KR102558660B1 - Primer set for the detection of geosmin synthase gene - Google Patents

Abstract

The present invention relates to a primer set capable of detecting geosmin synthesis genes, and a composition or method using the same. The present invention provides a sensitive, specific, and rapid process for predicting odorants in water and can contribute to proper management of water sources through early and intensive detection.

Description

Primer set for the detection of geosmin synthase gene}

The present invention relates to a primer set for detecting a geosmin synthesis gene and a composition or detection method comprising the same.

A variety of microorganisms, including blue-green algae, actinomycetes and some fungi, produce compounds with unpleasant odors (Zaitlin and Watson 2006). Among them, cyanobacteria are the main producers of odorous compounds that produce earthy or musty odors in water sources, and these odorants are secondary metabolites synthesized and secreted by cyanobacteria (John et al. 2018, Westerhoff et al. 2005, Wood et al. al. 2001). The major odorant in source water is geosmin (Juttner and Watson 2007), which has a significant effect on the quality of source water. Geosmin has a very low threshold (< 10 ng/L), so many consumers can perceive it, and sensitive people can perceive it even at less than 5 ng/L (Watson et al. 2008). Geosmin does not cause serious health effects to humans or aquatic animals, but people judge the water to be unsafe to drink when it has an unpleasant odor (Tsao et al. 2014). Many cases related to geosmin have been reported worldwide, and the trend has been gradually increasing over the past 30 years (Devi et al. 2021).

The Paldangho Lake in the Han River is the largest water supply (reservoir volume ~244 × 10 ⁶ tons) in Korea (Kim et al. 2013), located near the capital, Seoul, and serves as a major drinking water source for 25 million people (more than 50% of the population). The Ministry of Environment has set the water quality monitoring standard for Geosmin to 20 ng/L, and has been monitoring once a week since 1998 to manage blue-green algae and related odorous substances. However, despite these efforts, several algal blooms have been reported, including the 2012 algal bloom, and complaints about odorous substances have been continuously increasing in recent years (Lee et al. 2020). Blue-green algae that produce geosmin are fast-growing and algal blooms can occur within days (Tsao et al. 2014). In addition, geosmin is very stable in nature, does not decompose well, is difficult to remove even by boiling, and is difficult to effectively remove even by general water treatment processes such as chlorine, ozone, coagulation-sedimentation, and rapid sand filtration (John et al. 2018, Koch et al. 1992, Lin et al. 2003, Watson et al. 2016). Therefore, it is most important to accurately analyze geosmin-producing blue-green algae early and accurately for proper management of water source water.

Common analyzes of blue-green algae related to odorant production include morphological classification and cell counting using a microscope, and odorant concentration analysis using gas chromatography (GC). The former method is not only time-consuming, but also requires specially trained personnel as the results may differ from analyst to analyst, and it is difficult to distinguish odorant-producing species from non-producing species (Chiu et al. 2016). The latter analysis method, the GC method, can identify and quantify odorous substances in water, but this also requires unknown producer information and expensive special equipment (Jttner and Watson 2007).

Recently, genes involved in geosmin production have been discovered, and molecular biological methods such as quantitative PCR (qPCR) using genetic information target genes have been developed. and early on-site detection is possible, and interest is increasing. For proper molecular biological analysis, it is necessary to design specific primers that contain all the geosequence information related to geosmin production in the international database (NCBI GenBank), and consider geographical differences in the sequences registered in the database (Devi et al. 2021). Currently, despite the development of qPCR analysis methods for geosmin-producing blue-green algae, field application studies are lacking worldwide and non-existent in Korea. Therefore, the object of the present invention is to (i) develop a molecular biological method for quantifying geosmin production genes, and ii) evaluate odorant gene monitoring methods by applying them to source water sites.

Chang, H. (2008) Water Research 42(13), 3285-3304. Chiu, Y.-T., et al. (2016) Environmental Research 151, 618-627. Devi, A., et al. (2021) Water Research 188, 116478. Harris, T.D. and Graham, J.L. (2017) Lake and Reservoir Management 33(1), 32-48. Havens, K.E., et al. (2003) Environmental Science and Pollution Research 122(3), 379-390. Huang, X., et al. (2018) Environmental Science and Pollution Research 25(19), 19134-19142. John, N., et al. (2018) Water Research 136, 34-40. Juttner, F. and Watson, S.B. (2007) Applied and Environmental Microbiology 73(14), 4395-4406. Kim, J.Y., et al. (2013) Microbes and Environments 28(2), 187-194. Koch, B., et al. (1992) Water Science and Technology 25(2), 291-298. Lee, J.E., et al. (2020) Occurrence of cyanobacteria, actinomycetes, and geosmin in drinking water reservoir in Korea: a case study from an algal bloom in 2012. Water Supply. Lee, J.E., et al. (2022) Environmental Microbiology Reports 14(2), 197-202. Li, Z., et al. (2010) Harmful Algae 9(5), 481-488. Lin, T.-F., et al. (2003) Water Research 37(1), 21-26. Otten, T.G., et al. (2016) Applied and Environmental Microbiology 82(17), 5410-5420. Sotero-Santos, R.B., et al. (2008) Harmful Algae 7(5), 590-598. Su, M., et al. (2013) Water Research 47(10), 3444-3454. Tsao, H.-W., et al. (2014) Water Research 49, 416-425. Tung, S.-C., et al. (2008) Environmental Monitoring Assessment 145(1), 407-416. Wang, Z., et al. (2016) Journal of applied physiology 28(1), 325-333. Watson, S.B., et al. (2016) Harmful Algae 54, 112-127. Watson, S.B., et al. (2008) Canadian Journal of Fisheries and Aquatic Sciences 65(8), 1779-1796. Westerhoff, P., et al. (2005) Water Research 39(20), 4899-4912. Wood, S., et al. (2001) International Biodeterioration & Biodegradation 48(1), 26-40. Zaitlin, B. and Watson, S.B. (2006) Water Research 40(9), 1741-1753.

The present invention was derived from the above needs, and the present inventors identified a primer set for detecting a geosmin synthetic gene, a composition for detecting a geosmin synthetic gene, and a method for detecting a geosmin synthetic gene using the same or a method for predicting odorants in water completed the present invention.

In order to solve the above problems, the present invention provides a primer set for detecting geosmin synthetic gene comprising the primers of SEQ ID NO: 1 and SEQ ID NO: 2.

In one example of the present invention, the primer set is real-time polymerase chain reaction (real-time PCR), nested polymerase chain reaction (nested PCR), droplet digital polymerase chain reaction (droplet digital PCR) or recombinase polymerase amplification (recombinase polymerase amplification).

In another example of the present invention, the primer set may include a probe, wherein the probe is labeled with a fluorophore at the 5' end and labeled with a quencher at the 3' end, and the fluorophore is fluorescein ( fluorescein), 6-carboxyfluorescein (FAM, 6-carboxyfluorescein), hexachloro-6-carboxyfluorescein (HEX), tetrachloro-6-carboxyfluorescein (TET, tetrachloro- 6-carboxyfluorescein), 2-chloro-7-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC, 2-chloro-7-phenyl-1,4-dichloro-6-carboxyfluorescein), 2,7 -Dimethoxy-4,5-dichloro-6-carboxyfluorescein (JOE, 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein), 5-((2-aminoethyl)amino)naphthalene-1 -sulfonic acid (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid), coumarin and coumarin derivatives, cyanine-5 (Cy5, Cyanine-5), lucifer yellow, Texas red ( texas red), tetramethylrhodamine, Yakima Yellow (YG), and at least one selected from the group consisting of Cal Fluor Red 610 (CFR), wherein the quencher is tetra Methylrhodamine (TAMRA, tetramethylrhodamine), 4-(4-dimethylaminophenylazo)benzoic acid, 4-dimethylaminophenylazophenyl-4-maleimide (4-dimethylaminophenylazophenyl-4 -maleimide), carboxytetramethylrhodamine, and BHQ dyes.

The concentration of the primer or probe may be variously selected and used by a person skilled in the art according to experimental conditions, preferably 1 to 1000 nM each, more preferably 100 to 500 nM each, but limited thereto. it is not going to be

The step of performing the real-time polymerase chain reaction is 30 to 50 cycles, 30 to 46 cycles, 30 to 44 cycles, 33 to 50 cycles, 33 to 46 cycles, 33 to 44 cycles, 36 to 50 cycles, 36 to 46 cycles , 36 to 44 cycles, 38 to 50 cycles or 38 to 46 cycles, for example, it may be performed under conditions of 38 to 44 cycles, but is not limited thereto.

The present invention provides a composition for detecting a geosmin synthetic gene comprising the primer set.

In addition, the present invention provides a composition for predicting odorous substances in water containing the primer set.

In one example of the present invention, the composition may include an intercalating fluorescent dye, preferably SYBR Green, SYBR Blue, Boxto, or SYBR Gold. (SYBR Gold), Ethidium Bromide, Eva Green, Propidium Iodide, SYTOX Orange, NG-DCS1, POPO-1, POPO-3, YOYO -1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO -PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, YO-PRO-1, YO-PRO-3, LO-PRO-1, Pico Green ( PicoGreen), Pico 488, RiboGreen, SYBR Green Ⅰ, SYTO-40, SYTO-41, SYTO-42, SYTO-43, SYTO-44, SYTO-45, SYTO-13, SYTO-16, SYTO- 24, SYTO-21, SYTO-23, SYTO-12, SYTO-11, SYTO-20, SYTO-22, SYTO-15, SYTO-14, SYTO-25, SYTO-81, SYTO-80, SYTO-82, In the group consisting of SYTO-83, SYTO-84, SYTO-85, SYTO-64, SYTO-17, SYTO-59, SYTO-61, SYTO-62, SYTO-60, SYTO-63, Gelstar, Thiazole Orange and DRAQ5 It may be selected, more preferably SYBR Green (SYBR Green), but is not limited thereto.

In another example of the present invention, the sample for detection or prediction or the water is selected from the group consisting of ponds, rivers, valleys, lakes, surface water, fresh water, salt water, ground water, trickle water, process water, industrial water, agricultural water and drinking water It may be, but is not limited thereto.

In another example of the present invention, the odorant may be geosmin.

The present invention comprises the steps of preparing an isolated DNA or RNA sample; Amplifying by polymerase chain reaction using the primer set; And it provides a geosmin synthetic gene detection method comprising the step of obtaining an amplification result.

In addition, the present invention comprises the steps of preparing a DNA or RNA sample; Amplifying by polymerase chain reaction using the primer set; and acquiring an amplification result.

The primer set capable of detecting the geosmin synthetic gene of the present invention and the composition or method using the same provides a sensitive, specific, and rapid process for detecting odorous substances in water, and enables proper management of water sources through early and powerful detection. will be able to contribute

1 shows a water sampling location in the basin of the Bukhangang River in Korea according to an embodiment of the present invention.
Figure 2 shows a standard curve for real-time qPCR of geo gene using the SYBR Green real-time qPCR system according to an embodiment of the present invention.
Figure 3 shows the time course monitoring results of geo gene copy number, geosmin concentration, and blue-green algae cell number at four sampling sites according to an embodiment of the present invention. (▲, gene copy number by qPCR) (●, musty odor compound by GC/MS), (◆, Cyanobacteria cells by microscope).
4 shows a comparison of geo gene copy numbers for four different sampling locations according to an embodiment of the present invention. The horizontal dotted line within each box represents the mean. Bars represent standard deviations. Circles represent the 5th/95th percentile.
5 shows the relationship between geo gene copy number and geosmin concentration (A), and blue-green algae cell number and geosmin concentration (B) with a 95% confidence interval (dotted line) according to an embodiment of the present invention.
6 shows a Pearson correlation coefficient (R) matrix of geo genes and environmental factors according to an embodiment of the present invention. (* p < 0.05, ** p < 0.01)

Hereinafter, preferred embodiments of the present invention will be described in detail. In addition, in the following description, many specific details such as specific components are shown, which are provided to help a more general understanding of the present invention, and it is common in the art that the present invention can be practiced without these specific details. It will be self-evident to those who have the knowledge of And, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description will be omitted.

<Example 1> Monitoring sites and water sampling

Samples for odorant genetic monitoring were obtained from Paldang Lake and Bukhangang River. The Bukhangang River is the main stream flowing into Paldang Lake and is the main source of odorous substances in Paldang Lake. Uiam Dam (UA), Cheongpyeong Dam (CP), Sambong-ri (SB), and finally Paldang Lake Paldang Dam (P2) were selected as the study sites (Fig. 1). Sampling was conducted weekly from July 2020 to October 2020, and the odorant (geosmin) was observed along with the odorant (geosmin) production gene ( geo ) in cyanobacteria.

<Example 2> Primers design for real-time qPCR

Primers for quantification of geosmin synthetic genes using qPCR were designed based on gene sequences from the NCBI database. The referenced sequences were aligned using Bioedit software to select regions with high detectability (Table 1). The target DNA fragment for geosmin detection was contained in gsy1, a putative geosmin synthase sequence reported by (Tsao et al. 2014) for the geosmin synthesis operon of Anabaena ucrainica . In the OligoCalc program (biotools.nubic.nothwastern.edu/Oligocalc.html), the GC content (%) and the possibility of secondary structure formation in the selected region were confirmed.

Target
(gene) Primers Sequences 5'→3' Product
size (bp) Tm
(℃) Geosmin
( gsy 1 ^a ) 2014F GCC CTT TTC GAC GAT TTC AA
(SEQ ID NO: 1) 150 59 2163R GGA AGC ACT ATT ACG CAA TTC AAA
(SEQ ID NO: 2)

^b gsy1(geo) - putative geosmin synthase gene

<Example 3> Quantification of geosmin synthase gene by qPCR

Quantification of the geo gene was performed using a 7500 real-time PCR (Applied Biosystems, Thermo Fisher Scientific) instrument with 7500 software v2.3. SYBR Green was applied as an intercalator for fluorescence of double-stranded DNA in real-time PCR analysis. The PCR reaction was performed in a 20 μL volume containing 3 μL of template DNA sample, 1 μL of 10 pM specific primer, 10 μL of PowerUp ^TM SYBR ^TM Green Master Mix (Applied Biosystems, USA) and 5 μL of sterile deionized water. Real-time PCR conditions were pre-incubated at 95 ° C for 10 minutes, followed by denaturation at 95 ° C for 15 seconds and annealing / extension at 59 ° C for 30 seconds 40 times for the geo reaction. Then, the temperature was raised from 60 °C to 95 °C, and melting temperature analysis of the PCR product was performed to confirm the specific reaction of the primer.

As a reference for geo gene quantification, a plasmid cloned using the TOPcloner ^TM TA kit (Enzynomics, Republic of Korea) was used to target the target fragment for the 2014F/2163R primer of Anabaena sp. For each qPCR assay, an 8-point standard curve was prepared with a 10-fold serial dilution of the cloned plasmid. The geo genes in the samples were quantified with an external standard method using a standard curve. The canonical copy number was calculated through the following equation.

C _DNA and _AN are the DNA concentration (g/μL) and Avogadro number (bp/mole) detected using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, USA) in the standard solution, respectively. L _plasmid is the length (bp) of the plasmid into which the geosmin synthase gene is inserted, 3957 bp, and MW _bp represents the molecular weight of base pairs (660 g/mole).

<Example 4> Extraction of water cyanobacteria DNA

Cyanobacteria were concentrated by filtering 100–300 ml of field water samples using 0.45 μm pore size membrane filters (Merck Milipore, France). DNA of the filtered blue-green algae was extracted using the DNeasy PowerWater Kit (QIAGEN, Germany) according to the manufacturer's protocol. An additional heating procedure for cell lysis was included in a water bath at 65 °C for 10 minutes, and DNA was finally extracted with 30 μL of Solution EB (elution buffer) and stored in a deep freezer at -70 °C until qPCR analysis.

<Example 5> Analysis of odorous compounds

Geosmin of in situ samples was measured by headspace solid phase microextraction (HS-SPME) coupled to an Agilent 5977B GC/MS. A 10 mL sample to which 3 g NaCl was added was adsorbed to an activated SPME fiber (50/30 um DVB/CAR/PDMS, Supelco) for 30 min at 70° C. with stirring at 400 rpm. After absorption, the target component of the fiber was desorbed at 270° C. for 4 minutes and injected into GC/MS using a DB-5MS column for analysis. A standard mixture (47525-U, Supelco) for quantification of geosmin in the sample was analyzed simultaneously with the sample group and quantified by an external standard method.

<Example 6> Statistical analysis

The relationship between odorant gene copy number, geosmin compound concentration, and cyanobacteria cell number was evaluated using regression analysis (SigmaPlot, ver.10.0; Systat Software Inc., San Jose, CA, USA). P-value was analyzed using Student t-test, and p <0.05 was considered statistically significant. Correlations between odorant gene copy number and environmental factors were evaluated using Student t-test. The Person correlation coefficient (r) represents a linear relationship. (SPSS ver.20; IBM Corp, Armonk, New York, USA).

<Test Example 1> Standard curves for real-time qPCR

Several blue-green algae related to odorant production were isolated from Lake Paldang and cultivated in pure water. The sequence of the odorant-producing gene was registered in GenBank and received GenBank numbers MT360266, MT515744, ON365769, and ON365771. Among these blue-green algae, Anabaena crassa (#ON365770), a major producer of geosmin, was used to establish real-time qPCR of the SYBR Green system for the geo gene. A standard calibration curve was prepared using DNA extracted from this strain. After DNA extraction, the DNA was amplified with the designed primers (Table 1), and the PCR product was cloned using TOPcloner ^TM TA kit (Enzynomics, Korea). After cloning, plasmids were weighed, copy number calculated, diluted in 10-fold serial dilution steps (10 to 10 ⁷ copies/μL), and quantified by real-time qPCR.

All of the standard curves shown in Fig. 2 show high linearity with a high correlation coefficient R ² = 0.9999. Real-time qPCR efficiency (E) was calculated based on the equation E = (10 ^1/S -1) and S = slope of the standard curve (Chiu et al. 2016, Tsao et al. 2014). The real-time qPCR efficiency (E) was found to be 99.9% for the geo gene (Fig. 2).

<Test Example 2> Application of qPCR-based monitoring of drinking water reservoirs

In order to quantify the genes of blue-green algae that produce geosmin in the Han River, which is a major water source, real-time qPCR was applied and monitored. In 2020, samples were collected from upstream (sites UA, CP, and SB) to downstream (PD) from July to October, when blue-green algae growth was active. Geosmin production gene ( geo ), geosmin (GC-MS) concentration and the number of blue-green algae cells (microscopic) were analyzed in water samples through qPCR analysis.

3 shows the results of monitoring the geo gene copy number quantified by qPCR, the geosmin concentration measured by GC-MS, and the number of blue-green algae cells measured by a microscope over time. The similarity between geo gene and geosmin concentration was higher than that in cyanobacteria cell numbers. Geo gene copy numbers and geosmin concentrations increased from June to July and then decreased at all sites, ranging from 3 × 10 ² to 6.5 × 10 ⁵ copies mL ^-1 and 5 to 32 ng L ^-1 . The measurement of geosmin of the present invention is similar to that observed in Harris and Graham 2017, and the concentration of geosmin was highest in June and July, and the number of blue-green algae cells was also the highest in water source research data over 14 years. Tsao et al. 2014 investigated geosmin concentration and geo DNA copy number in Myponga Reservoirs, South Australia, and found that geo copy number was closely related to geosmin concentration and ranged from approximately 10 ² to 10 ⁵ mL ^-1 .

Figure 4 shows the geo gene copy number range for each point during the sampling period. Interestingly, the gene copy number at the four points had different concentrations depending on the location, which decreased from upstream to downstream. As shown in FIG. 4, the geosmin gene copy number was high in the upstream UA and CP (maximum log 5.82 copies mL ^-1 ), and the copy number concentration tended to decrease as it went downstream. The two main tributaries of the Han River, the Namhan River and the Bukhangang River, join at Paldang Dam. In other studies on water quality, concentrations of most parameters tend to decrease from the upper reaches of the Han River to the lower reaches of the Han River (Chang 2008). In particular, in Paldang Dam, which is the most downstream in this study, the water quality related parameters may be diluted due to the increase in inflow due to the confluence of the Bukhan River and the Namhan River.

<Test Example 3> Regression analysis of gene copies, odorant compounds, and cyanobacteria cells

The relationship between the three parameters gene copy number, odorant concentration and cyanobacteria cell number was evaluated. In Fig. 5(A), a positive relationship was confirmed between geo gene copy number and geosmin concentration (R ² = 0.601, p < 0.001) using the regression equation (y = 0.199 x + 0.19), log-log regression Depending on the analysis, geosmin production can be estimated to be between 1 and 186 fg per geo gene copy number. Su et al. 2013 reported geosmin production potential at concentrations ranging from 1-40 fg per geosmin gene copy. Another study showed that the average geosmin production of Anabaena was about 100 fg cell-1 (Li et al. 2010). In a previous study, comparison of GSG gene copy number and geosmin concentration showed a significant correlation (R ² = 0.694, p < 0.001), and environmental samples showed a broad distribution pattern (Su et al. 2013). The number of blue-green algae cells and the concentration of geosmin showed a low correlation in FIG. 5(B) (R ² = 0.2013, p = 0.05). Another study showed no correlation between gene and cell number by qPCR (Otten et al. 2016). Among blue-green algae, Anabaena is well known as a geosmin-producing species and has been reported to account for 46% of geosmin generation (Sotero-Santos et al. 2008). A previous study reported that the relationship between geosmin concentration and Anabaena cell density showed a good correlation (R ² = 0.97) (Tsao et al. 2014). However, since the present invention measured the total number of blue-green algae including geosmin-producing and non-producing species, the correlation between the number of blue-green algae cells and the concentration of geosmin would be lower compared to the study analyzing only Anabaena sp., which is known for producing geosmin. can

<Test Example 4> Environmental factors

The odorant concentration and gene copy number were compared with environmental factors including temperature, dissolved oxygen, Chl-a, NH ₃ -N and PO ₄ -P, and these environmental factors were correlated and expressed as a matrix.

In Figure 6, geo gene copy number showed a strong positive correlation with geosmin concentration (R = 0.94, ** p < 0.01), and in the environmental analysis result, only water temperature was correlated with geosmin gene (R = 0.46 ). Although environmental factors are important for the growth of blue-green algae and subsequent release of odorants (Huang et al. 2018), clear patterns between geosmin production and environmental factors (light, temperature, nutrients, dissolved oxygen) are still lacking. (Watson et al. 2016).

Claims (12)

Primer set for detecting geosmin synthesis gene including the primers of SEQ ID NO: 1 and SEQ ID NO: 2 The method of claim 1, wherein the primer set is real-time polymerase chain reaction (real-time PCR), nested polymerase chain reaction (nested PCR), droplet digital polymerase chain reaction (droplet digital PCR) or recombinase polymerase amplification ( Primer set for detection of geosmin synthesis gene for use in recombinase polymerase amplification) delete Composition for detecting geosmin synthetic gene comprising the primer set of any one of claims 1 or 2 According to claim 4, wherein the composition comprises intercalating (intercalating) fluorescent dye geosmin synthetic gene detection composition The composition for detecting geosmin synthetic genes according to claim 5, wherein the intercalating fluorescent dye is SYBR Green. A composition for predicting odorous substances in water comprising the primer set of any one of claims 1 or 2 The composition for predicting odorous substances in water according to claim 7, wherein the odorant is geosmin. The composition for predicting odorous substances in water according to claim 7, wherein the composition contains an intercalating fluorescent dye The composition for predicting odorous substances in water according to claim 7, wherein the water is selected from the group consisting of ponds, rivers, valleys, lakes, surface water, fresh water, salt water, ground water, trickle water, process water, industrial water, agricultural water, and drinking water. preparing an isolated DNA or RNA sample;
Amplifying by polymerase chain reaction using the primer set of any one of claims 1 or 2; and
Geosmin synthetic gene detection method comprising obtaining an amplification result preparing an isolated DNA or RNA sample;
Amplifying by polymerase chain reaction using the primer set of any one of claims 1 or 2; and
Method for predicting odorous substances in water comprising obtaining an amplification result