TWI516603B - Primer set, test kit and method for detecting frog virus 3 - Google Patents

Description

Detection of frog virus type 3 introduction group, inspection kit and method thereof

The invention relates to a primer group, a test kit and a method thereof for detecting a frog virus type 3, in particular to a primer group, a test kit and a method thereof for detecting a frog virus type 3 by a polymerase chain reaction.

At present, due to over-exploitation, sea area pollution, climate change and biodiversity reduction, the world's fishery resources have been depleted due to the imbalance of the marine environment, and the dependence on aquaculture fisheries has been insufficient to fill the supply. Aquaculture fisheries have always played an important role in Taiwan's social development. However, in Taiwan, where the land area is small, aquaculture fisheries tend to be high-density intensive farming, which leads to an increase in the incidence of various diseases and has a great impact on industrial economic losses.

Frog virus 3 (Frog Virus 3) belonging Iridoviridae (Iridoviridae) virus genus Rana (Frog Virus), a diameter of the straight icosahedron 120 to 300nm double-stranded DNA virus. The gene body is about 100 to 210 Kb in size, arranged in a circular array and terminally repeated. The frog virus type 3 was first isolated from the North American leopard frog, and the virus strain was isolated from other amphibious and reptilian hosts. The frog virus type 3 infection was also found in at least seven teleost fish hosts.

When the host is infected with the frog virus type 3, there is usually no obvious lesion on the naked eye. When the infection is found, the host has been killed a lot. This shows that the rapid, convenient and accurate diagnosis of the frog virus type 3 is critical. .

Therefore, the present invention provides a primer set, a test kit and a method thereof for detecting a frog virus type 3, which can detect a frog virus type 3 from an unknown sample quickly and specifically.

One aspect of the present invention provides a primer set for detecting a frog virus type 3, comprising an oligonucleotide represented by SEQ ID NO: 1 and an oligonucleotide represented by SEQ ID NO: 2.

Another aspect of the present invention provides a method of detecting a frog virus type 3 comprising the steps of providing DNA of an unknown sample as a template. The template was subjected to polymerase chain reaction with the oligonucleotide shown by SEQ ID NO: 1 and the oligonucleotide represented by SEQ ID NO: 2 to obtain a polymerase chain reaction product. Finally, it is detected whether the polymerase chain reaction product has a sequence or a partial sequence as shown in SEQ ID NO: 4.

Thereby, the primer set of the present invention and the method thereof use the polymerase chain reaction for the specific DNA fragment of the frog virus type 3 to rapidly and accurately detect the frog virus type 3, and rapidly diagnose the pathogen infection for early prevention and treatment.

A further aspect of the invention provides a method for detecting frog virus type 3 The test kit is applied to the fluorescent probe polymerase chain reaction, and the test kit comprises the oligonucleotide represented by sequence identification number 1 and the oligonucleotide represented by sequence identification number 2 for polymerase chain reaction. And a fluorescent probe having a sequence as shown in Sequence Identification No. 3.

According to an embodiment of the invention, wherein the fluorescent probe polymerase chain reaction is an insulated isothermal PCR.

Yet another aspect of the present invention is to provide a method of detecting a frog virus type 3 comprising: providing an unknown sample of deoxyribonucleic acid as a template. The template was ligated with the oligonucleotide represented by SEQ ID NO: 1, the oligonucleotide represented by SEQ ID NO: 2, and the fluorescent probe having the sequence of SEQ ID NO: 3 for fluorescent probe polymerase chain reaction. reaction. Finally, it is checked whether there is a fluorescent signal value.

According to an embodiment of the invention, the fluorescent probe polymerase chain reaction is a real-time polymerase chain reaction (Real-time PCR) or an insulated isothermal polymerase chain reaction (insulated isothermal PCR).

Thereby, the test kit and the detection method thereof of the invention can detect the presence or absence of the frog virus type 3 in an unknown sample quickly and only in a single step, in particular, the detection method of the chain reaction of the thermal insulation isothermal polymerase is needed, The simple bottom temperature control and the replacement of the mechanical temperature rise and fall by the natural temperature gradient can make the machine lighter, avoid the mechanical failure caused by the repeated lifting and lowering of the machine, and save the time required for the temperature to rise and fall. Therefore, the present invention More suitable for the detection of field aquaculture fisheries.

The above summary is intended to provide a simplified summary of the disclosure The reader has a basic understanding of the disclosure. This Summary is not an extensive overview of the disclosure, and is not intended to be an

The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Electrophoresis analysis chart.

Figure 2 is a graph showing the colloidal electrophoresis analysis of the thermal insulation isothermal polymerase chain reaction of the test kit of the present invention.

The disclosure of the present specification provides a primer set, a test kit and a detection method for rapidly detecting frog virus type 3. It is designed with a specific primer pair and a fluorescent probe in the highly reserved region of the frog virus type 3. Using this primer pair to carry out a polymerase chain reaction with an unknown sample. When there is a polymerase chain reaction product, the unknown sample is characterized by the presence of the frog virus type 3; or the test set containing the primer pair and the fluorescent probe, and The unknown sample was subjected to a fluorescent probe polymerase chain reaction. When there was a fluorescent signal, the unknown sample was characterized by the presence of frog virus type 3.

The term "insulating isothermal polymerase chain reaction" as used in the present invention refers to a single point temperature control and utilizing the principle of thermal convection to complete the deamination of the DNA double-strand structure during the polymerase chain reaction process, and the adhesion of the primer. A three-step cycle such as annealing and extension. When heating the bottom of the container filled with liquid, the liquid with a higher specific gravity on the upper surface will move downward by gravity; the liquid heated below will move upward due to the lower specific gravity, which naturally forms a circulation of the liquid. When the hotter liquid flows to the surface, it will be cooled by the heat dissipation of the environment. When the cold liquid flows to the bottom, it will heat up due to heat, so this cycle can continue for a long time, thus forming a stable temperature gradient. . At this time, if the container is designed and the optimum diameter and height are used, and the time of convection and the rate of heat dissipation are skillfully controlled, an environment suitable for the polymerase chain reaction can be formed. The polymerase chain reaction reagent is placed in a specially designed container, and the heating temperature at the bottom of the control is between 92 and 95 ° C. When the reagent flows to the bottom, the reaction of dissociating the DNA double-strand structure can be performed; when the liquid is circulated At the top of the vessel and at the end of the temperature, the primer bonding reaction can be carried out; then it is recycled to the hotter zone for elongation. In the process of uninterrupted heat convection, the polymerase chain reaction is also completed.

The term "fluorescent probe" as used in the present invention refers to a molecule comprising at least 8 nucleotides in succession, which can be heterozygously reacted with a target DNA under heterozygous conditions, and combined with two energy levels at both ends. Fluorescent substances with a lot of difference, the 5' end is a reporter fluorescent reporter, and the 3' end is a fluorescent inhibitor (quencher), which is based on the principle of fluorescence resonance energy transfer (FRET). The fluorescent probe, upon binding to the polymerase chain reaction product, causes a change in the wavelength of the emission light. When the fluorescent probe is free, the fluorescent signal of the reporter fluorescent group is absorbed by the fluorescent inhibitor; when the DNA is replicated, the fluorescent probe is hydrolyzed by the enzyme to make The reporter fluorophore is separated from the fluorochrome dye so that the fluorophore system can receive fluorescent signals from the reporter fluorophore.

The following test examples are intended to illustrate the present invention and are intended to facilitate the general use of the present invention in the art to which the present invention pertains, and the present invention may be fully utilized and practiced without undue interpretation. Limitations of the scope of the invention, but the materials and methods used to practice the invention.

Test Example 1: Design of the primer

The primer pair of the present invention is designed for the major capsid protein (MCP) gene of the frog virus type 3, and the major outer sheath protein has a sequence as shown in GenBank No. U36913.1. The primer pair is designed according to the following rules: (1) The size of the polymerase chain reaction product to be amplified should be less than 150 base pairs. (2) primers Tm value (melting temperature) between 58 to 62 ℃. (3) Avoid more than 4 consecutive identical bases. (4) The 5 bases at the 3' end of the primer are prevented from containing more than 3 G+C. (5) The ratio of the primer G+C should be between 20 and 80%. (6) The total length of the primer should be between 15 and 25 bases. The pair of primers designed are a forward primer as shown in sequence identification number 1, and a reverse primer as shown in sequence identification number 2. The amplified polymerase chain reaction product has a size of 90 bp, and its sequence is shown in SEQ ID NO: 4.

The present invention also provides a fluorescent probe designed for the major outer sheath protein gene of frog virus type 3, and the fluorescent probe is designed according to the following rules: (1) 5' end reporting fluorescent group using FAM (520 nm) or JOE, VIC (550 nm). (2) The 3'-end fluorescence suppressing dye uses black hole quencher (BHQ1) or minor groove binder (MGB). (3) The Tm value of the fluorescent probe is between 68 and 72 ° C, and is higher than the Tm value of the primer by 10 ° C. (4) There is at least one base distance between the fluorescent probe and the primer to avoid stereo interference. (5) Avoid more than 4 consecutive identical bases. (6) The ratio of G+C is between 40 and 80%. (7) The total length of the fluorescent probe is between 15 and 30 bases. (8) The bonding strength between the 5' end of the fluorescent probe and the template is strong. (9) Avoid designing a stable area in the secondary structure of the template. The designed fluorescent probe is shown as sequence identification number 3.

Test Example 2: Specific primer polymerase chain reaction

First, a plastid containing the major outer sheath protein gene of the frog virus type 3 was constructed as a DNA template of the positive control group. The plastids were extracted by commercial kit or conventional extraction method, and diluted in a 10-fold sequence, and the number of copies of the positive control group samples was adjusted to 10 ¹ and 10 ² , followed by a positive primer as shown in sequence identification number 1. And a reverse primer as shown in SEQ ID NO: 2 for a specific primer polymerase chain reaction. The reactants and reagents were carried out in the following ratios: 5 μl of 10X Taq buffer, 1 μl of 10 mM dNTP mixture, 1 μl of 10 μM primer, 1 μl of Taq DNA polymerase (5 U/μl), and a replica number of 10 ¹ or 10 ² . The body is a DNA template, and finally, the sterilized secondary deionized water is added, and the total volume is adjusted to 50 μl, and the solution is uniformly mixed to carry out a polymerase chain reaction. The reaction conditions were denaturation at 95 ° C for 2 minutes, followed by 35 cycles of thermal cycling, including denaturation at 95 ° C for 1 minute, adhesion at 60 ° C for 1 minute, extension at 72 ° C for 1 minute, followed by a final extension of 72 ° C for 2 minutes. The termination temperature was 4 °C. The amplified polymerase chain reaction product was separated by colloidal electrophoresis.

Please refer to Fig. 1 for a colloidal electrophoresis analysis of a specific polymerase chain reaction of the primer pair of the present invention. Wherein M is the molecular weight marker of the nucleic acid. Lanes 1 to 3 are positive control groups with a replica number of 10 ² , lanes 4 to 5 are positive control groups with a copy number of 10 ¹ , and lane 6 is a negative control group without DNA template. The results show that the positive control group shown in sequence identification number 1 and the reverse primer as shown in sequence identification number 2 can increase the size by 90 bp regardless of the positive control group with the number of replicas of 10 ¹ or 10 ² . The polymerase chain reaction product. No negative control group of DNA template, no polymerase chain reaction product

Test Example 3: Intermittent temperature isothermal polymerase chain reaction

First, the plastid containing the main outer sheath protein gene of the frog virus type 3 was extracted by commercial kit or conventional extraction method, and used as a DNA template of the positive control group, and diluted by 10 times, and the number of copies of the positive control group sample was determined. Adjusted to 10 ¹ and 10 ² , followed by a positive primer as shown in sequence identification number 1, a reverse primer as shown in sequence identification number 2, and a fluorescent probe as shown in sequence identification number 3 for temperature isolation. The same temperature polymerase chain reaction. The reagents and reagents were performed in the following ratios: 0.5 μM forward primer, 0.5 μM reverse primer, 0.15 μM fluorescent probe, 1X Uni-ii HS buffer (GeneReach), 25 units of Taq DNA polymerase, and replicas. The plastid with a number of 10 ¹ or 10 ² is a DNA template, and finally sterilized secondary deionized water is added to adjust the total volume to 50 μl. The solution is mixed and transferred to R-tube ^TM (GeneReach), and the iiPCR device is used. Temperature amplification. The reaction conditions were a denaturation at 93 ° C for 15 seconds, followed by a thermal cycle of 40 cycles, and a reaction at 65 ° C for 1 minute. The reaction time is about 50 minutes. The isothermal thermopolymerase chain reaction product is further separated by colloidal electrophoresis to ensure the accuracy of the present invention.

Please refer to Table 1 and Figure 2 below for the results of the temperature-isolated polymerase chain reaction of the test kit of the present invention.

Table 1 shows the results of fluorescence signal analysis, and Figure 2 shows the gel electrophoresis pattern. Wherein M is the molecular weight marker of the nucleic acid. Lanes 1 to 3 are positive control groups with a replica number of 10 ² , lanes 4 to 5 are positive control groups with a copy number of 10 ¹ , and lane 6 is a negative control group without DNA template. In the positive control group with a replica number of 10 ¹ or 10 ² , the forward primer shown by sequence identification number 1, the reverse primer as shown in sequence identification number 2, and the fluorescent probe shown in sequence identification number 3 are used. It can increase the polymerase chain reaction product with a size of 90 bp, and all of them can detect fluorescent signals at a wavelength of 520 nm. The negative control group without the DNA template had no fluorescent signal value, and no polymerase chain reaction product was confirmed by colloidal electrophoresis. The results show that the test kit of the present invention can detect whether there is a frog virus type 3 in an unknown sample, whether by fluorescence detection or colloidal electrophoresis analysis.

Test Example 4: Immediate polymerase chain reaction

First, the plastid containing the main outer sheath protein gene of the frog virus type 3 was extracted by commercial kit or conventional extraction method, and used as a DNA template of the positive control group, and diluted by 10 times, and the number of copies of the positive control group sample was determined. Adjusted to 10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ^{5 ,} and 10 ⁶ , followed by a forward primer as shown in sequence identification number 1, a reverse primer as shown in sequence identification number 2, and a sequence identification number The fluorescent probe shown in Figure 3 performs an instant polymerase chain reaction. The reagents and reagents were performed in the following ratios: 0.5 μM forward primer, 0.5 μM reverse primer, 0.15 μM fluorescent probe, 1X Uni-ii HS buffer (GeneReach), 25 units of Taq DNA polymerase, and replicas. A plastid having a number of 10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ or 10 ⁶ is a DNA template. The water was hydrated to a final reaction volume of 20 μL. The above experimental materials were placed one by one into a 200 μL eight-row PCR tube and capped, and mixed, and the rotation was stopped and placed for immediate polymerase chain reaction. The reaction conditions were as follows: heating at 98 ° C for 2 minutes followed by a thermal cycle of 40 cycles including denaturation at 95 ° C for 15 seconds and adhesion at 60 ° C for 60 seconds.

Please refer to the following Table 2 for the results of the fluorescence signal value analysis of the instant polymerase chain reaction of the test kit of the present invention. Where a is the average of 3 measurements and N is the value of the fluorescent signal not detected.

The results show that in the positive control group with the number of replicas from 10 ¹ to 10 ⁶ , the forward primer shown in sequence identification number 1, the reverse primer as shown in sequence identification number 2, and the firefly shown in sequence identification number 3 are used. The light probe can detect the fluorescence signal value, and when the initial concentration of the control group DNA is higher, the threshold value can be reached with fewer reaction cycles.

According to the above, the primer set, the test kit and the detection method thereof are rapid, sensitive, accurate and specific by using a molecular biology technique to utilize a polymerase chain reaction for a specific DNA fragment of the frog virus type 3, which is rapid, sensitive, accurate and specific. Detecting the presence of frog virus type 3 in unknown samples and rapidly diagnosing pathogen infection for early prevention and treatment. Among them, the detection method of the chain reaction of heat-insulating isothermal polymerase is used, and the total reaction time can be less than 60 minutes to know the detection result, and the machine is lighter, which is more suitable for the detection of aquaculture fisheries in the field.

The present invention has been disclosed in the above embodiments, but it is not intended to limit the invention, and the present invention can be modified and modified without departing from the spirit and scope of the invention. The scope is subject to the definition of the scope of the patent application.

<110> Ricky Marine Biotechnology Co., Ltd.

<120> Detection of frog virus type 3 introduction group, inspection kit and method thereof

<160> 4

<210> 1

<211> 17

<212> DNA

<213> Artificial sequence

<220> primer

<223> For the PCR amplification of frog virus deoxyribonucleic acid (forward)

<400> 1

<210> 2

<211> 21

<212> DNA

<213> Artificial sequence

<223> Introduction of PCR amplification of frog virus deoxyribonucleic acid (reverse)

<400> 2

<210> 3

<211> 17

<212> DNA

<213> Artificial sequence

<220> primer

<223> TaqMan probe for PCR amplification of frog virus deoxyribonucleic acid

<400> 3

<210> 4

<211> 89

<212> DNA

<213> Frog virus 3

<220> CDS

<223> Increased frog virus DNA fragment

<400> 4

Claims (7)

A primer set for detecting a frog virus type 3, comprising an oligonucleotide represented by SEQ ID NO: 1 and an oligonucleotide represented by SEQ ID NO: 2. A method for detecting a frog virus type 3, comprising: providing a DNA of an unknown sample as a template; and performing the chain reaction of the template on the primer group described in claim 1 to obtain a polymerase chain reaction a reaction product; and detecting whether the polymerase chain reaction product has a sequence as shown in SEQ ID NO: 4. A test kit for detecting a frog virus type 3, which is applied to a fluorescent probe polymerase chain reaction, the test kit comprising the primer set according to claim 1 and a fluorescent probe, wherein the fluorescent probe The needle has a sequence as shown in Sequence Identification Number 3. The test kit of claim 3, wherein the fluorescent probe polymerase chain reaction is an insulated isothermal PCR. A method for detecting a frog virus type 3, comprising: providing a DNA of an unknown sample as a template; and performing a fluorescent probe on the template according to claim 3 a polymerase chain reaction; and detecting whether it has a fluorescent signal value. The method for detecting a frog virus type 3 according to claim 5, wherein the fluorescent probe polymerase chain reaction reaction is a real-time polymerase chain reaction (Real-time PCR). The method for detecting a frog virus type 3 according to claim 5, wherein the fluorescent probe polymerase chain reaction is an insulated isothermal PCR.