental protection, and particularly relates to a method for controlling formation of red tide and simultaneously reducing abundance of resistance genes and virulence factors by using natural flash zinc ore. The method comprises the following steps: first, preparing a microscale natural flash zinc ore suspension with photocatalytic activity; then, adding the suspension into seawater containing red tide algae; and finally, culturing under suitable conditions for the algae to survive, so as to achieve the purposes of controlling formation of red tide and inhibiting spread of resistance genes and virulence factors. The method for controlling formation of red tide and simultaneously reducing abundance of resistance genes and virulence factors provided in the present application utilizes visible light to drive the nano natural flash zinc ore to generate H2O2 and e ‑ and other various free radicals to attack microbial cells, so as to inhibit propagation of algae and pathogenic bacteria hosts in water, and reduce abundance of resistance genes and virulence factor genes in the process of algal bloom, thereby solving the technical problem of lack of simultaneous control of red tide, pathogenic bacteria and resistance genes in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of environmental protection technology, specifically relating to a method for controlling red tide formation and synergistically reducing the abundance of resistance genes and virulence factors using natural zinc sphalerite. Background Technology

[0002] Antibiotic resistance gene (ARG) pollution and harmful algal blooms (HABs) are serious ecological problems facing the 21st century. The rapid increase in global antibiotic use has led to the widespread dissemination of various antibiotic resistance genes (ARGs), with the ocean serving as a global reservoir and natural source of these genes, playing a crucial role in their spread. Furthermore, large numbers of human pathogens, such as *Vibrio spp.*, have been detected in the ocean, primarily affecting marine organisms and human health through virulence factors (VFGs). However, research on the impact of red tides on marine resistance genes and virulence factors remains in its early stages. Changes in marine environmental conditions, such as eutrophication, and the interaction of hydrological, meteorological, and seawater physicochemical factors, have created favorable conditions for the proliferation of phytoplankton, leading to HABs. It has been reported that from 2003 to 2020, the spatial distribution and frequency of harmful algal blooms in global coastal areas increased by 13.2% and 59.2%, respectively. The malignant proliferation of harmful algal blooms in nearshore areas disrupts the structure and function of marine ecosystems, kills other marine organisms, restricts nearshore economic development, and threatens public health; therefore, the management of harmful algal blooms has always been a major concern. In addition, harmful algal blooms cause dysbiosis in aquatic microbiota. Existing research shows that antibiotic resistance in aquatic environments is driven by changes in bacterial communities, thus there is a necessary link between the occurrence of harmful algal blooms and the spread of resistance genes. The algal sphere formed by the proliferation of large numbers of phytoplankton can provide bacteria with a unique, nutrient-rich ecological niche, thereby recruiting a large number of algae-related bacteria and potentially promoting inter-bacterial communication, such as horizontal gene transfer. Therefore, it is necessary to pay attention to changes in ARG and VFG while controlling harmful algal blooms.

[0003] Among the many technologies currently available for controlling harmful algal blooms, traditional physicochemical methods suffer from drawbacks such as long processing time, high cost, and the potential for secondary pollution. Photocatalysis, on the other hand, is highly efficient and does not produce secondary pollution, thus attracting widespread attention. However, there are no reports on using photocatalysts to simultaneously kill algae and inhibit the spread of resistance genes. Therefore, it is necessary to investigate the impact of photocatalysis on the development of harmful algal blooms and the spread of resistance genes.

[0004] Natural magnetic zinc sphalerite can effectively remove marine red tide algae (Carya spp.) under visible light irradiation. Furthermore, zinc sphalerite can kill various drug-resistant E. coli and inhibit the frequency of bacterial conjugation and transfer, showing great potential in reducing the spread of resistance genes. However, there are no reports on simultaneously inhibiting the spread of resistance genes during the algae-killing process using photocatalysts. Therefore, this invention selects a novel, efficient, and common photocatalyst—natural zinc sphalerite—to investigate the impact of photocatalysis on the development of harmful algal blooms and the spread of resistance genes. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention provides a method for controlling red tide formation using natural zinc sphalerite and synergistically reducing the abundance of resistance genes and virulence factors. When applied to the control of marine red tides, pathogens, and resistance genes, it can achieve the purpose of controlling red tide formation and inhibiting the spread of resistance genes and virulence factors.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides the application of natural zinc sphalerite in controlling red tide formation and synergistically reducing the abundance of resistance genes and virulence factors.

[0008] Preferably, the method for controlling red tide formation and synergistically reducing the abundance of resistance genes and virulence factors using natural sphalerite specifically includes the following steps:

[0009] S1. Add natural zinc sphalerite to an aqueous solution, disperse and mix well to obtain a micron-sized particle suspension with photocatalytic activity;

[0010] S2. Mix the micron-sized particle suspension from S1 with environmental seawater containing red tide algae, and then place it in a column-type photobioreactor to obtain environmental seawater containing photocatalytically active micron-sized particles.

[0011] S3. By maintaining suitable conditions for algae survival in seawater containing micron-sized particles with photocatalytic activity, the formation of red tides can be controlled and the spread of resistance genes and virulence factors can be inhibited after cultivation.

[0012] More preferably, the particle size of the natural zinc sphalerite is 20-150 μm.

[0013] More preferably, the concentration of natural sphalerite in ambient seawater is 0.1-100 mg / L.

[0014] More preferably, suitable conditions for algae survival include a suitable temperature of 22-25℃; a light-dark cycle of 12h:12h; a light intensity of 3300-3700 lux; and the use of LED or xenon lamps as the light source.

[0015] More preferably, the culture time is 7-21 days.

[0016] More preferably, S1 is dispersed and mixed using ultrasound for 10-30 minutes.

[0017] Preferably, the cultivation process further includes extracting representative samples during the pre-emergence, outbreak, and decline phases of algal blooms to quantitatively detect the abundance of 16S rRNA genes, resistance genes, and virulence factor genes, in order to assess the effectiveness of the cultivation.

[0018] More preferably, the method for extracting representative samples is to collect algal bacteria and planktonic bacteria samples separately, filter 100-300 mL of seawater using a polycarbonate core-pore etched filter membrane with a pore size of 3-5 μm to separate algal bacteria, and then filter the filtrate again using a filter membrane with a pore size of 0.22-0.45 μm to obtain planktonic bacteria.

[0019] More preferably, the quantitative detection is performed using the Wafergen Smartchip ultra-high-throughput real-time PCR system. The experimental procedure is as follows: sample preparation → using Smartchip → MSND solution addition → high-throughput qPCR → data analysis; the PCR reaction program is: pre-denaturation 95℃, 10 min; annealing 95℃, 30 s; extension 60℃, 30 s, for a total of 40 cycles.

[0020] Furthermore, the quantitative detection was performed using the Wafergen Smartchip ultra-high-throughput real-time PCR system to quantify the 16S rRNA gene, 93 resistance genes, and 29 virulence factor genes. The PCR reaction mixture was first added to the microarray using a nano-scale multi-sample spotting device (MSND) in 60 (samples) × 80 (assays) mode, followed by qPCR reaction on a cyclist. The experimental procedure was as follows: sample preparation → using Smartchip → MSND solution addition → high-throughput qPCR → data analysis.

[0021] The 100 nL qPCR reaction system is as follows:

[0022] reagents Amount added (nL) 1×LightCycler480SYBRGreenIMaster 50 500nMeachprimer 5 DNA template 2 ng / μL 20 <![CDATA[ddH2O]]> Replenish to 100nL

[0023] qPCR results were automatically analyzed using qPCR software. t＝31 Define the detection domain; if the fit analysis is satisfied, the region is considered detected.

[0024] Methods for calculating absolute gene abundance:

[0025] Gene copy number (intermediate value) = 10 (31-Ct) / (10 / 3) ;

[0026] Relative abundance = (ARG / VFG Gene copy number) / (16S rRNS Gene copy number);

[0027] 16S rRNA absolute gene copy number (copies / μL) = 10 (39.153-Ct) / 3.8821 ;

[0028] ARG / VFG absolute gene copy number (copies / μL) = 16S rRNA absolute gene copy number × relative abundance;

[0029]

[0030] The sample filtration volume is 100-300 mL.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] This invention discloses a method for controlling red tide formation and synergistically reducing the abundance of resistance genes and virulence factors using natural zinc sphalerite. First, a micron-sized natural zinc sphalerite suspension with photocatalytic activity is prepared. Then, the suspension is added to seawater containing red tide algae. After cultivation under suitable algal survival conditions, the method achieves the goal of controlling red tide formation and inhibiting the spread of resistance genes and virulence factors. The method provided in this application utilizes visible light to drive the micron-sized natural zinc sphalerite to produce H₂O₂ and e₂. - Various free radicals attack microbial cells, thereby inhibiting the reproduction of algae and pathogen hosts in aquatic bodies, reducing the abundance of resistance genes and virulence factor genes during algal blooms, thus solving the technical problem of lack of synergistic control over red tides, pathogens and resistance genes in existing technologies. Attached Figure Description

[0033] Figure 1 The inactivation effect of natural zincblende on bacterial abundance at different stages of algal blooms is shown. FL represents planktonic bacteria, PA represents algal bacteria; L: 0.5 mg / L, see Example 1 for details; H: 25 mg / L, see Example 2 for details; CK represents Comparative Example 1.

[0034] Figure 2 The effect of natural sphalerite on reducing resistance genes and virulence factors at different stages of algal bloom. Detailed Implementation

[0035] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0036] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0037] Example 1:

[0038] This embodiment provides a method for inhibiting red tide formation and reducing the abundance of resistance genes and virulence factors using photocatalytically active micron-sized particles. The method includes the steps of preparing environmental seawater containing photocatalytically active micron-sized particles, a method for culturing red tide algae, and a method for detecting resistance genes and virulence factor genes.

[0039] The steps for preparing environmental seawater containing photocatalytically active micron-sized particles include: adding natural zinc sphalerite (from the Huangshaping deposit in Hunan Province, where the natural zinc sphalerite particles are ground into powder and passed through a 340-mesh sieve, with a mineral particle size ≤40μm) to an ultrapure aqueous solution to achieve a concentration of 100mg / mL; and then sonicating the solution for 15 minutes to ensure uniform dispersion of the micron-sized natural zinc sphalerite, resulting in a natural zinc sphalerite suspension. Simultaneously, environmental seawater from the Pearl River estuary in Guangdong Province, containing various red tide algae, is collected. The prepared micron-sized natural zinc sphalerite suspension is then added to a column-type photobioreactor (Shanghai Guangyu Technology) containing 20L of environmental seawater to obtain environmental seawater containing photocatalytically active micron-sized particles. The final concentration of natural zinc sphalerite in the prepared environmental seawater is 0.5mg / L.

[0040] The steps for applying light to the column photobioreactor and maintaining the conditions for red tide algae cultivation included: turning on the LED lamps, temperature controller, and hydraulic circulation module of the column photobioreactor, maintaining a light intensity of 3500 lux, a temperature of 23±1℃, a light-dark cycle ratio of 12h:12h, and maintaining water circulation. The culture lasted for 15 days, and the algal density was recorded daily. Algal attached (PA) and planktonic bacteria (FL) samples were obtained at the pre-emergence, outbreak, and post-emergence stages. High-throughput qPCR was used for quantitative analysis of resistance genes and virulence factor genes.

[0041] Example 2:

[0042] This embodiment provides a method for inhibiting red tide formation and reducing the abundance of resistance genes and virulence factors using photocatalytically active micron-sized particles. The method includes the steps of preparing environmental seawater containing photocatalytically active micron-sized particles, a method for culturing red tide algae, and a method for detecting resistance genes and virulence factor genes.

[0043] The steps for preparing environmental seawater containing photocatalytically active micron-sized particles include: adding natural zinc sphalerite (from the Huangshaping deposit in Hunan Province, where the natural zinc sphalerite particles are ground into powder and passed through a 340-mesh sieve, with a mineral particle size ≤40μm) to an ultrapure aqueous solution to achieve a concentration of 100mg / mL; and then sonicating the solution for 15 minutes to ensure uniform dispersion of the micron-sized natural zinc sphalerite, resulting in a natural zinc sphalerite suspension. Simultaneously, environmental seawater containing various algae is collected from the Pearl River estuary in Guangdong Province. The prepared micron-sized natural zinc sphalerite suspension is then added to a column-type photobioreactor (Shanghai Guangyu Technology) containing 20L of environmental seawater to obtain environmental seawater containing photocatalytically active micron-sized particles. The final concentration of natural zinc sphalerite in the prepared environmental seawater is 25mg / L.

[0044] The steps for applying light to the column photobioreactor and maintaining the conditions for red tide algae cultivation included: turning on the LED lamps, temperature controller, and hydraulic circulation module of the column photobioreactor, maintaining a light intensity of 3500 lux, a temperature of 23±1℃, a light-dark cycle ratio of 12h:12h, and maintaining water circulation. The culture lasted for 15 days, and the algal density was recorded daily. Algal attached (PA) and planktonic bacteria (FL) samples were obtained at the pre-emergence, outbreak, and post-emergence stages. High-throughput qPCR was used for quantitative analysis of resistance genes and virulence factor genes.

[0045] Comparative Example 1:

[0046] In this comparative study, environmental seawater containing red tide algae was placed in a column-type photobioreactor. The red tide algae were cultured under a light intensity of 3500 lux, a temperature of 23±1℃, a light-dark cycle ratio of 12h:12h, and a water circulation system. The culture lasted for 15 days, and the algal density was recorded daily. Algal attached (PA) and planktonic bacteria (FL) samples were obtained at the pre-emergence, during-emergence, and post-emergence stages. High-throughput qPCR was used to quantitatively analyze resistance genes and virulence factor genes.

[0047] Experimental example:

[0048] The effectiveness of the methods used in Examples 1-2 and Comparative Example 1 to inhibit red tide formation while reducing the abundance of resistance genes and virulence factors was verified.

[0049] The process for verifying the effectiveness was as follows: Representative samples of algal bacteria and planktonic bacteria extracted from Comparative Example 1 and Examples 1-2 were collected during the pre-emergence, outbreak, and decline phases of algal blooms and quantitative analysis was performed. Specifically, 100-300 mL of algal solution was collected from the culture vessel in triplicate, and a 5 μm pore size polycarbonate core-porous etched filter membrane (Whatman, USA) was used to separate algal bacteria and planktonic bacteria. Immediately afterwards, 100-300 mL of the filtrate was filtered again using a 0.22 μm aqueous filter membrane to obtain planktonic bacteria, and the filter membrane was stored at -20°C.

[0050] The quantitative analysis steps included: 16S rRNA gene and 93 resistance genes. 1 29 virulence factors 2 Gene quantification was performed using the Wafergen Smartchip ultra-high-throughput real-time PCR system (Anhui Yuanzai Biotechnology Co., Ltd.). (For details of the 93 resistance genes and 29 virulence factors detected, please refer to the literature "Guo, Y.;Liu, M.;Liu, L.;Liu, X.;Chen, H.;Yang, J., The antibiotic resistome of free-living and particle-attached bacteria under a reservoir cyanobacterial bloom. Environment International 2018, 117, 107-115. Xie, S.-T.;Ding, L.-J.;Huang, F.-Y.;Zhao, Y.;An, X.-L.;Su, ​​J.-Q.;Sun, G.-X.;Song, Y.-Q.;Zhu, Y.-G., VFG-Chip: A high-throughput qPCR microarray for profiling virulence factor genes from the environment. Environment International) 2023,172,107761.”), the PCR reaction mixture was first added to the microarray using a nanoscale multisample dispenser (MSND) in 60 (samples) × 80 (assays) mode, and then qPCR was performed on a cycler. The experimental procedure was as follows: sample preparation → using Smartchip → MSND solution addition → high-throughput qPCR → data analysis.

[0051] The 100 nL reaction system is as follows:

[0052] reagents Amount added (nL) 1×LightCycler480SYBRGreenIMaster 50 500nMeachprimer 5 DNA template 2 ng / μL 20 <![CDATA[ddH2O]]> Replenish to 100nL

[0053] The PCR reaction program was as follows: pre-denaturation at 95℃ for 10 min; annealing at 95℃ for 30 s; extension at 60℃ for 30 s, for a total of 40 cycles. qPCR results were automatically analyzed using qPCR software. t＝31 Define the detection domain; if the fit analysis is satisfied, the region is considered detected.

[0054] Methods for calculating absolute gene abundance:

[0055] Gene copy number (intermediate value) = 10 (31-Ct) / (10 / 3) ;

[0056] Relative abundance = (ARG / VFG Gene copy number) / (16S rRNS Gene copy number);

[0057] 16S rRNA absolute gene copy number (copies / μL) = 10 (39.153-Ct) / 3.8821 ;

[0058] ARG / VFG absolute gene copy number (copies / μL) = 16S rRNA absolute gene copy number × relative abundance;

[0059]

[0060] The sample filtration volume is 100-300 mL.

[0061] The results are as follows Figure 1 As shown, using the 16S rRNA gene to detect bacterial abundance in different treatment groups revealed that, during the algal bloom and decline phases, the absolute abundance of the 16S rRNA gene in the 0.5 mg / L mineral treatment group of Example 1 decreased by 68.98% and 42.12% compared to Control Example 1, respectively; the absolute abundance of the 16S rRNA gene in the 25 mg / L mineral treatment group of Example 2 decreased by 62.23% and 63.61% compared to Control Example 1. During the algal bloom and decline phases, the absolute abundance of the 16S rRNA gene in the 0.5 mg / L mineral treatment group of Example 1 decreased by 21.67% and increased by 15.88% compared to Control Example 1; the absolute abundance of the 16S rRNA gene in the 25 mg / L mineral treatment group of Example 2 decreased by 53.18% and 60.78% compared to Control Example 1.

[0062] like Figure 2As shown in A and B, during the outbreak phase of each group of planktonic bacteria, the total ARG decreased by 67.23% and 85.18% in absolute abundance compared to Comparison Example 1 in Examples 1 and 2, respectively. Among them, the major gene chloramphenicol decreased by 50.98% and 76.63%, respectively; fluoroquinolones decreased by 54.65% and 82.32%, respectively; MLSBs decreased by 85.59% and 94.17%, respectively; and multidrugs decreased by 73.93% and 60.24%, respectively. During the regression phase, the total ARG decreased by 13.61% and 21.05% in absolute abundance compared to Comparison Example 1 in Examples 1 and 2, respectively. Among them, the major gene fluoroquinolones decreased by 57.51% and 73.08%, respectively; and MLSBs decreased by 28.42% and 84.85%, respectively. In the algal community, during the outbreak phase of each group, the total ARG decreased by 56.81% and 97.41% in absolute abundance compared to Comparison 1 in Examples 1 and 2, respectively. Specifically, the major gene chloramphenicol decreased by 6.65% and 72.45%, fluoroquinolones by 31.83% and 98.70%, MLSBs by 92.81% and 99.56%, and peptides by 70.75% and 98.42%, respectively. During the regression phase, the total ARG decreased by 41.14% and 45.69% in absolute abundance compared to Comparison 1 in Examples 1 and 2, respectively. Specifically, the major gene chloramphenicol decreased by 58.89% and 13.71%, and fluoroquinolones by 30.46% and 84.90%, respectively. The specific gene names detected are shown in Table 1.

[0063] Table 1. Quantitative detection of major resistance genes

[0064]

[0065] like Figure 2As shown in C and D, in the outbreak phase of planktonic bacteria, the absolute abundance of total VFG in Examples 1 and 2 decreased by 39.95% and 91.61% compared to Comparative Example 1, respectively. Among them, the abundance of major adhesion genes decreased by 16.82% and 91.75%, respectively; the abundance of ion absorption genes decreased by 71.51% and 85.29%, respectively; the abundance of nutrient factor genes decreased by 31.53% and 88.72%, respectively; and the abundance of serum resistance genes decreased by 90.40% and 98.06%, respectively. In the regression phase, the absolute abundance of total VFG in Examples 1 and 2 decreased by 31.82% and 60.35% compared to Comparative Example 1, respectively. Among them, the abundance of major adhesion genes decreased by 56.11% and 82.29%, respectively; the abundance of ion absorption genes decreased by 4.71% and 26.96%, respectively; and the abundance of secretion system genes decreased by 58.04% and 80.62%, respectively. In the algal community, during the outbreak phase of each group, the absolute abundance of total VFG in Examples 1 and 2 decreased by 70.15% and 98.48% compared to Comparison Example 1, respectively. Specifically, the abundance of major adhesion genes decreased by 76.35% and 99.59%, ion-absorbing genes by 70.92% and 92.04%, secretion system genes by 69.59% and 91.15%, and serum resistance genes by 83.47% and 100%, respectively. During the decline phase, the absolute abundance of total VFG in Examples 1 and 2 decreased by 56.69% and 48.36% compared to Comparison Example 1, respectively. Specifically, the abundance of major ion-absorbing genes decreased by 43.08% and 92.32%, nutrient factor genes by 58.43% and 36.08%, and serum resistance genes by 77.04% and 86.21%, respectively. The specific gene names detected are shown in Table 2.

[0066] The above results demonstrate that the method provided by this invention for inhibiting the growth of red tide algae while reducing the abundance of resistance genes and virulence factors can utilize a series of active free radicals (including ·O2, ·OH, H2O2, and e-) generated by nanoparticles under photocatalysis. - (etc.) play a role in inactivating bacteria. Among them, H2O2 and e - The invention plays a decisive role in the inactivation of bacteria in water, enabling a significant reduction in the abundance of ARGs in water through bacterial inactivation.

[0067] Table 2. Quantitative detection of major virulence factor genes

[0068] Virulence factor type Virulence factor genotypes Adhesive ompA(d), csgG(d), shdA(d), mrkA(d), fimA(d)[Klebs], fimB Ion absorption bauA2(d), entB, fepB(d) endocrine system prgJ(d), ler(d), clpV / tssH(d) Serum resistance wbbM、wzm Nutritional factors allS(d)

[0069] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. The application of natural sphalerite in controlling red tide formation and synergistically reducing the abundance of resistance genes and virulence factors, characterized in that, The method of using natural zinc sphalerite to control red tide formation and synergistically reduce the abundance of resistance genes and virulence factors specifically includes the following steps: S1. Add natural zinc sphalerite to an aqueous solution, disperse and mix well to obtain a micron-sized particle suspension with photocatalytic activity; S2. Mix the micron-sized particle suspension from S1 with environmental seawater containing red tide algae, and then place it in a column-type photobioreactor to obtain environmental seawater containing photocatalytically active micron-sized particles. S3. Maintain the temperature of the seawater containing photocatalytically active micron-sized particles at 22-25℃; use LED or xenon lamp light sources with a light-dark cycle of 12h:12h; light intensity of 3300-3700 lux; culture for 7-21 days. After culture, the formation of red tides can be controlled and the spread of resistance genes and virulence factors can be inhibited.

2. The application according to claim 1, characterized in that, The grain size of natural sphalerite is 20-150 μm.

3. The application according to claim 1, characterized in that, Use ultrasonic dispersion to mix the mixture for 10-30 minutes.

4. The application according to claim 1, characterized in that, The concentration of natural sphalerite in environmental seawater is 0.1-100 mg / L.

5. The application according to claim 1, characterized in that, The cultivation process also includes extracting representative samples during the pre-emergence, outbreak, and decline phases of algal blooms to quantitatively detect the abundance of 16S rRNA genes, resistance genes, and virulence factor genes, in order to assess the effectiveness of the cultivation.

6. The application according to claim 5, characterized in that, The method for extracting representative samples is to collect algal bacteria and planktonic bacteria samples separately, filter 100-300 mL of seawater using a polycarbonate core-pore etched filter membrane with a pore size of 3-5 μm to separate algal bacteria, and then filter the filtrate again using a filter membrane with a pore size of 0.22-0.45 μm to obtain planktonic bacteria.

7. The application according to claim 5, characterized in that, The quantitative detection was performed using the Wafergen Smartchip ultra-high-throughput real-time PCR system. The experimental procedure was as follows: sample preparation → using Smartchip → MSND solution addition → high-throughput qPCR → data analysis; the PCR reaction program was: pre-denaturation 95℃, 10 min; annealing 95℃, 30 s; extension 60℃, 30 s, for a total of 40 cycles.

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