JP2009165371A - Double label fusion pcr immunochromatography - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for quickly and simultaneously detecting a plurality of genes by a simple method in high precision and in high sensitivity. <P>SOLUTION: The method for simultaneously detecting a plurality of genes in a sample comprises (1) a process for fusing a plurality of gene DNA fragments to prepare one fused DNA fragment and doubly labeling the fused DNA fragment by a first label and a second label different from the first label, (2) a process for amplifying the doubly labeled fused DNA fragment, (3) a process for subjecting the amplified double labeled fused DNA fragment to immunochromatography and (5) a process for observing the stationary phase of immunochromatography and judging the presence of the plurality of gene DNA fragments. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for simultaneously detecting the presence of a plurality of genes.

  Specific and simple screening of genes that are risk factors for pathogenesis or disease (for example, drug-resistant bacteria, carcinogenesis and lifestyle-related diseases high-risk gene SNPs set, etc.) Therefore, it is considered to be very useful in the medical field.

  For example, drug resistant bacteria to which antibiotics have not been effective have been regarded as a problem in recent years as the cause of opportunistic infections and hospital infections. Drug-resistant bacteria acquire resistance through mutation of their genes or incorporation of foreign genes. The drug resistance traits thus generated will rapidly proliferate and spread as pathogens, for example, by selective pressure due to the use of antibiotics in hospitals. Therefore, rapid discovery and control of the development and growth of drug resistant bacteria is an important issue in the medical field.

Conventionally, as a highly sensitive drug-resistant bacteria identification method, a method of amplifying a pathogen gene using polymerase chain reaction (PCR) and detecting it by gel electrophoresis or the like has been used. For example, a method is known in which a drug resistance gene mecA of methicillin-resistant Staphylococcus aureus (MRSA) is amplified by PCR, labeled, and detected by ELISA (Non-patent Document 1). A method for identifying pathogenic genes by combining PCR and immunochromatography is also known (Patent Document 1). However, the types of drug-resistant bacteria are very diverse and often have multiple resistance genes. Therefore, in order to rapidly detect and identify various resistant bacteria, a method capable of simultaneously detecting a plurality of genes is required.

As a method for simultaneously detecting a plurality of genes, a multiplex PCR method (Non-patent Document 2) is known. This is a method of performing PCR of multiple DNA templates simultaneously using multiple primer sets, and multiple genes in a sample can be detected by a single PCR, which can speed up testing. it can. On the other hand, this method has a problem in that it is difficult to set appropriate experimental conditions including selection of a primer set for target-specific detection.
Japanese Patent Laid-Open No. 2003-194817 Ubukata et al., J. Clin. Microbiol. (1992), vol. 30: 1728-1733 Cebula et al., J. Clin. Microbiol. (1995), 33: 248-250

  An object of the present invention is to provide a method for simultaneously detecting a plurality of genes with high accuracy and high sensitivity in a simple manner.

  In view of such circumstances, the present inventors examined a simpler method for simultaneously detecting a plurality of genes. As a result, if a nucleic acid amplification reaction using specific primers is used, a plurality of target gene fragments can be fused to one fragment, and this can be double-labeled and amplified. We found that it is possible to obtain double-labeled fusion fragments of multiple genes in one step, and that this amplified fragment can be detected by one-step immunochromatography, so that multiple genes can be detected simultaneously by a simple method. The invention has been completed.

That is, the present invention includes the following steps:
(1) A step of fusing DNA fragments of a plurality of genes to produce one fusion DNA fragment and double-labeling the fusion DNA fragment with a first label and a second label different from the first label;
(2) amplifying the double-labeled fusion DNA fragment;
(3) subjecting the amplified double-labeled fusion DNA fragment to immunochromatography;
(4) observing the stationary phase of the immunochromatograph to determine the presence or absence of DNA fragments of the plurality of genes,
The present invention relates to a method for simultaneously detecting a plurality of genes in a sample.

  By using the present invention, pathogenicity and disease risk factors (for example, multidrug-resistant bacteria, specific SNPs set, etc.) caused by multiple genes, or characteristic genes among microorganisms of the same species or animal individuals are retained. Those (for example, drug-resistant bacteria, genes that predispose to specific diseases such as carcinogenesis and lifestyle-related diseases, etc.) can be screened specifically and simply. Therefore, the present invention is considered to greatly contribute to the control of infectious diseases, the hygiene management of the medical environment, and the promotion of preventive medicine to promote the health of the people.

The present invention provides a method for simultaneously detecting a plurality of genes in a sample. Hereinafter, the method of the present invention will be described in detail. The method of the present invention is performed according to the following steps:
(1) A step of fusing DNA fragments of a plurality of genes to produce one fusion DNA fragment and double-labeling the fusion DNA fragment with a first label and a second label different from the first label;
(2) amplifying the double-labeled fusion DNA fragment;
(3) subjecting the amplified double-labeled fusion DNA fragment to immunochromatography;
(4) A step of observing the stationary phase of the immunochromatograph to determine the presence or absence of DNA fragments of the plurality of genes.

(1. Target gene)
Examples of target genes of the present invention include those derived from all animals, plants, microorganisms (bacteria, fungi, parasites, etc.), viruses, etc., and their complementary strands, as well as artificially created genes and their complementary strands. Can be mentioned. A plurality of genes to be targeted can be arbitrarily selected according to the purpose. For example, in order to detect drug-resistant bacteria, a combination of an individual species-specific gene and a drug resistance gene can be selected. Alternatively, when detecting pathogenic and disease risk factors (for example, multidrug-resistant bacteria or specific SNPs set) caused by a plurality of genes, a combination of the plurality of genes can be selected. It is also possible to select further combinations of the above (eg species specific genes + multiple drug resistance genes or specific SNPs set). Here, “multiple genes” means 2 genes or more, 3 genes or more, 4 genes or more, 5 genes or more, 5 to 10 genes or more, or 10 genes or more. To do. The “DNA fragment of a plurality of genes” used or detected in the method of the present invention may be the entire sequence or a partial sequence of the target gene. The sequence of the target gene can be obtained from literatures or public databases (eg, GenBank, EMBL, DDBJ, etc.).

The sample used in the present invention includes any sample that can contain a target gene. Specific examples of samples that can contain the target gene include animal and plant cells, tissues, organs, body fluids (blood, plasma, serum, urine, feces, saliva, lymph, cerebrospinal fluid, intercellular fluid, etc.), etc. Or those derived from cultures thereof; microorganisms such as fungi or bacteria or those derived from cultures thereof; pathogens (fungi, bacteria, parasites, viruses, etc.); and specimens that may contain these .

The sample is pretreated as necessary before being subjected to the method of the present invention. For example, the sample may be used after appropriately diluted with purified water or the like. Further, it is possible to perform a process for removing impurities from the sample and collecting only the target cells. Alternatively, it is possible to perform a process for extracting and purifying only the nucleic acid component containing the gene in the sample in advance. Target gene is DNA
If not, a reverse transcription reaction can be further performed to produce cDNA of the target gene. These pretreatment methods are well known in the art (see, eg, Sambrook & Russell, Molecular Cloning, A Laboratory Manual, 3rd. Ed., 2001).

(2. Target gene DNA fusion and double labeling)
The sample that can contain the DNA fragment of the target gene thus obtained is subjected to step (1) of the method of the present invention, so that one fusion DNA can be obtained from a plurality of DNA fragments of the target gene that can be contained in the sample. A fragment is prepared, and the prepared fusion DNA fragment is double-labeled with a first label and a second label different from the first label. This step is performed by polymerase chain reaction (PCR) using the following primers (see, for example, Sambrook et al., 2001, above).
A sequence complementary to the sequence at the 3 ′ end of one strand of the DNA fragment of one gene of the plurality of genes, and one strand of the DNA sequence of the gene fused to the DNA fragment of the one gene. A primer (A) having a sequence substantially identical to the sequence at the 3 ′ end;
A sequence complementary to the sequence of the 3 ′ end of one strand of the DNA fragment of the gene to be fused, and a sequence substantially identical to the sequence of the 3 ′ end of one strand of the DNA fragment of the one gene A primer (B) having
A primer (L1) having a sequence complementary to the sequence at the 3 ′ end of the other strand of the fusion DNA fragment and labeled at the 5 ′ end with the first label;
A primer (L2) having a sequence complementary to the sequence at the 3 ′ end of the other strand of the fusion DNA fragment and labeled at the 5 ′ end with the second label.

Here, in the present invention, the term “complementary” means that the sequence is not limited to a complete complementary sequence of a given sequence, and is at least 70% or more, preferably at least 80% or more, with a completely complementary sequence. More preferably at least 90% or more, and even more preferably at least 95% or more of the nucleotide sequence, and can hybridize with the given sequence under stringent conditions (Sambrook et al., 2001, mentioned above). Means. In the present invention, “substantially identical” of sequences means that two base sequences are at least 70% or more, preferably at least 80% or more, more preferably at least 90% or more, more preferably at least 95% or more. It means having sex. Sequence identity can be calculated by the Lipman-Pearson method (Science, 227, 1435, (1985)). Specifically, it is calculated by performing an analysis with Unit size to compare (ktup) set to 2 using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development).

PCR is performed using the primers and the DNA fragments of the plurality of target genes as templates. The progress of steps (1) to (2) of the method of the present invention when PCR is used is schematically shown in FIG. In the first cycle, two PCR reactions proceed. One is primer (L1) and (L2)
A DNA fragment having the same sequence as that of the template DNA fragment and having the 5 ′ end of one strand labeled with either the first label or the second label Is synthesized (reaction 1). Another reaction in the first cycle is an amplification reaction of the template DNA fragment with the primers (A) and (B). Here, the D of one gene among the plurality of target genes.
A fragment in which the terminal region of the DNA fragment of the gene to be fused is extended from the end of the NA fragment, and a fragment in which the terminal region of the one gene is extended from the end of the DNA fragment of the gene to be fused. Synthesized (Reaction 2). Here, the 3 ′ end region of the one strand of the extended fragment of the one gene and the 3 ′ end region of the one strand of the extended fragment of the fused gene are complementary to each other. It is.

In the second cycle of PCR, four different PCR reactions can proceed. Two of them are reactions from a labeled fragment (reaction 1 product) having the same sequence as the template synthesized in the first cycle, and the same DNA fragment as the template and an extended DNA fragment as in the first cycle. And are synthesized. At this time, these synthesized DNA fragments are labeled with the first label or the second label (reactions 3 and 4). The remaining two PCR reactions are reactions from the extended DNA fragment (reaction 2 product) synthesized in the first cycle, one of which is a replication reaction of the extended DNA fragment,
The product of this reaction can be labeled with primers (L1) and (L2) (Reaction 5). The other is a fusion reaction between the extended DNA fragments (reaction 6). Among the extended DNA fragments, the 3 ′ end region of a fragment containing one strand of one of the target genes and the 3 ′ end region of a fragment containing one strand of a gene to be fused therewith Because they are complementary to each other, they can pair with each other. Once paired, DNA synthesis is started from the polymerase, and as a result, a fused DNA fragment of the target gene DNA fragment is synthesized. The product of reaction 6 is unlabeled, but is double labeled by reaction with primers (L1) and (L2) in subsequent PCR cycles.

  In the third cycle of PCR, in addition to the PCR reaction similar to the above reactions 3 to 6, a DNA fusion reaction between the labeled extended DNA fragments generated in reactions 3 and 5 can proceed (reaction 7). This fusion reaction produces a double-labeled fusion DNA fragment.

Two different labels are used as labeling substances for double labeling. One of these is the first label for visualizing the fusion DNA to be detected. The antigen used for the first label is not particularly limited as long as it is stable to PCR and can directly or indirectly visualize the labeled DNA. Examples of the first label include an antigen and a biotin system. The antigen used for labeling is preferably a hapten antigen, and specific examples include digoxigenin, dinitrophenol, and fluorescein. These are targets for affinity substances such as antibodies or avidin (eg, streptavidin) labeled with a visualization substance, for example, colloidal gold, colored latex particles, a fluorescent substance or the like. Another labeling substance is a second label for immobilizing the fusion DNA on the immunochromatographic stationary phase. The second label is selected from those that are stable to PCR and can bind with high affinity to affinity substances such as antibodies and other proteins in an immunochromatographic stationary phase, and are different from the first label. The Specific examples include antigens and biotins that can be used as the first label.

(3. Amplification of double-labeled fusion DNA fragment)
In step (2) of the method of the present invention, the double-labeled fusion DNA fragment obtained in the above step (1) is amplified. Amplification of the double-labeled fusion DNA is performed by PCR using the primers (L1) and (L2). This amplification step may be performed as a separate step after isolating the fusion DNA from the PCR reaction product in the above step (1) according to a conventional method, but preferably as a single step, in the PCR reaction solution in the above step (1) Thus, it is performed in parallel with the step (1).

When performed as one step, the amplification reaction of the fusion DNA fragment in step (2) is performed after the fourth cycle of PCR following the above-described PCR cycle. After the fourth cycle of PCR, the replication reaction of the double-labeled fusion DNA fragment proceeds together with the reactions described in the second and third cycles of PCR (Reaction 8). As primers (L1) and (L2) used in this reaction, those remaining after step (1) may be used, so that it is not necessary to add a new primer. As the PCR cycle progresses, the amount of double-labeled fusion DNA fragments increases, while fragments other than the fusion DNA are also labeled and amplified. However, since only the fusion DNA is double-labeled among them, as described later, it is possible to specifically detect only the fusion DNA by immunochromatography.

(5. PCR conditions)
The PCR reaction conditions in the method of the present invention will be described. Examples of the thermostable DNA polymerase used in the PCR of the present invention include TaKaRa Taq (TaKaRa), TaKaRa Ex Taq (TaKaRa), TaKaRa LA Taq (TaKaRa), TaKaRa Z-Taq (TaKaRa), TaKaRa PrimeSTAR ^™ HS ( Commercially available DNA polymerases such as TaKaRa), AmpriTaq (Perkin-Elmer), AmpriTaqGold (Perkin-Elmer), and AGS Gold (Hybaid) can be used. PCR temperature and time conditions are set according to a standard method based on the length of target DNA, GC content, and the like. The number of PCR cycles can be changed according to the expected amount of gene DNA in the sample, but it may be about 30 to 40 cycles.
About 0 to 35 cycles are more preferable.

As a sample containing a template, as described above, a liquid obtained by suspending a viable bacterial solution, a cultured cell colony, or a clinical sample as it is in sterile water can be used. These suspensions need not be composed solely of target cells. This is because even if multiple types of cells are mixed, only the target DNA can be amplified by the specificity of PCR. If the concentration of the cell suspension is about 1 × 10 ² CFU / μl or more, it can be used in the method of the present invention, preferably about 1 × 10 ³ CFU / μl or more, and about 1 × 10 ⁴ CFU / μl. The above is more preferable. It is also possible to further purify DNA from these samples and use it as a template. The concentration range of purified DNA varies depending on the organism from which the DNA is derived, but is usually 0.01 ng / μl to 10 ng / μl, more preferably 0.1 to 1 ng / μl.

Primers used in PCR in the method of the present invention are the above primers (A) and (B)
Such as those used for fusion of target genes (fusion primer set) and those used for amplification and labeling of fusion DNA such as primers (L1) and (L2) above (labeled primer set) It can be divided roughly. The number of fusion primer sets varies depending on the number of target genes to be detected. That is, the number of fusion primer sets is (number of target genes-1).

The primer concentration may be set in the same manner as in a normal PCR reaction. However, when DNA fusion, labeling and amplification are performed as a single step as described above, the concentration of the labeled primer is set to the same as in the normal PCR reaction (about 0.1 to 0.5 μM), and the concentration of the fusion primer is The concentration of the labeled primer is desirably about 1/10 to 1/2. This is because once the fusion DNA fragment is prepared, the amplification can be performed with a labeled primer, and also to prevent non-specific amplification reaction via the fusion primer. The concentration of each fusion primer set (primers (A) and (B)) should be usually the same, but the length of overlapping sites between the primer and target DNA fragment, the difference in GC content between target DNA fragments, etc. Therefore, if the amplification rate of each DNA fragment and the fusion efficiency of each fusion site are expected to differ, a difference of about 1 to several times in the concentration is made between the primers (A) and (B). be able to.

The primer is designed in consideration of its base length, length, Tm value, GC content, complementarity with the template, and the like. In the case of a fusion primer, the base length of the primer is usually 30 to 35, preferably 35 to 40, more preferably 40 to 45, while in the case of a labeled primer, it is usually 20 to 25, preferably It is 25-30, More preferably, it is 30-35. Preferably, in the fusion primer, the length of the overlapping portion of the extended DNA fragment synthesized in step (1) with another DNA fragment is about 20 to 25, more preferably 25 to 3
It is designed to be about zero. Complementarity with the template does not need to be completely (100%) complementary. For example, it is at least 70% or more, preferably at least 80% or more, more preferably at least 90%, with a completely complementary nucleotide sequence. % Or more, more preferably at least 95% or more. Primer design is well known in the art, and may be performed manually or using a computer program (eg, LASERGENE, PrimerSelect, DNAStar, etc.).

The PCR conditions of the present invention vary depending on the type of sample (culture, clinical specimen, presence or absence of purification, etc.), the amount of target gene DNA in the sample, the length of the target gene, the GC content, and the like. The reaction conditions can be set according to a conventional method (for example, Sambrook et al., 2001 described above), but a preliminary test for searching for optimum conditions can also be performed. In the preliminary test, the conditions under which the target is most efficiently amplified are determined by stepwise changing the template (sample) and primer concentrations or by adjusting the number of cycles about 5 to 10 times. Such a preliminary test is usually performed in the field and can be appropriately performed by those skilled in the art.

  The reaction conditions for carrying out steps (1) to (2) of the present invention in one step are shown in Table 1 below, taking a methicillin-resistant Staphylococcus aureus (MRSA) detection system as an example.

(6. Immunochromatography)
The PCR reaction solution obtained in the above steps (1) to (2) is subjected to immunochromatography to detect the presence or absence of the double-labeled fusion DNA (step (3)). As a procedure for immunochromatography, a known method such as that described in JP-A-2003-194817 can be basically used. That is, by capturing the double-labeled fusion DNA in the PCR reaction solution with an antibody immobilized on a known membrane such as a nitrocellulose membrane, the presence or absence of a plurality of target genes can be determined.

Immunochromatography is performed using immunochromatography strips as conventionally used. The immunochromatography strip is a sample pad (a fibrous inert support capable of absorbing and holding moisture, such as filter paper), a conjugate that is a sample visualization substance holding part closely attached to the sample pad. It has a structure consisting of a gate pad (a support made of a material similar to the sample pad) and a chromatographic development film (such as a nitrocellulose film) in close contact with the conjugation pad (see, for example, FIG. 2). A region (stationary phase) for fixing and detecting the target sample exists ahead of the development membrane in the sample moving direction. The stationary phase may be subjected to a blocking treatment for suppressing nonspecific adsorption. The immunochromatographic strip may be further supported by another inert support such as a plate. The size of the strip, the amount of visualization substance to be retained, the size of the stationary phase, etc. can be appropriately adjusted according to the amount of sample.

More specifically, first, the PCR reaction solution obtained in the steps (1) to (2) is soaked into a sample pad of an immunochromatography strip. The PCR reaction solution can be used as it is without any treatment after PCR. Here, three types of labeled DNA fragments may be present in the PCR reaction solution penetrating the pad. One is a fusion DNA fragment of the target gene, which is double-labeled by the first label and the second label described above. The remaining two are unfused DNA fragments that are bound to only one of the first and second labels. The reaction solution soaked into the sample pad then permeates the conjugation pad. The conjugation pad contains substances (visualization substances) that can bind to the first label, such as antibodies labeled with colloidal gold or colored latex particles, avidin, or streptavidin, where these visualization substances are the first. It binds to the label to form a complex (DNA-visualizer complex). For example, when the first label is an antigen, an antibody binds, and when the first label is a biotin system, avidin or streptavidin binds to form a DNA-visualizing substance complex. At this stage, only two of the three types of labeled DNA fragments, that is, a double-labeled fusion DNA fragment and a non-fusion fragment labeled with the first label, can form a complex with the visualization substance.

The sample that has passed through the conjugation pad permeates the developed membrane in close contact with the pad by capillary action, and starts moving from one end where the permeation has started to the other end. A substance (immobilized substance) that can bind to the second label, such as an antibody of the second labeled antigen, avidin, or streptavidin, is fixed at a position ahead of the sample penetration site in the spreading membrane. A DNA fragment that is adsorbed locally (eg, on a spot or in a line) (stationary phase) and has a second label is captured here. On the other hand, the substance to which the second label is not bound passes here without being fixed to the stationary phase. Only the DNA fragment to which the second label is bound and the fusion DNA-visualizing substance complex to which the second label is bound are immobilized on the stationary phase.

(7. Detection of target gene)
If the fusion DNA of the target gene is present in the PCR reaction, it is visualized via the first label at the conjugation pad of the immunochromatographic strip and then captured to the stationary phase via the second label, resulting in: Deposits densely on the stationary phase. As a result, the stationary phase of the nitrocellulose membrane is dyed red in the case of gold colloid or in that color in the case of colored latex particles. By observing the staining, the presence of the fusion DNA in the PCR reaction can be detected. On the other hand, when no fusion DNA is present in the PCR reaction product, all DNA fragments are labeled only with either the first or second label. If the fragment is labeled only with the first label, it cannot be deposited on the stationary phase even if it is visualized. If the fragment is labeled only with the second label, it is not visualized even if it is deposited on the stationary phase. As a result, the stationary phase is not stained in either case. The stationary phase is stained only when double-labeled fusion DNA is present in the PCR reaction.

The presence of the fusion DNA in the PCR reaction means that the target genes were all present in the sample. Therefore, by observing the staining of the immunochromatographic stationary phase, the presence of a plurality of genes in the sample can be detected simultaneously. Such a method of the present invention using PCR and immunochromatography is referred to as a double-labeled fusion PCR immunochromatography method. The double-labeled fusion PCR immunochromatography method of the present invention is far more rapid in that multiple genes can be detected in a single PCR step compared to the conventional method of individually amplifying DNA of genes. , Less work is required. Compared with the simultaneous detection by the conventional multiplex method, the experimental conditions can be easily set, so that it can be easily applied to various genes. In addition, the method of the present invention is simple in that it is not necessary to separately label a plurality of genes for detection.

  The method of the present invention can be applied to various purposes. For example, by the method of the present invention, individuals possessing a characteristic gene can be easily selected from individuals of the same species. Alternatively, the method of the present invention makes it possible to easily detect the presence of pathogens and disease risk factors caused by a plurality of genes, thereby making it easier to determine the etiology of a patient and the risk of an individual disease. . Specific examples of the application of the present invention include detection of drug-resistant bacteria; detection of multidrug-resistant bacteria; detection of predisposing genes such as cancer and lifestyle-related diseases; detection of genes causing genetic diseases; detection of genes involved in drug metabolism Detection of genes that can cause other treatment resistance; DNA identification such as recognition of related relationships; SNPs screening; expression profiling.

(8. Kit)
A kit for simultaneous detection of multiple genes using the method of the present invention is also provided. The kit of the present invention comprises a fusion primer set (ie, primer (A), (B)) and a labeled primer set (ie, required for fusion, amplification and labeling of a specific combination of multiple genes to be targeted. Primers (L1) and (L2)) and an immunochromatographic strip for detecting the produced double-labeled fusion DNA. The immunochromatography strip includes a conjugation pad containing a visualization substance corresponding to the first label of the labeled primer, and a stationary phase on which an immobilization substance corresponding to the second label is immobilized. The kit of the present invention may also contain a manual for determining the presence or absence of the target gene from the result of immunochromatography (the staining state of the immobilized phase). The kit of the present invention may further contain other reagents for PCR reaction (polymerase, PCR buffer, etc.).

Examples of the present invention using a double-label fusion PCR immunochromatography method will be described below, but the present invention is not limited to these examples.

(1. Reagents and materials)
1-1) Preparation of colloidal gold-labeled avidin solution All reagents such as salts, surfactants, pH buffering agents, etc. are of special grade or biochemical grade, and were purchased from Wako Pure Chemicals, Nacalai Tesque, Katayama Chemical. Bovine serum albumin for blocking protein is albumin from bovine serum, ≧ 97% agarose gel electrophoresis (SIGMA, A7511-5G), and gold colloid for marker is gold colloid (British BioCell International Ltd.
, 30 nm, EMGC30).
1-2) Preparation of immunochromatographic strip Chromatographic strip is prepared using Membrane Test Platforms Immunopore FP, Absorbent 31ET (Whatman), and detection antibody is Anti-Digoxigenin [sheep] (Roche, 11 333 089 001). It was.
1-3) PCR
For DNA amplification, PrimeSTAR ^™ HS DNA Polymerase (TaKaRa) and PCR thermal cycler (HYBAID, PCR Express) were used. All oligonucleotide primers used were outsourced to Hokkaido System Science. The sequence is shown in Table 2.

1-4) Agarose gel electrophoresis Agarose, pH buffering agents, dyes and the like for electrophoresis are all of the special grade or biochemical / molecular biology grade, and were purchased from Wako Pure Chemical, Kanto Chemical, Nacalai Tesque, etc.
1-5) Bacterial culture For staphylococcal culture, tryptone, yeast extract (Nacalai Tesque), sodium chloride,
Using LB agar prepared with agar (Wako Pure Chemical Industries), it was aerobically cultured at 37 ° C.
1-6) The strains used were as follows.
S. aureus (methicillin resistant strain, MRSA); BCL5, BCL6, BCL7, BCL8, BCL9, BCL10, BCL11, BCL12, BCL13, BCL14, TY4, TY24, TY26, TY29, TY30, TY34, TY36, TY38, TY41, TY42 , TY45, TY47, TY49, TY54, HP1, HP2, HP3, HP4, HP17, HP19, HP20, HP21, HP47, HP48, HP125, HP233, HP271, HP395, HP397
S. aureus (methicillin sensitive strain, MSSA); TY25, TY27, TY28, TY31, TY32, TY33, TY35, TY37, TY39, TY40, HP8, HP15, HP16, HP22, HP23, HP28, HP42, HP43, HP44
P. aeruginosa ; PAO1
S. enteritidis ； IFO3316
S. Typhimurium; χ3306 strain
B. cereus ； IFO3001
B. subtilis ； ATCC6633
M. luteus ; IFO12708
A. xylosoxydans; S-6,8 (I -) resistant
K. pneumoniae ； ATCC4332
E. coli ; HME3, IFO12713, JM109
S. epidermidis ^T ； GTC289
S. intermedius ^T ; NCDO2227.

(2. Method)
2-1) Preparation of gold colloid labeled avidin solution Gold colloid labeled avidin solution was prepared in the following manner. All glassware used (beakers, stirrer chips, etc.) was treated with aqua regia, then washed and dried with ultrapure water. A 0.2 mg / ml avidin solution was prepared with 2 mM sodium tetraborate buffer [pH 9.0] and dialyzed against 2 L of the same buffer. Next, a 30 nm gold colloid solution was adjusted to pH 9.0 with 200 mM NaHCO ₃ . Eppendorf tube with 2 mM Borax 30, 28, 26, 24, 22, 20, 18, 16, 14, 12 μl, and 0.2 mg / ml
The avidin solution was added with 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 μl, and further 200 μl of colloidal gold, and the total amount of each 10 was 230 μl. After 5 minutes, 20% 10% NaCl in each tube
Preliminary titration was performed by adding μl each and letting it stand for 1 minute to observe color change. Since the tube which does not change from red to blue is the tube containing the minimum amount of avidin, the amount of avidin per 1 ml of colloidal gold was calculated based on this, and the amount of avidin solution actually used was determined. After adding 130 μl of Borax to the avidin solution at the dose determined by the pre-titration, add 1 ml of colloidal gold solution while stirring and let stand for 1 hour, and then PBS (137 mM NaCl, 8.1 mM Na ₂ HPO ₄ · 12H ₂ O 100% of 10% bovine serum albumin (BSA) dissolved in 2.68 mM KCl, 1.47 mM KH ₂ PO ₄ ) was added and stirred again for 10 minutes. After centrifugation (8000 × rpm, 30min, 4 ℃) with a centrifuge (micro high speed cooling centrifuge MX-100, TOMY), the precipitate contains Buffer A (1% BSA, 0.1% sodium azide, 0.1% Tween20) It was resuspended in 20 mM Tris-HCl [pH 8.2]) and used for the following experiment.

2-2) Preparation of immunochromatography strip The immunochromatography strip was prepared as follows. An immunochromatographic strip was prepared using Membrane Test Platforms Immunopore FP in which a nitrocellulose membrane was bonded to the center of the polymer support carrier as a chromatographic developing membrane and Absorbent 31ET, a cellulose material (FIG. 2). Membrane Test Platforms Immunopore FP was cut to a width of 2 mm, and then adsorbed 31ET having a width of 2 mm was adhered to the upper end of the adhered nitrocellulose development membrane (including the stationary phase) so as to overlap by 1 to 2 mm (water absorbing portion). The stationary phase (detection spot) was blotted with 1 μl of 0.2 mg protein / ml anti-digoxigenin antibody solution in dots and incubated at 37 ° C. for 10 minutes. A desiccator with Membrane Test Platforms Immunopore FP, which was uniformly dropped onto 2 mm x 5 mm Absorbent 31ET, and 15 μl of the colloidal gold-labeled avidin solution prepared by “Preparation of colloidal gold-labeled avidin solution” in Section 2-1). 1mm at the bottom of the nitrocellulose membrane
They were adhered so as to overlap (conjugation pad; gold colloid-labeled avidin holding part). Further, Absorbent 31ET having a width of 2 mm was adhered to the lower end of the colloidal gold labeled avidin portion so as to overlap 1 mm (sample pad; sample absorbing portion). The immunochromatographic strip was sealed in a nylon bag with silica gel and stored at 4 ° C. until use.

2-3) Double-label fusion PCR
2-3-1) Detection system for methicillin-resistant Staphylococcus aureus (MRSA) A gene having a sequence specific to Staphylococcus aureus (ferredoxin-dependent glutamate synthase; Ref. Martineau F. , Picard FJ, Roy PH, Ouellette M., Bergeron MG (1998) J. Clin. Microbiol., 36, 618-623) as a species-specific marker, Sa1406-f Bw (fusion primer Bw) was used. With this primer set, an amplified fragment of 268 bp is obtained. Further, in order to specifically amplify mecA , which is a methicillin resistance gene sequence, the primer sets of Table 1: mecA-f Fw (fusion primer Fw) and mecA1448Bw were used. With this primer set, an amplified fragment of 397 bp can be obtained. In order to fuse these two amplified fragments, Sa1406-f Bw (fusion primer Bw) and mecA-f Fw (fusion primer Fw) are designed as a “fusion primer set” having an overlapping portion of about 20 bp. Due to this structure, the fused fragment exhibits 645 bp. In order to detect the marker gene product with an immunochromatography strip, Sa442-2Fw and mecA1448Bw were labeled with digoxigenin and biotin, respectively, and used for the subsequent PCR reaction.

2-3-2) Specificity test
PCR volume is 50 μl / tube system (17.6 μl of purified water, 5 x PrimeSTAR ^TM Buffer (Mg ²⁺ plus)
10 μl, 2.5 mM dNTP Mixture 5 μl, 5 μM fusion primer Fw 1 μl, 1 μM fusion primer Bw 4 μl, 10 μM Bio / mecA1448Bw 1 μl, 10 μM Dig / Sa442-2Fw 1 μl, TaKaRa PrimeSTAR ^TM HS DNA Polymerase 0.4 μl, Use 10 μl of the live bacterial solution of the test bacterium as a template (sterile water containing 1 × 10 ⁵ CFU), or half of it in a 25 μl / tube system [the same enzyme amount is used, and the other final composition concentrations are the same] It was. The reaction was first treated at 95 ° C. for 10 minutes, and then 3 cycles of 95 ° C. for 5 seconds, 58 ° C. for 5 seconds and 72 ° C. for 20 seconds were repeated 35 cycles.

2-3-3) Sensitivity test
PCR 50 μl / tube system (25.6 μl of purified water, 10 μl of 5 × PrimeSTAR ^TM Buffer (Mg ²⁺ plus), 5 μl of 2.5 mM dNTP Mixture, 1 μl of 5 μM fusion primer Fw, 1 μM fusion primer
4 μl of Bw, 1 μl of 10 μM Bio / mecA1448Bw, 1 μl of 10 μM Dig / Sa442-2Fw, 0.4 μl of TaKaRa PrimeSTAR ^™ HS DNA Polymerase, live bacterial solution containing various numbers of bacteria (2 x 10 ² to 2 x 10 ⁸ ) (Template solution) 2 μl]. The PCR conditions were the same as in the specificity test, and only the cycle number was changed in PCR using the cycle number as a variable.

2-4) Agarose gel electrophoresis
Confirmation of the amplified fragment by PCR was performed by agarose gel electrophoresis using a minigel electrophoresis tank (Mupid, Cosmo Bio). Agarose was mixed with electrophoresis buffer (TBE; containing 44.5 mM Tris, 44.5 mM boric acid, 1.0 mM EDTA) so as to be 2%, heated and melted, and then solidified in a gel maker. A sample for electrophoresis was prepared by mixing 3 μl of gel loading buffer (0.04% BPB, 40% glycerol, 5 mM EDTA) per 1 μl of DNA solution. As a marker, 100 bp DNA Ladder (BioLabs or TaKaRa) was used. Use TBE as the running buffer, run at a constant voltage of 100V, soak the gel after soaking in 1mg / ml ethidium bromide, and use UV irradiation imaging equipment (AE-6915, ATTO). The band was confirmed and photographed.

2-5) Development and detection on immunochromatography strip The immunochromatography strip consists of four parts: a sample absorption part, a colloidal gold-labeled avidin holding part, a detection part on a nitrocellulose film coated with an anti-digoxigenin antibody, and a water absorption part. (FIG. 2). The sample absorption part is a part that is directly immersed in a reaction solution containing the target gene fragment amplified by PCR using a primer labeled with digoxigenin and biotin. Here, the debris in the reaction solution is filtered to obtain a solution phase. Only the capillarity is sucked up. In the gold colloid avidin holding part, the solution prepared according to the above item 2-1) (“Preparation of gold colloid labeled avidin solution”) is applied and dried, and the avidin labeled with gold colloid is dissolved in the reaction solution. The biotin moiety of the double-labeled amplified fragment that has risen binds to form a complex, and further penetrates into the deployment membrane. Anti-digoxigenin antibody is applied and immobilized on the spreading membrane. If the target fusion amplification product labeled with biotin and digoxigenin at both ends is present in the reaction solution, the amplification product binds to the antibody immobilized on this detection section. To do. As a result, the colloidal gold is deposited on the detection part via the amplification product, and the spot is colored red. This coloring is visually confirmed to determine a positive reaction. The development of the immunochromato strip used this time shown in FIG. 2 is completed if there is a reaction solution of 25 μl or more.

(3. Results and discussion)
(Detection and sensitivity of MRSA by double-label fusion PCR immunochromatography)
1) Specificity
Fusion of a part of the chromosomal region specifically possessed by S. aureus (sequence specific to S. aureus of ferredoxin-dependent glutamate synthase gene), the methicillin resistance gene ( mecA ) possessed by MRSA, and fragments thereof MRSA clinical isolates 29 as test strains using `` fusion primer set '' Dig / Sa442-2Fw and Sa 1406-f Bw (fusion primer Bw) and Bio / mecA 1448 Bw and mecA-f Fw (fusion primer Fw) PCR was performed using 11 strains listed in Section 1-6) (“Test Bacteria”) as well as 19 MSSA clinical isolates. 3 and 4 show the results of confirming the species specificity, and FIGS. 5 and 6 show typical amplification results in 10 strains of MRSA and MSSA. As is apparent from each figure, only the S. aureus fdgs gene ( safdgs ) was amplified in MSSA, and a safdgs and mecA gene and a 645 bp PCR product fused with them were confirmed by electrophoresis in MRSA ( 3A, 4A, 5A, 6A). Therefore, as a result of developing 25 μl of the PCR reaction solution on the immunochromatographic strip, a positive reaction was confirmed only for MRSA (FIGS. 3B, 4B, 5B, 6B). In addition, we examined the specificity of MRSA test specimens with several representative fungi that could be mixed from the environment. As shown in Figs. was detected.
From these results, it was considered that the method of the present invention had sufficient specificity to perform MRSA examination of clinical specimens and environmental specimens.

2) Detection sensitivity
When PCR was performed with the above primer set using MRSA BCL6 strain, the number of cycles was 30.
In the case of the cycle, detection was possible if there were 2 × 10 ⁵ CFU or more cells in the PCR reaction solution, and in the case of 35 cycles, it was confirmed by electrophoresis with 2 × 10 ⁴ CFU cells or more ( 7A, 8A). Therefore, when the PCR product was developed on an immunochromatographic strip, a positive reaction was confirmed in the sample in which the amplified fragment was confirmed by electrophoresis (FIGS. 7B and 8B). In addition, the sensitivity increased as the number of cycles was increased (FIG. 9), but 30 to 35 cycles seem to be desirable considering non-specific amplification.
From the above results, it was confirmed that the method of the present invention has a detection sensitivity equivalent to the fluorescence detection after electrophoresis used conventionally.

3) Immunochromato strip sensitivity
After electrophoresis of the PCR reaction solution, only a PCR fragment (645 bp) fused with a part of S. aureus fdgs and MRSA mecA was excised and purified, and its dilution series was prepared and developed on an immunochromatographic strip. Positive spots were confirmed in the concentration range of 0.1 μg / ml or more. Since strip development was performed with 25 μl of PCR solution, it could actually be detected with 1.25-2.5 ng of MRSA PCR product (data not shown). This was an amount corresponding to DNA that would be amplified when 31 to 32 cycles of ideal PCR were performed when one MRSA cell was present in the PCR reaction solution. Therefore, in the future, it was speculated that if the reaction efficiency of the double-labeled fusion PCR was further improved, even the strip used this time could be measured with higher sensitivity.
From these results, it is considered that the present invention can be universally and sufficiently applied not only to the MRSA test system shown here but also to simple screening of organisms having various complex inheritances.

Schematic diagram of steps (1) to (2) of the method of the present invention Basic structure of the immunochromatography strip of the present invention (applicable to 25 μl PCR) Detection of PCR products by the method of the present invention. A: Detection of safdgs , mecA and fusion gene fragments by agarose gel electrophoresis. B: MRSA detection result by the method of the present invention. Lane 1: MRSA BCL6 strain, Lane 2: MSSA HP23 strain, Lane 3: S. intermedius NCDO2227 strain, Lane 4: Sterile water (background), Lane M: DNA100 bp ladder marker. Specific detection of MRSA by the method of the present invention. A: Detection of safdgs , mecA and fusion gene fragments by agarose gel electrophoresis. B: Detection of safdgs , mecA and fusion gene fragments by the method of the present invention. Lane 1; S. aureus (methicillin resistant) BCL6 strain, lane 2; S. aureus (methicillin sensitive) HP23 strain, lane 3: S. enteritidis IFO3316 strain, lane 4: S. Typhimurium χ3306 strain, lane 5: E. coli HME3 strain, lane 6: A. xylosoxydans S-6,8 (I-) resistant strain, lane 7: B. cereus IFO3001 strain, lane 8: B. subtilis ATCC6633 strain, lane 9: K. pneumoniae ATCC4332 strain, lane 10 : M. luteus IFO12708 strain, lane 11: P. aeruginosa PAO1 strain, lane 12: S. epidermidis type strain, lane 13: E. coli JM109 strain, lane 14: purified water. Detection of MRSA clinical strains by the method of the present invention. A: Detection of safdgs , mecA and fusion gene fragments by agarose gel electrophoresis. B: Detection of MRSA clinical strain by the method of the present invention. From left to right, markers (A only), BCL5, BCL6, BCL7, BCL8, BCL9, BCL10, BCL11, BCL12, BCL13, BCL14 strain. Detection of MSSA clinical strains by the method of the present invention. A: Detection of safdgs , mecA and fusion gene fragments by agarose gel electrophoresis. B: Detection of MSSA clinical strain by the method of the present invention. From left to right, markers (A only), TY25, TY27, TY28, TY31, TY32, TY33, TY34, TY37, TY39, and TY40 strains. MRSA detection sensitivity according to the method of the present invention (30 cycles PCR). A: Detection of safdgs , mecA and fusion gene fragments by agarose gel electrophoresis. B: Detection of MRSA clinical strain by the method of the present invention. Lane 1: 2 × 10 ³ CFU, Lane 2: 2 × 10 ⁴ CFU, Lane 3: 2 × 10 ⁵ CFU, Lane 4: 2 × 10 ⁶ CFU, Lane 5: 2 × 10 ⁷ CFU (using MRSA BCL6 strain) . MRSA detection sensitivity according to the method of the present invention (35 cycles PCR). A: Detection of safdgs , mecA and fusion gene fragments by agarose gel electrophoresis. B: Detection of MRSA clinical strain by the method of the present invention. Lane 1: 2 × 10 ³ CFU, Lane 2: 2 × 10 ⁴ CFU, Lane 3: 2 × 10 ⁵ CFU, Lane 4: 2 × 10 ⁶ CFU, Lane 5: 2 × 10 ⁷ CFU (using MRSA BCL6 strain) . MRSA detection sensitivity according to the method of the present invention (40 cycles PCR). A: Detection of safdgs , mecA and fusion gene fragments by agarose gel electrophoresis. B: Detection of MRSA clinical strain by the method of the present invention. Lane 1: 2 × 10 ² CFU, Lane 2: 2 × 10 ³ CFU, Lane 3: 2 × 10 ⁴ CFU, Lane 4: 2 × 10 ⁵ CFU, Lane 5: 2 × 10 ⁶ CFU, Lane 6: 2 × 10 ⁷ CFU, Lane 7: 2 × 10 ⁸ CFU (using MRSA BCL6 strain).

Claims (5)

The following steps:
(1) A step of fusing DNA fragments of a plurality of genes to produce one fusion DNA fragment and double-labeling the fusion DNA fragment with a first label and a second label different from the first label;
(2) amplifying the double-labeled fusion DNA fragment;
(3) subjecting the amplified double-labeled fusion DNA fragment to immunochromatography;
(4) observing the stationary phase of the immunochromatograph to determine the presence or absence of DNA fragments of the plurality of genes,
A method for simultaneously detecting a plurality of genes in a sample, comprising: Preparation of the fusion DNA fragment and double labeling
A sequence complementary to the sequence of the 3 ′ end of one strand of the DNA fragment of one gene of the plurality of genes, and one strand of the DNA fragment of the gene fused to the DNA fragment of the one gene A primer (A) having a sequence substantially identical to the sequence at the 3 ′ end;
A sequence complementary to the sequence of the 3 ′ end of one strand of the DNA fragment of the gene to be fused, and a sequence substantially identical to the sequence of the 3 ′ end of one strand of the DNA fragment of the one gene A primer (B) having
A primer (L1) having a sequence complementary to the sequence at the 3 ′ end of the other strand of the fusion DNA fragment and labeled at the 5 ′ end with the first label; and the other of the fusion DNA fragment A primer (L2) having a sequence complementary to the sequence at the 3 ′ end of the strand and labeled at the 5 ′ end with the second label,
The method according to claim 1, wherein the amplification reaction of the double-labeled fusion DNA fragment is carried out in parallel with each other. The immunochromatography visualizes the fusion DNA via the first label on the double-labeled fusion DNA, and immobilizes the fusion DNA to the stationary phase of the immunochromatograph via the second label. A method according to claim 1 or 2, characterized. The method according to any one of claims 1 to 3, wherein the plurality of genes are a ferredoxin-dependent glutamate synthase gene of Staphylococcus aureus and a methicillin resistance gene mecA .   Means for immobilizing the primers (A), (B), (L1) and (L2) according to claim 2 and the first and second labels according to claim 1 to the stationary phase of the visualization and immunochromatograph, respectively. A kit for simultaneously detecting a plurality of genes in a sample.