KR20150105102A - Primer set, kit and method for detecting Toxoplasma gondii using loop-mediated isothermal amplification reaction - Google Patents

Abstract

The present invention relates to a primer set for detecting toxoplasma gondii using a loop-mediated isothermal amplification reaction, and a kit and a method for detecting toxoplasma gondii. A primer composition for detecting toxoplasma gondii using a loop-mediated isothermal amplification reaction according to the present invention comprising: a pair of primers having SEQ ID NO: 1 and 2; and a pair of internal primers having SEQ ID NO: 3 and 4 which has high susceptibility at least 100 times than an existing PCR diagnostic method and can directly carry out diagnosis and detection of toxoplasma gondii on the spot where diseases occur without the need of high-cost equipment as well as has excellent specificity of detecting only toxoplasma gondii can be effectively used in diagnosing and detecting toxoplasma gondii.

Description

TECHNICAL FIELD [0001] The present invention relates to a primer set for isothermal amplification reaction for detecting toxoplasmosis plasma, a kit for detecting toxoplasmosis comprising the same, and a detection method,

The present invention relates to a primer set for isothermal amplification reaction for detection of toxoplasmosis, a kit for detecting toxoplasmosis comprising the same, and a detection method.

Toxoplasma gondii is a single-celled parasite, a dominant animal belonging to the feline family, and is a representative acceptor common organism with various types of mammals including humans and some algae as intermediate hosts. Toxoplasma parasites multiply in cells surrounding the digestive tract of humans and can spread to all organs, including the brain, skeletal muscle, cardiac muscle, eyes, lungs, and lymph nodes. About 90% of patients with toxoplasmosis infection with normal immune defense systems do not recognize the infection because they do not feel any symptoms, but people with weakened immune systems, especially those with acquired immunodeficiency, often have symptoms of toxoplasmosis, It is strongly associated with. Toxoplasma is distributed worldwide and in many developed countries and in some parts of the country, the positive rate of toxoplasma antibodies has been found to be a risk factor for public health.

Immunological methods such as Latex Agglutination Test and enzyme-linked immunosorbent assay (ELISA) are mainly used for the conventional methods for diagnosing toxoplasmosis. However, There is a problem that the presence of the antibody can be changed and the agreement rate between the diagnostic methods is lowered. Nested PCR and real-time PCR have also been used to diagnose toxoplasmosis DNA in animals, water, soil, and clinical specimens. These molecular diagnostics are highly sensitive and require expensive mechanical and experimental knowledge and techniques, so there are many limitations on the field.

The isothermal amplification reaction to replace this is similar to the conventional PCR method, but the conventional PCR method repeatedly performs three steps of denaturation, splicing, and elongation to amplify the gene, While the isothermal amplification reaction has the advantage of being able to bond and stretch at a fixed constant temperature. This is because, instead of using the Taq DNA polymerase used in the conventional PCR method, the Bst DNA polymerase having the exonuclease function of the nucleic acid is used to induce denaturation of the double helical structure of the DNA without being dependent on heat It is because. Therefore, isothermal amplification does not require temperature changes during gene amplification, making it possible to easily amplify genes at fixed temperatures without specialized equipment.

Therefore, the present inventors have developed a primer set for isothermal amplification reaction for detection and diagnosis of toxoplasmosis plasma. When the above primer set is used, the problems caused by the conventional method are overcome, and when compared with the PCR diagnosis method, The present invention has been accomplished by confirming that it is highly sensitive, has excellent specificity for detecting only toxoplasmosis, and can be performed directly at a site where diseases are not generated because it requires no expensive equipment.

An object of the present invention is to provide a primer set for isothermal amplification reaction for detection of toxoplasmosis plasma, a kit for detecting toxoplasmosis plasma containing the same, and a detection method.

In order to achieve the above object, the present invention provides a primer pair of SEQ ID NOS: 1 and 2; And an inner primer pair of SEQ ID NOS: 3 and 4; and a primer set for detecting the toxoplasmosis plasma.

The present invention also provides a primer pair of SEQ ID NOS: 1 and 2; And an inner primer pair of SEQ ID NOs: 3 and 4.

The present invention also relates to a method for detecting a protein, comprising: (a) separating a genomic DNA (gDNA) of a sample; (b) a pair of primers of SEQ ID NOS: 1 and 2, using the separated genomic DNA as a template; And an internal primer pair of SEQ ID NOS: 3 and 4, and amplifying the target sequence by performing an isothermal amplification reaction using the primer set; And (c) detecting the amplification product. The present invention also provides a method for detecting toxoplasmosis using an isothermal amplification reaction.

Primer pairs of SEQ ID NOS: 1 and 2 according to the present invention; And internal primer pairs of SEQ ID NOS: 3 and 4, the primer composition for isothermal amplification reaction for detection of toxoplasmosis is more than 100 times more sensitive than the conventional PCR diagnosis method, and only the toxoplasmosis is detected And it can be directly carried out in a site where a disease occurs. Therefore, it can be effectively used for diagnosis and detection of toxoplasma.

FIG. 1 is a graph showing an electrophoresis result confirming an optimal reaction temperature (A), a dNTP concentration (B) and a time condition (C) for performing an isothermal amplification reaction using a primer set according to the present invention.
Figure 2 after performing the isothermal amplification reaction, and conventional PCR (1 × 10 1 × 10 0 copies / ㎕ from ^9), unlike the substrate dilution amount is a diagram showing the electrophoresis results.
FIG. 3 is a graph showing the results of electrophoresis after isothermal amplification reaction against toxoplasmosis infected tissue and Neospora caninum tachyzoites. FIG.
FIG. 4 shows fluorescence detection and electrophoresis results using SYBR Green I and GeneFinder under daylight and ultraviolet light conditions. FIG.

The present invention relates to a primer pair of SEQ ID NOS: 1 and 2; And an inner primer pair of SEQ ID NOS: 3 and 4; and a primer set for detecting the toxoplasmosis plasma.

A pair of primers of SEQ ID NOS: 1 and 2; And an internal primer pair of SEQ ID NO: 3 and SEQ ID NO: 4 is the sequence in the toxoplasma gene. 1 and 2 is a primer corresponding to both ends of the product to be amplified, wherein the primer represented by the nucleotide sequence of SEQ ID NO: 1 is a forward primer and the primer represented by the nucleotide sequence of SEQ ID NO: 2 is a reverse primer It is a primer. In addition, the primer pairs of SEQ ID NOS: 3 and 4 are primers located inside the product to be amplified, the primers represented by the nucleotide sequence of SEQ ID NO: 3 are forward inner primers (FIP) Is a backward inner primer (BIP).

In the present invention, the term "primer" refers to a single-stranded oligonucleotide sequence complementary to a nucleic acid strand to be amplified and can serve as a starting point for synthesis of a primer extension product. The length and sequence of the primer should allow the synthesis of the extension product to begin. The specific length and sequence of the primer will depend on the primer usage conditions such as temperature and ionic strength, as well as the complexity of the desired DNA or RNA target.

In the present invention, an oligonucleotide used as a primer may comprise a nucleotide analogue such as phosphorothioate, alkylphosphorothioate or peptide nucleic acid, And may include an intercalating agent.

The present invention also provides a primer pair of SEQ ID NOS: 1 and 2; And an inner primer pair of SEQ ID NOs: 3 and 4.

The kit may include a DNA polymerase, dNTPs, a buffer, and the like.

In addition,

(a) separating the genomic DNA (gDNA) of the sample;

(b) a pair of primers of SEQ ID NOS: 1 and 2, using the separated genomic DNA as a template; And an internal primer pair of SEQ ID NOS: 3 and 4, and amplifying the target sequence by performing an isothermal amplification reaction using the primer set; And

 and (c) detecting the amplification product. The present invention also provides a method for detecting toxoplasmosis using an isothermal amplification reaction.

Hereinafter, each step will be described in detail.

The step (a) is a step of isolating the genomic DNA from the sample. At this time, a method known in the art may be used for isolating the genomic DNA, but the method is not limited thereto. The sample in step (a) may be tissue, blood, saliva, urine, body fluids, or the like, but any sample capable of detecting toxoplasmosis may be used, but is not limited thereto.

The step (b) comprises using the isolated genomic DNA as a template and the pair of primers of SEQ ID NOS: 1 and 2; And an internal primer pair of SEQ ID NOS: 3 and 4; and amplifying the target sequence by performing an isothermal amplification reaction using the primer set. The isothermal amplification reaction in step (b) may be performed preferably at 50 to 70 ° C.

The target sequence amplified in step (b) may be labeled with a detectable labeling substance. In one embodiment, the labeling material may be a fluorescent, phosphorescent or radioactive substance, but is not limited thereto. Preferably, the labeling substance is Cy-5 or Cy-3. When the target sequence is amplified, Cy-5 or Cy-3 is labeled at the 5'-end of the primer and the target sequence can be labeled with a detectable fluorescent labeling substance. When radioactive isotopes such as ³² P or ³⁵ S are added to the amplification reaction solution, amplification products may be synthesized and the radioactive substance may be incorporated into the amplification product and the amplification product may be labeled as radioactive.

The step (c) is a step of detecting an amplification product. The amplification product can be detected using a method known in the art such as DNA chip, gel electrophoresis, radioactive measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. The gel electrophoresis can be performed by agarose gel electrophoresis or acrylamide gel electrophoresis according to the size of the amplification product. In the fluorescence measurement method, when Cy-5 or Cy-3 is labeled at the 5'-end of the primer and amplification is carried out, the target sequence is labeled with a fluorescent label capable of detecting the target, and the labeled fluorescence is measured using a fluorescence analyzer Method. In the above radioactive measurement method, a radioactive isotope such as ³² P or ³⁵ S is added to an amplification reaction solution to mark the amplification product, and then a radioactive measurement instrument, for example, a Geiger counter or a liquid scintillation counter the radioactivity can be measured using a liquid scintillation counter.

The detection of the amplified product can be performed using fluorescent dye materials commonly used in the art, but preferably SYBR Green I, GeneFinder and phenol red can be used, and more preferably, SYBR Green I and GeneFinder can be used . The fluorescent dye material is added in order to make it possible to read out the result without requiring additional experiment using the property of binding to DNA double strand. In particular, the SYBR Green I or GeneFinder is a material that is inserted into a double strand of DNA and expresses green fluorescence upon irradiation with ultraviolet light. Therefore, when the toxoplasmosis plasma is amplified by the LAMP method according to the present invention, SYBR Green I or GeneFinder is added to the amplified LAMP product, and then the ultraviolet light is irradiated, green fluorescence appears. That is, the present invention means that when green fluorescence is expressed in SYBR Green I or GeneFinder staining after LAMP amplification experiment without any additional experiment such as electrophoresis, it means that the disease is infected. If green fluorescence is not expressed, It is possible to visually confirm whether or not the virus has been infected with high sensitivity and promptly.

When the toxoplasmosis is detected using the above method, the sensitivity is more than 100 times as high as that of the conventional PCR diagnosis method, and the specificity of detecting only toxoplasmosis is excellent. In addition, And can be effectively used for diagnosis and detection of toxoplasmosis.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the present invention as defined by the appended claims. It will be obvious to you.

Example 1 Production of a Specific Primer Set for Isothermal Amplification Reaction for Detection of Toxoplasma Plasma

Primer Explorer (http://primerexplorer.jp/e/index.html), which is an isothermal amplification reaction primer design software, was used as a primer for the isothermal amplification reaction specific for toxoplasmosis based on the registered Toxoplasma gondii B1 gene (AF179871) Respectively.

The forward primer (5'-ACCAGCAGCAGAGGAGTG-3 ', SEQ ID NO: 1) and the reverse primer (5'-CAGACGAATCAACGGAACT-3', SEQ ID NO: 2) were designed to be located outside the inner primer, 19 nt in size. A reverse internal primer (BIP) (5'-AACAAGAGACGTGCCGCATGTTTTTACTGTGCCATTTTCTGAGCA-3 ', SEQ ID NO: 4) is a forward internal primer (FIP) (5'-CCTCCTCTTCGCGAAACCTCAATTTTAGATGCCTAGAGGAGACACA-3' A complementary base sequence of the plasma antisense sequence, a portion of the sequence forming the loop and a TTTT spacer were designed. The sequence numbers of SEQ ID NO: 3 and SEQ ID NO: 4 were 46 nt and 45 nt, respectively. The above primers were manufactured by Bioneer Co. (Korea) and internal primers were purified from PAGE.

 Example 2. Determination of optimum conditions for isothermal amplification reaction using the primer set of the present invention

The optimum reaction temperature, dNTP concentration and time conditions for performing the isothermal amplification reaction using the primer set prepared in Example 1 were confirmed.

First, in order to confirm the optimal reaction temperature, a PCR reaction was carried out in which 40 pmoles of forward and reverse primers (SEQ ID NOs: 1 and 2), FIP and BIP primers (SEQ ID NOs: 3 and 4), 5 pmole of 10 mM dNTPs and 8U Bst DNA polymerase large fragment 1 mM Tris-HCl, 10 mM KCL, 10 mM (NH ₄ ) ₂ SO ₄ , 2 mM MgSO ₄ , 0.1% Triton X-100) and 1 μl of toxoplasma DNA in a total volume of 50 μl A PCR reaction solution was prepared. The reaction mixture was denatured at 95 ° C for 10 minutes, subjected to DNA synthesis reaction at 55 to 65 ° C for 60 minutes and heat-treated at 80 ° C for 10 minutes to perform isothermal amplification reaction . The results are shown in Fig. 1 (A). M is a 100 bp ladder, 65 ° C for lane 1, 64.3 ° C for lane 2, 63.1 ° C for lane 3, 61.3 ° C for lane 4, 59 ° C for lane 5, 57.3 ° C for lane 6, Lane is 56.0 ℃, lane 8 is 55 ℃ and lane 9 is negative control.

As shown in Fig. 1 (A), it was confirmed that the optimum annealing temperature was 60 占 폚.

Experiments were carried out under optimal temperature conditions of 60 ° C with dNTP concentration varying from 2.5 to 40 mM in order to determine optimal dNTP concentration conditions. 10 pmole of forward and reverse primers (SEQ ID NOs: 1 and 2), FIP and BIP primers (SEQ ID NOs: 3 and 4), 2.5 to 40 mM dNTPs, 8 U Bst DNA polymerase large fragment (New England Biolabs, USA) and 1X ThermoPol Reaction Buffer (20 mM Tris-HCl, 10 mM KCL, 10 mM (NH ₄ ) ₂ SO ₄ , 2 mM MgSO ₄ , 0.1% Triton X-100) and 1 μl of toxoplasmosis DNA at a total volume of 50 ° C. Isothermal amplification reaction was carried out using denaturation at 95 ° C for 10 minutes, DNA synthesis at 60 ° C for 60 minutes and heat treatment at 80 ° C for 10 minutes . The results are shown in Fig. 1 (B). M is a 100 bp ladder, 2.5 mM of lane 1, 5 mM of lane 2, 10 mM of lane 3, 20 mM of lane 4, 30 mM of lane 5, 40 mM of lane 6, and negative control of lane 7 .

As shown in Fig. 1 (B), it was confirmed that the optimal dNTP concentration was 10 mM.

Experiments were carried out with varying reaction time from 10 to 60 minutes to determine optimal reaction time conditions. 40 pmole of FIP and BIP primers (SEQ ID NOS: 3 and 4), 10 mM dNTPs, 8 U Bst DNA polymerase large fragment (New England Biolabs, USA) and 1X ThermoPol reaction buffer (20 mM Tris-HCl, 10 mM KCL, 10 mM (NH ₄ ) ₂ SO ₄ , 2 mM MgSO ₄ , 0.1% Triton X-100) and 1 μl of toxoplasmosis DNA at a total volume of 50 ° C. Isothermal amplification reaction was carried out using denaturation at 95 ° C for 10 minutes, DNA synthesis reaction at 60 ° C for 10 to 60 minutes and heat treatment at 80 ° C for 10 minutes. The results are shown in Fig. 1 (C). M is 100bp ladder, lane 1 is 60 minutes, lane 2 is 40 minutes, lane 3 is 30 minutes, lane 4 is 20 minutes, lane 5 is 10 minutes, lane 6 is a positive control, and 7 Brenane is a negative control.

As shown in Fig. 1 (C), it was confirmed that diagnosis of toxoplasmosis was possible even if the reaction time was shortened to at least 30 minutes.

Example 3. Confirmation of sensitivity in isothermal amplification reaction using the primer set of the present invention

When the isothermal amplification reaction was carried out using the primer set prepared in Example 1, experiments were carried out by varying the dilution amount of the substrate in order to measure the detection limit. More specifically, DNA was extracted from Toxoplasma gondii tachyzoite (RH strain), and was diluted stepwise from 1 × 10 ⁹ to 1 × 10 ⁰ copies / μl to perform isothermal amplification reaction using the template. Sensitivity was compared with conventional PCR results. To perform the conventional PCR, a PCR reaction solution was prepared with 5 pmole each of forward and reverse primers (SEQ ID NOs: 1 and 2) and TOPsimple ™ DryMIX-HOT (Enzynomics, Korea), followed by denaturation (94 ° C., PCR was performed under the conditions of extension (72 ° C, 10 min) and repeated 35 cycles (94 ° C for 20 seconds, 60 ° C for 30 seconds and 72 ° C for 30 seconds). In order to perform isothermal amplification reaction, each 5 pmole of forward and reverse primers (SEQ ID NOs: 1 and 2), 40 pmole of FIP and BIP primers (SEQ ID NOs: 3 and 4), 10 mM dNTPs, 8 U Bst DNA polymerase large fragment Biolabs, USA) and 1 × ThermoPol reaction buffer (20mM Tris-HCl, 10mM KCL , 10mM (NH4) 2 SO 4, 2mM MgSO 4, 0.1% Triton X-100) , and Toxoplasma gondii 1㎕ total volume 50㎕ the tachyzoite DNA To prepare a PCR reaction solution. Using the reaction solution, denaturation at 95 DEG C for 10 minutes, 60 DEG C for 60 minutes Isothermal amplification reaction was performed by DNA synthesis reaction and heat treatment at 80 ° C for 10 minutes . The results are shown in Fig. (A) of Fig. 2 is a 1-10 lanes from 1 × 10 ⁹ 1 × 10 ⁰ copies / ㎕ as a result using an isothermal amplification reaction, Lane No. 11 is a negative control. Fig. 2 (B) shows the result of using conventional PCR. The lanes 1 to 10 are 1 × 10 ⁹ to 1 × 10 ⁰ copies / μl, and lane 11 is a negative control.

As shown in FIG. 2, when the isothermal amplification reaction was performed using the primer set of the present invention, detection was possible from 1 × 10 ⁹ to 1 × 10 ² copies / μl, indicating high sensitivity. On the other hand, when conventional PCR was used, it was confirmed that the detection reactivity decreased gradually as the number of copies decreased, and it was confirmed that detection was possible up to 1 × 10 ⁴ copies / μl. Therefore, when the isothermal amplification reaction is performed using the primer set of the present invention, it can be confirmed that the sensitivity to toxoplasmosis is 100 times higher than that of conventional PCR.

Example 4. Confirmation of specificity during isothermal amplification reaction using the primer set of the present invention

A comparative experiment using Neospora caninum tachyzoites belonging to the genus such as toxoplasma which was considered to be toxoplasma in the 1990s due to morphological similarity in order to confirm the specificity of the isothermal amplification reaction using the primer set prepared in Example 1 of the present invention Respectively. 40 pmole of FIP and BIP primers (SEQ ID NOS: 3 and 4), 10 mM dNTPs, 8 U Bst DNA polymerase large fragment (New England Biolabs, USA), 5 pmoles of forward and reverse primers USA) and 1X ThermoPol Reaction Buffer (20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4) 2SO4, 2 mM MgSO4, 0.1% Triton X-100) and 1 μl of each comparative DNA A reaction solution was prepared. Isothermal amplification reaction was carried out using denaturation at 95 ° C for 10 minutes, DNA synthesis at 60 ° C for 60 minutes and heat treatment at 80 ° C for 10 minutes. The results are shown in Fig. The lane 1 in FIG. 3 is the DNA of the liver of the toxoplasmosis infected cat, the lane 2 is the muscle DNA of the cat infected with toxoplasmosis, the lane 3 is the spleen DNA of the cat infected with toxoplasmosis, the lane 4 is the kidney DNA , Lane 5 is the lung DNA of the toxoplasmosis infected cat, lane 6 is the toxoplasmosis DNA, lane 7 is the negative control, and lane 8 is Neospora caninum tachyzoites DNA.

As shown in FIG. 3, when the isothermal amplification reaction was performed using the primer set of the present invention, Neospora caninum tachyzoites belonging to the same genus were not reacted, but all of the tissues of toxoplasma-infected cats were reacted to show excellent specificity Respectively.

Example 5. Confirmation of Detection Using Fluorescent Reagent

After the isothermal amplification reaction, X100 SYBR Green I (Sigma, USA) or GeneFinder (China) was added to the amplification product in order to confirm the application possibility of the isothermal amplification reaction using the primer set prepared in Example 1, And fluorescence detection was performed under daylight and ultraviolet light conditions. The results are shown in Fig. Fig. 4 (A) shows SYB Green I added to the amplification product, 1 SYB Green I added without the amplification product, 2 P added with GeneFinder to the amplification product, and 2 N added with GeneFinder without the amplification product under the condition of daylight under the condition of daylight. Fig. 4 (B) shows the addition of SYBR Green I to the amplification product and 1 N of SYBR Green I addition without amplification products under the ultraviolet light condition. FIG. 4C shows the result of the amplification by electrophoresis, wherein P represents a positive control group with amplification products and N represents a negative control group without amplification products.

As shown in Fig. 4, the addition of SYBR Green I and GeneFinder to the amplification product confirmed that the mixture exhibited strong green fluorescence. On the other hand, in the control group without amplification products, the mixture turned orange.

<110> Animal and plant quaratine agency <120> Primer set, kit and method for detecting Toxoplasma gondii using          환형 <130> 1.46P <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 1 accagcagca gaggagtg 18 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> backward primer <400> 2 cagacgaatc aacggaact 19 <210> 3 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> forward inner primer, FIP <400> 3 cctcctcttc gcgaaacctc aattttagat gcctagagga gacaca 46 <210> 4 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> backward inner primer, BIP <400> 4 aacaagagac gtgccgcatg tttttactgt gccattttct gagca 45

Claims (6)

A pair of primers of SEQ ID NOS: 1 and 2; And a pair of internal primers of SEQ ID NOS: 3 and 4. The primer composition for use in the Loop-Mediated Isothermal Amplification reaction for detecting Toxoplasma gondii . A pair of primers of SEQ ID NOS: 1 and 2; And an internal primer pair of SEQ ID NOs: 3 and 4. The kit for detecting toxoplasmosis plasma according to claim 1, 3. The method of claim 2,
Wherein the kit comprises at least one selected from the group consisting of a DNA polymerase, dNTPs, and a buffer. (a) separating the genomic DNA (gDNA) of the sample;
(b) a pair of primers of SEQ ID NOS: 1 and 2, using the separated genomic DNA as a template; And an internal primer pair of SEQ ID NOS: 3 and 4, and amplifying the target sequence by performing an isothermal amplification reaction using the primer set; And
(c) detecting the amplification product. The method of detecting toxoplasmosis using isothermal amplification reaction. 5. The method of claim 4,
Wherein the sample of step (a) is at least one selected from the group consisting of tissue, blood, saliva, urine, and body fluids. 5. The method of claim 4,
Wherein the isothermal amplification reaction in step (b) is performed at 50 to 70 ° C.

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