CN101052720B - Method of detecting H5 or H7 avian influenza virus - Google Patents

Abstract

The present invention provides an oligonucleotide that specifically hybridizes to an arbitrary nucleotide sequence designed based on the nucleotide sequence of hemagglutinin of H5 or H7 avian influenza virus, a nucleic acid amplification method using the primer, and a nucleic acid amplification method based on the nucleic acid amplification method. A diagnostic method for the newly detected H5 or H7 avian influenza virus infection and a diagnostic kit for influenza.

Description

Detection method of H5 or H7 avian influenza virus

technical field

The present invention relates to the detection method of H5 type or H7 type avian influenza virus, more specifically relate to the oligonucleotide primer for detecting H5 type or H7 type avian influenza virus, the detection of H5 type or H7 type avian influenza virus using this primer Method, method for diagnosing influenza and kit for diagnosing influenza.

Background technique

Influenza is an epidemic viral respiratory infectious disease, which affects a wide range of age groups from infants to the elderly, and is often fatal. Currently, the H5 avian influenza virus that infects poultry and causes problems does not originally infect humans. However, infection in humans was discovered in Hong Kong in 1997, and 6 of 18 patients died in this epidemic. Fortunately, no further human infections were found, but in 2004, human infections were found in Thailand and Vietnam, with 8 deaths in Thailand and 16 in Vietnam.

In addition to the H5 type, the highly pathogenic avian influenza virus also has the H7 type, and the H7 type is roughly divided into the European type and the American type according to its sequence. In 2003, one death was reported in the Netherlands, and the H7 avian influenza virus was also reported in the United States from 2003 to 2004.

Currently, the detection of avian influenza virus uses a rapid diagnostic kit for human influenza A virus. However, identification of the subtype of the infected virus requires more detailed analyzes such as antigenic analysis and genetic testing of the isolated virus.

Diagnosis using virus isolation and culture, which can obtain reliable results, cannot be performed quickly because it takes several days. There are several methods that can diagnose more quickly than virus isolation, and among them, RT-PCR method is considered to have higher detection sensitivity than other methods. However, for the currently disclosed RT-PCR method, it has been reported that it cannot detect the virus with high sensitivity compared with the infection rate of the virus. Even if it is negative in the inspection using the RT-PCR method, the infection of avian influenza cannot be ruled out. Therefore, the existence of an inspection method capable of rapidly and highly sensitively detecting H5 and H7 avian influenza viruses is desired.

Patent Document 1: Specification of European Patent Application Publication No. 1310565

Patent Document 2: Japanese Patent Publication No. 2004-509648

Non-Patent Document 1: Lau LT. et al., Biochem. Biophys. Res. Commun., vol.313, p.336-342 (2004)

Non-Patent Document 2: Shang S. et al., Biochem. Biophys. Res. Commun., vol.302, p.377-383 (2003)

Non-Patent Document 3: Collins RA. et al., Biochem. Biophys. Res. Commun., vol.300, p.507-515 (2003)

Non-Patent Document 4: Lee MS. et al., J. Virol. Methods, vol.97, p.13-22 (2001)

Non-Patent Document 5: Munch M. et al., Arch. Virol., vol.146, p.87-97 (2001)

disclosure of invention

In order to solve the above-mentioned problems, the present inventors have found after earnest research that an oligonucleotide primer that hybridizes to a nucleotide sequence specific to H5 or H7 type avian influenza virus is made, and the primers are used by LAMP (loop-mediated constant temperature amplification, loop- mediated isothermal amplification) method to amplify the base sequence specific to H5 or H7 type avian influenza virus, can detect H5 or H7 type avian influenza virus with high sensitivity, thereby completing the present invention.

That is, the present invention provides the following (1) to (8).

(1) An oligonucleotide designed based on any base sequence selected from the 693rd to 959th base sequence of the hemagglutinin base sequence of the H5 type avian influenza virus represented by SEQ ID NO: 1 or a base sequence complementary thereto nucleotide primers.

(2) The oligonucleotide primer according to (1), comprising an oligonucleotide selected from (a) to (c) below:

(a) An oligonucleotide containing at least 15 consecutive bases selected from the base sequence represented by sequence number 2-7 or the base sequence complementary thereto;

(b) an oligonucleotide capable of hybridizing to the oligonucleotide described in (a) above under stringent conditions;

(c) An oligonucleotide having a primer function comprising a base sequence in which one to several bases are substituted, deleted, inserted or added in the oligonucleotide described in (a) or (b) above .

(3) The oligonucleotide primers as described in (1) or (2), characterized in that they are selected from the 3' terminal side of the target nucleic acid of the hemagglutinin of the H5 type avian influenza virus and are denoted as F3c, F2c, For the nucleotide sequence region of F1c, the nucleotide sequence regions described as B3, B2, and B1 are selected from the 5' terminal side, and the respective complementary nucleotide sequences are described as F3, F2, F1 and B3c, B2c, and B1c. It consists of the base sequence of (a) to (d) below:

(a) having the F2 region of the target nucleic acid on the 3' terminal side and the base sequence of the F1c region of the target nucleic acid on the 5' terminal side;

(b) has the base sequence of the F3 region of the target nucleic acid;

(c) having the B2 region of the target nucleic acid on the 3' terminal side and the base sequence of the B1c region of the target nucleic acid on the 5' terminal side;

(d) has the base sequence of the B3 region of the target nucleic acid.

(4) The oligonucleotide primer as described in (1)～(3), is characterized in that, can amplify the nucleotide sequence specific to H5 type avian influenza virus, from 5 ' end to 3 ' end, by Base sequence composition selected from the following (a) to (b):

(a) 5'-(base sequence complementary to the base sequence of SEQ ID NO: 2)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 3)-3';

(b) 5'-(base sequence of SEQ ID NO: 5)-(arbitrary base sequence of 0 to 50 bases)-(base sequence complementary to the base sequence of SEQ ID NO: 6)-3'.

(5) A detection method for H5 avian influenza virus, characterized in that the amplification reaction of the target nucleic acid region of H5 avian influenza virus is carried out using the oligonucleotide primers described in (1) to (4).

(6) The method for detecting the H5 avian influenza virus as described in (5), wherein the amplification reaction of the target nucleic acid region of the H5 avian influenza virus adopts the LAMP method.

(7) A diagnostic method for influenza, characterized in that, by using the oligonucleotide primers described in (1) to (4), the amplification of the target nucleic acid region of the H5 type avian influenza virus is detected to diagnose whether there is an H5 type avian influenza virus. Infection with influenza virus.

(8) A kit comprising the oligonucleotide primers described in (1) to (4) for diagnosing influenza.

The present invention also provides the following (9) to (16).

(9) An oligonucleotide designed based on any base sequence selected from the 19th to 220th base sequence of the hemagglutinin base sequence of the H5 type avian influenza virus represented by SEQ ID NO: 1 or a base sequence complementary thereto nucleotide primers.

(10) The oligonucleotide primer according to (9), comprising an oligonucleotide selected from (a) to (c) below:

(a) An oligonucleotide containing at least 15 consecutive bases selected from the base sequence represented by sequence number 13-18 or the base sequence complementary thereto;

(b) an oligonucleotide capable of hybridizing to the oligonucleotide described in (a) above under stringent conditions;

(c) An oligonucleotide having a primer function comprising a base sequence in which one to several bases are substituted, deleted, inserted or added in the oligonucleotide described in (a) or (b) above .

(11) The oligonucleotide primer as described in (9) or (10), characterized in that it is selected from the 3' terminal side of the target nucleic acid of the hemagglutinin of the H5 type avian influenza virus and is denoted as F3c, F2c, For the nucleotide sequence region of F1c, the nucleotide sequence regions described as B3, B2, and B1 are selected from the 5' terminal side, and the respective complementary nucleotide sequences are described as F3, F2, F1 and B3c, B2c, and B1c. It consists of the base sequence of (a) to (d) below:

(a) having the F2 region of the target nucleic acid on the 3' terminal side and the base sequence of the F1c region of the target nucleic acid on the 5' terminal side;

(b) has the base sequence of the F3 region of the target nucleic acid;

(c) having the B2 region of the target nucleic acid on the 3' terminal side and the base sequence of the B1c region of the target nucleic acid on the 5' terminal side;

(d) has the base sequence of the B3 region of the target nucleic acid.

(12) The oligonucleotide primer as described in (9) to (11), characterized in that it can amplify the base sequence specific to the H5 type avian influenza virus, from the 5' end to the 3' end, by Base sequence composition selected from the following (a) to (b):

(a) 5'-(base sequence complementary to the base sequence of SEQ ID NO: 13)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 14)-3';

(b) 5'-(base sequence of SEQ ID NO: 16)-(arbitrary base sequence of 0 to 50 bases)-(base sequence complementary to the base sequence of SEQ ID NO: 17)-3'.

(13) A method for detecting H5 avian influenza virus, characterized in that the amplification reaction of the target nucleic acid region of H5 avian influenza virus is carried out using the oligonucleotide primers described in (9) to (12).

(14) The method for detecting H5 avian influenza virus as described in (13), wherein the amplification reaction of the target nucleic acid region of the H5 avian influenza virus adopts the LAMP method.

(15) A diagnostic method for influenza, characterized in that, by using the oligonucleotide primers described in (9) to (12), the amplification of the target nucleic acid region of the H5 type avian influenza virus is detected to diagnose whether there is an H5 type avian influenza virus. Infection with influenza virus.

(16) A kit comprising the oligonucleotide primers described in (9) to (12) for diagnosing influenza.

The present invention also provides the following (17) to (24).

(17) An oligonucleotide designed based on any base sequence selected from the 114th to 333rd base sequence of the hemagglutinin base sequence of the H5 type avian influenza virus represented by SEQ ID NO: 1 or a base sequence complementary thereto nucleotide primers.

(18) The oligonucleotide primer according to (17), comprising an oligonucleotide selected from (a) to (c) below:

(a) An oligonucleotide containing at least 15 consecutive bases selected from the base sequence represented by sequence number 24-29 or the base sequence complementary thereto;

(b) an oligonucleotide capable of hybridizing to the oligonucleotide described in (a) above under stringent conditions;

(c) An oligonucleotide having a primer function comprising a base sequence in which one to several bases are substituted, deleted, inserted or added in the oligonucleotide described in (a) or (b) above .

(19) The oligonucleotide primer as described in (17) or (18), characterized in that it is selected from the 3' terminal side of the target nucleic acid of the hemagglutinin of the H5 type avian influenza virus and is denoted as F3c, F2c, For the nucleotide sequence region of F1c, the nucleotide sequence regions described as B3, B2, and B1 are selected from the 5' terminal side, and the respective complementary nucleotide sequences are described as F3, F2, F1 and B3c, B2c, and B1c. It consists of the base sequence of (a) to (d) below:

(a) having the F2 region of the target nucleic acid on the 3' terminal side and the base sequence of the F1c region of the target nucleic acid on the 5' terminal side;

(b) has the base sequence of the F3 region of the target nucleic acid;

(c) having the B2 region of the target nucleic acid on the 3' terminal side and the base sequence of the B1c region of the target nucleic acid on the 5' terminal side;

(d) has the base sequence of the B3 region of the target nucleic acid.

(20) The oligonucleotide primer as described in (17) to (19), characterized in that it can amplify the base sequence specific to the H5 type avian influenza virus, from the 5' end to the 3' end, by Base sequence composition selected from the following (a) to (b):

(a) 5'-(base sequence complementary to the base sequence of SEQ ID NO: 24)-(arbitrary base sequence of 0 to 50 bases)-(base sequence of SEQ ID NO: 25)-3';

(b) 5'-(base sequence of SEQ ID NO: 27)-(arbitrary base sequence having 0 to 50 bases)-(base sequence complementary to the base sequence of SEQ ID NO: 28)-3'.

(21) A method for detecting H5 avian influenza virus, characterized in that the amplification reaction of the target nucleic acid region of H5 avian influenza virus is carried out using the oligonucleotide primers described in (17) to (20).

(22) The method for detecting H5 avian influenza virus as described in (21), characterized in that the amplification reaction of the target nucleic acid region of the H5 avian influenza virus adopts the LAMP method.

(23) A method for diagnosing influenza, which is characterized in that, by using the oligonucleotide primers described in (17) to (20), the amplification of the target nucleic acid region of the H5-type avian influenza virus is detected to diagnose whether there is an H5-type avian influenza virus. Infection with influenza virus.

(24) A kit comprising the oligonucleotide primers described in (17) to (20) for diagnosing influenza.

The present invention also provides the following (25) to (32).

(25) An oligonucleotide designed based on any base sequence selected from the 874th to 1065th base sequence of the hemagglutinin base sequence of the H5 type avian influenza virus represented by SEQ ID NO: 1 or a base sequence complementary thereto nucleotide primers.

(26) The oligonucleotide primer according to (25), comprising an oligonucleotide selected from (a) to (c) below:

(a) An oligonucleotide containing at least 15 consecutive bases selected from the base sequence represented by sequence number 35-40 or the base sequence complementary thereto;

(b) an oligonucleotide capable of hybridizing to the oligonucleotide described in (a) above under stringent conditions;

(c) An oligonucleotide having a primer function comprising a base sequence in which one to several bases are substituted, deleted, inserted or added in the oligonucleotide described in (a) or (b) above .

(27) The oligonucleotide primer as described in (25) or (26), characterized in that it is selected from the 3' terminal side of the target nucleic acid of the hemagglutinin of the H5 type avian influenza virus and is denoted as F3c, F2c, For the nucleotide sequence region of F1c, the nucleotide sequence regions described as B3, B2, and B1 are selected from the 5' terminal side, and the respective complementary nucleotide sequences are described as F3, F2, F1 and B3c, B2c, and B1c. It consists of the base sequence of (a) to (d) below:

(a) having the F2 region of the target nucleic acid on the 3' terminal side and the base sequence of the F1c region of the target nucleic acid on the 5' terminal side;

(b) has the base sequence of the F3 region of the target nucleic acid;

(c) having the B2 region of the target nucleic acid on the 3' terminal side and the base sequence of the B1c region of the target nucleic acid on the 5' terminal side;

(d) has the base sequence of the B3 region of the target nucleic acid.

(28) The oligonucleotide primer as described in (25) to (27), characterized in that it can amplify the base sequence specific to the H5 type avian influenza virus, from the 5' end to the 3' end, by Base sequence composition selected from the following (a) to (b):

(a) 5'-(base sequence complementary to the base sequence of SEQ ID NO: 35)-(arbitrary base sequence of 0 to 50 bases)-(base sequence of SEQ ID NO: 36)-3';

(b) 5'-(base sequence of SEQ ID NO: 38)-(arbitrary base sequence of 0 to 50 bases)-(base sequence complementary to the base sequence of SEQ ID NO: 39)-3'.

(29) A method for detecting H5 avian influenza virus, characterized in that the amplification reaction of the target nucleic acid region of H5 avian influenza virus is carried out using the oligonucleotide primers described in (25) to (28).

(30) The method for detecting H5 avian influenza virus as described in (29), characterized in that the amplification reaction of the target nucleic acid region of the H5 avian influenza virus uses the LAMP method.

(31) A method for diagnosing influenza, which is characterized in that, by using the oligonucleotide primers described in (25) to (28), the amplification of the target nucleic acid region of the H5-type avian influenza virus is detected to diagnose whether there is an H5-type avian influenza virus. Infection with influenza virus.

(32) A kit comprising the oligonucleotide primers described in (25) to (28) for diagnosing influenza.

The present invention also provides the following (33) to (40).

(33) An oligonucleotide designed based on any base sequence selected from the 1016-1225 base sequence of the hemagglutinin base sequence of the H7 type avian influenza virus represented by SEQ ID NO: 48 or a base sequence complementary thereto nucleotide primers.

(34) The oligonucleotide primer according to (33), comprising an oligonucleotide selected from (a) to (c) below:

(a) An oligonucleotide containing at least 15 consecutive bases selected from the base sequence represented by sequence number 49-54 or the base sequence complementary thereto;

(b) an oligonucleotide capable of hybridizing to the oligonucleotide described in (a) above under stringent conditions;

(c) An oligonucleotide having a primer function comprising a base sequence in which one to several bases are substituted, deleted, inserted or added in the oligonucleotide described in (a) or (b) above .

(35) The oligonucleotide primer as described in (33) or (34), characterized in that it is selected from the 3' terminal side of the target nucleic acid of the hemagglutinin of the H7 type avian influenza virus and is denoted as F3c, F2c, For the nucleotide sequence region of F1c, the nucleotide sequence regions described as B3, B2, and B1 are selected from the 5' terminal side, and the respective complementary nucleotide sequences are described as F3, F2, F1 and B3c, B2c, and B1c. It consists of the base sequence of (a) to (d) below:

(a) having the F2 region of the target nucleic acid on the 3' terminal side and the base sequence of the F1c region of the target nucleic acid on the 5' terminal side;

(b) has the base sequence of the F3 region of the target nucleic acid;

(c) having the B2 region of the target nucleic acid on the 3' terminal side and the base sequence of the B1c region of the target nucleic acid on the 5' terminal side;

(d) has the base sequence of the B3 region of the target nucleic acid.

(36) The oligonucleotide primer as described in (33) to (35), characterized in that it can amplify the base sequence specific to the H7 type avian influenza virus, from the 5' end to the 3' end, by Base sequence composition selected from the following (a) to (b):

(a) 5'-(base sequence complementary to the base sequence of SEQ ID NO: 49)-(arbitrary base sequence of 0 to 50 bases)-(base sequence of SEQ ID NO: 50)-3';

(b) 5'-(base sequence of SEQ ID NO:52)-(arbitrary base sequence of 0 to 50 bases)-(base sequence complementary to the base sequence of SEQ ID NO:53)-3'.

(37) A method for detecting H7 avian influenza virus, characterized in that the amplification reaction of the target nucleic acid region of H7 avian influenza virus is performed using the oligonucleotide primers described in (33) to (36).

(38) The method for detecting H7-type avian influenza virus as described in (37), wherein the amplification reaction of the target nucleic acid region of the H7-type avian influenza virus adopts the LAMP method.

(39) A method for diagnosing influenza, characterized in that, by using the oligonucleotide primers described in (33) to (36), the amplification of the target nucleic acid region of H7-type avian influenza virus is detected to diagnose whether there is H7-type avian influenza virus Infection with influenza virus.

(40) A kit comprising the oligonucleotide primers described in (33) to (36) for diagnosing influenza.

If adopt the present invention, by making the oligonucleotide primer that hybridizes selectively with the nucleotide sequence specific to H5 or H7 type avian influenza virus, use LAMP method to amplify the H5 or H7 type avian influenza virus specificity The base sequence can detect H5 or H7 avian influenza virus with high sensitivity and rapidly.

A brief description of the drawings

Fig. 1 is the figure that shows the result of the specificity test of primer set for H5 type avian influenza virus, (a), (b), (c) and (d) respectively represent when using primer set A, B, C and D As a result, NC indicates negative control, H1 indicates New Caledonia subtype, H3 indicates Panama subtype, and PC indicates positive control (H5 type plasmid DNA).

Fig. 2 is a graph showing the results of the sensitivity test of the primer sets for the H5 type avian influenza virus, (a), (b), (c) and (d) respectively show the results when using the primer sets A, B, C and D , 10 ³ to 10 ⁶ represent the dilution rate of the RNA extract.

Figure 3 is a graph showing the electrophoresis results of the product amplified with the primer set for the H5 type avian influenza virus. Swimming lane 1 is a 100bp ladder marker, swimming lane 2 is a sample of the LAMP product, and swimming lane 3 is obtained by processing the LAMP product with Dde I of samples.

Figure 4 is a graph showing the results of the crossover test of the primer set for the H5 type avian influenza virus, B-sd means B/Shandong/07/97, B-sh means B/Shanghai/361/2002, AIV-H1 etc. means poultry Influenza virus H1 etc.

Fig. 5 is a graph showing the results of a reactivity confirmation test of the primer set for the H7 avian influenza virus, 250, 100, and 50 indicate the amount of tRNA added (copy/test), and NC indicates a negative control.

Figure 6 is a graph showing the electrophoresis results of the product amplified with the primer set for the H7-type avian influenza virus. Swimming lane M is a 100bp ladder marker, swimming lane 1 is a sample of the LAMP product, and swimming lane 2 is obtained by processing the LAMP product with Pst I of samples.

Figure 7 is a graph showing the results of the crossover test of the primer sets for H7 avian influenza virus, H1 and H3 represent human influenza virus H1 and H3, B-sd represents B/Shandong/07/97, and B-sh represents B/Shanghai/361/2002, AIV-H1 etc. means avian influenza virus H1 etc., PC means positive control, NC means negative control.

Figure 8 is a graph showing the results of the reactivity of the H7-type avian influenza virus primer set to various strains, (a) shows A/Netherlands/219/2003, (b) shows A/Netherlands/33/2003, 10 ⁵ ~ ¹⁰⁸ represents the dilution ratio of the RNA extract.

Fig. 9 is a graph showing the results of the reactivity of the primer set for H7 avian influenza virus to various strains, (a) shows A/mallard/Netherlands/12/00, (b) shows A/water mallard/Osaka/1 /2001, 10 ⁵ to 10 ⁸ represents the dilution rate of the RNA extract.

The best way to practice the invention

Examples of samples used in the present invention include sputum, bronchoalveolar cleansing fluid, nasal fluid, nasal aspiration fluid, nasal cavity cleaning fluid, nasal cavity swab, throat swab, mouthwash, saliva, blood, and serum , blood plasma, cerebrospinal fluid, urine, feces, tissue, etc. come from living samples of humans or other animals suspected of being infected with avian influenza virus. In addition, as the sample, virus-containing samples such as cells isolated from cells used in infection experiments and their culture fluids, specimens derived from living bodies, cultured cells, and the like may be used. These samples can be subjected to pretreatments such as separation, extraction, concentration, and purification.

Such a nucleic acid can be amplified by a loop-mediated constant temperature amplification method called the LAMP method, a new nucleic acid amplification method that does not require temperature control, which is indispensable in the PCR method developed by Nafu et al. (International Publication No. 00/ Text No. 28082) to achieve this. In this method, by annealing its own 3' end to the nucleotide as a template to serve as the starting point of complementary chain synthesis, a primer that anneals to the loop formed at this time can be combined to realize a complementary chain synthesis reaction at a constant temperature nucleic acid amplification method. In addition, the LAMP method is a highly specific nucleic acid amplification method using at least 4 primers recognizing 6 regions.

The oligonucleotide primers used in the LAMP method are at least 4 kinds of primers that recognize the nucleotide sequence of a total of 6 regions of the nucleotide sequence of the template nucleic acid, and the 6 regions are denoted as F3c, F2c, The region of F1c and the regions denoted as B3, B2, and B1 from the 5' terminal side are referred to as inner primers F and B and outer primers F and B, respectively. In addition, the complementary sequences of F3c, F2c, and F1c are respectively referred to as F3, F2, and F1, and the complementary sequences of B3, B2, and B1 are respectively referred to as B3c, B2c, and B1c. An internal primer refers to a "specific nucleotide sequence region" that recognizes a target nucleotide sequence, and has a nucleotide sequence at the 3' end that serves as the origin of synthesis, and at the 5' end has a base sequence for the primer as a starting point. An oligonucleotide with a base sequence complementary to any region of the nucleic acid synthesis reaction product. Here, the primer containing the "base sequence selected from F2" and the "base sequence selected from F1c" is called an internal primer F (hereinafter abbreviated as FIP), and will contain the "base sequence selected from B2" The primer with "base sequence selected from B1c" is called internal primer B (hereinafter abbreviated as BIP). On the other hand, an external primer is one that recognizes "a specific nucleotide sequence region that exists closer to the 3' end than the region recognized by the internal primer" on the base sequence of interest, and has a base that serves as the origin of synthesis. base sequence oligonucleotides. Here, the primer containing the "base sequence selected from F3" is called external primer F (hereinafter abbreviated as F3), and the primer containing the "base sequence selected from B3" is called external primer B (hereinafter abbreviated as F3). for B3). Here, F in each primer represents a primer that complementarily binds to the sense strand of the target base sequence and provides a synthesis origin; on the other hand, B represents a primer that complementarily binds to the antisense strand of the target base sequence and provides a synthesis origin primers. Here, the length of the oligonucleotides used as primers is more than 10 bases, preferably more than 15 bases, and can be chemically synthesized or natural oligonucleotides, and each primer can be a single oligonucleotide, Mixtures of multiple oligonucleotides are also possible.

In the LAMP method, other primers, ie, loop primers, can be used in addition to the inner primers and outer primers. Loop primer refers to the case where the complementary sequences generated on the same strand of the amplification product based on the LAMP method anneal to each other to form a loop, and the 3' end contains a base sequence complementary to the sequence in the loop. Two types of primers (one for each strand constituting the double strand). Using this primer increases the starting point of nucleic acid synthesis, shortening the reaction time and improving detection sensitivity (International Publication No. 02/24902).

Oligonucleotides can be produced by known methods, for example, they can be chemically synthesized. Alternatively, a natural nucleic acid may be cut with a restriction enzyme or the like, and altered or linked into a desired nucleotide sequence structure. Specifically, it can be synthesized using an oligonucleotide synthesizer or the like. In addition, a method known per se, such as a synthesis method of an oligonucleotide having a base sequence in which one to several bases are substituted, deleted, inserted, or added, can be used. For example, the oligonucleotides can be synthesized by using site-specific mutation introduction method, gene homologous recombination method, primer extension method or PCR method alone or in appropriate combination.

Generally known conditions can be selected as "stringent hybridization conditions" in the present specification. As stringent conditions, for example, the following conditions can be exemplified: in the presence of 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt’s solution, 10 % dextran sulfate and 20 μg/ml DNA, hybridized overnight at 42°C, washed once in 2×SSC·0.1% SDS at room temperature, and then washed with 0.1×SSC·0.1% at about 65°C Wash twice in % SDS.

Influenza viruses are RNA viruses. When the template is RNA in the LAMP method, nucleic acid amplification reaction can be similarly performed by adding reverse transcriptase to the reaction solution when the template is DNA (RT-LAMP method).

After earnest research on the primer base sequence and combination thereof of the LAMP method that can rapidly amplify the base sequence specific to the H5 type avian influenza virus, the base sequence of the hemagglutinin of the H5 type avian influenza virus (Base sequence represented by SEQ ID NO: 1), the following four sets of A, B, C, and D were selected as primer sets. In addition, the sequences of these primers are completely different from the sequences of primers for NASBA (Nucleic Acid Sequence-Based Amplification) reported (for example, Patent Document 2) for the detection of H5 avian influenza virus.

(Primer Set A)

FIP19c: 5'-ACCATATTCCAACTCACTTTTCATAATTTCATTGCTCCAGAATATGC-3' (SEQ ID NO: 8)

BIP5: 5'-CAAACTCCAATGGGGGCATGGTGAGAGGGTGTAT-3' (SEQ ID NO: 9)

F3m6: 5'-GGAGTTTCTTCTGGACAA-3' (SEQ ID NO: 4)

B3m: 5'-GTCGCAAGGACTAATCT-3' (SEQ ID NO: 10)

LF24: 5'-GAGTCCCCCTTTCTTGACAAT-3' (SEQ ID NO: 11)

LB1: 5'-GATAAACTCTAGTATGCCA-3' (SEQ ID NO: 12)

(Primer Set B)

FIP: 5'-GGGCATGTGTAACAGTAACGTTAAACAACTCGACAGAGCA-3' (SEQ ID NO: 19)

BIP: 5'-TGGAAAAGACACACAATGGGAACATCCAGCTACACTACAATC-3' (SEQ ID NO: 20)

F3: 5'-CAGATTTGCATTGGTTACCA-3' (SEQ ID NO: 15)

B3: 5'-CGTCACACATTGGGTTTC-3' (SEQ ID NO: 21)

LF: 5'-TTCCATTATTGTGTCAACC-3' (SEQ ID NO: 22)

LB8: 5'-CGATCTAGATGGAGTGAAGC-3' (SEQ ID NO: 23)

(Primer Set C)

FIP: 5'-CACATTGGGTTTCCGAGGAGATCTAGATGGAGTGAAGCC-3' (SEQ ID NO: 30)

BIP: 5'-TTCATCAATGTGCCGGAATGGGTTGAAATCCCCTGGGTA-3' (SEQ ID NO: 31)

F3: 5'-GGAAAAGACACACAATGGG-3' (SEQ ID NO: 26)

B3: 5'-GCTCAATAGGTGTTTCAGTT-3' (SEQ ID NO: 32)

LF6: 5'-CCAGCTACACTACAATCTCT-3' (SEQ ID NO: 33)

LB6: 5'-TCCAGCCAATGACCTCTG-3' (SEQ ID NO: 34)

(Primer Set D)

FIP: 5'-TCGCAAGGACTAATCTGTTTGACATACACCCTCTCACCAT-3' (SEQ ID NO: 41)

BIP: 5'-TACCCCTCAAAGAGAGAGAAGATCCTCCCTCTATAAAACCTG-3' (SEQ ID NO: 42)

F3: 5'-TCTAGTATGCCATTCCACAA-3' (SEQ ID NO: 37)

B3: 5'-ACCATCTACCATTCCCTG-3' (SEQ ID NO: 43)

LF8: 5'-TCACATATTTGGGGCATTCC-3' (SEQ ID NO: 44)

LB8: 5'-AGAGAGGACTATTTGGAGCT-3' (SEQ ID NO: 45)

In addition, after earnest research on the primer base sequence and combination thereof of the LAMP method that can rapidly amplify the base sequence specific to the H7-type avian influenza virus, the base of the hemagglutinin of the H7-type avian influenza virus Base sequence (base sequence represented by SEQ ID NO: 1), the following primer set E was selected.

(Primer Set E)

22FIP: 5'-ACCACCCATCAATCAAACCTTCTATTTGGTGCTATAGCGG-3' (SEQ ID NO: 55)

22BIP: 5'-TTCAGGCATCAAAATGCACAAGCCTGTTATTTGATCAATTGCTG-3' (SEQ ID NO: 56)

22F3m: 5'-TTCCCGAAATCCCAAA-3' (SEQ ID NO: 51)

22B3: 5'-GGTTAGTTTTTTTCTATAAGCCG-3' (SEQ ID NO: 57)

22-9LF: 5'-CCCATCCATTTTCAATGAAAC-3' (SEQ ID NO: 58)

22-9LB: 5'-ACTGCTGCAGATTACAAAAG-3' (SEQ ID NO: 59)

The enzyme used for nucleic acid synthesis is not particularly limited as long as it is a template-dependent nucleic acid synthetase having strand displacement activity. Such an enzyme may, for example, be Bst DNA polymerase (large fragment), Bca (exo-) DNA polymerase, Klenow fragment of E. coli DNA polymerase I, etc., preferably Bst DNA polymerase (large fragment) .

The reverse transcriptase used in the RT-LAMP method is not particularly limited as long as it has the activity of synthesizing cDNA using RNA as a template. Examples of such enzymes include AMV, cloned AMV, reverse transcriptase of MMLV, Superscript II, ReverTraAce, Thermoscript, etc., preferably reverse transcriptase of AMV or cloned AMV. In addition, if an enzyme having both reverse transcriptase activity and DNA polymerase activity like Bca DNA polymerase is used, the RT-LAMP reaction can be performed with a single enzyme.

Enzymes and reverse transcriptases used in nucleic acid synthesis can be purified from viruses or bacteria, etc., or produced by genetic recombination techniques. In addition, these enzymes can undergo changes such as fragmentation or amino acid substitution.

A known technique can be used for detection of the nucleic acid amplification product after the LAMP reaction. For example, detection can be performed using a labeled oligonucleotide that specifically recognizes the amplified base sequence or a fluorescent intercalator method (Japanese Patent Application Laid-Open No. 2001-242169 ), or the reaction solution after the reaction can be directly It is easily detected by agarose gel electrophoresis. By agarose gel electrophoresis, the LAMP amplification product was detected as a ladder of various bands with different base lengths. In addition, in the LAMP method, a large amount of substrate is consumed due to the synthesis of nucleic acid, and pyrophosphate ions as a by-product react with coexisting magnesium ions to generate magnesium pyrophosphate, and the reaction solution becomes cloudy enough to be recognized by the naked eye. Therefore, by using a measuring instrument that can optically observe the turbidity rise after the reaction or during the reaction, the turbidity can be confirmed, for example, the change in absorbance at 400 nm can be confirmed using a conventional spectrophotometer, and the nucleic acid amplification reaction can also be detected (International Publication No. 01/83817).

Various reagents required for detection of nucleic acid amplification using the primers of the present invention can be combined in advance to form a kit. Specifically, various oligonucleotides required for primers or loop primers of the present invention, four kinds of dNTPs as substrates for nucleic acid synthesis, DNA polymerase for nucleic acid synthesis, enzymes with reverse transcription activity, and A buffer and salts suitable for the conditions of the enzyme reaction, a protective agent for stabilizing the enzyme and the template, and, if necessary, reagents required for the detection of the reaction product are provided in the form of a kit.

Example

Hereinafter, examples are given to illustrate the present invention in detail, but the present invention is not limited by these examples.

Example 1: Confirmation of Reactivity of Primer Set for H5 Type Avian Influenza Virus

Confirmation of the reactivity of the primers was performed by the following method. The composition of the reaction solution used for nucleic acid amplification by the LAMP method is as follows. In addition, the synthesis of primers was entrusted to QIAGEN, and the resulting products were purified using OPC (reverse-phase column chromatography).

20mM Tris-HCl pH8.8

10mM KCl

8mM MgSO ₄

1.4mM dNTPs

10mM(NH ₄ ) ₂ SO ₄

0.8M Betaine (SIGMA)

0.1%Tween20

1.6 μM FIP

1.6 μM BIP

0.2 μM F3

0.2 μM B3

0.8 μM LF

0.8 μM LB

AMV Reverse Transcriptase 2U (Finnzyme)

Bst DNA Polymerase 16U(NEB)

10 ⁴ copies of H5 plasmid DNA (HK/213/03; provided by the National Institute of Infectious Diseases) were added to the above reaction solution, and RT-LAMP reaction was performed at 62.5° C. for 60 minutes. The reaction was detected in real time using a real-time nephelometer LA-320C (Eiken Chemical). As a result, four primer sets (primer sets A, B, C, and D) confirmed amplification.

In addition, for these four groups, specificity tests were carried out using RNAs extracted from New Caledonian subtype (H1N1) and Panama subtype (H3N2) as cultured viruses as templates. Fig. 1 is a graph showing the results of a specificity test. As shown in FIG. 1 , no amplification of the H1 type and the H3 type was observed when each primer set was used. From the above results, it can be seen that all the primer sets have high specificity for the H5 type.

Next, a sensitivity test was performed using RNA extracted from Vietnam/JP1203/04 (H5N1) as a cultured virus. Since the amount of template for extracting RNA was unknown, RNA diluted 10 ³ to 10 ⁶ times with RNase-free sterile water was used as a template sample. Fig. 2 is a graph showing the results of a sensitivity test. When primer set A was used, amplification was confirmed even in a ¹⁰⁵ -fold diluted sample, and the sensitivity was the highest.

Example 2: Confirmation of the Product Amplified with the Primer Set for H5 Type Avian Influenza Virus

The LAMP product amplified with the primer set A was confirmed by electrophoresis and restriction enzyme DdeI. Fig. 3 is a graph showing the results of electrophoresis. The trapezoidal pattern peculiar to the LAMP product can be clearly confirmed from the lane 2 of FIG. 3 . In addition, digestion was confirmed for the sample treated with DdeI (lane 3). From the above results, it can be seen that the target sequence was specifically amplified.

Example 3: Evaluation of primer set for H5 type avian influenza virus (crossover test)

A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2), B/Shandong/07/97 and B/Shanghai/361/2002 of human influenza viruses and avian influenza A total of 18 types of viruses H1 to H15 (excluding H5) were used as samples. RNA was extracted from the cultured virus with QIAamp Viral RNA Kit (QIAGEN), and 5 μL of the extract was used for RT-LAMP reaction (primer set A was used as primers).

It can be seen from Figure 4(a) and (b) that no amplification was found in all samples by RT-LAMP method. Therefore, it was confirmed that the specificity of the RT-LAMP method was high.

Example 4: Evaluation of Primer Sets for H5 Type Avian Influenza Virus (Sensitivity Test)

The avian influenza H5 strain (CH/Yamaguchi 7/04) confirmed to be infected in Yamaguchi Prefecture in 2004 and the H5 strain (VN/JP1203/04) confirmed to be infected in Vietnam in 2004 were used as template samples. Separately dilute the RNA extracted from the cultured virus with ribonuclease-free sterilized water (10 ⁴ ～10 ⁸ ), use 10 μL dilution solution for RT-PCR method, and RT-LAMP method (use primer set A as primer) Use 5 μL of diluent.

The RT-PCR method was performed by modifying the conditions posted on the website of the Infectious Disease Information Center of the National Institute of Infectious Diseases. That is, using a commercially available kit (TaKaRa One Step RNA PCR Kit (AMV)), reverse transcription reaction was performed at 50°C for 30 minutes, followed by 94°C for 2 minutes, and repeated 30 times at 94°C for 1 minute and 45°C for 1 minute. minute, 72°C for 1 minute cycle, then elongation reaction at 72°C for 10 minutes, and storage at 4°C.

The primers and the composition of the reaction solution used in the RT-PCR method are as follows.

Primers (length of PCR product: 708bp)

H5515f: 5'-CATACCCAACAATAAAGAGG-3' (SEQ ID NO: 46)

H51220r: 5'-GTGTTCATTTTGTTAATGAT-3' (SEQ ID NO: 47)

reaction solution

RNA extraction solution 10μL

10×One Step RNA PCR Buffer 5μL

10mM dNTP 5μL

25mM _MgCl2 10μL

RNase inhibitor 1 μL

AMV reverse transcriptase 1 μL

AMV-optimized Taq 1 μL

H5 515f (10μM) 2μL

H5 1220r (10 μM) 2 μL

Add RNase-free sterilized water appropriately to adjust the reaction solution to 50 μL

Table 1 summarizes the results of amplification by the RT-PCR method and the RT-LAMP method. In Table 1, the column of the RT-LAMP method indicates the proportion of samples whose amplification was confirmed. In addition, the amplification in RT-PCR was visually confirmed as a result of electrophoresis (in Table 1, ○ indicates that amplification can be detected, and × indicates that amplification cannot be detected).

[Table 1]

When comparing the RT-LAMP method and the RT-PCR method, all the strains had a sensitivity 10 to 100 times higher than that of the RT-LAMP method.

Example 5: Confirmation of Reactivity of Primer Set for H7 Type Avian Influenza Virus

Confirmation of the reactivity of the primer set E was performed by the following method. The composition of the reaction solution used for nucleic acid amplification by the LAMP method is as follows. In addition, the synthesis of primers was entrusted to QIAGEN, and the resulting products were purified using OPC (reverse-phase column chromatography).

20mM Tris-HCl pH8.8

10mM KCl

8mM MgSO ₄

1.4mM dNTPs

10mM(NH ₄ ) ₂ SO ₄

0.8M Betaine (SIGMA)

0.1%Tween20

1.6 μM FIP

1.6 μM BIP

0.2 μM F3

0.2 μM B3

0.8 μM LF

0.8 μM LB

AMV Reverse Transcriptase 2U (Finnzyme)

Bst DNA Polymerase 16U(NEB)

The nucleotide sequence (fragment including the nucleotide sequence shown in SEQ ID NO: 1) of A/Netherlands/219/2003 (H7N7) (a strain isolated when a fatal case occurred in the Netherlands) was recombined into a cloning vector, and a transcription reaction was carried out. Prepare tRNA. The prepared tRNA was added to the above reaction solution in an amount of 250, 100 or 50 copies/test, reacted at 62.5°C for 35 minutes, and performed amplification reaction and detection using a real-time turbidimetry device LA-200 (Teramex).

The real-time detection results of the tRNA addition amount of 250, 100 and 50 copies/test and the negative control are shown in FIG. 5 . From this result, it was found that amplification was detected up to 50 copies/test.

Example 6: Confirmation of products amplified with primer set for H7 type avian influenza virus

The LAMP product amplified with the primer set E was confirmed by electrophoresis and restriction enzyme Pst I. Fig. 6 is a graph showing the results of electrophoresis. M represents the 100bp ladder marker, lane 1 is the sample of untreated LAMP product, and lane 2 is the sample obtained by digesting the LAMP product with Pst I. The trapezoidal pattern peculiar to the LAMP product can be clearly confirmed from the lane 1 of FIG. 6 . In addition, digestion was confirmed for the sample treated with Pst I (lane 2). From the above results, it can be seen that the target sequence was specifically amplified.

Embodiment 7: Evaluation of primer sets for H7 type avian influenza virus (crossover test)

A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2), B/Shandong/07/97 and B/Shanghai/361/2002 of human influenza viruses and avian influenza A total of 18 types of viruses H1 to H15 (excluding H7) were used as samples, and the specificity of the primers was examined. RNA was extracted from the cultured virus with QIAamp Viral RNA Kit (QIAGEN), and the extracted RNA diluted 100 times was used for RT-LAMP reaction. In addition, the positive control (PC) used the tRNA prepared in Example 5, and the negative control used distilled water.

It can be seen from Figure 7 that no amplification was found in all samples. Therefore, it was confirmed that the primer set of the present invention has high specificity.

Example 8: Evaluation of primer sets for H7-type avian influenza virus (reactivity to various strains)

The genomes of four H7-type avian influenza viruses were used as template samples to investigate the sensitivity to various viruses. The RNA extracted from the cultured virus was diluted stepwise (10 ⁵ -10 ⁸ ) with ribonuclease-free sterilized water, and 5 μL of the dilution was used.

The results of real-time detection when each viral genome was used as a template sample are shown in FIGS. 8 and 9 . For A/Netherlands/219/2003(H7N7) and A/Netherlands/33/2003(H7N7), 10 ⁷ -fold dilutions can be detected (refer to Figure 8), for A/Mallard/Netherlands/12/2003(H7N3) and A/water mallard/Osaka/1/2001 (H7N7) can be detected up to 10 ⁶ -fold dilution (see Figure 9). From the above results, it can be seen that the primer set of the present invention reacts with the above four strains.

Possibility of industrial use

If the present invention is adopted, H5 or H7 avian influenza virus can be detected rapidly and with high sensitivity.

sequence listing

<110> EIKEN KAGAKU KABUSHIKI KAISHA

  National Institute of Infectious Diseases

<120> Detection method of H5 or H7 avian influenza virus

<130>FP05-0385-00

<160>59

<170>PatentIn version 3.1

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gcatcatgtt tcatacttct ggccattgca atgggccttg tcttcatatg tgtgaagaat 1680

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<220>

<223> Primer

<400>52

ttcaggcatc aaaatgcaca ag 22

<210>53

<211>22

<212>DNA

<213> Artificial

<220>

<223> Primer

<400>53

cagcaattga tcaaataaca gg 22

<210>54

<211>22

<212>DNA

<213> Artificial

<220>

<223> Primer

<400>54

cggcttatag aaaaaactaa cc 22

<210>55

<211>40

<212>DNA

<213> Artificial

<220>

<223> Primer

<400>55

accaccccatc aatcaaacct tctatttggt gctatagcgg 40

<210>56

<211>44

<212>DNA

<213> Artificial

<220>

<223> Primer[

<400>56

ttcaggcatc aaaatgcaca agcctgttat ttgatcaatt gctg 44

<210>57

<211>22

<212>DNA

<213> Artificial

<220>

<223> Primer

<400>57

ggttagtttt ttctataagc cg 22

<210>58

<211>21

<212>DNA

<213> Artificial

<220>

<223> Primer

<400>58

cccatccatt ttcaatgaaa c 21

<210>59

<211>20

<212>DNA

<213> Artificial

<220>

<223> Primer

<400>59

actgctgcag attacaaaag 20

Claims (6)

1. oligonucleotide primer set, is characterized in that, contains 4 kinds of primers that are made up of the nucleotide sequence of following (i)-(iv) record, and this primer can amplify the base specific for H5 type avian influenza virus sequence: (i) the base sequence of sequence number 8, or the base sequence complementary to the base sequence of sequence number 8; (ii) the base sequence of sequence number 9, or the base sequence complementary to the base sequence of sequence number 9; (iii) the base sequence of SEQ ID NO: 4, or a base sequence complementary to the base sequence of SEQ ID NO: 4; (iv) the nucleotide sequence of SEQ ID NO: 10, or a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 10. 2. oligonucleotide primer set as claimed in claim 1, is characterized in that, also contains 2 kinds of primers that are made up of the base sequence of following (v)-(vi): (v) the base sequence of SEQ ID NO: 11; or the base sequence complementary to the base sequence of SEQ ID NO: 11; (vi) the nucleotide sequence of SEQ ID NO: 12; or the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 12. 3. the purposes of claim 1 or 2 described oligonucleotide primer sets, it is characterized in that, be used for the reagent of preparation detection H5 type avian influenza virus, this virus is to carry out the target nucleic acid region of H5 type avian influenza virus Characterized by an amplification reaction. 4. The use according to claim 3, characterized in that the amplification reaction of the target nucleic acid region of the H5 type avian influenza virus adopts the LAMP method. 5. The use of the oligonucleotide primer set according to claim 1 or 2, characterized in that it is used to prepare a reagent for diagnosing whether there is H5 type avian influenza virus infection. 6. The kit, characterized in that it comprises the oligonucleotide primer set according to claim 1 or 2 for diagnosing influenza.

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