Method for controlling red tide formation and cooperatively reducing abundance of resistance genes and virulence factors by using natural sphalerite

Abstract

The invention belongs to the technical field of environmental protection, and particularly relates to a method for controlling red tide formation and cooperatively reducing abundance of resistance genes and virulence factors by using natural sphalerite. The method comprises the following steps: firstly preparing a micron-sized natural sphalerite suspension with photocatalytic activity, then adding the suspension into environmental seawater containing red-tide algae, and culturing under the condition suitable for the survival of the algae, thereby achieving the purposes of controlling the formation of the red tide and inhibiting the propagation of resistance genes and virulence factors. According to the method for controlling the formation of the red tide and cooperatively reducing the abundance of the resistance gene and the virulence factor, provided by the invention, visible light is used for driving nano natural sphalerite to generate H2O2, e <-> and other various free radicals to attack microbial cells, so that the reproduction of algae and pathogenic bacteria hosts in a water body is inhibited; the abundance of resistance genes and virulence factor genes in the algae blooming process is reduced, so that the technical problem of lack of cooperative control on red tide, pathogenic bacteria and resistance genes in the prior art is solved.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental protection, and in particular relates to a method for controlling the formation of red tides and synergistically reducing the abundance of resistance genes and virulence factors by using natural sphalerite. Background Art

[0002] Antibiotic resistance gene (ARG) pollution and harmful algal blooms (HABs) are serious ecological problems facing the 21st century. Due to the sharp increase in the use of antibiotics worldwide, different types of antibiotic resistance genes (ARGs) have been widely spread. The ocean is a global reservoir and natural source of antibiotic resistance genes and plays an important role in the spread of ARGs. In addition, a large number of human pathogens have been detected in the ocean, such as Vibrio spp., which mainly affect marine life and human health through virulence factors (VFGs). However, research on the impact of red tides on marine resistance genes and virulence factors is still in its early stages. Due to changes in marine environmental conditions, such as eutrophication of water bodies, the interactive effects of hydrometeorological and seawater physical and chemical factors, suitable conditions have been obtained for the massive reproduction of phytoplankton, leading to the HABs phenomenon. It is reported that from 2003 to 2020, the occurrence space and frequency of harmful algal blooms in coastal areas around the world increased by 13.2% and 59.2%, respectively. The malignant proliferation of harmful algal blooms in offshore areas can destroy the structure and function of marine ecology, kill other marine organisms, restrict offshore economic development, and threaten public health. Therefore, the treatment of harmful algal blooms has always attracted much attention. In addition, the occurrence of harmful algal blooms can cause the imbalance of water flora. Existing studies have shown that the antibiotic resistance group in the aquatic environment is driven by changes in bacterial communities, so there is an inevitable connection between the occurrence of harmful algal blooms and the spread of resistance genes. The algal circle formed by the reproduction of a large number of phytoplankton can provide bacteria with a unique nutrient-rich ecological niche, thereby recruiting a large number of algae-related bacteria and may promote exchanges between bacteria, such as horizontal gene transfer. Therefore, it is necessary to pay attention to the changes in ARGs and VFGs while controlling harmful algal blooms.

[0003] Among the many technologies currently used to control harmful algal blooms, traditional physical and chemical methods have the disadvantages of being time-consuming, costly, and prone to secondary pollution, while photocatalytic technology has the characteristics of being highly efficient and non-polluting, and therefore has received widespread attention. However, there have been no reports on the simultaneous killing of algae and inhibition of the spread of resistance genes using photocatalysts, so it is necessary to explore the effects of photocatalysis on the development of harmful algal blooms and the spread of resistance genes.

[0004] Natural magnetic sphalerite can effectively remove marine red tide algae - marine cardinal algae under visible light irradiation, and sphalerite can kill multiple drug-resistant Escherichia coli and inhibit the frequency of conjugation transfer between bacteria. It has great potential in reducing the spread of resistance genes. However, there are no reports on the simultaneous inhibition of resistance gene propagation in the process of algae killing using photocatalysts. For this reason, the present invention selects an emerging, efficient and common photocatalyst - natural sphalerite to explore the effect of photocatalysis on the development of harmful algal blooms and the spread of resistance genes. Summary of the invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the present invention provides a method for using natural sphalerite to control the formation of red tides and synergistically reduce the abundance of resistance genes and virulence factors. It is used to control marine red tides, pathogens and resistance genes, so as to achieve the purpose of controlling the formation of red tides and inhibiting the spread of resistance genes and virulence factors.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

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

[0008] Preferably, the method of using natural sphalerite to control the formation of red tide and synergistically reduce the abundance of resistance genes and virulence factors specifically comprises the following steps:

[0009] S1. adding natural sphalerite into an aqueous solution, dispersing and mixing to obtain a micron-sized particle suspension having photocatalytic activity;

[0010] S2, mixing the micron-sized particle suspension of S1 with ambient seawater containing red tide algae species, and then placing the mixture into a column-type photobioreactor to obtain ambient seawater containing micron-sized particles with photocatalytic activity;

[0011] S3. Maintaining conditions suitable for the survival of algae in the environmental seawater containing micron-sized particles with photocatalytic activity can achieve the purpose of controlling the formation of red tides and inhibiting the spread of resistance genes and virulence factors after cultivation.

[0012] More preferably, the particle size of the natural 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, conditions suitable for algae survival include a suitable temperature of 22-25°C; a light-dark cycle of 12h:12h; a light intensity of 3300-3700lux; and a light source of LED or xenon lamp.

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

[0016] More preferably, S1 is dispersed and mixed using ultrasound, and the ultrasound time is 10-30 min.

[0017] Preferably, after the cultivation, the method further includes the step of extracting representative samples to quantitatively detect the abundance of 16SrRNA genes, resistance genes and virulence factor genes in the pre-outbreak, outbreak and subsidence periods of the algal bloom to detect the effect of the cultivation.

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

[0019] More preferably, the quantitative detection is performed using the Wafergen Smartchip ultra-high-volume fluorescence quantitative PCR system, and the experimental operation process is as follows: sample preparation→use of Smartchip→MSND liquid addition→high-throughput qPCR→data analysis; the PCR reaction program is: pre-denaturation at 95°C, 10 min; annealing at 95°C, 30s; extension at 60°C, 30s, for a total of 40 cycles.

[0020] Furthermore, the quantitative detection is to use the Wafergen Smartchip ultra-high-volume fluorescence quantitative PCR system to quantitatively detect 16S rRNA genes, 93 resistance genes, and 29 virulence factor genes. The PCR reaction mixture is first added to the microwell chip using the 60 (samples) × 80 (assays) mode of the nanoliter multi-sample detector (MSND), and then the qPCR reaction is performed on the cycler. The experimental process is: sample preparation → use Smartchip → MSND liquid addition → high-throughput qPCR → data analysis.

[0021] Among them, the 100nL qPCR reaction system is as follows:

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

[0023] qPCR results were automatically analyzed using qPCR software. t＝31 Set as the detection domain, and those that meet the fitting analysis are judged as detection.

[0024] Calculation method of absolute gene abundance:

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

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

[0027] 16SrRNA 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] Among them, the sample filtering volume is: 100-300mL.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention discloses a method for controlling the formation of red tides and synergistically reducing the abundance of resistance genes and virulence factors using natural sphalerite. First, a micron-sized natural sphalerite suspension with photocatalytic activity is prepared, and then the suspension is added to the environmental seawater containing red tide algae species. After culturing under conditions suitable for the survival of algae, the formation of red tides can be controlled and the spread of resistance genes and virulence factors can be inhibited. The method for controlling the formation of red tides and synergistically reducing the abundance of resistance genes and virulence factors provided by the present application uses visible light to drive micron-sized natural sphalerite to produce H2O2 and e - Various free radicals such as red tide and pathogens attack microbial cells, thereby inhibiting the reproduction of algae and pathogenic bacteria in the water body and reducing the abundance of resistance genes and virulence factor genes in the algal bloom process, thereby solving the technical problem of lack of coordinated control of red tide, pathogens and resistance genes in the existing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The inactivation effect of natural sphalerite on bacterial abundance at different stages of algal blooms. 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 results show that natural sphalerite has a reducing effect on resistance genes and virulence factors in different stages of algal blooms. DETAILED DESCRIPTION

[0035] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0036] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0037] Embodiment 1:

[0038] The present embodiment provides a method for inhibiting the formation of red tides by using micron-sized particles with photocatalytic activity while reducing the abundance of resistance genes and virulence factors. The method includes the steps of preparing environmental seawater containing micron-sized particles with photocatalytic activity, a method for culturing red tide algae, and a method for detecting resistance genes and virulence factor genes.

[0039] The steps of preparing environmental seawater containing micron-sized particles with photocatalytic activity include: adding natural sphalerite (from the Huangshaping deposit in Hunan Province, grinding the natural sphalerite particles into powder and passing through a 340-mesh sieve, with a mineral particle size of ≤40 μm) to an ultrapure aqueous solution to a concentration of 100 mg / mL, and using an ultrasonic instrument to sonicate for 15 minutes to evenly disperse the micron-sized natural sphalerite to obtain a natural sphalerite suspension; at the same time, collecting environmental seawater at the mouth of the Pearl River in Guangdong Province, which contains a variety of red tide algae species; adding the prepared micron-sized natural sphalerite suspension to a columnar photobioreactor (Shanghai Guangyu Technology) filled with 20L of environmental seawater to obtain environmental seawater containing micron-sized particles with photocatalytic activity. The final concentration of natural sphalerite in the prepared environmental seawater is 0.5 mg / L.

[0040] The steps of applying light to the column photobioreactor and maintaining the conditions for red tide algae cultivation include: turning on the LED lamps, temperature controller, and hydraulic circulation module of the column photobioreactor, maintaining the light intensity at 3500 lux, controlling the temperature at 23±1°C, and the light-dark cycle ratio at 12h:12h, and maintaining the water body in circulation. The culture was carried out for 15 days, and the density of algae was recorded every day. Algae attached (PA) and planktonic bacteria (FL) samples were obtained in the early stage, outbreak period, and late stage of the algal bloom, and high-throughput qPCR was used for quantitative analysis of resistance genes and virulence factor genes.

[0041] Embodiment 2:

[0042] The present embodiment provides a method for inhibiting the formation of red tides by using micron-sized particles with photocatalytic activity while reducing the abundance of resistance genes and virulence factors. The method includes the steps of preparing environmental seawater containing micron-sized particles with photocatalytic activity, a method for culturing red tide algae, and a method for detecting resistance genes and virulence factor genes.

[0043] The steps of preparing environmental seawater containing micron-sized particles with photocatalytic activity include: adding natural sphalerite (from the Huangshaping deposit in Hunan Province, grinding the natural sphalerite particles into powder and passing through a 340-mesh sieve, with a mineral particle size of ≤40 μm) to an ultrapure aqueous solution to a concentration of 100 mg / mL, and using an ultrasonic instrument to sonicate for 15 minutes to evenly disperse the micron-sized natural sphalerite to obtain a natural sphalerite suspension; at the same time, collecting environmental seawater at the mouth of the Pearl River in Guangdong Province, which contains a variety of algae; adding the prepared micron-sized natural sphalerite suspension to a columnar photobioreactor (Shanghai Guangyu Technology) filled with 20L of environmental seawater to obtain environmental seawater containing micron-sized particles with photocatalytic activity. The final concentration of natural sphalerite in the prepared environmental seawater is 25 mg / L.

[0044] The steps of applying light to the column photobioreactor and maintaining the conditions for red tide algae cultivation include: turning on the LED lamps, temperature controller, and hydraulic circulation module of the column photobioreactor, maintaining the light intensity at 3500 lux, controlling the temperature at 23±1°C, and the light-dark cycle ratio at 12h:12h, and maintaining the water body in circulation. The culture was carried out for 15 days, and the density of algae was recorded every day. Algae attached (PA) and planktonic bacteria (FL) samples were obtained in the early stage, outbreak period, and late stage of the algal bloom, and high-throughput qPCR was used for quantitative analysis of resistance genes and virulence factor genes.

[0045] Comparative Example 1:

[0046] In this comparative example, the collected environmental seawater containing red tide algae is placed in a column photobioreactor, and the red tide algae are cultured with a light intensity of 3500 lux, a temperature controlled at 23±1°C, a light-dark cycle ratio of 12h:12h, and a water body maintained in a circulating flow. The culture is carried out for 15 days, and the density of the algae is recorded every day. Algae attached state (PA) and planktonic bacteria (FL) samples are obtained in the early stage, outbreak period and late stage of the algal bloom, and high-throughput qPCR is used for quantitative analysis of resistance genes and virulence factor genes.

[0047] Experimental example:

[0048] The effectiveness of the method of inhibiting the formation of red tides while reducing the abundance of resistance genes and virulence factors in Examples 1-2 and Comparative Example 1 was verified.

[0049] The process of effect verification is as follows: representative samples of algal bacteria and planktonic bacteria extracted from Comparative Example 1 and Examples 1-2 were collected at the pre-bloom, outbreak and subsidence stages and quantitatively analyzed. That is, 100-300 mL of algal liquid was collected from the incubator in triplicate, and a polycarbonate nucleopore etched filter membrane with a pore size of 5 μm (Whatman, USA) was used to separate algal bacteria and planktonic bacteria, and then 100-300 mL of the filtrate was re-filtered using a 0.22 μm water filter membrane to obtain planktonic bacteria, and the filter membrane was stored in a -20°C refrigerator.

[0050] Quantitative analysis steps include: 16S rRNA gene, 93 resistance genes 1 , 29 virulence factors 2 Gene quantitative detection was completed using the Wafergen Smartchip ultra-high-throughput fluorescence quantitative PCR system (Anhui Yuanzai Biological Company) (the 93 resistance genes and 29 virulence factors detected are specifically described in 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 profilingvirulence factor genes from the environment. Environment International 2023,172,107761."), the PCR reaction mixture was first added to the microwell chip using the 60 (samples) × 80 (assays) mode of the nanoliter multi-sample detector (MSND), and then the qPCR reaction was performed on the cycler. The experimental process is: sample preparation → use Smartchip → MSND liquid addition → high-throughput qPCR → data analysis.

[0051] Among them, the reaction system of 100nL is as follows:

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

[0053] The PCR reaction program was: pre-denaturation at 95°C for 10 min; annealing at 95°C for 30 s; extension at 60°C for 30 s, for a total of 40 cycles. The qPCR results were automatically analyzed using qPCR software. t＝31 Set as the detection domain, and those that meet the fitting analysis are judged as detection.

[0054] Calculation method of absolute gene abundance:

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

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

[0057] 16SrRNA 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] Among them, the sample filtering volume is: 100-300mL.

[0061] The results are as follows Figure 1 As shown, it can be seen that the 16S rRNA gene was used to detect the bacterial abundance of different treatment groups. It was found that in the planktonic bacteria during the algal bloom and retreat periods, the absolute abundance of the 16S rRNA gene in the 0.5 mg / L mineral treatment group of Example 1 was reduced by 68.98% and 42.12% respectively compared with the comparative example 1; the absolute abundance of the 16S rRNA gene in the 25 mg / L mineral treatment group of Example 2 was reduced by 62.23% and 63.61% compared with the comparative example 1. In the algal bacteria during the algal bloom and retreat periods, the absolute abundance of the 16S rRNA gene in the 0.5 mg / L mineral treatment group of Example 1 was reduced by 21.67% and increased by 15.88% compared with the comparative example 1; the absolute abundance of the 16S rRNA gene in the 25 mg / L mineral treatment group of Example 2 was reduced by 53.18% and 60.78% compared with the comparative example 1.

[0062] like Figure 2As shown in A and B, in the planktonic bacteria, in the outbreak period of each group, the absolute abundance of total ARG in Example 1 and Example 2 was reduced by 67.23% and 85.18% respectively compared with that in Comparative Example 1, among which the main gene chloramphenicol decreased by 50.98% and 76.63%, fluoroquinolones decreased by 54.65% and 82.32, MLSB decreased by 85.59% and 94.17%, and multidrugs decreased by 73.93% and 60.24% respectively; in the subsidence period, the absolute abundance of total ARG in Example 1 and Example 2 was reduced by 13.61% and 21.05% respectively compared with that in Comparative Example 1, among which the main gene fluoroquinolones decreased by 57.51% and 73.08, and MLSB decreased by 28.42% and 84.85%, respectively. In the algal bacteria, in the outbreak period of each group, the absolute abundance of total ARG in Example 1 and Example 2 was reduced by 56.81% and 97.41% respectively compared with the comparison example 1, among which the main gene chloramphenicol decreased by 6.65% and 72.45%, fluoroquinolones decreased by 31.83% and 98.70%, MLSB decreased by 92.81% and 99.56%, and polypeptides decreased by 70.75 and 98.42% respectively; in the subsidence period, the absolute abundance of total ARG in Example 1 and Example 2 was reduced by 41.14% and 45.69% respectively compared with the comparison example 1, among which the main gene chloramphenicol decreased by 58.89% and 13.71%, and fluoroquinolones decreased by 30.46% and 84.90%. The specific names of the genes detected are shown in Table 1.

[0063] Table 1 Quantitative detection of major resistance genes

[0064]

[0065] like Figure 2As shown in Figures C and D, in the planktonic bacteria, in the outbreak period of each group, the absolute abundance of total VFG in Example 1 and Example 2 was reduced by 39.95% and 91.61% respectively compared with the comparison ratio 1, among which the genes of the main gene adhesion class decreased by 16.82% and 91.75, respectively, the ion absorption genes decreased by 71.51% and 85.29%, respectively, the nutritional factor genes decreased by 31.53% and 88.72%, respectively, and the serum resistance genes decreased by 90.40% and 98.06%, respectively; in the disappearance period, the absolute abundance of total VFG in Example 1 and Example 2 was reduced by 31.82% and 60.35% respectively compared with the comparison ratio 1, among which the genes of the main gene adhesion class decreased by 56.11% and 82.29%, respectively, the ion absorption genes decreased by 4.71% and 26.96%, respectively, and the secretion system genes decreased by 58.04% and 80.62%, respectively. In the algae bacteria, in the outbreak period of each group, the absolute abundance of total VFG in Example 1 and Example 2 was reduced by 70.15% and 98.48 respectively compared with the comparison ratio 1, among which the main gene adhesion genes decreased by 76.35% and 99.59% respectively, the ion absorption genes decreased by 70.92% and 92.04% respectively, the secretion system genes decreased by 69.59% and 91.15% respectively, and the serum resistance genes decreased by 83.47% and 100% respectively; in the disappearance period, the absolute abundance of total VFG in Example 1 and Example 2 was reduced by 56.69% and 48.36% respectively compared with the comparison ratio 1, among which the main gene ion absorption genes decreased by 43.08% and 92.32% respectively, the nutritional factor genes decreased by 58.43% and 36.08% respectively, and the serum resistance genes decreased by 77.04% and 86.21% respectively. The specific names of the genes detected are shown in Table 2.

[0066] The above results show that the method for inhibiting the growth of red tide algae and reducing the abundance of resistance genes and virulence factors provided by the present invention can utilize a series of active free radicals (including ·O2, ·OH, H2O2 and e - etc.) play a role in inactivating bacteria. - It plays a decisive role in the inactivation of bacteria in water bodies, so that the present invention can achieve a significant decrease in the abundance of ARGs in water bodies by inactivating bacteria.

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

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

[0069] The embodiments of the present invention are 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 of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. Application of natural sphalerite in controlling the formation of red tide and synergistically reducing the abundance of resistance genes and virulence factors.

2. The use according to claim 1, characterized in that: The method of using natural sphalerite to control the formation of red tide and synergistically reduce the abundance of resistance genes and virulence factors specifically includes the following steps: S1. adding natural sphalerite into an aqueous solution, dispersing and mixing to obtain a micron-sized particle suspension having photocatalytic activity; S2, mixing the micron-sized particle suspension of S1 with ambient seawater containing red tide algae species, and then placing the mixture into a column-type photobioreactor to obtain ambient seawater containing micron-sized particles with photocatalytic activity; S3. Maintaining conditions suitable for the survival of algae in the environmental seawater containing micron-sized particles with photocatalytic activity can achieve the purpose of controlling the formation of red tides and inhibiting the spread of resistance genes and virulence factors after cultivation.

3. The use according to claim 2, characterized in that: The particle size of natural sphalerite is 20-150μm.

4. The use according to claim 2, characterized in that: Use ultrasonic dispersion and mixing, and the ultrasonic time is 10-30min.

5. The use according to claim 2, characterized in that: The concentration of natural sphalerite in ambient seawater is 0.1-100 mg / L.

6. The use according to claim 2, characterized in that: Suitable conditions for algae survival include a suitable temperature of 22-25°C; a light-dark cycle of 12h:12h; a light intensity of 3300-3700lux; and a light source of LED or xenon lamp.

7. The use according to claim 2, characterized in that: The cultivation time is 7-21 days.

8. The use according to claim 2, characterized in that: After the cultivation, the method also includes the steps of extracting representative samples in the pre-outbreak, outbreak and decline periods of the algal bloom to quantitatively detect the abundance of 16S rRNA genes, resistance genes and virulence factor genes to detect the effect of the cultivation.

9. The use according to claim 8, characterized in that: The method for extracting representative samples is to collect samples of phycocyanobacteria and planktonic bacteria respectively, filter 100-300 mL of seawater using a polycarbonate nucleopore etched filter membrane with a pore size of 3-5 μm to separate the phycocyanobacteria, and then re-filter the filtrate using a 0.22-0.45 μm filter membrane to obtain the planktonic bacteria.

10. The use according to claim 8, characterized in that: The quantitative detection was completed using the Wafergen Smartchip ultra-high-volume fluorescence quantitative PCR system. The experimental operation process was as follows: sample preparation → use of Smartchip → MSND liquid addition → high-throughput qPCR → data analysis; the PCR reaction program was: pre-denaturation at 95°C, 10 min; annealing at 95°C, 30 s; extension at 60°C, 30 s, for a total of 40 cycles.

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