JP2007236215A - Method for detecting biological sample - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique for simply detecting a specimen, in which it is feared that contain a biological sample in slight amount, in a short time and high sensitivity. <P>SOLUTION: The method for detecting the biological sample comprises (1) a step for non-specifically amplifying a genome DNA in the biological sample contained in the specimen in the presence of DNA polymerase and random hexamer enabling a MDA (Multiple Displacement Amplification) method and (2) a step for carrying out PCR analysis by using the resultant amplified product as a template. The detection method is suitably used for detecting or monitoring a wood-decaying fungus, a sanitary insect pest, a fungus, a stored grain insect pest each contained in a minute amount in housing construction materials, bedding, grains, soil, fruit trees, etc., in high sensitivity. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for detecting a biological sample from a specimen with high sensitivity, and more particularly to a novel high-sensitivity detection method that combines non-specific amplification of genomic DNA and PCR analysis.

  There are a myriad of living organisms and living tissues that are harmful to humans, animals, or assets owned by humans but difficult to see and difficult to judge. Looking at it in the environment closest to humans, a large number of mites, their remains and excrement, or fungal hyphae and spores are deposited and deposited in the house. They cause allergies such as asthma, rhinitis, ocular allergies, atopic eczema and bites in humans. Bacteria and drug-resistant bacteria that cause nosocomial infections and airborne infections may enter hospitals and public facilities. Looking at cultural heritage such as wooden second-hand houses and shrines and temples, they are subject to erosion by termites and wood-rotting fungi. When a wider living area is found, soil pathogens and crop pests inhabit soils and crops in fields and the like, and the harvest of crops is threatened. In golf courses and arable land, there are concerns about the growth of pesticide-resistant bacteria that do not work with pesticides and environmental pollution caused by excessive use of pesticides. If these living organisms or living tissues propagate or spread to such an extent that humans can detect them, they will cause serious damage to human health and property.

  Although it is meaningful to detect these biological samples early and with high sensitivity, the current situation is difficult. This will be explained in detail by taking an example of wood-rotting fungi. Wood-destroying fungi are harmful bacteria that cause deterioration of the strength of wooden structures and are one of the major causes of damage and collapse of wooden houses due to earthquakes. Wood decay fungi are a kind of basidiomycete, and are roughly classified into brown decay fungi that decompose cellulose and hemicellulose in wood and leave lignin, and white decay fungi that degrade all components in wood. Surface-contaminating bacteria that blacken the surface of wood, such as ascomycetes, are also known. Their erosive power is so great that white and brown rot fungi destroy trees, while the surface pollutants are as slight as the surface layer.

  Monitoring the behavior of wood-rotting fungi in the living environment over a long period is important for the maintenance of wooden houses. In particular, it is significant to detect white / brown rot fungi, which have a large impact on wood, at the beginning of decay, and to distinguish them from surface-contaminated fungi that cause minor damage, in taking appropriate measures against wood decay.

  Brown rot fungus prefers to be moist, well ventilated and badly exposed to the sun, while white rot fungus grows in cold and direct sunlight more intensely and repeatedly in dry and wet places than in brown rot fungus However, it can be said that it is very difficult to distinguish them morphologically in the early stage of decay, although the surface-contaminating bacteria have different propagation conditions such as growth under normal mold growth conditions.

  In the past, fungi collected from rotting wood were planted on trees and grown over many weeks to mushrooms and fungi, and the rotting fungi were judged by their morphological characteristics. This takes too much time and effort. As another method, a method is employed in which the collected bacteria are cultured in a petri dish medium, the cultured bacteria are observed under a microscope, and the decaying bacteria are identified from their bacteriological characteristics. However, culturing is complicated and there is a risk of culturing exogenous bacteria, and it is difficult even for an expert to determine the bacterial species from the mycological form.

  Recently, a method using molecular biological techniques has been proposed for the detection and identification of decaying fungi (Wood Preservation Vol.30 (2004) No.5 p192-203, edited by Japan Wood Conservation Association, Patent Document 1). One of the most widely used techniques is the PCR (polymerase chain reaction) method, which can amplify a part of a DNA region in a short time and easily in large quantities. Here, a specific DNA region amplified in large quantities is further subjected to genetic analysis to identify decaying fungi.

The PCR method is generally used as a method for amplifying a specific DNA region in a large amount from a small amount of template DNA. Nevertheless, in order to perform an effective PCR reaction, a relatively large amount of sample (for example, 20 to 50 ng in terms of DNA amount, or 1 × 10 ^{2 to 5 in} terms of the number of spores) is required. This makes it difficult to determine the initial stage of decay with a low amount of bacteria. On the other hand, securing a large amount of specimens for collecting a large amount of bacteria greatly damages buildings and the like, and is not practical.

  Therefore, as a pretreatment for the PCR method, generally, a living tissue in a specimen is cultured and DNA is further extracted. However, culturing is time-consuming and expensive for a period of two weeks or more, and involves the risk of increasing unknown ectobacterium.

  To extract DNA from hard cell walls such as fungi even if a large amount of culture or DNA can be obtained, freeze the culture and then grind it in a mortar, etc., in a surfactant, organic solvent, etc. It is necessary to go through a process of suspending and repeating the extraction, which takes time and labor.

  Furthermore, it is difficult to amplify all the biological samples contained in the specimen by culturing. Therefore, there is a problem that the analysis result by culturing does not reflect the actual decayed state.

  Hokkaido Forestry Experiment Station Report Vol. 17 No. 2 (March 2003) pp6-11 (Non-patent Document 2) contains proteinase K when extracting template DNA of decaying fungi from decayed wood (Todomatsu) that has decayed It is described that the decaying bacteria were identified by adopting the relatively quick and simple technique used and performing PCR analysis of the ITS region of rDNA contained in the DNA extract. However, this method still has a problem as a method for monitoring bacteria contained in trace amounts in wood at the early stage of decay.

The challenges of detecting biological samples contained in trace amounts in specimens as described in rot fungi in a short time, simply and with high sensitivity are not limited to rot fungi. The same applies to the detection of. Therefore, even now, there is a need for a method that can easily and highly sensitively detect a specimen that is likely to contain a trace amount of these biological samples in a short time.
Wood Preservation Vol.30 (2004) No.5 p192-203 Hokkaido Forest Products Laboratory Report Vol.17 No.2 (March 2003) pp6-11 Figure 5

  As a result of intensive studies on the above problems, the present inventors have found that the above problems can be solved by two-step amplification comprising DNA non-specific amplification and specific PCR, and have completed the present invention. Specifically, the present invention includes (1) a step of non-specifically amplifying genomic DNA in a biological sample contained in a specimen in the presence of a DNA polymerase capable of MDA (Multiple Displacement Amplification) method and a random hexamer, and (2) The present invention provides a method for detecting a biological sample, comprising a step of performing PCR analysis using the obtained amplification product as a template. The method of the present invention is clearly different from the conventional method in that DNA non-specific amplification is actively used and PCR analysis is performed without culturing a biological sample from a specimen.

  In this specification, the term “biological sample” is used to collectively refer to a minute living body or the whole or a part of a tissue that is difficult to visually recognize or cannot be morphologically determined. To do. Biological samples include, for example, bacteria, fungi and their spores, follicular spores, mycelia, fruiting bodies, micro animals and their eggs, larvae, shells and excreta. Biological samples referred to in the present invention include adults that can be visually recognized as minute animals such as cockroaches and termites, but that are difficult to determine such as eggs and larvae, or those that are difficult to determine such as remains and fragments. It is. Similarly, even if the grown fruit bodies (mushrooms) are morphologically clear, those that are difficult to determine, such as mycelia and spores, are included in the biological sample referred to in the present invention.

  The specimen contains a biological sample in various forms. For example, if the specimen is solid, it exists in a state of inclusion, adhesion, invasion, erosion, and the like, and if it is a liquid and gas, it exists in a state of deposition, suspension, or the like.

  In the method of the present invention, (1) in the non-specific amplification step, after adjusting the amount of the sample so that the amount of DNA of the biological sample contained in the sample becomes 1.0 to 10 ng, the amount of DNA of the biological sample is changed to 1.0 to Amplification to 10 μg is preferred.

  In the method of the present invention, (2) PCR analysis step preferably uses ITS region amplification by PCR, ethanol precipitation, ligation, transformation, colony PCR, miniprep, and DNA sequence.

  In the method of the present invention, it is preferable to use the PCR-RFLP method in the (2) PCR analysis step.

  In the method of the present invention, the biological sample is preferably derived from at least one of wood decay fungi, sanitary pests, bacteria, fungi, stored grain pests, pesticide resistant fungi, crop pests, soil pathogens, and antagonistic microorganisms.

  The specimen is any of tree, wood, residential building material, woodwork product, clothing, bedding, shellfish, processed food, soil, fruit tree, crop, plant, ocean, river, lake, pool, bathhouse, indoor air It is preferable.

  The method of the present invention is useful for diagnosis of decay and prediction of useful life by determining wood decay fungi that erode trees, wood, residential building materials or woodwork products.

  The method of the present invention is useful for use in detecting human allergens or selecting insecticides / bactericides by determining sanitary pests and / or fungi adhering to clothing or bedding.

  The method of the present invention is useful for performing storage management by determining stored pests contaminated in shellfish or dry processed food.

  The method of the present invention is useful for pesticide management of these by determining pesticide-resistant bacteria, crop diseases and / or soil pathogens that are mixed or attached to soil, fruit trees, crops or plants.

  The present invention uses a reagent for non-specific amplification of genomic DNA of a biological sample contained in a specimen in the presence of a DNA polymerase capable of MDA method and a random hexamer, and the obtained amplification product as a template. A kit for sensitive detection of a biological sample from an analyte is also provided, which includes reagents for performing a PCR reaction.

  The detection method of the present invention is characterized in that (1) a non-specific amplification step of genomic DNA in a specimen is employed instead of a conventional biological sample culturing step and the step of extracting DNA is omitted. Have. This feature makes it possible to amplify the DNA of all biological samples including those that cannot be cultivated, such as organisms and carcasses that have been difficult to culture.

  Since the method of the present invention does not perform culturing and extraction, it enables detection of a living tissue in a short time with a simple operation compared to the conventional method. Specifically, the culture and extraction operations that conventionally required two weeks or more are replaced with nonspecific DNA amplification of about 4 to 11 hours. As a result, the total time required for detection is shortened within 3 days.

  The detection method of the present invention can (1) amplify the DNA amount of a biological sample from 1.0 to 10 ng to 1.0 to 10 μg in a non-specific amplification step. When the amount of DNA in a biological sample is referred to as spores, even a trace amount of about 1 to 5 spores can be used as a starting amount for non-specific amplification. This is extremely effective when only a very small amount of sample can be collected as in the early stage of decay of wood or when it is not desired to damage the specimen as in cultural heritage.

  The adjustment of the DNA starting amount to be subjected to non-specific amplification is also effective for a sample (for example, decayed wood) whose collected sample has an amount of DNA that can be subjected to PCR analysis. Since the sample contains a large amount of PCR-inhibiting components such as spears, the reaction will not be carried out efficiently if subjected to PCR analysis as it is. By adjusting the amount of the sample so that the amount of DNA in the biological sample becomes 1.0 to 10 ng by the method of the present invention and performing non-specific amplification therefrom, only the nucleic acid portion is amplified, and the PCR-inhibiting component is not amplified. The so-called SN ratio is improved by 100 to 10,000 times. That is, (1) the non-specific amplification step also plays a role of purifying the biological sample in the specimen, and (2) improves the accuracy of the PCR analysis step. The detection method of the present invention can be applied to all specimens regardless of the amount of DNA of the biological sample in the specimen, and exhibits effects unique to the present invention.

  In the method of the present invention, (2) in the PCR analysis step, amplification of the ITS region by PCR, ethanol precipitation, ligation, transformation, colony PCR, miniprep, and DNA sequencing are employed to perform genome amplification and PCR analysis. The total processing time is reduced from 2 weeks or more of the conventional method to 3 days or less.

  The method of the present invention can further reduce the determination time (for example, 8 hours) by employing PCR-RFLP analysis in the (2) PCR analysis step.

  According to the method of the present invention, biological samples that were not easily detected by conventional methods, in particular, wood decay fungi, sanitary pests, bacteria, fungi, stored grain pests, pesticide-resistant fungi, crop pests, soil pathogens, antagonistic microorganisms Biological samples derived from at least one species such as trees, wood, residential building materials, woodwork products, clothing, bedding, shellfish, processed food, soil, fruit trees, crops, plants, oceans, rivers, lakes, pools, baths, indoor air Can be detected with high sensitivity from a sample such as

  The detection method of the present invention can be applied to various applications. It is used to determine wood decay fungi that erode specimens consisting of trees, timber, residential building materials or woodwork products, and to use clothing or equipment used to diagnose decay or predict the service life of the trees, wood, housing materials or woodwork products. Stored pests contaminated with samples of shellfish or dried processed food, used to determine sanitary pests and / or fungi adhering to samples made of bedding, and to detect allergens in humans and animals or to select pesticides and fungicides And determine pesticide-resistant bacteria, crop pests and / or soil pathogens that are mixed in or attached to specimens made of soil, fruit trees, crops or plants used to store and manage the shellfish or dry processed food In addition, the industrial applicability is very high, such as use for pesticide management of the soil, fruit trees, agricultural crops or plants.

  Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. As shown in FIG. 1, the method of the present invention comprises (1) a non-specific amplification step of genomic DNA in a specimen, and (2) a PCR analysis of the amplification product.

(1) Non-specific amplification step First, biological samples and specimens that are preferably handled by the detection method of the present invention will be described. Among biological samples, wood-destroying fungi, sanitary pests, golf field pests, pathogenic bacteria, fungi harmful to humans and animals, pesticide-resistant fungi, crop pests and soil pathogens, antagonistic microorganisms and the like are suitable. Specific examples are listed below.

  Specific examples of the wood-rotting fungi include Ginkgo biloba, Idodake, Ozuradake, Kikaigaratake, Namitake, Matsuooji (above, brown rot fungus), Kaidatake, Kawaratake, Suhirotake, Hiirotake, Hoshigetake (above, white rot fungus), Ketomi Ascomycetes (generally classified as surface-contaminating bacteria).

  Specific examples of the sanitary pests include dust mites, staghorn mites, mites, house dust mites (more than mites), oyster mites, mosquito mosquitoes, mosquito mosquitoes, mushrooms, mosquito mosquitoes (more than mosquitoes), mosquito flies, mosquito flies, large flies, large flies, Fleas (and above, flies), human fleas, cat fleas, dog fleas, European mouse minamis, European rat minnows, mountain rat minamis (meat rats) (my fleas), body lice, head lice, white lice, bed bugs (and above, lices) ), German cockroach, Black cockroach, American cockroach, Japanese cockroach (Over, cockroach), Japanese termite, Japanese termite, American termite, Japanese termite, Japanese termite, Thai termite, die Termites, Caterpillars, Saturn Termites, Long-tailed Termites, Kodama Termites, Kushimoto Termites, Crested Termites, Termites, Amphic Termites, Termites And tiger beetle.

  Specific examples of the golf field pests include hirataookogane, uschakogane, koichakogane, chibi-sakuragane, sedaragagane, beangane, himekogane, osakasakokogane, shibao weevil, shibata toga, tsujikiriyoto, and tadpole mushroom.

  Specific examples of pathogenic bacteria include Salmonella, pathogenic Escherichia coli, enterohemorrhagic Escherichia coli, Legionella, resistant Staphylococcus aureus, Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Bacillus cereus, Bacillus cereus, Campylobacter, Examples include Vibrio parahaemolyticus, Salmonella typhi, Salmonella paratyphi, Shigella and Cholera.

  Specific examples of fungi that are harmful to humans and animals include Suscabi, Tuciamobi, Kumonosukabi, Kekabi, Saphaerosperma, Penicillium, Fusarium, Eurotium, Humanplasma, Imidis, Cocciosides, Neoformans, Cryptococcus, Fumigatus, Aspergillus, Candida, Examples include albicans, rubrum, mentagul phytes, trichofteon, violaceum, tsukiyotake, kakimeji, kusaurabenita, garlic, sardine and white agaric.

  Specific examples of stored-grain pests include: Ittenkuga, Omeno-Kirihira-Tameshi, Guyi-myme-nosed beetle, Guymite moth Riga, Guymyde-kiosui, Kakumunehira-mushi, Kashino-no-Mameiga, Kashmir Kokunustomodoki, Kuromome-shidamashi, Kokuzoumushi Bark beetle, Pterocarpus longiflora, Rice-bellied beetle, Rice-nosed beetle, Rice-nosed-eye moth, Ginseng-bamboo, Sujiko-no-sama-mae-gai, Suda-no-madagai, Tobacco beetle, Citrus terrestris, B. elegans Beetle, stag beetle, scotch beetle, cabbage weevil, beetle weevil, bean weevil, lobster beetle Bushimushi, scallop moth Riga, scallop moth, oak weevil, scotch weevil, sorghum weevil, ginseng weevil, scallop moth, tobacco scallop, chamadara moth, scallop moth, harajiro katsushi sushi, sword worm Examples include worms, cocknuts, cinna beetles, cigarette beetles, cigarette beetle moth chamadarameiga, narcissus leopard hornworm, yellow-bellied beetle, long-horned beetle, long-horned beetle, and weevil weevil.

  Specific examples of pesticide-resistant bacteria, crop diseases and soil pathogens include gray mold, red mold, periwinkle black spot fungus, green coconut black spot fungus, rice blast fungus, leaf blight anthracnose fungus, cyclamen anthracnose fungus, black spot root Rot fungus, Strawberry root rot fungus, Tomato root rot fungus, Burdock wilt fungus, Soybean wilt fungus, Flower ginger wilt fungus, Lily wilt fungus, Tomato wilt fungus, Tomato half wilt fungus, Eggplant half wilt fungus Fungus, cyclamen wilt fungus, stock wilt fungus, spinach wilt fungus, tomato bacterial wilt fungus, strawberry wilt fungus, radish wilt fungus, cucumber vine split fungus, yugao vine split fungus, watermelon vine split fungus, garlic dry rot fungus , Onion dry rot fungus, rakkyo dry rot fungus, leek dry rot fungus, konjac dry rot fungus, lily dry rot fungus, leek white silk Bacteria, soybean white silk fungus, Schlumbergera Rot, peacock bar cactus Rot, Cymbidium Rot, Dendrobium Rot, Gibberella fujikuroi, Sclerotinia sclerotiorum, Fusarium, Rhizoctonia fungi include Fitofura bacteria and Pythium.

  Specific examples of antagonistic microorganisms include Bacillus subtilis, Actinomyces, Pseudomonas, Trichoderma, Agrobacterium, Lactic acid bacteria, Yeast, Aspergius, Penicillium, Rhizopus (above, antibacterial microorganism), Pseudomonas florescence, Pseudomonas gladioli (above, root) Bacterium), VA mycorrhizal fungi, truffle rhizobial fungi, and matsutake fungi (above, mycorrhizal fungi).

  Although there are all objects and places related to the human sphere as specimens including the biological sample, the following subjects are particularly prioritized. It includes trees, wood, housing materials (cultural heritage such as general houses, shrines and temples), woodworking products (cooking / cooking utensils, tableware), furniture (clogging boxes, closets, sofas, chairs, stuffed animals, tatami mats, wallpaper), houses Equipment (bathroom, air conditioner, toilet), bedding (futon, blanket, bed, mattress, bed sheet, pillow, cushion, curtain, carpet, carpet), clothing, footwear, leather products, cereals (rice, wheat, beans), Dry processed food, soil (fields, fields, forests, fields, golf courses, soil contaminated with dioxin / mineral oil), fruit trees, crops, plants, oceans, rivers, lakes, ponds, pools, baths (public baths, hot springs) Facilities), water use facilities (water and sewage treatment plants, factory wastewater treatment facilities, cooling towers, hydroponic cultivation facilities), indoor air (hospitals, food processing plants).

  When the correlation between the specimen and the biological sample is shown, a tree, wood, residential building material or woodwork product and wood decaying fungus, clothing or bedding that erodes the sanitary pest and / or fungus, shellfish or Dry processed food and stored grain pests, soil, fruit trees, crops or plants and pesticide-resistant bacteria, crop diseases and / or soil pathogens, indoor air and hot springs, and pathogens contained therein Relationships like sex bacteria and fungi that are harmful to people and animals.

  A specimen presumed to contain a biological sample needs to expose DNA to some extent in order to be subjected to a non-specific growth process of genomic DNA described later. However, it is not necessary to extract DNA. Specifically, after crushing the specimen as appropriate, the cells of the living tissue may be crushed with a cell crusher.

  The amount of DNA of the biological sample subjected to the non-specific amplification step is preferably 1.0 to 10 ng, particularly preferably 2.0 to 5.0 ng. On the other hand, the amount of the non-specific amplification product may be an amount sufficient for the PCR analysis in the step (2), and is usually 1.0 to 10 μg. Even if the amount of DNA in the sample is already a PCR-capable amount, it is preferable to perform non-specific amplification after adjusting the above starting concentration. As a result, the nucleic acid portion is amplified, and PCR-inhibiting components such as spears are not amplified. The analysis accuracy of PCR analysis described later is improved by 100 to 10,000 times in the so-called SN ratio. Therefore, in a more preferred embodiment of the present invention, the step of adjusting the amount of the sample so that the amount of DNA of the biological sample contained in the collected sample becomes 1.0 to 10 ng, the genomic DNA of the biological sample contained in the sample, Non-specific amplification in the presence of DNA polymerase and random hexamer that can be processed until the amount of DNA reaches 1.0 to 10 μg, and PCR analysis using the obtained amplification product as a template. It is a detection method.

  The disrupted sample obtained above is amplified non-specifically with genomic DNA using a DNA polymerase capable of MDA (Multiple Displacement Amplification) method and a random hexamer. Details of the MDA method are described in PNAS April 16, 2002 vol. 99 No. 8 pp5261-5266. The polymerase used for non-specific amplification of the genome is not particularly limited as long as it is a DNA polymerase that can be subjected to the MDA method. Currently, a DNA polymerase that can be applied to this method is a DNA polymerase derived from Bacillus subtilis phage phi29. (Hereinafter referred to as Phi29 DNA polymerase). This polymerase has strand displacement and forward synthetic specificity and also has a proofreading exonuclease activity in the 3 'to 5' direction.

  A genomic DNA amplification kit containing Phi29 DNA polymerase and random hexamer primers is commercially available from GE Healthcare Biosciences (formerly Amersham Biosciences) as the product name GenomiPhi DNA Apmliphication Kit. Below, the non-specific amplification process using this amplification kit is demonstrated.

  First, in order to initiate an isothermal polymerase reaction in the presence of Phi29 DNA polymerase and random hexamer, the biological sample pulverized solution and random hexamer are mixed, heat-denatured at 95 ° C for 3 minutes, and then rapidly cooled to 4 ° C. Later, dNTPs and Phi29 DNA polymerase are added and placed at a temperature of 30 ° C. After being allowed to stand, the primer anneals to a plurality of locations on the genomic DNA, and Phi29 DNA polymerase starts replication on the single-stranded linear DNA all at once. By the strand displacement function of the polymerase, the synthesis proceeds while peeling the strand, and the synthesis reaction also proceeds from the peeled portion. As a result, genomic DNA is exponentially amplified under isothermal conditions. While the reaction time depends on the starting concentration, it is generally 3 to 15 hours, preferably 4 to 11 hours.

(2) PCR analysis step The non-specific amplification product of genomic DNA obtained above is subjected to PCR analysis. PCR reaction is performed by adding specific primers and thermostable DNA polymerase to a buffer containing genomic DNA, (1) denaturation of double-stranded DNA into single strands at 90-95 ° C, and (2) annealing temperature. A specific base sequence is exponentially repeated by repeating a series of reactions of DNA strand and primer annealing, and (3) complementary strand synthesis by DNA polymerase at a temperature higher than the annealing temperature, usually about 20-50 times. It is a technique to amplify.

  For PCR analysis, it is necessary to prepare primers specific to the biological sample to be identified. Selection of the primer can be performed by a conventional method, for example, by clustalw analysis of DNA registered in the NCBI database.

  When the biological sample to be identified is a eukaryote, it is advantageous to synthesize it based on the sequence information of the ITS (Internal Transcribed Spacer) region. The ITS region refers to a non-transcribed region existing between all eukaryotic rDNAs as shown in FIG. In FIG. 2, 18S rDNA, 5.8S rDNA, and 28S rDNA on both sides of the ITS region are highly conserved, but the ITS region is likely to undergo sequence variation with evolution. Therefore, identification of organism species is facilitated by the difference in the sequence of the ITS region. Specifically, when trying to discriminate filamentous fungi (including basidiomycetes and ascomycetes), a primer specific to the filamentous fungus is designed based on the base sequence registered in the NCBI database or the like. In order to discriminate basidiomycetes, a primer specific to basidiomycetes may be designed by the same search.

  The length of the base sequence of the primer can be appropriately designed according to the length of the specific base sequence in the genomic DNA of the biological tissue to be detected. The length is usually 10 to 50 bp, preferably 15 to 30 bp.

  Selection and amount of thermostable DNA polymerase when selectively replicating a specific base sequence in the genomic DNA by PCR reaction using the species-specific primer and a non-specific amplification product (template DNA), genomic DNA The amount, primer amount, reaction time, temperature, etc. are appropriately determined.

  As a method for easily determining whether or not the amplified DNA fragment is a presumed species, it is effective to perform gel electrophoresis of the PCR amplification product. That is, the replicated PCR product is subjected to electrophoresis on an agarose gel to which ethidium bromide is added, or after electrophoresis, ethidium bromide staining or other DNA staining is performed. As a result, when a band based on a base sequence specific to living tissue appears, genomic DNA derived from the predicted living tissue is included in the PCR product, and the expected biological sample exists in the specimen. Will be.

  For identification with higher accuracy, the PCR amplification product may be subcloned and the DNA sequence determined by sequencing. The steps leading up to the sequencing of the PCR product are in the order of, for example, ethanol precipitation, ligation, transformation, colony PCR, miniprep, and sequencing. The sequence obtained in the sequence is subjected to Blast search and the like to identify the species of the biological tissue.

  When the detection method of the present invention is performed up to the sequence, the processing time is summarized in Table 1. According to the method of the present invention, instead of conventional time-consuming culture and extraction operations, only DNA non-specific amplification is performed. It is possible to process within 3 days.

  In the detection method of the present invention, the steps from ethanol precipitation to sequencing may be replaced with PCR-RFLP (Restriction Fragment Length Polymorphism). RFLP is based on the fact that the length of a DNA fragment cleaved by a restriction enzyme differs between individuals within the same species (ie, becomes polymorphic). Since non-specifically amplified DNA can be subjected to PCR, DNA cleavage, electrophoresis, and DNA visualization with UV light in about 2 hours, the total time required for identification can be reduced to about 8 hours. . Further, according to this fragment analysis, various mixed samples can be identified simultaneously.

The detection method of the present invention can also determine the abundance ratio of a plurality of biological tissues contained in a specimen. Specifically, the absorbance (A 2 ₆₀ ) of multiple types of PCR amplification products measured simultaneously and the ratio of fluorescence intensity during gel electrophoresis are measured.

  The detection method of the present invention can also quantify biological tissues contained in a specimen. Specifically, prepare multiple samples with different biological tissue concentrations, measure the absorbance and fluorescence intensity of the target amplification product after non-specific amplification of the genome and PCR reaction under certain conditions, and then create a calibration curve. create. The sample to be measured is amplified by the method of the present invention, and then applied to a calibration curve to determine the concentration of the sample in the specimen.

  In the method of the present invention, the steps from ethanol precipitation to sequencing may be replaced with a DNA array. A DNA array is obtained by spotting a plurality of DNA (annotated) of biological tissue to be identified on a substrate. Non-specific amplification of genomic DNA of a biological sample, and PCR amplification of the amplified product are further stained with a fluorescent substance to produce labeled DNA.

  The present invention also collects a sample presumed to contain a biological sample, a reagent for non-specific amplification of DNA in the sample in the presence of a DNA polymerase capable of MDA method and a random hexamer, and Provided is a kit for detecting a biological sample from a specimen with high sensitivity, including a reagent for performing a PCR reaction using the obtained amplification product as a template.

Hereinafter, the detection method of the present invention will be described in detail using examples, but the present invention is not limited to only the examples.
[Example 1]
(1) Collection of specimens Mycelia and spores were collected from various strains (Table 2) obtained from the National Institute of Agrobiological Sciences, cultured on potato dextrose agar at 26.5 ° C for 7 to 14 days. FIG. 9 shows a photograph of the culture state.

  The mycelia of sample Nos. 2, 4, 5 and 6 were collected by picking the mycelium on the agar medium with tweezers. The spores of Samples No. 1 and 3 were obtained by removing the mycelium by culturing the spores after culturing, suspending them in water, and filtering. They were freeze-dried and pulverized with a cell crusher (product name: Multi-Bead Shocker, manufactured by Yasui Instruments Co., Ltd.).

  No. 7 decayed wood flour was obtained by grinding sterilized beech wood, placing 2.8 g of the wood flour and 7.2 ml of water in a 45 ml container, inoculating Flammulina populicola strain, and allowing to stand at a temperature of 26.5 ° C. for 30 days. . As shown in FIG. 9, this wood powder was in a decayed state so that the wood powder was white. The decayed wood flour was collected in a volume of about 2 ml and further pulverized with a cell crusher (beater).

  The decayed wood pieces of Nos. 8 to 13 were placed in a container with sterilized cedar wood pieces (1 × 1 × 2 cm), inoculated with the standard strains listed in Table 2, and maintained at a temperature of 26 ° C. for 7 days (No. 8, 10, 12) or 14 days (No. 9, 11, 13). As shown in FIG. 9, the degree of decay of this specimen was so mild that it could not be discriminated at all whether or not it was decayed visually. This piece of wood was collected in a volume of about 2 ml and further pulverized with a cell crusher (beater).

(2) Non-specific amplification of genomic DNA with Phi29 DNA polymerase The ground sample obtained above is transferred to TE buffer (10 mM Tris-HCI (pH 8.0), 1 mM EDTA)), and the DNA becomes approximately <1 ng / μl. Suspended in a quantity to prepare a suspension solution of each sample.

  1 μl of each sample suspension was collected in a PCR tube. According to the instructions attached to the GenomiPhi DNA Amplification Kit, random hexamers are added, heat-denatured at 95 ° C. for 3 minutes, rapidly cooled to 4 ° C., and then added with appropriate amounts of Phi29 DNA polymerase and dNTPs at a temperature of 30 ° C. × 18 Nonspecific amplification of the genome of time was performed.

  After completion of the reaction, 1 μl of non-specific amplification product is mixed with 0.5 μl of loading buffer and electrophoresed on a 1.0% agarose gel with ethidium bromide added (electrophoresis: Mupid-EX (ADVANCE), buffer: 1 × TBE) After that, the DNA band was observed on a UV transilluminator. The results are shown in FIG. 3 (M is a marker).

  As shown in Fig. 3, P.ch spores, T.vi spores, F.po mycelia, F.po wood flour, and S.la mycelia can be used for non-specific amplification of genomic DNA using GenomiPhi. there were. That is, it was possible to non-amplify DNA regardless of the difference between basidiomycetes and ascomycetes, and in the form of spores, hyphae, and wood flour. From these results, it can be seen that GenomiPhi can be used to amplify genomic DNA by simple operations without the need for culture or DNA extraction.

(Non-specific amplification over time)
Sample No. 1 (P.ch spore) and sample no. 3 (T.vi spores) were each amplified for 5 spores (corresponding to <1 pg of DNA) and non-specifically amplified at different times. FIG. 4 shows the result of gel electrophoresis after the reaction in the same manner as described above.

  From FIG. 4, it can be seen that the concentration of the amplification product increases with the passage of the amplification time, and that the plateau is reached in about 4 hours. In another experiment, even a single spore could be amplified nonspecifically, and finally a DNA amount of several μg could be secured. From the above results, according to the non-specific amplification using the Genomiphi kit, even with a very small amount of biological sample of about 1 to 5 spores, the amount is sufficient for PCR analysis in a short time of 4 to 5 hours. Samples can be obtained.

(3) PCR analysis (ITS region PCR amplification)
1 μg of the sample non-specifically amplified as described above was subjected to PCR analysis. Primers used in the PCR reaction were synthesized by selecting a homology search from the NCBI database and selecting three types of primers shown in Table 3 from the ITS region of rDNA.

Table 4 shows the sizes predicted from the sequence data of PCR products using these primers.

* Since T. vi is an ascomycete, it is not amplified when basidiomycetes-specific R (basidiomycetes) primers are used.

The PCR reaction contains 1 μl of template DNA solution and 0.5 units of KOD DNA polymerase (product name: KOD-Plus, manufactured by TOYOBO). The final concentration is 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM. The reaction was performed in 25 μl of a reaction solution prepared in MgCl ₂ and 0.2 mM dNTPs.

  PCR amplification conditions are as follows: (1) heat denaturation (94 ° C) x 30 seconds → (2) annealing (53 ° C) x 30 seconds → (3) extension reaction (68 ° C) x 1 minute. A thermal cycler (product name: i-Cycler, manufactured by BIO-RAD) was used. After cycling, it was left at 68 ° C. for 1 minute.

(Distinction between basidiomycetes and ascomycetes)
FIG. 5 and FIG. 6 show the results of gel electrophoresis in the same manner as described above after the PCR reaction. In FIG. 5, basidiomycetes and pups are detected in accordance with PCR using primer pairs specific to filamentous fungi (including basidiomycetes and ascomycetes) [F (universal), R (universal)]. Both amplifications of cysts were confirmed. In each lane, a DNA band corresponding to the size predicted from the sequence was observed.

  On the other hand, in FIG. 6, amplification was confirmed only for basidiomycetes, corresponding to PCR performed using primer pairs specific to basidiomycetes [F (universal), R (basidiomycetes)]. The size predicted from the sequence was also consistent.

  As shown in FIG. 5 and FIG. 6, basidiomycetes (P.ch hyphae and S.la mycelia) and ascomycetes (T.vi hyphae) could be distinguished by the detection method of the present invention. The method first determines whether or not there is a decaying fungus in the specimen, and if there is a decaying fungus, it is a serious white / brown decaying fungus, or it is a minor surface contamination. It means that you can make a secondary judgment of whether there is any.

(Sequence)
Sample No. 1 (P.ch mycelium) and sample no. Sequencing was performed to identify the PCR amplification product of 5 (S.la mycelium). The specific procedure is as follows.

(Ligation)
Take 1 μl of the PCR solution in a new eppen, and use the following reagents: 1 μl of sterilized water, Salt Solution (1.2M NaCl, 0.06M MgCl ₂ ) 0.5 μl Plasmid vector Zero Blunt® TOPO® PCR Cloning Kit for Sequencing (Invitrogen ) 0.5 μl was added and left at room temperature for 5 minutes.

(transformation)
3 μl of the above ligation reaction solution was added to 27 μl of an E. coli suspension obtained by warming and melting commercially available E. coli JM109 Competent Cells (TAKARA BIO). After being left on ice for 2 minutes, a heat shock was applied at 42 ° C. for 45 seconds. Next, 270 μl of SOC culture solution (2% Tryptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl ₂ , 10 mM MgSO ₄ , 20 mM Glucose) was added, and cultured with shaking for 60 minutes. Spread the culture on an agar medium and spread with a spreader. Incubate overnight at 37 ° C.

(Colony PCR)
Pick up where the colonies of the culture grew with a toothpick, PCR master mix (sterile water, buffer, 1.5 mM MgCl ₂ , 0.2 mM dNTPs, 0.5 μM forward primer, 0.5 μM reversal primer, 0.25 U / μl Taq DNA polymerase) was suspended in 10 μl, and the toothpick was pulled out and rubbed against the master plate. A PCR tube was set in the thermal cycler, and [94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 1 minute] × 35 cycles of PCR reaction was performed. The PCR product was electrophoresed on a 1.0% agarose gel. After gel staining with ethidium bromide, it was checked whether the band that appeared was the desired length. Colonies with correct bands were checked against the master plate to determine the plasmid to be sequenced.

(Miniprep)
10 ml of the plasmid solution was transferred to a tube, collected by centrifugation at 3000 g for 5 minutes, the LB medium was completely sucked out, and the pellet was dissolved in 500 μl of TE. 1000 μl of 0.2N NaOH / 1% SDS solution was added and mixed gently to lyse. 750 μl of 3M potassium acetate, 11.5% acetic acid was added to neutralize, and left on ice for 5 minutes. After centrifuging at 5000 g for 5 minutes, the supernatant was transferred to another tube. After isopropyl alcohol precipitation and rinsing with 70% ethanol, centrifugation was performed at 15000 rpm for 5 minutes, and then the precipitate was dried.

(Sequence)
The mini-prepared amplified fragments were sequenced by a one-color dye terminator method using an automatic sequencer (product name: SQ5500E, manufactured by HITACHI). The sequence data was subjected to Blast search and the like to identify the species of biological tissue. An overview of the sequence results is shown in FIG. As shown in FIG. 1 (P.ch mycelium) and sample no. 5 (S.la mycelium) were confirmed to be Phanerochaete chrysosporium and Serpula lacrymans, respectively.

  Figure 8 shows samples taken from decayed wood pieces of samples Nos. 8-13, non-specific amplification of the genome using the GenomiPhi kit, and subsequent PCR amplification of the ITS region using filamentous fungus-specific (F, R) primers. It is a gel electrophoresis photograph of the product obtained by 1. The electrophoretograms show reproducible results for samples 8 and 9, 10 and 11, and 12 and 13. Furthermore, bacteria were identified in the sequence of amplification products. Thereby, according to the method of the present invention, it can be seen that the presence or absence of decay can be reliably determined even with very slight decay as shown in FIG.

  The method of the present invention has various uses as a method for detecting a biological sample in a simple and highly sensitive manner from a specimen or environment in which a biological sample that is difficult or impossible to distinguish (whether it is alive or dead) is present even in a trace amount. This is exemplified below according to the relationship between the biological tissue to be identified and the specimen containing it, but is not limited thereto.

  When the specimen is a tree, wood, house building material, or woodwork product, the decay diagnosis and the service life are predicted by determining the wood decay fungus that erodes the specimen. In cultural heritage, etc., it helps to protect cultural heritage by identifying wood-rotting fungi in the early stages of decay.

  When the specimen is clothes or bedding, by detecting the hygienic pests and / or fungi attached to the specimen, detection of allergens in the human body, assistance in improving the living environment, selection of appropriate insecticides / bactericides Etc. are useful.

  When the specimen is shellfish or dried processed food, it is useful for storage management and quality control of shellfish or dried processed food by determining the stored pests contaminated by the specimen.

  If the specimen is soil, fruit trees, crops or plants, the contamination state of the field or cultivated land by detecting pesticide-resistant bacteria, crop pathogens and / or soil pathogens mixed in or attached to them at an early stage and even in trace amounts Contributes to early diagnosis and early disease control. In addition, appropriate pesticide management becomes possible, which also develops into organic and reduced pesticide cultivation.

  There are many types of fungi that are useful in the living sphere of people. For example, pesticide-free cultivation is possible by detecting useful antagonistic microorganisms contained in the soil. By detecting dioxin-degrading bacteria (for example, white rot fungi) and oil-degrading bacteria contained in soil, it can be used for bioremediation of soil contaminated with dioxin or mineral oil. The method of the present invention is also useful for searching for artificial reproduction and detecting truffle mycorrhizal fungi and matsutake fungi (a kind of ectomycorrhizal fungi) contained in soil.

  When the specimen is indoor air, hot springs, etc., it is useful for the prevention of hospital infection and hygiene management by detecting pathogenic bacteria contained therein and fungi harmful to humans and animals.

It is a flowchart which shows the process of the detection method of this invention. FIG. 4 is a positional relationship diagram of primers targeting non-transcribed regions (ITS) existing between nuclear rDNAs. It is a gel electrophoretic diagram (drawing substitute photograph) of the nonspecific amplification product of genomic DNA of the method of the present invention. It is an electrophoretic diagram (drawing substitute photograph) which shows a time-dependent change of the nonspecific amplification of the genomic DNA of the method of this invention. FIG. 3 is a gel electrophoresis diagram (drawing substitute photograph) of a product obtained by PCR amplification of P.ch, S.la and T.vi using filamentous fungus-specific (F, R) primers according to the method of the present invention. Gel electrophoresis diagram of products obtained by PCR amplification of P.ch, S.la and T.vi using filamentous fungus-specific (F) and basidiomycetes-specific (R) primers according to the method of the present invention (drawing substitute) Photo). It is a figure which shows the sequence result of the PCR amplification product of FIG. It is a gel-electrophoresis diagram (drawing substitute photograph) of the product which PCR-amplified the decayed wood piece (No.8-13) using the filamentous fungus-specific (F, R) primer according to the method of this invention. It is a figure (drawing substitute photograph) of the external appearance of the decaying fungi used in the Example of the method of the present invention, decayed wood flour, and decayed wood fragments.

Claims (11)

(1) A step of non-specific amplification of genomic DNA of a biological sample contained in a specimen in the presence of a DNA polymerase capable of MDA method and a random hexamer, and (2) PCR using the obtained amplification product as a template. A method for detecting a biological sample, comprising a step of performing an analysis.   (1) In the non-specific amplification step, after adjusting the amount of the sample so that the amount of DNA of the biological sample contained in the sample becomes 1.0 to 10 ng, the amount of DNA of the biological sample is amplified to 1.0 to 10 μg The method for detecting a biological sample according to claim 1.   (2) The PCR analysis step comprises amplification of an ITS region by PCR, ethanol precipitation, ligation, transformation, colony PCR, miniprep, and DNA sequence, The biological sample according to claim 1 or 2, Detection method.   (2) The method for detecting a biological sample according to claim 1 or 2, wherein the PCR analysis step is PCR-RFLP.   The biological sample is derived from at least one of wood decay fungi, sanitary pests, bacteria, fungi, stored grain pests, pesticide-resistant fungi, crop pests, soil pathogens, and antagonistic microorganisms. The method for detecting a biological sample according to any one of the above.   The specimen is any of tree, wood, residential building material, woodwork product, clothing, bedding, shellfish, processed food, soil, fruit tree, crop, plant, ocean, river, lake, pool, bathhouse, indoor air The method for detecting a biological sample according to any one of claims 1 to 5, wherein:   The detection of the biological sample in the specimen is carried out by determining a wood decay fungus that erodes a specimen composed of a tree, wood, a housing construction material or a woodwork product, and performing a diagnosis of the decay of the tree, wood, a housing construction material or a woodworking product and a prediction of a useful life. The method for detecting a biological sample according to claim 1, wherein the method is used for performing.   The detection of the biological sample in the specimen is characterized by determining sanitary pests and / or fungi adhering to a specimen composed of clothing or bedding, and used for detection of human or animal allergens or selection of insecticides / bactericides. The method for detecting a biological sample according to any one of claims 1 to 5.   The detection of the biological sample in the specimen is characterized in that it is used to determine a stored grain pest contaminated by a specimen made of shells or dried processed food, and to perform storage management of the shells or dried processed food. Item 6. A biological sample detection method according to any one of Items 1 to 5.   The detection of the biological sample in the specimen is carried out by determining pesticide-resistant bacteria, crop pathogens and / or soil pathogens mixed in or attached to a specimen comprising soil, fruit trees, crops or plants, and detecting the soil, fruit trees, crops or plants. The method for detecting a biological sample according to any one of claims 1 to 5, which is used for pesticide management.   Reagents for nonspecific amplification of genomic DNA of biological samples contained in specimens in the presence of DNA polymerase and random hexamer capable of MDA method, and PCR reaction using the obtained amplification product as a template A kit for detecting a biological sample from a specimen with high sensitivity, including a reagent for the detection.