KR20160129436A - Real-time PCR assays for quantitative detection of Viral hemorrhagic septicemia virus - Google Patents

Abstract

The present invention relates to a quantitative detection method of viral hemorrhagic septicemia virus (VHSV) and, more specifically, a quantitative detection method of VHSV, having excellent sensitivity and specificity and high reproducibility. The latency of the virus and viral diseases at an early stage of infection can be diagnosed in a rapid manner, by quantitatively analyzing the VHSV using a polymerase chain reaction method. A primer set includes a primer of SEQ ID NO: 1 to SEQ ID NO: 4. A probe set include a probe of SEQ ID NO: 5.

Description

[0001] The present invention relates to a method for quantitative detection of viral hemorrhagic septicemia virus by real-time polymerase chain reaction

The present invention relates to a method for quantitatively detecting viral hemorrhagic septicemia virus (VHSV), and more particularly, to quantitative analysis of viral hemorrhagic sepsis virus using a real-time PCR method, To a method for quantitative detection of viral hemorrhagic septicemia viruses capable of rapidly diagnosing diseases, having high reproducibility, and excellent in sensitivity and specificity.

With the recent increase in aquaculture and the emergence of a variety of marine biological diseases, the importance of marine biosecurity, monitoring and monitoring has increased in the world. It is urgent to develop an early detection technology for viral infection in order to combat viral diseases which are becoming the biggest problem in fish culture. Because of the degradation of the traits in the fish farms produced by the self-seedling due to long-term aquaculture, it is inevitable that fishes with excellent traits will move from region to region because of growth, It is a problem that is becoming a big issue because it is strictly limited. In addition, there is an urgent need to develop a rapid virus detection technology for prevention of massive death caused by viral diseases and prevention of vertical and horizontal infection of viruses in the production or breeding of seedlings by preliminary examination of virus infection of siblings and crocodiles. Furthermore, as imports of freshwater fish species from foreign countries increase rapidly, it is necessary to develop a rapid diagnosis technology for intractable virus diseases using genetic engineering techniques to protect the domestic aquaculture industry from indiscreet introduction of foreign species and their pathogens.

In Korea, viruses such as VHSV and IHNV have been harmful to fish farming, and many of the salmon eggs have been imported from the United States and Japan, and the salmon returning to the East Coast has been mixed with American and Japanese salmon in the North Pacific. It is very likely that these viruses are present in the salmon, given the fact that they come and go.

Viral haemorrhagic septicemia virus (VHSV) is the most common virus among marine biological diseases. VHSV is a pathogen causing severe viral diseases infected with freshwater salmon and fish including rainbow trout. At present, VHSV and IHNV are designated as diseases controlled by the aquatic animal health regulations of the World Animal Health Organization (OIE), and they are classified as a legal infectious disease in Korea. If infectious diseases are detected, transportation restriction and disinfection measures It is a disease in which action is taken.

On the other hand, as the damage caused by VHSV spreads worldwide, efforts are being made to produce virus free stock in the seed production stage to avoid VHSV infection, but it is now known that it is impossible to completely block the virus. Therefore, there is a growing interest in research to minimize the spread of VHSV and minimize the damage caused by virus infection.

VHSV is a virus that can enter the aquaculture environment and cause diseases. In order to manage fishery resources, millions of marine species are released to the coast every year, so it is necessary to optimize virus detection methods in cultured fish and wild fish . Enzyme immunoassay, Western blotting, and electron microscopy for VHSV detection have difficulties in rapidly diagnosing latent and early viral diseases, and polyclonal antibodies have been reported to continue to have antibodies of the same nature And thus it is difficult to apply it to the VHSV detection test method.

In order to secure such disadvantages, it has been attempted to establish a test method for quantitative detection of viruses and virus removal test using PCR, but general PCR, which is an end-point analysis, There is a problem that it is difficult to quantitatively analyze it on an agarose gel.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a primer for detecting a viral hemorrhagic septic virus having high reproducibility, sensitivity and specificity, Thereby providing a probe set.

A second object of the present invention is to provide a kit for real-time PCR for detecting viral hemorrhagic sepsis virus.

A third problem to be solved by the present invention is to provide a method for detecting viral hemorrhagic sepsis virus having sensitivity and specificity for detecting rapidly or quantitatively.

In order to achieve the first object of the present invention, there is provided a primer set and a probe set for real-time PCR for detecting viral hemorrhagic sepsis virus, wherein the primer set comprises primers of SEQ ID NOS: And the probe set is a probe set including the probe of SEQ ID NO: 5. In the primer set,

Specifically, the present invention relates to an optimized real-time PCR method using a primer and a probe set. By performing optimized real-time PCR using the primer and the probe set, the presence or absence of a viral hemorrhagic sepsis virus can be rapidly It is possible to detect it accurately.

In the present invention, real-time PCR using a TaqMan probe was performed as a method for quickly and accurately detecting viral hemorrhagic sepsis virus infection.

That is, according to one embodiment of the present invention, a TaqMan real-time PCR condition was optimized by preparing a primer set capable of detecting a VHSV gene having high specificity and sensitivity (see Example 4) (see Example 6).

According to another embodiment of the present invention, the real-time PCR conditions such as annealing temperature and MgCl ₂ concentration were optimized using the selected primer and probe set, and the sensitivity of the established assay was 8.1010 ^-1 TCID ₅₀ / mL . In order to verify the reproducibility of the established VHSV assay, real-time PCR was performed on different days and the crossing point values were compared. As a result, the crossing point value (VHSV log titer log titer (log ₁₀ TCID ₅₀ / mL; x) y), the coefficient of determination (r2) was 0.99 or more, indicating that the regression coefficient was high as well as the reproducibility (see Example 7).

Through the above results, we performed optimized real-time PCR using a primer set consisting of the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 4 and a probe composed of SEQ ID NO: 5 to detect the viral hematopoietic sepsis virus And it was confirmed that there was an excellent effect for the purpose.

The term " primer " in the present invention refers to a nucleic acid sequence having a short free 3 'hydroxyl group capable of forming a base pair with a complementary template and serving as a starting point for template strand copying The primers can initiate DNA synthesis in the presence of reagents for polymerization and four different nucleoside triphosphates at the appropriate buffer solution and temperature.

The primer used in the present invention is hybridized or annealed at one site of the template to form a double-stranded structure. Conditions for nucleic acid hybridization suitable for forming such a double-stranded structure can be found in Nucleic Acid Hybridization, Hayes, BD, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, A Practical Approach, IRL Press, Washington, DC (1985).

The primer set of the present invention is used to identify and quantitate the presence of a specific virus using a PCR method, and a site having a conserved sequence in which the genetic mutation is not severe in the virus should be selected. The selected sequence should exist only in a specific virus and have high specificity.

A primer set for real-time PCR for detecting the viral hemorrhagic sepsis virus of the present invention is a primer set designed to detect viral hemorrhagic sepsis virus with high specificity and sensitivity, and is characterized in that a specific primer set for VHSV To detect the presence or absence of a virus, thereby providing excellent specificity and sensitivity for quantitative detection.

When PCR is performed with the primers having the nucleotide sequences of SEQ ID NOS: 1 and 2 and SEQ ID NOS: 3 and 4 provided in the present invention, viral hemorrhagic sepsis virus can be detected with high specificity and sensitivity. 'Sensitivity' refers to the rate at which a person with a disease is judged to have a disease, and 'specificity' means a rate at which a normal person is judged to be normal.

Specifically, as can be seen from Fig. 1, for the sensitivity measurement of a primer set for quantitative detection of VHSV As a result of SYBR Green Real-time PCR, VHSV-F2 and VHSV-R2 primer sets were found to be the most sensitive.

According to a preferred embodiment of the present invention, the probe may include a fluorescent substance at the 5 'end and the 3' end.

The term "probe" in the present invention is a single-stranded nucleic acid molecule and includes a sequence complementary to a target nucleotide sequence. The probe of the present invention can be modified to the extent that the hybridization specificity is not impaired. For example, a reporter fluorescent material or a quencher can be tagged to the end of the oligonucleotide that is the probe.

In the method using the real-time PCR reaction of the present invention, the probe uses a probe capable of complementarily binding to a partial sequence within the base sequence amplified by the primer set.

A reporter-fluorescent substance is tagged at the 5'-end of the probe used in the real-time PCR method of the present invention, and a quencher is tagged at the 3'-end.

In the present invention, the reporter fluorescent substance and the fluorescence inhibition substance are not limited to specific substances. For example, the reporter fluorescent substance may be 6-FAM, JOY, TET, 6-JOE, HEX, Cy3, Cy5, VIC, EDD, And BHQ-1, BHQ-2, BHQ-3, a dichroic dark fluorescence inhibiting substance or ROX can be used as the fluorescence inhibitor.

The probe of the present invention does not emit fluorescence at the 5'-terminal reporter fluorescent substance due to the action of the fluorescence inhibiting substance present at the 3'-terminal. However, the 5'-3'exonuclease activity of the Taq DNA polymerase in the next step of nucleic acid amplification, the extension step, degrades the probe hybridized to the template and the fluorescent substance at the 5'-end from the probe And the fluorescence inhibition by the fluorescence inhibiting substance is released to release the fluorescence.

Suitable lengths of the probes can be designed to have hybridization temperatures that are typically about 20-35 nucleotides and are about 5-10 higher than the hybridization temperature of the primers, depending on various factors such as primer hybridization temperature and length, The specificity can be increased.

According to another preferred embodiment of the present invention, the fluorescent substance at the 5'-terminal is selected from the group consisting of 6-carboxyfluorescein, hexachloro-6-carboxyfluorescein, tetrachloro-6-carboxyfluorescein, Tetrachloro-6-carboxyfluorescein, 5-carboxy fluorescein, HEX (2 ', 4', 5 ', 7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein) and Cy5 (cyanine-5), and the like.

According to another preferred embodiment of the present invention, the fluorescent substance at the 3 'end is selected from the group consisting of 6-carboxytetramethyl-rhodamine, TAMRA (5-Carboxytetramethylrhodamine), BHQ 1,2 and 3 black hole quencher 1, 2, 3).

In order to achieve the second object, the present invention provides a real-time PCR reaction kit for detecting viral hemorrhagic sepsis virus comprising the primer and the probe set as an active ingredient.

The composition of the kit may be prepared by a kit including, but not limited to, a primer having a sequence of the present invention and a probe, and a primer and a probe as an internal control. In order to detect and quantify viral hemorrhagic sepsis virus, , Potassium chloride, magnesium chloride or dNTP, and the like.

When the kit of the present invention is applied to a PCR amplification process, the kit of the present invention may optionally comprise a reagent necessary for PCR amplification, such as a buffer, a DNA polymerase (e.g., Thermus aquaticus (Taq), Thermus thermophilus a heat-stable DNA polymerase obtained from a DNA polymerase, filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu), DNA polymerase joinder, dNTP mixture consisting of dATP, dCTP, dGTP, dTTP and dUTP and UDG (Uracil DNA Gylcosylase) can do.

In order to achieve the third object, the present invention provides a method for detecting viral hemorrhagic sepsis virus comprising the step of detecting a viral hemorrhagic sepsis virus gene of a separated biological sample using the primer and the probe set do.

The detection method of the present invention can be applied to a sample expected to be infected with a viral hemorrhagic sepsis virus. In the present invention, the sample to be detected is preferably a biological sample, preferably a viral hemorrhagic sepsis virus, a tissue suspected of being infected or a viral hemorrhagic sepsis virus vaccine, tissues, cells, whole blood, But it is not limited thereto, and more preferably, it is a sample obtained from the blood of a suspect individual of viral hemorrhagic sepsis virus.

Specifically, the viral hemorrhagic septicemia virus (VHSV) is a fish infectious virus belonging to Rhabdoviridae and Novirhabdovirus which has negative single strand RNA and has an optimum temperature of 9 to 12, (Smail, 1999; OIE).

The target fishes to be detected include Paralichthys olivaceus, Seriola quinqueradiata, Pagrus major, Takifugu rubripes, Seriola aureovittata, trout (eg Oncorhyncus nykiss), Seriola dumerili, Such as Pseudocaranx dentex, Epinephelus septemfasciatus, Thunnus thynnus, Cyprinus carpio, Danino rerio, Catfish (e.g., Clarias gariepinus), Tilapia (Oreochronis niloticus), Salmon Salmo Salar or Oryzias latipes.

Since the present invention detects the presence or absence of a virus using specific primers and probe sets for viral hemorrhagic septicemia virus (VHSV), the specificity for quantitative detection is excellent. In addition, it is possible to detect pathogens in a short time, has high detection sensitivity, and can analyze a large number of samples in a short time, thereby saving time and expense as compared with a biological test method.

FIG. 1 is a graph showing sensitivity of a primer set for quantitative detection of VHSV. FIG. 1 (A) is a graph of SYBR Green Real-time PCR and FIG. 1 (B) The results of the melting curve analysis are shown in Fig.
8.10 x 10 ¹ TCID ₅₀ / mL,?: 8.10 x 10 ³ TCID ₅₀ / mL,?: 8.10 x 10 ² TCID ₅₀ / mL,?: 8.10 x 10 ¹ TCID ₅₀ / ⁰ TCID ₅₀ / mL)
FIG. 2 shows the results of verification of the reliability of the TaqMan probe real-time PCR for quantitative detection of VHSV. The crossing point value (y) for (A) sensitivity and (B) VHSV log titer (log10 TCID ₅₀ / ) As a result of the measurement.
FIG. 3 is a graph showing the reproducibility of the TaqMan probe real-time PCR for quantitative detection of VHSV.
Fig. 4 is a graph showing the results of verifying the specificity of the TaqMan probe real-time PCR for quantitative detection of VHSV.
Bovine herpesvirus type 1 (BHV), Bovine parvovirus (BPV), Bovine viral diarrhea virus (BVDV), Mimute virus of mice (MVM), Bovine parainfluenza3 virus (BPIV-3 ), Porcine rotavirus (ProV), Reovirus-3 (REO-3), Hepatitis A virus (HAV), Porcine epidemic diarrhea virus (PEDV), Transmissible gastroenteritis coronavirus (TGEV), Bovine adenovirus ), Infectious hematopoietic necrosis virus (IHNV), Infectious Pancreatic Necrosis Virus (IPNV), Negative control)

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

[Example]

Example 1: Culture of viral hemorrhagic sepsis virus (VHSV)

Epithelial papilloma of carp (EPC) cells (purchased from Prof. Park, Jeongwoo professor, University of Ulsan) were used for culture and quantification of VHSV (from the laboratory of Professor Park, Jeongwoo University of Ulsan). EPC cells were cultured in CO ₂ incubator 5% CO ₂ , 19 with a minimal medium (Minimum Essential Medium (MEM: WELGENE, Korea)) supplemented with 10% bovine serum (Gibco BRL, USA) The cytopathic effect (CPE) was observed periodically after infecting the monocytic cells cultured on the T-150 flask with the virus. When the CPE was clearly observed, the culture medium and cells were collected, and centrifuged at 2000 rpm for 3 minutes to separate the supernatant and re-suspend the pellet. The pellet was disrupted by freezing and thawing twice, followed by centrifugation at 400 xg for 5 minutes to obtain supernatant. The centrifuged supernatant was mixed, filtered through a 0.45 m filter, and subdivided and stored at 70 ° C.

Example 2: Quantification of VHSV

The titer of the infectious virus was expressed as 50% tissue culture infectious dose (TCID ₅₀ ) for quantification of VHSV. VHSV was diluted 7 times with MEM medium supplemented with 2% fetal bovine serum (FBS) and 0.25 mL was inoculated into cells cultured on a 24-well plate. As a negative control, 0.25 mL of cell culture medium was inoculated. The cells were then cultured in CO ₂ incubator at 5% CO ₂ , 14, and CPE was continuously observed under a microscope.

Example 3: RNA isolation of VHSV

The virus culture solution stored at -70 was dissolved in ice, and RNA was isolated. RNA isolation was performed using the QuickGene RNA tissue kit S (KURABO INDUSTRIES LTD, Japan) according to the manufacturer's instructions. A total of 100 L of RNA was eluted from 150 L of virus culture.

Example 4: Selection of primer sets for quantitative detection of VHSV

4-1. Design of primer

The oligonucleotide primers and probe sequences used to amplify the VHSV gene were amplified using Primer3 Input 4.0 software (Applied Biosystems) based on the VHSV isolate FYeosu05 complete genome (KF477302.1) reported in the NCBI data base as shown in Table 1 Two pairs of VHSV specific primers were designed.

Name of primer and probe Base sequence SEQ ID NO: Location of the base sequence Amplification product size (bp) VHSV-1-F ACC TCA TGG ACA TCG TCA AGG One 534 - 634 ^a 101 VHSV-1-R CTC CCC AAG CTT CTT GGT GA 2 VHSV-2-F ACT TGG AGG ACC CCA GAC TG 3 331 - 510 ^a 180 VHSV-2-R AGT TCC CTC ATC GGC ATA AG 4

* Genbank no .: KF477302.1

4-2. SYBR Green Real-time RT-PCR for primer selection

SYBR Green Real-time RT-PCR was performed to select an optimal primer among the two pairs of primers prepared in Example 4-1.

CDNA was synthesized using Topscript cDNA Synthesis Kit (Enzynomics, KOREA) with the RNA isolated by the method of Example 3 as a template. For the synthesis of cDNA, 10 l of template RNA and 2 l of 10 pmol specific primer (reverse primer) were mixed and reacted at 70 for 5 min. Then, 2 L of TOPscript ™ RT buffer, 1 L of TOPscript ™ Reverse Transcriptase, 2 L of dNTP mixture, Inhibiter 0.5 L and distilled water 2.5 L were mixed and reacted at 50 ° C. PCR was performed using the cDNA obtained by the reverse transcription reaction of VHSV as a template. 6 L of Nuclease free water (Promega, USA) was added to 2 L of VHSV cDNA, 1 L of 10 pmol forward primer, 1 L of 10 pmol reverse primer and 10 L of SYBR green Realtime PCR Master Mix (TOYOBO, Japan) To a final volume of 20L. Nucleic acid amplification using a StepOnePlus real-time PCR system from Applied Biosystems, Inc. was performed at 95 cycles of 10 minutes for pre-incubation, 95 seconds for denaturation for 20 seconds, and 30 cycles of annealing for 30 cycles. After the last cycle, melting curve analysis was performed in the range of 72 to 95 for all reactants. DNA sequencing of the PCR products was performed to confirm that the amplified DNA was the desired product.

4-3. Sensitivity measurement of selected primer sets

SYBR Green Real-time PCR was performed to confirm the sensitivity of each primer set. Primer set was diluted 10-fold in succession, and SYBR Green Real-time PCR was performed with VHSV having a Titer of 10 ⁴ TCID ₅₀ / mL, 10 ³ TCID ₅₀ / mL, 10 ² TCID ₅₀ / mL, and 101 TCID ₅₀ / mL as a sample And the results are shown in Fig.

As shown in Fig. 1, the sensitivity of VHSV-F2 and VHSV-R2 among the primer sets was the highest.

Example 5: VHSV probe design

TaqMan probe In order to perform real-time PCR, the probe was designed to have FAM (5-carboxy fluorescein) at the 5 'end and black hole quencher 1 (BHQ1) as the quencher at the 3' end.

Name of primer and probe Base sequence SEQ ID NO: Location of the base sequence VHSV-2-probe FAM - GAG ATC TGG AGG CAA AGT GC - BHQ1 5 402 - 421 ^a

* Genbank no .: KF477302.1

Example 6: VHSV detection test using TaqMan probe real-time PCR

Real-time RT-PCR conditions were established using Premix Ex Taq (Takara, Japan) to optimize the real-time PCR for VHSV quantitation.

6-1. TaqMan probe for VHSV detection Optimal annealing temperature for real-time polymerase chain reaction

Real-time RT-PCR conditions were established using Premix Ex TaqTM (TaKaRa, Japan) for VHSV quantitation. For VHSV quantitation, a StepOnePlus real-time PCR system from Applied Biosystems was used. For real-time PCR reaction, 5.6 l of Nuclease free water (Promega, USA) was added to 2 L of VHSV cDNA, 1 L of 10 pmol forward primer, 1 L of 10 pmol reverse primer, 0.4 L of ROX and 10 L of Premix Ex Taq The final volume was adjusted to 20 L. Nucleic acid amplification was performed by pre-incubation at 95 ° C for 10 min, denaturation at 95 ° C for 3 sec, annealing for 30 sec (real-time PCR at 52, 54, 56, 58 and 60 for annealing temperature optimization) Respectively.

As can be seen in FIG. 2, the annealing temperature of the PCR reaction was optimized using VHSV-F2 as the forward primer and VHSV-R2 as the reverse primer.

As a result, VHSV having a Titer of 10 ³ TCID ₅₀ / mL, 10 ² TCID ₅₀ / mL, 10 ¹ TCID ₅₀ / mL, and 10 ⁰ TCID ₅₀ / mL was used as a sample and the annealing temperature was 52, The real time RT-PCR was carried out with the TaqMan probe for VHSV detection because the cycle threshold value according to the temperature change was not large and the nonspecific reaction of the primer was lowered as the temperature increased. Optimal annealing temperature was set at 60 ℃ for polymerase chain reaction.

TCID ₅₀ / mL Annealing temperature 52 54 56 58 60 8.1010 ³ 24.41 ^a 24.06 24.84 24.82 24.71 25.13 25.29 24.91 23.73 24.02 8.1010 ² 27.64 27.62 27.71 27.79 27.61 28.19 28.46 28.26 27.45 27.63 8.1010 ¹ 30.96 30.88 31.55 31.04 31.17 31.46 31.87 31.60 30.45 30.67 8.1010 ⁰ 34.62 34.75 35.02 34.66 34.51 35.07 34.91 35.43 34.34 34.04 Negative control N / A ^b N / A N / A N / A N / A N / A N / A N / A N / A N / A

^a Values indicating cycle threshold (Ct) value

^b N / A, Not Applicable; real-time PCR signals were not detected

6-2. TaqMan probe for VHSV detection Optimal chloride concentration in real-time PCR

TaqMan probe for VHSV detection The optimal threshold for the MgCl2 concentration in the real-time PCR was 60 mM MgCl ₂ at 1 mM, 2 mM, and 3 mM at an annealing temperature of 60 ° C. Respectively.

And then with the sample to be measured Titer Titer dilution 10 ⁴ TCID ₅₀ / a mL of VHSV in sequence 10 ⁰ TCID ₅₀ / mL to 10-fold, the standard curve for quantification by performing real-time RT-PCR Respectively. The amount of VHSV RNA in the sample was quantified by substituting the standard curve. The standard curve was prepared by converting the cycle threshold value detected by real-time PCR to TCID ₅₀ equivalent / mL according to the concentration of VHSV. The cycle threshold represents the number of cycles in which the PCR cycle enters the exponential phase.

As shown in Table 4 below, the PCR conditions were optimized by varying the MgCl ₂ concentration at the optimal annealing temperature.

As a result, as shown in Table 4, Titer is _{^{10 3 TCID 50 / mL, 10}} 2 TCID 50 / mL, 10 1 TCID 50 / mL, 10 0 TCID / mL of VHSV sample to 1 mM of MgCl ₂ concentration, 2 mM and 3 mM, respectively, and no difference in cycle threshold was observed between real-time RT-PCR and TaqMan probe for detection of VHSV by the absence of MgCl ₂ . The optimum MgCl ₂ concentration was set.

TCID ₅₀ / mL MgCl ₂ concentration Control 1 mM 2 mM 3 mM 8.1010 ³ 23.73 ^a 24.02 24.87 24.94 24.02 24.37 23.73 23.66 8.1010 ² 27.45 27.63 27.93 28.50 27.55 27.42 26.45 26.87 8.1010 ¹ 30.45 30.67 31.18 31.72 30.71 30.66 30.30 30.17 8.1010 ⁰ 34.34 34.04 34.53 31.72 33.87 33.24 34.28 34.27 Negative control N / A ^b N / A N / A N / A N / A N / A N / A N / A

^a Values indicating cycle threshold (Ct) value

^b N / A, Not Applicable; real-time PCR signals were not detected

Example 7 Verification of Reliability of Real-Time Polymerase Chain Reaction for VHSV Quantification

The sensitivity, reproducibility, detection limit, specificity and specificity of established laboratory methods to ensure the reliability of the real-time PCR detection assays for established VHSV quantitation The robustness was verified.

7-1. Sensitivity verification

To measure the sensitivity of the detection test method, real-time PCR was performed after diluting the VHSV 10-fold in succession. The results are shown in Fig.

As shown in FIG. 2, increase in fluorescence according to the number of real-time PCR cycles was observed for each sample. As a result, it was confirmed that the sensitivity was 8.1010 ^-1 TCID ₅₀ / mL (FIG. 2A), VHSV log titer _The coefficient of determination (r2) between the crossing point values (y) for ₁₀ TCID ₅₀ / mL; x was found to be highly regressive between the 0.994 VHSV log titer and the crossing point value (FIG. 2B).

7-2. Reproducibility Verification

To verify the reproducibility of established VHSV assays, RNA was extracted from VHSV standard samples on different days and real-time RT-PCR was performed and the cycle threshold values were compared. The results are shown in Fig.

As shown in FIG. 3, the standard regression equation between the crossing point values (y) for the VHSV log titer (log ₁₀ TCID ₅₀ / mL; x) is y = -3.250x + 33.811 for the first day (determination coefficient r ² = 0.994 ), for a second day, ^{y = -3.512x + 33.845 (r 2} = 0.993), when the third day of ^{y = -3.266x + 33.328 (r 2} = 0.996), the fourth day for y = -3.325x + 33.716 ( r ² = 0.997), the fifth day for y = -3.390x + 34.759 (r2 = 0.993), when the sixth day ^{y = -3.201x + 333.676 (r 2} = 0.992), when the seventh day of y = -3.370x +34.637 (r ² = 0.996), when the eighth day of the found to be ^{y = -3.388x + 34.793 (r 2} = 0.998).

Therefore, the standard regression equation between the crossing point values (y) for the VHSV log titer log titer (log ₁₀ TCID _{50 /} mL; x) after the real-time PCR was compared with the determination coefficient r ² ) were not less than 0.99 and showed high reproducibility as well as reproducibility.

7-3. Verification of detection limit

VHSV cDNA, 8.1010 ⁰ TCID ₅₀ / mL, was diluted 4-fold with 2-fold to verify the detection limit of the detection test method. Diluted 8.1010 ⁰ TCID ₅₀ / mL, 4.0510 ⁰ TCID ₅₀ / mL, 2.0310 ⁰ TCID ₅₀ / mL, 1.0110 ⁰ TCID ₅₀ / mL and 5.0110 ^-1 TCID ₅₀ / mL were repeatedly tested for 8 days.

As shown in Table 5 below, it was confirmed that the detection limit was 1.0110 ⁰ TCID ₅₀ / mL.

Run TCID ₅₀ / mL Detection limit
(Limit of detection) 8.10 x 10 ⁰ 4.05 x 10 ⁰ 2.03 x 10 ⁰ 1.01 x 10 ⁰ 5.01 x 10 ^-1 One + + + + + + + + + + + + - - + 1.01 x 10 ⁰ 2 + + + + + + + + + + + + + - + 1.01 x 10 ⁰ 3 + + + + + + + + + + + + - + + 1.01 x 10 ⁰ 4 + + + + + + + + + + + + + + + 5.01 x 10 ^-1 5 + + + + + + + + + + + + + + + 5.01 x 10 ^-1 6 + + + + + + + + + + + + + + + 5.01 x 10 ^-1 7 + + + + + + + + + + + + + + + 5.01 x 10 ^-1 8 + + + + + + + + + + + + + + + 5.01 x 10 ^-1

7-4. Specificity verification

(IHNV, IPNV) and other viruses (BHV, BPV, BVDV, BPIV-3, PRoV, PRV, PEDV, MVM, Reo-3, HAV, TGEV and bADV) Cross-reactivity was measured in the subjects.

As can be seen from FIG. 4, the specificity of the other fish viruses was tested. As a result, the increase of the fluorescence value was observed only in the case of VHSV, and in the other viruses, the increase of the fluorescence value I could not observe it. These results confirm that the real-time PCR method is an experimental method specific to VHSV.

7-5. Waning validation

Comparison of primer manufacturer and PCR instrument was performed for the waning test of the detection test method.

As shown in Table 6 below, in order to verify the solubility of the primers, three companies were prepared with the same base sequences as the VHSV primers, and 8.1010 ³ TCID ₅₀ / mL, 8.1010 ² TCID ₅₀ / mL, 8.1010 ¹ TCID ₅₀ / mL, and 8.1010 ⁰ TCID ₅₀ / mL.

As a result, there was no difference in the cycle threshold value among the three companies.

TCID ₅₀ / mL Ct values obtained with different primer sets Vendor Vendor B Vendor C Mean of Cp SD of Cp CV
(%) 8.1010 ³ 23.37 ^a 23.76 23.89 24.16 24.56 24.33 25.36 24.79 25.04 24.36 0.605 2.481 8.1010 ² 27.55 27.45 27.54 28.06 27.96 27.92 28.28 28.30 28.42 27.94 0.340 1.218 8.1010 ¹ 30.87 30.86 30.95 31.30 31.21 31.37 31.76 31.59 31.74 31.29 0.334 1.067 8.1010 ⁰ 34.47 34.40 33.99 34.72 34.28 34.82 34.38 34.83 34.82 34.52 0.277 0.803 8.1010 ^-1 36.74 37.89 36.99 36.25 37.39 36.74 36.69 36.20 36.58 36.83 0.505 1.371 Negative Control N / A ^b N / A N / A N / A N / A N / A N / A N / A N / A

^a Values indicating cycle threshold (Ct) value

^b N / A, Not Applicable; real-time PCR signals were not detected

CV (%): Coefficient of variance % = (SD of Cp / Mean of C t) x 100

Vendor A: Bioneer, Vender B: Neoprobe, Vender C: Macrogen

Real-time PCR was performed using three companies to compare PCR devices.

As a result, as shown in Table 7, in the case of AB7500 (applied biosystems, USA) and SteponeplusTM (applied biosystems, USA) has been detected up to _{^{8.1010 -1 TCID 50 / mL, EcoTM}} (illumina, USA) is 8.1010 ^-1 One TCID ₅₀ / mL was not detected in any of the three samples, indicating that the sensitivity was lower than that of the other instruments. There was no difference in cycle threshold values between AB7500 (applied biosystems, USA) and StepOnePlusTM Real-time PCR System (Applied Biosystems, USA).

TCID ₅₀ / mL Ct values obtained with different primer sets Vendor Vendor B Vendor C Mean of Ct SD of Ct CV
(%) 8.1010 ³ 25.10 ^a 24.73 24.16 24.09 24.11 23.75 24.66 24.56 24.68 24.43 0.398 1.629 8.1010 ² 28.34 29.09 28.73 27.14 27.42 27.01 28.10 28.11 27.94 27.99 0.660 2.357 8.1010 ¹ 31.95 32.05 31.47 30.35 30.55 30.35 31.36 31.41 31.35 31.20 0.607 1.946 8.1010 ⁰ 35.77 35.58 33.10 33.80 33.51 33.98 34.96 34.90 34.40 34.44 0.871 2.530 8.1010 ^-1 38.96 38.47 N / A 36.69 36.45 37.24 36.57 37.27 36.19 37.23 0.932 2.503 Negative Control N / A ^b N / A N / A N / A N / A N / A N / A N / A N / A

^a Values indicating cycle threshold (Ct) value

^b N / A, Not Applicable; real-time PCR signals were not detected

CV (%): Coefficient of variance% = (SD of Ct / Mean of C t) x 100

^{Vender A: Eco TM (illumina,} USA), Vender B: AB7500 (applied biosystems, USA), Vender C: Steponeplus TM (applied biosystems, USA)

In general, the present invention relates to a TaqMan probe real-time PCR test method capable of rapidly and quantitatively detecting VHSV. In the virus detection test method, the sensitivity and reproducibility are very important with the accuracy of analysis, , And it was confirmed that the quantitative method established in the present invention is excellent in sensitivity and reproducibility in quantifying VHSV.

In addition, the VHSV quantitative assay method established by the present invention is an excellent method for quickly diagnosing latency and viral diseases in the early stage of infection, and is a substitute for the existing test method for detecting viruses in cultured fish and wild fish The effect can be excellent.

<110> Hannam University Institute for Industry-Academia Cooperation <120> Real-time PCR assays for quantitative detection of Viral          hemorrhagic septicemia virus <130> 20150429 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> forword primer for VHSV-1 <400> 1 acctcatgga catcgtcaag g 21 <210> 2 <211> 20 <212> DNA <213> Reverse primer for VHSV-1 <400> 2 ctccccaagc ttcttggtga 20 <210> 3 <211> 20 <212> DNA <213> forword primer for VHSV-2 <400> 3 gagatctgga ggcaaagtgc 20 <210> 4 <211> 20 <212> DNA <213> reverse primer for VHSV-2 <400> 4 agttccctca tcggcataag 20 <210> 5 <211> 20 <212> DNA <213> probe for VHSV-2 primer set <400> 5 gagatctgga ggcaaagtgc 20

Claims (7)

A primer set and a probe set for real-time PCR reaction for detecting viral hemorrhagic sepsis virus, wherein the primer set is a primer set comprising primers of SEQ ID Nos: 1 to 4, and the probe set comprises a probe of SEQ ID NO: 5 Wherein the probe set is a probe set.
The method according to claim 1,
Wherein the virus is a fish infectious virus.
The method according to claim 1,
Wherein the probe comprises a fluorescent substance at a 5 'end and a 3' end.
The method according to claim 1,
The fluorescent substance at the 5'-terminal is selected from the group consisting of 6-carboxyfluorescein, hexachloro-6-carboxyfluorescein, tetrachloro-6-carboxyfluorescein, -carboxyfluorescein, 5-carboxy fluorescein, HEX (2 ', 4', 5 ', 7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein) and Cy5 (cyanine-5) A set of real-time polymerase chain reaction primers for detecting viral hemorrhagic septicemia virus, which is a fluorescent substance.
The method according to claim 1,
The fluorescent substance at the 3 'end was composed of 6-carboxytetramethyl-rhodamine, TAMRA (5-Carboxytetramethylrhodamine), BHQ 1, 2 and 3 (black hole quencher 1, 2, Wherein the primer set is a real-time PCR primer set for detecting a viral hemorrhagic sepsis virus, which is a fluorescent substance selected from the group consisting of:
A kit for real-time polymerase chain reaction for detecting viral hemorrhagic sepsis virus comprising the primer and probe set of any one of claims 1 to 5 as an active ingredient.
A method for detecting a viral hemorrhagic sepsis virus comprising the step of detecting a viral hemorrhagic sepsis virus gene of a separated biological sample using the primer and the probe set of any one of claims 1 to 5.

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