JP2011135799A - Method of detecting and/or identifying biopolymer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a detection technology which solves problems in a method of detecting and/or identifying a biopolymer, is quick, simple and inexpensive, further can easily achieve high throughput and is capable of processing numerous samples. <P>SOLUTION: The method of detecting and/or identifying a target biopolymer in a sample includes the following: (1) the step of causing a probe having a molecular weight smaller than that of the target biopolymer and specifically coupling with the target biopolymer to specifically react with the target biopolymer included in the sample to form a complex; (2) the step of separating the complex from the probe by molecular cutoff means; (3) the step of collecting the separated complex; and (4) the step of detecting the probe included in the collected complex. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

The present invention relates to a method for detecting and / or identifying biopolymers such as nucleic acids (polynucleotides) and proteins, and in particular, a probe that specifically binds to the biopolymer using the principle of molecular weight fractionation. The present invention relates to a method for specifically detecting and / or identifying a nucleic acid in a specimen quickly and easily by the difference in the molecular weight of the target nucleic acid.

Detection and / or identification of biopolymers is an important technique in the medical community, industry, and the like, and establishment of an inexpensive, quick and simple technique is required. In the case of nucleic acid, the base sequence is decoded to detect and identify microorganisms, but usually it takes about 2 days.

In addition to this, a method for detecting, identifying, and quantifying microorganisms based on the presence or absence of amplification, the amount of amplification, or the difference in the molecular weight of the amplification product by PCR or real-time PCR using probes or primers specific to the target microorganism or gene marker (Non-Patent Document 1, Non-Patent Document 2). In addition, as a quick and simple method that does not involve PCR, RNaseH can be used to hybridize rRNA and probe, and then add RNaseH to cleave RNA in a sequence-specific manner to specifically detect, identify, and quantify microorganisms. There are sequence-specific cleavage methods (Non-patent Document 3 and Patent Document 1).

Furthermore, in Patent Document 2, a complex that can form a complex with a binding nucleic acid and is a polymer higher than the nucleic acid is formed in a solution containing the binding nucleic acid and the non-binding nucleic acid. A method is described in which such a substance (specifically, β-1,3-glucan) is added to form a polymer complex, and the binding nucleic acid complex is separated by a molecular weight fractionation filter.

JP 2005-065535 A JP 2005-112828 JP

Song et al. ,, FEMS Lett, vol. 187, 167-173, (2000) Fanget al., JCM, vol. 40, No. 1, 287-291 (2002) Uyenoet al., AEM, vol. 70, No. 6, 3650-3663 (2004)

The above conventional method using PCR is highly sensitive, but requires two or more kinds of primers or probes, and requires a great amount of labor for setting experimental conditions. Furthermore, it is known that the quantification result does not match the abundance in the sample due to PCR bias (due to the fact that the amplification efficiency is not constant, etc.).

In addition, the conventional RNaseH sequence-specific cleavage method does not require an external standard and is quick and simple. However, since a single base mismatch cannot be identified, the probe design conditions become very severe. There is also a problem that it is difficult to target nucleic acids other than rRNA.

The present invention solves such problems in conventional detection and / or identification methods for biopolymers, is rapid, simple and inexpensive, and can easily achieve high throughput, and can handle multiple samples. It is an object of the present invention to provide a detection technique that makes it possible.

As a result of research to solve the above problems, the present inventor uses molecular weight fractionation means based on the difference in molecular weight between a biopolymer such as a nucleic acid and a protein and a probe that specifically binds to the biopolymer. Thus, the present inventors have found that a biopolymer in a specimen can be detected and / or identified quickly and easily, and the present invention has been completed.

That is, the present invention relates to each aspect shown below.
[Aspect 1]
A method for detecting and / or identifying a target biopolymer in an analyte comprising the following steps:
(1) a step of forming a complex by specifically reacting a probe having a molecular weight smaller than that of a target biopolymer and specifically binding to the target biopolymer with the target biopolymer contained in a specimen;
(2) separating the complex from the probe by molecular weight fractionation means;
(3) a step of recovering the separated complex, and (4) a step of detecting the probe contained in the recovered complex.
[Aspect 2]
The separation in the step (3) allows the reaction liquid containing the target biopolymer, the probe, and the complex to pass through a molecular weight fractionation membrane that is a molecular weight fractionation means, and the target biopolymer and the complex. The method according to embodiment 1, which is carried out by capturing at least a part of the molecular weight on the molecular weight fractionation membrane.
[Aspect 3]
A method according to embodiment 2, wherein the molecular weight cutoff membrane has a molecular weight cut-off of at least 50,000 MW.
[Aspect 4]
The method according to any one of aspects 1 to 3, wherein the target biopolymer is a polynucleotide.
[Aspect 5]
The method according to embodiment 4, wherein the polynucleotide is DNA or RNA.
[Aspect 6]
The method of embodiment 5, wherein the DNA is a PCR product.
[Aspect 7]
The method according to any one of Embodiments 1 to 6, wherein the molecular weight of the probe is 3,000 to 20,000.
[Aspect 8]
The method according to embodiment 7, wherein the probe is an oligonucleotide having a base length of 10 to 50 bp.
[Aspect 9]
The method according to any one of aspects 1 to 6, wherein the probe is labeled.
[Aspect 10]
The method of embodiment 9, wherein the probe is labeled with a fluorescent material.
[Aspect 11]
A kit used for carrying out the method according to any one of embodiments 1 to 10, wherein the probe has a molecular weight smaller than that of the target biopolymer and specifically binds to the target biopolymer, and a molecular weight fraction The kit comprising a drawing means.

According to the method of the present invention, the target biopolymer can be detected and / or identified in an extremely short time of about 1 hour. Furthermore, the use of a molecular weight fractionation film, which is an inexpensive molecular weight fractionation means, makes it possible to make the cost required for detection very low compared to other detection techniques. Furthermore, since it is possible to easily increase the throughput, multi-sample processing is possible.

An outline of an example of the method of the present invention is shown. The method of this invention using a molecular weight fractionation membrane is shown. In Example 3, the result of capillary electrophoresis according to the applied 16S rRNA gene sample (PCR product) is shown. In Example 4, the result of capillary electrophoresis for each applied 16S rRNA sample (artificial synthetic RNA) is shown.

The method for detecting and / or identifying a target biopolymer in a specimen according to the present invention includes the following steps.
(1) a step of forming a complex by specifically reacting a probe having a molecular weight smaller than that of a target biopolymer and specifically binding to the target biopolymer with the target biopolymer contained in a specimen;
(2) separating the complex from the probe by molecular weight fractionation means;
(3) a step of recovering the separated complex, and (4) a step of detecting the probe contained in the recovered complex.

In this specification, there is no restriction | limiting in particular in the structure of "biopolymer" used as the object of a detection and / or identification, For example, various polynucleotides (nucleic acid), protein (polyester), such as DNA, RNA, and those various modifications Peptide), lipoprotein, glycoprotein, glycolipid, polysaccharide, and complexes thereof. Accordingly, there is no particular limitation on the molecular weight of the biopolymer, and for example, it can range from several tens of thousands to several millions. Detection can be performed as qualitative, semi-quantitative and quantitative measurements.

Biopolymers are derived from materials originally contained in broadly defined organisms such as humans, animals, plants, microorganisms and viruses, as well as genetic engineering means such as DNA that is a PCR product and chemical synthesis. Also includes artificially prepared materials.

“Sample” means any sample containing a biopolymer to be detected and / or identified, such as various body fluids or extracts such as blood and urine collected from various biological species, Further, it means various samples containing microorganisms collected from natural environments such as soil, river water and seawater, and samples containing biopolymers prepared artificially.

As the molecular weight fractionating means used in step (2) of the method of the present invention, any appropriate means known to those skilled in the art can be used as long as the complex and the probe can be effectively separated based on the difference in molecular weight. Can be used. That is, at least a part of the target biopolymer and / or its complex with the probe, preferably about 50% or more, more preferably 95% or more is different from the probe (for example, “B / F separation”). It is desirable to separate them as B (Bound) fractions).

Representative examples of molecular weight fractionating means include various molecular weight fractionated membranes such as ultrafiltration membranes. The fractional molecular weight of the molecular weight fractionation membrane is appropriately determined by those skilled in the art according to the type and size of the biopolymer to be detected and / or identified, the type and size of the probe, and the operating conditions such as separation. You can choose. Usually, about 50,000 MW or more is suitable.

On the other hand, depending on the type and size of the probe and the target biopolymer, those skilled in the art can appropriately select a compound that specifically reacts therewith, and the type and structure are not particularly limited. When the target biopolymer is a polynucleotide, a probe comprising an oligonucleotide is preferred. Even when the target biopolymer is a protein, an oligonucleotide probe can be used as an “aptamer”.

The probes have a significantly smaller molecular weight relative to them, such that the probes can be separated from the target biopolymer or a complex of the target biopolymer and the probe based on molecular weight differences. For example, the molecular weight of the probe is usually preferably about 3,000 to 20,000. When the probe is an oligonucleotide, it is usually preferable that the probe has a base length of about 10 to 50 bp.

In order to sufficiently exert the effects of the present invention, the molecular weight fractionating means and the molecular weight of the probe are selected in an appropriate combination, so that preferably 90% or more, more preferably 99.5% or more of the probe is targeted. It is desirable that the complex of the biopolymer and the probe is separated as a separate fraction (for example, “F (Free) fraction in B / F separation”).

For example, when Millipore Microcon YM-100 is used as a molecular weight fractionation means, the membrane can recover only 1% of a substance having a molecular weight of about 10,000, whereas a substance having a molecular weight of about 150,000 is about 95% or more. Can be collected (Millipore's public data). Thus, for example, when 16S rRNA (molecular weight of about 500,000) is to be detected as the target biopolymer, it is preferable to first use a fluorescently labeled gene probe (molecular weight of about 7,000) of about 20 bases. A probe is reacted (hybridized / hybridized) with the RNA in a solution, and then applied to Microcon YM-100, which is a kind of molecular weight fractionation membrane. Most of the probes permeate YM-100 and are hardly recovered by YM-100, whereas RNA has a molecular weight larger than the fractional molecular weight of YM-100. Since the probe hybridized with the RNA binds to the RNA to form a complex, it is trapped (captured) together with the RNA on the molecular weight fractionation membrane.

In order to detect the probe contained in the complex, the probe is preferably labeled with any substance known to those skilled in the art. Examples of such labeling substances include radiolabels such as ³² P, cyanines such as Cy2, Cy3 and Cy5, fluorescent substances represented by rhodamine dyes such as fluorescein (FITC) and tetramethylrhodamine (TRITC), and , Chemiluminescent substances, etc. can be raised. The probe can be labeled by any method known to those skilled in the art.

The recovery of the separated complex in the step (3) and the detection of the probe contained in the recovered complex in the step (4) are performed by repairing the target biopolymer, the probe, the molecular weight fractionation means, etc. Depending on the properties and the like, it can be carried out by any method known to those skilled in the art.

  A feature of the method of the present invention is that the molecular weight fractionation separates them quickly and easily based on the difference between the molecular weight of the target biopolymer and the molecular weight of the probe. An outline of an example of the method of the present invention is shown in FIG. Furthermore, as a more specific example, the method of the present invention (FIG. 2) using a molecular weight fractionation membrane is outlined below.

Step (1):
Prior to carrying out step (1), a specimen containing the target biopolymer is appropriately subjected to appropriate pretreatments known to those skilled in the art, such as grinding, crushing, dissolution and extraction operations, depending on the kind of specimen. Can be prepared in a form suitable for the subsequent reaction, for example, as a solution containing the target biopolymer. Similarly, the probe to be used is usually preferably prepared as a solution with an appropriate concentration using an appropriate buffer or the like.

There is no particular limitation on the type of specific reaction between the target biopolymer and the probe, and various reaction modes can be taken according to their structure and properties. For example, when the target biopolymer is a polynucleotide and the probe is an oligonucleotide, specific binding occurs between them by a reaction called a hybridization reaction.
The above specific reaction can be carried out under reaction conditions such as an appropriate temperature and time depending on the type and amount of the target biopolymer and the probe, the type of reaction system, and the like. For example, in the case of a hybridization reaction, it is usually preferable to react at 30 to 60 ° C. for about 2 to 10 minutes.

Step (2):
In order to separate the complex of the target biopolymer and the probe from the probe, for example, the reaction solution that has been reacted in the step (1) is applied on the molecular weight fractionation membrane and subjected to appropriate conditions such as centrifugation or suction filtration. The membrane is obtained by allowing the reaction solution to permeate by, for example, dropping a suitable washing solution onto the molecular weight fractionation membrane as necessary, and allowing the washing solution to permeate under suitable conditions such as centrifugation or suction filtration. By washing, the complex of the target biopolymer and the probe can be effectively separated from the probe based on B / F separation.

Step (3):
The complex trapped on the molecular weight fractionation membrane can be recovered as an appropriate solution, for example, by subjecting the molecular weight fractionation membrane treated in the step (2) to reverse centrifugation under suitable conditions. .

Step (4):
The probe contained in the complex recovered in the step (3) may be any suitable detection device known to those skilled in the art, such as a fluorescence spectrophotometer, depending on its properties, for example, the characteristics of the labeling substance. It can be easily detected by using means. Prior to detection, the complex can be detected after being separated by an appropriate separation means such as electrophoresis.

The kit used for carrying out the above-described method of the present invention includes a probe having a molecular weight smaller than that of the target biopolymer and specifically binding to the target biopolymer, and a molecular weight fractionating means. Furthermore, depending on the purpose of use, for example, any appropriate probe detection device / means known to those skilled in the art, buffer solution, vial, and other necessary other substances / instruments / devices are included as appropriate. I can do it.

  The present invention will be described in detail with reference to the following examples, which are merely provided for explaining the present invention. Accordingly, these examples do not limit or limit the scope of the invention disclosed herein. It is easily understood by those skilled in the art that various embodiments based on the technical idea described in the claims can be made in the present invention.

1. Evaluation of permeation performance of oligonucleotide probe by molecular weight fractionation membrane used 1.1. Outline of Experiment Dropping fluorescently labeled oligonucleotide probes with different base lengths onto a molecular weight fractionation membrane (YM series), centrifuging at 14,000 g, inverting the molecular weight fractionation membrane, and then centrifuging to reverse the molecular weight The probe trapped on the membrane was collected.

1.2. Experimental Method 1.2.1 Experimental Procedures ・ Fluorescently labeled oligonucleotide probe (Table 1) was adjusted to about 1000 fmol / μL (1000 nM).
・ 100 μL of the probe solution was dropped onto the molecular weight fractionation membrane (YM series), and the molecular weight fractionation membrane unit was centrifuged at 14,000 × g (YM-3: 20 min, YM-10: 6 min, YM-30, 50, 100: 3min).
-The filtrate was discarded, and Milli Q was dropped onto a 500 μL molecular weight fractionation membrane and centrifuged at 14,000 × g (YM-10: 30 min, YM-30, 50, 100: 12 min) (washing).
・ Invert the molecular weight fractionation membrane, drop 10 μL of Milli Q, and centrifuge at 1,000 × g (3 min) (reverse centrifugation)

1.2.2 Measurement and Evaluation Method The fluorescence-labeled oligonucleotide probe solution adjusted to about 1000 nM and the solution collected after reverse centrifugation were measured with a fluorescence spectrometer (NanoDrop ND-3300). The detection sensitivity of ND-3300 is 5 fmol (2.5 nM) when 2 μL of sample is used, and the detection range is 2.5 to 3000 nM.
Assuming that 100 μL of the probe solution passes through the molecular weight fractionation membrane and that the probe remaining on the molecular weight fractionation membrane is recovered in 10 μL of the solution after reverse centrifugation and concentrated 10 times, the molecular weight fractionation membrane permeability of the probe (%) Was evaluated by the following formula.

1.3. Summary of results Table 2 summarizes the transmittance of the molecular weight fractionated membranes (YM series) of the oligonucleotide probes classified by base length in the molecular weight fractionated membranes (YM series). In the molecular weight fractionation membrane YM-100, the transmittance of probes having base lengths of 10, 18, and 25 was 99.5% or more.
Moreover, the transmittance | permeability of 99.5% or more was similarly obtained also in the system which contained 0-1,000 mM NaCl and 0-6M urea in 1000nM Eco327-18.

2. Evaluation of YM-100 permeation performance of PCR products or artificially synthesized RNA 2.1. Outline of Experiment Drop PCR products or artificially synthesized RNAs with different base lengths onto a molecular weight fractionation membrane (YM-100), perform centrifugation at 3,000 g, invert the molecular weight fractionation membrane, and reverse-centrifuge to obtain a molecular weight The PCR product or artificially synthesized RNA trapped on the fractionation membrane was recovered.

2.2. Experimental method 2.2.1 Experimental procedure (PCR product)
1) PCR products with different base lengths were obtained using genomic DNA extracted from E. coli K12 as template DNA and a primer set specific to the 16S rRNA gene (Table 3).
2) 100 μL of the PCR product diluted to an appropriate concentration (about 0.15 ng / μL) was dropped on the molecular weight fractionation membrane (YM-100) and centrifuged at 500 × g (10 min).
3) The filtrate was discarded, and Milli Q was dropped on a 500 μL molecular weight fractionation membrane and centrifuged at 500 × g (46 min).
4) The molecular weight fractionation membrane was inverted, 10 μL of Milli Q was added dropwise, and centrifuged at 1,000 × g (3 min) (reverse centrifugation).

2.2.2 Experimental procedure (artificial synthetic RNA)
1) PCR products with different base lengths were obtained using a primer with the T7 promoter (5'-TAA TAC GAC TCA CTA TAG GG-3 ') added to the 5' end of the forward primer as in 2.2.1 .
2) RNA was synthesized (transcription reaction) with T7-derived RNA polymerase to obtain artificially synthesized RNAs with different base lengths.
3) 100 μL of artificially synthesized RNA diluted to an appropriate concentration (about 0.5 ng / μL) was dropped onto the molecular weight fractionation membrane (YM-100) and centrifuged at 500 × g (10 min).
The following is the same as 3.2.1 and 4) of 2.2.1.

2.3 Measurement and Evaluation Method For highly sensitive nucleic acid stains (for PCR products: PCR products or artificially synthesized RNA diluted to an appropriate concentration (0.1-0.2 ng / μL) and solutions collected after each reverse centrifugation. In the case of Sybr (trademark) Green I, artificially synthesized RNA: RiboGreen (trademark)), it was stained and measured with a fluorescence spectrometer (NanoDrop ND-3300). The detection range of ND-3300 is 0.002-2 ng / μL for Sybr ™ Green I and 0.02-2 ng / μL for RiboGreen ™.
Assuming that 100 μL of PCR product or artificially synthesized RNA solution remains on the molecular weight fractionation membrane and is recovered in 10 μL of the solution after reverse centrifugation and concentrated 10-fold, the PCR product or artificially synthesized RNA permeates through the molecular weight fractionation membrane. The rate (%) was evaluated by the following formula.

2.4 Summary of Results Table 4 summarizes the approximate molecular weights of PCR products or artificially synthesized RNAs by molecular length and the molecular weight fractionated membrane recovery rate in the molecular weight fractionated membrane (YM-100) (based on a recovery rate of 50% as a reference). Evaluation). The nominal molecular fraction of YM-100 is 100,000 MW.
It was confirmed that 50% or more of all PCR products or artificially synthesized RNAs with different base lengths were trapped on the molecular weight fractionation membrane and recovered.

3. 3. Detection of Escherichia coli 16S rRNA gene using Eco327-18-D3 probe 3.1. Outline of experiment
The almost full-length 16S rRNA gene of Escherichia coli or Methanococcus maripaludis (approximately 1500 bases) and the fluorescently labeled probe (Eco327-18-D3) specific for E. coli 16S rRNA (Eco327-18 as a fluorescent substance (Beckman Dye3)) The labeled probe) was hybridized in the solution, applied to a molecular weight fractionation membrane (YM-100), and the excess probe was removed by centrifugation. Thereafter, the probe specifically hybridized with the 16S rRNA gene of E. coli remaining on YM-100 was detected by capillary electrophoresis (CEQ8000).

3.2. Experimental method 3.2.1 Sample preparation (PCR product)
Using the genomic DNA extracted from E. coli K12 and M. maripaludis S2 as template DNA, and using a primer set specific to 16S rRNA gene (EUB8f-UNIV1500r or ARC21f-UNIV1500r), PCR product of almost full length of 16S rRNA gene Obtained.

3.2.2 Cross in liquid
The 16S rRNA gene sample and the Eco327-18-D3 probe were reacted at 46 ° C. for 5 minutes in a total volume of 15 μL of hybridization buffer (20 mM Tris-HCl, 100 mM NaCl, 1M urea). The whole reaction solution was transferred to 135 μL of hybridization buffer and reacted at 46 ° C. for 1 minute.

3.2.3 Washing of probe surplus with molecular weight fractionation membrane The whole amount of the cross-reaction solution in liquid was applied onto the molecular weight fractionation membrane (YM-100) and centrifuged at 500 × g (3 min). Furthermore, 500 μL of hybridization buffer was applied onto YM-100 and centrifuged at 500 × g (45 min). Thereafter, the probe specifically crossed with the 16S rRNA gene remaining on YM-100 and the 16S rRNA gene of E. coli was collected in 10 μL by reverse centrifugation.

3.2.4 Analysis by capillary electrophoresis Mix 1 μL of the solution collected in 3.2.3 with Sample Loading Solution: 39.8 μL and DNA size standard 80 kit: 0.2 μL, and perform electrophoresis for 16 min at 6 kV with CEQ8000. The fluorescence-labeled probe (Eco327-D3) was analyzed by the fragment analysis software of the CEQ8000 system.

3.3 Experimental Results The results of capillary electrophoresis for each applied 16S rRNA gene sample are shown in FIGS. 3 (a)-(c). Fig. 3 (a) shows only E. coli 16S rRNA gene (about 280ng), Fig. 3 (b) shows only M. maripaludis 16S rRNA gene (about 300ng), and Fig. 3 (c) shows half of both (E coli: about 140 ng, M. maripaludis: about 150 ng). In the M. maripaludis-only system, no peak due to Eco327-18-D3 was detected (Fig. 3 (b)), and a system in which half of both E. coli and M. maripaludis were mixed (Fig. 3 (c)). Then, it can be seen that the peak area (3469) due to Eco327-18-D3 is almost half the peak area (6630) of the E. coli-only system (Fig. 3 (a)).

4). 4. Detection of Escherichia coli 16S rRNA using Eco327-18-D3 probe 4.1. Outline of experiment
An approximately full-length 16S rRNA (approximately 1500 bases) of Escherichia coli or Methanococcus maripaludis and a fluorescently labeled probe (Eco327-18-D3) specific for E. coli 16S rRNA were hybridized in the solution, and molecular weight fractionation membrane (YM -100) and the excess of the probe was removed by centrifugation. Thereafter, the probe specifically hybridized to the 16S rRNA of E. coli remaining on YM-100 was detected by capillary electrophoresis (CEQ8000).

4.2. Experimental method 4.2.1 Sample preparation (PCR product)
PCR of almost full length of 16S rRNA gene using genomic DNA extracted from E. coli K12 and M. maripaludis S2 as template DNA and primer set with T7 specific to 16S rRNA gene (T7EUB8f-UNIV1500r or T7ARC21f-UNIV1500r) The product was obtained and almost full length 16S rRNA was synthesized by T7-derived RNA polymerase.

4.2.2 Cross in liquid
The 16S rRNA sample and Eco327-D3 probe were reacted at 46 ° C. for 5 minutes in a total volume of 15 μL of hybridization buffer (20 mM Tris-HCl, 100 mM NaCl, 1M urea). The whole reaction solution was transferred to 135 μL of hybridization buffer and reacted at 46 ° C. for 1 minute.

4.2.3 Washing of probe surplus with molecular weight fractionation membrane All the in-liquid hybridization reaction solution was applied onto the molecular weight fractionation membrane (YM-100) and centrifuged at 500 × g (3 min). Subsequently, 500 μL of hybridization buffer was applied onto YM-100 and centrifuged at 500 × g (45 min). Further, 100 μL of hybridization buffer was applied onto YM-100 and centrifuged at 500 × g (10 min). Probes specifically hybridized to 16S rRNA remaining on YM-100 and E. coli 16S rRNA were recovered in 10 μL by reverse centrifugation.

4.2.4 Analysis by capillary electrophoresis 1μL of the solution collected in 2.3 is mixed with Sample Loading Solution: 39.8μL and DNA size standard 80 kit: 0.2μL, and electrophoresis is performed with CEQ8000 at 6kV for 16min. The fluorescently labeled probe (Eco327-D3) was analyzed with the fragment analysis software of the CEQ8000 system.

4.3 Experimental results Fig. 4 shows the results of capillary electrophoresis for each applied 16S rRNA sample. Fig. 4 (a) shows E. coli 16S rRNA (approx. 320ng) only, Fig. 4 (b) shows M. maripaludis 16S rRNA (approx. 340ng) only, and Fig. 4 (c) shows half of both (E. coli). : About 160 ng, M. maripaludis: about 170 ng). In the M. maripaludis-only system, no peak due to Eco327-18-D3 was detected (Fig. 4 (b)), and a half-volume mixture of both E. coli and M. maripaludis (Fig. 4 (c)). Then, it can be seen that the peak area (20630) by Eco327-18-D3 is almost half of the peak area (42387) of the E. coli-only system (Fig. 4 (a)).

By utilizing the present invention, for example, about 1 microorganism out of 100 (sensitivity 1%) (DNA or RNA), which is a practical detection limit in the field of environmental microorganisms, can be detected. The method of the present invention can also be applied to diagnostic methods based on various gene sequence information. Furthermore, the method of the present invention can also be used in SNIPS (single nucleotide mutation) detection and mixed nucleic acid systems.

Claims (11)

A method for detecting and / or identifying a target biopolymer in an analyte comprising the following steps:
(1) a step of forming a complex by specifically reacting a probe having a molecular weight smaller than that of a target biopolymer and specifically binding to the target biopolymer with the target biopolymer contained in a specimen;
(2) separating the complex from the probe by molecular weight fractionation means;
(3) a step of recovering the separated complex, and (4) a step of detecting the probe contained in the recovered complex. The separation in the step (3) allows the reaction liquid containing the target biopolymer, the probe, and the complex to pass through a molecular weight fractionation membrane that is a molecular weight fractionation means, and the target biopolymer and the complex. The method according to claim 1, wherein the method is carried out by capturing at least a part of the protein on the molecular weight fractionation membrane. The method according to claim 2, wherein the molecular weight cut-off membrane has a molecular weight cut-off of at least 50,000 MW. The method according to any one of claims 1 to 3, wherein the target biopolymer is a polynucleotide. The method according to claim 4, wherein the polynucleotide is DNA or RNA. 6. The method according to claim 5, wherein the DNA is a PCR product. The method according to any one of claims 1 to 6, wherein the probe has a molecular weight of 3,000 to 20,000. The method according to claim 7, wherein the probe is an oligonucleotide having a base length of 10 to 50 bp. The method according to any one of claims 1 to 6, wherein the probe is labeled. The method of claim 9, wherein the probe is labeled with a fluorescent material. It is a kit used in order to implement the method as described in any one of Claims 1-10, Comprising: The probe whose molecular weight is smaller than a target biopolymer, and specifically couple | bonds with this target biopolymer, and molecular weight Said kit comprising fractionation means.