KR102172810B1 - A composition for simultaneous detecting of H5 highly pathogenic avian influenza virus clades and uses thereof - Google Patents

Abstract

The present invention relates to: a composition for simultaneous detection of H5 highly pathogenic avian influenza (HPAI) virus, and clades of H5 HPAI; and uses thereof. The composition for detecting HPAI according to the present invention is capable of detecting three influenza virus characteristics, which are avian influenza virus confirmation, high pathogenic verification, and H5 HPAI clade verification, in a single reaction by using a small amount of samples. The composition according to the present invention can quickly and accurately detect avian influenza virus, as well as can quickly identify information about clades with high specificity and sensitivity, thereby being able to be usefully used for diagnosis of avian influenza virus infection and more efficient disinfection response to HPAI.

Description

A composition for simultaneous detecting of H5 highly pathogenic avian influenza virus clades and uses thereof}

The present invention relates to a composition for simultaneous detection of H5 HPAI, as well as detection of a highly pathogenic avian influenza A virus (HPAI) virus, and a use thereof, and in more detail, hemagglutinin ( HA) Real-time PCR based real-time PCR capable of simultaneously detecting H5 HPAI and the H5 HPAI strains of 2.3.4.4 and 2.3.2.1c, which are currently prevalent in a small amount of samples, using conserved protein sequences. It relates to a marker for molecular diagnosis.

Avian Influenza (AI) virus is classified into Low Pathogenic Avian Influenza (LPAI) and Highly Pathogenic Avian Influenza (HPAI) according to pathogenicity, and HPAI is caused only by H5 and H7 types. . In Korea, H5N1 type HPAI occurred four times from the first occurrence in 2003 to 2010, the first H5N8 type HPAI occurred in 2014, and in 2016, H5N6 and H5N8 type viruses were simultaneously prevalent. In particular, the 2.3.4.4 clade virus, which occurred in 2014 and 2016, occurred on a larger scale than before, causing enormous economic damage.

The avian influenza virus was originally known to not be transmitted directly from birds to humans, but after the outbreak of bird flu in Hong Kong in 1997, various cases of avian influenza infection have been reported in the Netherlands in 2003 and Vietnam and Thailand in 2004. HPAI has a mortality rate of more than 60% when infected with humans, and if it becomes possible to spread from person to person, it can lead to an influenza pandemic such as the Spanish flu of 1918, which has a very high public health risk. In addition, highly pathogenic avian influenza viruses have a high mutation rate and are evolving into various phylogenetic groups, so there is a limit to distinguishing them genetically and serologically. Therefore, in order to minimize damage that may be caused by the avian influenza virus, there is an urgent need to develop a method capable of rapidly detecting avian influenza virus and further discriminating various strains at the same time.

Currently, in Korea, virus detection and identification using reverse transcriptase-polymerase chain reaction (RT-PCR) techniques are being conducted for faster detection of avian influenza virus, and a recommended test by the World Animal Health Organization. Antigen detection methods such as hemagglutination test, serotype identification, and pathogenicity identification after inoculation, which are methods, are used in parallel. However, there is no commercialized product that can simultaneously diagnose the 2.3.4.4 and 2.3.2.1c strains of H5 HPAI and H5 HPAI, which are frequently introduced into Korea. In addition, conventional influenza virus multiple detection methods use primers/probe sets suitable for each target, and information on the phylogeny can be confirmed through gene sequencing analysis, but as a number of primers are used, primers There is a disadvantage in that the sensitivity and specificity of detection are degraded due to mutual interference and competition between the two, and it takes an enormous amount of time and cost according to gene sequencing analysis.

Therefore, a new molecular diagnosis technique that can detect avian influenza virus by a small sample and a simple method, and detect different phylogenies, including H5 HPAI 2.3.4.4 and 2.3.2.1c, which are the most prevalent in Korea, at the same time. I need this.

The present inventors secured the HA gene sequences of the 2.3.4.4 and 2.3.2.1c phylogenies of the domestic and foreign HPAI H5 subtypes, and based on this, one primer set was used and the 2.3.4.4 and 2.3.2.1c phylogenetic group specific probes were obtained. The present invention was completed by confirming that it is possible to quickly detect different phylogenies, including the 2.3.4.4 and 2.3.2.1c phylogenies of H5 HPAI, without gene sequencing.

Accordingly, the object of the present invention is (a) a primer pair of SEQ ID NOs: 1 and 2 for specifically amplifying the HA gene of H5 avian influenza virus; And (b) a probe of SEQ ID NOs: 3 to 5; It is to provide a composition for detecting a virus containing H5 highly pathogenic avian influenza (H5 highly pathogenic avian influenza A virus, HPAI) virus.

Another object of the present invention is to provide a simultaneous detection kit capable of simultaneously detecting different strains of H5 highly pathogenic avian influenza A virus (HPAI) virus comprising a primer pair and a probe according to the present invention. To provide.

Another object of the present invention is to use a reverse transcriptase chain reaction (RT-PCR), using a primer pair and a probe according to the present invention to each other of H5 highly pathogenic avian influenza A virus (HPAI) viruses. It is to provide a method of simultaneously detecting different phylogenies.

In order to achieve the above object, the present invention (a) a primer pair of SEQ ID NOs: 1 and 2 for specifically amplifying the HA gene of H5 avian influenza virus; And (b) a probe of SEQ ID NOs: 3 to 5; It provides a composition for detecting a virus containing H5 highly pathogenic avian influenza (H5 highly pathogenic avian influenza A virus, HPAI) virus.

In addition, the present invention provides a kit for simultaneous detection that can simultaneously detect different strains of H5 highly pathogenic avian influenza A virus (HPAI) virus comprising a primer pair and a probe according to the present invention. .

In addition, the present invention uses reverse transcription polymerase chain reaction (RT-PCR), using the primer pair and probe according to the present invention, H5 highly pathogenic avian influenza (H5 highly pathogenic avian influenza A virus, HPAI) different strains of virus It provides a method of simultaneously detecting groups.

The composition for detecting H5 highly pathogenic avian influenza virus according to the present invention is capable of verifying three kinds of influenza virus characteristics: avian influenza virus confirmation, highly pathogenicity verification, and H5 HPAI family group verification with a single reaction using a small sample. The composition according to the present invention not only enables rapid and accurate detection of avian influenza virus, but also allows rapid and high specificity and sensitivity to grasp information on the phylogenetic group. It can be usefully used for

1 is a diagram showing the results of verifying the specificity of the composition for detection according to the present invention for the 2.3.4.4 and 2.3.2.1c phylogenies of H5 HPAI and H5 HPAI subtypes using recombinant DNA.
Figure 2 is a diagram showing the sensitivity verification results of the composition for detection according to the present invention for the 2.3.4.4 and 2.3.2.1c phylogenetic groups of H5 HPAI and H5 HPAI subtypes using recombinant DNA.
3 is a verification of the specificity of the composition for detection according to the present invention for the 2.3.4.4 and 2.3.2.1c phylogenies of H5 HPAI and H5 HPAI subtypes using various animal-derived influenza viruses and highly pathogenic and low pathogenic avian influenza viruses. It is a diagram showing the results.
4 is a diagram showing the results of confirming the specificity of the composition for detection according to the present invention by performing agarose gel electrophoresis.
5 is a result of verification of the sensitivity of the detection composition according to the present invention for the 2.3.4.4 and 2.3.2.1c phylogenies of H5 HPAI and H5 HPAI subtypes using various animal-derived influenza viruses and highly pathogenic and low pathogenic avian influenza viruses. Is a diagram showing.
6 is a diagram showing the results of verifying the specificity of the composition for detection according to the present invention for the 2.3.4.4 and 2.3.2.1c phylogenies of H5 HPAI and H5 HPAI subtypes using outdoor clinical samples.
7 is a composition for detection according to the present invention for the 2.3.4.4 and 2.3.2.1c phylogenies of H5 HPAI and H5 HPAI subtypes identified using field clinical samples using an Influenza Research Database (IRD). This is a diagram confirming that the specificity verification result of and the gene sequencing analysis result are identical.

Hereinafter, the present invention will be described in detail.

The present invention (a) a primer pair of SEQ ID NOs: 1 and 2 for specifically amplifying the HA gene of H5 avian influenza virus; And (b) a probe of SEQ ID NOs: 3 to 5; It provides a composition for detecting a virus containing H5 highly pathogenic avian influenza (H5 highly pathogenic avian influenza A virus, HPAI) virus.

In the present invention, the term "avian influenza virus" means a virus that can cause bird flu in chickens, ducks, wild birds, and the like. The virus has a variety of serotypes, such as 16 types of HA and 9 types of NA, depending on the genes of HA and NA in the outer membrane, and it is known that there is no cross-protection between different serotypes. It is known that all of the highly pathogenic avian influenza (HPAI) occurring to date are caused by H5 or H7 serotypes. The avian influenza virus can also infect humans through contact with infected birds, and it is known that the main infection vector is excrement of infected birds. When avian influenza virus infects humans, respiratory symptoms such as cough and shortness of breath may be accompanied by symptoms throughout the body such as fever, chills, and muscle pain, and gastrointestinal symptoms such as diarrhea, headache, and loss of consciousness. Symptoms related to the central nervous system may be accompanied, and in some cases, only gastrointestinal symptoms or symptoms related to the central nervous system appeared without respiratory symptoms.

In the present invention, the term "clade" refers to a classification criterion determined to distinguish viral strain branches, and the present invention is different from each other including H5 HPAI 2.3.4.4 and 2.3.2.1c phylogenies that are popular at home and abroad. An object of the present invention is to provide a composition for simultaneous detection of phylogenetic groups.

Specifically, in the 2.3.4.4 lineage group C, the H5N6 type virus, which belongs to group C, was recently isolated from poultry such as quail, and has high homology with viruses found in wild birds in East Asia such as Korea and Japan from 2016 to 2017. It has been reported that there is a possibility that the H5N6 virus occurring in China could be introduced into Korea through wild birds as in 2016. 2.3.2.1c The phylogenetic group shows genetic differences depending on whether the segregation area is in the southeast of China (Yunnan, Hubei, Zhejiang, etc.) or the northeast (Henan, Zhejiang, Jiangsu, etc.), of which the northeastern region of China is between Korea and South Korea. It has been reported that there is a possibility that the virus could be introduced directly due to the existence of a migration path for migratory birds.

The H5 HPAI virus of the 2.3.4.4 and 2.3.2.1c lineage groups has been introduced into Korea through wild birds, and many cases of infection with wild birds have been reported to date, and there is a limit to distinguishing them genetically and serologically. There is a need for a new molecular diagnosis technique that can detect this at the same time.

Accordingly, the composition for detecting highly pathogenic H5 avian influenza virus according to the present invention is characterized in that it comprises one primer set and a probe for each of the H5 HPAI and H5 HPAI 2.3.4.4 and 2.3.2.1c phylogenies, which are frequently introduced into Korea. .

First, the primer set was composed of forward and reverse primers based on the HA gene sequence (conservative sequence) of the domestic and foreign HPAI H5 2.3.4.4 and 2.3.2.1c phylogenies. Specifically, the forward nucleotide sequence is 5'-ACC CAG CCA ATG ACC TYT GT-3' (SEQ ID NO: 1), and the reverse nucleotide sequence is 5'-GRT AAG CCA YAC CAC ATT TCT G-3' (SEQ ID NO: 2) It consisted of. Here, Y is T/U or C, and R is G or A.

Next, in the case of the probe, based on the conserved nucleotide sequence of each phylogenetic group, it was composed of polynucleotides matching a certain nucleotide sequence. Specifically, the 2.3.4.4 lineage detection probe is 5'- TAC CCA GGG AVC CTC AAT GA-3' (SEQ ID NO: 3), and the 2.3.2.1c lineage detection probe is 5'- TCC CTG GTA TGA ACA TGC TGC GCT- 3'(SEQ ID NO: 4), H5 HPAI detection probe was composed of 5'- TGA RGA AYT GAA ACA CCT ATT GAG CAG- -3' (SEQ ID NO: 5), and 5'of the corresponding nucleotide sequence Labels used for detection were further conjugated to the and 3'ends, respectively. Where V is A or G or C.

In the present invention, the term "marker" refers to a specific nucleotide sequence that can be diagnosed by dividing H5 HPAI and H5 HPAI family groups of 2.3.4.4 and 2.3.2.1c. The detection composition according to the present invention is tested according to the hybridization of the PCR amplification product obtained by performing PCR amplification using the primer pairs of SEQ ID NOs: 1 and 2 on the H5 avian influenza virus gene sample and the probes of SEQ ID NOs: 3 to 5 It can accurately confirm the target avian influenza virus, high pathogenicity, and H5 HPAI strain diagnosis with high sensitivity.

In the present invention, the term "primer" is a nucleic acid sequence having a short free 3'hydroxyl group, which can form a complementary template and a base pair, and is the starting point for copying the template strand It refers to a short nucleic acid sequence that functions as. Primers can initiate DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer and temperature. The PCR conditions, the length of the sense and antisense primers can be modified based on those known in the art.

In the present specification, the term "primer set" refers to a set consisting of a forward primer and a reverse primer used to amplify a desired gene through polymerase chain reaction, and is compatible with the term "primer pair".

The primer may incorporate additional features that do not change the basic properties of the primer, which serves as a starting point for DNA synthesis. In addition, the primer may contain a label detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means, if necessary. Examples of labels include enzymes (alkaline phosphatase), radioactive isotopes (eg 32P), fluorescent molecules, and chemical groups (eg biotin).

In the present invention, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to a short number of bases to several hundreds of bases capable of specific binding to a gene or mRNA, and an oligonucleotide probe , Single-stranded DNA (single stranded DNA) probe, double-stranded DNA (double stranded DNA) probe, it can be produced in the form of an RNA probe.

In the probe, a label used for detection may be further conjugated to the 5′ and 3′ ends of the corresponding nucleotide sequence, and specifically, a reporter or a quencher may be conjugated to the 5′ and 3′ ends, respectively. . Accordingly, when a reporter and a quencher are present in the probe in a combined form, light emission is suppressed due to mutual interference, and when the probe is decomposed through a polymerase chain reaction, the interference between the reporter and quencher labeled on the probe disappears, resulting in light emission. Is done. Through such a reaction, the presence or absence of avian influenza virus can be detected from the sample, and the phylogenetic group of avian influenza virus can be diagnosed.

Reporters that can be labeled at the 5'end include 6-carboxyfluorescein (FAM), texas red, fluorescein, 5-hexachloro-fluorescein (HEX), and fluorescein chlorotriazinyl. ), rhodamine green, rhodamine red, tetramethylrhodamine, FITC (fluorescein isothiocyanate), oregon green, alexa fluor, JOE (6) -Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX(6-Carboxyl-X-Rhodamine), TET(Tetrachloro-Fluorescein), TRITC(tertramethylrodamine isothiocyanate), TAMRA(6-carboxytetramethyl- rhodamine), NED (N-(1-Naphthyl) ethylenediamine), cyanine-based dyes and thiadicarbocyanine may be any one or more selected from the group consisting of, but may be used as a reporter in the art. Any known substance can be used. In one embodiment of the present invention, the reporter may be FAM, HEX or Texas Red.

In addition, the quencher that can be labeled at the 3'end can also be used without limitation, a quencher commonly used in the art, for example, TAMRA (6-carboxytetramethyl-rhodamine), BHQ1 (black hole quencher 1). , BHQ2 (black hole quencher 2), BHQ3 (black hole quencher 3), NFQ (nonfluorescent quencher), dabcyl, Eclipse, DDQ (Deep Dark Quencher), Blackberry Quencher and Iowa black ) It may be any one or more selected from the group consisting of. In an embodiment of the present invention, the quencher may be BHQ-1 or BHQ-2.

When performing real-time polymerase chain reaction using a fluorescent substance-labeled probe, the amplified product can be monitored in real time, enabling accurate quantification of DNA and RNA. Also, it does not require electrophoresis, so it is quick, simple and accurate detection. It has the advantage that it is possible.

The primer or probe can incorporate additional features that do not change the basic properties. That is, as long as it exhibits the effect of simultaneously detecting the 2.3.4.4 and 2.3.2.1c phylogenies of the HPAI H5 subtype of the present invention, the oligonucleotide sequence can be modified using conventional means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution of nucleotides with one or more homologs and modifications between nucleotides, such as uncharged linkers (e.g. methylphosphonate, phosphotriester, phosphoroami Date, carbamate, etc.) or a charged linker (e.g. phosphorothioate, phosphorodithioate, etc.). In addition, nucleic acids include nucleases, toxins, antibodies, signal peptides, proteins such as poly L lysine, intercalating agents such as acridine or psoralen, chelating agents such as metals, radioactive metals, iron oxidizing metals, and mancalizing agents. It may have one or more additional covalently bonded moieties.

The composition according to the present invention is characterized in that it can simultaneously detect different strains of H5 avian influenza virus. In particular, the composition is characterized in that it can simultaneously detect the 2.3.4.4 and 2.3.2.1c strains of H5 avian influenza virus.

In an embodiment of the present invention, using the composition for detection according to the present invention, H5 HPAI and H5 HPAI strains of 2.3.4.4 and 2.3.2.1c are specifically detected, and real-time RT in agarose gel electrophoresis experiments -It was confirmed that the same results as those of the PCR result appeared. Furthermore, in the case of the highly pathogenic H5N1 avian influenza virus, specific fluorescence was not detected for the H5 HPAI lineages of 2.3.4.4 and 2.3.2.1c, but it was confirmed that specific fluorescence was detected for H5 HPAI. According to the detection composition, it was confirmed that it can detect not only H5 HPAI 2.3.4.4 and 2.3.2.1c lineages, but also H5 HPAI included in other lineages.

In addition, the present invention provides a kit for simultaneous detection that can simultaneously detect different strains of H5 highly pathogenic avian influenza A virus (HPAI) virus comprising a primer pair and a probe according to the present invention. .

In the present invention, the kit may be characterized in that it is for reverse transcription polymerase chain reaction (RT-PCR).

In addition, the kit according to the present invention may further include a composition, solution, or device as one kind or more other components suitable for the analysis method. Preferably, the kit may further include essential elements necessary for performing reverse transcription and RT-PCR.

Since the kit according to the present invention uses the above-described primer pair and probe according to the present invention, descriptions in common are omitted in order to avoid excessive complexity of the present specification due to repeated description.

In addition, the present invention uses reverse transcription polymerase chain reaction (RT-PCR), using the primer pair and probe according to the present invention, H5 highly pathogenic avian influenza (H5 highly pathogenic avian influenza A virus, HPAI) different strains of virus It provides a method of simultaneously detecting groups.

In the present invention, the method may be characterized in that it comprises the step of extracting the nucleic acid from the sample and amplifying it.

The amplifying step is 45 to 55 ℃, preferably 47 to 53 ℃, more preferably at 50 ℃ 10 to 20 minutes, preferably 13 to 17 minutes, more preferably 15 minutes and 90 to 100 ℃, A first step of cycling once at 93 to 97°C, more preferably at 95°C for 10 to 20 minutes, preferably 13 to 17 minutes, more preferably 15 minutes; And 90 to 100°C, preferably 94°C for 5 seconds, 50 to 60°C, preferably 56°C for 20 to 40 seconds, preferably 30 seconds, 55 to 65°C, preferably 60°C for 5 seconds. It may include a second step of cycling during 35 to 40 times, preferably 40 times. In the present invention, the first step is a reverse transcription process, the second step is a gene amplification process, and the gene amplification process consists of a dissociation-binding-extension step.

The sample may be a sample isolated from an animal suspected of being infected with avian influenza virus, and the animal may be selected from the group consisting of birds, livestock and humans. In addition, the sample may be a clinical sample or an environmental sample, for example, the clinical sample may be obtained from tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine.

In the present invention, the reverse transcription polymerase chain reaction is not limited thereto, but may be characterized in that it is a one-step real-time reverse transcription polymerase chain reaction.

In the present invention, when performing RT-PCR, detection of an amplification product may be performed through a DNA chip, gel electrophoresis, capillary electrophoresis, radiometric measurement, fluorescence measurement, or phosphorescence measurement, but is not limited thereto.

In one embodiment of the present invention, using QIAGEN's One-Step RT-PCR Kit, and attaching a reporter to the 5'end of the probe and a quencher to the 3'end of the probe to obtain avian influenza through hybridization reaction between the amplified nucleic acid and the probe. The presence or absence of the virus was detected and its phylogenetic group was diagnosed.

The detection method using a primer pair and a probe according to the present invention enables efficient detection of avian influenza virus with simple steps than conventional methods, and at the same time in real time, H5 highly pathogenic avian influenza A virus (HPAI) virus Different phylogenies can be detected.

Terms not otherwise defined in the specification have the meanings commonly used in the art to which the present invention belongs.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Example 1. Preparation of the composition for detection according to the present invention

Primers according to the present invention by analyzing the nucleotide sequence of the 2.3.4.4 and 2.3.2.1c phylogenies of the highly pathogenic H5 avian influenza virus and HPAI H5 subtypes occurring at home and abroad, and securing the conservative nucleotide sequence of each clade /Designed the probe. First, the HA gene sequences of 2081 low pathogenic H5 avian influenza viruses and 4301 highly pathogenic H5 avian influenza viruses were obtained from NCBI. The obtained nucleotide sequence was aligned, and based on this, a common H5 subtype forward/reverse primer and a detection probe specific to each of the 2.3.4.4 and 2.3.2.1c phylogenies and HPAI H5 were prepared. For simultaneous detection, each strain was prepared to contain fluorescent dyes of 5-Carboxyfluorescein (FAM) and 5-hexachloro-fluorescein (HEX), and fluorescent dyes of Texas Red for HPAI H5. . Primers/probes related to M gene detection were identified in previous studies (J Clin Microbiol. 2002 Sep;40(9):3256-60.). All primers and probes used in the present invention are shown in Table 1 below.

primer / Probe designation Base sequence (5'-3') Amplicon size Sequence number Avian H5 HA primer Forward primer ACC CAG CCA ATG ACC TYT GT 203 bp One Reverse primer GRT AAG CCA YAC CAC ATT TCT G 2 Avian H5 probe Clade 2.3.4.4 probe HEX-TAC CCA GGG AVC CTC AAT GA-BHQ1 3 Clade 2.3.2.1c probe FAM-TCC CTG GTA TGA ACA TGC TGC GCT-BHQ1 4 HPAI probe Texas red-TGA RGA AYT GAA ACA CCT ATT GAG CAG-BHQ2 5 Influenza A virus Forward primer AGA TGA GTC TTC TAA CCG AGG TCG 100 bp 6 Reverse primer TGC AAA AAC ATC TTC AAG TCT CTG 7 M gene probe Cy5-TCA GGC CCC CTC AAA GCC GA-BHQ2 8

Example 2. primer / Probe Standard plasmid construction for efficacy verification

A recombinant plasmid was constructed to verify the primary specificity and sensitivity of the primer/probe according to the present invention. Specifically, partial HA protein region of A/environment/Korea/W150/2006 (H5 HPAI) having a binding site for H5 HPAI and H5 HPAI phylogeny detection primers and probes of 2.3.4.4 and 2.3.2.1c Sequence (29-778) and partial sequencer (1-749) of the HA protein region of A/wild_bird/Jiangsu/H184/2015 (2.3.2.1c phylogeny) and A/environment/Korea/W541/2016 (2.3. 4.4 phylogenetic group) of the partial sequencer (59-1116) of the HA protein region was inserted into each plasmid vector (pMD20-T vactor, Takara) and transformed into E.coli (DH5α) cells to amplify the plasmid and extract it. After that, it was used for specificity and sensitivity analysis.

Example 3. Using recombinant plasmid DNA primer / Probe Specificity verification

In order to verify the specificity of the primer/probe prepared in Example 1, the purified recombinant plasmids for the 2.3.2.1c (2.61×10 ¹⁰ copies/mL) and 2.3.4.4 (3.25×10 ¹⁰ copies/mL) lineages were used. Ready. Each 20 µl of the reaction was 2 µl of template, 4 µl of primer (0.5 pM), 4 µl of 0.25 uM mixed probe (including H5 HPAI, 2.3.2.1c, 2.3.4.4 and M gene probe), 10 µl of 2X It was configured with One-Step PCR Mix (Takara) to perform real-time PCR. Real-time PCR was repeated 40 cycles of denaturation at 94°C for 2 minutes, 5 seconds at 94°C, 30 seconds at 56°C, and 5 seconds at 72°C. Thermocycling was performed using the LightCycler 96 System (Roche, USA), and the positive result was estimated by analysis of the fluorescence curve derived from each probe within 40 cycles. During real-time PCR, the intensity of fluorescence (FAM, HEX, and Texas Red) emitted from the hydrolyzed probe was measured every cycle, and the result was a quantification cycle value (Cq value) based on the measured amplification curve. Is shown. The results are shown in FIG. 1.

As shown in Fig. 1, it was confirmed that fluorescence was specifically detected for H5 HPAI and the H5 HPAI lineages of 2.3.4.4 and 2.3.2.1c.

Example 4. Using recombinant plasmid DNA primer / Probe Sensitivity verification

To measure the detection limits of primers and probes according to the present invention, purified recombinant plasmids were prepared for the 2.3.2.1c (2.61×10 ¹⁰ copies/µl) and 2.3.4.4 (3.25×10 ¹⁰ copies/µl) lineages. Then, the plasmid template was diluted 10-fold with distilled water without nuclease-free. Real-time PCR was performed using the 10-fold diluted plasmid as a single template or a mixed template. During real-time PCR, the intensity of fluorescence (FAM, HEX, and Texas Red) emitted from the hydrolyzed probe was measured every cycle, and the result was expressed as a quantitative cycle value based on the measured amplification curve. The results are shown in FIG. 2.

As shown in Fig. 2, the Cq value obtained from the diluted plasmid was all 1.0 for each recombinant plasmid, and it was confirmed that there was a significant correlation with the number of copies of each plasmid. On the other hand, it was confirmed that the negative control (distilled water) did not show any amplification curves.

Example 5. Using virus primer / Probe Specificity verification

Specificity analysis was performed on the developed H5 HPAI and the primers/probes for detecting H5 HPAI lineage groups of 2.3.4.4 and 2.3.2.1c using various viruses secured by the present inventors. To verify the specificity, influenza viruses of various animals including humans and highly pathogenic and low pathogenic avian influenza viruses were used, and a total of 15 viruses used are as follows:

A/California/04/2009 (human H1N1), A/wild bird/Korea/SK14/2014(H1N1, LPAI), A/swine/Korea/CAN04/2005(swine H3N2), A/canine/Korea/01/ 2007(canine H3N2), A/aquatic bird/South Korea/sw006/2016(avian H3N3), A/equine/Kyonggi/SA1/2011(equine H3N8), A/environment/Korea/W150/2006(H5N1, HPAI) , A/duck/Vietnam/NCVD-1584/2012(H5N1, HPAI), A/aquatic bird/Korea/CN2/2009(H5N2 LPAI), A/aquatic bird/ South Korea/sw007/2015(H5N3 LPAI), A /Anas platyrhynchos/Korea/W612/2017(H5N6 HAPI), A/Common Teal/Korea/W555/2017(H5N8 HPAI), A/aquatic bird/ South Korea/sw001/2015(H7N1, LPAI), A/Chicken/ Korea/MS96/1996(H9N2, LPAI), A/Aquatic bird/South Korea/SW1/2018(H10N1 LPAI)

Real-time reverse transcription PCR (rRT-PCR) was performed using the QIAGEN One-Step RT-PCR Kit (QIAGEN Inc., Valencia, CA, USA). Each 20 µl of reaction contains 4 µl of real-time RT-PCR buffer, 2 µl of RNA template, 4 µl of primer (0.5 pM), and 4 µl of 0.25 µM mixed probe (H5 HPAI, 2.3.2.1c, 2.3.4.4). And M gene probe), 0.8 µl of an enzyme mixture, 0.8 µl of dNTP, and 4.4 µl of distilled water. Real-time RT-PCR was repeated 40 cycles at 50° C. for 30 minutes, followed by reverse transcription at 95° C. for 15 minutes, 94° C. for 5 seconds, 56° C. for 30 seconds, and 60° C. for 5 seconds. Thermocycling was performed using the LightCycler 96 System (Roche, USA), and the positive result was estimated by analysis of the fluorescence curve derived from each probe within 40 cycles. The results are shown in FIGS. 3 and 4.

As shown in FIG. 3, fluorescence was specifically detected for H5 HPAI and the H5 HPAI lineage of 2.3.4.4 and 2.3.2.1c, whereas fluorescence was not detected for other subtypes of influenza and low pathogenic avian influenza. Confirmed. In addition, as shown in FIG. 4, it was confirmed that the same results as those of the real-time RT-PCR were shown in the agarose gel electrophoresis experiment. In particular, in the case of the highly pathogenic H5N1 avian influenza virus, specific fluorescence was not detected for the H5 HPAI strains of 2.3.4.4 and 2.3.2.1c, but specific fluorescence was detected for H5 HPAI.

Through the above results, it was confirmed that the use of the primers/probes according to the present invention can specifically detect H5 HPAI and the H5 HPAI family of 2.3.4.4 and 2.3.2.1c. In addition, from the above results, it was confirmed that the primer/probe according to the present invention can detect not only the two H5 HPAI strains, which are analyzed to be highly likely to be introduced into Korea, but also H5 HPAI included in other strains.

Example 6. Using virus primer / Probe Sensitivity verification

Sensitivity analysis was performed on the developed H5 HPAI and the primers/probes for detection of H5 HPAI lineage groups of 2.3.4.4 and 2.3.2.1c using various viruses secured by the present inventors. A/duck/Vietnam/NCVD-1584/2012 (H5N1 HAPI, clade 2.3.2.1c), A/Common Teal/Korea/W555/2017 (H5N8 HPAI clade 2.3.4.4) viruses recently isolated at home and abroad for sensitivity analysis Was used.

For sensitivity analysis, the virus was diluted 10-fold with Phosphate Buffered Saline (PBS, pH 7.4), and real-time RT-PCR Kit (QIAGEN Inc., Valencia, CA, USA) was used to dilute the virus 10 times. -PCR (real-time reverse transcription PCR, rRT-PCR) was performed. Real-time RT-PCR was performed in the same manner as in Example 5. During real-time PCR, the intensity of fluorescence (FAM, HEX, and Texas Red) emitted from the hydrolyzed probe was measured every cycle, and the result was expressed as a quantitative cycle value based on the measured amplification curve. The results are shown in FIG. 5.

As shown in Fig. 5, the Cq value obtained from the diluted viral RNA template had a R2 value of 1.0 for each RNA template, confirming that there was a significant correlation between each RNA template. On the other hand, it was confirmed that the negative control (distilled water) did not show any amplification curves.

Example 7. Using outdoor clinical samples primer / Probe Specificity verification

Detection of the developed H5 HPAI and H5 HPAI phylogenies of 2.3.4.4 and 2.3.2.1c using fecal samples collected from poultry (chicken) secured by the National University of Vietnam Agricultural Sciences, which is conducting international joint research with this research team. Specificity analysis for the primer/probe was performed. A/environment/Korea/W150/2006 (H5N1 HPAI), A/Environment/Korea/W541/2016 (H5N6 HPAI), and A/Common Teal/Korea/W555/2017 (H5N8 HPAI) were used as positive controls, and Newcastle disease Virus (Newcastle disease virus) was used as a negative control.

Real-time reverse transcription PCR (rRT-PCR) was performed using the QIAGEN One-Step RT-PCR Kit (QIAGEN Inc., Valencia, CA, USA). Real-time RT-PCR was performed in the same manner as in Example 5. The results are shown in FIGS. 6 and 7.

As shown in FIGS. 6 and 7, 36 influenza positive samples were analyzed by M gene-specific RT-qPCR analysis (J Clin Microbiol. 2002 Sep;40(9):3256-60) in a total of 2010 poultry feces samples. Of the obtained influenza-positive samples, fluorescence was specifically detected for H5 HPAI and H5 HPAI strains of 2.3.4.4 and 2.3.2.1c in a total of 22 samples, whereas specific fluorescence was detected in the remaining 14 samples. It was confirmed that it was not detected. In addition, as a result of comparing the results with the Influenza Research Database ( https://www.fludb.org/brc/home.spg?decorator=influenza ), specific fluorescence using the primer/probe according to the present invention It was confirmed that the detected 22 H5 HPAIs and samples of 2.3.4.4 and 2.3.2.1c and gene sequencing results were consistent with high pathogenicity and lineage.

Through the above results, as the composition according to the present invention comprises a primer pair for specifically amplifying the HA gene of H5 avian influenza virus and a probe specific to 2.3.4.4 and 2.3.2.1c phylogenies and HPAI H5, Using a small amount of samples, it was confirmed that three types of influenza virus characteristics can be verified with a single reaction: avian influenza virus confirmation, high pathogenicity verification, and H5 HPAI strain verification. Furthermore, the specificity of the primer/probe according to the present invention was additionally verified using an outdoor clinical sample. In particular, the composition according to the present invention not only enables rapid and accurate detection of avian influenza virus, but also allows rapid and high specificity and sensitivity to grasp information on the phylogenetic group. It has been confirmed that it can be usefully used in coping with quarantine.

Although the present invention has been described as a preferred embodiment mentioned above, it is possible to make various modifications or variations without departing from the spirit and scope of the invention. In addition, the appended claims include such modifications or variations that fall within the gist of the present invention.

<110> Korea Research Institute of Bioscience and Biotechnology <120> A composition for simultaneous detecting of H5 highly pathogenic avian influenza virus clades and uses thereof <130> KRIBB1-36 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Avian H5 HA primer_F <220> <221> misc_feature <222> (17) <223> Y is T/U or C <400> 1 acccagccaa tgacctytgt 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Avian H5 HA primer_R <220> <221> misc_feature <222> (2) <223> R is G or A <400> 2 grtaagccay accacatttc tg 22 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Avian H5 probe Clade 2.3.4.4 <220> <221> misc_feature <222> (11) <223> V is A or G or C <400> 3 tacccaggga vcctcaatga 20 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Avian H5 probe Clade 2.3.2.1c <400> 4 tccctggtat gaacatgctg cgct 24 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Avian H5 probe HPAI <220> <221> misc_feature <222> (4) <223> R is G or A <220> <221> misc_feature <222> (8) <223> Y is T/U or C <400> 5 tgargaaytg aaacacctat tgagcag 27 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Influenza A virus_F <400> 6 agatgagtct tctaaccgag gtcg 24 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Influenza A virus_R <400> 7 tgcaaaaaca tcttcaagtc tctg 24 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Influenza A virus M gene <400> 8 tcaggccccc tcaaagccga 20

Claims (13)

(a) a primer pair of SEQ ID NOs: 1 and 2 for specifically amplifying the HA gene of H5 avian influenza virus; And
(b) the probe of SEQ ID NO: 3 to 5; H5 highly pathogenic avian influenza (H5 highly pathogenic avian influenza A virus, HPAI) virus detection composition comprising:
Here, the composition for detection of the H5 highly pathogenic avian influenza A virus (HPAI) virus is capable of simultaneously detecting different phylogenies.
According to claim 1, wherein the probe is characterized in that the reporter is further conjugated to the 5'end thereof, the composition.
The composition of claim 1, wherein the probe is further conjugated to a quencher at its 3'end.
The method of claim 2, wherein the reporter is FAM (6-carboxyfluorescein), Texas red (texas red), fluorescein (fluorescein), HEX (5-hexachloro-fluorescein), fluorescein chlorotriazinyl (fluorescein chlorotriazinyl), Rhodamine green, rhodamine red, tetramethylrhodamine, FITC (fluorescein isothiocyanate), oregon green, alexa fluor, JOE (6-Carboxy) -4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX (6-Carboxyl-X-Rhodamine), TET (Tetrachloro-Fluorescein), TRITC (tertramethylrodamine isothiocyanate), TAMRA (6-carboxytetramethyl-rhodamine) , NED (N-(1-Naphthyl) ethylenediamine), cyanine (Cyanine) series dye and thiadicarbocyanine (thiadicarbocyanine), characterized in that at least one selected from the group consisting of, the composition.
The method of claim 3, wherein the quencher is TAMRA (6-carboxytetramethyl-rhodamine), BHQ1 (black hole quencher 1), BHQ2 (black hole quencher 2), BHQ3 (black hole quencher 3), NFQ (nonfluorescent quencher), dassil ( dabcyl), Eclipse, DDQ (Deep Dark Quencher), Blackberry Quencher (Blackberry Quencher) and Iowa Black (Iowa black), characterized in that at least one selected from the group consisting of, the composition.
delete The composition of claim 1, wherein the composition is capable of simultaneously detecting 2.3.4.4 and 2.3.2.1c phylogenies of H5 avian influenza virus.
The composition of claim 1, wherein the primer pair is a primer pair for reverse transcriptase chain reaction (RT-PCR).
A kit for simultaneous detection that can simultaneously detect different strains of H5 highly pathogenic avian influenza A virus (HPAI) virus comprising the primer pair and probe according to claim 1.
The kit of claim 9, wherein the kit is for reverse transcription polymerase chain reaction (RT-PCR).
Using reverse transcription polymerase chain reaction (RT-PCR), different strains of H5 highly pathogenic avian influenza A virus (HPAI) virus are simultaneously detected using the primer pair and probe according to claim 1 How to.
The method of claim 11, wherein the method comprises the step of extracting nucleic acid from a sample and amplifying it.
The method of claim 11, wherein the reverse transcription polymerase chain reaction is a one-step real-time reverse transcription polymerase chain reaction.

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