KR20130102431A - Kit for detecting influenza a, h1n1 - Google Patents

Abstract

PURPOSE: A kit for detecting influenza A virus subtype H1N1 is provided to detect influenza A virus subtype H1N1 with high sensitivity and specificity and to early diagnose and treat H1N1 infectious diseases. CONSTITUTION: A primer set contains a polynucleotide sequence which is able to amplify 679th to 842th bases of hemagglutinin (HA) gene of influenza A virus subtype H1N1 to detect H1N1. A composition for diagnosing H1N1 infectious diseases contains the primer set. A kit for diagnosing H1N1 infectious diseases contains the primer set. The kit additionally contains a probe, DNA polymerase, reverse transcriptase, or a combination thereof. A method for providing information for diagnosing H1N1 infectious diseases comprises the step of detecting the HA gene of H1N1 from a sample of a patient who is suspected to have H1N1 infectious diseases using the primer set, the composition, or the kit. [Reference numerals] (AA) Diagnostic specificity analysis of primer set for diagnosing the H1N1 influenza; (BB,CC) Positive control group

Description

A kit for detecting influenza A virus H1N1 {kit for detecting Influenza A, H1N1}

More particularly, the present invention relates to a kit for detecting influenza A virus H1N1 (Influenza A, H1N1), a H1N1 infectious disease comprising the primer set A kit for diagnosing H1N1 infectious disease comprising the primer set, and a method for providing information for diagnosing H1N1 infectious disease using the composition or kit.

Influenza A virus H1N1 (Influenza A, H1N1) is a new type of influenza virus that combines the genetic material of humans, birds and swine influenza virus. According to the results reported so far, the swine flu virus spreads at a high rate, but it causes mild symptoms in most patients and also shows a similar mortality rate to that of seasonal influenza. However, since most people do not have immunity to the new influenza virus and can not rule out the possibility of massive epidemics, countries around the world and the World Health Organization are taking measures to deal with it. Symptoms of swine influenza usually present with typical influenza-like illness (ILI), but in addition to typical ILI, they may manifest clinical manifestations of various spectra ranging from severe pneumonia, sepsis, and multiple organ dysfunction, There are also cases. Patients with confirmed H1N1 flu have symptoms such as fever, chills, headache, upper respiratory symptoms (cough, sore throat, runny nose, shortness of breath), muscle pain, arthralgia and tiredness. Unlike seasonal influenza, It is known to accompany the symptoms of digestive system such as. Most healthy people with influenza A virus H1N1 are cured within a week without antiviral therapy. The mortality rate of patients with influenza A virus H1N1 varies from country to country but is currently very low at less than 0.2% in developed countries. Most cases of severe illness and death (60 to 70%) are chronic diseases, pregnant women, Lt; / RTI > The main pathway for the influenza A virus H1N1 is known as respiratory droplet spreading, contact propagation, etc., spreading through air transmission similar to that of seasonal influenza virus

At present, the infection diagnosis of the influenza A virus H1N1 is performed through a method of isolating and identifying viruses, a method of detecting a virus nucleic acid, and the like. For example, the isolation and identification method of influenza A virus H1N1 can be carried out by inoculating a patient's sample into a Madin-Darby canine kidney (MDCK) cell to identify a hemagglutinating virus In order to quickly and accurately diagnose virus-infected patients, real-time PCR (RT-PCR) is used to detect viral nucleic acid. Among them, RT-PCT method has been used for rapid diagnosis because of the disadvantage that it is difficult to diagnose and isolate virus rapidly and it requires a lot of time and experts until the typing of virus is required and quick diagnosis is difficult. And probe sequences are known. However, when such RT-PCR is used, there is a disadvantage in that the diagnostic success rate is relatively low, because the probability of a false negative reaction is high. That is, even if primers and probes specific for H1N1 are used, there is a high possibility that the results of viruses having other kinds of similar sequences are misdetected. To this end, efforts have been made to develop primers that exhibit high specificity for H1N1.

Under these circumstances, the present inventors have made extensive efforts to develop a primer exhibiting high specificity for H1N1. As a result, it has been found that when a primer capable of amplifying a specific region of a gene encoding a hemagglutinin protein of H1N1 is used, H1N1 Sensitivity, and completed the present invention.

One object of the present invention is to provide a set of primers that can be used for the detection of influenza A virus H1N1 (Influenza A, H1N1).

Another object of the present invention is to provide a composition for diagnosing H1N1 infectious disease comprising the above primer set.

It is still another object of the present invention to provide a kit for the diagnosis of H1N1 infectious disease comprising the primer set.

It is still another object of the present invention to provide a method for providing information for diagnosing H1N1 infectious disease using the composition or kit.

In one embodiment for achieving the above object, the present invention provides a primer set which can be used for the detection of influenza A virus H1N1 (Influenza A, H1N1).

The term "Influenza A (H1N1) virus of the present invention" refers to a type (HA) of Ha (hemagglutinin) protein among 16 HA subtypes out of type A influenza (H1) Neureminidase) refers to a virus whose protein is type 1 (N1). For purposes of the present invention, the H1N1 can be used as an object that can be detected through the primer set provided in the present invention, but is not particularly limited thereto.

The term "primer" of the present invention refers to a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming base pairs with a complementary template and having a starting point for template strand copying ≪ / RTI > The primers can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures.

The term "primer set" of the present invention means a set consisting of a sense primer and an antisense primer used for amplifying a desired gene through a polymerase chain reaction. For the purpose of the present invention, the primer set may preferably be composed of forward and reverse primers capable of amplifying the hemagglutinin (HA) gene of H1N1, more preferably the 5'-terminal May be composed of sense primers 679 to 704 and antisense primers 816 to 842, most preferably, a sense primer of SEQ ID NO: 1 and an antisense primer of SEQ ID NO: 2, But is not limited thereto.

The present inventors compared the HA full-length genes of known H1N1, H3N2 and H5N1 influenza strains to select regions 679 to 842 as regions having no gene homology with other subtypes (Fig. 1) A primer set containing the amplifiable primers H1N1-HA-F (SEQ ID NO: 1) and H1N1-HA-R (SEQ ID NO: 2) was prepared (FIG. The prepared primers H1N1-HA-F and H1N1-HA-R were prepared such that the GC content was 48.2% and 44.4%, respectively (Table 1).

On the other hand, as a result of evaluating the clinical utility using the prepared primer sets, 89 of the 89 positive samples were found to be positive by the primer set described above and showed 100% diagnostic sensitivity (FIGS. 5A to 6B) , H1N1 influenza viruses, and Adenovirus, Rhinovirus, Respiratory syncytial virus (RSV), and Metapneumovirus, which are the major viruses causing respiratory diseases, were all diagnosed as negative and highly specific for H1N1 (Figs. 7A and 7B).

As another embodiment for achieving the above object, the present invention provides a composition for diagnosing H1N1 infectious disease comprising the primer set.

The term "H1N1 infectious disease" of the present invention means that H1N1 enters the animal in various ways and causes not only typical influenza-like illness (ILI) but also fever, chills, headache, , Dyspnea), muscular pain, arthralgia, tiredness, etc., and means a disease that can develop due to severe pneumonia, sepsis, multiple organ failure, and the like.

In the present invention, the term "diagnosis" means any action that identifies the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis may be interpreted as any action to ascertain the likelihood of developing an H1N1 infectious disease, but is not limited thereto.

The term "diagnostic composition" of the present invention means a main means used for diagnosis of a desired disease. For the purpose of the present invention, the diagnostic composition preferably comprises a primer set for RT-PCR for diagnosing H1N1 infectious disease, more preferably a sense primer from 679th to 704th from the 5'- And 816 to 842 antigens, and most preferably more preferably, a primer set consisting of the primers of SEQ ID NOS: 1 and 2 for diagnosing H1N1 infectious disease, It is not limited.

As another embodiment for achieving the above object, the present invention provides a kit for the diagnosis of H1N1 infectious disease comprising the primer set.

The term "diagnostic kit " of the present invention means a kit containing auxiliary means other than the main means used for diagnosis of the desired disease. For the purpose of the present invention, the diagnostic kit may be a kit for RT-PCR for diagnosing H1N1 infectious disease, preferably a RT-PCR primer set for the diagnosis of H1N1 infectious disease, a probe, Enzyme, and reverse transcriptase, and more preferably a primer set consisting of sense primers 679 to 704 and antisense primers 816 to 842 from the 5'-end of the HA gene, a probe, DNA A DNA polymerase, a reverse transcriptase, or the like, which is composed of the primers of SEQ ID NOS: 1 and 2 for diagnosing H1N1 infectious disease, But is not particularly limited thereto.

In another aspect of the present invention, the present invention provides a method for providing information for diagnosing H1N1 infectious disease using the composition or kit.

Specifically, the method for providing information for diagnosing H1N1 infectious disease of the present invention comprises detecting HA gene of H1N1 from a sample of a patient suspected of having H1N1 infectious disease.

The term "patient sample " of the present invention refers to a sample such as tissue, cell, whole blood, serum, plasma, saliva, sputum, runny nose, cerebrospinal fluid or urine expressing HA gene of H1N1, Do not.

Using the primer set of the present invention, Influenza A virus H1N1 (Influenza A, H1N1) can be detected with high sensitivity and specificity, and thus it can be widely used for the early diagnosis and treatment of infectious diseases of H1N1.

Figure 1 is a graph showing the results of comparing gene sequences of influenza subtypes for H1N1-specific primer design.
Figure 2 shows the H1N1 specific primer design.
Figure 3 shows a template DNA (plasmid) used as a positive control for real-time PCR.
4A to 4C show results of setting optimal conditions for the real-time PCR using the H1N1 diagnostic primer set.
5A to 5C are graphs showing the results of evaluation of the clinical utility of the primer set in a domestic H1N1 influenza infection sample.
6A and 6B are graphs showing the results of examining a specimen diagnosed as positive in the H1N1 voice diagnostic specimen.
7A and 7B are graphs showing the results of analyzing the diagnostic specificity of the H1N1 influenza diagnostic primer set.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Primer production for HA region of H1N1 influenza virus

HA full-length nucleotides of H1N1, H3N2, and H5N1 influenza reported from NCBI's database until January 2011 were listed and set to be free of gene homology with other subtypes (Fig. 1).

In addition, among the nucleotide sequence portions set so as not to be homologous with the HA gene of H3N2 and H5N1 influenza, the most stable portion of the reported H1N1 influenza virus HA gene sequence and the least mutation of each individual is set, and the set site is amplified (Fig. 2).

The primer set includes a sense primer (H1N1-HA-F) corresponding to the 679th to 704th positions from the 5'end of the H1N1 influenza virus HA gene and an antisense primer (H1N1 -HA-R). The H1N1-HA-F and H1N1-HA-R were prepared such that the Tm (占 폚) values were 68.3 and 67.9, respectively, and the GC contents were 48.2% and 44.4%, respectively.

H1N1-HA-F: 5'-AAAGGGAAAGAAGTCCTCGTGCTATGG-3 '(SEQ ID NO: 1)

H1N1-HA-R: 5'-TTGATCCCTCACTTTGGGTCTTATTGC-3 '(SEQ ID NO: 2)

Example 2: Construction of template DNA for H1N1 influenza diagnosis positive control

The template DNA (SEQ ID NO: 3) of the corresponding region was synthesized (COSMO GENETEC, Korea) to use the base sequence region amplified by real-time PCR using the primer set prepared in Example 1 as a positive control, The template DNA was cloned into pUC57 plasmid (Figure 3).

Example 3: RT-PCR reaction

RT-PCR was performed using the primer set synthesized in Examples 1 and 2 and template DNA for positive control. At this time, the concentration of the template was diluted stepwise to 10 ^-6 to 1 ng and the primer concentration was set to 1 uM, 0.5 uM or 0.2 uM, and real-time PCR was performed using Bio-Rad Realtime PCR CFX96 kit.

RT-PCR was performed at 95 ° C for 3 minutes, at 95 ° C for 10 seconds, and at 72 ° C for 14 seconds at 30 cycles. For all reactions, the template DNA was replaced with distilled water as a negative control (No Template Control) and duplicate (Figs. 4A to 4C and Table 1).

As shown in Table 1 and FIGS. 4A to 4C, under the condition of using a primer concentration of 1 uM, amplification was carried out with a product specific to the HA gene of H1N1 influenza in all the conditions except for the negative control, and the detection was possible stably. When the standard graph of the standard template real-time PCR was prepared, the slope was -3.3 and the R2 value was 0.994, indicating that real-time PCR was more stable at different primer concentrations. In addition, the diagnosis was not made at increasing concentrations as 0.5um and 0.2um.

Example 4 Evaluation of Clinical Usefulness of Primer Set

Example 4-1: Diagnostic sensitivity analysis

Using the primer set prepared in Example 1, the clinical utility of the H1N1 influenza virus-infected sample was evaluated (provided by Kangwon National University Hospital). RNA was extracted from the patient's swab sample in VTM (Viral transfer media) using an RNA extraction kit (MACHEREY-NAGEL, Germany). 5 ng (5 ng) of the extracted RNA was reverse-transcribed using ReverTra Ace qPCR RT Kit (Toyobo, Japan) to synthesize cDNA. The synthesized cDNA was used for the diagnosis of H1N1 influenza specimen and real-time PCR was performed under the conditions optimized in Example 3 (primer concentration, 1 uM). 90 swab specimens diagnosed with H1N1 influenza positive and 17 swab specimens diagnosed by negative molecular diagnosis kit (Seegene, Bioneer), which were distributed in the market, were used for the real - time PCR using the above primer set.

Of the 90 positive specimens tested, 89 samples were positive and one specimen was negative (FIG. 5A) through the primer set described above. Real-time PCR was performed using the molecular diagnostic kit used in the diagnosis and the molecular diagnostic primer set described above in order to verify the samples diagnosed by the voice. Both of the primer sets were used as a control for the experiment by including the positive diagnosed specimens. As a result, it was confirmed that the positive specimens which were not amplified as shown in FIG. 5C were false positive. Consequently, 89 of the 89 positive specimens were confirmed as positive by the primer set and 100% of the diagnostic specimens were shown.

On the other hand, 13 of the 17 swab specimens diagnosed as negative were identified as negative and 4 swab specimens were positive (FIG. 5c).

In order to examine the four swab specimens diagnosed as positive, the amplification product obtained by real-time PCR was cloned into pGEM-T Easy Vector (Promega, USA), and the DNA sequence was determined and the determined sequence was assigned to NCBI BLAST The virus was identified as H1N1 influenza virus. From this, four false-positive diagnoses among 17 swab specimens diagnosed by voice using commercially available conventional diagnostic kits (Seegene, Bioneer) were positively confirmed by the primer set of the present invention (FIGS. 6A and 6B).

Example 4-2: Analysis of diagnostic specificity

In order to analyze the diagnostic specificity of the primer set prepared in Example 1, in addition to H1N1 influenza virus, swab specimens infected with Adenovirus, Rhinovirus, RSV (respiratory syncytial virus), Metapneumovirus which are major viruses causing respiratory diseases RT-PCR was performed in the same manner as in Example 4-1, except that the primers (SEQ ID NOs: 1, 2, and 3) were used (Figs. 7A and 7B). As shown in FIGS. 7A and 7B, each of the swab specimens was diagnosed negative in real-time PCR using the present H1N1 diagnostic primer set, indicating that the primer set of the present invention was highly specific for H1N1 diagnosis.

The results of Example 4 demonstrate that the primer set provided in the present invention can detect HA gene of H1N1 with high sensitivity and specificity and thus can diagnose H1N1 infectious disease more effectively.

<110> Wonkwang University Center for Industry-Academy Cooperation <120> Kit for detecting Influenza A, H1N1 <130> PA120143 / KR <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> H1N1-HA-F primer <400> 1 aaagggaaag aagtcctcgt gctatgg 27 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> H1N1-HA-R primer <400> 2 ttgatccctc actttgggtc ttattgc 27 <210> 3 <211> 201 <212> DNA <213> Artificial Sequence <220> <223> template DNA <400> 3 cccaaagctc agcaaatcct acattaatga taaagggaaa gaagtcctcg tgctatgggg 60 cattcaccat ccatctacta gtgctgacca acaaagtctc tatcagaatg cagatgcata 120 tgtttttgtg gggacatcaa gatacagcaa gaagttcaag ccggaaatag caataagacc 180 caaagtgagg gatcaagaag g 201

Claims (8)

A primer set having a polynucleotide sequence capable of amplifying regions 679 to 842 of HA (Hemagglutinin) gene of H1N1 so that it can be used for detection of Influenza A virus H1N1 (Influenza A, H1N1).
The method of claim 1,
A primer set comprising sense primers 679 to 704 and antisense primers 816 to 842 from the 5'-end of the HA gene.
The method of claim 1,
A primer set comprising the polynucleotide sequences of SEQ ID NOS: 1 and 2.
A composition for the diagnosis of H1N1 infectious disease comprising the primer set of claim 1.
A kit for the diagnosis of H1N1 infectious disease comprising the primer set of claim 1.
The method of claim 5,
A probe, a DNA polymerase, a reverse transcriptase, or a combination thereof.
Detecting the HA gene of H1N1 from a sample of a patient suspected of H1N1 infectious disease using the primer set of any one of claims 1 to 3, the composition of claim 4 or the kit of claim 5 or 6. Method for providing information for diagnosing an H1N1 infectious disease comprising.
The method according to claim 6,
Wherein said sample of said patient is a tissue, cell, whole blood, serum, plasma, saliva, sputum, runny nose, cerebrospinal fluid or urine expressing HA gene of H1N1.

Images