CN104531899B - The GeXP rapid detection kit of avian influenza virus and its H6 hypotypes and N1 hypotypes - Google Patents

The GeXP rapid detection kit of avian influenza virus and its H6 hypotypes and N1 hypotypes Download PDF

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CN104531899B
CN104531899B CN201410827498.9A CN201410827498A CN104531899B CN 104531899 B CN104531899 B CN 104531899B CN 201410827498 A CN201410827498 A CN 201410827498A CN 104531899 B CN104531899 B CN 104531899B
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CN104531899A (en
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谢芝勋
罗思思
谢志勤
邓显文
刘加波
谢丽基
黄莉
黄娇玲
曾婷婷
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Guangxi Veterinary Research Institute
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Abstract

The invention discloses avian influenza virus and its GeXP rapid detection kit of H6 hypotypes and N1 hypotypes.GeXP rapid detection kit provided by the present invention includes primer set 1, primer set 2, primer set 3 or primer set 4；Primer set 1 includes H6, N1 and M；Primer set 2 includes H6 and M；Primer set 3 includes N1 and M；Primer set 4 includes H6 and N1；H6 is made up of the single stranded DNA shown in SEQ ID No.1 and SEQ ID No.2；N1 is made up of the single stranded DNA shown in SEQ ID No.3 and SEQ ID No.4；M is made up of the single stranded DNA shown in SEQ ID No.5 and SEQ ID No.6.It is demonstrated experimentally that the primer set of the present invention can be used to identify avian influenza virus and its H6 hypotypes and N1 hypotypes.

Description

The GeXP rapid detection kit of avian influenza virus and its H6 hypotypes and N1 hypotypes
Technical field
The present invention relates to the examination of the GeXP quick detections of avian influenza virus in biological technical field and its H6 hypotypes and N1 hypotypes Agent box.
Background technology
Avian influenza virus (Avian Influenza Virus, AIV) is according to surface glycoprotein (Hemagglutinin, HA) It is divided into different hypotypes with neuraminidase (Neuraminidase, NA) antigenic specificity, has had now been found that 16 kinds of HA hypotypes (H1-H16) and 9 kinds of NA hypotypes (N1-N9), HA and NA combination can produce at least 135 kinds hypotypes, wherein, in H5 and H7 hypotypes Part combination (H5N1, H7N7 and H7N9 etc.) be referred to as highly pathogenic AIV, the mankind can be infected and lethal.Low pathogenicity AIV infects Although without the Non-typical symptoms that any symptom or performance are gentle, slight, not having menace to the health of livestock and poultry and the mankind, working as During the low pathogenicity AIV strain mixed infection livestock and poultry of different subtype, gene can be intercoursed in animal body, so as to produce nothing Method predicts pathogenic and communicable new variant, and this will have very big threat to birds and human health.
H6 hypotypes AIV belongs to low pathogenicity AIV, but have been reported that show 1997 Hong Kong break out infection people it is highly pathogenic H5N1 virus, in addition to HA genes, remaining 7 genetic fragment is all viral highly with A/teal/Hong Kong/W312/97 (H6N1) It is similar, therefore H6 hypotypes AIV is likely to the genetic donor as highly pathogenic H5 hypotypes AIV.In June, 2013, Taiwan is found that First case people infection type H6N1 hypotype AIV, are a kind of LPAIVs, and its HA gene is by Taiwan H6 hypotype AIV branch It is interior to evolve.At present, H6 hypotypes AIV is widely paid close attention to, and has important influence to aviculture and public health.
Antidiastole AIV traditional conventional method, mainly includes Antigen isolation and identification and serological test etc., but these sides Method is often limited by clinical pathological material of disease freshness, pollution level or the course of disease, and operation is also very cumbersome time-consuming, to multiple mixed infection Antidiastole is just increasingly difficult.In recent years, with the development of molecular biology, round pcr has been widely used in these breathings The detection of road disease.But the multiplex PCR detection technique set up based on Standard PCR technology, needs several combined to primer while competing Expand with striving, can produce and interfere between primer, increase pair of primers more, sensitiveness is lower.PCR primer is needed by fine jade Lipolysaccharide electrophoresis ability observable result, the electrophoresis is difficult to differentiate between the band within 50bp-100bp, typically only accomplishes 2-4 weights reluctantly PCR, it is difficult to accomplish that high flux is detected.Multiple fluorescence PCR typically only detects 3 weights, because probe will mark different emission wavelengths Fluorophor, such as mark too many fluorophor, can mutually produce interference, cause testing result inaccurate.And multiple Exist simultaneously in PCR reaction systems several to primer so that the probability for forming complicated primer dimer is greatly increased, while detection Target gene limited amount so that multiplex PCR does not reach the target of high flux quick detection and analysis also.
The content of the invention
The technical problems to be solved by the invention are how to identify avian influenza virus, and determine whether its HA hypotype is H6 sub- Whether type and its NA hypotypes are N1 hypotypes.
In order to solve the above technical problems, drawing present invention firstly provides identification or auxiliary identification the complete of avian influenza virus Thing.
The primer set provided by the present invention for identifying or aiding in identify avian influenza virus is primer set 1, primer set 2nd, primer set 3 or primer set 4；
The PCR primer of the primer set 1 including entitled H6 is to, entitled N1 PCR primer pair and entitled M PCR primer pair；
The H6 is made up of H6-F and H6-R；The H6-F is 19-38 cores for including SEQ ID No.1 in sequence table The single stranded DNA of thuja acid；The H6-R is the single stranded DNA for including SEQ ID No.2 20-39 nucleotides in sequence table；
The N1 is made up of N1-F and N1-R；The N1-F is 19-38 cores for including SEQ ID No.3 in sequence table The single stranded DNA of thuja acid；The N1-R is the single stranded DNA for including SEQ ID No.4 20-38 nucleotides in sequence table；
The M is made up of M-F and M-R；The M-F is 19-38 nucleotides for including SEQ ID No.5 in sequence table Single stranded DNA；The M-R is the single stranded DNA for including SEQ ID No.6 20-39 nucleotides in sequence table；
The primer set 2 includes the H6 and M；
The primer set 3 includes the N1 and M；
The primer set 4 includes the H6 and N1.
In above-mentioned primer set, the H6 can be as the single stranded DNA and SEQ ID shown in SEQ ID No.1 in sequence table Single stranded DNA composition shown in No.2；The N1 can be as the single stranded DNA shown in SEQ ID No.3 in sequence table and SEQ ID No.4 Shown single stranded DNA composition；The M can be as shown in the single stranded DNA shown in SEQ ID No.5 in sequence table and SEQ ID No.6 Single stranded DNA composition.
In above-mentioned primer set, the primer set 1 can be made up of the H6, the N1, the M and primer pair G；It is described G is as the single stranded DNA and SEQ ID No.2 1-19 cores shown in SEQ ID No.1 1-18 nucleotides in sequence table Single stranded DNA composition shown in thuja acid；
The primer set 2 can be made up of the H6, the M and the G；
The primer set 3 can be made up of the N1, the M and the G；
The primer set 4 can be made up of the H6, the N1 and the G.
Above-mentioned primer set can also be primer set 5, primer set 6 or primer set 7；
The primer set 5 is made up of the M and the G；
The primer set 6 is made up of the H6 and the G；
The primer set 7 is made up of the N1 and the G.
In above-mentioned primer set, the G can use fluorochrome label, concretely CY5.In one embodiment of the present of invention In, one article of single stranded DNA (single stranded DNA shown in SEQ ID No.1 1-18 nucleotides) of the G is marked for 5 ' ends by CY5 The single stranded DNA of note.
In order to solve the above technical problems, present invention also offers PCR primer pair.
PCR primer pair provided by the present invention, for identify or aid in identify avian influenza virus PCR primer to M, identification or The primer pair H6 of auxiliary identification H6 subtype avian influenza virus or identification or the primer pair N1 of auxiliary identification N1 subtype avian influenza virus；
The H6 is made up of H6-F and H6-R；The H6-F is 19-38 cores for including SEQ ID No.1 in sequence table The single stranded DNA of thuja acid；The H6-R is the single stranded DNA for including SEQ ID No.2 20-39 nucleotides in sequence table；
The N1 is made up of N1-F and N1-R；The N1-F is 19-38 cores for including SEQ ID No.3 in sequence table The single stranded DNA of thuja acid；The N1-R is the single stranded DNA for including SEQ ID No.4 20-38 nucleotides in sequence table；
The M is made up of M-F and M-R；The M-F is 19-38 nucleotides for including SEQ ID No.5 in sequence table Single stranded DNA；The M-R is the single stranded DNA for including SEQ ID No.6 20-39 nucleotides in sequence table.
In above-mentioned primer pair, the H6 can be as the single stranded DNA shown in SEQ ID No.1 in sequence table and SEQ ID No.2 Shown single stranded DNA composition；The N1 can be as shown in the single stranded DNA shown in SEQ ID No.3 in sequence table and SEQ ID No.4 Single stranded DNA composition；The M can be as the list shown in the single stranded DNA shown in SEQ ID No.5 in sequence table and SEQ ID No.6 Chain DNA is constituted.
In order to solve the above technical problems, present invention also offers identification or the system of auxiliary identification avian influenza virus.
It is provided by the present invention to identify or aid in identify that the system of avian influenza virus is any of following H-Q：
H, the system containing the primer set 1；
I, the system containing the primer set 2；
J, the system containing the primer set 3；
K, the system containing the primer set 4；
L, the system containing the primer set 5；
M, the system containing the primer set 6；
N, the system containing the primer set 7；
O, the system containing the M；
P, the system containing the H6；
Q, the system containing the N1.
Above-mentioned identification or auxiliary identification avian influenza virus system, may also include identified by GeXP genetic analysis systems or Reagent and instrument needed for auxiliary identification avian influenza virus.Specifically, identify or aid in the system of identification avian influenza virus can Including the primer set 1, the primer set 2, the primer set 3, the primer set 4, the primer set 5, institute State its required for primer set 6, the primer set 7, the M, the H6 or described N1 and GeXP genetic analysis systems Its reagent and instrument.
The system of above-mentioned identification or auxiliary identification avian influenza virus can also only include the primer set 1, described complete draw Thing 2, the primer set 3, the primer set 4, the primer set 5, the primer set 6, the primer set 7, institute State M, the H6 or described N1.
The primer set 1, the primer set 2, the primer set 3, the primer set 4, the primer set 5th, each primer pair in the primer set 6 and the primer set 7 and other examinations required for GeXP genetic analysis systems Agent can independent packaging.The M, the H6 and other reagents required for N1 the and GeXP genetic analysis systems can be only Vertical packaging.
In the primer set of above-mentioned identification or auxiliary identification avian influenza virus, the H6, the N1, the M and the G Two single stranded DNAs in every kind of primer pair can independent packaging.
Other reagents required for GeXP genetic analysis systems may include DNA Polymerase and/or MgCl2And/or 10 × buffer and/or dNTP and/or Random Primer (9mer) and/or dNTP Mixture and/or Ribonuclease Inhibitor and/or Reverse Transcriptase XL (AMV) and/or DNA size standard Kit-400Base Pairs and/or formamide and/or dissociating buffer.
In said system, the H also includes recording following H1) and carrier H2)：
H1) in same PCR reaction systems, using the nucleic acid of biological sample to be measured, (DNA or RNA turn by template of RNA Record obtained cDNA) it is template, entering performing PCR amplification with the primer set 1 obtains PCR primer；
H2) detecting step H1) the obtained size of PCR primer, if in the PCR primer containing 194bp, 249bp and 164bp DNA fragmentation, avian influenza virus or candidate that the testing sample contains H6N1 hypotypes contain the bird flu of H6N1 hypotypes Virus；If the DNA fragmentation containing 194bp and 164bp in the PCR primer, the testing sample contains the fowl stream of H6 hypotypes Influenza Virus or candidate contain the avian influenza virus of H6 hypotypes；It is described if not containing 194bp DNA fragmentation in the PCR primer Testing sample does not contain the avian influenza virus of H6 hypotypes or candidate does not contain the avian influenza virus of H6 hypotypes；If the PCR productions DNA fragmentation containing 249bp and 164bp in thing, the avian influenza virus or candidate that the testing sample contains N1 hypotypes contains N1 The avian influenza virus of hypotype；If not containing 249bp DNA fragmentation in the PCR primer, it is sub- that the testing sample does not contain N1 The avian influenza virus of type or candidate do not contain the avian influenza virus of N1 hypotypes；If the DNA containing 164bp in the PCR primer Fragment, the testing sample contains avian influenza virus or candidate contains avian influenza virus；If do not contained in the PCR primer 164bp DNA fragmentation, the testing sample is free of avian influenza virus without avian influenza virus or candidate.
In said system, H1) PCR system in, every 25 μ L PCR reaction systems can contain：25mM MgCl22.5 μ L, 10 The μ L of 2.5 μ L, 10mM dNTP of × buffer 1.0, primer is (single-stranded shown in SEQ ID No.1 19-38 nucleotides Single stranded DNA shown in DNA, SEQ ID No.2 20-39 nucleotides, SEQ ID No.3 19-38 nucleotides institutes The single stranded DNA shown, the single stranded DNA shown in SEQ ID No.4 20-38 nucleotides, 19-38 of SEQ ID No.5 Single stranded DNA shown in nucleotides and the single stranded DNA shown in SEQ ID No.6 20-39 nucleotides), DNA The μ L of Polymerase 0.5, concentration is the 10ng/ μ L μ L of viral nucleic acid to be measured 1, supplies 25 μ L with sterile ultra-pure water, makes each bar The final concentration of primer is 1 μM.The response procedures of the PCR are as follows：50 DEG C of 30min, 94 DEG C of 2min；94 DEG C of 30s, 50-60 DEG C 30s, 72 DEG C of 30s, 30 circulations；72℃10min.
In said system, H1) PCR system in, every 25 μ L PCR reaction systems can contain：25mM MgCl22.5 μ L, 10 Single stranded DNA shown in μ L, the SEQ ID No.1 of 2.5 μ L, 10mM dNTP of × buffer 1.0, it is single-stranded shown in SEQ ID No.3 Single stranded DNA shown in DNA, SEQ ID No.5, the single stranded DNA shown in SEQ ID No.2 is single-stranded shown in SEQ ID No.4 Single stranded DNA shown in DNA, SEQ ID No.6, the single stranded DNA shown in SEQ ID No.1 1-18 nucleotides, SEQ ID Single stranded DNA shown in No.2 1-19 nucleotides, the μ L of DNA Polymerase 0.5, concentration is 10ng/ μ L disease to be measured The malicious μ L of nucleic acid 1, supply 25 μ L with sterile ultra-pure water, make the final concentration of single stranded DNA shown in SEQ ID No.1,2,3,4,5 and 6 It is 1 μM, 1-19 nucleotides of single stranded DNA and SEQ ID No.2 shown in SEQ ID No.1 1-18 nucleotides The final concentration of shown single stranded DNA is 10 μM.The response procedures of the PCR are as follows：95 DEG C of pre-degeneration 10min；95 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 10 circulations；95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 10 circulations；95 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 20 circulations；72 DEG C of extension 10min；4 DEG C of terminations.
In said system, the I also includes recording following I1) and carrier I2)：
I1) in same PCR reaction systems, using the nucleic acid of biological sample to be measured, (DNA or RNA turn by template of RNA Record obtained cDNA) it is template, entering performing PCR amplification with the primer set 2 obtains PCR primer；
I2) detecting step I1) the obtained size of PCR primer, if containing 194bp's and 164bp in the PCR primer DNA fragmentation, avian influenza virus or candidate that the testing sample contains H6 hypotypes contain the avian influenza virus of H6 hypotypes；If institute State the DNA fragmentation that 194bp is not contained in PCR primer, the testing sample do not contain H6 hypotypes avian influenza virus or candidate not Avian influenza virus containing H6 hypotypes；If the DNA fragmentation containing 164bp in the PCR primer, the testing sample contains fowl Influenza virus or candidate contain avian influenza virus；It is described to treat test sample if not containing 164bp DNA fragmentation in the PCR primer Product are free of avian influenza virus without avian influenza virus or candidate.
In said system, I1) PCR system in, every 25 μ L PCR reaction systems can contain：25mM MgCl22.5 μ L, 10 The μ L of 2.5 μ L, 10mM dNTP of × buffer 1.0, primer is (single-stranded shown in SEQ ID No.1 19-38 nucleotides Single stranded DNA shown in DNA, SEQ ID No.2 20-39 nucleotides, SEQ ID No.5 19-38 nucleotides institutes The single stranded DNA shown in single stranded DNA and SEQ ID No.6 20-39 nucleotides shown), the μ L of DNA Polymerase 0.5, Concentration is the 10ng/ μ L μ L of viral nucleic acid to be measured 1, supplies 25 μ L with sterile ultra-pure water, the final concentration for making each bar primer is 1 μM.The response procedures of the PCR are as follows：50 DEG C of 30min, 94 DEG C of 2min；94 DEG C of 30s, 50-60 DEG C of 30s, 72 DEG C of 30s, 30 are followed Ring；72℃10min.
In said system, I1) PCR system in, every 25 μ L PCR reaction systems can contain：25mM MgCl22.5 μ L, 10 Single stranded DNA shown in μ L, the SEQ ID No.1 of 2.5 μ L, 10mM dNTP of × buffer 1.0, it is single-stranded shown in SEQ ID No.5 Single stranded DNA shown in DNA, SEQ ID No.2, the single stranded DNA shown in SEQ ID No.6, SEQ ID No.1 1-18 cores Single stranded DNA shown in thuja acid, the single stranded DNA shown in SEQ ID No.2 1-19 nucleotides, DNA Polymerase 0.5 μ L, concentration is the 10ng/ μ L μ L of viral nucleic acid to be measured 1, supplies 25 μ L with sterile ultra-pure water, makes SEQ ID No.1,2,5 and 6 The final concentration of shown single stranded DNA is 1 μM, single stranded DNA and SEQ ID shown in SEQ ID No.1 1-18 nucleotides The final concentration of single stranded DNA shown in No.2 1-19 nucleotides is 10 μM.The response procedures of the PCR are as follows：95℃ Pre-degeneration 10min；95 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 10 circulations；95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 10 are followed Ring；95 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 20 circulations；72 DEG C of extension 10min；4 DEG C of terminations.
In said system, the J also includes recording following J1) and carrier J2)：
J1) in same PCR reaction systems, using the nucleic acid of biological sample to be measured, (DNA or RNA turn by template of RNA Record obtained cDNA) it is template, entering performing PCR amplification with the primer set 3 obtains PCR primer；
J2) detecting step J1) the obtained size of PCR primer, if containing 249bp's and 164bp in the PCR primer DNA fragmentation, avian influenza virus or candidate that the testing sample contains N1 hypotypes contain the avian influenza virus of N1 hypotypes；If institute State the DNA fragmentation that 249bp is not contained in PCR primer, the testing sample do not contain N1 hypotypes avian influenza virus or candidate not Avian influenza virus containing N1 hypotypes；If the DNA fragmentation containing 164bp in the PCR primer, the testing sample contains fowl Influenza virus or candidate contain avian influenza virus；It is described to treat test sample if not containing 164bp DNA fragmentation in the PCR primer Product are free of avian influenza virus without avian influenza virus or candidate.
In said system, J1) PCR system in, every 25 μ L PCR reaction systems can contain：25mM MgCl22.5 μ L, 10 The μ L of 2.5 μ L, 10mM dNTP of × buffer 1.0, primer is (single-stranded shown in SEQ ID No.3 19-38 nucleotides Single stranded DNA shown in DNA, SEQ ID No.4 20-38 nucleotides, SEQ ID No.5 19-38 nucleotides institutes The single stranded DNA shown in single stranded DNA and SEQ ID No.6 20-39 nucleotides shown), the μ L of DNA Polymerase 0.5, Concentration is the 10ng/ μ L μ L of viral nucleic acid to be measured 1, supplies 25 μ L with sterile ultra-pure water, the final concentration for making each bar primer is 1 μM.The response procedures of the PCR are as follows：50 DEG C of 30min, 94 DEG C of 2min；94 DEG C of 30s, 50-60 DEG C of 30s, 72 DEG C of 30s, 30 are followed Ring；72℃10min.
In said system, J1) PCR system in, every 25 μ L PCR reaction systems can contain：25mM MgCl22.5 μ L, 10 Single stranded DNA shown in μ L, the SEQ ID No.3 of 2.5 μ L, 10mM dNTP of × buffer 1.0, it is single-stranded shown in SEQ ID No.5 Single stranded DNA shown in DNA, SEQ ID No.4, the single stranded DNA shown in SEQ ID No.6, SEQ ID No.1 1-18 cores Single stranded DNA shown in thuja acid, the single stranded DNA shown in SEQ ID No.2 1-19 nucleotides, DNA Polymerase 0.5 μ L, concentration is the 10ng/ μ L μ L of viral nucleic acid to be measured 1, supplies 25 μ L with sterile ultra-pure water, makes SEQ ID No.3,4,5 and 6 The final concentration of shown single stranded DNA is 1 μM, single stranded DNA and SEQ ID shown in SEQ ID No.1 1-18 nucleotides The final concentration of single stranded DNA shown in No.2 1-19 nucleotides is 10 μM.The response procedures of the PCR are as follows：95℃ Pre-degeneration 10min；95 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 10 circulations；95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 10 are followed Ring；95 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 20 circulations；72 DEG C of extension 10min；4 DEG C of terminations.
In said system, the K also includes recording following K1) and carrier K2)：
K1) in same PCR reaction systems, using the nucleic acid of biological sample to be measured, (DNA or RNA turn by template of RNA Record obtained cDNA) it is template, entering performing PCR amplification with the primer set 4 obtains PCR primer；
K2) detecting step K1) the obtained size of PCR primer, if the DNA pieces containing 194bp in the PCR primer Section, avian influenza virus or candidate that the testing sample contains H6 hypotypes contain the avian influenza virus of H6 hypotypes；If the PCR 194bp DNA fragmentation is not contained in product, the testing sample does not contain the avian influenza virus of H6 hypotypes or candidate does not contain H6 The avian influenza virus of hypotype；If the DNA fragmentation containing 249bp in the PCR primer, the testing sample contains N1 hypotypes Avian influenza virus or candidate contain the avian influenza virus of N1 hypotypes；If not containing 249bp DNA fragmentation in the PCR primer, The testing sample does not contain the avian influenza virus of N1 hypotypes or candidate does not contain the avian influenza virus of N1 hypotypes.
In said system, K1) PCR system in, every 25 μ L PCR reaction systems can contain：25mM MgCl22.5 μ L, 10 The μ L of 2.5 μ L, 10mM dNTP of × buffer 1.0, primer is (single-stranded shown in SEQ ID No.1 19-38 nucleotides Single stranded DNA shown in DNA, SEQ ID No.2 20-39 nucleotides, SEQ ID No.3 19-38 nucleotides institutes Single stranded DNA shown in the single stranded DNA H and SEQ ID No.4 shown 20-38 nucleotides), the μ of DNA Polymerase 0.5 L, concentration is the 10ng/ μ L μ L of viral nucleic acid to be measured 1, supplies 25 μ L with sterile ultra-pure water, makes the final concentration of each bar primer equal For 1 μM.The response procedures of the PCR are as follows：50 DEG C of 30min, 94 DEG C of 2min；94 DEG C of 30s, 50-60 DEG C of 30s, 72 DEG C of 30s, 30 Individual circulation；72℃10min.
In said system, K1) PCR system in, every 25 μ L PCR reaction systems can contain：25mM MgCl22.5 μ L, 10 Single stranded DNA shown in μ L, the SEQ ID No.1 of 2.5 μ L, 10mM dNTP of × buffer 1.0, it is single-stranded shown in SEQ ID No.3 Single stranded DNA shown in DNA, SEQ ID No.2, the single stranded DNA shown in SEQ ID No.4, SEQ ID No.1 1-18 cores Single stranded DNA shown in thuja acid, the single stranded DNA shown in SEQ ID No.2 1-19 nucleotides, DNA Polymerase 0.5 μ L, concentration is the 10ng/ μ L μ L of viral nucleic acid to be measured 1, supplies 25 μ L with sterile ultra-pure water, makes SEQ ID No.1,2,3 and 4 The final concentration of shown single stranded DNA is 1 μM, single stranded DNA and SEQ ID shown in SEQ ID No.1 1-18 nucleotides The final concentration of single stranded DNA shown in No.2 1-19 nucleotides is 10 μM.The response procedures of the PCR are as follows：95℃ Pre-degeneration 10min；95 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 10 circulations；95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 10 are followed Ring；95 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 20 circulations；72 DEG C of extension 10min；4 DEG C of terminations.
In said system, the L also includes recording following L1) and carrier L2)：
L1) in same PCR reaction systems, using the nucleic acid of biological sample to be measured, (DNA or RNA turn by template of RNA Record obtained cDNA) it is template, entering performing PCR amplification with the primer set 5 obtains PCR primer；
L2) detecting step L1) the obtained size of PCR primer, if the DNA pieces containing 164bp in the PCR primer Section, the testing sample contains avian influenza virus or candidate contains avian influenza virus；If do not contained in the PCR primer 164bp DNA fragmentation, the testing sample is free of avian influenza virus without avian influenza virus or candidate.
In said system, L1) PCR system in, every 25 μ L PCR reaction systems can contain：25mM MgCl22.5 μ L, 10 Single stranded DNA shown in μ L, the SEQ ID No.5 of 2.5 μ L, 10mM dNTP of × buffer 1.0, it is single-stranded shown in SEQ ID No.6 Single stranded DNA shown in DNA, SEQ ID No.1 1-18 nucleotides, shown in SEQ ID No.2 1-19 nucleotides Single stranded DNA, the μ L of DNA Polymerase 0.5, concentration be 10ng/ μ L the μ L of viral nucleic acid to be measured 1, with sterile ultra-pure water 25 μ L are supplied, the final concentration for making single stranded DNA shown in SEQ ID No.5 and 6 is 1 μM, SEQ ID No.1 1-18 nucleosides The final concentration of single stranded DNA shown in acid and the single stranded DNA shown in SEQ ID No.2 1-19 nucleotides is 10 μM.Institute The response procedures for stating PCR are as follows：95 DEG C of pre-degeneration 10min；95 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 10 circulations；95℃ 30s, 60 DEG C of 30s, 72 DEG C of 1min, 10 circulations；95 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 20 circulations；72 DEG C of extensions 10min；4 DEG C of terminations.
In said system, the M also includes recording following M1) and carrier M2)：
M1) in same PCR reaction systems, using the nucleic acid of biological sample to be measured, (DNA or RNA turn by template of RNA Record obtained cDNA) it is template, entering performing PCR amplification with the primer set 6 obtains PCR primer；
M2) detecting step M1) the obtained size of PCR primer, if the DNA pieces containing 194bp in the PCR primer Section, avian influenza virus or candidate that the testing sample contains H6 hypotypes contain the avian influenza virus of H6 hypotypes；If the PCR 194bp DNA fragmentation is not contained in product, the testing sample does not contain the avian influenza virus of H6 hypotypes or candidate does not contain H6 The avian influenza virus of hypotype.
In said system, M1) PCR system in, every 25 μ L PCR reaction systems can contain：25mM MgCl22.5 μ L, 10 Single stranded DNA shown in μ L, the SEQ ID No.1 of 2.5 μ L, 10mM dNTP of × buffer 1.0, it is single-stranded shown in SEQ ID No.2 Single stranded DNA shown in DNA, SEQ ID No.1 1-18 nucleotides, shown in SEQ ID No.2 1-19 nucleotides Single stranded DNA, the μ L of DNA Polymerase 0.5, concentration be 10ng/ μ L the μ L of viral nucleic acid to be measured 1, with sterile ultra-pure water 25 μ L are supplied, the final concentration for making single stranded DNA shown in SEQ ID No.1 and 2 is 1 μM, SEQ ID No.1 1-18 nucleosides The final concentration of single stranded DNA shown in acid and the single stranded DNA shown in SEQ ID No.2 1-19 nucleotides is 10 μM.Institute The response procedures for stating PCR are as follows：95 DEG C of pre-degeneration 10min；95 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 1min, 10 circulations；95℃ 30s, 60 DEG C of 30s, 72 DEG C of 1min, 10 circulations；95 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 20 circulations；72 DEG C of extensions 10min；4 DEG C of terminations.
In said system, the N also includes recording following N1) and carrier N2)：
N1) in same PCR reaction systems, using the nucleic acid of biological sample to be measured, (DNA or RNA turn by template of RNA Record obtained cDNA) it is template, entering performing PCR amplification with the primer set 7 obtains PCR primer；
N2) detecting step N1) the obtained size of PCR primer, if the DNA pieces containing 249bp in the PCR primer Section, avian influenza virus or candidate that the testing sample contains N1 hypotypes contain the avian influenza virus of N1 hypotypes；If the PCR 249bp DNA fragmentation is not contained in product, the testing sample does not contain the avian influenza virus of N1 hypotypes or candidate does not contain N1 The avian influenza virus of hypotype.
In said system, N1) PCR system in, every 25 μ L PCR reaction systems can contain：25mM MgCl22.5 μ L, 10 Single stranded DNA shown in μ L, the SEQ ID No.3 of 2.5 μ L, 10mM dNTP of × buffer 1.0, it is single-stranded shown in SEQ ID No.4 Single stranded DNA shown in DNA, SEQ ID No.1 1-18 nucleotides, shown in SEQ ID No.2 1-19 nucleotides Single stranded DNA, the μ L of DNA Polymerase 0.5, concentration be 10ng/ μ L the μ L of viral nucleic acid to be measured 1, with sterile ultra-pure water 25 μ L are supplied, the final concentration for making single stranded DNA shown in SEQ ID No.3 and 4 is 1 μM, SEQ ID No.1 1-18 nucleosides The final concentration of single stranded DNA shown in acid and the single stranded DNA shown in SEQ ID No.2 1-19 nucleotides is 10 μM.Institute The response procedures for stating PCR are as follows：95 DEG C of pre-degeneration 10min；95 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 10 circulations；95℃ 30s, 60 DEG C of 30s, 72 DEG C of 1min, 10 circulations；95 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 20 circulations；72 DEG C of extensions 10min；4 DEG C of terminations.
In said system, the O also includes recording following O1) and carrier O2)：
O1) in same PCR reaction systems, using the nucleic acid of biological sample to be measured, (DNA or RNA turn by template of RNA Record obtained cDNA) it is template, entering performing PCR amplification with the M obtains PCR primer；
O2) detecting step O1) the obtained size of PCR primer, if the DNA pieces containing 164bp in the PCR primer Section, the testing sample contains avian influenza virus or candidate contains avian influenza virus；If do not contained in the PCR primer 164bp DNA fragmentation, the testing sample is free of avian influenza virus without avian influenza virus or candidate.
In said system, O1) PCR system in, every 25 μ L PCR reaction systems can contain：25mM MgCl22.5 μ L, 10 The μ L of 2.5 μ L, 10mM dNTP of × buffer 1.0, the primer (single stranded DNA shown in SEQ ID No.5 19-38 nucleotides With the single stranded DNA shown in SEQ ID No.6 20-39 nucleotides), DNA Polymerase0.5 μ L, concentration is 10ng/ μ The L μ L of viral nucleic acid to be measured 1, supply 25 μ L, the final concentration for making each bar primer is 1 μM with sterile ultra-pure water.The PCR's Response procedures are as follows：50 DEG C of 30min, 94 DEG C of 2min；94 DEG C of 30s, 50-60 DEG C of 30s, 72 DEG C of 30s, 30 circulations；72℃ 10min。
In said system, the P also includes recording following P1) and carrier P2)：
P1) in same PCR reaction systems, using the nucleic acid of biological sample to be measured, (DNA or RNA turn by template of RNA Record obtained cDNA) it is template, entering performing PCR amplification with the H6 obtains PCR primer；
P2) detecting step P1) the obtained size of PCR primer, if the DNA pieces containing 194bp in the PCR primer Section, avian influenza virus or candidate that the testing sample contains H6 hypotypes contain the avian influenza virus of H6 hypotypes；If the PCR 194bp DNA fragmentation is not contained in product, the testing sample does not contain the avian influenza virus of H6 hypotypes or candidate does not contain H6 The avian influenza virus of hypotype.
In said system, P1) PCR system in, every 25 μ L PCR reaction systems can contain：25mM MgCl22.5 μ L, 10 The μ L of 2.5 μ L, 10mM dNTP of × buffer 1.0, the primer (single stranded DNA shown in SEQ ID No.1 19-38 nucleotides With the single stranded DNA shown in SEQ ID No.2 20-39 nucleotides), DNA Polymerase0.5 μ L, concentration is 10ng/ μ The L μ L of viral nucleic acid to be measured 1, supply 25 μ L, the final concentration for making each bar primer is 1 μM with sterile ultra-pure water.The PCR's Response procedures are as follows：50 DEG C of 30min, 94 DEG C of 2min；94 DEG C of 30s, 50-60 DEG C of 30s, 72 DEG C of 30s, 30 circulations；72℃ 10min。
In said system, the Q also includes recording following Q1) and carrier Q2)：
Q1) in same PCR reaction systems, using the nucleic acid of biological sample to be measured, (DNA or RNA turn by template of RNA Record obtained cDNA) it is template, entering performing PCR amplification with the N1 obtains PCR primer；
Q2) detecting step Q1) the obtained size of PCR primer, if the DNA pieces containing 249bp in the PCR primer Section, avian influenza virus or candidate that the testing sample contains N1 hypotypes contain the avian influenza virus of N1 hypotypes；If the PCR 249bp DNA fragmentation is not contained in product, the testing sample does not contain the avian influenza virus of N1 hypotypes or candidate does not contain N1 The avian influenza virus of hypotype.
In said system, Q1) PCR system in, every 25 μ L PCR reaction systems can contain：25mM MgCl22.5 μ L, 10 The μ L of 2.5 μ L, 10mM dNTP of × buffer 1.0, the primer (single stranded DNA shown in SEQ ID No.3 19-38 nucleotides With the single stranded DNA shown in SEQ ID No.4 20-38 nucleotides), DNA Polymerase0.5 μ L, concentration is 10ng/ μ The L μ L of viral nucleic acid to be measured 1, supply 25 μ L, the final concentration for making each bar primer is 1 μM with sterile ultra-pure water.The PCR's Response procedures are as follows：50 DEG C of 30min, 94 DEG C of 2min；94 DEG C of 30s, 50-60 DEG C of 30s, 72 DEG C of 30s, 30 circulations；72℃ 10min。
In order to solve the above technical problems, present invention also offers the preparation method of primer set.
The preparation method of the primer set provided by the present invention, including by the institute of each primer pair in the primer set State the step of two single stranded DNAs are individually packed.
In order to solve the above technical problems, present invention also offers the preparation method of PCR primer pair.
The preparation method of PCR primer pair provided by the present invention, including it is single-stranded by described two of the PCR primer centering The step of DNA is individually packed.
In the present invention, the M, the H6 of primer set 1 are when the N1 is used together, the molal quantity of every kind of primer pair It is identical；When the M, the H6, the N1 together with the G in use, the molal quantity of these four primer pairs can be 1:1:1: 10.When the M and the H6 of primer set 2 are used together, the molal quantity of every kind of primer pair is identical；As the M, the H6 and The G is together in use, the molal quantity of these three primer pairs can be 1:1:10.The M and the N1 of primer set 3 are together In use, the molal quantity of every kind of primer pair is identical；When the M, the H6 together with the G in use, these three primer pairs Molal quantity can be 1:1:10.When the H6 and the N1 of primer set 4 are used together, the molal quantity phase of every kind of primer pair Together；When the H6, the N1 together with the G in use, the molal quantity of these three primer pairs can be 1:1:10.Primer set 5 M is together with the G in use, the molal quantity of both primer pairs can be 1:10.H6 described in primer set 6 and institute G is stated together in use, the molal quantity of both primer pairs can be 1:10.The N1 of primer set 7 makes together with the G Used time, the molal quantity of both primer pairs can be 1:10.
In the present invention, the mol ratio of two single stranded DNAs in every kind of primer pair can be 1：1.
DNA Polymerase in the present invention concretely Sigma Co., USA's product, article No. is D4184-1.5KU； 25mM MgCl2Concretely Sigma Co., USA's product, article No. is M8787-1.5ML；The μ L of 10 × buffer 2.5 specifically may be used For Sigma Co., USA's product, article No. is P2317-1VL；Random Primer (9mer) concretely Dalian TaKaRa companies Product, catalog number (Cat.No.) is 3802；DNTP Mixture concretely Dalian TaKaRa Products, catalog number (Cat.No.) is 4019； Ribonuclease Inhibitor concretely Dalian TaKaRa Products, catalog number (Cat.No.) is 2313A；Reverse Transcriptase XL (AMV) concretely Dalian TaKaRa Products, catalog number (Cat.No.) is 2621.DNA size Standard Kit-400Base Pairs concretely Beckman Coulter Inc. of U.S. products, article No. is 608098；Formyl Amine concretely Beckman Coulter Inc. of U.S. product, article No. is 608082；Dissociating buffer concretely U.S.'s Beckman Products, article No. is 608012.
It is demonstrated experimentally that the primer set identification avian influenza virus and its H6 hypotypes and N1 hypotypes of the present invention have higher spirit Sensitivity and specificity：The primer set of the present invention can unique identification avian influenza virus, H6N1 subtype avian influenza virus, H6 hypotype fowl Influenza virus and N1 subtype avian influenza virus；The primer set of the present invention can detect the μ L of 100 copies/25 H6N1 hypotypes AIV. The primer pair M identification avian influenza virus of the present invention has higher sensitivity and specificity：The primer pair M of the present invention can specifically reflect Determine avian influenza virus；The primer pair M of the present invention can detect the μ L of 100 copies/25 AIV.The primer pair H6 identifications H6 of the present invention Subtype avian influenza virus has higher sensitivity and specificity：The primer pair H6 of the present invention can unique identification H6 subtype avian influenzas Virus；The primer pair H6 of the present invention can detect the μ L of 100 copies/25 H6 hypotypes AIV.The primer pair N1 identifications N1 of the present invention is sub- Type avian influenza virus has higher sensitivity and specificity：The primer pair N1 of the present invention can unique identification N1 subtype avian influenzas disease Poison；The primer pair N1 of the present invention can detect the μ L of 100 copies/25 N1 hypotypes AIV.It is demonstrated experimentally that the primer set of the present invention It can be used to identify avian influenza virus and its H6 hypotypes and N1 hypotypes；The primer pair H6 of the present invention can be used to identify H6 subtype avian influenzas Virus；The primer pair N1 of the present invention can be used to identify N1 subtype avian influenza virus；The primer pair M of the present invention can be used to identify that fowl is flowed Influenza Virus.
Brief description of the drawings
Fig. 1 is as primer, with H6N1 subtype avian influenza virus using the primer set for identifying or aiding in identify avian influenza virus The capillary electrophoresis analysis results of PCR primer that obtain for template of cDNA.
Fig. 2 is to identify or aid in the primer set for identifying avian influenza virus to be flowed as primer, with H6Ny (y ≠ 1) hypotype fowl The cDNA of Influenza Virus is the capillary electrophoresis analysis result for the PCR primer that template is obtained.
Fig. 3 is to identify or aid in the primer set for identifying avian influenza virus to be flowed as primer, with HxN1 (x ≠ 6) hypotype fowl The cDNA of Influenza Virus is the capillary electrophoresis analysis result for the PCR primer that template is obtained.
Fig. 4 is to identify or aid in identify the primer set of avian influenza virus as primer, with the HxNy (hypotypes of x ≠ 6, y ≠ 1) The cDNA of avian influenza virus is the capillary electrophoresis analysis result for the PCR primer that template is obtained.
In Fig. 1-4, abscissa is the base number of pcr amplification product, and ordinate is fluorescence signal value.
Embodiment
The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining The bright present invention, the scope being not intended to be limiting of the invention.
Experimental method in following embodiments, is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
H1N1 subtype avian influenza virus, H2N3 subtype avian influenza virus, H3N2 subtype avian influenzas disease in following embodiments Poison, H5N1 subtype avian influenza virus, H7N2 subtype avian influenza virus, H8N4 subtype avian influenza virus, H9N2 subtype avian influenzas disease Poison and H12N5 subtype avian influenza virus (Yi Peng.et al.Visual detection of H3subtype avian influenza viruses by reverse transcription loop-mediated isothermal ampl ification assay.Virology Journal,2011,8:337) public can be from Veterinary Institute of Guangxi Zhuang Autonomous Region (i.e. applicant) is obtained, and the biomaterial is only attached most importance to used in the related experiment of duplicate invention, can not be used as other purposes.
H6N1 subtype avian influenza virus, H6N2 subtype avian influenza virus, H6N5 subtype avian influenzas disease in following embodiments Poison, H6N6 subtype avian influenza virus and H6N8 subtype avian influenza virus (Zhou Chenyu, H6 subtype avian influenza virus pathogenic surveillance and The research of quick determination method, Guangxi University's master's thesis in 2012,2012-06-01.) public can from Zhuang nationality in Guangxi from Area's veterinary institute (i.e. applicant) acquisition is controlled, the biomaterial is only attached most importance to used in the related experiment of duplicate invention, can not conduct Other purposes are used.
H4N5 subtype avian influenza virus, H10N3 subtype avian influenza virus and H11N9 subtype avian influenzas in following embodiments (Luo Sisi, Xie Zhixun, Liu Jiabo wait the foundation of .H7N9 hypotype AIV dual real-time fluorescence quantitative RT-PCR detecting methods to virus [J] animal medicines are in progress, 2013,34 (12)：1-5.) public can be from Veterinary Institute of Guangxi Zhuang Autonomous Region (i.e. applicant) Obtain, the biomaterial is only attached most importance to used in the related experiment of duplicate invention, can not be used as other purposes.
H13N5 subtype avian influenza virus (Xie Z, Pang Y S, Liu J, et al.A in following embodiments multiplex RT-PCR for detection of type a influenza virus and differentiation Of avian H5, H7and H9hemagglutinin subtypes [J] .Mol Cell Probes, 2006,20 (3-4)： 245-249.) public can be obtained from Veterinary Institute of Guangxi Zhuang Autonomous Region (i.e. applicant), and the biomaterial is only attached most importance to duplicate Used in the related experiment of invention, it can not be used as other purposes.
F48E9 plants of Newcastle Disease poison strain, Mukteswar plants, C in following embodiments30Strain, V4 plants, Ulster plants, (Chen Anli, Xie Zhixun, Zhou Chenyu wait the strong and weak poison of NDVs by Losota, GX1/00, GX2/00, GX6/02 and GX7/02 Strain LAMP differentiates the Chinese veterinary sciences of foundation [J] of detection method, 2011,41 (09):917-922.) public can be from Guangxi Zhuang Autonomous region of race veterinary institute (i.e. applicant) is obtained, and the biomaterial is only attached most importance to used in the related experiment of duplicate invention, can not Used as other purposes.
Infectious bronchitis virus strain in following embodiments:Massachussetts 41、Connecticut (Conn), Arkansas (Ark), Delaware (DE/2686/92), PA, JMK, H52 and Guangxi separation strains GXIB/02 (Xie Zhi Duty, Xie Zhixun, Lv Hua has just waited .30 plants of avian infectious bronchitis virus type strains and the clone of separation strains S1 genes and sequence point Analyse the agriculture journal in [J] southwest, 2008,21 (6):1733-1736.) public can be from Veterinary Institute of Guangxi Zhuang Autonomous Region (i.e. Applicant) obtain, the biomaterial is only attached most importance to used in the related experiment of duplicate invention, can not be used as other purposes.
Infectious Laryngotracheitis Virus Beijing strain, the strain of Taiwan vaccine strain, Unite States Standard in following embodiments, Israel's epidemic disease (such as Xie Zhixun, Xie Zhiqin, Pang Yaoshan is using two Wen Shi PCR amplifications chicken trachitis disease for Miao Zhu, Guangxi separation strains The Chinese animal doctor's science and technology of research [J] of malicious TK genes, 2000,30 (9):5-7) public can study from Guangxi Zhuang Autonomous Region animal doctor Institute (i.e. applicant) is obtained, and the biomaterial is only attached most importance to used in the related experiment of duplicate invention, can not be used as other purposes.
DNA Polymerase in following embodiments are Sigma Co., USA's product, and article No. is D4184-1.5KU；Under State the 25mM MgCl in embodiment2For Sigma Co., USA's product, article No. is M8787-1.5ML；10 in following embodiments The μ L of × buffer 2.5 are Sigma Co., USA's product, and article No. is P2317-1VL；Random Primer in following embodiments (9mer) is Dalian TaKaRa Products, and catalog number (Cat.No.) is 3802；25mM dNTP Mixture in following embodiments are Dalian TaKaRa Products, catalog number (Cat.No.) is 4019；Ribonuclease Inhibitor in following embodiments are Dalian TaKaRa Products, catalog number (Cat.No.) is 2313A；Reverse Transcriptase XL (AMV) in following embodiments are Dalian TaKaRa Products, catalog number (Cat.No.) is 2621.
DNA size standard Kit-400Base Pairs in following embodiments are public for U.S.'s Beckman Kurt Product is taken charge of, article No. is 608098.
Formamide in following embodiments is Beckman Coulter Inc. of U.S. product, and article No. is 608082.
Dissociating buffer in following embodiments is U.S.'s Beckman Products, and article No. is 608012.
Embodiment 1, the primer set for preparing identification or auxiliary identification avian influenza virus
Identification or auxiliary identification avian influenza virus (AIV) primer set, by entitled H6 PCR primer to, it is entitled N1 PCR primer is to, entitled M PCR primer pair and entitled G PCR primer to composition；H6 SEQ in sequence table Single stranded DNA (H6-R) composition shown in single stranded DNA (H6-F) and SEQ ID No.2 shown in ID No.1, can be from the fowl of H6 hypotypes The DNA fragmentation that size is 194bp is amplified in influenza virus；The N1 is as the single stranded DNA shown in SEQ ID No.3 in sequence table (N1-F) single stranded DNA (N1-R) composition and shown in SEQ ID No.4, can amplify size from the avian influenza virus of N1 hypotypes For 249bp DNA fragmentation；The M is as the single stranded DNA (M-F) shown in SEQ ID No.5 in sequence table and SEQ ID No.6 institutes Single stranded DNA (M-R) composition shown, can amplify the DNA fragmentation that size is 164bp from avian influenza virus；The G is marked by CY5 Single stranded DNA shown in the SEQ ID No.1 of note 1-18 nucleotides and shown in SEQ ID No.2 1-19 nucleotides Single stranded DNA composition, primer pair G be GeXP universal primers pair.
Wherein, SEQ ID No.1 1-18 nucleotides, SEQ ID No.3 1-18 nucleotides and SEQ ID No.5 1-18 nucleotides are consistent；SEQ ID No.2 1-19 nucleotides, SEQ ID No.4 1-19 cores Thuja acid is consistent with SEQ ID No.6 1-19 nucleotides.
In the primer set of above-mentioned identification or auxiliary identification avian influenza virus, the H6, the N1, the M and the G are equal Independent packaging.The mol ratio of two single stranded DNAs in every kind of primer pair is 1：1.
The specificity experiments of the primer set of embodiment 2, the identification of embodiment 1 or auxiliary identification avian influenza virus
First, the preparation of template
The virus used in experiment be 17 plants of avian influenza virus (H6N1 hypotypes, H6N2 hypotypes, H6N5 hypotypes, H6N6 hypotypes, H6N8 hypotypes, H1N1 hypotypes, H2N3 hypotypes, H3N2 hypotypes, H4N5 hypotypes, H5N1 hypotypes, H7N2 hypotypes, H8N4 hypotypes, H9N2 Hypotype, H10N3 hypotypes, H11N9 hypotypes, H12N5 hypotypes and H13N5 hypotypes), 10 plants of NDVs (F48E9 plants, Mukteswar plants, C30Strain, V4 plants, Ulster plants, Losota, GX1/00, GX2/00, GX6/02 and GX7/02), 8 plants of infectiousness Bronchitis poison strain (Massachussetts 41, Connecticut (Conn), Arkansas (Ark), Delaware (DE/2686/92), PA, JMK, H52 and Guangxi separation strains GXIB/02) and 5 plants of ILTV strain (Beijing Strain, the strain of Taiwan vaccine strain, Unite States Standard, Israel's vaccine strain and Guangxi separation strains).
MiniBEST Viral RNA/DNA Extraction Kit Ver.4.0 are utilized according to the specification of kit (Dalian TaKaRa companies, production code member is DV819A), extracts and obtains from the chick embryo allantoic liquid of following virus stain respectively 15 plants of avian influenza virus (H6N1, H6N2, H6N5, H6N6, H6N8, H1N1, H2N3, H3N2, H4N5, H8N4, H9N2, H10N3, H11N9, H12N5 and H13N5), 10 plants of NDVs (F48E9 plants, Mukteswar plants, C30Strain, V4 plants, Ulster plants, Losota, GX1/00, GX2/00, GX6/02 and GX7/02) and 8 plants of infectious bronchitis virus strains (Massachussetts 41、Connecticut(Conn)、Arkansas(Ark)、Delaware(DE/2686/92)、PA、 JMK, H52 and Guangxi separation strains GXIB/02) total serum IgE, to extract the chick embryo allantoic liquid total serum IgE that negative chick embryo allantoic liquid is obtained For negative control.H5N1 and H7N2 avian influenza virus (H5N1 and H7N2 avian influenza virus is inactivation of viruses) RNA is by U.S.'s health Nie Dige states university give.Respectively to above-mentioned total serum IgE Random Primer (9mer), dNTP Mixture, Ribonuclease Inhibitor and Reverse Transcriptase XL (AMV) carry out reverse transcription, obtain above-mentioned virus CDNA and chick embryo allantoic liquid cDNA.
Using blood tissues cellular genome extracts kit, (TIANGEN Biotech (Beijing) Co., Ltd., article No. is DP304), according to kit specification, the disease of the chick embryo allantoic liquid of following 5 plants of ILTV strains is extracted respectively Virus gene group DNA：Beijing Strain, Taiwan vaccine strain, Unite States Standard strain, Israel's vaccine strain and Guangxi separation strains, respectively obtain north Capital strain, the strain of Taiwan vaccine strain, Unite States Standard, the DNA of Israel's vaccine strain and Guangxi separation strains, to extract negative chick embryo allantoic liquid The chick embryo allantoic liquid DNA of acquisition is negative control.
2nd, primer pair M specificity experiments
1st, PCR is expanded
With the primer pair M and primer pair G of embodiment 1 respectively with 17 kinds of subtype avian influenza virus strains of step one, 10 kinds The cDNA samples of Newcastle Disease poison strain and 8 plants of infectious bronchitis virus strains are that template enters performing PCR amplification, with step one Chick embryo allantoic liquid cDNA be negative control, obtain primer pair M PCR primer；With G points of the primer pair M and primer pair of embodiment 1 Do not enter performing PCR amplification by template of the DNA sample of 5 plants of ILTV strains of step one, with the chicken of step one Embryo allantoic liquid DNA is negative control, obtains primer pair M PCR primer.Reaction system and response procedures are as follows：
Reaction system (25 μ L)：25mM MgCl22.5 μ L, the μ L of 10 × buffer 2.5, the μ L of 25mM dNTP 1.0, primer To M (final concentration of the primer pair M every primer in reaction system is 1 μM), (primer pair G every primer exists primer pair G Final concentration in reaction system is 10 μM), the μ L of DNA Polymerase 0.5, the viral cDNA or DNA that step one is obtained (a kind of viral cDNA or DNA or chick embryo allantoic liquid cDNA or DNA in each reaction system) 2 μ L, plus sterilized water is to 25 μ L.Often Individual reaction sets 3 repetitions.
Response procedures：95 DEG C of pre-degeneration 10min；95 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 1min, 10 circulations；95 DEG C of 30s, 60 DEG C 30s, 72 DEG C of 1min, 10 circulations；95 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 20 circulations；72 DEG C of extension 10min；4 DEG C of ends Only.
2nd, Capillary Electrophoresis
The primer pair M obtained using GenomeLab GeXP genetic analysis systems to step 1 PCR primer carries out capillary Electrophoresis detection, concrete operation step is as follows：
By DNA size standard Kit-400Base Pairs, (Beckman Coulter Inc. of the U.S., article No. is 608098) (608082) Beckman Coulter Inc. of the U.S., article No. is by DNA size standard Kit- with formamide 400Base Pairs：Formamide=1:100 volume ratio adds 39 μ L in sample panel after mixing in every hole.And backward sample The primer pair M of the step 1 μ L of PCR primer 1 (per a kind of PCR primer in hole) are added on plate in every hole, piping and druming is mixed, finally in every hole A dropstone wax oil is instilled to be closed.Every hole adds 2/3 dissociating buffer on buffer solution plate, carries out Capillary Electrophoresis.Hair The condition of cons electrophoresis is as follows：Capillary heats up：Temperature 50 C；Denaturation：90℃,120s；Sample is injected into capillary： 2.0KV, 30s；Separation：6.0KV, 35min.The primer obtained using GenomeLab GeXP genetic analysis systems analytical procedure 1 To M PCR primer.
As a result show, 17 plants of avian influenza virus (H6N1 hypotypes, H6N2 hypotypes, H6N5 hypotypes, H6N6 hypotypes, H6N8 hypotypes, H1N1 hypotypes, H2N3 hypotypes, H3N2 hypotypes, H4N5 hypotypes, H5N1 hypotypes, H7N2 hypotypes, H8N4 hypotypes, H9N2 hypotypes, H10N3 Hypotype, H11N9 hypotypes, H12N5 hypotypes and H13N5 hypotypes) the amplifiable PCR primer for obtaining 164bp of cDNA samples, it is right These PCR primers are sequenced, and sequencing result shows, 17 plants of avian influenza virus (H6N1 hypotypes, H6N2 hypotypes, H6N5 hypotypes, H6N6 hypotypes, H6N8 hypotypes, H1N1 hypotypes, H2N3 hypotypes, H3N2 hypotypes, H4N5 hypotypes, H5N1 hypotypes, H7N2 hypotypes, H8N4 Hypotype, H9N2 hypotypes, H10N3 hypotypes, H11N9 hypotypes, H12N5 hypotypes and H13N5 hypotypes) PCR primer be primer pair M Target sequence.10 plants of NDVs (F48E9 plants, Mukteswar plants, C30Strain, V4 plants, Ulster plants, Losota, GX1/00, GX2/00, GX6/02 and GX7/02), 8 plants of infectious bronchitis virus strains (Massachussetts 41, Connecticut (Conn), Arkansas (Ark), Delaware (DE/2686/92), PA, JMK, H52 and Guangxi separation strains ) and 5 plants of ILTV strains (Beijing Strain, Taiwan vaccine strain, Unite States Standard strain, Israel's vaccine strains GXIB/02 With Guangxi separation strains) and negative control without any amplified production.As a result show, primer pair M specificity can specifically reflect very well Determine avian influenza virus.
3rd, primer pair H6 specificity experiments
1st, PCR is expanded
According in step 21 method, by " the primer pair M " of embodiment 1 replace with " the primer pair H6 " of embodiment 1, its His step is constant, obtains primer pair H6 PCR primer.
2nd, Capillary Electrophoresis
According in step 22 method, " primer pair M PCR primer " is replaced with to " the primer pair H6 PCR productions of step 1 Thing ", other steps are constant, the primer pair H6 obtained using GenomeLab GeXP genetic analysis systems analytical procedure 1 PCR Product.
As a result show, 5 plants of avian influenza virus (H6N1 hypotypes, H6N2 hypotypes, H6N5 hypotypes, H6N6 hypotypes and H6N8 hypotypes) The amplifiable PCR primer for obtaining 194bp of cDNA samples, these PCR primers are sequenced, sequencing result shows, 5 plants of fowl The PCR primer of influenza virus (H6N1 hypotypes, H6N2 hypotypes, H6N5 hypotypes, H6N6 hypotypes and H6N8 hypotypes) is primer pair H6 Target sequence.(H1N1 hypotypes, H2N3 hypotypes, H3N2 hypotypes, H4N5 hypotypes, H5N1 hypotypes, H7N2 are sub- for 12 plants of avian influenza virus Type, H8N4 hypotypes, H9N2 hypotypes, H10N3 hypotypes, H11N9 hypotypes, H12N5 hypotypes and H13N5 hypotypes), 10 plants of NDVs (F48E9 plants, Mukteswar plants, C30Strain, V4 plants, Ulster plants, Losota, GX1/00, GX2/00, GX6/02 and GX7/02), 8 plants of infectious bronchitis virus strains (Massachussetts 41, Connecticut (Conn), Arkansas (Ark), Delaware (DE/2686/92), PA, JMK, H52 and Guangxi separation strains GXIB/02) and 5 plants of ILTV poison Strain (Beijing Strain, Taiwan vaccine strain, Unite States Standard strain, Israel's vaccine strain and Guangxi separation strains) and negative control expand without any Increase production thing.As a result show, primer pair H6 specificity very well, can unique identification H6 subtype avian influenza virus.
4th, primer pair N1 specificity experiments
1st, PCR is expanded
According in step 21 method, by " the primer pair M " of embodiment 1 replace with " the primer pair N1 " of embodiment 1, its His step is constant, obtains primer pair N1 PCR primer.
2nd, Capillary Electrophoresis
According in step 22 method, " primer pair M PCR primer " is replaced with to " the primer pair N1 PCR productions of step 1 Thing ", other steps are constant, the primer pair N1 obtained using GenomeLab GeXP genetic analysis systems analytical procedure 1 PCR Product.
As a result show, the cDNA samples of 3 plants of avian influenza virus (H6N1 hypotypes, H1N1 hypotypes and H5N1 hypotypes) are amplifiable 249bp PCR primer is obtained, these PCR primers are sequenced, sequencing result shows, 3 plants of avian influenza virus (H6N1 hypotypes, H1N1 hypotypes and H5N1 hypotypes) cDNA samples primer pair N1 PCR primer be primer pair N1 target sequence.14 plants of fowl streams (H6N2 hypotypes, H6N5 hypotypes, H6N6 hypotypes, H6N8 hypotypes, H2N3 hypotypes, H3N2 hypotypes, H4N5 hypotypes, H7N2 are sub- for Influenza Virus Type, H8N4 hypotypes, H9N2 hypotypes, H10N3 hypotypes, H11N9 hypotypes, H12N5 hypotypes and H13N5 hypotypes), 10 plants of NDVs (F48E9 plants, Mukteswar plants, C30Strain, V4 plants, Ulster plants, Losota, GX1/00, GX2/00, GX6/02 and GX7/02), 8 plants of infectious bronchitis virus strains (Massachussetts 41, Connecticut (Conn), Arkansas (Ark), Delaware (DE/2686/92), PA, JMK, H52 and Guangxi separation strains GXIB/02) and 5 plants of ILTV poison Strain (Beijing Strain, Taiwan vaccine strain, Unite States Standard strain, Israel's vaccine strain and Guangxi separation strains) and negative control expand without any Increase production thing.As a result show, primer pair N1 specificity very well, can unique identification N1 subtype avian influenza virus.
5th, the specific detection of the primer set of the identification of embodiment 1 or auxiliary identification avian influenza virus
1st, PCR is expanded
With the identification of embodiment 1 or the primer set of auxiliary identification avian influenza virus (AIV) respectively with 17 plants of step one The cDNA samples of fowl influenza virus strain, 10 plants of Newcastle Disease poison strains and 8 plants of infectious bronchitis virus strains are template Enter performing PCR amplification, the chick embryo allantoic liquid cDNA using step one obtains the PCR primer of primer set as negative control；Use embodiment 1 identification or the primer set and primer pair G of auxiliary identification avian influenza virus (AIV) are respectively with 5 plants of infectiousness larynxs of step one The DNA sample of bronchitis virus strain is that template enters performing PCR amplification, and the chick embryo allantoic liquid DNA using step one is obtained as negative control To the PCR primer of primer set.Reaction system and response procedures are as follows：
Reaction system (25 μ L)：25mM MgCl22.5 μ L, the μ L of 10 × buffer 2.5, the μ L of 25mM dNTP 1.0, primer To M (final concentration of the primer pair M every primer in reaction system is 1 μM), primer pair H6 (primer pair H6 every primer Final concentration in reaction system is 1 μM), primer pair N1 (final concentrations of the primer pair N1 every primer in reaction system Be 1 μM), primer pair G (final concentration of the primer pair G every primer in reaction system is 10 μM), DNA The μ L of Polymerase 0.5, viral cDNA or DNA that step one is obtained (in each reaction system a kind of viral cDNA or DNA or chick embryo allantoic liquid cDNA or DNA) 2 μ L, plus sterilized water is to 25 μ L.Each reaction sets 3 repetitions.
Response procedures：95 DEG C of pre-degeneration 10min；95 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 10 circulations；95 DEG C of 30s, 60 DEG C 30s, 72 DEG C of 1min, 10 circulations；95 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 20 circulations；72 DEG C of extension 10min；4 DEG C of ends Only.
2nd, Capillary Electrophoresis
According in step 22 method, " primer pair M PCR primer " is replaced with to " the PCR productions of primer set of step 1 Thing ", other steps are constant, the PCR of the primer set obtained using GenomeLab GeXP genetic analysis systems analytical procedure 1 Product.
As a result show, the purpose peak containing 194bp in the PCR primer of the primer set of H6N1 subtype avian influenza virus, 249bp purpose peak and 164bp purpose peak (Fig. 1)；H6N2 hypotypes, H6N5 hypotypes, H6N6 hypotypes and H6N8 subtype avian influenzas Purpose peak containing 194bp and 164bp purpose peak (Fig. 2) in the PCR primer of the primer set of virus；H1N1 hypotypes and H5N1 Purpose peak containing 249bp and 164bp purpose peak (Fig. 3) in the PCR primer of the primer set of subtype avian influenza virus；H2N3 Hypotype, H3N2 hypotypes, H4N5 hypotypes, H7N2 hypotypes, H8N4 hypotypes, H9N2 hypotypes, H10N3 hypotypes, H11N9 hypotypes, H12N5 are sub- 164bp purpose peak (Fig. 4) is comprised only in the PCR primer of the primer set of type and H13N5 subtype avian influenza virus；10 plants new City epidemic disease poison (F48E9 plants, Mukteswar plants, C30Strain, V4 plants, Ulster plants, Losota, GX1/00, GX2/00, GX6/02 and GX7/02), 8 plants of infectious bronchitis virus strains (Massachussetts 41, Connecticut (Conn), Arkansas (Ark), Delaware (DE/2686/92), PA, JMK, H52 and Guangxi separation strains GXIB/02) and 5 plants of infectiousness Laryngotracheitis poison strain (Beijing Strain, Taiwan vaccine strain, Unite States Standard strain, Israel's vaccine strain and Guangxi separation strains) and feminine gender Control is without any amplified production.As a result show, the identification of embodiment 1 or complete the drawing of auxiliary identification avian influenza virus (AIV) Thing can be used to identify avian influenza virus and its HA hypotypes and NA hypotypes：If have in PCR primer size be respectively 194bp, 249bp and 164bp purpose peak, it is H6N1 subtype avian influenza virus to show the virus；If in PCR primer only size be respectively 194bp and 164bp purpose peak, it is H6Ny (y ≠ 1) subtype avian influenza virus to show the virus；If only size is respectively in PCR primer 249bp and 164bp purpose peak, it is HxN1 (x ≠ 6) subtype avian influenza virus to show the virus；If only size in PCR primer For 164bp purpose peak, it is the HxNy (subtype avian influenza virus of x ≠ 6, y ≠ 1) to show the virus；If without any mesh in PCR primer Peak, show the viral non-avian influenza virus.The experimental result illustrates that the identification of embodiment 1 or auxiliary identify avian influenza virus Primer set can specifically detect H6N1 subtype avian influenza virus, H6 subtype avian influenza virus and N1 subtype avian influenza virus.
The sensitivity experiment of the primer set of embodiment 3, the identification of embodiment 1 or auxiliary identification avian influenza virus
1st, the structure of recombinant vector
CDNA using the H6N1 subtype avian influenza virus of embodiment 2 enters performing PCR as template with the primer pair H6 of embodiment 1 Amplification, obtained PCR primer is connected on pGEM-T Easy Vector (being Promega Products), restructuring is obtained and carries Body, H6-T is named as by the recombinant vector of the target sequence containing primer pair H6.
According to the method described above, will " primer pair H6 " replaces with that " primer pair N1 ", other steps are constant, obtain containing primer To the recombinant vector N1-T of N1 target sequence.
According to the method described above, will " primer pair H6 " replaces with that " primer pair M ", other steps are constant, obtain containing primer To the recombinant vector M-T of M target sequence.
2nd, primer pair H6 sensitivity
It is respectively 10 that the H6-T of step 1 is diluted into concentration gradient with sterilized water7、106、105、104、103、102With 101Copy Shellfish/μ L H6-T solution.
According in the step 2 of embodiment 21 method, by " the primer pair M " of embodiment 1 replaces with the " primer pair of embodiment 1 H6 ", and by " it is respectively 10 that the viral cDNA or DNA " that step one is obtained replace with above-mentioned concentration gradient respectively7、106、105、 104、103、102With 101Copy/μ L H6-T solution, other steps are constant, respectively obtain the 10 of H67PCR primer, H6 106The 10 of PCR primer, H65The 10 of PCR primer, H64The 10 of PCR primer, H63The 10 of PCR primer, H62PCR primer and H6's 101PCR primer.
According in the step 2 of embodiment 22 method, " primer pair M PCR primer " is replaced with above-mentioned H6's respectively 107The 10 of PCR primer, H66The 10 of PCR primer, H65The 10 of PCR primer, H64The 10 of PCR primer, H63PCR primer, H6 102The 10 of PCR primer and H61PCR primer, other steps are constant, using in the analysis of GenomeLab GeXP genetic analysis systems State obtained primer pair H6 PCR primer.
As a result show, the 10 of H67The 10 of PCR primer, H66The 10 of PCR primer, H65The 10 of PCR primer, H64PCR primer, The 10 of H63The 10 of PCR primer and H62There is 194bp purpose peak in PCR primer, show, primer pair H6 most low energy detects dense Spend for 102Copy/25 μ L H6 subtype avian influenza virus.
3rd, primer pair N1 sensitivity
It is respectively 10 that the N1-T of step 1 is diluted into concentration gradient with sterilized water7、106、105、104、103、102With 101Copy Shellfish/μ L N1-T solution.
According in the step 2 of embodiment 21 method, by " the primer pair M " of embodiment 1 replaces with the " primer pair of embodiment 1 N1 ", and by " it is respectively 10 that the viral cDNA or DNA " that step one is obtained replace with above-mentioned concentration gradient respectively7、106、105、 104、103、102With 101Copy/μ L N1-T solution, other steps are constant, respectively obtain the 10 of N17PCR primer, N1 106The 10 of PCR primer, N15The 10 of PCR primer, N14The 10 of PCR primer, N13The 10 of PCR primer, N12PCR primer and N1's 101PCR primer.
According in the step 2 of embodiment 22 method, " primer pair M PCR primer " is replaced with above-mentioned N1's respectively 107The 10 of PCR primer, N16The 10 of PCR primer, N15The 10 of PCR primer, N14The 10 of PCR primer, N13PCR primer, N1 102The 10 of PCR primer and N11PCR primer, other steps are constant, using in the analysis of GenomeLab GeXP genetic analysis systems State obtained primer pair N1 PCR primer.
As a result show, the 10 of N17The 10 of PCR primer, N16The 10 of PCR primer, N15The 10 of PCR primer, N14PCR primer, The 10 of N13The 10 of PCR primer and N12There is 249bp purpose peak in PCR primer, show, primer pair N1 most low energy detects dense Spend for 102Copy/25 μ L N1 subtype avian influenza virus.
4th, primer pair M sensitivity
It is respectively 10 that the M-T of step 1 is diluted into concentration gradient with sterilized water7、106、105、104、103、102With 101Copy Shellfish/μ L M-T solution.
According in the step 2 of embodiment 21 method, by " the viral cDNA or DNA " that step one is obtained are replaced with respectively Above-mentioned concentration gradient is respectively 107、106、105、104、103、102With 101Copy/μ L M-T solution, other steps are constant, Respectively obtain the 10 of M7The 10 of PCR primer, M6The 10 of PCR primer, M5The 10 of PCR primer, M4The 10 of PCR primer, M3PCR primer, M 102The 10 of PCR primer and M1PCR primer.
According in the step 2 of embodiment 22 method, " primer pair M PCR primer " is replaced with above-mentioned M's respectively 107The 10 of PCR primer, M6The 10 of PCR primer, M5The 10 of PCR primer, M4The 10 of PCR primer, M3The 10 of PCR primer, M2PCR is produced The 10 of thing and M1PCR primer, other steps are constant, analyze obtained above using GenomeLab GeXP genetic analysis systems Primer pair M PCR primer.
As a result show, the 10 of M7The 10 of PCR primer, M6The 10 of PCR primer, M5The 10 of PCR primer, M4PCR primer, M 103The 10 of PCR primer and M2There is 164bp purpose peak in PCR primer, show, it is 10 that primer pair M most low energy, which detects concentration,2 Copy/25 μ L avian influenza virus.
5th, the sensitivity of primer set
It is diluted after H6-T, N1-T and M-T equimolar concentration of step 1 are mixed with sterilized water, obtains H6-T, N1-T Concentration with M-T is 106Copy/μ L plasmid mixed liquor, H6-T, N1-T and M-T concentration are 105Copy/μ L plasmid Mixed liquor, H6-T, N1-T and M-T concentration are 104Copy/μ L plasmid mixed liquor, H6-T, N1-T and M-T concentration are equal For 103Copy/μ L plasmid mixed liquor, H6-T, N1-T and M-T concentration are 102Copy/μ L plasmid mixed liquor and H6-T, N1-T and M-T concentration is 101Copy/μ L plasmid mixed liquor.
According in the step 5 of embodiment 21 method, by " the viral cDNA or DNA " that step one is obtained are replaced with respectively Above-mentioned H6-T, N1-T and M-T concentration are 106Copy/μ L plasmid mixed liquor, H6-T, N1-T and M-T concentration are 105 Copy/μ L plasmid mixed liquor, H6-T, N1-T and M-T concentration are 104Copy/μ L plasmid mixed liquor, H6-T, N1-T Concentration with M-T is 103Copy/μ L plasmid mixed liquor, H6-T, N1-T and M-T concentration are 102Copy/μ L plasmid Mixed liquor and H6-T, N1-T and M-T concentration are 101Copy/μ L plasmid mixed liquor, other steps are constant, respectively To 106PCR primer, 105PCR primer, 104PCR primer, 103PCR primer, 102PCR primer and 101PCR primer.
According in the step 2 of embodiment 22 method, " primer pair M PCR primer " is replaced with above-mentioned 10 respectively6PCR is produced Thing, 105PCR primer, 104PCR primer, 103PCR primer, 102PCR primer and 101PCR primer, other steps are constant, utilize GenomeLab GeXP genetic analysis systems analyze each PCR primer obtained above.
As a result show, 106PCR primer, 105PCR primer, 104PCR primer, 103PCR primer and 102Have in PCR primer Size is respectively 194bp, 249bp and 164bp purpose peak, is shown, the identification of embodiment 1 or auxiliary identify avian influenza virus The concentration for the H6N1 subtype avian influenza virus that primer set most low energy is detected is 102Copy/25 μ L.

Claims (4)

1. identification or the primer set of auxiliary identification avian influenza virus, the PCR primer of the primer set including entitled H6 to, The PCR primer pair of entitled N1 PCR primer pair and entitled M；The H6 is as single-stranded shown in SEQ ID No.1 in sequence table Single stranded DNA composition shown in DNA and SEQ ID No.2；The N1 as the single stranded DNA shown in SEQ ID No.3 in sequence table and Single stranded DNA composition shown in SEQ ID No.4；The M is as the single stranded DNA and SEQ ID shown in SEQ ID No.5 in sequence table Single stranded DNA composition shown in No.6.
2. primer set according to claim 1, it is characterised in that：The primer set also includes entitled G primer It is right；The G is as the single stranded DNA and SEQ ID No.2 1- shown in SEQ ID No.1 1-18 nucleotides in sequence table Single stranded DNA composition shown in 19 nucleotides.
3. identification or the kit of auxiliary identification avian influenza virus, it is characterised in that：The kit contains described in claim 1 Primer set.
4. the preparation method of the primer set described in claim 1, including by each primer in the primer set described in claim 1 To two single stranded DNAs individually pack the step of.
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