KR101414714B1 - Method of detecting a target nucleic acid by using a competitive amplification - Google Patents

Abstract

The present invention relates to a method for detecting a target nucleic acid using a competitive amplification reaction.

Competitive amplification reactions, PCR, probes

Description

[0001] The present invention relates to a method for detecting a target nucleic acid using a competitive amplification reaction,

The present invention relates to a method for detecting a target nucleic acid using a competitive amplification reaction.

Methods for detecting a target nucleic acid using an amplification reaction include a method using an external control nucleic acid and a method using an internal control nucleic acid. Methods using external control nucleic acids are described, for example, in U.S. Patent No. 6,358,679. U.S. Patent No. 6,358,679 discloses a method for detecting a target nucleic acid sequence comprising: (a) amplifying a target nucleic acid with a primer extension reagent in a first set of one or more reaction vessels, and amplifying the target nucleic acid with a primer extension reagent in a second set of one or more reaction vessels Amplifying an outer control nucleic acid, wherein the primer extension reagent comprises a forward primer, a reverse primer, one or more detectable probes, a polymerase, and at least one nucleotide 5'-triphosphate, wherein the forward primer and the detectable probe 0-5 nucleotides are separated when hybridized to the external control nucleic acid or its complement, and the reverse primer and the detectable probe are separated by 0-5 nucleotides when hybridized to the external control nucleic acid or the complement thereof, The control nucleic acid can be used as a single strand Wherein the amplification process is initiated and the external control nucleic acid is less than the length of the target nucleic acid, and (b) detecting a signal from the one or more detectable probes.

Methods using internal control nucleic acids include those disclosed in U.S. Patent No. 5,667,974 and Japanese Patent Laid-Open No. 10-66597. For example, Japanese Patent Application Laid-Open No. 10-66597 discloses a method for detecting a hepatitis B virus genome using a primer designed based on the nucleotide sequence of SEQ ID NO: 1, which is the 5 'terminal untranslated region of hepatitis G virus genome, A method for quantitatively determining hepatitis G virus by competitive PCR method characterized in that the amount of hepatitis G virus is quantitatively measured by competitive amplification with a known amount of an internal standard cDNA and comparing the quantities of amplified products derived from both Lt; / RTI > Herein, it is disclosed that the internal standard is a plasmid in which a cDNA in which a part of the amplification region is deficient is inserted.

Even with this conventional technique, there is still a demand for a method that can more simply and quickly detect a target nucleic acid using a competitive amplification method.

It is an object of the present invention to provide a method for measuring the amount of a target nucleic acid sequence in a sample using a competitive amplification reaction.

The present invention relates to a method for measuring the amount of a target nucleic acid sequence in a sample, comprising the steps of: (a) using the sample and the internal standard nucleic acid as a template and using a primer set common to the target nucleic acid and the internal standard nucleic acid in the sample as a primer Competing the internal standard nucleic acid with the target nucleic acid, wherein the internal standard nucleic acid has the same length as the target nucleic acid sequence and the target nucleic acid and sequence are identical except for the site to which the detectable probe binds, Wherein the amplification reaction is performed in the presence of the detectable probe and the detectable probe comprises a target probe specific for the target nucleic acid and an internal standard probe specific for the internal standard nucleic acid, ≪ / RTI > (b) detecting a signal originating from the target probe and the internal standard probe; (c) determining a ratio of a target signal generated from the target probe to a standard signal generated from the internal standard probe; And (d) if the target signal is larger than the standard signal, determining that the target nucleic acid sequence is higher than the concentration of the internal standard nucleic acid used in the amplification reaction, and if the target signal is the same as the standard signal The target nucleic acid sequence is determined to be equal to the concentration of the internal standard nucleic acid used in the amplification reaction, and when the target signal is smaller than the standard signal, And determining that the concentration of the nucleic acid is less than the concentration of the standard nucleic acid.

The method of the present invention comprises the steps of: (a) using a sample and an internal standard nucleic acid as a template, and using a target nucleic acid in the sample and a primer set common to the internal standard nucleic acid as a primer, Wherein the internal standard nucleic acid has a length identical to the target nucleic acid sequence and a concentration at which the sequence is substantially the same as that of the target nucleic acid except for a site to which a detectable probe binds is known, Wherein the amplification reaction is performed in the presence of the detectable probe and the detectable probe comprises a target probe specific for the target nucleic acid and an internal standard probe specific for the internal standard nucleic acid.

The amplification reaction may be a PCR amplification reaction. PCR is widely known in the art and is an amplification method for amplifying a target nucleic acid by repeatedly circulating a reaction solution containing a template, a forward primer, a reverse primer, a polymerase, and a dNTP at a temperature corresponding to denaturation, annealing, .

   The sample may be any one including a target nucleic acid. For example, it may be a biological sample such as blood, urine, tissue, and biopsy sample. The nucleic acid may include DNA, RNA, and analogs thereof. The "target sequence" means a polynucleotide to which a primer or probes are hybridized.

"Amplicon" means a polynucleotide sequence that is amplified in a target sequence and whose terminal is defined by two primer binding sites.

In step (a), the internal standard nucleic acid has the same length as the target nucleic acid sequence, and the concentration is known to be substantially the same as that of the target nucleic acid except for a site to which a detectable probe binds. That is, since the internal standard nucleic acid has the same sequence and the same length as the target nucleic acid and the primer binding site, the amplified target and the length of the internal standard "Amplicon" are also the same. Thus, changes in the amount of amplification product due to kinetic variation due to differences in length during amplification are minimized. In addition, the internal standard nucleic acid is substantially identical in sequence to the target nucleic acid except for a portion to which the standard probe binds or a complementary portion thereof. As used herein, the term " substantially indentical " includes not only identical sequences but also sequences that differ in sequence to such an extent that they have little effect on the amplification efficiency of ampicillin even if the sequences are different.

The target probe and the standard probe may bind to the target nucleic acid or the standard nucleic acid at a position adjacent to or away from the primer binding site.

The target probe and the internal standard probe may bind to each other at positions corresponding to the target nucleic acid and the internal standard nucleic acid, respectively.

"Detectable probe" means an oligonucleotide capable of forming a double stranded structure, or a higher order structure, for example, a triple helix and emitting a detectable signal by complementary base pairing with a sequence of a target nucleic acid . The detectable probe may be labeled with a fluorescent dye.

The detectable probe may be a self-absorbing fluorescent probe comprising a reporter dye and a quencher. "Self-quenching fluorescence probe (SQP)" is labeled with a pair of labels consisting of a quencher that reacts with the fluorescent reporter dye by energy transfer (FRET).

As used herein, the term "label" refers to an oligonucleotide, nucleotide or nucleotide 5'-triphosphate which is capable of binding (i) providing a detectable signal and (ii) a first or second label, Refers to any moiety that reacts with a second marker to alter the detectable signal provided by the marker, and (iii) functions to stabilize the hybridization and, for example, function to form double strands.

"Quenching" refers to the reduction of the fluorescence of a first moiety (reporter dye) caused by a second moiety (quencher) regardless of mechanism. "Self-quenching" means intramolecular, energy transfer effect, for example, fluorescence resonance energy transfer (FRET). Fluorescent reporter dyes and absorbers are used to transfer energy from a fluorophore to a light absorbing absorber Lt; RTI ID = 0.0 > fluorescence < / RTI > by the fluorescent dye. The self-absorption effect may be reduced or lost by the hybridization of the probe to the complement or by the cleavage of the fluorescent reporter dye due to the action of the 5 'nuclease and separation of the fluorescent reporter dye and the light absorbing absorber.

In step (a), the polymerase used in the polymerization reaction may be a step of separating the reporter dye from the light absorbing absorber by cutting the reporter dye from the self-absorbing fluorescent probe during amplification. The polymerase may be a thermostable polymerase having 5 ' nuclease activity.

The reporter dye may be a xanthene dye, such as fluorescein. The reporter dye is usually separated from the light absorbing absorbent by 12 nt or more. The reporter dye binds to the 5 'end or the 3' end of the SQP, and the light absorbing absorber can bind to the opposite end of the SQP. For example, SQP can be that the fluorescent reporter dye is labeled 5 'nucleotides and the light absorbing label is labeled 3' nucleotides. The light absorbing material may be a non-fluorescent material or a fluorescent material for dividing the reporter dye according to the wavelength. The reporter dye-probe comprising a light absorbing absorption character pairs, commonly referred to as TaqMan ^TM, and require a 5 'nuclease activity. The probe is cut off during amplification and emits a fluorescence signal proportional to the amount of double stranded DNA present (see U.S. Patent Nos. 5,538,848, 5,804,375, 6,358,679).

Fluorescent dyes that can be used in probe labels include, but are not limited to, fluorescein, rhodamine (e.g., U. S. Patents 5,366,960, 5,936,087, 6,051,719), cyanines (U. S. Patent No. 6,080, WO 97/45539), metal porphyrin complexes (WO 88/04777). Examples of fluororesin dyes include 6-carboxyl fluorosine (6-FAM) 1, 2 ', 4', 1,4, -tetrachlorofluorescein (TET) 2 and 2 ', 4' ', 7', 1,4-hexachlorofluorescein (HEX) 3 (US Patent No. 5,654,442), 2 ', 7'-dimethoxy-4', 5'-dichloro- Fused phenyl-1,4-dichloro-6-carboxyfluorescein 5 (U.S. Pat. Nos. 5,188,934 and 5,885,778), 2'-chloro-5'-fluoro-7 ' '-Chloro-7'-phenyl-1,4-dichloro-6-carboxyfluorescein 6 (US Patent No. 6,008,379) (see formulas 1-6 below). The fluorescein and rhodamine dyes may have 1,4-dichloro substituents (lower ring as shown in the following formulas 5-6). The reporter dye may be Cy3 or Cy5.

In the formula, X is a portion connected to the probe.

Another label used for probe labeling is the fluorescent light absorbing moiety. The light absorbing absorber includes (i) a rhodamine fluorescent dye selected from the group consisting of tetramethyl-6-carboxyhodamine (TAMRA) 7, tetrapropano-6-carboxyhodamine (ROX) 8, and But are not limited to, cyanine dyes including a crude compound such as 9-11, and (iii) 11, anthraquinone, malachite green, nitrothiazole, and nitroimidazole compounds.

In the formula, X is a portion connected to the probe.

The reporter dye of the target probe may be distinguished from the reporter dye of the standard probe.

In the amplification process, the reporter dye is cleaved from the SQP by the 5 'nuclease activity of the polymerase and is no longer absorbed by the absorber, but emits fluorescence. As a result, the increase in fluorescence increases directly in proportion to the increase in the copy of the amplification product. The amplification products can be measured and quantified through end point analysis or real time analysis.

As used herein, the term " end point analysis "refers to data collection occurring only after the reaction is completed. The end point analysis of the PCR means that the fluorescent dye is measured when the thermocycling and amplification are completed. "Real time analysis" means periodically monitoring during PCR. Certain systems, such as the ABI 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif.) Perform monitoring during each thermal cycle at preset points or user defined points.

In the present specification, the SQP is preferably 10 to 40 nt in length, but is not limited thereto.

 In the present specification, the length of the target nucleic acid and / or the internal standard nucleic acid is not particularly limited, but is preferably 30 to 110 nt, 50 to 70 nt. In addition, the primer may be 10 to 40 nt in length, but is not limited thereto.

In the method of the present invention, in the step (a), the amplification reaction may be performed under the restriction condition of the primer.

In the present specification, the term " restriction condition of the primer "means that the concentration of the primer is 1/2 or less, preferably 1/5 or less, more preferably 1/10 or less, more preferably 1 / 20 or less. In PCR, the restriction conditions of the primer may vary depending on the concentration of the template and the number of cycles. Preferably, the restriction condition of the primer may be that the concentration of the primer is preferably 0.1 μM to 5 μM, more preferably 0.1 μM to 2 μM, and most preferably 0.1 μM to 1 μM. Under the restriction of the primer, when the amplification reaction, for example, PCR is carried out, the ratio of the amplification product to the target nucleic acid and the concentration of the internal standard nucleic acid becomes larger.

The method of the present invention includes the steps of: (b) detecting a signal originating from the target probe and the internal standard probe. The detection of the signal may be performed by any signal detection means known in the art depending on the type of the signal used. For example, in the case of a fluorescence signal, a fluorescence detector may be used. It is preferable that the target probe and the internal standard probe generate fluorescence signals of different wavelengths. By detecting this signal, the amount of the target nucleic acid and the internal standard nucleic acid initially contained can be estimated.

The method of the present invention includes the step of: (c) determining a ratio of a target signal originating from the target probe to a standard signal originating from the internal standard probe.

(D) determining that the target nucleic acid sequence is higher than the concentration of the internal standard nucleic acid used in the amplification reaction when the target signal is larger than the standard signal; and Determining that the target nucleic acid sequence is equal to the concentration of the internal standard nucleic acid used in the amplification reaction when the target signal is equal to the size of the standard signal and if the target signal is smaller than the standard signal, Is less than the concentration of the internal standard nucleic acid used in the amplification reaction.

Means that the target signal is greater than, equal to, or smaller than the standard signal, when the target signal and the target signal are generated from the same label, the absolute value is used as a reference, yield are different from each other, this means a signal obtained by reflecting the quantum yield. That is, the ratio of signals obtained by dividing the obtained signal by the unit quantum yield.

By doing so, it is possible to quantify whether the concentration of the target nucleic acid is higher or lower than the concentration of the internal standard nucleic acid, which knows the concentration at the beginning of the amplification reaction. The concentration of the internal standard nucleic acid introduced at the initial stage of the reaction may preferably be included at a concentration that is a standard for legal regulation or judgment.

According to the method of the present invention, the presence or absence of the target nucleic acid in the sample can be simply detected. In addition, the number of reaction vessels used in the reaction is reduced, and the amount of fluorescent light is not necessarily measured in real time, so that the optical measuring instrument can be simplified.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One: primer  Concentration is competitive PCR Effect on

In this example, the ratio of the concentration of the target nucleic acid having the nucleotide sequence of SEQ ID NO: 1 to the internal standard nucleic acid having the nucleotide sequence of SEQ ID NO: 2 is different and the ratio of the nucleotide sequence of SEQ ID NO: 3 (forward primer) PCR was performed using oligonucleotide as a primer. The target nucleic acid, the internal standard nucleic acid, the primer, and the probe used in this example were synthesized from Bioneer (Korea).

Specifically, the concentration ratio of the target nucleic acid and the internal standard nucleic acid was set to 10: 1 (10 ⁶ : 10 ⁵ copy / μl), and the concentration of each primer was 1 μM, 1/3 μM, 1/9 μM and 1 / Respectively. The reaction solution contained 5 μM of the target probe of SEQ ID NO: 5 and 5 μM of the standard probe of SEQ ID NO: 6 together with the above-mentioned concentrations of the target nucleic acid, the internal standard nucleic acid, the oligonucleotide primer of SEQ ID NO: 3 (forward primer) and the oligonucleotide primer of SEQ ID NO: , 250 μM of dNTP, and 0.1 unit of Taq polymerase, and a total volume of 1 μl. The target probe is labeled with Cy3 at the 5 'end and labeled with EHQ2 (black hole quencher) at the 3' end. In the standard probe, Cy5 is labeled at the 5 'end and EHQ2 (black hole quencher) is absorbed at the 3' end.

The PCR was repeated 30 times at initial denaturation at 94 캜 for 30 seconds, denaturation at 94 캜 for 10 seconds, and annealing and polymerization at 60 캜 for 30 seconds. After the completion of the PCR reaction, the fluorescence values generated from Cy3 and Cy5 in the amplification products, that is, the absorbances at wavelengths of 532 nm (Cy3) and 635 nm (Cy5) were measured.

Brief Description of the Drawings Fig. 1 is a diagram schematically showing a target nucleic acid and an internal standard nucleic acid. As shown in Fig. 1, the target nucleic acid and the internal standard nucleic acid have the same sequence except for the probe binding site. Thus, the same forward and reverse primers can bind to either the target nucleic acid or the internal standard nucleic acid. In the probe, the target probe and the standard probe are modified at the 5 'ends with Cy3 and Cy5, respectively, so that amplified target nucleic acid and standard nucleic acid can be distinguished by fluorescence. The target nucleic acid corresponds to a sequence of nucleotides 1849 to 1929 of the entire genome of lambda DNA.

FIG. 2 is a graph showing the result of competitive PCR in which the concentration of primer is different for a template having the same concentration ratio. FIG. As shown in Fig. 2, the lower the concentration of the primer, the more the template with the higher concentration was amplified. Therefore, when PCR is performed under the constraint of the primer, it is possible to better predict which template in the template contains more templates from the PCR result. In Figure 2, the abscissa is the concentration of the primer and the ordinate is the ratio of fluorescence generated from the fluorescent / amplified standard nucleic acid arising from the amplified target nucleic acid. In FIG. 2, each experimental result shows the result of two repeated experiments.

As a control, when the concentration of the target nucleic acid and the internal standard nucleic acid was 1: 1, the amount of fluorescence at the termination of the PCR was approximately 1.04: 1. This means that the ratio of the template nucleic acid is 1: 1, which is much better than that of the template nucleic acid.

Example  2: primer  Under the concentration-limiting conditions, PCR Effect on

In this example, the ratio of the concentration of the target nucleic acid having the nucleotide sequence of SEQ ID NO: 1 to the internal standard nucleic acid having the nucleotide sequence of SEQ ID NO: 2 is different and the ratio of the nucleotide sequence of SEQ ID NO: 3 (forward primer) PCR was performed using oligonucleotide as a primer.

In this example, the concentration ratios of the target nucleic acid and the internal standard nucleic acid are 10: 1, 5: 1, 2: 1, 1: 1, 1: 2, 1: 5, 1:10, Was equivalent to 10 < ⁵ > copy / [mu] l), and the PCR and fluorescence measurement conditions were the same as in Example 1 except that the concentration of the primer was 1/9 [mu] M.

FIG. 3 is a graph showing the result of competitive PCR in which the ratio of the template concentration is varied at the primer concentration limiting condition. 3, the left vertical axis is Cy5 / Cy3 (internal standard nucleic acid / target nucleic acid) and the right vertical axis is Cy3 / Cy5 (target nucleic acid / internal standard nucleic acid). As shown in FIG. 3, when the ratio of the target nucleic acid and the internal standard nucleic acid is 1, the target nucleic acid is amplified more when the ratio is greater than 1, and the internal standard nucleic acid is more amplified when the ratio is smaller than 1.

Therefore, it is possible to qualitatively analyze whether a target nucleic acid exists in a larger amount or a smaller amount in a sample by using an internal standard nucleic acid having a known concentration through competitive PCR under the condition of primer concentration limitation have.

According to the method of the present invention, the concentration of the target nucleic acid in the sample is qualitatively analyzed . For example, two pathogens Chlamydia In the case of the detection of Trachomatis (CT) and Neisseria gonorrhoeae (NG), there are three kinds of reactions including the reaction involving the CT sample and the internal standard nucleic acid, the reaction involving the NG sample and the internal standard nucleic acid, The presence or absence of a certain concentration of pathogens can be detected. In this case, PCR amplification is assumed to proceed according to the embodiment of the present invention. Assuming that the concentration of CT and NG in the blood is 10 ³ cells / ml, the concentration of the internal standard nucleic acid used should be a concentration corresponding to 10 ³ cells / ml. When the value of the target nucleic acid fluorescence value Cy3 / standard nucleic acid fluorescence value Cy5 obtained from the PCR is as follows, the following discrimination can be performed.

Table 1.

number Discrimination PCR results One If CT / NG is present in the blood at a much higher than reference concentration (eg, 10 ⁵ cells / ml) Dark red 2 When CT / NG is present in the blood slightly higher than the reference concentration (eg, 10 ³ -10 ⁴ cell / ml) Red 3 When CT / NG is present at the reference concentration (10 ³ cell / ml) purple 4 If CT / NG is present in the blood slightly below the reference concentration (eg, 10 ³ -10 ² cells / ml) blue 5 If CT / NG is present in the blood very low (eg 10-0 cell / ml) than the reference concentration Dark blue 6 DNA purification error Colorless 7 Errors in reagents and preparation steps Negative control color

As described above, according to the method of the present invention, the presence or absence of pathogens can be simply detected through only three reactions. In addition, the number of reaction vessels used in the reaction is reduced, and the amount of fluorescent light is not measured at the rear time, so that the optical measuring instrument can be simplified.

Brief Description of the Drawings Fig. 1 is a diagram schematically showing a target nucleic acid and an internal standard nucleic acid.

FIG. 2 is a graph showing the result of competitive PCR in which the concentration of primer is different for a template having the same concentration ratio. FIG.

FIG. 3 is a graph showing the result of competitive PCR in which the ratio of the template concentration is varied at the primer concentration limiting condition.

<110> Samsung Electronics Co., Ltd. <120> Method of detecting a target nucleic acid by using a competitive          amplification <130> PN075821 <160> 6 <170> Kopatentin 1.71 <210> 1 <211> 81 <212> DNA <213> Lambda virus <220> <221> misc_feature &Lt; 222 > (1) .. (81) <223> target nucleic acid <400> 1 agcggaaaga gcattattca gcgcccgttc ctgaccgtgt ggcttacctg accgccggta 60 tcgactccca gctggaccgc t 81 <210> 2 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> internal standard nucleic acid <400> 2 agcggaaaga gcattattca gcgcccgaca gaacccgcag aacaacaacc cccgccggta 60 tcgactccca gctggaccgc t 81 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 3 agcggaaaga gcattattca g 21 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 4 agcggtccag ctgggagt 18 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> A target probe: 5 'hydroxyl group is modified with Cy3 and 3'          hydroxyl group is modified with EHQ2 <400> 5 ttcctgaccg tgtggcttac ctga 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> A standard probe: 5 'hydroxyl group is modified with Cy3 and 3'          hydroxyl group is modified with EHQ2 <400> 6 acagaacccg cagaacaaca accc 24  

Claims (10)

As a method for measuring the amount of a target nucleic acid sequence in a sample, (a) competitively amplifying the target nucleic acid and the internal standard nucleic acid using the sample and the internal standard nucleic acid as a template and using a primer set common to the target nucleic acid in the sample and the internal standard nucleic acid as a primer Wherein the internal standard nucleic acid is the same in length as the target nucleic acid sequence and the sequence is identical to the target nucleic acid except for the site to which the detectable probe binds and the concentration used is known, Wherein the detectable probe comprises a target probe specific for the target nucleic acid and an internal standard probe specific for the internal standard nucleic acid; (b) detecting a signal originating from the target probe and the internal standard probe; (c) determining a ratio of a target signal generated from the target probe to a standard signal generated from the internal standard probe; And (d) determining that the concentration of the target nucleic acid sequence is higher than the concentration of the internal standard nucleic acid used in the amplification reaction when the target signal is larger than the standard signal, In the same case, the concentration of the target nucleic acid sequence is determined to be equal to the concentration of the internal standard nucleic acid used in the amplification reaction, and when the target signal is smaller than the standard signal, Is lower than the concentration of the internal standard nucleic acid used in the step (a). 2. The method of claim 1, wherein the amplification reaction is a polymerase chain reaction (PCR). The method according to claim 1, wherein in the step (a), the amplification reaction is performed under a condition that the primer is restricted. 4. The method according to claim 3, wherein the conditions under which the primer is restricted are that the concentration of the primer is 0.1 mu M to 1 mu M. 2. The method of claim 1, wherein the target probe and the internal standard probe bind to each other at the same position relative to the target nucleic acid and the internal standard nucleic acid. The method of claim 1, wherein the detectable probe is a self-absorbing fluorescent probe comprising a reporter dye and a quencher. 7. The method of claim 6, wherein the polymerase used in the amplifying step is to remove the reporter dye from the absorbance absorber by cutting the reporter dye from the self-absorbing fluorescent probe during amplification. 8. The method of claim 7, wherein the polymerase is a thermostable polymerase having 5 ' nuclease activity. 7. The method of claim 6, wherein in step (b), the reporter dye of the target probe differs in fluorescence wavelength from the reporter dye of the standard probe. 7. The method of claim 6, wherein the reporter dye is Cy3 or Cy5.

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