KR20140110138A

KR20140110138A - Lyophilized Reagent For PCR with UDG System to Protect Cross-Contamination

Info

Publication number: KR20140110138A
Application number: KR1020130022772A
Authority: KR
Inventors: 윤기홍; 명현군; 이병철
Original assignee: 우송대학교 산학협력단; 솔젠트 (주)
Priority date: 2013-03-04
Filing date: 2013-03-04
Publication date: 2014-09-17
Also published as: WO2014137093A1

Abstract

The present invention relates to a freeze-dried reagent composition for polymerase chain reaction and a method for producing the same. The freeze-dried reagent composition for polymerase chain reaction of the present invention can improve stability and storage at room temperature by freeze-drying the polymerase chain reaction components. When the nucleic acid amplification reaction is performed by adding distilled water at room temperature to the lyophilized reagent composition, the efficacy of the polymerase chain reaction and the ability to identify the specimen can be maintained. In addition, in the case of a freeze-dried reagent composition for polymerase chain reaction containing UDG (Uracil DNA Glycosylase), problems and risks of diagnostic errors that may occur due to cross-contamination during nucleic acid amplification reaction can be minimized.

Description

(Lyophilized Reagent For PCR with UDG System to Protect Cross-Contamination) containing a cross-contamination-inhibiting UDG

The present invention relates to a freeze-drying reagent composition for polymerase chain reaction, and more particularly, to a freeze-dried reagent composition for polymerase chain reaction containing a UDG (Uracil DNA Gylcosylase) enzyme having cross- And a method for producing the same.

Molecular diagnosis based on polymerase chain reaction (PCR) is to identify a disease or genotype by amplifying a specific DNA using PCR method. In vitro diagnosis is used for the purpose of determining disease infestation It is a test that detects or quantitatively analyzes a specific indicator substance using a sample derived from human body such as blood, urine, or saliva. Molecular diagnostics are based on techniques that can detect specific genes following symptom diagnosis and immunodiagnosis. Molecular diagnostics are a very useful diagnostic method if culture tests are difficult or impossible, or if they are difficult to diagnose quickly through culture tests or if they have a high rate of transmission and frequent occurrence of strains such as avian influenza virus. Therefore, it is important to develop biomarkers capable of detecting specific genotypes suitable for the diagnosis of pathogenic microorganisms or viruses, which are based on genome sequences. PCR, which is used for molecular diagnostics, is an inspection method that is currently used for almost all processes of manipulating genetic material and amplifying a specific target genetic material to be detected. By polymerase chain reaction, it is possible to amplify large quantities of the same genetic material in a base sequence from a small amount of genetic material. For molecular diagnosis, there are three stages of testing. First, nucleic acid is extracted from various biological samples. This should prevent cross-contamination between nucleic acid purity, yield, stability and the sample's genome. Second, the gene amplification process amplifies a specific gene using a detection oligonucleotide composed of a specific nucleotide sequence. In this case, the PCR conditions are adjusted to amplify the gene, and the amplified gene fragment is examined in the final step through electrophoresis or real time PCR. In some cases, the genetic multiplicity can be analyzed by determining the base sequence of the amplified gene.

PCR is basically the polymerization of a gene by a heat-resistant enzyme called Taq polymerase in a solution state. Even in the case of a heat-resistant DNA polymerase, the protein gradually loses its function as an enzyme due to the action of proteolytic enzymes and the oxidation of various free radicals in an aqueous solution at room temperature. Therefore, in order to develop a kit for molecular diagnosis, It should be stored and circulated in a frozen state in the solution. Therefore, the refrigeration state is inevitably required when the product is distributed, so that it is a reality that additional logistics costs such as dry ice packaging and refrigeration box are relatively large. In particular, since the user prefers a convenient method, in the form of premix, that is, a method in which the existing user directly mixes the reaction solution, all the basic reaction liquids are already supplied in a mixed state so that the user can obtain the minimum substrate, template DNA , Primers, and distilled water alone, so that the PCR reaction can be performed. Furthermore, since diagnostic reagents are becoming more active in overseas markets, development of dry type diagnostic premixes is required to solve problems in distribution process.

In the case of diagnostic products using PCR technology, relatively premixed products are preferred in the market because the results are completely different due to the user's mistakes and thus the possibility of diagnosis errors increases. Despite the advantages of the PCR-related enzyme-free pre-products, it is pointed out that the stability of the mixture is deteriorated due to the large disadvantages of the premix product and it is more susceptible to the external environment such as temperature than the conventional product.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

U.S.A. Pat No. 5,834,254

The present inventors have endeavored to develop a technique for manufacturing a dry type product of a PCR kit for diagnosis, which can be applied to a wide range of industrial fields such as medical, food, chemical, and environment. As a result, it was confirmed that the PCR reaction solution containing sugars as a cryopreservative was lyophilized and then distilled water was added to the PCR reaction to maintain the same level of detection and detection efficiency by the PCR reaction. Thereby completing the invention.

Accordingly, it is an object of the present invention to provide a lyophilized reagent composition for polymerase chain reaction.

Another object of the present invention is to provide a method for preparing the lyophilized reagent composition for the polymerase chain reaction.

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a lyophilized reagent composition for a polymerase chain reaction comprising lyophilized following components: (i) a polymerase chain reaction buffer, (ii) dNTPs (dATP, dGTP , dCTP, and dTTP), (iii) DNA polymerase, (iv) primer, and (v) sugar.

In the present invention, the sugar acts as a cryopreservation agent during lyophilization of polymerase chain reaction components.

According to an embodiment of the present invention, in the reagent composition of the present invention, the sugar may be glucose, xylose, xylitol, sucrose, cellobiose, Is a sugar selected from the group consisting of maltotriose, mannose, melibiose, trehalose and stachyose.

According to another embodiment of the present invention, the sugar is selected from the group consisting of maltotriose, mannose, melibiose, trehalose and stachyose.

According to another embodiment of the present invention, the sugar is trehalose or stachyose.

According to another embodiment of the present invention, the sugar is trehalose.

According to one embodiment of the present invention, the sugar is contained in the reagent composition at a concentration of 10-40% by weight.

According to another embodiment of the present invention, the sugar is contained in the reagent composition at a concentration of 16-40% by weight.

According to another embodiment of the present invention, the sugar is contained in the reagent composition at a concentration of 16-30% by weight.

According to another embodiment of the present invention, the sugar is contained in the reagent composition at a concentration of 20-30% by weight.

According to another embodiment of the present invention, the sugar is contained in the reagent composition at a concentration of 20-25% by weight.

According to an embodiment of the present invention, the DNA polymerase is a DNA-dependent DNA polymerase, an RNA-dependent DNA polymerase, or a mixture thereof.

According to another embodiment of the present invention, the DNA polymerase is Taq DNA polymerase or Pfu DNA polymerase.

According to one embodiment of the present invention, the reagent composition of the present invention further comprises UDG (Uracil DNA Gylcosylase) and dUTP.

According to one aspect of the present invention, the present invention provides a method for preparing a lyophilized reagent composition for a polymerase chain reaction comprising the steps of: (a) (i) a polymerase chain reaction buffer, (ii) dNTPs dATP, dGTP, dCTP, and dTTP), (iii) DNA polymerase, (iv) primer, (v) sugar, and (vi) distilled water. ; And (b) lyophilizing the prepared reagent composition.

The content of the lyophilized reagent composition in the above production method of the present invention is the same as that described in the above reagent composition, and thus is not described in duplicate.

According to one embodiment of the present invention, in the above production method, the sugar of step (a) is selected from the group consisting of glucose, xylose, xylitol, sucrose, cellobiose, maltotriose, mannose, melibiose, trehalose and stachyose Lt; / RTI >

The freeze-dried reagent composition for polymerase chain reaction of the present invention can improve stability and storage at room temperature by freeze-drying the polymerase chain reaction components. When the nucleic acid amplification reaction is performed by adding distilled water at room temperature to the lyophilized reagent composition, the efficacy of the polymerase chain reaction and the ability to identify the specimen can be maintained. In addition, in the case of a freeze-dried reagent composition for polymerase chain reaction containing UDG (Uracil DNA Glycosylase), problems and risks of diagnostic errors that may occur due to cross-contamination during nucleic acid amplification reaction can be minimized.

Figure 1 shows the results of comparing the PCR efficiencies of the lyophilized Taq premix for PCR reaction (top panel) and the Taq premix for the general dried PCR reaction (bottom panel).
FIG. 2 shows the results of comparing the PCR efficiencies of the Taq premix for PCR reaction according to the type and concentration of the added sugars. A; Glucose, B; Xylose, C; Xylitol, D; Maltotriose, E; Sucrose, F; Cellobiose, G; Mannos, H; Melibiose, I; Starkios, J; Trehalose. The concentration of the template was 50 ng, 10 ng and 2 ng, respectively, from the left lane.
FIG. 3 is a view showing a state after lyophilization and freeze-drying of a Taq premix for PCR reaction. FIG.
FIG. 4 shows the results of comparing the activity of the PCR pre-mix kit after drying with different kinds and concentrations of added sugars. The addition amount of each sugar from the left lane is 10, 20, 30, and 40%, respectively.
FIG. 5 shows the results of comparing the activity of the one-step RT-PCR pre-mix kit before and after drying. The left panel is the result of using the liquid type kit before drying, and the right panel is the result of using the kit prepared by freeze drying. The amount of mold used is 1; 50ng, 2; 5 ng, 3; 0.5 ng, 4; 0.05 ng, 5; 0.005 ng, 6; 0.0005 ng.
FIG. 6 shows the results of comparing the activity of the single-step RT-PCR pre-mix kit before (A) and after (B) the lyophilization.
FIG. 7 shows the results of measuring the stability of the PCR premix lyophilization kit according to the addition amount of trehalose. The addition concentration of trehalose was 0% (top panel), 10% (middle panel) and 20% (bottom panel). Blue is the result of a kit in which glycerol is added as a control, not trehalose, and black is a freeze-thawed kit (0 times repeated) as a trehalose added kit, and pink is freezing-thawing Freeze-thawing is repeated 5 times. Blue is repeated 10 times for freeze-thawing. Green is repeated 15 times for freeze-thawing. Red is freeze-thawing. It is the result of repeating 20 times.
Fig. 8 shows the result of comparing the gene amplification activities of the PCR premix containing UDG (Uracil DNA Glycosylase) before drying (lane 1) and after drying (lane 2). The number underlined is the sample number for each sample.
FIG. 9 shows the results of comparing amplification activities of genes before and after drying of a multiplex PCR premix with UDG (Uracil DNA Glycosylase). The amount on the underline is the amount of the template sample dielectric used. For each template, the left shows the result of using the pre-mix kit before drying, and the right shows the result of using the dried pre-mix kit.
10 shows the results of the comparison of the efficacy of pfu premix with trehalose (25%). The left-hand gel in the drawing is a liquid-phase gel and the right gel is a dry-type premix. The dielectric amount of the mold used was 1; 50ng, 2; 10 ng, 3; 2ng, 4; 0.4ng, 5; 0.08 ng, 6; 0.016 ng. NTC is a control group that does not use the template's genome.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Measurement of drying efficiency by drying method

The freeze-drying method has an advantage of high stability because it does not lose activity during the drying process, but it is not suitable for industrial enzymes because it needs to use a freeze dryer and its production scale is limited. In general drying, mass production is easy and production cost is relatively low compared to freeze drying. However, since drying time is long and may affect enzyme stability, there is a difference in drying degree between products There is a problem in that there is a great possibility that the uniformity of the product quality is likely to occur. Molecular diagnostic PCR premixes contain inorganic salts and sensitive detection oligonucleotides and nucleotides at high temperatures, which can affect diagnostic efficacy when using conventional hot air drying methods. Freeze drying is not suitable for drying relatively inexpensive industrial enzyme products, but has little impact on the cost of molecular PCR primers. Therefore, in order to compare the performance of the dried Taq premix using the general drying method and the freeze drying method, the PCR reaction was carried out using the Escherichia coli genome as a template. As a result, as shown in Fig. 1, the amount of amplified DNA was observed in the case of using 0.4 ng of the template DNA in the premix prepared by the freeze-drying method. However, in the pre-mix prepared by the general drying method, 10 ng template It was observed that the amplification efficiency was about 25 times higher than that of the control. From these results, it was confirmed that the freeze drying method is more suitable for the PCR pre-mix.

Example 2: Effect of added sugar on the drying efficiency of PCR premix

Glycerol is used as a cryoprotectant for storage at -20 ° C in liquid phase PCR premix. However, glycerol has a disadvantage in that it acts as a substance which interferes with drying when preparing a dry-type premix, resulting in an increase in the viscosity of the composition and a complete drying. Therefore, in order to prepare a dry type premix, it is necessary to select an additive material suitable for maintaining the activity and stability of the enzyme upon lyophilization. In this example, 10 sugars among monosaccharides, disaccharides and trisaccharides were selected, added to the premix kit, and then lyophilized to compare the activities before and after freeze-drying.

To determine the effect of the addition of sugar on the efficiency of the PCR primer, PCR was carried out with the sugar concentration of 10% added, and the amplified DNA fragments were subjected to agarose gel electrophoresis Respectively. As a result, as shown in FIG. 2, there was a significant difference in the PCR efficiency depending on the type and amount of sugar added. When the glucose, xylose and xylitol were added, the efficiency of the premix was the lowest, while the sucrose and cellobiose were slightly more efficient. Maltotriose, mannose and melibiose were found to be highly effective when added, and trehalose and stachyose were found to be most effective when added.

A premix kit was prepared by adding 5 sugars, maltose resource, mannose, melibiose, trehalose and stachyose, which were confirmed to be low in the degree of hindering the reaction efficiency of PCR premix, to 10, 20, 30 and 40% The difference in activity after freeze drying was analyzed. The freeze-drying time was observed for 2 days. The degree of freeze-drying was visually observed, and when the lyophilized reaction solution was dissolved, it was confirmed that the final volume was completely equal to the amount of distilled water added. .

After the PCR was performed using the dried PCR primer, the amplified product was analyzed by agarose gel electrophoresis. As shown in FIG. 4, the PCR reaction was not normally performed in the premix without the sugar. This is presumably due to the inactivation of enzyme activity during freezing and drying. However, in the case of addition of sugar, the PCR efficiency was higher than that in the case of no addition, and the addition of trehalose was the highest. When the concentration was 20%, the maintenance of activity was overall high. The activity of tastant lutein stachyose was lower than that of trehalose but remained more active than other sugars at 20% or more (Fig. 4).

Example 3: Activity analysis of PCR premix containing reverse transcriptase

In order to diagnose RNA virus disease, it is required to develop a dry type product for PCR premix containing RTase as well as DNA polymerase. For this purpose, a one-step RT-PCR premix kit was dried and its activity was examined. As a result, as shown in FIG. 5, the activity was slightly lowered after drying than in the case of performing the PCR in the liquid form before drying, but it was confirmed that it was possible to develop a molecular diagnostic kit for detecting RNA virus in dry form . In addition, when the activity of the dry type RT-PCR primer was compared with that of the detection primer, it was confirmed that the activity remained almost the same before and after drying. Thus, the dry type RT-PCR premix kit containing trehalose at 25% (Fig. 6).

Example 4: Activity analysis by concentration of trehalose in real-time PCR

In order to investigate the effect of trehalose on the stability in the freezing of PCR premix, PCR efficiency was investigated by real - time PCR while repeating freezing and thawing with different concentration. As a result, the amplification efficiency of the premix without trehalose was decreased by repeating the freeze thawing more than 5 times and the amplification efficiency was decreased by repeating 20 freeze thaw cycles. However, when 10% of trehalose was added, the amplification efficiency decreased at 20 times of freezing thawing. However, when 20% of thawing was added at 20 times of freezing thawing, PCR amplification efficiency decreased slightly. From this, it was judged that trehalose prevents deterioration of the premixed effect by freezing during the freeze-drying process (Fig. 7).

Example 5: Preparation of dry type PCR pre-mix with UDG for cross-contamination prevention

In molecular diagnostics, cross-contamination between samples is the most likely to cause problems in the determination of samples from the sampling stage to the PCR reaction stage. In the step of collecting the specimen, it is necessary to take care to prevent the inclusion of substances other than the specimen to be inspected. Generally, cross-contamination between the specimens may occur in the PCR process in which several specimens are handled at once. In this case, it becomes difficult to analyze the PCR amplification products, so PCR products using UDG (uracil DNA glycosylase) system are advantageous in order to reduce cross-contamination. However, the UDG system has a problem that the UDG effect is very vulnerable to freezing and drying. Thus, in this example, a dry PCR primmix was prepared by adding UDG to the PCR composition and its efficacy was analyzed. As a result, as shown in FIG. 8, it was confirmed that the amount of amplified DNA was almost the same before and after the drying without any difference in the PCR efficiency, so that the efficacy was not deteriorated in the freeze-drying process (FIG. 8). In addition, since it is more accurate to determine the sample by amplifying several gene targets simultaneously, the multiplex PCR primmix with UDG was prepared and its efficacy was analyzed. The amount of template samples used was higher than when UDG was not added. As a result, as shown in the results of FIG. 9, the polymorphisms of DNA amplified before and after freeze drying were observed in the same manner, and it was confirmed that UDG could be added to prevent cross-contamination in the development of PCR- (Fig. 9).

Example 6 Effect of PCR Premix Kit Including Dry Pfu DNA Polymerase

Pfu DNA polymerase has a very low probability of error in the amplified DNA during the polymerization process, amplifying the same gene fragment as the genomic sequence of the template. Molecular diagnostic premixes are used not only for the detection of disease but also for the determination of the genotype of living organisms including human body. When determining the nucleotide sequence of the amplified genome, mutation phenomenon during the amplification process should be minimized. Therefore, it is advantageous to use Pfu DNA polymerase rather than Taq DNA polymerase. To investigate the possibility of applying the drying technique of PCR Premix Kit to pre-mix using Pfu DNA polymerase, gene amplification efficiency was examined before and after drying of Pfu PCR premix. As a result, it was confirmed that the amplified gene fragments were the same in size and almost the same in amplification amount by using the pre-mix before lyophilization or after drying after lyophilization (FIG. 10). Therefore, it has been confirmed that Trehalose can be used for the development of dry products of premixes using Pfu DNA polymerase.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims

(I) a polymerase chain reaction buffer solution, (ii) dNTPs (dATP, dGTP, dCTP, and dTTP), (iii) a DNA polymerase, (Iv) primers and (v) sugars.

The method of claim 1, wherein the sugar is selected from the group consisting of glucose, xylose, xylitol, sucrose, cellobiose, maltotriose, mannose, ), Melibiose, trehalose, and stachyose. &Lt; Desc / Clms Page number 13 >

The reagent composition according to claim 1, wherein the sugar is contained in a concentration of 10-40 wt%.

The reagent composition according to claim 1, wherein the DNA polymerase is a DNA-dependent DNA polymerase, an RNA-dependent DNA polymerase, or a mixture thereof.

The reagent composition according to claim 1, wherein the DNA polymerase is Taq DNA polymerase or Pfu DNA polymerase.

The reagent composition of claim 1, wherein the reagent composition further comprises UDG (Uracil DNA Gylcosylase) and dUTP.

A method for preparing a lyophilized reagent composition for a polymerase chain reaction comprising the steps of:
(a) a polymerase chain reaction buffer solution, (d) dNTPs (dATP, dGTP, dCTP and dTTP), (iii) DNA polymerase, (iv) primer, (v) sugar, And (vi) preparing a reagent composition for polymerase chain reaction comprising distilled water; And
(b) lyophilizing the prepared reagent composition.

The method according to claim 7, wherein the sugar is selected from the group consisting of glucose, xylose, xylitol, sucrose, cellobiose, maltotriose, mannose, melibiose, trehalose and stachyose.

The method according to claim 7, wherein the sugar is contained at a concentration of 10-40% by weight.

The method according to claim 7, wherein the DNA polymerase is a DNA-dependent DNA polymerase, an RNA-dependent DNA polymerase, or a mixture thereof.

8. The method according to claim 7, wherein the DNA polymerase is Taq DNA polymerase or Pfu DNA polymerase.

8. The method of claim 7, wherein the reagent composition further comprises UDG (Uracil DNA Gylcosylase) and dUTP.