WO2023120453A1

WO2023120453A1 - Method for detecting bacteria and kit for detecting bacteria

Info

Publication number: WO2023120453A1
Application number: PCT/JP2022/046584
Authority: WO
Inventors: 直杉田; 聡子中野; 則夫清水; 博高瀬; 學望月; 崇鈴木; 靖浩外丸
Original assignee: 国立大学法人大分大学; 国立大学法人東京医科歯科大学; 学校法人東邦大学; 日本テクノサービス株式会社; 直杉田
Priority date: 2021-12-22
Filing date: 2022-12-19
Publication date: 2023-06-29
Also published as: JP2023093129A

Abstract

According to the present invention, nucleic acids of bacteria are efficiently extracted and detected by PCR. A method for detecting bacteria comprises: a decomposition step for performing the reaction of a proteolytic enzyme in a sample mixture solution of a biological sample and a PCR buffer solution containing a proteolytic enzyme; a deactivation step for deactivating the proteolytic enzyme; and a detection step for adding at least a part of the sample mixture solution after the deactivation step to a PCR reaction composition to amplify a PCR product and detecting the PCR product.

Description

Method for detecting bacteria and kit for detecting bacteria

The present invention relates to a method for detecting bacteria and a kit for detecting bacteria.

Various technologies have been proposed to detect pathogens. Among them, the PCR method using the polymerase chain reaction (PCR) is widely used because of its excellent detection sensitivity and detection accuracy.

For example, Patent Document 1 discloses a kit that includes a DNA polymerase and a PCR primer pair in the same tube. In addition, Patent Document 1 proposes a technique of lysing viruses with a PCR buffer containing a surfactant.

Japanese Patent Application Laid-Open No. 2020-198809

The technology disclosed in Patent Document 1 can usually be applied when pathogens are present in liquids such as blood or body fluids. On the other hand, when a pathogen is present in a tissue piece such as the cornea, the nucleic acid of the pathogen must first be separated from the tissue piece in order to carry out the PCR method. However, the technique of Patent Document 1 has difficulty in isolating nucleic acids of pathogens from tissue pieces, and the types of applicable biological samples are limited.

In addition, the technology of Patent Document 1 mainly targets viruses, and when targeting bacteria, it is unknown whether the bacterial wall can be efficiently dissolved for nucleic acid extraction.

One aspect of the present invention aims to realize a method for efficiently extracting bacterial nucleic acids from various biological samples and detecting them by PCR.

In order to solve the above-mentioned problems, a method for detecting bacteria according to an aspect of the present invention provides a sample mixture obtained by mixing a biological sample that may contain bacteria and a PCR buffer containing a protease, and Degradation step for advancing enzymatic reaction, deactivation step for deactivating the proteolytic enzyme contained in the specimen mixed solution, and adding at least part of the specimen mixed solution after the deactivation step to the PCR reaction composition a detection step of adding to amplify the PCR product and detecting the PCR product.

In order to solve the above-mentioned problems, a kit for detecting bacteria according to one aspect of the present invention comprises a PCR buffer solution containing a protease and a PCR reaction composition, wherein the PCR reaction composition contains bacteria infected with bacteria. PCR primer pairs for detecting at least one of the pathogens that cause disease.

According to one aspect of the present invention, it is possible to realize a method for efficiently extracting bacterial nucleic acids from various biological samples and detecting them by PCR.

[Outline of the present invention]
Various methods have been used in techniques for amplifying and detecting bacterial nucleic acids. For example, PCR methods utilizing PCR, transcription-reverse transcription concerted reaction (TRC) methods, transcription-mediated amplification (TMA) methods, spreading sequence-based amplification (NASBA) methods, loop-mediated isothermal amplification (LAMP) methods, The Smart Amplification Process (SMAP) method, the Isothermal Chimera Primer-Initiated Nucleic Acid Amplification (ICAN) method, and the like are known.

Among them, the PCR method is widely applied in research and clinical practice due to its many advantages. Advantages of the PCR method include, for example, the ability to selectively amplify specific DNA fragments, the ability to work with extremely small amounts of sample, the relatively short time required for amplification, and the simple process, such as and the ability to amplify with an automated tabletop device.

The PCR method is also used, for example, in diagnosing ophthalmic bacterial infections. In order to detect bacteria that cause ophthalmologic bacterial infections from ophthalmologic specimens of which the amount of specimens to be obtained is limited, the PCR method capable of rapidly detecting nucleic acids from minute amounts of specimens is preferably used.

The PCR method requires sample pretreatment. A biological sample collected from a subject usually contains a large amount of a substance that inhibits an enzymatic reaction. Since a substance that inhibits this enzymatic reaction inhibits PCR, it is necessary to remove the substance that inhibits the enzymatic reaction in advance. Thus, in order to detect bacteria by the PCR method, pretreatments such as removing substances that inhibit enzymatic reactions in biological samples and purifying nucleic acids in biological samples were required.

For example, Ampdirect (registered trademark) technology is known as a technology that reduces the complexity of such pretreatment and enables detection of pathogens by PCR without pretreatment of biological samples (Ann Clin Biochem. 37, 674-680, 2000). Using such techniques, nucleic acids extracted from biological samples containing bacteria can be used as they are for PCR.

For nucleic acid extraction, there is a method using a PCR buffer containing a surfactant, as in Patent Document 1 above. However, such a method assumes a liquid specimen such as blood, and it is difficult to extract bacterial nucleic acids from a tissue piece or the like. As a method for isolating bacterial nucleic acids from a tissue piece, a method is known in which the tissue piece is added to a buffer solution containing a protease to digest the protein of the tissue piece, since the tissue piece is mainly composed of protein. . However, since protease also digests DNA polymerase, protease and DNA polymerase cannot coexist.

Therefore, the proteolytic enzyme and the DNA polymerase must be kept in separate reaction vessels, and it is difficult to add the tissue pieces directly to the PCR buffer solution containing the DNA polymerase to analyze bacterial nucleic acids by the PCR method. is.

In addition, the composition of the buffer containing protease and the PCR buffer containing DNA polymerase is usually different. Therefore, the addition of the sample after the reaction with the protease may cause a change in the composition of the PCR buffer containing the DNA polymerase, and the subsequent PCR may not proceed well. Therefore, conventionally, the amount of sample added to a PCR buffer containing DNA polymerase after reaction with a protease was limited. To increase the amount of bacterial nucleic acids in the PCR buffer, for example, it was necessary to increase the amount of sample.

A method for detecting a bacterium according to an embodiment of the present invention (hereinafter sometimes referred to as "one detection method of the present invention") can detect bacteria regardless of whether a biological sample is liquid or solid. Nucleic acids can be efficiently extracted from biological samples that may contain them. One detection method of the present invention will be described below.

[One detection method of the present invention]
One detection method of the invention includes a decomposition step, a deactivation step, and a detection step.

(Decomposition process)
The decomposition step is a step in which a reaction of a protease is allowed to proceed with respect to a specimen mixed solution obtained by mixing a biological sample that may contain bacteria and a PCR buffer solution containing a protease.

Bacteria are not particularly limited, but include, for example, bacteria that cause bacterial infections. A subject to be infected with bacteria may be a human, a non-human mammal, or a non-mammal animal. Such bacteria include, for example, respiratory pathogens of ophthalmic bacterial infections. Respiratory pathogens of ophthalmic bacterial infections include, for example, Enterococcus, Klebsiella, Nocardia, Streptococcus, Staphylococcus, Pseudomonas aeruginosa, and Escherichia coli.

Bacteria belonging to the genus Enterococcus include, for example, Enterococcus faecalis and Enterococcus agalactiae. Examples of bacteria belonging to the genus Klebsiella include Klebsiella pneumoniae. Bacteria of the genus Streptococcus include, for example, group A streptococci, group B hemolytic streptococci, and Streptococcus pneumoniae. Bacteria belonging to the genus Staphylococcus include, for example, Staphylococcus aureus and staphylococci other than Staphylococcus aureus. Examples of staphylococci other than Staphylococcus aureus include Staphylococcus epidermidis such as Staphylococcus epidermidis.

A biological sample that may contain bacteria is a sample obtained from a living body that is subject to detection of the presence or absence of bacterial infection, such as suspected bacterial infection, and is not particularly limited. A living body may be a human, a non-human mammal, or a non-mammal animal. Biological samples obtained from these living organisms include, for example, ophthalmic specimens containing at least a portion of an eyeball or ocular appendage.

A part of the eyeball includes, for example, a part of the cornea, vitreous body, lens, sclera, uvea and ciliary body. Aqueous humor present in the eyeball may also be included as part of the eyeball. Examples of ocular appendages include ocular appendages and secretions such as tears secreted from the eyeball or ocular appendages. Parts of the ocular appendages include, for example, parts of the conjunctiva, lacrimal apparatus, ocular muscles and eyelids.

Since it is difficult to collect a large amount of such ophthalmic specimens, the amount of biological samples that can be subjected to PCR is likely to be limited. One detection method of the present invention can detect nucleic acids derived from bacteria from minute amounts of biological samples. Therefore, one detection method of the present invention can be particularly suitably used for biological samples such as ophthalmic specimens, which tend to be collected in very small amounts. The biological sample may be other than an ophthalmic specimen, such as blood or skin.

Such a biological sample is mixed with a PCR buffer containing protease (hereinafter sometimes referred to as "pretreatment solution"). The pretreatment solution does not contain DNA polymerase. For example, a biological sample that may contain bacteria may be added to the pretreatment solution by soaking a cotton swab or the like with the biological sample attached in the pretreatment solution. A pretreatment solution to which a biological sample has been added is referred to as a specimen mixed solution.

The composition of the PCR buffer is not particularly limited, and conventionally known PCR buffers can be used. The PCR buffer can be, for example, a Tris buffer containing KCl, _MgCl2 and dNTP mix (a mixture of dATP, dGTP, dCTP and dTTP).

The protease contained in the PCR buffer is not particularly limited, but examples include serine protease, cysteine protease, threonine protease, aspartic protease, glutamic protease, metalloprotease and asparagine peptide lyase. Among them, proteinase K, which is a type of serine protease, is preferable from the viewpoint of proteolytic activity, availability, and the like.

The amount of the biological sample added to the pretreatment solution is 0.5 mg to 5 mg, more preferably 0.5 mg to 3 mg, and still more preferably 0 when the biological sample is solid such as a keratoconjunctival scraping. .75 mg to 2 mg. Also, when the biological sample is liquid such as aqueous humor, the volume is preferably 12 μL to 20 μL, but may be less than 12 μL.

Allow the reaction of the proteolytic enzyme to proceed with the obtained sample mixture. From the viewpoint of efficiently decomposing the protein with the protease, it is preferable to heat the specimen mixture at a temperature of 37° C. or higher and 60° C. or lower in the reaction. In this case, the heating time may be until almost all of the proteins in the sample mixture are decomposed, and may be, for example, 30 minutes or more and 60 minutes or less.

(Deactivation step)
The deactivation step is a step of deactivating the protease contained in the sample mixture after the reaction with the protease. The method for inactivating the protease is not particularly limited, but examples include methods such as heating the specimen mixture, adding a protease inhibitor to the specimen mixture, and autolyzing the protease. . Among them, deactivation by heating is preferable from the viewpoint of simply and reliably deactivating the protease.

When the protease is deactivated by heating, for example, it is preferable to heat the sample mixture at a temperature of 90°C or higher and 100°C or lower. In this case, the heating time may be until the protease is almost completely deactivated, and may be, for example, 5 minutes or more and 10 minutes or less.

(Detection process)
The detection step is a step of adding at least part of the specimen mixed solution after the deactivation step to the PCR reaction composition, amplifying the PCR product, and detecting the PCR product.

The PCR reaction composition may contain a DNA polymerase and at least one PCR primer pair. By adding the sample mixed solution to such a PCR reaction composition and performing PCR, if the sample mixed solution contains the target nucleic acid corresponding to the PCR primer pair, the PCR product is amplified.

The DNA polymerase is not particularly limited as long as it is a thermostable DNA polymerase, but may be, for example, Taq, Tth, KOD, Pfu and mutants thereof. A hot-start DNA polymerase may be used from the viewpoint of avoiding non-specific amplification of nucleic acids by the DNA polymerase. Hot-start DNA polymerases include, for example, DNA polymerases to which an anti-DNA polymerase antibody is bound, DNA polymerases in which the enzyme active site is chemically modified with heat sensitivity, and the like.

PCR primer pairs are not particularly limited. mentioned.

PCR primer pairs for detecting bacteria may be appropriately designed according to the type of bacteria. Such a PCR primer pair includes, for example, a PCR primer pair for detecting at least one respiratory pathogen of ophthalmologic bacterial infection. In this case, the PCR primer pair may be designed so as to be able to amplify nucleic acids having sequences unique to each of the bacteria mentioned above. Such design can be carried out, for example, based on base sequence information obtained from known sequence databases (GenBank, etc.).

PCR primer pairs for detecting drug resistance genes possessed by bacteria may be appropriately designed according to the types of bacteria and drug resistance genes possessed. The term "drug resistance gene" as used herein refers to a gene that bacteria do not originally possess, and includes, for example, a transmissible drug resistance gene such as a plasmid gene. In other words, the "drug resistance gene" as used herein may be different from the later-described "mutation of a bacterial gene that exhibits drug resistance".

The drug resistance genes possessed by bacteria are not particularly limited, but include, for example, methicillin resistance genes (mecA, etc.), vancomycin resistance genes (vanA, vanB, vanC, etc.), and quinolone resistance genes (qnr, etc.). That is, the PCR primer pair for detecting drug resistance genes possessed by bacteria may be for detecting at least one of methicillin resistance gene, vancomycin resistance gene and quinolone resistance gene.

PCR primer pairs for detecting the presence or absence of gene mutations that are bacterial genes that indicate drug resistance may be appropriately designed according to the type of gene mutation. Such genetic mutations include, for example, quinolone-tolerant genetic mutations in at least one of the DNA gyrase gene and the topoisomerase IV gene. More specifically, for example, gene mutations that cause quinolone resistance by occurring in the gyrA gene, the gyrB gene, the parC gene, the parE gene, and the like can be mentioned.

From the viewpoint of simultaneously detecting a plurality of bacterial nucleic acids, two or more PCR reaction compositions containing at least one PCR primer pair may be used. In this case, at least a portion of the specimen mixed solution after the deactivation step is added to each of these two or more types of PCR reaction compositions, and PCR is performed, whereby a plurality of bacterial nucleic acids can be detected simultaneously. A plurality of nucleic acids to be detected simultaneously may be, for example, nucleic acids for each of a plurality of types of bacteria, or nucleic acids for detecting bacteria and nucleic acids for detecting drug resistance genes possessed by the bacteria.

In addition, a multiplex PCR method may be used for PCR. The multiplex PCR method is known as a method for simultaneously amplifying multiple types of nucleic acids while saving the amount of sample mixture (Sugita S, et al. Br J Ophthalmol. 2008;92:928-932, Sugita S 2013;120:1761-1768). The multiplex PCR method is a method for simultaneously amplifying multiple nucleic acid regions by using multiple types of PCR primer pairs in a single PCR reaction system. This method has the advantage of being able to simultaneously detect a plurality of bacterial nucleic acids, in addition to being able to save the amount of sample mixture. When using the multiplex PCR method, it is preferable to optimize the sequence of each primer and the reaction conditions so that the amplification of the target nucleic acid by each PCR primer pair proceeds well in a single PCR reaction system. .

As described above, in one detection method of the present invention, when "the PCR reaction composition includes two or more PCR primer pairs", it includes either or both of the following (a) and (b): good. (a) when two or more types of PCR primer pairs are contained in a single PCR reaction composition, (b) one detection method of the present invention is performed using a plurality of PCR reaction compositions containing one type of PCR primer pair. if you do.

When a plurality of bacterial nucleic acids are detected simultaneously by one detection method of the present invention, the PCR primer pair is for detecting at least one of a methicillin resistance gene, a vancomycin resistance gene and a quinolone resistance gene, and exhibits quinolone resistance. It may include both for detecting the presence or absence of gene mutation. According to such a configuration, bacterial drug resistance caused by a plurality of factors can be detected at once. Moreover, it is preferable to include both a PCR primer pair for detecting a quinolone resistance gene and a PCR primer pair for detecting the presence or absence of a gene mutation exhibiting quinolone resistance. According to such a configuration, quinolone resistance in bacteria can be comprehensively detected both due to transmissible quinolone resistance genes and due to genetic mutations such as DNA gyrase in bacteria.

The PCR reaction composition may be a solution or a solid prepared by freeze-drying or the like. If the PCR reaction composition is solid, PCR can be started simply by mixing the specimen mixed solution after the deactivation step with the PCR reaction composition, so that one detection method of the present invention can be performed with a simple operation. . In this case, storage of the PCR reaction composition is also facilitated.

From the viewpoint of detection accuracy, the PCR reaction composition preferably contains at least one fluorescently labeled probe. Fluorescent-labeled probes are oligonucleotide probes labeled with fluorescent dyes for fluorescence detection of PCR products. If the PCR reaction composition contains one PCR primer pair, one fluorescent dye may also be used. On the other hand, when the PCR reaction composition contains two or more PCR primer pairs, it is preferable to use two or more different fluorescent dyes for detection of each PCR product.

Examples of such fluorescent dyes include 6-caroxyfluorescein (FAM), 6-carboxy-X-rhodamine (ROX), Alexa Fluor (registered trademark) dyes (e.g., ALEXA594), and cyanine dyes (e.g., Cy5). and 4,7,2',4',5',7'-hexachloro-6-carboxyfluorescein (HEX). The base sequence of the fluorescence-labeled probe may be appropriately designed based on the base sequence of the nucleic acid to be detected.

PCR proceeds by mixing the PCR reaction composition and the specimen mixture after the deactivation step and performing thermal cycling. The PCR conditions (temperature, time and number of cycles) may be appropriately set depending on the sequence of the nucleic acid derived from the assumed bacterium. A PCR product can be amplified if a nucleic acid derived from bacteria is contained in the sample mixture. Moreover, when the bacterium is a drug-resistant bacterium, a PCR product corresponding to the presence or absence of a drug-resistant gene or gene mutation indicating drug resistance can be amplified.

Next, the amplified PCR products are detected. Methods for detecting PCR products include, for example, electrophoresis using agarose gel, detection by thermal melting curve analysis, fluorescence detection, and the like. From the viewpoint of detection speed, a PCR product detection method called real-time measurement is preferred.

Real-time measurement of PCR products is also called real-time PCR. Real-time PCR generally detects PCR products by fluorescence detection. Methods of fluorescence detection in real-time PCR include, for example, methods using intercalating fluorescent dyes or fluorescently labeled probes. Intercalating fluorescent dyes include, for example, SYBR (registered trademark) Green. An intercalating fluorescent dye binds to double-stranded DNA synthesized by PCR. The amount of PCR product produced can be measured by irradiating the fluorescent dye accumulated by binding to the double-stranded DNA with excitation light and measuring the fluorescence intensity.

　Fluorescent-labeled probes used for real-time measurement include, for example, hydrolysis probes, Molecular Beacons, and cycling probes. The hydrolysis probe may be an oligonucleotide probe modified at the 5' end with a fluorescent dye and modified at the 3' end with a quencher substance. The fluorescent dye used for the hydrolysis probe is not particularly limited, and may be, for example, the fluorescent dyes described above. Quencher substances include, for example, TAMRA®, Black Hole Quencher (BHQ®) 1, BHQ2, MGB-Eclipse® and DABCYL. In order to distinguish and detect two or more different PCR products, it is preferable to perform PCR using two or more fluorescently labeled probes (e.g., hydrolysis probes) labeled with different fluorescent dyes from the viewpoint of detection accuracy. preferred from

In the case of real-time measurement of PCR products, the progress of PCR can be confirmed in real time by monitoring the amplification curve of the PCR product using a fluorescent filter that corresponds to the fluorescent dye used. If the fluorescence intensity increases with the number of PCR cycles, it may be determined that the PCR product is amplified.

In one detection method of the present invention, it is preferable to carry out the detection step including the condition that part of the specimen mixed solution after the deactivation step is added to the control PCR reaction composition. A control PCR reaction composition may have, for example, a positive control nucleic acid and a PCR primer pair corresponding to the positive control nucleic acid in addition to the PCR reaction composition described above. The positive control nucleic acid is used as an indicator that the PCR reaction was performed normally, an indicator that the nucleic acid derived from the biological sample was added correctly to the PCR reaction composition, and the like.

When used as an indicator that the PCR reaction was performed normally, the positive control nucleic acid may be a nucleic acid that is considered to be contained in a biological sample, or an artificial sequence that is artificially synthesized.

When using a nucleic acid that is considered to be contained in a biological sample as a positive control nucleic acid, it is more preferable to use a housekeeping gene whose expression level is less likely to fluctuate. Housekeeping genes include, for example, TATA-binding protein (TBP) gene, glyceraldehyde-3-phophate dehydrogenase (GAPDH) gene, β-actin gene, β2-microglobulin gene, hypoxanthine phosphoribosyl transferase 1 (HPRT1) gene, 18S rRNA gene , 5-aminolevulinate synthase (ALAS) gene, β-globin gene, glucose-6-phophate dehydrogenase (G6PD) gene, β-glucuronidase (GUSB) gene, importin 8 (IPO8) gene, porphobilinogen deaminase (PBGD ) gene, phosphoglycerate kinase 1 (PGK1) gene, peptidylprolyl isomerase A (PPIA) gene, ribosomal protein L13a (RPL13A) gene, ribosomal protein large P0 (RPLP0) gene, succinate dehydrogenase subunit A (SDHA) gene, transferrin receptor (TFRC) genes, 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta (YWHAZ) gene, and the like.

The positive control nucleic acid is also useful for quantification of nucleic acids derived from bacteria by quantifying the nucleic acid copy number (absolute or relative quantification). The quantification result of nucleic acids derived from bacteria can be used as an indicator of the number of bacteria in a biological sample, for example. When performing absolute quantification, for example, by creating a calibration curve based on the measurement results of a positive control nucleic acid of known concentration, nucleic acid derived from bacteria of unknown concentration can be quantified with high accuracy. In this case, the positive control nucleic acid is preferably an artificial sequence that is artificially synthesized and is not contained in the biological sample.

When performing relative quantification, for example, the number of cycles required for the PCR product to reach a certain amount may be compared between the positive control nucleic acid and the target nucleic acid. By this comparison, it is possible to calculate the relative concentration difference of the target nucleic acid with respect to the positive control nucleic acid, based on the characteristic of PCR that in principle the PCR product is doubled in each cycle.

The positive control nucleic acid may be one of these, or two or more. A configuration using two or more types of positive control nucleic acids is preferable from the viewpoint of increasing the reliability of nucleic acid quantification. As a positive control nucleic acid, for example, GAPDH and TBP may be used in combination.

According to one detection method of the present invention, nucleic acid derived from a bacterium can be extracted from a minute amount of a biological sample without the need for complicated operations when testing for the presence of bacteria and/or the presence or absence of drug resistance of the bacterium. can be used for PCR. Therefore, it is possible to efficiently carry out inspections on bacteria. According to such a configuration, through the promotion of popularization by simplification of testing for bacterial infectious diseases, for example, it contributes to the achievement of Goal 3 of the Sustainable Development Goals (SDGs) "Good health and well-being for all." can.

[Detection kit]
A bacterium detection kit according to an embodiment of the present invention (hereinafter sometimes referred to as "one kit of the present invention") comprises a PCR buffer containing a protease and a PCR reaction composition. A PCR reaction composition included in one kit of the present invention includes a PCR primer pair for detecting at least one respiratory pathogen of ophthalmologic bacterial infection. In addition, one kit of the present invention preferably further contains the aforementioned control PCR reaction composition. For each configuration included in one kit of the present invention, the description of [one detection method of the present invention] described above can be used.

A kit of the present invention may comprise, for example, a specimen collection container containing a PCR buffer solution containing a protease, and a PCR reaction container containing a PCR reaction composition. Moreover, a plurality of PCR reaction containers may be provided for each type of PCR primer pair included in the PCR reaction composition. In this case, the user of the one kit of the present invention can carry out the decomposition step and the deactivation step in the sample collection container as it is by adding the collected biological sample to the sample collection container. In addition, the user can directly perform the detection process in each PCR reaction container by adding at least a portion of the specimen mixed solution after the deactivation step to each PCR reaction container.

Both the sample collection container and the PCR reaction container are preferably containers with a shape compatible with the thermal cycler used for PCR. Such containers include, for example, a tube strip with a plurality of tubes connected together or a well plate for PCR with a plurality of wells.

In addition, one kit of the present invention may contain reagents other than those described above, such as protease inhibitors. The term "include" used in the description of a kit of the present invention can mean the state of inclusion in any of the individual containers that constitute the kit. A kit of the present invention may also include instructions for carrying out a detection method of the present invention.

According to the one kit of the present invention, when a small amount of biological sample is collected and the presence or absence of bacteria and/or the presence or absence of drug resistance of the bacteria is examined according to the one detection method of the present invention, testing can be performed efficiently. .

〔summary〕
A method for detecting bacteria according to an aspect of the present invention includes a decomposition step of allowing the reaction of the protease to proceed with respect to a sample mixed solution obtained by mixing a biological sample that may contain bacteria and a PCR buffer solution containing a protease. , a deactivation step of deactivating the protease contained in the specimen mixture, and adding at least part of the specimen mixture after the deactivation step to a PCR reaction composition to amplify a PCR product; and a detection step of detecting the PCR product.

In the method for detecting bacteria according to one aspect of the present invention, the biological sample may be an ophthalmic specimen containing at least part of an eyeball or an eyeball appendage.

In the method for detecting bacteria according to one aspect of the present invention, the PCR reaction composition may contain a PCR primer pair for detecting at least one respiratory pathogen of ophthalmologic bacterial infection.

In the method for detecting bacteria according to one aspect of the present invention, the respiratory pathogen is at least selected from the group consisting of Enterococcus, Klebsiella, Nocardia, Streptococcus, Staphylococcus, Pseudomonas aeruginosa, and Escherichia coli. It may be one type.

In the method for detecting bacteria according to one aspect of the present invention, the PCR reaction composition may contain a PCR primer pair for detecting at least one of a methicillin resistance gene, a vancomycin resistance gene and a quinolone resistance gene.

In the method for detecting bacteria according to one aspect of the present invention, the PCR reaction composition includes a PCR primer pair for detecting the presence or absence of a gene mutation exhibiting quinolone resistance in at least one of a DNA gyrase gene and a topoisomerase IV gene. may contain

In the method for detecting bacteria according to one aspect of the present invention, the PCR reaction composition comprises a PCR primer pair for detecting at least one of a methicillin resistance gene, a vancomycin resistance gene, and a quinolone resistance gene, and the quinolone resistance. and a PCR primer pair for detecting the presence or absence of gene mutation.

A kit for detecting bacteria according to one aspect of the present invention comprises a PCR buffer solution containing a protease and a PCR reaction composition, wherein the PCR reaction composition contains at least one respiratory pathogen of bacterial infection. including PCR primer pairs for the detection of

The present invention is not limited to the above-described embodiments, but can be modified in various ways within the scope of the claims, and can be obtained by appropriately combining technical means disclosed in different embodiments. is also included in the technical scope of the present invention.

[1. Detection Performance Evaluation of PCR Primer Pairs and Fluorescent Labeled Probes]
(1-1. Confirmation of PCR product amplification)
A PCR product amplification experiment was performed using a PCR primer pair and a fluorescence-labeled probe used in one detection method of the present invention. The PCR primer pairs used in this experiment are shown below:
For GAPDH gene detection (forward) 5'-tgtgctcccactcctgatttc-3' (SEQ ID NO: 1)
(reverse) 5'-cctagtcccagggctttgatt-3' (SEQ ID NO: 2)
For TBP gene detection (forward) 5'-gcaccactccactgtatccc-3' (SEQ ID NO: 3)
(Reverse) 5'-cccagaactctccgaagctg-3' (SEQ ID NO: 4)
For detection of Enterococcus (forward) 5'-agcctcggaattgagaatga-3' (SEQ ID NO: 5)
(Reverse) 5'-gaccttagctggtggtctgg-3' (SEQ ID NO: 6)
For Klebsiella detection (forward) 5'-aaccaggcgtcgataat-3' (SEQ ID NO: 7)
(reverse) 5'-gtttacggcgcaatcc-3' (SEQ ID NO: 8)
For detection of group B hemolytic streptococci (forward) 5'-ctctagtggctggtgcattg-3' (SEQ ID NO: 9)
(Reverse) 5'-ccatttgctgggcttgatta-3' (SEQ ID NO: 10)
For detection of group A hemolytic streptococci (forward) 5'-aggcggacatgcctttgtta-3' (SEQ ID NO: 11)
(Reverse) 5'-tgcctacaacagcactttgg-3' (SEQ ID NO: 12)
For detection of Streptococcus pneumoniae (forward) 5'-cacgcaatctagcagatgaagca-3' (SEQ ID NO: 13)
(Reverse) 5'-tcgtgcgttttaattccagct-3' (SEQ ID NO: 14)
For detection of Pseudomonas aeruginosa (forward) 5'-gccgaggtcatggaattc-3' (SEQ ID NO: 15)
(Reverse) 5'-atccgcgccatcatcttc-3' (SEQ ID NO: 16)
For detection of Staphylococcus epidermidis (forward) 5'-ccagaacgtgaYtctgacaaa-3' (SEQ ID NO: 17)
(Reverse) 5'-tcaccRactttgatttgacca-3' (SEQ ID NO: 18)
For Escherichia coli detection (forward) 5'-catgccgcgtgtatgaagaa-3' (SEQ ID NO: 19)
(Reverse) 5'-cgggtaacgtcaatgagcaaa-3' (SEQ ID NO: 20)
For detection of Staphylococcus aureus (forward) 5'-catcggaaacattgtgttctgtatg-3' (SEQ ID NO: 21)
(Reverse) 5'-tttggctggaaaatataactctcgta-3' (SEQ ID NO: 22)
For Nocardia detection (Forward) 5'-ccggaaaacctgcagagatgt-3' (SEQ ID NO: 23)
(Reverse) 5'-tacgcgctggcaacataaga-3' (SEQ ID NO: 24)
For vanA gene detection (forward) 5'-ggatagctactcccgccttt-3' (SEQ ID NO: 25)
(Reverse) 5'-cagcctgctcaattaagattttg-3' (SEQ ID NO: 26)
For mecA gene detection (forward) 5'-cctctgctcaacaagttcca-3' (SEQ ID NO: 27)
(Reverse) 5'-aacgttgtaaccaccccaag-3' (SEQ ID NO: 28)
For gyrA gene mutation detection (forward) 5'-gaacaaggtatgacgcccgata-3' (SEQ ID NO: 29)
(Reverse) 5'-ggccattcttaccattgcttca-3' (SEQ ID NO: 30).

The fluorescence-labeled probes used in this experiment were ROX-labeled, FAM-labeled or Alexa Fluor (registered trademark) 594-labeled at the 5' end and BHQ-labeled at the 3' end. The nucleotide sequence and 5' end label of the fluorescently labeled probe are shown below:
For GAPDH gene detection
5'-aaaagagctaggaaggacaggcaacttggc-3' (SEQ ID NO: 31) (FAM label)
For TBP gene detection
5'-acccccatcactcctgccacgc-3' (SEQ ID NO: 32) (ROX label)
for enterococcal detection
5'-cccatctcgggttaccgaattcaga-3' (SEQ ID NO: 33) (FAM label)
for Klebsiella detection
5'-acaggaaaagacaagactatgcagacc-3' (SEQ ID NO: 34) (ROX label)
For detection of group B hemolytic streptococci
5′-catgctgatcaagtgacaactccaca-3′ (SEQ ID NO: 35) (FAM label)
For detection of group A hemolytic streptococci
5′-tggtggcggcgcaggcggcttcaac-3′ (SEQ ID NO: 36) (ROX labeled)
for detection of Streptococcus pneumoniae
5′-ctccctgtatcaagcgttttcggc-3′ (SEQ ID NO: 37) (FAM label)
for Pseudomonas aeruginosa detection
5′-cgacaaccgcaaggaagccga-3′ (SEQ ID NO: 38) (ROX label)
for detection of Staphylococcus epidermidis
5'-ccattcatgatgccagttgaggacg-3' (SEQ ID NO: 39) (FAM label)
for Escherichia coli detection
5'-tattaactttactcccttcctccccgctgaa-3' (SEQ ID NO: 40) (ROX labeled)
for detection of Staphylococcus aureus
5′-aagccgtcttgataatctttagtagtaccgaagctggt-3′ (SEQ ID NO: 41) (FAM label)
for Nocardia detection
5′-agtcccgcaacgagcgcaaccctt-3′ (SEQ ID NO: 42) (ROX label)
For vanA gene detection
5′-tcctgtttttgttaagccggcgc-3′ (SEQ ID NO: 43) (FAM label)
for mecA gene detection
5'-aatcgatggtaaaggttggcaaaaaga-3' (SEQ ID NO: 44) (ROX labeled)
Wild-type (C allele) 5'-ttgaagaatcacc-3' (SEQ ID NO: 45) for gyrA gene mutation detection (FAM label)
Mutant (T allele) 5'-tgaaaaatcaccatga-3' (SEQ ID NO: 46) (ALEXA594 labeled) Mutant (A allele) 5'-tgaataatcaccatga-3' (SEQ ID NO: 47) (ALEXA594 labeled).

A test plasmid in which the PCR amplification region containing the target nucleic acid of these PCR primer pairs and the surrounding sequence is inserted is used as a sample, and each is added to a pretreatment solution (PCR buffer containing protease), and the sample mixture and Three conditions of 10 ⁶ copies, 10 ⁴ copies and 10 ² copies were set for each reaction for the copy number of the test plasmid. A negative control (NC) condition without test plasmid was also set up. The composition of the pretreatment solution before addition was 200 μg/mL proteinase K, 0.05% (w/v) nonionic detergent, 1.5 mM MgCl ₂ , 35 mM KCl and dNTP Mix (200 μM each of dATP, dGTP , dCTP and dTTP). 20 μL of the sample mixed solution after the decomposition step and the inactivation step by heating were dispensed into each tube of the 8-tube strip containing the solid composition for PCR reaction. A solid composition for a PCR reaction in a strip tube contains a DNA polymerase, a fluorescently labeled probe and a PCR primer pair. The types of fluorescently labeled probes and PCR primer pairs vary from tube to tube.

After the deactivation step, the sample mixture was monitored for PCR reaction by the hydrolysis probe method using a real-time PCR device. As PCR conditions, initial denaturation was performed at 95° C./10 seconds, followed by 45 cycles of PCR at 95° C./5 seconds-60° C./20 seconds. The presence or absence of PCR product amplification was determined based on the Cq value (cycle number at which the amplification curve of the PCR product crosses the threshold line). The results are shown in Table 1 below.

Note that A to H in Table 1 indicate tube names. Experiments were also conducted under each condition of n=3, and Table 1 shows the average and standard deviation of the Cq values as the results. The PCR reaction solid composition also contains a GAPDH test plasmid as a positive control nucleic acid. Therefore, in this experiment, only the GAPDH PCR product is also amplified in NC.

As shown in Table 1, amplification of the PCR product from the target nucleic acid was confirmed for any combination of PCR primer pairs and fluorescently labeled probes. In addition, in tube F, amplification was also observed in NC. However, this was a phenomenon that was observed only once in three trials, and it is thought that amplification was caused by contamination from the environment. Under all conditions other than NC, amplification of the PCR product was confirmed with good reproducibility in three out of three trials.

(1-2. Confirmation of detection performance from sample mixed with target nucleic acid)
Next, a sample mixed solution was prepared in which the test plasmids containing each target nucleic acid were present individually or in a mixed state. The copy number of the test plasmid containing each target nucleic acid was 50 copies per reaction. A detection method of the present invention was carried out under the same conditions as (1-1. Confirmation of PCR product amplification) above. The results are shown in Table 2 below. Table 2 shows the resulting Cq values.

As shown in Table 2, amplification of PCR products from samples mixed with test plasmids containing each target nucleic acid was confirmed for any combination of PCR primer pairs and fluorescently labeled probes.

[2. Detection of nucleic acid derived from bacteria]
A biological sample obtained from a patient suspected of having an ophthalmologic infection was used as a specimen to perform an experiment to detect nucleic acids derived from bacteria. A detection method of the present invention was carried out under the same conditions as (1-1. Confirmation of PCR product amplification) except for the specimen. However, tube F in this experiment used a PCR reaction solid composition that did not contain a PCR primer pair and fluorescently labeled probe for Nocardia detection. The results are shown in Table 3 below. Table 3 shows Cq values as results.

As shown in Table 3, it was shown that nucleic acids derived from bacteria can be detected even when ophthalmic specimens such as anterior aqueous humor, vitreous or corneal scrapings obtained from patients are used as biological samples. . As described above, according to one detection method of the present invention, the presence or absence of bacteria, the presence or absence of drug resistance genes, and the presence or absence of gene mutations indicating drug resistance can be easily detected from biological samples such as ophthalmic specimens, which are difficult to collect in large amounts. It is possible.

The present invention can be used, for example, for testing for bacterial infections.

Claims

a decomposition step of allowing the reaction of the protease to proceed with respect to a specimen mixed solution obtained by mixing a biological sample that may contain bacteria and a PCR buffer solution containing a protease;
a deactivation step of deactivating the protease contained in the sample mixture;
a detection step of adding at least part of the specimen mixed solution after the deactivation step to a PCR reaction composition to amplify a PCR product, and detecting the PCR product.
The method for detecting bacteria according to claim 1, wherein the biological sample is an ophthalmic specimen containing at least part of an eyeball or an ocular appendage.
The method for detecting bacteria according to claim 1 or 2, wherein the PCR reaction composition contains a PCR primer pair for detecting at least one respiratory pathogen of ophthalmologic bacterial infection.
The bacterium according to claim 3, wherein the respiratory pathogen is at least one species selected from the group consisting of Enterococcus, Klebsiella, Nocardia, Streptococcus, Staphylococcus, Pseudomonas aeruginosa and Escherichia coli. Detection method.
The method for detecting bacteria according to any one of claims 1 to 3, wherein the PCR reaction composition contains a PCR primer pair for detecting at least one of a methicillin resistance gene, a vancomycin resistance gene and a quinolone resistance gene. .
6. The PCR reaction composition according to any one of claims 1 to 5, wherein the PCR reaction composition contains a PCR primer pair for detecting the presence or absence of a gene mutation exhibiting quinolone resistance in at least one of the DNA gyrase gene and the topoisomerase IV gene. The method for detecting bacteria according to .
The PCR reaction composition comprises a PCR primer pair for detecting at least one of a methicillin resistance gene, a vancomycin resistance gene, and a quinolone resistance gene, and a PCR primer pair for detecting the presence or absence of a gene mutation exhibiting the quinolone resistance. The method for detecting bacteria according to claim 6, comprising:
A PCR buffer containing a protease and a PCR reaction composition,
A kit for detecting bacteria, wherein the PCR reaction composition comprises a PCR primer pair for detecting at least one pathogen causing bacterial infection.