CN101052720A - Method of detecting H5 or H7 avian influenza virus - Google Patents
Method of detecting H5 or H7 avian influenza virus Download PDFInfo
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Abstract
An oligonucleotide primer capable of specifically hybridizing with an arbitrary base sequence designed from the base sequence of hemagglutinin of H5 or H7 avian influenza virus; a method of nucleic acid amplification using the primer; a method of diagnosing any infection with H5 or H7 avian influenza virus through detection of nucleic acid amplification; and a kit for influenza diagnosis.
Description
Technical field
The present invention relates to the detection method of H5 type or H7 type avian influenza virus, more specifically relate to the Oligonucleolide primers that is used to detect H5 type or H7 type avian influenza virus, the H5 type that uses this primer or H7 type avian influenza virus detection method, influenza diagnostic method and be used to diagnose the test kit of influenza.
Background technology
Influenza is epidemic viral respiratory organs transmissible disease, and the trouble patient has the wide age level from infant to the elderly, usually is fatal.At present, infected poultry and the H5 type avian influenza virus that forms problem did not infect the mankind originally.But, found infection in Hong Kong in 1997 to the people, this time be 6 death of 18 patients in the groove.Do not find infection after fortunately again, but enter 2004, found infection to the people 8 people's death are arranged, 16 people are arranged in Vietnam in Thailand in Thailand and Vietnam to the people.
Highly Pathogenic Avian Influenza Virus (HPAIV) also has the H7 type except that the H5 type, the H7 type roughly is divided into Europe class and american type according to its sequence.2003 in Holland popular time report has 1 people's death, also reported the popular of H7 type avian influenza virus in 2003 to 2004 in the U.S..
At present, the detection end user A type influenza virus quick diagnosis reagent kit of avian influenza virus.But, more detailed analyses such as the antigen analysis of the isolating virus of evaluation needs of the hypotype of institute's infective virus and gene test.
Therefore the diagnosis of employing virus separation and Culture that can obtain reliable result can't be diagnosed fast owing to need a few days.Can separate the method diagnose more quickly than virus have some kinds, and RT-PCR method wherein is considered to that to compare detection sensitivity higher with other method.But, for present disclosed RT-PCR method, have and report that it is compared and can't detect virus with highly sensitive with the infection valency of virus, even be negative in the inspection of employing RT-PCR method, can not get rid of the infection of bird flu.Therefore, wish occurring can be rapidly and detect the test procedure of H5 and H7 type avian influenza virus in high sensitivity.
Patent documentation 1: european patent application discloses specification sheets No. 1310565
Patent documentation 2: the public table of Japanese Patent 2004-509648 communique
Non-patent literature 1:Lau LT. etc., Biochem.Biophys.Res.Commun., vol.313, p.336-342 (2004)
Non-patent literature 2:Shang S. etc., Biochem.Biophys.Res.Commun., vol.302, p.377-383 (2003)
Non-patent literature 3:Collins RA. etc., Biochem.Biophys.Res.Commun., vol.300, p.507-515 (2003)
Non-patent literature 4:Lee MS. etc., J.Virol.Methods, vol.97, p.13-22 (2001)
Non-patent literature 5:Munch M. etc., Arch.Virol., vol.146, p.87-97 (2001)
The announcement of invention
In order to solve above-mentioned problem, the inventor conscientiously studies the back and finds, make and the oligonucleotide primer that the base sequence of H5 or H7 type avian influenza virus specific is hybridized, by LAMP (loop-mediated isothermal amplification, loop-mediated isothermal amplification) the method amplification is to the base sequence of H5 or H7 type avian influenza virus specific, can detect H5 or H7 type avian influenza virus in high sensitivity, thereby finish the present invention.
That is, the invention provides following (1)～(8).
(1) according to be selected from any base sequence of 693～959 base sequence of the hemagglutinin base sequence of the H5 type avian influenza virus of sequence numbering 1 expression or with the Oligonucleolide primers of its complementary base sequence design.
(2), wherein, comprise the oligonucleotide that is selected from following (a)～(c) as (1) described Oligonucleolide primers:
(a) be selected from the base sequence of sequence numbering 2～7 expression or with the oligonucleotide that contains 15 bases of successive at least of its complementary base sequence;
(b) can with the oligonucleotide of aforementioned (a) described oligonucleotide hybridize under stringent condition;
(c) comprise aforementioned (a) or (b) 1 in the described oligonucleotide～several base base sequence of being replaced, lack, insert or adding, oligonucleotide with primer function.
(3) as (1) or (2) described Oligonucleolide primers, it is characterized in that, 3 ' end side from the target nucleic acid of the hemagglutinin of H5 type avian influenza virus selects note to do the base sequence zone of F3c, F2c, F1c, select note to do the base sequence zone of B3, B2, B1 from 5 ' end side, when complementary base sequence note is separately made F3, F2, F1 and B3c, B2c, B1c, constitute by the base sequence that is selected from following (a)～(d):
(a) have the F2 zone of target nucleic acid at 3 ' end side, have the base sequence in the F1c zone of target nucleic acid at 5 ' end side;
(b) has the base sequence in the F3 zone of target nucleic acid;
(c) have the B2 zone of target nucleic acid at 3 ' end side, have the base sequence in the B1c zone of target nucleic acid at 5 ' end side;
(d) has the base sequence in the B3 zone of target nucleic acid.
(4) as the described Oligonucleolide primers in (1)～(3), it is characterized in that, can increase, to 3 ' end, constitute by the base sequence that is selected from following (a)～(b) from 5 ' end to the base sequence of H5 type avian influenza virus specific:
(a) 5 '-(with the base sequence complementary base sequence of sequence numbering 2)-(base sequence arbitrarily of base several 0～50)-(base sequence of sequence numbering 3)-3 ';
(b) 5 '-(base sequence of sequence numbering 5)-(base sequence arbitrarily of base several 0～50)-(with the base sequence complementary base sequence of sequence numbering 6)-3 '.
(5) detection method of H5 type avian influenza virus is characterized in that, uses the described Oligonucleolide primers in (1)～(4), carries out the amplified reaction in the target nucleic acid zone of H5 type avian influenza virus.
As the detection method of (5) described H5 type avian influenza virus, it is characterized in that (6) amplified reaction in the target nucleic acid zone of H5 type avian influenza virus adopts the LAMP method.
(7) diagnostic method of influenza is characterized in that, by using the described Oligonucleolide primers in (1)～(4), detects the amplification in the target nucleic acid zone of H5 type avian influenza virus, and whether diagnosis has the infection of H5 type avian influenza virus.
(8) test kit is characterized in that, comprises the described Oligonucleolide primers in (1)～(4) that is used to diagnose influenza.
The present invention also provides following (9)～(16).
(9) according to be selected from any base sequence of 19～220 base sequence of the hemagglutinin base sequence of the H5 type avian influenza virus of sequence numbering 1 expression or with the Oligonucleolide primers of its complementary base sequence design.
(10), wherein, comprise the oligonucleotide that is selected from following (a)～(c) as (9) described Oligonucleolide primers:
(a) be selected from the base sequence of sequence numbering 13～18 expression or with the oligonucleotide that contains 15 bases of successive at least of its complementary base sequence;
(b) can with the oligonucleotide of aforementioned (a) described oligonucleotide hybridize under stringent condition;
(c) comprise aforementioned (a) or (b) 1 in the described oligonucleotide～several base base sequence of being replaced, lack, insert or adding, oligonucleotide with primer function.
(11) as (9) or (10) described Oligonucleolide primers, it is characterized in that, 3 ' end side from the target nucleic acid of the hemagglutinin of H5 type avian influenza virus selects note to do the base sequence zone of F3c, F2c, F1c, select note to do the base sequence zone of B3, B2, B1 from 5 ' end side, when complementary base sequence note is separately made F3, F2, F1 and B3c, B2c, B1c, constitute by the base sequence that is selected from following (a)～(d):
(a) have the F2 zone of target nucleic acid at 3 ' end side, have the base sequence in the F1c zone of target nucleic acid at 5 ' end side;
(b) has the base sequence in the F3 zone of target nucleic acid;
(c) have the B2 zone of target nucleic acid at 3 ' end side, have the base sequence in the B1c zone of target nucleic acid at 5 ' end side;
(d) has the base sequence in the B3 zone of target nucleic acid.
(12) as the described Oligonucleolide primers in (9)～(11), it is characterized in that, can increase, to 3 ' end, constitute by the base sequence that is selected from following (a)～(b) from 5 ' end to the base sequence of H5 type avian influenza virus specific:
(a) 5 '-(with the base sequence complementary base sequence of sequence numbering 13)-(base sequence arbitrarily of base several 0～50)-(base sequence of sequence numbering 14)-3 ';
(b) 5 '-(base sequence of sequence numbering 16)-(base sequence arbitrarily of base several 0～50)-(with the base sequence complementary base sequence of sequence numbering 17)-3 '.
(13) detection method of H5 type avian influenza virus is characterized in that, uses the described Oligonucleolide primers in (9)～(12), carries out the amplified reaction in the target nucleic acid zone of H5 type avian influenza virus.
As the detection method of (13) described H5 type avian influenza virus, it is characterized in that (14) amplified reaction in the target nucleic acid zone of H5 type avian influenza virus adopts the LAMP method.
(15) diagnostic method of influenza is characterized in that, by using the described Oligonucleolide primers in (9)～(12), detects the amplification in the target nucleic acid zone of H5 type avian influenza virus, and whether diagnosis has the infection of H5 type avian influenza virus.
(16) test kit is characterized in that, comprises the described Oligonucleolide primers in (9)～(12) that is used to diagnose influenza.
The present invention also provides following (17)～(24).
(17) according to be selected from any base sequence of 114～333 base sequence of the hemagglutinin base sequence of the H5 type avian influenza virus of sequence numbering 1 expression or with the Oligonucleolide primers of its complementary base sequence design.
(18), wherein, comprise the oligonucleotide that is selected from following (a)～(c) as (17) described Oligonucleolide primers:
(a) be selected from the base sequence of sequence numbering 24～29 expression or with the oligonucleotide that contains 15 bases of successive at least of its complementary base sequence;
(b) can with the oligonucleotide of aforementioned (a) described oligonucleotide hybridize under stringent condition;
(c) comprise aforementioned (a) or (b) 1 in the described oligonucleotide～several base base sequence of being replaced, lack, insert or adding, oligonucleotide with primer function.
(19) as (17) or (18) described Oligonucleolide primers, it is characterized in that, 3 ' end side from the target nucleic acid of the hemagglutinin of H5 type avian influenza virus selects note to do the base sequence zone of F3c, F2c, F1c, select note to do the base sequence zone of B3, B2, B1 from 5 ' end side, when complementary base sequence note is separately made F3, F2, F1 and B3c, B2c, B1c, constitute by the base sequence that is selected from following (a)～(d):
(a) have the F2 zone of target nucleic acid at 3 ' end side, have the base sequence in the F1c zone of target nucleic acid at 5 ' end side;
(b) has the base sequence in the F3 zone of target nucleic acid;
(c) have the B2 zone of target nucleic acid at 3 ' end side, have the base sequence in the B1c zone of target nucleic acid at 5 ' end side;
(d) has the base sequence in the B3 zone of target nucleic acid.
(20) as the described Oligonucleolide primers in (17)～(19), it is characterized in that, can increase, to 3 ' end, constitute by the base sequence that is selected from following (a)～(b) from 5 ' end to the base sequence of H5 type avian influenza virus specific:
(a) 5 '-(with the base sequence complementary base sequence of sequence numbering 24)-(base sequence arbitrarily of base several 0～50)-(base sequence of sequence numbering 25)-3 ';
(b) 5 '-(base sequence of sequence numbering 27)-(base sequence arbitrarily of base several 0～50)-(with the base sequence complementary base sequence of sequence numbering 28)-3 '.
(21) detection method of H5 type avian influenza virus is characterized in that, uses the described Oligonucleolide primers in (17)～(20), carries out the amplified reaction in the target nucleic acid zone of H5 type avian influenza virus.
As the detection method of (21) described H5 type avian influenza virus, it is characterized in that (22) amplified reaction in the target nucleic acid zone of H5 type avian influenza virus adopts the LAMP method.
(23) diagnostic method of influenza is characterized in that, by using the described Oligonucleolide primers in (17)～(20), detects the amplification in the target nucleic acid zone of H5 type avian influenza virus, and whether diagnosis has the infection of H5 type avian influenza virus.
(24) test kit is characterized in that, comprises the described Oligonucleolide primers in (17)～(20) that is used to diagnose influenza.
The present invention also provides following (25)～(32).
(25) according to be selected from any base sequence of 874～1065 base sequence of the hemagglutinin base sequence of the H5 type avian influenza virus of sequence numbering 1 expression or with the Oligonucleolide primers of its complementary base sequence design.
(26), wherein, comprise the oligonucleotide that is selected from following (a)～(c) as (25) described Oligonucleolide primers:
(a) be selected from the base sequence of sequence numbering 35～40 expression or with the oligonucleotide that contains 15 bases of successive at least of its complementary base sequence;
(b) can with the oligonucleotide of aforementioned (a) described oligonucleotide hybridize under stringent condition;
(c) comprise aforementioned (a) or (b) 1 in the described oligonucleotide～several base replaced, lacked the base sequence of husband, insertion or interpolation, oligonucleotide with primer function.
(27) as (25) or (26) described Oligonucleolide primers, it is characterized in that, 3 ' end side from the target nucleic acid of the hemagglutinin of H5 type avian influenza virus selects note to do the base sequence zone of F3c, F2c, F1c, select note to do the base sequence zone of B3, B2, B1 from 5 ' end side, when complementary base sequence note is separately made F3, F2, F1 and B3c, B2c, B1c, constitute by the base sequence that is selected from following (a)～(d):
(a) have the F2 zone of target nucleic acid at 3 ' end side, have the base sequence in the F1c zone of target nucleic acid at 5 ' end side;
(b) has the base sequence in the F3 zone of target nucleic acid;
(c) have the B2 zone of target nucleic acid at 3 ' end side, have the base sequence in the B1c zone of target nucleic acid at 5 ' end side;
(d) has the base sequence in the B3 zone of target nucleic acid.
(28) as the described Oligonucleolide primers in (25)～(27), it is characterized in that, can increase, to 3 ' end, constitute by the base sequence that is selected from following (a)～(b) from 5 ' end to the base sequence of H5 type avian influenza virus specific:
(a) 5 '-(with the base sequence complementary base sequence of sequence numbering 35)-(base sequence arbitrarily of base several 0～50)-(base sequence of sequence numbering 36)-3 ';
(b) 5 '-(base sequence of sequence numbering 38)-(base sequence arbitrarily of base several 0～50)-(with the base sequence complementary base sequence of sequence numbering 39)-3 '.
(29) detection method of H5 type avian influenza virus is characterized in that, uses the described Oligonucleolide primers in (25)～(28), carries out the amplified reaction in the target nucleic acid zone of H5 type avian influenza virus.
As the detection method of (29) described H5 type avian influenza virus, it is characterized in that (30) amplified reaction in the target nucleic acid zone of H5 type avian influenza virus adopts the LAMP method.
(31) diagnostic method of influenza is characterized in that, by using the described Oligonucleolide primers in (25)～(28), detects the amplification in the target nucleic acid zone of H5 type avian influenza virus, and whether diagnosis has the infection of H5 type avian influenza virus.
(32) test kit is characterized in that, comprises the described Oligonucleolide primers in (25)～(28) that is used to diagnose influenza.
The present invention also provides following (33)～(40).
(33) according to be selected from any base sequence of 1016～1225 base sequence of the hemagglutinin base sequence of the H7 type avian influenza virus of sequence numbering 48 expression or with the Oligonucleolide primers of its complementary base sequence design.
(34), wherein, comprise the oligonucleotide that is selected from following (a)～(c) as (33) described Oligonucleolide primers:
(a) be selected from the base sequence of sequence numbering 49～54 expression or with the oligonucleotide that contains 15 bases of successive at least of its complementary base sequence;
(b) can with the oligonucleotide of aforementioned (a) described oligonucleotide hybridize under stringent condition;
(c) comprise aforementioned (a) or (b) 1 in the described oligonucleotide～several base base sequence of being replaced, lack, insert or adding, oligonucleotide with primer function.
(35) as (33) or (34) described Oligonucleolide primers, it is characterized in that, 3 ' end side from the target nucleic acid of the hemagglutinin of H7 type avian influenza virus selects note to do the base sequence zone of F3c, F2c, F1c, select note to do the base sequence zone of B3, B2, B1 from 5 ' end side, when complementary base sequence note is separately made F3, F2, F1 and B3c, B2c, B1c, constitute by the base sequence that is selected from following (a)～(d):
(a) have the F2 zone of target nucleic acid at 3 ' end side, have the base sequence in the F1c zone of target nucleic acid at 5 ' end side;
(b) has the base sequence in the F3 zone of target nucleic acid;
(c) have the B2 zone of target nucleic acid at 3 ' end side, have the base sequence in the B1c zone of target nucleic acid at 5 ' end side;
(d) has the base sequence in the B3 zone of target nucleic acid.
(36) as the described Oligonucleolide primers in (33)～(35), it is characterized in that, can increase, to 3 ' end, constitute by the base sequence that is selected from following (a)～(b) from 5 ' end to the base sequence of H7 type avian influenza virus specific:
(a) 5 '-(with the base sequence complementary base sequence of sequence numbering 49)-(base sequence arbitrarily of base several 0～50)-(base sequence of sequence numbering 50)-3 ';
(b) 5 '-(base sequence of sequence numbering 52)-(base sequence arbitrarily of base several 0～50)-(with the base sequence complementary base sequence of sequence numbering 53)-3 '.
(37) detection method of H7 type avian influenza virus is characterized in that, uses the described Oligonucleolide primers in (33)～(36), carries out the amplified reaction in the target nucleic acid zone of H7 type avian influenza virus.
As the detection method of (37) described H7 type avian influenza virus, it is characterized in that (38) amplified reaction in the target nucleic acid zone of H7 type avian influenza virus adopts the LAMP method.
(39) diagnostic method of influenza is characterized in that, by using the described Oligonucleolide primers in (33)～(36), detects the amplification in the target nucleic acid zone of H7 type avian influenza virus, and whether diagnosis has the infection of H7 type avian influenza virus.
(40) test kit is characterized in that, comprises the described Oligonucleolide primers in (33)～(36) that is used to diagnose influenza.
If adopt the present invention, by making and the oligonucleotide primer that the base sequence of H5 or H7 type avian influenza virus specific is optionally hybridized, with LAMP method amplification base sequence to H5 or H7 type avian influenza virus specific, can highly sensitive and promptly detect H5 or H7 type avian influenza virus.
The simple declaration of accompanying drawing
Fig. 1 is the figure of expression H5 type avian influenza virus with the result of the specificity test of primer sets, (a) and (b), (c) and (d) result when primer sets A, B, C and D are used in expression respectively, NC represents negative control, H1 represents the New Caledonia hypotype, H3 represents Panamanian hypotype, and PC represents positive control (H5 type plasmid DNA).
Fig. 2 is the figure of expression H5 type avian influenza virus with the result of the sensitivity test of primer sets, (a) and (b), (c) and (d) result when expression use primer sets A, B, C and D respectively, 10
3～10
6The thinning ratio of expression RNA extract.
Fig. 3 is for representing that with the figure of H5 type avian influenza virus with the electrophoresis result of the product of primer sets amplification, swimming lane 1 is a 100bp ladder marker, and swimming lane 2 is the sample of LAMP product, and swimming lane 3 is for handling the sample that obtains with the LAMP product with Dde I.
Fig. 4 is the figure of expression H5 type avian influenza virus with the result of the cross matching of primer sets, and B-sd represents B/ Shandong/07/97, and B-sh represents B/ Shanghai/361/2002, expression such as AIV-H1 avian influenza virus H1 etc.
Fig. 5 is the figure of expression H7 type avian influenza virus with the result of the reactive validation test of primer sets, the addition (copy/test) of 250,100,50 expression tRNA, and NC represents negative control.
Fig. 6 is for representing that with the figure of H7 type avian influenza virus with the electrophoresis result of the product of primer sets amplification, swimming lane M is a 100bp ladder marker, and swimming lane 1 is the sample of LAMP product, and swimming lane 2 is for handling the sample that obtains with the LAMP product with Pst I.
Fig. 7 is the figure of expression H7 type avian influenza virus with the result of the intercrossing test of primer sets, H1 and H3 represent human influenza virus H1 type and H3 type, B-sd represents B/ Shandong/07/97, B-sh represents B/ Shanghai/361/2002, expression such as AIV-H1 avian influenza virus H1 etc., PC represents positive control, and NC represents negative control.
Fig. 8 is the expression H7 type avian influenza virus figure of primer sets for reactive result of various strains, (a) expression A/ Holland/219/2003, (b) expression A/ Holland/33/2003,10
5～10
8The thinning ratio of expression RNA extract.
Fig. 9 is the expression H7 type avian influenza virus figure of primer sets for reactive result of various strains, (a) expression A/ wild duck/Holland/12/00, (b) expression A/ water wild duck/Osaka/1/2001,10
5～10
8The thinning ratio of expression RNA extract.
The best mode that carries out an invention
As the sample that uses among the present invention, can exemplify the sample that for example sputum, bronchovesicular scavenging solution, nose liquid, nasal cavity liquid draw, nasal cavity scavenging solution, nasal cavity wiping liquid, pharynx wiping liquid, collutory, saliva, blood, serum, blood plasma, cerebrospinal fluid, urine, ight soil, tissue etc. come from the live body of the people of infection avian influenza virus under a cloud or other animal.In addition, sample also can adopt the sample that contains virus of the sample that separates cell and its nutrient solution used in the self-infection experiment etc. or come from live body and culturing cell etc. etc.These samples can separate, extracting, concentrate, pre-treatment such as purifying.
The amplification of such nucleic acid can indispensable temperature controlled new nucleic acid TRAP---the loop-mediated isothermal amplification method (the international text that discloses No. 00/28082) that is called as the LAMP method realizes by not needing to receive in the PCR method of exploitation such as richness.This method is to be annealed to as on the Nucleotide of template and as complementary strand synthetic starting point the time by the 3 ' end that makes self, and combination is annealed to the primer on the ring that at this moment forms, and can realize the nucleic acid amplification of the complementary strand synthesis reaction under the constant temperature.In addition, the LAMP method is to use the high nucleic acid amplification of specificity of 4 primers in 6 zones of identification at least.
Used Oligonucleolide primers is at least 4 kinds of primers of base sequence in totally 6 zones of the base sequence of recognition template nucleic acid in the LAMP method, the zone of B3, B2, B1 is done for the zone of making F3c, F2c, F1c from the note of 3 ' end side with from the note of 5 ' end side in these 6 zones, is called as inner primers F and B and outside primers F and B respectively.In addition, the complementary sequence of F3c, F2c, F1c remembered respectively make F3, F2, F1, the complementary sequence of B3, B2, B1 is remembered respectively made B3c, B2c, B1c.Inner primer is meant " the nucleotides sequence column region that certain is specific " on the recognition objective base sequence, and have the base sequence that is made for synthetic starting point at 3 ' end, have for the oligonucleotide of this primer at 5 ' end simultaneously as the arbitrary region complementary base sequence of the nucleic acid building-up reactions resultant of starting point.Here, the primer that will comprise " base sequence that is selected from F2 " and " being selected from the base sequence of F1c " is called inner primers F (following FIP slightly), and the primer that will comprise " base sequence that is selected from B2 " and " being selected from the base sequence of B1c " is called inner primer B (following BIP slightly).On the other hand, outside primer is meant " being present in than the zone of inner primer identification more near certain specific nucleotides sequence column region of 3 ' end side " on the recognition objective base sequence, and has the oligonucleotide of the base sequence that is made for synthetic starting point.Here, the primer that will comprise " base sequence that is selected from F3 " is called outside primers F (following F3 slightly), and the primer that will comprise " base sequence that is selected from B3 " is called outside primer B (following B3 slightly).Here, the F in each primer represents complementally to combine and provide with the sense strand of detecting target base sequence the primer of synthetic starting point; On the other hand, B represents complementally to combine and provide with the antisense strand of detecting target base sequence the primer of synthetic starting point.Here, more than 10 bases, better be more than 15 bases as the length of the oligonucleotide of primer, can be chemosynthesis or natural oligonucleotide, each primer can be single oligonucleotide, also can be the mixture of multiple oligonucleotide.
In the LAMP method, except that inner primer and outside primer, can also use other primer, promptly encircle primer.Ring primer (Loop Primer) is meant that the complementary sequence that produces on the same chain based on the amplification resultant of LAMP method is annealed mutually and forms under the situation of ring, comprises 2 kinds of primers (constituting each a kind on each double-stranded chain) with this intra-annular sequence complementary base sequence at its 3 ' end.If use this primer, then nucleic acid synthetic starting point increases, the shortening that can the realization response time and the rising (the international text that discloses No. 02/24902) of detection sensitivity.
Oligonucleotide can for example can carry out chemosynthesis by the known method manufacturing.Perhaps, also can change or connect to the structure of required base sequence with natural nucleic acid with shearings such as Restriction Enzymes.Specifically, can use oligonucleotide synthesizer etc. to synthesize.In addition, can use and have 1～the known method for making of the synthesis method of the oligonucleotide of the base sequence that several base is replaced, lacks, inserted or adds etc. itself.For example, can be separately or appropriate combination use locus specificity sudden change introductory technique, dna homolog recombination method, primer elongation method or PCR method, synthetic described oligonucleotide.
As " the strict hybridization conditions " in this specification sheets, can select generally well-known condition.As stringent condition, can exemplify for example following condition: containing 50% methane amide, 5 * SSC (150mM NaCl, the 15mM trisodium citrate), in the solution of the DNA of 50mM sodium phosphate (pH7.6), 5 * denhardt solution, 10% T 500 and 20 μ g/ml, 42 ℃ hybridization one evening after, at room temperature in 2 * SSC0.1%SDS, clean once, in about 65 ℃ are descended with 0.1 * SSC0.1%SDS, clean twice again.
Influenza virus is a RNA viruses.Template is under the situation of RNA in the LAMP method, by adding ThermoScript II in the reaction solution when template is DNA, can similarly carry out nucleic acid amplification reaction (RT-LAMP method).
The inventor is to increasing for the primer base sequence of the LAMP method of the base sequence of H5 type avian influenza virus specific and combination thereof conscientiously after the research apace, according to the base sequence (with the base sequence of sequence numbering 1 expression) of the hemagglutinin of H5 type avian influenza virus, these 4 groups of following A, B, C and D have been selected as primer sets.In addition, the sequence of these primers is different fully with the sequence of primer with the NASBA (based on the amplification of nucleotide sequence, Nucleic Acid Sequence-BasedAmplification) of the detection that is used for H5 type avian influenza virus of (for example patent documentation 2) reported.
(primer sets A)
FIP19c:5 '-ACCATATTCCAACTCACTTTTCATAATTTCATTGCTCCAGAATATGC-3 ' (sequence numbering 8)
BIP5:5 '-CAAACTCCAATGGGGGCATGGTGAGAGGGTGTAT-3 ' (sequence numbering 9)
F3m6:5 '-GGAGTTCTTCTGGACAA-3 ' (sequence numbering 4)
B3m:5 '-GTCGCAAGGACTAATCT-3 ' (sequence numbering 10)
LF24:5 '-GAGTCCCCTTTCTTGACAAT-3 ' (sequence numbering 11)
LB1:5 '-GATAAACTCTAGTATGCCA-3 ' (sequence numbering 12)
(primer sets B)
FIP:5 '-GGGCATGTGTAACAGTAACGTTAAACAACTCGACAGAGCA-3 ' (sequence numbering 19)
BIP:5 '-TGGAAAAGACACACAATGGGAACATCCAGCTACACTACAATC-3 ' (sequence numbering 20)
F3:5 '-CAGATTTGCATTGGTTACCA-3 ' (sequence numbering 15)
B3:5 '-CGTCACACATTGGGTTTC-3 ' (sequence numbering 21)
LF:5 '-TTCCATTATTGTGTCAACC-3 ' (sequence numbering 22)
LB8:5 '-CGATCTAGATGGAGTGAAGC-3 ' (sequence numbering 23)
(primer sets C)
FIP:5 '-CACATTGGGTTTCCGAGGAGATCTAGATGGAGTGAAGCC-3 ' (sequence numbering 30)
BIP:5 '-TTCATCAATGTGCCGGAATGGGTTGAAATCCCCTGGGTA-3 ' (sequence numbering 31)
F3:5 '-GGAAAAGACACACAATGGG-3 ' (sequence numbering 26)
B3:5 '-GCTCAATAGGTGTTTCAGTT-3 ' (sequence numbering 32)
LF6:5 '-CCAGCTACACTACAATCTCT-3 ' (sequence numbering 33)
LB6:5 '-TCCAGCCAATGACCTCTG-3 ' (sequence numbering 34)
(primer sets D)
FIP:5 '-TCGCAAGGACTAATCTGTTTGACATACACCCTCTCACCAT-3 ' (sequence numbering 41)
BIP:5 '-TACCCCTCAAAGAGAGAGAAGATCCTCCCTCTATAAAACCTG-3 ' (sequence numbering 42)
F3:5 '-TCTAGTATGCCATTCCACAA-3 ' (sequence numbering 37)
B3:5 '-ACCATCTACCATTCCCTG-3 ' (sequence numbering 43)
LF8:5 '-TCACATATTTGGGGCATTCC-3 ' (sequence numbering 44)
LB8:5 '-AGAGAGGACTATTTGGAGCT-3 ' (sequence numbering 45)
In addition, the inventor is to increasing for the primer base sequence of the LAMP method of the base sequence of H7 type avian influenza virus specific and combination thereof conscientiously after the research apace, according to the base sequence (with the base sequence of sequence numbering 1 expression) of the hemagglutinin of H7 type avian influenza virus, selected following primer sets E.
(primer sets E)
22FIP:5 '-ACCACCCATCAATCAAACCTTCTATTTGGTGCTATAGCGG-3 ' (sequence numbering 55)
22BIP:5 '-TTCAGGCATCAAAATGCACAAGCCTGTTATTTGATCAATTGCTG-3 ' (sequence numbering 56)
22F3m:5 '-TTCCCGAAATCCCAAA-3 ' (sequence numbering 51)
22B3:5 '-GGTTAGTTTTTTCTATAAGCCG-3 ' (sequence numbering 57)
22-9LF:5 '-CCCATCCATTTTCAATGAAAC-3 ' (sequence numbering 58)
22-9LB:5 '-ACTGCTGCAGATTACAAAAG-3 ' (sequence numbering 59)
The enzyme that uses during nucleic acid is synthetic is not particularly limited so long as have the active template dependency of strand displacement nucleic acid synthetic enzyme and get final product.As such enzyme, can exemplify the Klenow fragment of Bst archaeal dna polymerase (big fragment), Bca (exo-) archaeal dna polymerase, e. coli dna polymerase I etc., better be Bst archaeal dna polymerase (big fragment).
As the ThermoScript II of using in the RT-LAMP method, so long as have RNA is got final product as the active enzyme of the synthetic cDNA of template, be not particularly limited.As such enzyme, can exemplify ThermoScript II and Superscript II, ReverTraAce, the Thermoscript etc. of AMV, clone's AMV, MMLV, better be the ThermoScript II of AMV or clone's AMV.In addition, as the Bca archaeal dna polymerase, have reverse transcriptase activity and these two kinds of active enzymes of dna polymerase activity, then can carry out the RT-LAMP reaction with a kind of enzyme if use.
Enzyme and the ThermoScript II used during nucleic acid is synthetic can obtain by purifying from virus or bacterium etc., also can make by gene recombination technology.In addition, these enzymes can carry out changes such as fragmentation or amino acid whose displacement.
The detection of the reacted nucleic acid amplification product of LAMP can be used technique known.For example, can use the labeled oligonucleotide or the fluorescence intercalator method (the Japanese Patent spy opens the 2001-242169 communique) of the base sequence that specific recognition amplification obtains to detect, the reaction solution after also reaction can being finished directly carry out agarose gel electrophoresis and easily detect.By agarose gel electrophoresis, the LAMP amplified production is detected with the different multiple band of base length with being scalariform.In addition, in the LAMP method, because nucleic acid is synthetic, substrate is reacted and the generation magnesium pyrophosphate as the pyrophosphate ion of by product and the magnesium ion of coexistence by mass consumption, and the reaction solution gonorrhoea is to the also certifiable degree of naked eyes.Therefore, can finish turbidity in back or the reaction determining instrument that continue to carry out optical observation that rises to reaction and confirm this gonorrhoea by using, for example use conventional spectrophotometer to confirm that the absorbancy of 400nm changes, also can detect nucleic acid amplification reaction (the international text that discloses No. 01/83817).
All ingredients required when using primer of the present invention to carry out the detection of nucleic acid amplification can make up and test kitization in advance.Specifically, as primer of the present invention or the required various oligonucleotide of ring primer, as 4 kinds of dNTP of nucleic acid synthetic substrate, carry out nucleic acid synthetic archaeal dna polymerase, have the enzyme of reverse transcriptase activity, the damping fluid that the condition that is suitable for enzyme reaction is provided and salt, make the protective material of enzyme and form stableization and reagent that the detection of the resultant of reaction that adopts as required is required provides with the form of test kit.
Embodiment
Below, exemplify embodiment, the present invention is specifically described, but the present invention is not subjected to any qualification of these embodiment.
The reactive affirmation of primer sets of embodiment 1:H5 type avian influenza virus
Reactive affirmation of primer is undertaken by following method.Be used for being undertaken reaction soln composed as follows of nucleic acid amplification by the LAMP method.In addition, the synthetic trust QIAGEN company of primer, the product that uses OPC (reversed phase column chromatography) purifying to obtain.
20mM Tris-HCl pH8.8
10mM KCl
8mM MgSO
4
1.4mM dNTP
10mM(NH
4)
2SO
4
0.8M trimethyl-glycine (SIGMA)
0.1％Tween20
1.6μM FIP
1.6μM BIP
0.2μM F3
0.2μM B3
0.8μM LF
0.8μM LB
AMV ThermoScript II 2U (Finnzyme)
Bst archaeal dna polymerase 16U (NEB)
In above-mentioned reaction soln, add 10
4H5 plasmid DNA (the HK/213/03 of copy; Provide by National Institute of Communicable Diseases), carry out RT-LAMP reaction 60 minutes at 62.5 ℃.Use real-time turbidity measurement device LA-320C (Rong Yan chemistry), in real time reaction is detected.Consequently, 4 kinds of primer sets (primer sets A, B, C and D) are confirmed amplification.
In addition, for these 4 groups, use the RNA that extracts from as New Caledonia hypotype (H1N1) of cultivating virus and Panamanian hypotype (H3N2) to carry out the specificity test as template.Fig. 1 is the result's of expression specificity test figure.As shown in Figure 1, use under the situation of each primer sets, all do not find the amplification of H1 type and H3 type.By The above results as can be known, all primer sets are all high for the specificity of H5 type.
Then, use the RNA that extracts from as Vietnam/JP1203/04 (H5N1) that cultivates virus, carry out sensitivity test.Because it is not clear to extract the template amount of RNA, therefore use aqua sterilisa dilution 10 with deoxyribonuclease
3～10
6RNA doubly is as template samples.Fig. 2 is the result's of expression sensitivity test figure.When using primer sets A, even dilute 10
5Sample doubly also confirms amplification, and sensitivity is the highest.
Embodiment 2: the affirmation of the product that obtains with primer sets amplification with H5 type avian influenza virus
LAMP product for obtaining with primer sets A amplification uses the affirmation of electrophoresis and Restriction Enzyme Dde I.Fig. 3 is the figure of expression electrophoresis result.Swimming lane 2 by Fig. 3 can be confirmed the distinctive trapezoidal pattern of LAMP product significantly.In addition, the sample of having handled with Dde I (swimming lane 3) is confirmed to digest.As can be known from the above results, target sequence is increased specifically.
The evaluation (intercrossing test) of primer sets of embodiment 3:H5 type avian influenza virus
With 18 kinds of the totals of human influenza virus's A/ New Caledonia/20/99 (H1N1), A/ Panama/2007/99 (H3N2), B/ Shandong/07/97 and B/ Shanghai/361/2002 and avian influenza virus H1～H15 (except that H5) as sample.Carry out the extracting of RNA from cultivating virus with QIAamp Viral RNA Kit (QIAGEN company) respectively, 5 μ L extracts are used for RT-LAMP reaction (using primer sets A as primer).
By Fig. 4 (a) and (b) as can be known, do not find amplification by all samples of RT-LAMP method.Therefore, confirm the specificity height of RT-LAMP method.
The evaluation (sensitivity test) of primer sets of embodiment 4:H5 type avian influenza virus
Use the bird flu H5 type strain (CH/ mountain pass 7/04) of confirming infection in 2004 in the mountain pass county and the H5 type strain of confirming to infect in Vietnam in 2004 (VN/JP1203/04) as template samples.Respectively will be from cultivating RNA that viral extracting obtains aqua sterilisa stepwise dilution (10 with deoxyribonuclease
4～10
8), the RT-PCR method is used 10 μ L diluents, and RT-LAMP method (using primer sets A as primer) is used 5 μ L diluents.
The condition of being published on the website of the transmissible disease information center of RT-PCR method modification National Institute of Communicable Diseases is carried out.Promptly, use commercially available test kit (TaKaRa One Step RNA PCR Kit (AMV)), carried out reverse transcription reaction 30 minutes at 50 ℃, 94 ℃ handle 2 minutes after, repeat 30 94 ℃ 1 minute, 45 ℃ 1 minute, 72 ℃ circulations of 1 minute, carried out lengthening reaction 10 minutes in 72 ℃ again, preserve at 4 ℃.
Primer that uses in the RT-PCR method and reaction soln composed as follows.
Primer (the length of PCR product: 708bp)
H5 515f:5 '-CATACCCAACAATAAAGAGG-3 ' (sequence numbering 46)
H5 1220r:5 '-GTGTTCATTTTGTTAATGAT-3 ' (sequence numbering 47)
Reaction soln
RNA extract 10 μ L
10 * One Step RNA PCR damping fluid, 5 μ L
10mM dNTP 5μL
25mM MgCl
2 10μL
Ribonuclease inhibitor 1 μ L
AMV ThermoScript II 1 μ L
AMV-optimizes Taq 1 μ L
H5515f(10μM)2μL
H51220r(10μM)2μL
The aqua sterilisa of deoxyribonuclease suitably adds and reaction solution is adjusted to 50 μ L
Adopt the result of the amplification of RT-PCR method and RT-LAMP method to gather and be shown in table 1.In the table 1, the ratio of the sample that increases is represented to confirm in the hurdle of RT-LAMP method.In addition, the amplification among the RT-PCR is confirmed electrophoretic result (in the table 1, zero expression can detect amplification, and * expression can't detect amplification) by naked eyes.
[table 1]
| Sample | Assay | Thinning ratio | |||||||||
| 10 4 | 10 5 | 10 6 | 10 7 | 10 8 | |||||||
| CH/ mountain pass 7/04 | RT- | 2/2 | 2/2 | 2/2 | 2/2 | 0/2 | |||||
| RT-PCR | ○ | ○ | × | × | × | ||||||
| VN/JP1203/04 | RT- | 2/2 | 2/2 | 2/2 | 2/2 | 1/2 | |||||
| RT-PCR | ○ | ○ | ○ | × | × | ||||||
When comparing RT-LAMP method and RT-PCR method, all strains all are the sensitivity of RT-LAMP method up to 10～100 times.
The reactive affirmation of primer sets of embodiment 5:H7 type avian influenza virus
Reactive affirmation of primer sets E is undertaken by following method.Be used for being undertaken reaction soln composed as follows of nucleic acid amplification by the LAMP method.In addition, the synthetic trust QIAGEN company of primer, the product that uses OPC (reversed phase column chromatography) purifying to obtain.
20mM Tris-HCl pH8.8
10mM KCl
8mM MgSO
4
1.4mM dNTP
10mM(NH
4)
2SO
4
0.8M trimethyl-glycine (SIGMA)
0.1％ Tween20
1.6μM FIP
1.6μM BIP
0.2μM F3
0.2μM B3
0.8μM LF
0.8μM LB
AMV ThermoScript II 2U (Finnzyme)
Bst archaeal dna polymerase 16U (NEB)
On A/ Holland/219/2003 (H7N7) base sequence (fragment that comprises the base sequence shown in the sequence numbering 1) cloning vector of recombinating of (separating the strain that obtains when death occurring), prepare tRNA by responsive transcription in Holland.The tRNA that makes is joined in the above-mentioned reaction soln with the amount of 250,100 or 50 copy/tests,, use real-time turbidity measurement device LA-200 (テ ラ メ Star Network ス), carry out amplified reaction and detection 62.5 ℃ of reactions 35 minutes.
The addition of tRNA is that the situation of 250,100 and 50 copy/tests and the real-time detected result of negative control are shown in Fig. 5.By this result as can be known, also detect amplification to 50 copy/tests.
Embodiment 6: the affirmation of the product that obtains with primer sets amplification with H7 type avian influenza virus
LAMP product for obtaining with primer sets E amplification uses the affirmation of electrophoresis and Restriction Enzyme Pst I.Fig. 6 is the figure of expression electrophoresis result.M represents the terraced marker of 100bp, and swimming lane 1 is the sample of untreated LAMP product, the sample of swimming lane 2 for the LAMP product is obtained with Pst I digestion.Swimming lane 1 by Fig. 6 can be confirmed the distinctive trapezoidal pattern of LAMP product significantly.In addition, the sample of having handled with Pst I (swimming lane 2) is confirmed to digest.As can be known from the above results, target sequence is increased specifically.
The evaluation (intercrossing test) of primer sets of embodiment 7:H7 type avian influenza virus
18 kinds of the totals of human influenza virus's A/ New Caledonia/20/99 (H1N1), A/ Panama/2007/99 (H3N2), B/ Shandong/07/97 and B/ Shanghai/361/2002 and avian influenza virus H1～H15 (except that H7) as sample, are investigated the specificity of primer.Carry out the extracting of RNA from cultivating virus with QIAamp Viral RNA Kit (QIAGEN company) respectively, the extracting RNA of diluting 100 times is used for the RT-LAMP reaction.In addition, positive control (PC) uses the tRNA of preparation among the embodiment 5, and negative control uses distilled water.
As shown in Figure 7, all samples are not all found amplification.Therefore, confirm the specificity height of primer sets of the present invention.
The evaluation (to the reactivity of various strains) of primer sets of embodiment 8:H7 type avian influenza virus
4 kinds of H7 type avian influenza virus genomes as template samples, are investigated the sensitivity to various viruses.Respectively will be from cultivating RNA that viral extracting obtains aqua sterilisa stepwise dilution (10 with deoxyribonuclease
5～10
8), use 5 μ L diluents.
Real-time detection with each viral genome during as template samples the results are shown in Fig. 8 and 9.For A/ Holland/219/2003 (H7N7) and A/ Holland/33/2003 (H7N7), can detect to 10
7Doubly dilution (with reference to Fig. 8) for A/ wild duck/Holland/12/2003 (H7N3) and A/ water wild duck/Osaka/1/2001 (H7N7), can detect to 10
6Doubly dilution (with reference to Fig. 9).By above result as can be known, primer sets of the present invention and above-mentioned 4 kinds of strains reaction.
The possibility of utilizing on the industry
If employing the present invention can high sensitivity and promptly detect the H5 type or H7 type avian influenza virus.
Sequence table
＜110〉Eiken Chemical (EIKEN KAGAKU KABUSHIKI KAISHA)
National Institute of Communicable Diseases (National Institute of Infectious Diseases)
＜120〉detection method of H5 type or H7 type avian influenza virus
<130>FP05-0385-00
<160>59
<170>PatentIn version 3.1
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<211>1674
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＜213〉avian influenza virus
<400>1
agtcttgtta aaagtgatca gatttgcatt ggttaccatg caaacaactc gacagagcag 60
gttgacacaa taatggaaaa gaacgttact gttacacatg cccaagacat attggaaaag 120
acacacaatg ggaagctctg cgatctagat ggagtgaagc ctctaatttt gagagattgt 180
agtgtagctg gatggctcct cggaaaccca atgtgtgacg aattcatcaa tgtgccggaa 240
tggtcttaca tagtggagaa ggccagtcca gccaatgacc tctgttaccc aggggatttc 300
aacgactatg aagaactgaa acacctattg agcagaataa accattttga gaaaattcag 360
atcatcccca aaagttcttg gtccaatcat gaagcctcat caggggtgag ctcagcatgt 420
ccatatcttg ggaagtcctc ctttttcaga aatgtggtat ggcttatcaa aaagaacagt 480
acatacccaa caataaagag gagctataat aataccaacc aagaagatct tttggtactg 540
tgggggattc accatcctaa tgatgcggca gagcagacaa agctctatca aaacccaacc 600
acctatattt ccgttggaac atcaacacta aaccagagat tggtaccaaa aatagctact 660
agatccaaag taaacgggca aagtggaaga atggagttct tctggacaat tttaaagccg 720
aatgatgcta tcaatttcga gagtaatgga aatttcattg ctccagaata tgcatacaaa 780
attgtcaaga aaggggactc agcaattatg aaaagtgaat tggaatatgg taactgcaac 840
accaagtgtc aaactccaat gggggcgata aactctagta tgccattcca caacatacac 900
cctctcacca tcggggaatg ccccaaatat gtgaaatcaa acagattagt ccttgcgact 960
ggactcagaa atacccctca aagagagaga agaagaaaaa agagaggact atttggagct 1020
atagcaggtt ttatagaggg aggatggcag ggaatggtag atggttggta tgggtaccac 1080
catagcaatg agcaggggag tggatacgct gcagacaaag aatccactca aaaggcaata 1140
gatggagtta ccaataaggt caactcgatc attgacaaaa tgaacactca gtttgaggcc 1200
gttggaaggg aatttaataa cttagaaagg agaatagaaa atttaaacaa gaagatggaa 1260
gacggattcc tagatgtctg gacttataat gctgaacttc tggttctcat ggaaaatgag 1320
agaactctag actttcacga ctcaaatgtc aagaaccttt acgacaaggt ccgactacag 1380
cttagggata atgcaaagga gctgggtaac ggctgtttcg agttctatca caaatgtgat 1440
aatgaatgta tggaaagtgt aaaaaacgga acgtatgact acccgcagta ttcagaagaa 1500
gcaagactaa acagagagga aataagtgga gtaaaattgg aatcaatggg aacttaccaa 1560
atactgtcaa tttattcaac agtggcgagt tccctagcac tggcaatcat ggtagctggt 1620
ctatctttat ggatgtgctc caatggatcg ttacaatgca gaatttgcat ttaa 1674
<210>2
<211>28
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>2
aattatgaaa agtgagttgg aatatggt 28
<210>3
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>3
tcattgctcc agaatatgc 19
<210>4
<211>17
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>4
ggagttcttc tggacaa 17
<210>5
<211>17
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＜213〉artificial
<220>
＜223〉primer
<400>5
caaactccaa tgggggc 17
<210>6
<211>17
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>6
atacaccctc tcaccat 17
<210>7
<211>17
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＜213〉artificial
<220>
＜223〉primer
<400>7
agattagtcc ttgcgac 17
<210>8
<211>47
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>8
accatattcc aactcacttt tcataatttc attgctccag aatatgc 47
<210>9
<211>34
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>9
caaactccaa tgggggcatg gtgagagggt gtat 34
<210>10
<211>17
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>10
gtcgcaagga ctaatct 17
<210>11
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＜213〉artificial
<220>
＜223〉primer
<400>11
<210>12
<211>19
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＜213〉artificial
<220>
＜223〉primer
<400>12
gataaactct agtatgcca 19
<210>13
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>13
aacgttactg ttacacatgc cc 22
<210>14
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>14
aaacaactcg acagagca 18
<210>15
<211>20
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>15
<210>16
<211>22
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>16
tggaaaagac acacaatggg aa 22
<210>17
<211>20
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>17
<210>18
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>18
gaaacccaat gtgtgacg 18
<210>19
<211>40
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>19
gggcatgtgt aacagtaacg ttaaacaact cgacagagca 40
<210>20
<211>42
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>20
tggaaaagac acacaatggg aacatccagc tacactacaa tc 42
<210>21
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>21
cgtcacacat tgggtttc 18
<210>22
<211>19
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＜213〉artificial
<220>
＜223〉primer
<400>22
ttccattatt gtgtcaacc 19
<210>23
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<220>
＜223〉primer
<400>23
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<212>DNA
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<220>
＜223〉primer
<400>24
<210>25
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>25
atctagatgg agtgaagcc 19
<210>26
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>26
ggaaaagaca cacaatggg 19
<210>27
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>27
ttcatcaatg tgccggaatg g 21
<210>28
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>28
tacccagggg atttcaac 18
<210>29
<211>20
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>29
<210>30
<211>39
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>30
cacattgggt ttccgaggag atctagatgg agtgaagcc 39
<210>31
<211>39
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>31
ttcatcaatg tgccggaatg ggttgaaatc ccctgggta 39
<210>32
<211>20
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>32
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＜213〉artificial
<220>
＜223〉primer
<400>33
<210>34
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>34
tccagccaat gacctctg 18
<210>35
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＜213〉artificial
<220>
＜223〉primer
<400>35
tcaaacagat tagtccttgc ga 22
<210>36
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＜213〉artificial
<220>
＜223〉primer
<400>36
catacaccct ctcaccat 18
<210>37
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<220>
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<400>37
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<211>22
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<220>
＜223〉primer
<400>38
tacccctcaa agagagagaa ga 22
<210>39
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<220>
＜223〉primer
<400>39
<210>40
<211>18
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<400>40
cagggaatgg tagatggt 18
<210>41
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＜223〉primer
<400>41
tcgcaaggac taatctgttt gacatacacc ctctcaccat 40
<210>42
<211>42
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>42
tacccctcaa agagagagaa gatcctccct ctataaaacc tg 42
<210>43
<211>18
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>43
accatctacc attccctg 18
<210>44
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<400>44
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<220>
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<400>45
<210>46
<211>20
<212>DNA
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<220>
＜223〉RT-PCR primer (H5 515f)
<400>46
<210>47
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<220>
＜223〉RT-PCR primer (H5 1220r)
<400>47
<210>48
<211>1737
<212>DNA
＜213〉avian influenza virus
<400>48
agcaaaagca ggggatacaa aatgaacact caaatcctgg tattcgctct ggtggcgagc 60
attccgacaa atgcagacaa gatctgcctt gggcatcatg ccgtgtcaaa cgggactaaa 120
gtaaacacat taactgagag aggagtggaa gtcgttaatg caactgaaac ggtggaacga 180
acaaacgttc ccaggatctg ctcaaaaggg aaaaggacag ttgacctcgg tcaatgtgga 240
cttctgggaa caatcactgg gccaccccaa tgtgaccaat tcctagaatt ttcggccgac 300
ttaattattg agaggcgaga aggaagtgat gtctgttatc ctgggaaatt cgtgaatgaa 360
gaagctctga ggcaaattct cagagagtca ggcggaattg acaaggagac aatgggattc 420
acctacagcg gaataagaac taatggaaca accagtgcat gtaggagatc aggatcttca 480
ttctatgcag agatgaaatg gctcctgtca aacacagaca atgctgcttt cccgcaaatg 540
actaagtcat acaagaacac aaggaaagac ccagctctga taatatgggg gatccaccat 600
tccggatcaa ctacagaaca gaccaagcta tatgggagtg gaaacaaact gataacagtt 660
gggagttcta attaccaaca gtcctttgta ccgagtccag gagcgagacc acaagtgaat 720
ggccaatctg gaagaattga ctttcattgg ctgatactaa accctaatga cacggtcact 780
ttcagtttca atggggcctt catagctcca gaccgtgcaa gctttctgag agggaagtcc 840
atgggaattc agagtgaagt acaggttgat gccaattgtg aaggagattg ctatcatagt 900
ggagggacaa taataagtaa tttgcccttt cagaacataa atagcagggc agtaggaaaa 960
tgtccgagat atgttaagca agagagtctg ctgttggcaa caggaatgaa gaatgttccc 1020
gaaatcccaa agaggaggag gagaggccta tttggtgcta tagcgggttt cattgaaaat 1080
ggatgggaag gtttgattga tgggtggtat ggcttcaggc atcaaaatgc acaaggggag 1140
ggaactgctg cagattacaa aagcacccaa tcagcaattg atcaaataac agggaaatta 1200
aatcggctta tagaaaaaac taaccaacag tttgagttaa tagacaacga attcactgag 1260
gttgaaaggc aaattggcaa tgtgataaac tggaccagag attccatgac agaagtgtgg 1320
tcctataacg ctgaactctt agtagcaatg gagaatcagc acacaattga tctggccgac 1380
tcagaaatga acaaactgta cgaacgagtg aagagacaac tgagagagaa tgccgaagaa 1440
gatggcactg gttgcttcga aatatttcac aagtgtgatg acgactgcat ggccagtatt 1500
agaaacaaca cctatgatca cagcaagtac agggaagaag caatacaaaa tagaatacag 1560
attgacccag tcaaactaag cagcggctac aaagatgtga tactttggtt tagcttcggg 1620
gcatcatgtt tcatacttct ggccattgca atgggccttg tcttcatatg tgtgaagaat 1680
ggaaacatgc ggtgcactat ttgtatataa gtttggaaaa acacccttgt ttctact 1737
<210>49
<211>21
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>49
aaggtttgat tgatgggtgg t 21
<210>50
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>50
ctatttggtg ctatagcgg 19
<210>51
<211>16
<212>DNA
＜213〉artificial
<220>
＜223〉primer
<400>51
ttcccgaaat cccaaa 16
<210>52
<211>22
<212>DNA
＜213〉artificial
<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
accacccatc 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
Claims (40)
1. Oligonucleolide primers is characterized in that, according to be selected from any base sequence of 693～959 base sequence of the hemagglutinin base sequence of the H5 type avian influenza virus of sequence numbering 1 expression or with its complementary base sequence design.
2. Oligonucleolide primers as claimed in claim 1 is characterized in that, comprises the oligonucleotide that is selected from following (a)～(c):
(a) be selected from the base sequence of sequence numbering 2～7 expression or with the oligonucleotide that contains 15 bases of successive at least of its complementary base sequence;
(b) can with the oligonucleotide of aforementioned (a) described oligonucleotide hybridize under stringent condition;
(c) comprise aforementioned (a) or (b) 1 in the described oligonucleotide～several base base sequence of being replaced, lack, insert or adding, oligonucleotide with primer function.
3. Oligonucleolide primers as claimed in claim 1 or 2, it is characterized in that, 3 ' end side from the target nucleic acid of the hemagglutinin of H5 type avian influenza virus selects note to do the base sequence zone of F3c, F2c, F1c, select note to do the base sequence zone of B3, B2, B1 from 5 ' end side, when complementary base sequence note is separately made F3, F2, F1 and B3c, B2c, B1c, constitute by the base sequence that is selected from following (a)～(d):
(a) have the F2 zone of target nucleic acid at 3 ' end side, have the base sequence in the F1c zone of target nucleic acid at 5 ' end side;
(b) has the base sequence in the F3 zone of target nucleic acid;
(c) have the B2 zone of target nucleic acid at 3 ' end side, have the base sequence in the B1c zone of target nucleic acid at 5 ' end side;
(d) has the base sequence in the B3 zone of target nucleic acid.
4. as each described Oligonucleolide primers in the claim 1～3, it is characterized in that, can increase, to 3 ' end, constitute by the base sequence that is selected from following (a)～(b) from 5 ' end to the base sequence of H5 type avian influenza virus specific:
(a) 5 '-(with the base sequence complementary base sequence of sequence numbering 2)-(base sequence arbitrarily of base several 0～50)-(base sequence of sequence numbering 3)-3 ';
(b) 5 '-(base sequence of sequence numbering 5)-(base sequence arbitrarily of base several 0～50)-(with the base sequence complementary base sequence of sequence numbering 6)-3 '.
5.H5 the detection method of type avian influenza virus is characterized in that, uses each described Oligonucleolide primers in the claim 1～4, carries out the amplified reaction in the target nucleic acid zone of H5 type avian influenza virus.
6. the detection method of H5 type avian influenza virus as claimed in claim 5 is characterized in that, the amplified reaction in the target nucleic acid zone of H5 type avian influenza virus adopts the LAMP method.
7. the diagnostic method of influenza is characterized in that, by using each described Oligonucleolide primers in the claim 1～4, detects the amplification in the target nucleic acid zone of H5 type avian influenza virus, and whether diagnosis has the infection of H5 type avian influenza virus.
8. test kit is characterized in that, comprises each the described Oligonucleolide primers of claim 1～4 that is used for diagnosing influenza.
9. Oligonucleolide primers is characterized in that, according to be selected from any base sequence of 19～220 base sequence of the hemagglutinin base sequence of the H5 type avian influenza virus of sequence numbering 1 expression or with its complementary base sequence design.
10. Oligonucleolide primers as claimed in claim 9 is characterized in that, comprises the oligonucleotide that is selected from following (a)～(c):
(a) be selected from the base sequence of sequence numbering 13～18 expression or with the oligonucleotide that contains 15 bases of successive at least of its complementary base sequence;
(b) can with the oligonucleotide of aforementioned (a) described oligonucleotide hybridize under stringent condition;
(c) comprise aforementioned (a) or (b) 1 in the described oligonucleotide～several base base sequence of being replaced, lack, insert or adding, oligonucleotide with primer function.
11. as claim 9 or 10 described Oligonucleolide primers, it is characterized in that, 3 ' end side from the target nucleic acid of the hemagglutinin of H5 type avian influenza virus selects note to do the base sequence zone of F3c, F2c, F1c, select note to do the base sequence zone of B3, B2, B1 from 5 ' end side, when complementary base sequence note is separately made F3, F2, F1 and B3c, B2c, B1c, constitute by the base sequence that is selected from following (a)～(d):
(a) have the F2 zone of target nucleic acid at 3 ' end side, have the base sequence in the F1c zone of target nucleic acid at 5 ' end side;
(b) has the base sequence in the F3 zone of target nucleic acid;
(c) have the B2 zone of target nucleic acid at 3 ' end side, have the base sequence in the B1c zone of target nucleic acid at 5 ' end side;
(d) has the base sequence in the B3 zone of target nucleic acid.
12., it is characterized in that as each described Oligonucleolide primers in the claim 9～11, can increase to the base sequence of H5 type avian influenza virus specific,, constitute to 3 ' end from 5 ' end by the base sequence that is selected from following (a)～(b):
(a) 5 '-(with the base sequence complementary base sequence of sequence numbering 13)-(base sequence arbitrarily of base several 0～50)-(base sequence of sequence numbering 14)-3 ';
(b) 5 '-(base sequence of sequence numbering 16)-(base sequence arbitrarily of base several 0～50)-(with the base sequence complementary base sequence of sequence numbering 17)-3 '.
13.H5 the detection method of type avian influenza virus is characterized in that, uses each described Oligonucleolide primers in the claim 9～12, carries out the amplified reaction in the target nucleic acid zone of H5 type avian influenza virus.
14. the detection method of H5 type avian influenza virus as claimed in claim 13 is characterized in that, the amplified reaction in the target nucleic acid zone of H5 type avian influenza virus adopts the LAMP method.
15. the diagnostic method of influenza is characterized in that, by using each described Oligonucleolide primers in the claim 9～12, detects the amplification in the target nucleic acid zone of H5 type avian influenza virus, whether diagnosis has the infection of H5 type avian influenza virus.
16. test kit is characterized in that, comprises each the described Oligonucleolide primers of claim 9～12 that is used for diagnosing influenza.
17. Oligonucleolide primers is characterized in that, according to be selected from any base sequence of 114～333 base sequence of the hemagglutinin base sequence of the H5 type avian influenza virus of sequence numbering 1 expression or with its complementary base sequence design.
18. Oligonucleolide primers as claimed in claim 17 is characterized in that, comprises the oligonucleotide that is selected from following (a)～(c):
(a) be selected from the base sequence of sequence numbering 24～29 expression or with the oligonucleotide that contains 15 bases of successive at least of its complementary base sequence;
(b) can with the oligonucleotide of aforementioned (a) described oligonucleotide hybridize under stringent condition;
(c) comprise aforementioned (a) or (b) 1 in the described oligonucleotide～several base base sequence of being replaced, lack, insert or adding, oligonucleotide with primer function.
19. as claim 17 or 18 described Oligonucleolide primers, it is characterized in that, 3 ' end side from the target nucleic acid of the hemagglutinin of H5 type avian influenza virus selects note to do the base sequence zone of F3c, F2c, F1c, select note to do the base sequence zone of B3, B2, B1 from 5 ' end side, when complementary base sequence note is separately made F3, F2, F1 and B3c, B2c, B1c, constitute by the base sequence that is selected from following (a)～(d):
(a) have the F2 zone of target nucleic acid at 3 ' end side, have the base sequence in the F1c zone of target nucleic acid at 5 ' end side;
(b) has the base sequence in the F3 zone of target nucleic acid;
(c) have the B2 zone of target nucleic acid at 3 ' end side, have the base sequence in the B1c zone of target nucleic acid at 5 ' end side;
(d) has the base sequence in the B3 zone of target nucleic acid.
20., it is characterized in that as each described Oligonucleolide primers in the claim 17～19, can increase to the base sequence of H5 type avian influenza virus specific,, constitute to 3 ' end from 5 ' end by the base sequence that is selected from following (a)～(b):
(a) 5 '-(with the base sequence complementary base sequence of sequence numbering 24)-(base sequence arbitrarily of base several 0～50)-(base sequence of sequence numbering 25)-3 ';
(b) 5 '-(base sequence of sequence numbering 27)-(base sequence arbitrarily of base several 0～50)-(with the base sequence complementary base sequence of sequence numbering 28)-3 '.
21.H5 the detection method of type avian influenza virus is characterized in that, uses each described Oligonucleolide primers in the claim 17～20, carries out the amplified reaction in the target nucleic acid zone of H5 type avian influenza virus.
22. the detection method of H5 type avian influenza virus as claimed in claim 21 is characterized in that, the amplified reaction in the target nucleic acid zone of H5 type avian influenza virus adopts the LAMP method.
23. the diagnostic method of influenza is characterized in that, by using each described Oligonucleolide primers in the claim 17～20, detects the amplification in the target nucleic acid zone of H5 type avian influenza virus, whether diagnosis has the infection of H5 type avian influenza virus.
24. test kit is characterized in that, comprises each the described Oligonucleolide primers of claim 17～20 that is used for diagnosing influenza.
25. Oligonucleolide primers is characterized in that, according to be selected from any base sequence of 874～1065 base sequence of the hemagglutinin base sequence of the H5 type avian influenza virus of sequence numbering 1 expression or with its complementary base sequence design.
26. Oligonucleolide primers as claimed in claim 25 is characterized in that, comprises the oligonucleotide that is selected from following (a)～(c):
(a) be selected from the base sequence of sequence numbering 35～40 expression or with the oligonucleotide that contains 15 bases of successive at least of its complementary base sequence;
(b) can with the oligonucleotide of aforementioned (a) described oligonucleotide hybridize under stringent condition;
(c) comprise aforementioned (a) or (b) 1 in the described oligonucleotide～several base base sequence of being replaced, lack, insert or adding, oligonucleotide with primer function.
27. as claim 25 or 26 described Oligonucleolide primers, it is characterized in that, 3 ' end side from the target nucleic acid of the hemagglutinin of H5 type avian influenza virus selects note to do the base sequence zone of F3c, F2c, F1c, select note to do the base sequence zone of B3, B2, B1 from 5 ' end side, when complementary base sequence note is separately made F3, F2, F1 and B3c, B2c, B1c, constitute by the base sequence that is selected from following (a)～(d):
(a) have the F2 zone of target nucleic acid at 3 ' end side, have the base sequence in the F1c zone of target nucleic acid at 5 ' end side;
(b) has the base sequence in the F3 zone of target nucleic acid;
(c) have the B2 zone of target nucleic acid at 3 ' end side, have the base sequence in the B1c zone of target nucleic acid at 5 ' end side;
(d) has the base sequence in the B3 zone of target nucleic acid.
28., it is characterized in that as each described Oligonucleolide primers in the claim 25～27, can increase to the base sequence of H5 type avian influenza virus specific,, constitute to 3 ' end from 5 ' end by the base sequence that is selected from following (a)～(b):
(a) 5 '-(with the base sequence complementary base sequence of sequence numbering 35)-(base sequence arbitrarily of base several 0～50)-(base sequence of sequence numbering 36)-3 ';
(b) 5 '-(base sequence of sequence numbering 38)-(base sequence arbitrarily of base several 0～50)-(with the base sequence complementary base sequence of sequence numbering 39)-3 '.
29.H5 the detection method of type avian influenza virus is characterized in that, uses each described Oligonucleolide primers in the claim 25～28, carries out the amplified reaction in the target nucleic acid zone of H5 type avian influenza virus.
30. the detection method of H5 type avian influenza virus as claimed in claim 29 is characterized in that, the amplified reaction in the target nucleic acid zone of H5 type avian influenza virus adopts the LAMP method.
31. the diagnostic method of influenza is characterized in that, by using each described Oligonucleolide primers in the claim 25～28, detects the amplification in the target nucleic acid zone of H5 type avian influenza virus, whether diagnosis has the infection of H5 type avian influenza virus.
32. test kit is characterized in that, comprises each the described Oligonucleolide primers of claim 25～28 that is used for diagnosing influenza.
33. Oligonucleolide primers is characterized in that, according to be selected from any base sequence of 1016～1225 base sequence of the hemagglutinin base sequence of the H7 type avian influenza virus of sequence numbering 48 expression or with its complementary base sequence design.
34. Oligonucleolide primers as claimed in claim 33 is characterized in that, comprises the oligonucleotide that is selected from following (a)～(c):
(a) be selected from the base sequence of sequence numbering 49～54 expression or with the oligonucleotide that contains 15 bases of successive at least of its complementary base sequence;
(b) can with the oligonucleotide of aforementioned (a) described oligonucleotide hybridize under stringent condition;
(c) comprise aforementioned (a) or base sequence that (b) 1～several base is replaced, lacks, inserted or adds in the described oligonucleotide, oligonucleotide with primer function.
35. as claim 33 or 34 described Oligonucleolide primers, it is characterized in that, 3 ' end side from the target nucleic acid of the hemagglutinin of H7 type avian influenza virus selects note to do the base sequence zone of F3c, F2c, F1c, select note to do the base sequence zone of B3, B2, B1 from 5 ' end side, when complementary base sequence note is separately made F3, F2, F1 and B3c, B2c, b1c, constitute by the base sequence that is selected from following (a)～(d):
(a) have the F2 zone of target nucleic acid at 3 ' end side, have the base sequence in the F1c zone of target nucleic acid at 5 ' end side;
(b) has the base sequence in the F3 zone of target nucleic acid;
(c) have the B2 zone of target nucleic acid at 3 ' end side, have the base sequence in the B1c zone of target nucleic acid at 5 ' end side;
(d) has the base sequence in the B3 zone of target nucleic acid.
36., it is characterized in that as each described Oligonucleolide primers in the claim 33～35, can increase to the base sequence of H7 type avian influenza virus specific,, constitute to 3 ' end from 5 ' end by the base sequence that is selected from following (a)～(b):
(a) 5 '-(with the base sequence complementary base sequence of sequence numbering 49)-(base sequence arbitrarily of base several 0～50)-(base sequence of sequence numbering 50)-3 ';
(b) 5 '-(base sequence of sequence numbering 52)-(base sequence arbitrarily of base several 0～50)-(with the base sequence complementary base sequence of sequence numbering 53)-3 '.
37.H7 the detection method of type avian influenza virus is characterized in that, uses each described Oligonucleolide primers in the claim 33～36, carries out the amplified reaction in the target nucleic acid zone of H7 type avian influenza virus.
38. the detection method of H7 type avian influenza virus as claimed in claim 37 is characterized in that, the amplified reaction in the target nucleic acid zone of H7 type avian influenza virus adopts the LAMP method.
39. the diagnostic method of influenza is characterized in that, by using each described Oligonucleolide primers in the claim 33～36, detects the amplification in the target nucleic acid zone of H7 type avian influenza virus, whether diagnosis has the infection of H7 type avian influenza virus.
40. test kit is characterized in that, comprises each the described Oligonucleolide primers of claim 33～36 that is used for diagnosing influenza.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP318214/2004 | 2004-11-01 | ||
| JP2004318214 | 2004-11-01 | ||
| JP148487/2005 | 2005-05-20 | ||
| JP2005148487 | 2005-05-20 | ||
| PCT/JP2005/019710 WO2006049061A1 (en) | 2004-11-01 | 2005-10-26 | Method of detecting h5 or h7 avian influenza virus |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009101601847A Division CN101613700B (en) | 2004-11-01 | 2005-10-26 | Method of detecting H5 or H7 avian influenza virus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101052720A true CN101052720A (en) | 2007-10-10 |
| CN101052720B CN101052720B (en) | 2012-12-26 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009101601847A Expired - Fee Related CN101613700B (en) | 2004-11-01 | 2005-10-26 | Method of detecting H5 or H7 avian influenza virus |
| CN2005800376057A Expired - Fee Related CN101052720B (en) | 2004-11-01 | 2005-10-26 | Method of detecting H5 or H7 avian influenza virus |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009101601847A Expired - Fee Related CN101613700B (en) | 2004-11-01 | 2005-10-26 | Method of detecting H5 or H7 avian influenza virus |
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| Country | Link |
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| CN (2) | CN101613700B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101575649B (en) * | 2008-06-02 | 2011-08-03 | 中国农业科学院兰州兽医研究所 | Quick detection technology for H9 type avian influenza virus |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111218529B (en) * | 2020-03-13 | 2023-06-23 | 湖南融健生物科技有限公司 | Primer composition, kit and method for detecting novel coronaviruses |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2424867C (en) * | 2000-10-05 | 2013-03-26 | Hong Kong Dna Chips Limited | A kit for detecting non-pathogenic or pathogenic influenza a subtype h5 virus |
-
2005
- 2005-10-26 CN CN2009101601847A patent/CN101613700B/en not_active Expired - Fee Related
- 2005-10-26 CN CN2005800376057A patent/CN101052720B/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101575649B (en) * | 2008-06-02 | 2011-08-03 | 中国农业科学院兰州兽医研究所 | Quick detection technology for H9 type avian influenza virus |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101613700A (en) | 2009-12-30 |
| CN101052720B (en) | 2012-12-26 |
| CN101613700B (en) | 2011-12-28 |
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