Nucleic acid detection method of microorganism, reagent composition, reagent kit, measurement system, and program

Abstract

To provide a nucleic acid detection method of a microorganism in which the concentration of target nucleic acid can be measured simply and rapidly with high accuracy, and a sufficient measurement range can be secured.SOLUTION: A nucleic acid detection method of a microorganism includes: a sample mixture preparation step of mixing probe solution being solution of gold nanoparticle probe in which gold nanoparticles are modified to DNA specifically binding to a target nucleic acid into extracted liquid obtained by executing nucleic acid extraction treatment to a sample, and furthermore adding at least sodium chloride and heating the liquid to prepare a sample mixture; a step of acquiring an absorption spectrum of probe solution; a step of acquiring the absorption spectrum of blank solution being solution which does not include the sample and is obtained by adding sodium chloride to probe solution and heating the solution; a step of acquiring the absorption spectrum of the sample mixture; and a calculation step of estimating the concentration of the target nucleic acid in the sample on the basis of the absorption spectra of the probe solution, blank solution and sample mixture.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for detecting microbial nucleic acid, a reagent composition, a reagent kit, a measurement system, and a program. [Background technology]

[0002] According to a report by the World Health Organization (WHO), in 2020, about one-quarter of the people in 138 countries around the world, or about 2 billion people, did not have access to safely managed drinking water facilities (Reference: “Our lifetime opportunity to enable water, sanitation and hygiene for all”, Joint WHO / UNICEF statement, <https: / / www.who.int / news / item / 22-03-2023-our-lifetime-opportunity-to-enable-water-sanitation-and-hygiene-for-all> Of these, about 1.2 billion people have only basic unsafe water supplies, about 282 million have limited supplies, about 367 million have unimproved sources, and about 122 million have surface water.

[0003] With regard to sanitation facilities such as toilets, only 54% of the world's population has access to safely managed facilities (facilities that allow for safe management of excrement inside or outside the facility), and 3.6 billion people use inadequate facilities. For these people to have access to safely managed water supply facilities and basic sanitation facilities by 2030, it is estimated that the current rate of progress in the spread of these facilities needs to be four times higher. The situation is even more severe in least developed countries and areas in fragile situations, where it is said that the current rate of progress needs to be increased by 10 and 23 times, respectively. In 2020, approximately 44% of wastewater from households was discharged into the environment without being safely treated, and inadequate sanitation facilities and wastewater treatment are sources of pathogens being discharged into the environment.

[0004] WHO estimates that unsafe drinking water supplies and lack of sanitation caused 870,000 related deaths worldwide in 2016. The African region had a related death rate of 45.9 per 100,000 people, four times higher than the global average (11.7 per 100,000). Europe was the least affected region (0.3 per 100,000), with the African region's mortality rate about 150 times higher, showing significant regional disparities.

[0005] In general, the greatest microbiological risk from drinking water is associated with the ingestion of water contaminated by human or animal (including avian) feces. Feces is likely to contain pathogenic bacteria, viruses, protozoa and helminths. The main symptoms of waterborne diseases are abdominal pain, diarrhea, vomiting, fever and headache. Diarrhea can cause dehydration, which can be fatal in developing countries with poor nutrition. The WHO has formulated guidelines for drinking water quality, in which it defines the microorganisms shown in Table 1 as pathogenic microorganisms (Reference: "WHO Guidelines for Drinking Water Quality, 4th Edition (Japanese version)", National Institute of Public Health,<https: / / www.mhlw.go.jp / file / 05-Shingikai-11121000-Iyakushokuhinkyoku-Soumuka / 0000195447.pdf> ).

[0006] [Table 1]

[0007] It is expected that by generally reducing the size and cost of water purification systems and using water resources efficiently, more people will be able to have access to safe water, especially in developing countries and localized areas. To achieve this, a system that can monitor water quality quickly on-site in various regions and environments is required. This will make the water purification process more efficient and enable water reuse in simple membrane filtration systems and reverse osmosis membrane filtration systems, promoting the sustainable use of water resources.

[0008] Known methods for monitoring microorganisms in environmental water include culture, PCR, latex agglutination, immunoassay, and PCR. For example, Patent Document 1 discloses an analysis method using a PCR method in which a nucleic acid isolation step of a pathogen contained in a tissue section and a PCR buffer preparation step are performed simultaneously. Patent Document 2 discloses a nucleic acid primer set for distinguishing and detecting Chlamydia bacteria, and a method for detecting Chlamydia bacteria using the primer set. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Patent Publication No. 2021-122186 [Patent Document 2] Patent Publication No. 2021-158989 [Non-patent literature]

[0010] [Non-Patent Document 1] Meri Nakajima et al., "Development of a paper analysis chip for detecting 16S rRNA of bacteria in sewage," Analytical Chemistry, Vol. 69, No. 12, pp. 715-722 (2020) [Non-Patent Document 2] Meri Nakajima et al., “Simple Assay for Colorimetric Quantification of Unamplified Bacterial 16S rRNA in Activated Sludge using Gold Nanoprobes”, Chemosphere, Vol. 263, January 2021, 128331 [Non-Patent Document 3] Hisashi Satoh et al., “Highly sensitive and homogeneous detection of unamplified RNA based on the light scattering properties of gold nanoparticle probes”, Biosensors and Bioelectronics: X, Vol. 12, December 2022, 100249 Summary of the Invention [Problem to be solved by the invention]

[0011] The culture method is a typical measurement method, but it takes a long time (e.g., 24 hours) to obtain results. In addition, there is a problem that it cannot detect microorganisms that are difficult to culture or viable but non-culturable (VBNC) microorganisms. In addition, as mentioned above, there are a wide variety of pathogenic microorganisms that cause waterborne infectious diseases, so a selective medium is required for each pathogenic microorganism, which is not realistic.

[0012] Testing methods that can replace the culture method include the PCR method and the quantitative (real-time) PCR method. The PCR method is a method for detecting and quantifying by amplifying nucleic acids using an enzyme (DNA polymerase) called a polymerase chain reaction (PCR), and has high sensitivity and specificity. However, the PCR method has problems such as complicated steps, requiring advanced operational capabilities, long analysis time (e.g., 3 hours), and the need for expensive consumables such as enzymes and expensive analytical equipment. The above-mentioned Patent Documents 1 and 2 also have similar problems because they use the PCR method.

[0013] Furthermore, although the immunoassay method using an antibody and the latex agglutination method are simple, they have a problem of being inferior in terms of sensitivity.

[0014] Therefore, it is extremely difficult to frequently analyze the biological risks of drinking water using these conventional methods, especially in developing countries and disaster areas. For these reasons, a system that is simpler yet has the same detection capabilities as the PCR method is desired.

[0015] The present inventors have focused on gold nanoparticles, designed DNA that specifically binds to the 16S rRNA gene of bacteria, and prepared a gold nanoparticle probe by modifying this with gold nanoparticles. Using this probe, they have progressed in the development of a simple, rapid, and highly accurate method and reagent composition for nucleic acid analysis (see Non-Patent Documents 1 to 3).

[0016] However, although the technique developed by the present inventors allows the concentration of a nucleic acid to be detected (target nucleic acid) to be measured simply, quickly and with high accuracy, there is a problem in that the measurement range (dynamic range) of the concentration is insufficient.

[0017] The present invention has been made in consideration of the above, and aims to provide a microbial nucleic acid detection method, a reagent composition, a reagent kit, a measurement system, and a program that can measure the concentration of a target nucleic acid simply, quickly, and with high accuracy while ensuring a sufficient measurement range (dynamic range). [Means for solving the problem]

[0018] In order to solve the above problems, a method for detecting nucleic acids in microorganisms according to one embodiment of the present invention includes the steps of: preparing a sample mixture by mixing an extract obtained by subjecting a sample to a nucleic acid extraction process with a probe solution, which is a solution of gold nanoparticle probes in which gold nanoparticles are modified with DNA that specifically binds to target nucleic acids, and then adding at least sodium chloride and heating the mixture; acquiring an absorption spectrum of the probe solution; acquiring an absorption spectrum of a blank solution, which is a solution containing no sample and is prepared by adding sodium chloride to the probe solution and heating the mixture; acquiring an absorption spectrum of the sample mixture; and estimating the concentration of the target nucleic acid in the sample based on the absorption spectra of the probe solution, the blank solution, and the sample mixture.

[0019] In the above-mentioned microbial nucleic acid detection method, the calculation step may estimate the concentration of the target nucleic acid in the sample by performing curve fitting based on the absorption spectra of the probe solution, the blank solution, and the sample mixture.

[0020] In the above-described microbial nucleic acid detection method, the calculation step may perform a least squares method so that a linear combination of a first term representing the absorption spectrum of the probe solution, a second term representing the absorption spectrum of the blank solution, and a third term representing the absorption spectrum of the sample mixture is closest to the third term representing the absorption spectrum of the sample mixture, and estimate the concentration of the target nucleic acid based on the coefficient of the third term calculated thereby.

[0021] In the above-mentioned method for detecting a microbial nucleic acid, the preparation step may further comprise adding bovine serum albumin.

[0022] In the above-mentioned method for detecting a microbial nucleic acid, the concentration of the bovine serum albumin in the sample mixture may be more than 0.01 mg / L and less than 1 mg / L.

[0023] Another embodiment of the present invention is a reagent composition used for detecting microorganisms contained in a sample, comprising a probe solution, which is a solution of gold nanoparticle probes in which gold nanoparticles are modified with DNA that specifically binds to a target nucleic acid, and a sodium chloride solution, and is used to prepare a sample mixed solution obtained by mixing the probe solution with an extract obtained by subjecting a sample to a nucleic acid extraction process, and further adding at least sodium chloride and heating the solution, and a blank solution, which is a solution not containing a sample, obtained by adding sodium chloride to the probe solution and heating the solution, and is used to estimate the concentration of the target nucleic acid in the sample by calculation based on the absorption spectra of the sample mixed solution, the blank solution, and the probe solution. The reagent composition may further comprise bovine serum albumin.

[0024] Another embodiment of the present invention is a reagent kit used for detecting microorganisms contained in a sample, comprising a probe solution, which is a solution of gold nanoparticle probes in which gold nanoparticles are modified with DNA that specifically binds to a target nucleic acid, and a sodium chloride solution, the probe solution and the sodium solution being sealed in predetermined amounts and used to prepare a sample mixed solution obtained by mixing the probe solution with an extract obtained by subjecting a sample to a nucleic acid extraction process, and further adding at least sodium chloride and heating the mixture, and a blank solution, which is a solution not containing a sample and is obtained by adding sodium chloride to the probe solution and heating the mixture, and to estimate the concentration of the target nucleic acid in the sample by calculation based on the absorption spectra of the sample mixed solution, the blank solution, and the probe solution. The reagent kit may further include a predetermined amount of encapsulated bovine serum albumin.

[0025] Another embodiment of the measurement system of the present invention comprises a measurement unit that acquires an absorption spectrum of a sample mixture obtained by mixing an extract obtained by subjecting a sample to a nucleic acid extraction process with a probe solution, which is a solution of gold nanoparticle probes in which gold nanoparticles are modified with DNA that specifically binds to a target nucleic acid, and further adding at least sodium chloride and heating the resulting sample mixture; and a calculation unit that estimates the concentration of the target nucleic acid in the sample based on the absorption spectrum of the probe solution, the absorption spectrum of a blank solution, which is a solution containing no sample obtained by adding sodium chloride to the probe solution and heating it, and the absorption spectrum of the sample mixture.

[0026] In the above measurement system, the measurement unit may further measure an absorption spectrum of the probe solution and an absorption spectrum of the blank solution, and the calculation unit may estimate the concentration of the target nucleic acid in the sample based on the absorption spectrum of the probe solution, the absorption spectrum of the blank solution, and the absorption spectrum of the sample mixture measured by the measurement unit.

[0027] The measurement system may further include a memory unit that stores at least one of the absorption spectrum of the probe solution and the absorption spectrum of the blank solution, and the calculation unit may read out at least one of the absorption spectrum of the probe solution and the absorption spectrum of the blank solution from the memory unit, and estimate the concentration of the target nucleic acid in the sample using the read out absorption spectrum.

[0028] In the above measurement system, the calculation unit may estimate the concentration of the target nucleic acid in the sample by performing curve fitting based on the absorption spectra of the probe solution, the blank solution, and the sample mixture.

[0029] Another aspect of the present invention is a program that causes a computer to execute the steps of: mixing an extract obtained by subjecting a sample to a nucleic acid extraction process with a probe solution, which is a solution of gold nanoparticle probes in which gold nanoparticles are modified with DNA that specifically binds to a target nucleic acid, and further acquiring an absorption spectrum of the sample mixture obtained by adding at least sodium chloride and heating it; and estimating the concentration of the target nucleic acid in the sample based on the absorption spectrum of the probe solution, the absorption spectrum of a blank solution, which is a solution not containing a sample obtained by adding sodium chloride to the probe solution and heating it, and the absorption step of the sample mixture. Effect of the Invention

[0030] According to the present invention, the concentration of a target nucleic acid to be detected can be measured simply, quickly and with high accuracy, while ensuring a sufficient measurement range (dynamic range). [Brief description of the drawings]

[0031] [Figure 1] 1 is a flowchart showing a basic nucleic acid detection method according to an embodiment of the present invention. [Diagram 2] FIG. 1 is a schematic diagram for explaining the principle of nucleic acid detection in an embodiment of the present invention. [Diagram 3] FIG. 1 is a schematic diagram for explaining the principle of nucleic acid detection in an embodiment of the present invention. [Figure 4] FIG. 1 is a schematic diagram for explaining a nucleic acid detection method according to a first embodiment of the present invention. [Diagram 5] 1 is a graph showing the results of Example 1. [Figure 6] FIG. 4 is a schematic diagram for explaining a nucleic acid detection method according to a second embodiment of the present invention. [Figure 7] 13 is a graph showing a comparative example of Example 2. [Figure 8] 13 is a graph showing the experimental results of Example 2. [Figure 9]FIG. 11 is a block diagram showing a schematic configuration of a measurement system according to a third embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Hereinafter, a method for detecting microbial nucleic acid, a reagent composition, a reagent kit, a measurement system, and a program according to embodiments of the present invention will be described with reference to the drawings. Note that the present invention is not limited to these embodiments. In addition, in the description of each drawing, the same parts are indicated by the same reference numerals.

[0033] The drawings referred to in the following description merely show the shape, size, and positional relationship in a schematic manner to the extent that the contents of the present invention can be understood. In other words, the present invention is not limited to the shape, size, and positional relationship exemplified in each drawing. In addition, there may be cases where the dimensional relationship and ratio of each part differs between the drawings.

[0034] The nucleic acid detection method for microorganisms according to the embodiment described below is a method for quantifying the microorganisms contained in a sample by extracting nucleic acid from a sample such as environmental water or sludge, mixing it with a gold nanoparticle probe solution, and measuring the absorbance of this solution. According to the embodiment described below, it is possible to quantify the nucleic acid simply, quickly, and with high accuracy without the need to amplify the extracted nucleic acid.

[0035] For the purposes of this application, microorganisms are intended to include prokaryotes, including bacteria and archaea, eukaryotes, including protists, algae, and fungi, and viruses.

[0036] (Principle of nucleic acid detection) FIG. 1 is a flow chart showing a basic nucleic acid detection method in each embodiment of the present invention. FIG. 2 and FIG. 3 are schematic diagrams for explaining the principle of nucleic acid detection in each embodiment of the present invention. In the following embodiment, a gold nanoparticle probe (hereinafter, also simply referred to as "probe") is prepared by modifying gold nanoparticles (AuNPs) on DNA complementary to the base sequence of the target nucleic acid to be detected, and a solution containing this gold nanoparticle probe is used as a reagent composition. The probe (1) shown in FIG. 2 is obtained by modifying gold nanoparticles (2) on complementary DNA (3) that specifically binds to the target nucleic acid. The target nucleic acid (target gene) is not particularly limited, and the principle of nucleic acid detection described below can be applied to various bacterial and viral RNA genes, such as the 16S rRNA gene of bacteria and the GI or GII type RNA gene of norovirus.

[0037] First, a process for extracting nucleic acid from a sample is performed (pretreatment step S10). Note that the method for extracting nucleic acid is not particularly limited, and any known method can be applied.

[0038] Next, a gold nanoparticle probe solution (hereinafter also simply referred to as "probe solution") is added to a nucleic acid extract from the sample (hereinafter also simply referred to as "extract"), and sodium chloride (NaCl) is further added and heated (preparation step S20). Heating conditions can be, for example, 95°C for 30 seconds. As a result, if the extract contains a target nucleic acid, the probe will hybridize with the target nucleic acid in the mixture.

[0039] Next, the absorption spectrum of the mixed solution is obtained (measurement step S30), and the nucleic acid concentration is estimated based on this absorption spectrum (calculation step S40).

[0040] Here, as shown in FIG. 3, when the probe is hybridized, the target nucleic acid covers the gold nanoparticles of the probe, inhibiting the aggregation of the gold nanoparticles by sodium (Na) ions. Therefore, when the concentration of the target nucleic acid in the extract is high (i.e., when the target (microorganism) is present in the sample at a high concentration), the original red color of the gold nanoparticles is maintained in the mixed solution. On the other hand, when the concentration of the target nucleic acid in the extract is low, the gold nanoparticles are easily aggregated by the Na ions and aggregate. Therefore, the original color of the gold nanoparticles is no longer observed. Therefore, by analyzing the spectroscopic spectrum in the mixed solution, the state of the gold nanoparticles, in other words, the degree of hybridization of the target nucleic acid to the probe, can be estimated.

[0041] (Reagent Composition) The reagent composition used in the embodiment described below is a reagent composition used to detect microorganisms contained in a sample, and includes a probe solution, which is a solution of gold nanoparticle probes in which gold nanoparticles are modified on DNA that specifically binds to target nucleic acids, and a sodium chloride solution. Such a reagent composition is used to prepare a sample mixture obtained by mixing the probe solution with an extract obtained by subjecting a sample to a nucleic acid extraction process, and further adding at least sodium chloride and heating the mixture. The reagent composition may further include bovine serum albumin.

[0042] The reagent composition may also be used to prepare a blank solution that is a solution that does not contain a sample and is obtained by adding sodium chloride to a probe solution and heating the solution. In this case, the reagent composition is used to estimate the concentration of the target nucleic acid in the sample by calculation based on the absorption spectra of the sample mixture, the blank solution, and the probe solution.

[0043] As described above, the type of target nucleic acid is not particularly limited, and a reagent composition can be composed of a gold nanoparticle probe in which gold nanoparticles are modified on complementary DNA that specifically binds to the target nucleic acid, targeting the RNA genes of various bacteria and viruses. By combining such a reagent composition with a general-purpose nucleic acid extractor, it is possible to realize a one-stop (no complicated operations required) and simple method of detecting nucleic acids of microorganisms that is equivalent to the PCR method.

[0044] (First embodiment) Next, a description will be given of a nucleic acid detection method according to a first embodiment of the present invention. Fig. 4 is a schematic diagram for explaining the nucleic acid detection method according to the first embodiment of the present invention. In the first embodiment, when sodium chloride is added to the mixture of the extract from the sample and the probe solution (see step S20), bovine serum albumin (BSA) is also added. In this case, the heating conditions can be 95°C for 30 seconds. Alternatively, after heating at 95°C for 30 seconds, the mixture can be further heated at 59°C for 1 minute.

[0045] The nucleic acid concentration can be estimated (step S40) using absorbance ratio. As mentioned above, when the probe is hybridized, the aggregation of gold nanoparticles by sodium (Na) ions is inhibited. Therefore, as shown in Figure 4, when the concentration of the target nucleic acid in the extract is high, the original red color of the gold nanoparticles is maintained in the sample mixture, and a peak in absorbance is observed around a wavelength of 550 nm.

[0046] On the other hand, as the concentration of the target nucleic acid in the extract decreases, the gold nanoparticles become more likely to aggregate due to Na ions, and the more they aggregate, the greater the absorbance at a wavelength of about 650 nm in the sample mixture.

[0047] From this, the absorbance A of the sample mixture at a wavelength of 650 nm is 650 The absorbance A at a wavelength of 550 nm 550 The absorbance ratio R divided by 650 / R 550 It can be said that the absorbance ratio represents the degree of aggregation of gold nanoparticles, in other words, the concentration of the target nucleic acid. That is, the higher the absorbance ratio, the lower the target nucleic acid concentration, and there is a negative correlation between the two.

[0048] Therefore, the absorbances at wavelengths of 550 nm and 650 nm are extracted from the spectrum acquired in step S30, and the absorbance ratio R 650 / R 550 The concentration of the target nucleic acid can be estimated by calculating the absorbance ratio and comparing it with a calibration curve obtained in advance or substituting the absorbance ratio into an estimation formula obtained in advance. In the first embodiment, since the wavelengths used are the above two wavelengths, in step S30, for example, a microplate reader may be used to obtain only these wavelengths.

[0049] Here, in the first embodiment, BSA is further added to the sample mixture. As a result, BSA molecules cling to the periphery of the probe, and aggregation of gold nanoparticles by Na ions is easily inhibited. Therefore, even if the concentration of the target nucleic acid is low, the peak of absorbance at a wavelength of about 550 nm in the mixture is higher than that in the case where BSA is not added. This makes it possible to measure the concentration even in a sample with a low concentration of target nucleic acid. In other words, the measurement range can be expanded to the low concentration side.

[0050] Example 1 A gold nanoparticle probe that specifically binds to a target nucleic acid was prepared, and an experiment was carried out to detect the target nucleic acid contained in a sample using this gold nanoparticle probe. In Example 1, the 16S rRNA gene was targeted, and a gold nanoparticle probe was prepared in which gold nanoparticles were modified on DNA complementary to the base sequence of the 16S rRNA gene.

[0051] 1. Preparation of Probe Solution The gold nanoparticle probes were prepared based on previous studies (see Liu et al., “Preparation of aptamer-linked gold nanoparticle purple aggregates for colorimetric sensing of analytes” Nat. Protoc. 1, 246e252. https: / / doi.org / 10.1038 / nprot.2006.38., X. Zhang et al., “Instantaneous and Quantitative Functionalization of Gold Nanoparticles with Thiolated DNA Using a pH-Assisted and Surfactant-Free Route” J. Am. Chem. Soc., 134, 7266 (2012) https: / / doi.org / 10.1021 / ja3014055).

[0052] First, a 100 mM acetate buffer with a pH of 5.2 was prepared by mixing 0.107 mL of acetic acid and 0.677 g of sodium acetate and diluting the mixture to 100 mL. Next, a 10 mM tris(2-carboxyethyl)phosphine (TCEP) solution was prepared by measuring out 1 mg to 3 mg of TCEP and dissolving it in ultrapure water (Milli-Q water). The TCEP solution is unstable, so it was prepared each time the probe was produced.

[0053] Using the above solutions, 100 mM acetate buffer and 10 mM TCEP solution were mixed in a ratio of 9:1 to prepare a deprotection buffer. 3 μL of 10 μM DNA solution was added to 27 μL of deprotection buffer and vortexed thoroughly (mixed using a vortex mixer (same below)). After spinning down, it was left at room temperature for 1 hour to deprotect the thiol groups of DNA.

[0054] 3 μL of 0.1 M hydrochloric acid (HCl) solution was added to 300 μL of the gold nanoparticle solution and vortexed. 20 μL of the deprotected probe DNA solution was added and vortexed. After spinning down, the solution was left to stand at room temperature for more than 15 hours to modify the DNA with gold nanoparticles. The gold nanoparticle probe after preparation was stored at room temperature out of direct sunlight. Next, the DNA that was not fixed was removed by centrifugation. After centrifugation to remove as much of the supernatant as possible, 0.1 M phosphate buffer (pH 7.0) was added to resuspend the gold nanoparticle probe.

[0055] 2. Preparation of Sample Mixture The probe solution prepared as described above was added to the extract obtained by subjecting the sample to a nucleic acid extraction treatment, and then a saturated aqueous solution of sodium chloride (NaCl) and bovine serum albumin (BSA) were added, followed by heating at 95°C to prepare a sample mixture. The concentrations of BSA were set to four levels: 0 mg / L (not added), 0.01 mg / L, 0.1 mg / L, and 1 mg / L, relative to the mixture (reagent composition in the assay system) containing the extract from the sample, the probe solution, the saturated aqueous solution of sodium chloride, and BSA. The nucleic acid concentrations of the samples were measured separately by an official method (PCR method).

[0056] 3. Measurement and evaluation of absorbance The absorbance spectrum of the heated sample mixture is measured, and the absorbance ratio R is calculated by dividing the absorbance at 650 nm by the absorbance at 550 nm. 650 / R 550 was calculated.

[0057] When the absorbance ratio without BSA was compared with the target nucleic acid concentration measured by the official method for the same sample, a strong correlation was found between the two, confirming that the detection method according to the first embodiment can quantify the target nucleic acid concentration in a sample.

[0058] 5 is a graph showing the experimental results of Example 1. The horizontal axis of FIG. 5 shows the concentration of the target nucleic acid contained in the sample, and the vertical axis shows the output of the sensor (absorbance ratio R 650 / R 550 As shown in FIG. 5, when BSA is not added, the measurable range of the target nucleic acid concentration is 10 9 ~10 6 In contrast, when the BSA concentration was 0.1 mg / L, the measurable range was 10 9 ~10 3 This allowed us to expand the range.

[0059] On the other hand, when the BSA concentration was 0.01 mg / L, the sensor output was not stable, and when the BSA concentration was 1 mg / L, the sensor output moved in the opposite direction to the nucleic acid concentration.

[0060] From the above results, it was found that the measurable range of the target nucleic acid concentration can be expanded by adding an appropriate amount of BSA. It was also found that the appropriate amount (concentration) of BSA added to the sample mixture is in the range of more than 0.01 mg / L and less than 1 mg / L, and preferably around 0.1 mg / L.

[0061] Second embodiment Next, a nucleic acid detection method according to a second embodiment of the present invention will be described below. Fig. 6 is a conceptual diagram for explaining the nucleic acid detection method according to the second embodiment.

[0062] In the second embodiment, a curve fitting technique is used when estimating the nucleic acid concentration (see step 40 in FIG. 1). Note that in the second embodiment, when sodium chloride is added to the mixture of the extract from the sample and the probe solution (see step S20 in FIG. 1), it is not essential to add BSA as in the first embodiment.

[0063] Here, the gold nanoparticle probe can be in three states: unagglutinated, aggregated, and hybridized. In these states, the gold nanoparticle probe has its own unique absorption spectrum. Therefore, when the target nucleic acid is present in the sample, in the mixture of the extract from the sample and the probe (sample mixture, see preparation step S20), the aggregation of the gold nanoparticles is inhibited by hybridization, causing a change in the absorption spectrum. Therefore, the absorption spectrum of the sample mixture is spectrally resolved by curve fitting, so that the absorption spectrum unique to the hybridized gold nanoparticle probe can be calculated.

[0064] Specifically, the absorption spectrum of the sample mixture can be expressed as a linear combination of the absorption spectra in three states, ie, non-agglutination, aggregation, and hybridization, as shown in the following formula (1). X(λ)=x×C(λ)+y×D(λ)+z×E(λ)…(1) In formula 1, X(λ) is the absorbance of the sample mixture at wavelength λ, D(λ) is the absorbance of the non-aggregated probe, C(λ) is the absorbance of the aggregated probe, and E(λ) is the absorbance of the hybridized probe. Also, x, y, and z are coefficients.

[0065] According to the Beer-Lambert law, the coefficients x, y, and z in the above formula (1) are considered to be proportional to the concentrations of the gold nanoparticle probes in the unagglutinated, agglutinated, and hybridized states, respectively. Therefore, by determining these coefficients x, y, and z, it is possible to quantify the concentration of the target nucleic acid in the extract. Specifically, the coefficients x, y, and z are determined so that the sum of the linear combination shown in the following formula (2) is minimized.

number

[0066] The actual method involves first measuring the absorption spectrum of (i) a solution of gold nanoparticle probes that does not contain any sample, NaCl, or BSA (hereinafter, the probe solution), (ii) a solution in which NaCl has been added to the probe solution without the sample and heated (hereinafter, the blank solution), and (iii) a solution in which the nucleic acid extract from the sample and the probe solution have been mixed, NaCl has been added, and the solution has been heated (sample mixture).

[0067] If the term representing the absorption spectrum of the probe solution (absorbance at wavelength λ) is A(λ), the term representing the absorption spectrum of the blank solution (same as above) is B(λ), and the term representing the absorption spectrum of the sample mixture (same as above) is S(λ), the linear combination X'(λ) of these three absorption spectra is expressed by the following equation (2). X'(λ)=a×A(λ)+b×B(λ)+h×S(λ) …(2) A(λ), B(λ), and h are determined using the least squares method so that this linear combination X'(λ) is closest to the absorption spectrum S(λ) of the sample mixture.

[0068] Here, as described above, the absorption spectrum of the sample mixture can be said to be a linear combination of the absorption spectra of the gold nanoparticle probes in the non-aggregated, aggregated, and hybridized states (see formula (1)). Then, the absorption spectra A(λ), B(λ), and S(λ) of the above probe solution, blank solution, and sample mixture can be expressed as the following formulas (3) to (5) using coefficients c, d, and e.

[0069] A(λ) = C(λ) … (3) B(λ) = c × C(λ) + D(λ) … (4) S(λ)=d×C(λ)+e×D(λ)+E(λ) …(5)

[0070] From equations (3) to (5), the linear combination X'(λ) can be expressed as in the following equation (6). X'(λ)=(x-yc-dz-cez)×A(λ) +(y-cz)×B(λ)+z×S(λ) …(6)

[0071] Therefore, the coefficient h in equation (2) is equal to the coefficient z in equation (1), and by calculating the coefficient h, the concentration of the hybridized gold nanoparticle probes in the sample mixture, in other words, the concentration of the target nucleic acid, can be estimated.

[0072] Example 2 Gold nanoparticles were used, 5 nm diameter, OD 1, stabilized in suspension in citrate buffer (Sigma-Aldrich). The probe DNA was T50-cCTO189F and T50-RT1R, which contain a T50 spacer, based on the base sequences of PCR primers (CTO189F A / B / C and RT1R) that detect the 16S rRNA gene of ammonia-oxidizing bacteria (AOB) of the Betaproteobacteria genus. A gold nanoparticle probe was created by modifying this probe DNA with gold nanoparticles.

[0073] A total of 51 μL of a mixture of 10 μL of T50-cCTO189F and 10 μL of T50-RT1R as gold nanoparticle probes, 1 μL of nucleic acid extract from the sample, and 30 μL of NaCl aqueous solution was heated at 95°C for 30 seconds and then heated at 59°C for 1 minute. This was used as a sample mixture, and the absorption spectrum of the sample mixture was measured using a UV-visible spectrophotometer. Note that, after measuring the absorption spectrum of each of the samples collected from multiple plants, the absorption spectrum of any one sample was selected and used for the subsequent calculations.

[0074] The absorption spectra of the probe solution and blank solution were also measured in the same manner. The blank solution was prepared and measured three times to determine the detection limit.

[0075] As described above, A(λ), B(λ), and h were determined using the least squares method so that the linear combination X'(λ) of equation (2) was closest to the absorption spectrum S(λ) of the sample mixture. The coefficient h was used as an index of hybridization and was compared with the target nucleic acid concentration of the sample measured by qPCR.

[0076] FIG. 7 is a graph showing a comparative example of Example 2, showing the relationship between the absorbance ratio calculated based on the absorbance spectrum measured for the sample mixture and the target nucleic acid concentration measured by qPCR for the same sample mixture. In FIG. 7, the horizontal axis shows the target nucleic acid concentration (copies / μL), and the vertical axis shows the absorbance ratio. FIG. 8 is a graph showing the experimental results of Example 2, showing the relationship between the coefficient calculated by the above-mentioned curve fitting and the target nucleic acid concentration measured by qPCR. In FIG. 8, the horizontal axis shows the target nucleic acid concentration, and the vertical axis shows the coefficient h.

[0077] As shown in Figure 7, the concentration is 1 × 10 7 When the concentration of the target nucleic acid was below 100 copies / μL, the correlation between the absorbance ratio and the target nucleic acid concentration became weak. This indicates that when the concentration of the target nucleic acid is low, quantification is difficult using the method based on the absorbance ratio.

[0078] On the other hand, as shown in FIG. 8, when the coefficient h is used, the target nucleic acid concentration is 1×10 7 A positive correlation was observed between the coefficient h and the target nucleic acid concentration when the target nucleic acid concentration was 0.8 (copies / μL) or less. When the target nucleic acid concentration was higher than this, the coefficient h ranged from 0.8 to 1.0.

[0079] Target nucleic acid concentration is 1×10 7 The coefficient h is considered to be roughly proportional to the target nucleic acid concentration in the range of 10000 copies / μL or less. In this region, the experimental results were approximated by a straight line, and the function shown in the following formula (7) was obtained. h=9.85×10 -8 ×(concentration)+0.010 …(7) Note that σ = 0.976831613. From this, the coefficient h is considered to be a value that follows the Beer-Lambert law.

[0080] For the blank solution (n=3), the coefficient h was calculated by curve fitting, and the average was 5.9×10 -6 The standard deviation (σ) was 0.086. The detection limit (3σ) was calculated as follows: coefficient h = 0.258, target concentration 2.9 × 10 6 From this, the dynamic range in which the target nucleic acid concentration can be quantified is at least 2.9 × 10 6 More than 10×10 6 It can be said that the range is as follows:

[0081] From the above results, it was found that the method of curve fitting of the absorbance spectrum in the second embodiment can quantify the target nucleic acid concentration more accurately and to a lower concentration range, compared to the conventional absorbance ratio.

[0082] In Example 2, BSA was not added to the sample mixture, but it is also possible to add BSA to the sample mixture and then quantify the target nucleic acid concentration using a curve fitting technique. In this case, BSA is also added to the blank solution. This allows the dynamic range to be further expanded to the low concentration side.

[0083] (Modification) The reagent composition used in the first or second embodiment may be a reagent kit for detecting microorganisms contained in a sample. The reagent kit includes a probe solution, which is a solution of gold nanoparticle probes in which gold nanoparticles are modified on DNA that specifically binds to a target nucleic acid, and a sodium chloride solution. The reagent kit may further include bovine serum albumin. The reagent kit may be a set in which these reagents are enclosed in predetermined amounts. Such a reagent kit can be used to prepare a sample mixture. The reagent kit can also be used to prepare a blank solution, and the probe solution included in the kit can be used directly to measure the absorption spectrum. Thereby, it becomes possible to estimate the concentration of the target nucleic acid in the sample by calculation based on the absorption spectra of the sample mixture, blank solution, and probe solution.

[0084] (Third embodiment) 9 is a block diagram showing a schematic configuration of a measurement system according to a third embodiment of the present invention. The measurement system 10 according to the third embodiment is a system for detecting nucleic acids of microorganisms contained in a sample, and includes a measurement unit 11, a display unit 12, an operation input unit 13, and an information processing device 14.

[0085] The measurement unit 11 is, for example, an absorptiometer, and measures the absorption spectrum of a liquid containing a sample. The wavelength range of the absorption spectrum may be in the range of 450 nm to 800 nm. Of course, scattered light may be measured instead of the absorption spectrum.

[0086] Alternatively, a microplate reader may be used as the measurement unit 11. In this case, only two wavelengths near 550 nm and near 650 nm may be measured. Alternatively, a monochromatic reader may be used to measure multiple desired wavelengths.

[0087] The display unit 12 is, for example, a liquid crystal display or an organic EL display, and displays the absorption spectrum input from the measurement unit 11, the concentration of the target nucleic acid in the sample estimated based on the absorption spectrum, and the like.

[0088] The operation input unit 13 is an input device such as a keyboard, a mouse, or a touch panel provided on the surface of the display unit 12, and receives operations by a user and inputs a signal according to the operation to a processor 17 described later.

[0089] The information processing device 14 is configured with a general-purpose arithmetic processing device such as a personal computer or a tablet terminal, and can be used as a measuring device for measuring the concentration of a target nucleic acid. As shown in FIG. 3, the information processing device 14 includes an external interface 15, a storage unit 16, and a processor 17.

[0090] The external interface 15 is an interface that connects the information processing device 14 to an external device and transmits and receives signals between the information processing device 14 and the external device. Examples of the external device that can be connected to the information processing device 14 include the measurement unit 11, the display unit 12, and the operation input unit 13. The external interface 11 functions as, for example, a receiving unit that receives input of information representing the absorption spectrum measured by the measurement unit 11.

[0091] The storage unit 16 is configured using a computer-readable storage medium such as a semiconductor memory such as a ROM or RAM, a hard disk, etc., and stores application programs that execute various functions, various parameters used during the execution of these programs, etc., in addition to an operating system program and a driver program. For example, the storage unit 16 stores a program 161 that estimates the concentration of the target nucleic acid in a sample based on the absorption spectrum represented by the information input from the measurement unit 11, and parameters used in this estimation calculation. In addition, the storage unit 16 may store at least one of the absorption spectrum of a solution of gold nanoparticle probes that does not contain any of the sample, NaCl, and BSA (probe solution), and the absorption spectrum of a blank solution in which NaCl (or NaCl and BSA) are added to the probe solution. In addition, the absorption spectra of various target nucleic acids may be stored as the absorption spectrum of the probe solution and the absorption spectrum of the blank solution, and the absorption spectrum to be used may be read out depending on the bacteria or viruses to be detected.

[0092] The processor 17 is configured using, for example, a CPU (Central Processing Unit) and a GPU (Graphics Processing Unit), and controls each part of the information processing device 14 in an integrated manner by reading various programs stored in the storage unit 16, and executes applications based on the various programs. In detail, when an absorption spectrum (or intensities at multiple wavelengths) relating to the sample (i.e., of the sample mixture) is input from the measurement unit 11, the calculation unit 171 of the processor 17 executes a process of estimating the concentration of the target nucleic acid in the sample based on the absorption spectrum (or intensities at multiple wavelengths).

[0093] As one type of calculation process, the calculation unit 171 can estimate the concentration of the target nucleic acid based on the absorbance ratio between the absorbances at 550 nm and 650 nm in the absorption spectrum of the sample mixture.

[0094] As another calculation process, the calculation unit 171 can estimate the concentration of the target nucleic acid based on the absorption spectrum of the probe solution, the absorption spectrum of the blank solution, and the absorption spectrum of the sample mixture. In detail, the calculation unit 171 can perform curve fitting based on the absorption spectra of the probe solution, the blank solution, and the sample mixture. In this case, the calculation unit 171 may acquire the absorption spectra of the probe solution, the blank solution, and the sample mixture measured by the measurement unit 11 from the measurement unit 11, and perform calculation based on these absorption spectra. Alternatively, the calculation unit 171 may acquire the absorption spectrum of the sample mixture from the measurement unit 11, while reading out the absorption spectra of the probe solution and the blank solution from the storage unit 16, and perform calculation based on these absorption spectra of the sample mixture, the probe solution, and the blank solution. Of course, the calculation unit 171 may read out the absorption spectrum of either the probe solution or the blank solution from the storage unit 16, and acquire the absorption spectrum of the other from the measurement unit 11 to perform calculation.

[0095] As described above, according to each embodiment of the present invention, the concentration of nucleic acid contained in a sample can be quantified. The sample is not particularly limited, and it is possible to test sewage, wastewater, environmental water such as lakes, rivers, and seas, food, specimens collected from humans and animals, etc. In addition, the type of microorganism is not particularly limited, and it is possible to detect the nucleic acid of various microorganisms shown in Table 1 above, for example.

[0096] Moreover, according to each embodiment of the present invention, the use of a gold nanoparticle probe allows the nucleic acid concentration to be estimated quickly and simply with the same accuracy as the PCR method. In particular, in each embodiment of the present invention, since there is no need to subject the nucleic acid extract from the sample to a nucleic acid amplification process, the test time can be significantly reduced. Specifically, the time required for the analysis reaction of the nucleic acid extract is only 10 minutes, and the total time including the nucleic acid extraction, which is a pretreatment process, can be reduced to about 20 minutes.

[0097] Furthermore, according to the first embodiment of the present invention, the dynamic range of measurable nucleic acid concentrations can be expanded to the low concentration side by adding BSA.

[0098] According to the second embodiment of the present invention, the accuracy of estimating the nucleic acid concentration can be improved by using a curve fitting technique, and the dynamic range of the measurable nucleic acid concentration can be expanded to the low concentration side. Furthermore, by adding BSA and using a curve fitting technique, the dynamic range on the low concentration side can be further expanded.

[0099] According to the first and second embodiments, the dynamic range of measurable nucleic acid concentration can be expanded to the low concentration side, and a sufficient range can be secured. Therefore, it may be possible to measure the presence or absence and concentration of microorganisms in environmental water and determine whether the water can be used for drinking or daily life. Therefore, it is possible to improve access to safe water, for example, in developing countries and disaster areas.

[0100] Furthermore, according to the first to third embodiments, since gold nanoparticle probes are used, there is no need to immunize animals compared to detection systems using antibodies, so production costs are low and stable production is possible because chemical synthesis is possible. Furthermore, since expensive enzymes and analytical equipment such as PCR are not required, testing is possible in cases where regular or frequent testing is required, such as microbial monitoring of environmental water, or in situations where facilities are not in place, such as developing countries and disaster areas.

[0101] According to the first to third embodiments, it is possible to detect various bacterial and viral genes as targets, in addition to bacterial 16S rRNA genes and norovirus GI or GII RNA genes, etc. In short, as long as a gold nanoparticle probe can be produced in which gold nanoparticles are modified on complementary DNA that specifically binds to a target nucleic acid, each embodiment of the present invention can be applied.

[0102] The present invention described above is not limited to the first to third embodiments and modifications, and various inventions can be formed by appropriately combining multiple components disclosed in the first to third embodiments and modifications. For example, some components may be removed from all the components shown in the first to third embodiments and modifications, or the components shown in the first to third embodiments and modifications may be appropriately combined.

[0103] (Appendix 1) A sample mixture is prepared by mixing a probe solution, which is a solution of gold nanoparticle probes in which gold nanoparticles are modified with DNA that specifically binds to target nucleic acids, with an extract obtained by subjecting a sample to a nucleic acid extraction process, and then adding sodium chloride and bovine serum albumin and heating the mixture; obtaining an absorption spectrum of the sample mixture; a calculation step of estimating the concentration of the target nucleic acid in the sample based on the absorption spectrum; A method for detecting nucleic acid of a microorganism, comprising:

[0104] (Appendix 2) The method for detecting microbial nucleic acid described in Appendix 1, wherein the concentration of bovine serum albumin in the sample mixture is greater than 0.01 mg / L and less than 1 mg / L.

[0105] (Appendix 3) 3. The method for detecting a microbial nucleic acid according to claim 1 or 2, wherein the calculation step includes acquiring absorbances of the sample mixture at 550 nm and 650 nm, and estimating a concentration of the target nucleic acid based on an absorbance ratio of the absorbance at 650 nm to the absorbance at 550 nm. [Explanation of symbols]

[0106] 1... gold nanoparticle probe (probe), 2... gold nanoparticle, 3... DNA, 10... measurement system, 11... measurement unit, 12... display unit, 13... operation input unit, 14... information processing device, 15... external interface, 16... memory unit, 17... processor, 161... program, 171... calculation unit

Claims

1. A sample mixture is prepared by mixing a probe solution, which is a solution of gold nanoparticle probes in which gold nanoparticles are modified with DNA that specifically binds to a target nucleic acid, with an extract obtained by subjecting a sample to a nucleic acid extraction process, and then adding sodium chloride and bovine serum albumin and heating the mixture; obtaining an absorption spectrum of the sample mixture; a calculation step of estimating the concentration of the target nucleic acid in the sample based on the absorption spectrum; Including, A method for detecting a nucleic acid of a microorganism, wherein the concentration of bovine serum albumin in the sample mixture is more than 0.01 mg / L and less than 1 mg / L.

2. 2. The method for detecting a microbial nucleic acid according to claim 1, wherein the calculation step comprises acquiring absorbances of the sample mixture at 550 nm and 650 nm, and estimating a concentration of the target nucleic acid based on an absorbance ratio of the absorbance at 650 nm to the absorbance at 550 nm.

3. 1. A reagent composition for use in detecting a microorganism in a sample, comprising: a probe solution which is a solution of gold nanoparticle probes in which gold nanoparticles are modified on DNA that specifically binds to a target nucleic acid; Sodium chloride solution; Bovine serum albumin, Including, The method is used to prepare a sample mixture obtained by mixing the probe solution with an extract obtained by subjecting a sample to a nucleic acid extraction treatment, and further adding sodium chloride and bovine serum albumin and heating the mixture, the sample mixture having a concentration of the bovine serum albumin in the sample mixture being greater than 0.01 mg / L and less than 1 mg / L; A reagent composition for obtaining an absorption spectrum of the sample mixture and estimating the concentration of the target nucleic acid in the sample based on the absorption spectrum.

4. A reagent kit for use in detecting a microorganism contained in a sample, comprising: a probe solution which is a solution of gold nanoparticle probes in which gold nanoparticles are modified on DNA that specifically binds to a target nucleic acid; Sodium chloride solution; Bovine serum albumin, Including, The probe solution, the sodium solution, and the bovine serum albumin are each enclosed in a predetermined amount, The method is used to prepare a sample mixture obtained by mixing the probe solution with an extract obtained by subjecting a sample to a nucleic acid extraction treatment, and further adding sodium chloride and bovine serum albumin and heating the mixture, the sample mixture having a concentration of the bovine serum albumin in the sample mixture being greater than 0.01 mg / L and less than 1 mg / L; A reagent kit for obtaining an absorption spectrum of the sample mixture and estimating the concentration of the target nucleic acid in the sample based on the absorption spectrum.

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