KR101919896B1 - Method for detecting multiple nucleic acid using a single fluorescence channel PCR machine - Google Patents

Abstract

The present invention provides a method for detecting multiple nucleic acid using a single fluorescence channel PCR machine using a TaqMan probe and a PNA probe. When using the PNA probe and the TaqMan probe of the present invention designed to have low melting temperatures (Tm) by mixing the same together, a single fluorescence channel PCR machine can simultaneously detect two genes. Therefore, the method of the present invention can reduce the cost of purchasing an expensive new multichannel PCR machine, thereby being economically used.

Description

[0001] The present invention relates to a method for detecting multiple nucleic acids using a single fluorescence channel gene amplifier,

The present invention relates to a method for detecting multiple nucleic acids in a single fluorescence channel gene amplification apparatus (PCR machine), and more particularly to a method for detecting multiple nucleic acids in a single fluorescence channel gene amplification apparatus (PCR machine) using a PNA probe and a TaqMan probe .

Real-time amplification and detection of target nucleic acid became possible with real-time gene amplification device (PCR machine), and as a result, it became a key tool for identification of target and biomarker and various applications such as in vitro diagnosis, food safety test, and drug genome . However, the biotechnology industry is struggling with the increasing demand for high-level multiple nucleic acid detection using existing real-time PCR techniques. A detection kit using an expensive real-time gene amplification apparatus (PCR machine) having fluorescence channels of four or more channels or a probe labeled with an expensive multi-fluorescence is purchased and analyzed using a melting curve analysis, And the detection is confirmed.

Techniques have been developed for detecting multiple target genes using a conventional real-time gene amplification apparatus (PCR machine), but a method for detecting multiple nucleic acids in a single fluorescence channel gene amplification apparatus (PCR machine) Development and melting temperature (Tm) change. Therefore, it is urgent to develop a multiplex nucleic acid detection method that can overcome the interference phenomenon in a channel in a single fluorescence channel gene amplification apparatus (PCR machine) which has been used previously.

Korean Patent Publication No. 10-2014-0091944 (Jul. 23, 2014). Korean Patent Publication No. 10-2016-0094288 (2016.08.09.)

PLoS ONE 2012, 7 (1), e30340 SCIENTIFIC REPORTS 2014, 4, 7439

The inventors of the present invention have studied a method for detecting two genes in a single fluorescence channel gene amplification apparatus (PCR machine). The PNA probe is subjected to a nucleic acid amplification process simultaneously with a TaqMan probe and amplified The first gene is detected in an amplification plot using a TaqMan probe, and the melting temperature (Tm) of the first gene is detected using a TaqMan probe. A method of detecting a second gene using a PNA probe designed to have a low value has been found.

 Accordingly, it is an object of the present invention to provide a method for detecting two genes in a single fluorescence channel gene amplification apparatus using a PNA probe and a TaqMan probe.

According to one aspect of the present invention, there is provided a method for detecting a gene comprising: (a) performing PCR using a TaqMan probe for a first gene and a PNA probe for a second gene; And (b) confirming the detection of the first gene by an amplification plot and confirming the detection of the second gene by Melting curve analysis. A method for detecting individual genes is provided.

In one embodiment, the concentration of the TaqMan probe of step (a) may be 1 to 10 pmoles / l.

In one embodiment, the PNA probe of step (a) may be characterized in that the melting temperature (Tm) value is between 30 and 60 < 0 > C.

In one embodiment, the PNA probe of step (a) may be used at a concentration of 5 to 50 pmoles / l.

In one embodiment, the ratio of the primer to the second gene of step (a) may be characterized as being 1: 1 for a forward primer and a reverse primer.

In one embodiment, the PCR of step (a) is performed at 95 ° C. for 3 minutes followed by 95 ° C. (30 seconds) [denaturation step] → 55 ° C. (30 seconds) [annealing step] → 72 ° C. 30 sec.) [Extension step] is repeated.

In one embodiment, the first gene of step (b) is identified by an amplification signal in the PCR extension step, and then the second gene is identified by Melting curve analysis. Detection method.

According to the present invention, a PNA probe and a TaqMan probe in a single fluorescence channel gene amplification apparatus (PCR machine) are found by finding a method of excluding interference phenomenon in a channel by using a PNA probe designed by lowering the melting temperature (Tm) A method for detecting two genes has been found. The method of simultaneously detecting two genes in a single fluorescence channel gene amplification apparatus (PCR machine) according to the present invention can utilize a conventional single-fluorescence channel PCR machine, PCR machine), it can be used economically.

1 is a schematic diagram for explaining a method of detecting two genes in a single fluorescence channel gene amplification apparatus (PCR machine) obtained according to the present invention.
2 is a photograph showing an example of a single fluorescence channel gene amplification apparatus (PCR machine) according to the present invention.
FIG. 3 shows a result (a) and a melting curve analysis result (b) of the amplification signal (Amplification plot) using a specific PNA probe in the PCR annealing step in Example 2. FIG.
FIG. 4 shows a result (a) and a melting curve analysis result (b) of amplification (Amplification plot) confirmation using a specific PNA probe in the PCR amplification step in Example 3. FIG.
5 shows a result (a) and a melting curve analysis result (b) of a amplification signal (Amplification plot) using a TaqMan probe in the PCR extension step in Example 4. FIG.
FIG. 6 shows the results of evaluating the interference of the specific PNA probe with the IS6110 amplified signal in the case of using the symmetric primer for the psbA gene in Example 5. As a result of confirming the amplification signal when the IS6110 gene alone was amplified a1) and Melting curve analysis results (a2); (B1) and Melting curve analysis result (b2) obtained by confirming the amplification signal when the IS6110 gene and psbA were simultaneously amplified; And (c) the result of merge of the two results.
FIG. 7 shows the result of evaluating whether or not the asymmetric primer for the psbA gene in Example 6 was used and the interference of the specific PNA probe against the IS6110 amplified signal against the psbA gene in the same amount as in Example 5, (A1) and Melting curve analysis result (a2) as a result of confirming the amplification signal in the case of the sample of the present invention; (B1) and Melting curve analysis result (b2) obtained by confirming the amplification signal when the IS6110 gene and psbA were simultaneously amplified; And (c) the result of merge of the two results.
8 is a result of evaluating whether or not the asymmetric primer for the psbA gene in Example 6 is used and the interference with the IS6110 amplified signal of the specific PNA probe against the psbA gene using the amount of 100 times in Example 7. As a result, (A1) and Melting curve analysis result (a2) obtained by confirming the amplification signal when only the gene was amplified; (B1) and Melting curve analysis result (b2) obtained by confirming the amplification signal when the IS6110 gene and psbA were simultaneously amplified; And (c) the result of merge of the two results.

(A) performing PCR using a TaqMan probe for a first gene and a PNA probe for a second gene; And (b) confirming the detection of the first gene in an amplification plot and confirming the detection of the second gene in Melting curve analysis. Thereby providing a method for detecting individual genes.

The gene detection method of the present invention comprises (a) performing PCR using a TaqMan probe for a first gene and a PNA probe for a second gene.

The TaqMan probe used in step (a) is a kind of hydrolysis probe. A reporter (FAM or the like) which is a fluorescent substance is bound to the 5 'end and a quencher, which is an offsetting substance, And the probe is specifically hybridized to a target nucleic acid in the annealing step during the PCR but the fluorescence of the fluorescence is inhibited by a quencher. However, when the TaqMan probe bound to the nucleic acid template was hydrolyzed by the exonuclease activity of the nucleic acid polymerase in the extension step, and the reporter which had been canceled by the quencher FAM, etc.) are liberated and fluorescence is developed.

In one embodiment, the concentration of the TaqMan probe can be determined according to the concentration of the plasmid DNA, the type of primer, gene amplification device (PCR instrument), and preferably 1 to 10 pmole / But is not limited thereto.

In addition, the PNA (Peptide Nucleic Acid) probe used in step (a) is a pure synthetic material that does not exist in vivo and has excellent complementary binding ability to DNA or RNA. In addition, the probe is characterized in that it hybridizes and fluoresces when there is a complementary sequence of the gene regardless of the activity of the exonuclease of the nucleic acid polymerase.

In one embodiment, the PNA probe can be designed to have a fusion temperature (Tm) value in the range of 30 to 60 占 폚, preferably in the range of 40 to 50 占 폚. The melting temperature (Tm) of the PNA probe can be designed to be lower than the melting temperature (Tm) of the TaqMan probe.

In one embodiment, the concentration of the PNA probe can be determined according to the concentration of the plasmid DNA, the type of primer, gene amplification apparatus (PCR apparatus), and preferably 5 to 50 pmoles / But is not limited thereto.

In one embodiment, the ratio of primers to the second gene to be detected by the PNA probe is not limited to 1: 1 for a forward primer and a reverse primer. Generally, a PNA probe amplifies gene amplification in asymmetric manner by using a larger amount of forward or reverse primer. However, in the present invention, when the amount of the forward primer and the reverse primer is the same The detection of the target nucleic acid can be easily performed. However, detection may be possible even when the amount of the reverse primer is larger.

In one embodiment, the PCR conditions are 95 ° C for 3 minutes followed by 95 ° C for 30 seconds [denaturation step] → 55 ° C for 30 seconds [annealing step] → 72 ° C for 30 seconds, [Extension] may be repeatedly performed, and may be performed at a repetition frequency that is normally performed. For example, 25 to 50 cycles, preferably 30 to 40 cycles may be performed , But is not limited thereto.

 The gene detection method of the present invention includes the steps of (b) confirming the detection of the first gene by an amplification signal and confirming the detection of the second gene by Melting curve analysis.

In step (b), the two genes may be confirmed by amplification in an amplification plot in the PCR extension step, followed by confirmation of amplification by Melting curve analysis. The intensity of the fluorescence signal when the PCR amplification product reaches a detectable amount by fluorescence is referred to as a threshold value and the number of amplification cycles corresponding to the threshold value in the amplification plot is referred to as a threshold cycle ; Ct) value. The gene from the TaqMan probe is detected by an amplification plot or a Ct value obtained from the amplification signal, and the gene from the PNA probe is also detected by Melting curve analysis.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

<Examples>

1. Design and construction of probe base sequence of specific PNA

The amplification signal by the PNA probe was designed so that the melting temperature (Tm) value was low so that it could be observed only in the annealing step in the PCR step, and the PNA probe and the primer set used in Table 1 were shown. The calculated melting temperature (Tm) may vary depending on the type of program used. The gene to be amplified used in the PNA probe test was spinbia (psbA).

The PCR primers and probes used in the TaqMan probe test were the nucleotide sequences used in the products developed to diagnose infection of Mycobacteria tuberculosis (Table 1), and the gene to be amplified was IS6110.

The target genes were amplified using a single fluorescence real-time PCR instrument (ABI7500, Applied Biosystems, Foster. City, CA, see Fig. 2), and the typical PCR reaction solution compositions are shown in Table 2 below. Primer and probe concentration Was changed according to each test.

For PNA probes For TaqMan probes forward primer 5'-AAT CTG TAA CCT TCA TTA GCA-3 ' 5'-CAA CCG GGA GCC CAG CCG CC-3 ' reverse primer 5'-CTG AGC ACA ACA TCC TTA-3 ' 5'-GGT CCT GCG GGC TTT GCC GC-3 ' Probe 5 'FAM-oo-GGT GCC ATT ATT CC-Lys Dabcyl 3' (Tm: 42 ° C) 5 'FAM-ATC TGG CCA CCT CGA TGC CC-BHQ1 3' (Tm: 63 ° C)

Components required for PCR reaction Usage (μl) 2 × PCR premixture 10.0 forward PCR primer 1.0 reverse PCR primer 1.0 PCR probe (10 pmole / l) 1.0 Nucleic acid 2.0 Distilled water 5.0 Total volume 20.0

2. Assessment of suitability in the annealing step of specific PNA probes by real-time PCR

In the PCR process, the melting temperature (Tm) value of the specific PNA probe was designed to be low so that the amplification signal could be detected only in the annealing step. The designed PNA probe was designed properly and the PCR amplification signal was well detected A test was conducted to confirm.

In general, PCR using PNA probes uses asymmetric amounts of forward and reverse primers. Therefore, the test was performed under three conditions: asymmetric two cases and symmetric one case. When the amount of the reverse primer is increased to 20: 3, the amount of the forward primer is increased to 20: 3 when the amount of the forward primer is equal to that of the reverse primer. Respectively. The conditions are 95 ° C for 3 minutes followed by 95 ° C for 30 seconds [Denaturation step] → 55 ° C for 30 seconds [Annealing step] → 72 ° C for 30 seconds [Extension step ] Was repeated for 35 cycles, and the result was measured by amplification plot and Melting curve analysis.

As shown in FIG. 3, amplification was confirmed by Melting curve analysis when the amount of reverse primer (A) was larger than that of the reverse primer among the three conditions (B) (C) could not confirm the amplification in the Melting curve analysis (FIG. 3) when the amount of the forward primer was increased. The reason is that the DNA strand that the PNA probes can bind to was not properly amplified. When asymmetry is used for the PCR primer, the DNA strand to which a larger amount of the inserted primer can bind can be amplified more.

In order to utilize the specific base sequence of the PNA probe designed according to the present invention, it was confirmed that a reverse primer should be used in a large amount or a reverse primer and a reverse primer should be used in the same amount.

3. Performing conformance test at the extension stage of a specific PNA probe through real-time PCR

A test was carried out to confirm whether or not a gene amplification signal was detected in the PCR extension process using the designed PNA probe under the same conditions as in Example 2. The results are shown in FIG.

As shown in FIG. 4, when the amplification plot was checked at the extension stage, the amplification plot was not detected under the three conditions (FIG. 4A), but the melting curve analysis ) Was confirmed to be amplified under the primer conditions (A) and (B) (FIG. 4B). In other words, although the gene was actually amplified, the fluorescence was not detected because the PNA probe was separated from the target sequence at the extension (72 ° C) step, and the gene was amplified in the melting curve analysis Fluorescence was detected.

4. Conformity test of TaqMan probe by real-time PCR

A primer and a probe for the ISB2 gene, IS6110, were used to test whether the TaqMan probe correctly detected the amplified signal during PCR. Real-time PCR was performed using three plasmids (1 ng, 100 pg, and 10 pg) of plasmid DNA containing IS6110 gene and PCR amplification was confirmed at the extension (72 ° C) The results are shown in Fig.

As shown in FIG. 5, the amplification plot was detected at a high concentration early according to the plasmid DNA concentration and was detected later at a low concentration to show the appearance of the amplification curve (FIG. 5A) Melting curve analysis did not confirm whether the gene was amplified (Figure 5b).

5. Using a symmetric primer against the psbA gene to determine if the specific PNA probes are interfering with the IS6110 amplified signal

When the fluorescence detection was set in the extension step in Example 3, detection by the PNA probe was not performed. However, since the amplification of the gene was confirmed through the melting curve analysis, the presence or absence of the interference was confirmed I want to. When the TaqMan probe and the designed specific PNA probe were mixed to amplify the gene at the same time, the presence of interference of the TaqMan probe by the specific PNA probe was confirmed.

PCR was performed by mixing 3 plasmids (1 ng, 100 pg, 10 pg) of IS6110 gene containing plasmid DNA and 9 pg of psbA plasmid DNA. Table 3 shows the results. In addition, PCR was carried out using only plasmid DNA containing IS6110 gene (the composition of the reaction solution is shown in Table 4 below).

The conditions are 95 ° C for 3 minutes followed by 95 ° C for 30 seconds [Denaturation step] → 55 ° C for 30 seconds [Annealing step] → 72 ° C for 30 seconds [Extension step ] Was repeated for 35 cycles of 55 ° C. (30 seconds) → 72 ° C. (30 seconds), and amplification signal detection was performed in an extension step. In addition, forward and reverse primers for the psbA gene were used at the same concentration (10 pmoles).

Components required for PCR reaction Usage (μl) 2 × PCR premixture 10.0 IS6110 forward primer 1.0 IS6110 reverse primer 1.0 IS6110 probe 1.0 psbA forward primer 1.0 psbA reverse primer 1.0 psbA PNA probe 1.0 psbA nucleic acid (9 pg / l) 2.0 IS6110 nucleic acid (3 concentrations) 2.0 Total volume 20.0

Components required for PCR reaction Usage (μl) 2 × PCR premixture 10.0 IS6110 forward primer 1.0 IS6110 reverse primer 1.0 IS6110 probe 1.0 IS6110 nucleic acid (3 concentrations) 2.0 Distilled water 5.0 Total volume 20.0

As a result, an amplification plot was confirmed in the PCR reaction using only the TaqMan probe (FIG. 6A1), but the detection was not confirmed by the melting curve analysis (FIG. 6A2). Amplification plots in the PCR reaction using the TaqMan probe and the PNA probe were not different from those in the PCR reaction using only the TaqMan probe (see FIGS. 6B1 and 6C). In addition, the fluorescence of the PNA probe could be confirmed by the melting curve analysis only in the PCR reaction using the TaqMan probe and the PNA probe (FIG. 6B2).

6. Using the asymmetric primer for the psbA gene to check whether the specific PNA probe is interfering with the IS6110 amplified signal

In Examples 2 and 3, when the reverse primer concentration was high and the forward and reverse primer concentrations were the same in the PCR reaction of the psbA gene using the specific PNA probe, The psbA gene was detected. Based on this, it was confirmed whether two genes could be detected simultaneously when the reverse primer concentration was high and the PCR reaction was performed by mixing IS6110 and psbA genes.

PCR was carried out by changing the concentration of the primer to the psbA gene in the PCR reaction solution composition of Example 5 (Table 3). 3 pmole / μl of psbA forward primer and 20 pmole / μl of psbA reverse primer were used, respectively, and the results are shown in FIG.

As shown in Fig. 7, Melting curve analysis by a specific PNA probe was not performed in the reaction in which two genes were simultaneously amplified (Fig. 7B2). Unlike the result of amplifying one gene, it was assumed that it would be interfered when two genes were amplified at the same time.

The PCR reaction was carried out by increasing the concentration of the psbA gene from 9 pg to 0.9 ng by 100 times under the same primer conditions. The results are shown in Fig. As shown in Fig. 8, when the concentration of the psbA gene was 0.9 ng, the psbA gene could be detected by Melting curve analysis using a specific PNA probe (Fig. 8B2).

Generally, when a PCR reaction is performed using a PNA probe, the primer is used asymmetrically. However, in the case of analyzing the amplification of two or more genes as in the present invention, it is 100 times smaller Of nucleic acids, indicating a singularity that shows better results.

Claims (7)

(a) using a TaqMan probe for the first gene and a PNA probe for the second gene, and performing PCR such that the ratio of the primer to the second gene is 1: 1 for the forward primer and the reverse primer; And
(b) the detection of the first gene is confirmed by an amplification signal in the PCR extension step, and the detection of the second gene is confirmed by Melting curve analysis after the first gene is identified step
A method for detecting two genes in a single fluorescence channel gene amplification apparatus. 2. The method according to claim 1, wherein the TaqMan probe of step (a) is used at a concentration of 1 to 10 pmole / l. The detection method according to claim 1, wherein the PNA probe of step (a) has a melting temperature (Tm) value of 30 to 60 ° C. 2. The method according to claim 1, wherein the PNA probe of step (a) is used at a concentration of 5 to 50 pmoles / l. delete The method according to claim 1, wherein the PCR of step (a) is carried out at 95 ° C for 3 minutes followed by 95 ° C for 30 seconds [denaturation step] → 55 ° C for 30 seconds [annealing step] (30 seconds) [Extension step] is repeated. delete

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