JP2012139191A - Diatoms detecting method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a safe and easy diatoms detecting method for detecting the presence or absence of diatoms frustule directly or indirectly from DNA trapping the diatoms frustule.SOLUTION: The diatoms detecting method includes putting silica coated beads or short-stranded DNA coated beads in a chaotropic ion presence solution together with the diatoms frustule, bonding them as they are in the case of the short-stranded DNA coated beads, while, in the case of silica coated beads, bonding them irreversibly to the diatoms frustule as they are or by adding long-stranded DNA, biotin/fluorescent labeled DNA, short-stranded DNA different in sequence from coating short-stranded DNA, or non-labeled DNA, carrying out B/F separation washing to stain DNA and to make a microscopic examination, or carrying out PCR amplification of short-stranded DNA or long-stranded DNA bonded to dissociated diatoms frustule to obtain an analytical curve and to quantitatively detect the diatoms frustule, or measuring the fluorescent intensity of biotin/fluorescent labeled DNA bonded to the dissociated diatoms frustule to quantitatively detect the diatoms frustule.

Description

The present invention relates to a method for detecting diatoms.

  Conventionally, as a method for observing and classifying diatoms, a method of observing the siliceous putamen characteristic of diatoms under a microscope has been used. In this siliceous shell, unique forms, shell surfaces, and patterns on the shell surface are formed for each species, and the genus and species are classified by observing them with a general optical microscope. However, the putamen is combined with the lunch box lid like a fruit, and it contains cell components such as nuclei and chloroplasts. It is difficult to distinguish the characteristics of the shells specific to the species. For this reason, in order to observe diatoms, it is common to first perform a cleaning operation in order to remove organic substances such as nuclei and chloroplasts contained in the putamen.

As a method of cleaning the putamen, a crushing method using concentrated sulfuric acid or nitric acid,
1294123916390_0.htm # Dichromate potassium,
1294123916390_1.htm # UV
,
1294123916390_2.htm # Shochu,
1294123916390_3.htm # Bleach,
There are 1294123916390_4.htm # pipe unish etc., which can be used according to the purpose and the situation of the equipment. In particular, the pipe unish method can be easily implemented by anyone, and there are no problems with safety, so it has been adopted in schools as practical training. After cleaning the putamen, it is developed and dried on a preparation, sealed with a suitable mounting agent, and observed. Since the putamen is the same vitreous as a preparation or a cover glass, it cannot be observed under an encapsulant having a low refractive index. Generally, high refractive index encapsulants such as Styrax, Hyrax, Pleurax are used.
Such observation methods for diatom shells are applied not only in the fields of biology and water and environmental chemistry, but also in various other fields. In particular, in the field of forensic medicine, there is a history that has been used extensively as a proof of drowning since ancient times.

The dying proof method in forensic medicine is based on the diagnosis that plankton present in drowned water passes through the alveolar wall, enters the general circulatory system, and moves to the organs of the whole body. Hearts, lungs, liver, kidneys, spleen, and other organs removed by dissection are heated with concentrated nitric acid to destroy organic matter, and diatom shells contained in the remaining minerals are observed under a microscope. Proving that you sucked in the water before life. However, in this method, it is often difficult to detect in organs other than the lung due to its low density, and there are many cases where only a few putamen are detected for each organ. In addition, there are often cases where observation under a microscope is difficult due to residual impurities such as charcoal powder and lipids. In addition, many problems have been pointed out, such as the fact that the diatom shell is glassy and it is difficult to observe on the slide.

Many studies have been conducted and reported to solve these problems, but the direction can be roughly divided into two.
The first is a method for detecting and verifying minute foreign substances other than diatoms contained in the brine. A method of culturing microorganisms such as bacteria contained in drinking water from major organs and blood (
1294123916390_5.Pubmed_RVAbstractPlus
,
1294123916390_6.Pubmed_RVAbstractPlus
,
1294123916390_7.Pubmed_RVAbstractPlus
et al. Forensic Sci Int 2008), sandwich enzyme immunoassay using surfactant protein D that exists specifically in the lung (Kamada S,
1294123916390_8.Pubmed_RVAbstractPlus
et al. Forensic Sci Int 2000).
The second is an improvement to the diatom detection method itself, such as solubilization of organs by protease treatment and improvement of the crushing method. In addition to this, research is being conducted to specifically amplify and verify plankton DNA such as diatoms by direct PCR.
However, although the above-mentioned proof methods for minute foreign substances other than diatoms are excellent methods, they are highly specific and often lack in versatility that can only be carried out by specific institutions. Furthermore, the improvement of the diatom detection method has not yet led to a radical solution to various problems.

<Conventional diatom detection method and its problems>
Conventionally, the morphological detection of diatoms has only been carried out after cleaning the shell and encapsulating it in a preparation as it is, and observing with a microscope. Therefore, it is impossible to eliminate the influence of impurities other than the shell. . In particular, even if a large amount of diatoms collected from rivers, lakes, and marine sediments are cleaned by pipe unish method, etc., foreign matter such as fine earth and sand is processed at the same time, so the shape of the putamen under the microscope There are many cases that obstruct observation.
In addition, in order to observe diatoms from water in summer and winter when floating diatoms decrease, it is necessary to concentrate a large amount of water by filtering or centrifuging the diatoms. Other suspended matter will be concentrated at the same time, resulting in the same result as from the sediment.
At present, there is no method for specifically and selectively extracting and purifying diatoms floating in water.
In addition to the direct measurement of diatom shells, as a method for quantifying the biogenic silicon content of marine sediments, separation method using particle density difference and X-ray diffraction method, molybdenum blue colorimetric method, alkali extraction method, etc. Various methods are used.
As a method for quantifying the amount of silicic acid in blood, a method of separating and quantifying silicic acid as a molybdenum complex by thin layer chromatography has been reported.
However, these quantification methods require a large amount of labor and are not only lacking in specificity, but also require a large amount of sample for analysis because of their low sensitivity. Therefore, it is difficult to detect and quantify diatoms from a small amount of water, or a dead body organ that sometimes contains only a few diatoms.

Therefore, the present invention provides a method for detecting a crushed diatom more easily and with higher accuracy. The greatest feature of diatoms is that they have a shell made of silicic acid and have excellent acid resistance. First, the disintegration method is a detection method using this characteristic. The inventor has paid attention to another characteristic of the siliceous shell.
A glassy material containing silicon dioxide (SiO ₂ ) has long been known to have a property of nonspecifically adsorbing DNA, RNA, some proteins, and the like in the presence of chaotropic ions. Furthermore, this adsorption has the property of easily dissociating in a low polarity environment such as water. Recently, a DNA purification method using glass fiber as a solid phase has been developed, and this basic principle is applied to many DNA extraction kits. The present invention utilizes the fact that these basic principles of glassy are also applied to diatom shells made of silicic acid as they are.

Although there are many methods for purifying DNA, there was no patent as a method for detecting diatoms.

In the present invention, first, after the cleaning operation conventionally performed for observing the diatom shell, the remaining sediment and inorganic matters are removed, and the shell is specifically and selectively purified. Provide a way to observe. Usually, in order to observe the putamen, a preparation is prepared through a washing process with water several times after cleaning. The washing operation at that time is to collect the shell with a high specific gravity at the bottom of the test tube by centrifugation, discard the supernatant, float the shell again, and at the same time dilute the cleaning solution and collect it again by centrifugation. It is a repetition of the process. However, this operation cannot remove impurities having the same degree as the shell or a higher specific gravity.
In order to make up for this drawback, the present invention specifically and selectively purifies only the putamen by utilizing the characteristic of siliceous putamen that DNA adsorbs specifically in the presence of chaotropic ions. Provide a method. This operation is performed together with silica-coated magnetic beads having the same properties, and is performed after the shell removal and washing operations. Specifically, the cleaned shell is placed in a solution containing a chaotropic ion with a known concentration, DNA and silica beads are added thereto, and the shell is bound to the solid phase via DNA in which silica beads are added as a solid phase. Let

The type of DNA adsorbed on the diatom shell or silica-coated beads in the presence of chaotropic ions is not limited. For example, human genomic DNA may be used, and commercially available products such as λ phage DNA and DNA markers for various electrophoresis can be used.
The silica beads used as the solid phase are not limited to magnetic beads. Any material having the property of adsorbing and dissociating DNA in the presence of chaotropic ions may be used. In general, commercially available beads having a particle size of about 0.1 to 10 μm can be used, and the beads can be operated with tweezers of about 2 to 4 mm.
On the other hand, this operation can also be carried out to collect diatoms floating in the water. Since the putamen is the outer shell of diatoms, trapping the diatom hulls floating in water on the magnetic beads using DNA concentrates the floating diatoms with impurities removed. Can be collected. The collected diatoms can be observed under a microscope by a conventional method after cleaning.

In addition, the shell is observed with an optical microscope. The cleaned and washed shell is developed on a preparation, dried and fixed, and then sealed with a suitable encapsulant. The shell is the same as a preparation or cover glass. Since it is glassy, it cannot be observed under an encapsulant with a small refractive index. Generally, high refractive index encapsulants such as Styrax, Hyrax, and Pleurax are used, but it is not easy to find and observe the putamen under a microscope even using these high refractive index encapsulants. In particular, in the case of forensic drowning proof, if the number of diatoms contained in the first place is small, it is difficult to find the putamen.
In order to solve this problem, it is only necessary to introduce a staining method frequently used in general microscopic observation. For example, if the putamen is stained with red, blue, or a fluorescent dye as in general tissue staining, even if the number is small, it will be easy to find and observe under a microscope.

Therefore, secondly, only the shell is specifically bound to the magnetic beads coated with silica, and then biotin and fluorescence-labeled DNA are bound again to the shell trapped on the beads, and biotin bound to the shell is bound. The present invention provides a method for observing the putamen in an indirectly stained state by staining fluorescently labeled DNA using an existing staining method.
If the DNA bound to the putamen and silica cotote beads in the presence of chaotropic ions is 1 Kbp or more, the binding is easily dissociated if the solution is replaced with a low polarity solution such as water. However, in the case of binding of short-chain DNA of 100 bp or less, the dissociation property is extremely low. By combining these DNA binding characteristics, DNA can be trapped on silica beads or eluted.
The kind of short-chain DNA of 100 bp or less is not ask | required. For example, commercially available salmon sperm DNA used as a blocking agent for membranes and DNA markers for various electrophoresis can be used, and markers pre-labeled with fluorescent dyes such as biotin and FITC are also commercially available. Yes. Furthermore, an oligonucleotide labeled with a fluorescent dye such as biotin or FITC can be synthesized and used.

Third, a method for quantitatively detecting diatom shells is provided. This is done by specifically binding DNA to the shell and then taking out only the shell with the DNA bound by silica-coated magnetic beads and quantitatively measuring the DNA bound to the shell. Is called. Quantification methods for bound DNA include enzyme-labeled antibody method, PCR method, real-time PCR method and the like. If the amount of DNA bound to the shell is constant, the amount of DNA bound to the shell is proportional to the measurement method.
Compared to the conventional quantitative method for measuring the amount of silicon released from silicon dioxide, the bound DNA can be measured by amplifying the DNA several million times by the PCR method. Can be measured even with a single molecule of putamen.

The basic technical condition that characterizes the present invention is that “the presence or absence of diatom shell is directly detected by trapping the diatom shell by interposing the DNA on a silica-coated bead in chaotropic ion-containing solution via DNA or different DNA. Or “a diatom detection method, wherein the diatom shell is indirectly detected from trapped DNA”.
Specific examples of the basic technical conditions are as follows (1) to (6).
(1) As shown in FIG. 1, silica-coated beads, long-chain DNA, and diatom shells are put in a chaotropic ion-containing solution to bind them, and then separated by B / F and washed to give an unreacted length. A method for detecting diatoms, comprising washing away strand DNA and impurities, then adding water to dissociate the long-chain DNA and diatom shell from the surface of the silica-coated beads, taking out only the diatom shell, and performing a microscopic examination.
Here, when the core of the silica-coated beads is made of a magnetic metal sphere, as shown in (4) of FIG. Only the shell is attached to the container, and the waste liquid containing unreacted long-chain DNA and impurities can be efficiently and rapidly discharged out of the container. In addition, after the hydrolytic dissociation, as shown in (7) and (8) of Fig. 1, only the silica-coated beads are held in the container and only the dissociated diatom shell is effectively removed from the container together with water. Can be taken out and examined.

(2) As shown in FIG. 2, silica-coated beads, long-chain DNA, and diatom shells are put in a chaotropic ion-containing solution to bind them, and then the first B / F separation washing is performed. After washing away unreacted long DNA and impurities, biotin / fluorescently labeled short DNA is added, and this is irreversibly bound to diatom shells and stained, and then the second B / F Separate and wash, then add water and dissociate the diatom shells from the surface of the silica-coated beads separately from the long-chain DNA in a state of binding to the biotin / fluorescent-labeled short-chain DNA. A method for detecting diatoms, comprising: observing diatom shells bound to the diatom.
Here, by making the core of the silica-coated bead into a magnetic metal sphere, when draining after the first and second B / F separation and washing, a long-chain DNA and a diatom shell are collected with a magnet. Alternatively, long-chain DNA, diatom shell, and short-chain DNA are attached, and only the silica-coated beads are held in the container, and substantially (almost) the entire amount of the drainage can be discharged quickly and efficiently. In addition, as shown in (11) and (12) of Fig. 2, after hydrolytic dissociation, the long-chain DNA, diatom shell and short-chain DNA dissociated from the beads are retained only in the silica-coated beads. Substantially (substantially) the entire amount can be efficiently taken out of the container together with water and observed with a microscope.

(3) As shown in FIG. 3, after the silica-coated beads, long-chain DNA, and diatom shells are put in a chaotropic ion-containing solution to bind them, the first B / F separation washing is performed. After washing away unreacted long-chain DNA and contaminants, adding unlabeled short-chain DNA, binding it to the diatom shell, washing the B / F for the second time, It is characterized by dyeing and watering the bound unlabeled short DNA, dissociating the long DNA from the silica-coated beads and the unlabeled short DNA bound to the diatom shell, taking them out and observing them under a microscope. To detect diatoms.
Here, by making the core of the silica-coated bead into a magnetic metal sphere, when draining after the first and second B / F separation and washing, a long-chain DNA and a diatom shell are collected with a magnet. Alternatively, it is possible to hold only silica-coated beads with long DNA, diatom shell, and unlabeled short DNA attached in the container, and to discharge almost (almost) all of the liquid out of the container efficiently and quickly. it can. In addition, as shown in FIGS. 3 (11) and (12), after hydrolytic dissociation, only the silica-coated beads are held in the container and the long-chain DNA dissociated from the beads, and the unlabeled short-chain DNA stained with the diatom shell. As a result, it is possible to efficiently take out substantially (substantially) the entire amount of the connected body together with water out of the container for microscopic observation.

(4) As shown in FIG. 4, silica-coated beads coated with short-chain DNA and a diatom shell are put in a chaotropic ion-containing solution to bind them, followed by B / F separation washing, Add long DNA, bind it to the diatom shell attached to the short DNA, then add water to dissociate and take out only the long DNA bound to the shell and PCR amplify it to obtain a calibration curve A method for detecting diatoms, characterized by quantitatively detecting diatoms.
Here, when the core of the silica-coated beads is made of a magnetic metal sphere, the diatom shell that is bonded to the short-chain DNA with a magnet is attached to the silica-coated beads when draining after the B / F separation washing. By holding only the beads in the container, substantially (almost) the entire amount of the drainage can be efficiently and quickly discharged out of the container. Also, as shown in FIGS. 4 (7) and (8), after hydrolytic dissociation, the silica-coated beads are held in a container together with the diatom shells bound to the short-chain DNA, so that the long-chain DNA dissociated from the beads is substantially removed. The (almost) whole amount can be efficiently taken out of the container together with water, PCR amplified to obtain a calibration curve, and diatoms can be detected quantitatively and accurately.

(5) As shown in FIG. 5, the first B / F separation is carried out after the diatom shell is put in silica coated beads coated with short-chain DNA in a solution containing chaotropic ions to bind them. After washing, and then adding short DNA having a different sequence from the short DNA and irreversibly binding it to the diatom shell, the second B / F separation washing was performed, and the silica-coated beads were added thereto. A method for detecting diatoms, comprising: extracting a short-chain DNA binding to an attached diatom shell and performing PCR amplification to obtain a calibration curve to quantitatively detect diatoms.
Here, when the core of the silica-coated beads is made of a magnetic metal sphere, the diatomaceous matter bound to the short-chain DNA with a magnet at the time of drainage after the first and second B / F separation washings. Only the silica-coated beads with the shell attached, or the silica-coated beads with the coated short-chain DNA, diatom shell, and the added short-chain DNA are held in the container, and the total amount of the drainage is almost (approximately). Can be efficiently and quickly discharged out of the container.

(6) As shown in FIG. 6, the first B / F separation is carried out after the diatom shells are put in silica coated beads coated with short DNA in a solution containing chaotropic ions and these are bound. Wash, and then add biotin / fluorescently labeled short-chain DNA to bind it irreversibly to the diatom shell, then perform a second B / F separation wash, then add substrate and stain A method for detecting diatoms, comprising: extracting biotin / fluorescently labeled short-chain DNA bound to diatom shells attached to silica-coated beads, and quantitatively detecting diatoms by measuring fluorescence intensity.
Here, when the core of the silica-coated bead is made of a magnetic metal sphere, the diatom shell put to the short-chain DNA is removed during the drainage after the first and second B / F separation washing. Hold only the silica-coated beads or the coated silica-coated beads with the coated short-chain DNA, biotin / fluorescent-labeled short-chain DNA, and diatom shells in the container with a magnet to drain the liquid. Substantially (almost) the entire amount can be discharged out of the container efficiently and quickly.

However, in the present invention, PCR amplification refers to a DNA amplification technique by a polymerase chain reaction utilizing a thermostable DNA polymerase of a thermophilic bacterium. (: Using the difference between denaturation and annealing due to the difference in DNA strand length, repeating DNA synthesis by simply repeating the temperature up and down to amplify the DNA)

In the present invention, B / F separation washing is Bound / Free separation washing. After the DNA binding reaction, first, the chaotropic solution (for example, 4 mol, guanidine hydrochloride) used for the binding is discarded, and then Add 10 times the amount of chaotropic solution of the same concentration to discard unreacted DNA and contaminants, and then wash twice with 70% ethanol, and then discard 70% ethanol completely, and only the beads bound to the shell Say leave.

In addition, in (1), (2) and (4) of the present invention, when water is added after B / F separation washing, the DNA bound to the silica beads and the shell is released (dissociated) by the addition. In (3), (5) and (6) above, when the water is not added, only the beads remain after the B / F separation and washing operation. Therefore, when performing PCR, the PCR reaction solution, biotin, In the case of fluorescence measurement, a buffer suitable for the measurement is added.

In the present invention, the term “staining” refers to a foilgen reaction used in general tissue staining, a nucleic acid staining method such as Nuclea Fast Red, a staining method using a general DNA staining agent such as Mupid ^(R) Blue, and various fluorescent methods. Other known staining methods such as a staining method using a staining agent and a silver staining method known as a highly sensitive staining method for proteins and DNA.

The method for detecting diatoms of the present invention pays attention to the fact that DNA easily binds to siliceous material, and this is used as a bonding agent with diatom shells to easily trap the diatom shells on DNA and diatoms. It is a simple, safe, quick, and inexpensive detection method that directly or indirectly detects the presence or absence of a clam shell from trapped DNA.

Table 1 summarizes the method of the present invention. The method of the present invention includes a diatom shell in a solution containing chaotropic ions.
A. Silica-coated beads and long-chain DNA are added and bound, followed by B / F separation washing. Alternatively, B. Silica-coated beads coated with short DNA are added and bound, followed by B / F separation washing.
Next, in the case of A, it is hydrolyzed as it is, or it is hydrolyzed by adding biotin / fluorescently labeled DNA. In the case of B, short-chain DNA or biotin / fluorescent-labeled DNA having a different sequence from long-chain DNA or coated short-chain DNA or unlabeled DNA is added to irreversibly bind the DNA to the diatom shell. B / F separation cleaning.
Next, in the case of A, the dissociated diatom shell is alone or the diatom shell bound to biotin / fluorescently labeled DNA is detected, and in the case of B, the DNA bound to the dissociated diatom shell is stained. And then amplifying the DNA by PCR and obtaining a calibration curve to detect the diatom shells quantitatively with high accuracy, or fluorescence of the biotin / fluorescence labeled DNA bound to the dissociated diatom shells The strength is measured and the diatom shell is quantitatively detected with high accuracy.

By this detection method, the following excellent specific effects are exhibited as compared with the conventional method.
1. Effect (meaning) of method (1)
Conventionally, after cleaning the diatom shell, it was only developed and dried directly on a slide and observed under a microscope. By adopting this detection method, diatoms that were difficult to see and detect due to residual contaminants were used. It became possible to observe the hulls in a clean state. In particular, in the proof of drowning, there are few things that hindered microscopic examinations such as charcoal powder and denatured fat.
In an experiment using a known number of shells, the recovery rate is almost 100%, so that the shells are almost completely bound to the silica-coated beads via the added DNA. In addition, it was considered that the putamen were completely released into the added water by the addition of water after the washing operation.
In particular, if magnetic beads are used for the silica-coated beads, the beads can be collected using a magnet for the cleaning operation in a state where the shell is trapped, so that loss due to the operation technique can be prevented.
This method is considered to be very useful because it is very easy to operate and can sufficiently exhibit its effects in a short time.

2. Effects (meaning) of methods (2) and (3)
Conventionally, after cleaning the diatom shell, it was only developed and dried directly on a slide and observed under a microscope. By adopting this detection method, diatoms that were difficult to see and detect due to residual contaminants were used. It became possible to observe the hulls in a clean state. In particular, in the proof of moribundity, not only can there be few things that hindered microscopic examination such as charcoal powder and denatured fat, but it is only necessary to look for stained putamen under a microscope, so This time is greatly shortened and the focus setting at the time of taking a picture is easy.
Biotin bound to the putamen and fluorescently labeled DNA can be observed in a state where the putamen is indirectly stained by staining using the existing staining method. The labeled biotin is stained with streptavidin labeled with an enzyme such as alkaline phosphatase, beta galactosidase or peroxidase, and a substrate corresponding to each enzyme. Not only biotin but also digoxigenin may be used for labeling the DNA.
Various fluorescent dyes can be used for fluorescent labeling of short-chain DNA, but FITC is particularly common, and in this case, observation with a fluorescence microscope is possible as it is.
In addition, even if unlabeled short DNA is bound to the putamen, the bound short DNA can be stained by a nucleic acid staining method such as Foilgen reaction or Nuclea Fast Red used in general tissue staining. . Furthermore, it can be stained not only by common DNA stains such as Mupid ^(R) Blue, but also various fluorescent stains can be used. Further, a silver staining method known as a high-sensitivity staining method for proteins and DNA can also be applied.
Since the putamen are already dyed, they can be stored as permanent specimens using common low refractive index mounting media.

3. Effect (meaning) of method (4)
It was possible to sandwich the shell using magnetic coated silica beads pre-coated with a sufficient amount of short DNA and long DNA of known sequence. This has the same principle as the non-competitive method in which an antigen is sandwiched and quantitatively detected using a polyclonal antibody or a plurality of types of monoclonal antibodies.
The amount of long-chain DNA with a known sequence that reacts secondarily increases in proportion to the amount of putamen bound to the magnetic beads serving as the solid phase. After removing unreacted long DNA by washing, the long DNA can be released into water by adding water. Since long-chain DNA has a known sequence, a primer can be synthesized in advance for a target region.
By performing a PCR reaction using the released long-chain DNA as a template and quantifying the amount of amplified DNA, the amount of putamen bound to the solid phase can be measured indirectly. At this time, the same operation is performed for a standard solution in which the amount of the putamen is quantified in advance by microscopic examination to prepare a calibration curve.
Using this method, it was possible to quantitatively detect several tens of shells even in experiments using a simple PCR method. If a more sensitive real-time PCR method or the like is introduced, it is possible to detect even several units, theoretically even one putamen.
As the magnetic silica beads serving as the solid phase, MagPrep (R) Silica manufactured by Merck Co., Ltd., which has the property of adsorbing and eluting the DNA by changing the pH, can also be used. This MagPrep (R) Silica has the property of adsorbing DNA under acidic conditions and releasing DNA under alkaline conditions of pH 8 or higher, so DNA will not be released even with neutral water. Suitable for washing and storage of solid phase.

4). Effect (meaning) of method (5)
To coat silica-coated beads (also called magnetic beads) coated with silica on magnetic metal spheres with a sufficient amount of short-chain DNA in advance, and using this short-chain DNA of known sequence, the shell is sandwiched with the coated short-chain DNA. Is possible. This has the same principle as the non-competitive method in which an antigen is sandwiched and quantitatively detected using a polyclonal antibody or a plurality of types of monoclonal antibodies.
The amount of short-chain DNA with a known sequence that reacts secondarily increases in proportion to the amount of putamen bound to the magnetic beads serving as the solid phase. After removing unreacted short DNA by washing, the short DNA secondaryly bound to the putamen trapped in the solid phase is transferred to another tube as it is, and PCR reaction is performed. Since secondary short-chain DNA has a known sequence, a primer can be synthesized in advance for a target region. In addition, a DNA having a completely different sequence from the short-chain DNA previously bound to the solid phase is used.
Since silica-coated beads coated with short-chain DNA used as a solid phase have a particle size of 2 to 4 μl, even if PCR reaction is performed together with these, the reaction is not hindered.
If the amount of DNA amplified by PCR is quantified, the amount of putamen bound to the solid phase indirectly can be measured. At this time, the same operation is performed for a standard solution in which the amount of the putamen is quantified in advance by microscopic examination to prepare a calibration curve.
Since the secondary short-chain DNA sandwiched has a smaller molecular weight (molecular structure) than the long-chain DNA, it has been demonstrated that the amount bound to the putamen is larger than that of the long-chain DNA. Therefore, if a more sensitive real-time PCR method or the like is introduced, it is possible to detect even several units, theoretically even one putamen.

5. Effect (meaning) of method (6)
It is possible to sandwich the shell by using a silica-coated magnetic bead pre-coated with a sufficient amount of short DNA, biotin, and fluorescently labeled short DNA. This has the same principle as the non-competitive method in which an antigen is sandwiched and quantitatively detected using a polyclonal antibody or a plurality of types of monoclonal antibodies.
The amount of secondary biotin and fluorescently labeled short-chain DNA increases in proportion to the amount of putamen bound to the magnetic beads serving as the solid phase. After removing unreacted biotin and fluorescently labeled short-chain DNA by washing, biotin labeled on the short-chain DNA secondarily bound to the putamen trapped on the solid phase, the fluorescence amount of the appropriate substrate, or Measure using a measuring device.
Biotin bound to the putamen and fluorescently labeled DNA can be measured using existing color development methods. The labeled biotin is used in combination with a streptavidin labeled with an enzyme such as alkaline phosphatase, beta galactosidase or peroxidase and a substrate corresponding to each enzyme. The fluorescence measurement method using a fluorescent substrate generally quantifies the amount of biotin and fluorescently labeled short-chain DNA that can be measured with higher sensitivity. Can be measured. At this time, the same operation is performed for a standard solution in which the amount of the putamen is quantified in advance by microscopic examination to prepare a calibration curve.
Further, as the magnetic silica beads serving as the solid phase, MagPrep (R) Silica manufactured by Merck Co., Ltd., which has the property of adsorbing and eluting DNA by changing the pH, can also be used. This MagPrep (R) Silica has the property of adsorbing DNA under acidic conditions and releasing DNA under alkaline conditions of pH 8 or higher, so DNA will not be released even with neutral water. Suitable for washing and storage of solid phase. In addition, short-chain DNA bound to the shell is not released into water, so the reaction is completed and measured under mild conditions without denaturing biotin, fluorescent dyes, or streptavidin labeled with various enzymes. be able to.

It is a schematic process drawing which shows the detection method of the diatom of Example 1 of this invention method (1). It is a schematic process drawing which shows the detection method of the diatom of Example 2 of this invention method (2). It is a schematic process drawing which shows the detection method of the diatom of Example 3 of this invention method (3). It is a schematic process drawing which shows the detection method of the diatom of Example 4 of this invention method (4). It is a schematic process drawing which shows the detection method of the diatom of Example 5 of this invention method (5). It is a schematic process drawing which shows the detection method of the diatom of Example 6 of this invention method (6).

Embodiments for carrying out the detection methods (1) to (6) of the present invention are as described in the following Table 1 and FIGS. Silica-coated beads are simply referred to as silica beads.

This example is an example of the method (1) with the above characteristics, and specific technical conditions and effects are introduced in Table 2.

This example is an example of the methods (2) and (3) of the above characteristics, and specific technical conditions and effects are introduced in Table 3.

This example is an example of the method (4) with the above characteristics, and specific technical conditions and effects are introduced in Table 4.

This example is an example of the method (5) with the above characteristics, and specific technical conditions and effects are introduced in Table 5.

This example is an example of the method (6) of the above feature, and specific technical conditions and effects are introduced in Table 6.

The detection method of the present invention has a great deal of use in and contributes to the detection of diatoms from organs indispensable for the verification of corpses in the forensic field. Specifically, the detection of diatoms from organs that had been broken in the past was often difficult due to the presence of contaminants, but the long-chain DNA of the present invention was used as an intervention agent for the putamen and silica beads. If the morphological observation method to be used is used, it is possible not only to produce a clean specimen by a simple operation but also to conduct microscopic inspection in a stained state. In particular, it does not require special techniques or expensive equipment, and can be used immediately as a good proof of death.
This method is not limited to the proof of drowning, but can be applied to the detection of all diatoms living in the sea, rivers, lakes, etc., so it can be applied to various fields such as biological experiments and environmental measurements. It is.
By utilizing the property that the putamen bind to silica beads via DNA, diatoms that have been concentrated and collected from a large amount of water by centrifugation can be specifically and selectively removed from the water. There is a possibility. This is particularly effective for collecting diatoms from water or seawater where the density of diatoms is low. When collecting diatoms from seawater, due to the high salt concentration, the centrifugal operation to replace the collected diatoms with fresh water must be repeated many times, but if this method is used, diatoms bound to silica beads are used. Can be easily washed while trapped in beads.
The method of immobilizing the putamen on a solid phase via DNA and sandwiching it with long and short DNAs with known sequences, biotin, and fluorescently labeled short DNAs is a new method for quantifying diatoms. It is expected to open up.
In particular, it seems that quantification of diatoms at low concentrations has not been envisaged so far, but the bound DNA can be amplified and detected tens of thousands of times using the PCR method, so only a small amount exists. It can be detected with high sensitivity even in a solution that does not. As is well known, DNA labeled with biotin or the like can be measured with high sensitivity by a combination of enzyme-labeled streptavidin and a specific substrate.
These technologies bring a new concept to the dying proof method, which has previously relied on morphological observation and proof, and its use is expected in the future.
Furthermore, if diatoms can be quantified from water and seawater with low density of diatoms, it will contribute to environmental measurements and biological measurements, which have conventionally required complicated operations and great efforts. There is something.

The methods of cleaning the putamen include the crushing method using concentrated sulfuric acid and nitric acid, the sulfuric acid-potassium dichromate bath , the ultraviolet irradiation method, the shochu method, the bleach stirring method, and the pipe unish method. You can use them according to your needs. In particular, the pipe unish method can be easily implemented by anyone, and there are no problems with safety, so it has been adopted in schools as practical training. After cleaning the putamen, it is developed and dried on a preparation, sealed with a suitable mounting agent, and observed. Since the putamen is the same vitreous as a preparation or a cover glass, it cannot be observed under an encapsulant having a low refractive index. Generally, high refractive index encapsulants such as Styrax, Hyrax, Pleurax are used.
Such observation methods for diatom shells are applied not only in the fields of biology and water and environmental chemistry, but also in various other fields. In particular, in the field of forensic medicine, there is a history that has been used extensively as a proof of drowning since ancient times.

Many studies have been conducted and reported to solve these problems, but the direction can be roughly divided into two.
The first is a method for detecting and verifying minute foreign substances other than diatoms contained in the brine. A method for culturing microorganisms such as bacteria contained in drowned water from major organs and blood (Kakizaki E, Takahama K, Seo Y et al. Forensic Sci Int 2008) This is the sandwich enzyme immunoassay (Kamada S, Seo Y et al. Forensic Sci Int 2000) .
The second is an improvement to the diatom detection method itself, such as solubilization of organs by protease treatment and improvement of the crushing method. In addition to this, research is being conducted to specifically amplify and verify plankton DNA such as diatoms by direct PCR.
However, although the above-mentioned proof methods for minute foreign substances other than diatoms are excellent methods, they are highly specific and often lack in versatility that can only be carried out by specific institutions. Furthermore, the improvement of the diatom detection method has not yet led to a radical solution to various problems.

Claims (7)

Trap the diatom shell by sandwiching it with silica-coated beads in a chaotropic ion-containing solution via DNA or with different DNA, and the presence or absence of diatom shell is directly or indirectly from the trapped DNA. A method for detecting diatoms, characterized by comprising:   Silica coated beads, long DNA, and diatom shells are put in a solution containing chaotropic ions to bind them, and then B / F separation washing is performed to wash away unreacted long DNA and impurities, and then add water. 2. The method for detecting diatoms according to claim 1, wherein long-chain DNA and diatom shells are dissociated from the surface of the silica-coated beads, and only the diatom shells are taken out and mirrored. Silica coated beads, long-chain DNA and diatom shells are put in a solution containing chaotropic ions to bind them, and the first B / F separation washing is performed to wash away unreacted long-chain DNA and impurities. Next, biotin / fluorescently labeled short-chain DNA is added, and this is irreversibly bound to diatom shells and dyed, followed by the second B / F separation washing, and then water is added to the silica-coated bead surface. The diatom shell is bound to biotin / fluorescence-labeled short DNA and dissociated separately from long-chain DNA, and the diatom shell is bound to biotin / fluorescence-labeled short DNA. The method for detecting diatoms according to claim 1, wherein: Silica coated beads, long-chain DNA and diatom shells are put in a solution containing chaotropic ions to bind them, and the first B / F separation washing is performed to wash away unreacted long-chain DNA and impurities. Next, unlabeled short DNA is added and bound to the diatom shell, and after the second B / F separation washing, the unlabeled short DNA bound to the diatom shell is stained and hydrolyzed. 2. The method for detecting diatoms according to claim 1, wherein long-chain DNA and unlabeled short-chain DNA bound to diatom shells are dissociated from the silica-coated beads, and these are taken out and microscopically observed. . A silica-coated bead coated with a short-chain DNA and a diatom shell are placed in a solution containing chaotropic ions and bonded to each other, followed by B / F separation and washing. Characterized by quantitatively detecting diatoms by binding to the bound diatom shell and then removing only the long-chain DNA bound to the shell by dissociation and PCR amplification. The method for detecting diatoms according to claim 1. A silica-coated bead coated with a short-chain DNA in a solution containing chaotropic ions is filled with a diatom shell and bonded thereto, followed by a first B / F separation and washing. After adding different short-chain DNA and irreversibly binding it to the diatom shell, the second B / F separation washing was performed, and the diatom shell attached to the silica-coated beads was shortly bonded. The method for detecting diatoms according to claim 1, wherein the diatoms are quantitatively detected by taking out the strand DNA and performing PCR amplification to obtain a calibration curve. A silica-coated bead coated with a short-chain DNA in a solution containing chaotropic ions is filled with a diatom shell and bonded thereto, followed by a first B / F separation washing, and then a biotin / fluorescent-labeled short-chain DNA After irreversibly binding to the diatom shell, the second B / F separation washing was performed, and then the substrate was added and stained to bind to the diatom shell attached to the silica-coated beads. 2. The method for detecting diatoms according to claim 1, wherein the biotin / fluorescently labeled short-chain DNA is taken out and the fluorescence intensity is measured to quantitatively detect diatoms.