JP2011072222A - Method for detecting target nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting a target nucleic acid of so-called lateral flow type, which is simpler, quicker straight line shaft and more specific and has an excellent visual judgment property while making the most of a nucleic acid amplification method and a nucleic acid hybridization method and a kit for performing the method. <P>SOLUTION: The method for detecting a target nucleic acid in a sample includes the following steps (1)-(4): the step (1) for amplifying a target biotin labeled single-stranded nucleic acid, the step (2) for developing the target single-stranded nucleic acid on a strip and hybridizing the target single-stranded nucleic acid with a prefixed complementary nucleic acid probe to catch the hybridization product, the step (3) for developing a metal colloid labeled streptavidin conjugate liquid on a strip and visualizing the caught nucleic acid, and the step (4) for developing a cleaning liquid on a strip. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method and kit for specifically detecting a target nucleic acid, particularly a viral nucleic acid, in a biological sample.

In recent years, research and diagnostic techniques relating to detection of nucleic acids have rapidly advanced, and several methods such as gene amplification methods and hybridization methods are known as methods for detecting specific genes in test samples.
Among them, gene amplification methods represented by PCR method (polymerase chain reaction method) and RT-PCR method (reverse transcriptase polymerase chain reaction method) are widely studied and used as methods having high detection sensitivity.

  In addition, a hybridization method in which a single strand or a double strand having a base sequence complementary to a target nucleic acid is labeled with a radioactive or non-radioactive labeling substance, and the target sequence is detected using complementarity with the target sequence. It is widely used as a method for detecting the presence of a target nucleic acid or a target nucleic acid amplification product. In addition, a liquid phase-solid phase hybridization method in which a probe is immobilized on a carrier and a sandwich type liquid phase-liquid phase hybridization method have been devised.

Recently, for the purpose of improving detection sensitivity and ease of operation, a method combining a gene amplification method such as a PCR method and a hybridization method, for example, an amplified single-stranded nucleic acid is immobilized on a strip in advance (or semi-immobilized). ) And a method for detection by hybridization with a first oligonucleotide probe and a labeled second oligonucleotide probe that have been detected (Patent Document 1).
However, this detection method has a problem that it is necessary to prepare a second labeled oligonucleotide probe for each target nucleic acid, which is troublesome, or requires a semi-immobilization operation on the strip.

On the other hand, influenza viruses belong to the Orthomyxoviridae family that causes influenza and contain a negative-strand single-stranded RNA wrapped in an envelope. There are three genera: A type, B type, and C type. To do. Influenza A virus infects mammals including birds and humans, while B and C infect only humans.
In the envelope of influenza virus, hemagglutinin (HA) and neuraminidase (NA) are present, and the combination of HA and NA subtypes is used to identify virus isolates. Influenza A viruses have particularly many HA and NA mutations, so far, 16 types of large mutations have been found in HA and 9 types in NA, and there can be as many subtypes as there are combinations thereof. Differences in subtypes are expressed by abbreviations such as H1N1 to H16N9.

  To date, H1N1 known as “A Soviet type”, H3N2, H1N2, and H2N2 known as “A Hong Kong type” are known to cause human influenza. In April 2009, a virus that was prevalent among pigs was transmitted directly from pigs to people on farms in April 2009, and then the epidemic was recognized among people. The influenza virus is type A, H1N1, and was declared a pandemic on June 12, 2009. In addition, there have been reported cases in which several types such as H9N1 and H5N1 known as highly pathogenic avian influenza have infected humans.

  Therefore, there is a strong demand for a method capable of detecting and determining the influenza virus at a gene level accurately, simply and quickly.

Japanese Patent No. 4268944

  The present invention implements a method for detecting a so-called lateral flow type target nucleic acid, which is simpler, quicker, specific and excellent in visual judgment, while taking advantage of the advantages of the nucleic acid amplification method and the nucleic acid hybridization method, and the method. It is related to providing a kit for.

  The present inventors amplify a labeled single-stranded nucleic acid from a biological sample, develop it on a strip by capillary action, and hybridize it with a complementary nucleic acid probe immobilized on the strip. It was found that by capturing and then visualizing it, the target nucleic acid can be detected easily and quickly by visual judgment.

That is, the present invention relates to the following 1) to 5).
1) A method for detecting a target nucleic acid in a sample, comprising the following steps (1) to (4):
(1) A step of amplifying a biotin-labeled single-stranded target nucleic acid (2) A step of developing the single-stranded target nucleic acid on a strip, hybridizing it with a complementary nucleic acid probe immobilized in advance, and capturing this ( 3) Step of developing a metal colloid-labeled streptavidin conjugate solution on the strip and visualizing the captured nucleic acid (4) Step of developing a washing solution on the strip 2) Amplification of single-stranded target nucleic acid, but uncontrolled PCR method The method for detecting a target nucleic acid according to claim 1, wherein
3) The method for detecting a target nucleic acid according to 2) above, wherein the non-control PCR method is performed using one or a plurality of primer sets shown below.
a) Primer consisting of the base sequence of SEQ ID NO: 1, primer consisting of the base sequence of SEQ ID NO: 2 b) Primer consisting of the base sequence of SEQ ID NO: 3, Primer consisting of the base sequence of SEQ ID NO: 4 4) Capture on multiple strips The method for detecting a target nucleic acid according to 1) to 3), wherein the target nucleic acid is simultaneously visualized using one metal colloid-labeled streptavidin conjugate solution.
5) A target nucleic acid detection kit for carrying out the method of 1) above, comprising at least a PCR amplification primer, a complementary nucleic acid probe-immobilized strip, and a metal colloid-labeled streptavidin conjugate solution.

  According to the method and kit for detecting a target nucleic acid of the present invention, a target nucleic acid in a biological sample, particularly an influenza virus, can be detected and determined visually at a gene level with high sensitivity, simply and quickly. Further, according to the method of the present invention, target nucleic acids captured on a plurality of strips can be visualized simultaneously using one metal colloid-labeled streptavidin conjugate solution, and a plurality of them can be visualized at one time by one visualization operation. The target nucleic acid can be detected.

Figure showing an example of the strip configuration The figure which showed the detection procedure of the target nucleic acid (A: Hybridization, B: Visualization, C: Washing)

(1) Step of amplifying biotin-labeled single-stranded target nucleic acid In this step, biotin-labeled single-stranded target nucleic acid is amplified from nucleic acid in a biological material using a known nucleic acid amplification reaction.

Amplification of a biotin-labeled single-stranded target nucleic acid is not particularly limited as long as it can selectively amplify a single-stranded nucleic acid containing a target nucleic acid among nucleic acids contained in a biological material. It is preferably carried out by asymmetric PCR using a biotin-labeled primer capable of amplifying the target nucleic acid sequence.
Asymmetric PCR is a method in which PCR is performed with the primer to be obtained as a single strand at a concentration of about 10 to 100 times that of one primer. The primer design and primer ratio to be used may be adjusted as appropriate. From the viewpoint of efficiency of single strand formation, for example, when a biotin-labeled primer is used as a forward primer, the primer concentration ratio with a non-labeled reverse primer (forward The primer: reverse primer) is preferably 3-6: 1, more preferably 5: 1.
Further, the number of PCR cycles is preferably 40 to 60, and a single strand can be efficiently formed by applying 60 cycles of PCR continuously.

  The asymmetric PCR may employ so-called multiplex PCR (MPCR) in which PCR is performed at once using a plurality of, for example, about 3 to 10 primer sets. By using this, various long and short base sequences can be amplified at a time, and there is also an advantage that a single-stranded nucleic acid can be easily obtained.

  The target nucleic acid is not particularly limited and may be appropriately selected depending on the intended purpose. A part of the base sequence that exists only in prokaryotes, the base sequence that exists only in eukaryotes (except for humans) It is preferable that the nucleic acid is selected from any one of the above and a part of the base sequence existing only in human. Specific examples include viral genes, bacterial genes, cancer-related genes, genetic disease-related genes, and the like. Among these, examples of the virus and bacterial gene include genes such as hepatitis C virus, hepatitis B virus, influenza virus, measles virus, HIV virus, mycoplasma, rickettsia, streptococci, and Salmonella.

As for influenza viruses, for example, Influenza A virus (A / Narita / 1/2009 (H1N1)) segment 4 hemagglutinin (HA) gene (ACCESSION: GQ165815), Influenza A virus (A / New Jersey / 8/76 (H1N1) ) segment 4 hemagglutinin (HA) gene (ACCESSION: VR-897), Influenza A virus (A / Aichi / 2/68 (H3N2)) segment 4 hemagglutinin (HA) gene (ACCESSION: VR-547), Influenza A virus ( A / Duck / PA / 10218/84 (H5N2)) segment 4 hemagglutinin (HA) gene (ACCESSION: VR-1331), Influenza B virus (B / Taiwan / 2/62) segment 4 hemagglutinin (HA) gene (ACCESSION: VR-295).
Examples of primers for amplifying Influenza A virus (A / Narita / 1/2009 (H1N1)) segment 4 hemagglutinin (HA) gene include those shown in Table 1 below.

  Examples of the biomaterial (specimen) containing the target nucleic acid include pathogens such as bacteria and viruses, blood separated from the living body, saliva, tissue disease pieces, and excreta such as manure. In addition, these samples are concentrated as a sediment directly or if necessary by centrifugation, etc., and then, for the purpose of revealing DNA derived from the target tissue, for example, enzyme treatment, heat treatment, surfactant treatment, What performed the cell destruction process by sonication etc. previously can be used.

(2) Step of developing the single-stranded amplified nucleic acid on a strip and hybridizing and capturing with a complementary nucleic acid probe immobilized in advance In this step, the nucleic acid amplified in step (1) is subjected to chromatography. Based on the principle, it is developed on the strip and brought into contact with and captured by a nucleic acid probe complementary to the target nucleic acid previously immobilized on the strip.

  The strip may be any solid-phase carrier capable of binding the capture oligonucleotide probe, but a porous membrane such as nitrocellulose or nylon is preferably used, and a covalently modified nylon membrane having a chemically modified carboxyl group or the like may be used. .

  A complementary nucleic acid probe is a single-stranded oligonucleotide having a sequence complementary to a single-stranded target nucleic acid (amplification product) to be detected. Such a nucleic acid can be prepared by a PCR method, a chemical synthesis method, or the like as described above. The length of the nucleic acid is not limited as long as it can be stably hybridized with the target molecule under normal hybridization conditions, but is preferably not longer than necessary, 10 to 50 bases, more preferably 10 to 30 bases, Preferably it is 15-25 bases.

  The complementary nucleic acid probe may be fixed to the strip by fixing the nucleic acid probe directly with the strip or indirectly. For example, when a chemically modified membrane is used, it is desirable that the complementary nucleic acid probe has a functional group that reacts therewith. For example, a membrane modified with a carboxyl group is aminated at the 5 ′ or 3 ′ end. Synthetic oligonucleotides are preferred.

  The form of binding of the capture oligonucleotide probe to the strip is not particularly limited, and may be any of a line shape or a circular spot shape, but a line shape is preferred. Furthermore, in order to simultaneously capture the target nucleic acid and the amplification product derived from the internal standard nucleic acid, each capture oligonucleotide probe can be bound to different positions on the same strip.

(3) Step of developing a metal colloid-labeled streptavidin conjugate solution on a strip and visualizing the captured nucleic acid In this step, the metal colloid-labeled streptavidin is compared with biotin of the single-stranded target nucleic acid captured above. By binding, the capture position of the target nucleic acid is visualized.
The metal colloid label in streptavidin is not particularly limited as long as it can provide a visually detectable signal and can be moved by capillary action. Specifically, for example, gold colloid, iron colloid, etc. And colloidal silver, and gold colloid is preferred from the standpoint of ease of handling and sensitivity. Furthermore, an alloy colloid composed of gold and platinum can also be suitably used. Avidin may be used instead of streptavidin.

  As a method for preparing the metal colloid-labeled streptavidin, it may be prepared using a known physical adsorption method, chemical bonding method or the like, and a commercially available product may be used.

Metal colloid-labeled streptavidin is used, for example, by appropriately diluting gold colloid-labeled streptavidin 40 nm (50 OD) with a 1% BSA solution to form a metal colloid-labeled streptavidin conjugate solution and spreading it on a strip. .
Note that one metal colloid-labeled streptavidin conjugate solution may be prepared, and the target nucleic acid captured on a plurality of strips can be simultaneously visualized with one conjugate solution.

(4) Step of developing the cleaning solution on the strip In this step, the step of developing the cleaning solution on the strip and removing the remaining metal colloid-labeled streptavidin is performed by capillary action on the test tube or plate well. Suck up and wash.
The washing solution used here is not limited as long as it can remove the metal colloid-labeled streptavidin, and examples thereof include a solution obtained by adding 0.1% Tween 20 to a 2 × PBS solution.

  By performing the steps (1) to (4) and confirming the visualized band, the presence of the target nucleic acid can be visually determined.

In the method of the present invention, when an internal standard nucleic acid is used, the target nucleic acid and the internal standard nucleic acid may be detected separately, but a co-detection method in which detection is performed simultaneously on the same strip is preferred.
Specifically, the determination of the result in detection is desirably performed as follows. If the target nucleic acid amount is equal to, or less than or equal to the expected target nucleic acid amount, compared to the color development amount of the internal standard nucleic acid that is co-amplified competitively or non-competitively and is co-detected Judgment is made visually. For example, when an internal standard nucleic acid that can be competitively amplified with the target nucleic acid is used, the color development amount of the amplification product obtained from the target nucleic acid is equivalent to the color development amount equivalent to the amplification product derived from the control internal standard nucleic acid. The amount of target nucleic acid is determined to be larger than that of the internal standard nucleic acid when the color of the internal standard nucleic acid as a control is slightly recognized or not, and only the color of the target nucleic acid is developed. On the other hand, when only the color of the internal standard nucleic acid as a control is developed or the color of the target nucleic acid is very small, it is determined that the amount of the target nucleic acid is small.

The target nucleic acid detection kit of the present invention comprises means for carrying out the series of steps (1) to (4), specifically, reverse transcriptase, PCR amplification primer, PCR Includes "Amplification reagent set" including enzyme solution, dNTP, UNG reagent, positive control, dH ₂ O, complementary nucleic acid probe immobilization strip, amplification reaction stop solution, denaturing solution, metal colloid-labeled streptavidin conjugate solution, etc. Examples of the “detection reagent set” include instruments such as a 96-well flat bottom microtiter plate and a micropipette, and reagents such as a washing solution.

Reference Example 1 Primer and probe design (1) Primer

(2) Probe

Reference Example 2 Preparation of nucleic acid sample A nucleic acid sample was prepared by the following procedure.
1. Add 25 μl of proteinase to a 1.5 ml microcentrifuge tube by pipet or Excel.
2. Add 200 μl of sample to the tube.
3. Add 200 μl extraction reagent and mix by vortex for 15 seconds.
4). Incubate for 15 minutes at 56 ° C in a heat block and spin down.
5. Add 250 μl pure ethanol, vortex for 15 seconds and spin down.
6). Transfer the entire volume to the column. Centrifuge at 6,000g for 1 minute.
7). Add 500 μl Buffer 1 to the column and centrifuge at 6,000 g for 1 minute. Discard the filtrate.
8). Add 500 μl Buffer 2 to the column and centrifuge at 6,000 g for 1 minute. Discard the filtrate.
9. Add 500 μl ethanol to the column and centrifuge at 6,000 g for 1 minute. Discard the filtrate.
10. The column is centrifuged at 20,000 g for 3 minutes and dried.
11. Incubate the column at 56 ° C for 3 minutes.
12. Add 50 μl RNase Free H ₂ O and centrifuge at 20,000 g for 1 minute.

Example 1. Asymmetric PCR
Asymmetric PCR was performed under the following conditions.
(1) Preparation of PCR master mixed solution
Add 32.5 μl of dH ₂ O, 10 μl of 10 × RT-PCR buffer, 2 μl of dNTPs, 0.5 μl of 20 μM FLAsw-R la, 2 μl of 25 μM FLAsw-F la bio, and 2 μl of Enzyme mix.
(2) In 49 μl of the PCR master mixed solution prepared in (1), 1 μl of the nucleic acid sample prepared in the above Reference Example 2 is placed in a PCR tube, heated at 95 ° C. for 15 minutes with ABI9700 (Applied Biosystems), The test was repeated 60 times in a cycle of 95 ° C., 30 seconds, 60 ° C., 30 seconds, 72 ° C., 30 seconds, heated at 72 ° C. for 3 minutes, and then allowed to stand at 4 ° C. for 1 hour.

Example 2 Multiplex asymmetric PCR
(1) Preparation of PCR master mixed solution
dH ₂ O 29.5 μl, 10 × RT-PCR buffer 10 μl, dNTPs 2 μl, 20 μM FLAsw-R la 0.5 μl, 25 μM FLAsw-F la bio 2 μl, 20 μM β354-F la 0.5 μl, 25 μM β455-R la bio 2 μl Enzyme mix 2 μl Add each one.
(2) In 49 μl of the PCR master mixed solution prepared in (1), 2 μl of the nucleic acid sample prepared in Reference Example 2 above is placed in a PCR tube, heated at 95 ° C. for 15 minutes with ABI9700 (Applied Biosystems), The test was repeated 60 times in a cycle of 95 ° C., 30 seconds, 60 ° C., 30 seconds, 72 ° C., 30 seconds, heated at 72 ° C. for 3 minutes, and then allowed to stand at 4 ° C. for 1 hour.

Example 3 Multiplex asymmetric PCR
(1) Preparation of PCR master mixed solution
dH ₂ O 14.5 μl, 2 × RT-PCR Master buffer 25 μl, 10 × Primer Mix 5 μl (20 μM FLAsw-R la 0.5 μl, 25 μM FLAsw-F la bio 2 μl, 20 μM β354-F la 0.5 μl, 25 μM β455-R la bio 2 μl) Add 0.5 μl of RT Enzyme mix.
(2) Into 49 μl of the PCR master mixed solution prepared in (1), 2 μl of the nucleic acid sample prepared in Reference Example 2 above is put into a PCR tube, and ABI9700 (Applied Biosystems) at 50 ° C. for 20 minutes at 95 ° C. After heating for 15 minutes, it was repeated 40 times in a cycle of 94 ° C., 45 seconds, 54 ° C., 75 seconds, heated at 72 ° C. for 3 minutes, and then allowed to stand at 4 ° C. for 1 hour.
This method also has performance equivalent to or better than that of the second embodiment.

Reference Example 3 Preparation of gold colloid-labeled streptavidin conjugate solution Gold-labeled streptavidin 40 nm (50 OD) was diluted 8-fold with 1% BSA solution and allowed to stand at room temperature.

Reference Example 4 Preparation of capture probe-immobilized strip A capture probe-immobilized strip was prepared according to the following procedure (FIG. 1).
1) Dissolve the probe to 100 μM with 10xSSC.
2) Dissolve the biotin-labeled primer in 50 μM with 10 × SSC.
3) On the Schleicher & Schuell PRIMA60 membrane, draw a control line and a probe line with a watercolor brush and leave at 80 ° C for 5 minutes. Repeat this 5 times, then leave it at 80 ° C for 30 minutes to immobilize.
4) Wash with 1 × PBS; 0.1% Tween20 solution (15 minutes), then with sterile water (10 minutes), and air dry the membrane (37 ° C., O / N).
5) Paste the solid-phased membrane on Arcare 9020 precut cards.
Affix filter paper (Whatman 17Chr) and a laminate film. Cut the finished card to 2mm width.

Example 3 Detection of Target Nucleic Acids Influenza A virus (A / Narita / 1/2009 (H1N1)) segment 4 hemagglutinin (HA) gene) was detected by the following operations 1) to 6).
1) Add 50 μL of PCR product (primer: SEQ ID NO: 1 to 4) to a polypropylene plate.
2) Add 8 μL of colloidal gold-labeled streptavidin conjugate solution (gold colloid-labeled streptavidin conjugate is diluted 8 times with TBS; 1% BSA solution) to the next well.
3) Add 100 μL of washing solution (2 × PBS 0.1% Tween20) to the next well.
4) Immerse the strip with immobilized capture probe in 50 μL of PCR product (FIG. 2A) and leave it for 10 minutes.
5) Transfer the strip to the colloidal gold labeled streptavidin conjugate well (FIG. 2B) and let stand for 3 minutes.
6) Move the strip to the wash well (FIG. 2C), leave it for 2 minutes, remove the strip and check the band.

Claims (5)

A method for detecting a target nucleic acid in a sample, comprising the following steps (1) to (4):
(1) A step of amplifying a biotin-labeled single-stranded target nucleic acid (2) A step of developing the single-stranded target nucleic acid on a strip and hybridizing it with a complementary nucleic acid probe immobilized in advance to capture it. 3) Step of developing a metal colloid-labeled streptavidin conjugate solution on the strip and visualizing the captured nucleic acid (4) Step of developing a washing solution on the strip   The method for detecting a target nucleic acid according to claim 1, wherein the amplification of the single-stranded target nucleic acid is performed by an uncontrolled PCR method. The method for detecting a target nucleic acid according to claim 2, wherein the non-control PCR method is performed using one or a plurality of primer sets shown below.
a) Primer consisting of the base sequence of SEQ ID NO: 1, primer consisting of the base sequence of SEQ ID NO: 2 b) Primer consisting of the base sequence of SEQ ID NO: 3, Primer consisting of the base sequence of SEQ ID NO: 4   The method for detecting a target nucleic acid according to any one of claims 1 to 3, wherein target nucleic acids captured on a plurality of strips are simultaneously visualized using one metal colloid-labeled streptavidin conjugate solution.   The kit for detecting a target nucleic acid for carrying out the method according to claim 1, comprising at least a PCR amplification primer, a complementary nucleic acid probe-immobilized strip, and a metal colloid-labeled streptavidin conjugate solution.