CN1687454A - Method for sceening viral nucleic acid of blood through isothermal amplification based on loop mediated technique - Google Patents

Abstract

This invention belongs to clinic domain, discloses a detecting method for screening viral nucleic acid of blood through isothermal amplification based on loop mediated technique, which is used to detecting nucleic acid virus selection of blood. We detect or select the nucleic acid consisting of HBV, HCV and HV by technology of molecular domain, this technology process is that: using special primer, using LMAP tech platform to amplify target gene under assistant of a series control system and inner comparison. This invent has the virtue of simple and convenient, economical and fast, delicacy and special, so that it has a expansive application.

Description

Method for sceening viral nucleic acid of blood through based on the isothermal amplification technique that encircles mediation

Technical field

The invention belongs to clinical medicine domain, relate to a kind of blood screening method, more specifically, the present invention relates to a kind of method for sceening viral nucleic acid of blood through of developing based on isothermal amplification technique (LAMP) principle of encircling mediation.

Background technology

Existing blood screen method mainly is enzyme linked immunosorbent detection (ELISA), but because the existence of window phase usually causes omission.The window phase average out to of known HCV antibody test 70 days, the window phase average out to of HIV antibody test 22 days, HBsAg then are 56 days, and the window phase omission of the inspection-free survey of enzyme is almost inevitable, are the current bottlenecks that blood safety further improves that influences.The result of study of area, the Shanghai blood safety evaluation of just having finished recently shows, the geographic blood safety in Shanghai is near developed country's level, enzyme exempt from loss well below 100,000/, under strict quality control ensured, de novo post-transfusion hepatitis was caused by the window phase omission.Therefore must in the blood screening system, introduce new screening method, shorten the detection window phase significantly, could further improve blood safety.

The experience of American-European countries shows that (Nucleic Acid Testing NAT) is applied to blood screening and can effectively shortens HCV antibody test window phase 72% detection of nucleic acids, shortens HIV-1 antibody test window phase 50%.Japan also report uses the NAT blood screening method of HBV DNA can further reduce the omission of HBsAg.NAT introduces blood screening, can shorten the window phase that virus detects greatly, improves blood safety.In March, 1999, the TMA technology of Chiron company and the AmpliScreen technology of Roche company are applied to blood screening under the new drug review procedure of U.S. FDA; In July, 1999, the European medical product standard council (CPMP) stipulates that also raw material, pilot product and the finished product of all blood plasma products all should carry out HCV PCR and detect; In October calendar year 2001, Japan and France apply to blood screening with the NAT technology; In March, 2002 and in October, 2002, the TMA technology of U.S. FDA official approval Chiron company and the AmpliScreen technology of Roche company applied to blood screening after having tried out 2 years under the new drug review procedure.China administrative department of public health stipulates that also after 1 day January in 2003, the former slip of all blood plasma products must carry out NAT and detect before operation.The Shanghai City Blood Center is assessed TMA technology Application feasibility in the blood screening of Shanghai of Chiron company in the period of 2001～2002, proves that the NAT technology has feasibility in China's application, and can improve the blood safety of China to greatest extent.

Isothermal duplication (the Loop-mediated Isothermal Amplification of ring mediation, LAMP) technology, be inexpensive, rapid, easy, the NAT method accurately of the alternative PCR (Polymerase Chain Reaction) that develops alone of Japanese Rong Yan chemistry, worldwide applied for technical patent.Be characterized in 4 kinds of primers are set at 6 positions of target gene, utilize strand replacement reaction under constant temperature, target gene efficiently to be increased.The LAMP technology has identical sensitivity with PCR, but its technology platform is more superior than PCR.Because its reaction is that multiple primer starts jointly, makes reaction result more special than PCR; Since need not reverse transcription just direct cloning RNA, the LAMP technology is more suitable for the augmentation detection of RNA viruses, saves time than PCR; Because what carry out is isothermal duplication, make that being reflected at constant water bath box just can finish, not only save instrument cost, and will make the NAT working method simpler, be applicable to that bedside diagnosis and a large amount of sample detect simultaneously.

What is more important, the LAMP amplification reaction system requires 4 pairs of primers to mate fully just and can increase, therefore other primers in the reaction system are very little to the interference of reaction, LAMP is more suitable for carrying out the duplex or the three joint inspections survey of virus than PCR, in blood screening, use and not only save time greatly, and can also reduce the detection cost significantly.Prove that through preliminary study utilization LAMP detects HBV, HCV can reach the identical detection sensitivity of Roche Amplicor.HBV, HCV and HIV three amplification systems are also tentatively set up, and its detection sensitivity and specificity all do not have bigger change, illustrates that in three amplification systems mutual interference effect is smaller between different primers and the target sequence.Easy, economic, quick with it, the sensitive and special characteristics of LAMP technology are more suitable for using in blood screening, clinical diagnosis than round pcr, and the LAMP blood screening reagent of developing has broad application prospects.

Summary of the invention

The examination detection method that the purpose of this invention is to provide a kind of rapid, easy, economic blood disease nucleic acid.

The present invention is achieved in that a kind of viral nucleic acid detection method that is applied to blood screening, utilize the isothermal amplification technique (LAMP) of ring mediation to carry out, it is by using Auele Specific Primer, utilize the specific region of LMAP technology platform amplified target gene, assisting down of positive and negative Quality Control and internal reference detection architecture, blood disease is carried out nucleic acid screening or detection from molecular level.

Further, the present invention utilizes the particular target nucleotide sequence zone of LAMP technology amplification HBV, HCV, HIV and res-1, thereby realizes carrying out nucleic acid screening or detection for the blood disease that contains HBV, HCV, HIV.

Further, in the examination detection method of blood disease nucleic acid of the present invention, in LAMP blood screening process, introduced the internal reference detection system, the internal reference quality control product is res-1 DNA or the RNA that is different from the amplicon virus target gene, and has used the temporal resolution nephelometry to detect internal reference and viral target gene amplified production simultaneously.

Further, in the examination detection method of blood disease nucleic acid of the present invention, described Auele Specific Primer is selected from:

HBV primer: SEQ ID NO:1-SEQ ID NO:50;

HCV primer: SEQ ID NO:51-SEQ ID NO:101;

HIV primer: SEQ ID NO:102-SEQ ID NO:153;

Res-1 primer: SEQ ID NO:154-SEQ ID NO:179.

Further, in the examination detection method of blood disease nucleic acid of the present invention, HBV, HCV and HIV merging can be carried out two inspections or three inspections, that is.Only need through nucleic acid extraction, amplification and a testing process, HBV, HCV and the HIV in the test sample simultaneously just arrives secondary detection with the blood screening process boil down to one that needs to detect three times originally.

In existing open source literature, LAMP is not used for the report of blood screening about the LAMP technology.The present invention is by using Auele Specific Primer, utilize the specific region of LMAP technology platform amplified target gene, assisting down of a series of Quality Controls and internal reference detection architecture, the blood disease nucleic acid that comprises HBV, HCV and HIV is carried out examination or detection from molecular level.

The present invention has strict discriminating sieving and diagnosis system, to guarantee the accuracy of detected result.This differentiates that the sieving and diagnosis system is made up of with negative quality control product, internal reference quality control product and relevant amplification and detection architecture positive.

Whether effectively the positive and the negative quality control product of differentiating the sieving and diagnosis system among the present invention are every batch of laboratory test results important symbols.For every batch of experiment, must artificially introduce the detection of a negative quality control product, to guarantee that reaction system false positive results can not occur; Every batch of experiment for single virus detection, must artificially introduce the detection of the positive quality control product of a virus to be measured, and survey for duplex or three joint inspections that many viruses detect simultaneously, then in every batch of experiment, detect the detection that all must introduce corresponding positive quality control product, to guarantee that reaction system false negative result can not occur at every virus.Negative quality control product is through HBV, the HCV that detects repeatedly and the human normal plasma of HIV nucleic acid feminine gender.At the positive quality control product of HBV, be the artificial constructed plasmid DNA that comprises HBV specificity target sequence to be checked.At the positive quality control product of HCV and HIV, be the corresponding RNA that obtains in in-vitro transcription by the artificial constructed plasmid that comprises HCV or HIV specificity target sequence to be checked.

Concrete preparation method's operation steps of HBV DNA positive quality control product is as follows:

A. use PCR method to obtain the dna fragmentation (Gene Bank ID:AY738847.1) of one section 421bp of coding HBV surface antigen

B. this fragment is cloned in the pBluescriptII plasmid (buying is from Invitrogene company) construction recombination plasmid with 5 ' BamHI restriction enzyme site and 3 ' PstI restriction enzyme site

C. recombinant plasmid transformed is gone in the DH5 α bacterium, extract plasmid DNA after 37 ℃ of aerated culture spend the night

D. with 0D260 plasmid DNA is carried out quantitatively, serial dilution is to 100Copies/ml and packing-80 ℃ preservation, as HBV DNA positive quality control product

With HCV is example, and concrete preparation method's operation steps of RNA positive quality control product is as follows:

A. use PCR method to obtain the dna fragmentation (Gene Bank ID:U94722.1) of one section 422bp in coding HCV 5 ' UTR district

B. this fragment is cloned in pBluescriptII SK (+) plasmid (buying is from Invitrogene company) construction recombination plasmid with EcoR V restriction enzyme site

C. recombinant plasmid transformed is gone in the DH5 α bacterium, extract plasmid DNA after 37 ℃ of aerated culture spend the night

D. with OD260 plasmid DNA is carried out quantitatively

E. use T3 or T7 RNA polymerase plasmid DNA to be transcribed external

F. transcription product is crossed column purification after Dnase I digestion

G. with OD260 gained RNA is carried out quantitatively, serial dilution is to 1000Copies/ml and packing-80 ℃ preservation, as HCV RNA positive quality control product

The present invention differentiates that the internal reference quality control product is the important indicator whether each amplified reaction of examination normally carries out in the sieving and diagnosis system.Internal reference quality control product among the present invention is res-1 DNA or the RNA that is different from the amplicon virus target gene, joins in each amplified reaction with the amplification threshold concentration, can utilize reaction conditions self to increase, but not influence the amplification of viral target gene.The appearance time of its amplified production is after 60 minutes, lags behind viral target gene amplified production far away; Peak height significantly is lower than viral target gene amplified production less than 0.3.If reaction result is the product peak that occurred later at 60 minutes less than 0.3, then represent to have only in the reaction system internal reference quality control product to increase, this detection result of specimen is negative; If reaction result is the product peak that occurred before 60 minutes greater than 0.3, represent that then viral target gene to be measured increases in the reaction system, this detection result of specimen is positive; If in the reaction result, the spawn peak does not appear, show that then other reasons such as reaction system gene-amplification inhibition causes reaction to fail normally to carry out, and detects failure; If occurred later on product peak in the reaction result at 60 minutes greater than 0.3, then might cause appearance time to be delayed for the copy number of virus to be measured is extremely low, also might be for reaction has taken place due to the non-specific amplification, detection reaction need repeat once.The introducing of internal reference quality control product has reduced the false negative and the false-positive incidence of detected result, has improved the accuracy of detected result, and is extremely important in the detection of nucleic acids field of blood screening.Differentiate the principle that nephelometry detects internal reference and viral target gene amplified production simultaneously duration of service in the internal reference detection architecture of the present invention that Here it is.

According to detecting the different of dna virus and RNA viruses, the internal reference quality control product also is divided into DNA and RNA two classes, and usually, the plasmid DNA of structure directly can be used as DNA internal reference quality control product, and RNA internal reference quality control product is then by the expression type Quality Control in-vitro transcription that makes up.Only needing to add a kind of RNA internal reference quality control product in duplex or three joint inspection survey systems gets final product.With HCV is example, and concrete preparation method's operation steps of internal reference quality control product is as follows:

A. with the res-1 dna fragmentation (Gene Bank ID:L09270.1) of 2692bp, be cloned in pBluescriptII SK (+) (buying is from the Invitrogene company) plasmid construction recombination plasmid with BamH I site

B. recombinant plasmid transformed is gone in the DH5 α bacterium, extract plasmid DNA after 37 ℃ of aerated culture spend the night

C. with OD260 plasmid DNA is carried out quantitatively

D. use T3 or T7 RNA polymerase plasmid DNA to be transcribed external

E. transcription product is crossed column purification after Dnase I digestion

F. with OD260 gained RNA is carried out quantitatively, serial dilution detects the internal reference quality control product to 50Copies/ml and packing-80 ℃ preservation as HCV

Specificity amplification primer in the differential diagnosis of the present invention system designs by the contriver, prepares by the nucleic acid synthesizer.The nucleic acid synthesizer can be selected the product of different manufacturers, different model for use, also can entrust relevant genome company synthetic.Specificity amplification primer among the present invention is by TAKARA company synthetic.All primers are according to LAMP design of primers principle, by Japanese Rong Yan chemistry website (www.eiken.co.

Jp) go up the primer-design software design.The nucleic acid specificity primer sequence of HBV, HCV, HIV virus is as follows:

(1) HBV primer, totally 10 covers

Numbering	Title	Sequence	Length
				?1	??FIP	AGGACAAACGGGCAACATACCTTCTTCATCCTGCTGCTATGC (SEQ?ID?NO：1)	42
	??BIP	TCCAGGAACATCAACCACCAGCAGGTTCCTTGAGCAGGAATC (SEQ?ID?NO：2)	42
					??F3	GCTATCGCTGGATGTGTCTG(SEQ?ID?NO：3)	20
	??B3	ACAGCAACAAGAGGGAAACA(SEQ?ID?NO：4)	20
					??Loop?F	CCAGAAGAACCAACAAGAAGATGA(SEQ?ID?NO：5)	24
?2	??FIP	AGGACAAACGGGCAACATACCTTCTTCATCCTGCTGCTATGC (SEQ?ID?NO：6)	42
					??BIP	TCCAGGAACATCAACCACCAGCAGGTTCCTTGAGCAGGAATC (SEQ?ID?NO：7)	42
	??F3	GCTATCGCTGGATGTGTCTG(SEQ?ID?NO：8)	20
					??B3	ACAGCAACAAGAGGGAAACA(SEQ?ID?NO：9)	20
	??Loop?F	GGTAGTCCAGAAGAACCAACAAG(SEQ?ID?NO：10)	23
				?3	??FIP	GATAAAACGCCGCAGACACATCCCCAACCTCTTGTCCTCCAA (SEQ?ID?NO：11)	42
	??BIP	CCTGCTGCTATGCCTCATCTTCTGACAAACGGGCAACATACCTT (SEQ?ID?NO：12)	44
					??F3	CAAAATTCGCAGTCCCCAAC(SEQ?ID?NO：13)	20
	??B3	GGTGGTTGATGTTCCTGGA(SEQ?ID?NO：14)	19
					??Loop?F	CAGCGATAGCCAGGACAAA(SEQ?ID?NO：15)	19
	??Loop?B	GTTGGTTCTTCTGGACTACC(SEQ?ID?NO：16)	20
				?4	??FIP	GATAAAACGCCGCAGACACATCCCCAACCTCTTGTCCTCCAA (SEQ?ID?NO：17)	42
	??BIP	CCTGCTGCTATGCCTCATCTTCTGACAAACGGGCAACATACCTT (SEQ?ID?NO：18)	44

	??F3	?CCAAAATTCGCAGTCCCCAA(SEQ?ID?NO：19)	20
					??B3	?GCAGGTTTTGCATGGTCC(SEQ?ID?NO：20)	18
	??Loop?F	?CAGCGATAACCAGGACAA(SEQ?ID?NO：21)	18
					??Loop?B	?TGTTGGTTCTTCTGGA(SEQ?ID?NO：22)	16
?5	??FIP	?GATTGGAGGTTGGGGACTGCGAATTTTCTAGGGGGAGCACC ?(SEQ?ID?NO：23)	41
					??BIP	?TGGCTATCGCTGGATGTGTCTGATGAGGCATAGCAGCAGGAT ?(SEQ?ID?NO：24)	42
	??F3	?AGAGTCTAGACTCGTGGTGG(SEQ?ID?NO：25)	20
					??B3	?GGGCAACATACCTTGGTAGT(SEQ?ID?NO：26)	20
?6	??FIP	?TGATTGGAGGTTGGGGACTGCAATTTTCTAGGGGGAGCACC ?(SEQ?ID?NO：27)	41
					??BIP	?TGGCTATCGCTGGATGTGTCTGATGAGGCATAGCAGCAGGAT ?(SEQ?ID?NO：28)	42
	??F3	?AGAGTCTAGACTCGTGGTGG(SEQ?ID?NO：29)	20
					??B3	?GGGCAACATACCTTGGTAGT(SEQ?ID?NO：30)	20
?7	??FIP	?GTGATTGGAGGTTGGGGACTGCAATTTTCTAGGGGGAGCACC (SEQ?ID?NO：31)	42
					??BIP	?GGCTATCGCTGGATGTGTCTGCATGAGGCATAGCAGCAGGAT (SEQ?ID?NO：32)	42
	??F3	?AGAGTCTAGACTCGTGGTGG(SEQ?ID?NO：33)	20
					??B3	?GGGCAACATACCTTGGTAGT(SEQ?ID?NO：34)	20
	??Loop?F	?GAATTTTGGCCAGGACACGT(SEQ?ID?NO：35)	20
				?8	??FIP	?AGGACAAACGGGCAACATACCTTCTTCATCCTGCTGCTATGC ?(SEQ?ID?NO：36)	42
	??BIP	?TCCAGGAACATCAACCACCAGCAGGTTCCTTGAGCAGGAATC ?(SEQ?ID?NO：37)	42
					??F3	?GCTATCGCTGGATGTGTCTG(SEQ?ID?NO：38)	20
	??B3	?ACAGCAACAAGAGGGAAACA(SEQ?ID?NO：39)	20
					??Loop?F	?GTCCAGAAGAACCAACAAGAAGATG(SEQ?ID?NO：40)	25
?9	??FIP	?AGGACAAACGGGCAACATACCTTCTTCATCCTGCTGCTATGC	42

		?(SEQ?ID?NO：41)
					??BIP	?TCCAGGAACATCAACCACCAGCAGGTTCCTTGAGCAGGAATC ?(SEQ?ID?NO：42)	42
	??F3	?GCTATCGCTGGATGTGTCTG(SEQ?ID?NO：43)	20
					??B3	?ACAGCAACAAGAGGGAAACA(SEQ?ID?NO：44)	20
	??Loop?F	?GTCCAGAAGAACCAACAAGAAGA(SEQ?ID?NO：45)	23
				?10	??FIP	?AGGACAAACGGGCAACATACCTTCTTCATCCTGCTGCTATGC ?(SEQ?ID?NO：46)	42
	??BIP	?TCCAGGAACATCAACCACCAGCAGGTTCCTTGAGCAGGAATC ?(SEQ?ID?NO：47)	42
					??F3	?GCTATCGCTGGATGTGTCTG(SEQ?ID?NO：48)	20
	??B3	?ACAGCAACAAGAGGGAAACA(SEQ?ID?NO：49)	20
					??Loop?F	?GGTAGTCCAGAAGAACCAACAAGAA(SEQ?ID?NO：50)	25

(2) HCV primer, totally 10 covers

Numbering	Title	Sequence	Length
				?1	??FIP	?GGTTKATCCAAGAAAGGACCCAGTCGCCATAGTGGTCTGCGGA ?(SEQ?ID?NO：51)	43
	??BIP	?CCGCAAGACTGCTAGCCGAGGCAAGCACCCTATCAGGC ?(SEQ?ID?NO：52)	38
					??F3	?GGCGTTAGTATGAGTGTCGTAC(SEQ?ID?NO：53)	22
	??B3	?CATGGTGCACGGTCTACG(SEQ?ID?NO：54)	18
					??Loop?B	?TTGGGTTGCGAAAGG(SEQ?ID?NO：55)	15
?2	??FIP	?CACCCTATCAGGCAGTACCAAGACTGCTAGCCGAGTAG ?(SEQ?ID?NO：56)	38
					??BIP	?GGGAGGTCTCGTAGACCGTCGTTTGGTTTTTCTTTGAGG ?(SEQ?ID?NO：57)	39
	??F3	?GGCGTTAGTATGAGTGTCGTAC(SEQ?ID?NO：58)	22
					??B3	?CTCCACCAACGATCTGAC(SEQ?ID?NO：59)	18
	??Loop?F	?CAAGGCCTTTCGCG(SEQ?ID?NO：60)	14

	????Loop?B	?CATGAGCACGAATCCTAAA(SEQ?ID?NO：61)	19
				??3	????FIP	?CACCCTATCAGGCAGTACCAAGACTGCTAGCCGAGTAG ?(SEQ?ID?NO：62)	38
	????BIP	?GGGAGGTCTCGTAGACCGTCGTTTGGTTTTTCTTTGAGG ?(SEQ?ID?NO：63)	39
					????F3	?GGCGTTAGTATGAGTGTCGTAC(SEQ?ID?NO：64)	22
	????B3	?CTCCACCAACGATCTGAC(SEQ?ID?NO：65)	18
					????Loop?F	?CAAGGCCTTTCGC(SEQ?ID?NO：66)	13
	????Loop?B	?CATGAGCACGAATCCTAAA(SEQ?ID?NO：67)	19
				?4	????FIP	?TGGAGGCTGCACGACACTCAACTGTCTTCACGCAGAAAGC ?(SEQ?ID?NO：68)	40
	????BIP	?GGAACCGGTGAGTACACCGGCCCAAATCTCCAGGCATTGA ?(SEQ?ID?NO：69)	40
					????F3	?CACTCCCCTGTGAGGAACT(SEQ?ID?NO：70)	19
	????B3	?ACTCGGCTAGCAGTCTCG(SEQ?ID?NO：71)	18
					????Loop?B	?AATTGCCAGGACGACCGG(SEQ?ID?NO：72)	18
?5	????FIP	?TGGAGGCTGCACGACACTCAACTGTCTTCACGCAGAAAGC ?(SEQ?ID?NO：73)	40
					????BIP	?GGAACCGGTGAGTACACCGGCCCAAATCTCCAGGCATTGA ?(SEQ?ID?NO：74)	40
	????F3	?CACTCCCCTGTGAGGAACT(SEQ?ID?NO：75)	19
					????B3	?ACTCGGCTAGCAGTCTCG(SEQ?ID?NO：76)	18
	????Loop?B	?ACGACCGGGTCCTTTCTTGG(SEQ?ID?NO：77)	20
				?6	????FIP	?TGGAGGCTGCACGACACTCAACTGTCTTCACGCAGAAAGC ?(SEQ?ID?NO：78)	40
	????BIP	?CCTCCCGGGAGAGCCATAGTGTCGTCCTGGCAATTCCG ?(SEQ?ID?NO：79)	38
					????F3	?CACTCCCCTGTGAGGAACT(SEQ?ID?NO：80)	19
	????B3	?CGGGTTGATCCAAGAAAGGA(SEQ?ID?NO：81)	20
				?7	????FIP	?TGGAGGCTGCACGACACTCAACTGTCTTCACGCAGAAAGC ?(SEQ?ID?NO：82)	40

	??BIP	?GGAACCGGTGAGTACACCGGAGCCCAAATCTCCAGGCAT ?(SEQ?ID?NO：83)	39
					??F3	?CACTCCCCTGTGAGGAACT(SEQ?ID?NO：84)	19
	??B3	?ACTCGGCTAGCAGTCTCG(SEQ?ID?NO：85)	18
					??Loop?B	?GACGACCGGGTCCTTTCT(SEQ?ID?NO：86)	18
?8	??FIP	?ACTATGGCTCTCCCGGGAGGACGCAGAAAGCGTCTAGC ?(SEQ?ID?NO：87)	38
					??BIP	?ACCGGTGAGTACACCGGAATTGGCACGCCCAAATCTCCAG ?(SEQ?ID?NO：88)	40
	??F3	?CCCCTGTGAGGAACTACTGT(SEQ?ID?NO：89)	20
					??B3	?ACCCAACACTACTCGGCTAG(SEQ?ID?NO：90)	20
	??Loop?F	?CACGACACTCATACTAACGCC(SEQ?ID?NO：91)	21
					??Loop?B	?AGGACGACCGGGTCCTTTC(SEQ?ID?NO：92)	19
?9	??FIP	?ACTCGGCTAGCAGTCTCGCGACCGGGTCCTTTCTTGGA ?(SEQ?ID?NO：93)	38
					??BIP	?AAAGGCCTTGTGGTACTGCCTGGGATTCGTACTCATGGTGCA ?(SEQ?ID?NO：94)	42
	??F3	?GAGTACACCGGAATTGCCA(SEQ?ID?NO：95)	19
					??B3	?CGGTTGGTGTTACGTTTGGT(SEQ?ID?N0：96)	20
?10	??FIP	?GCCTTTCGCGACCCAACACTACCCTGGAGATTTGGGCGTG ?(SEQ?ID?NO：97)	40
					??BIP	?CGGGAGGTCTCGTAGACCGTGCGGTTGGTGTTACGTTTG ?(SEQ?ID?NO：98)	39
	??F3	?TGGATCAACCCGCTCAATG(SEQ?ID?NO：99)	19
					??B3	?GGAACTTGACGTCCTGTGG(SEQ?ID?NO：100)	19
	??Loop?B	?GCACCATGAGTACGAATCCTAAACC(SEQ?ID?NO：101)	25

(3) HIV primer, totally 10 covers

Numbering	Title	Sequence	Length
				1	??FIP	?CGTAACACTAGGCAAAGGTGGCTTCTCTACAATACTTGGCACTAGC	46

		?(SEQ?ID?NO：102)
					????BIP	?CCCAGAAGACCAAGGGCCACCAGCTTCATTCTTAAGCTCCTCT ?(SEQ?ID?NO：103)	43
	????F3	?AGCAGGACATAACAAGGTAGGA(SEQ?ID?NO：104)	22
					????B3	?GCCATGGAGCCAAATCCT(SEQ?ID?NO：105)	18
	????Loop?B	?GCCACACAATGAATGGACACTAGAG(SEQ?ID?NO：106)	25
				?2	????FIP	?CCCCAATCCCCCCTTTTCTTTTAATGAACATCTTAAGACAGCAGTA ?C(SEQ?ID?NO：107)	47
	????BIP	?AGTGCAGGGGAAAGAATAGTAGACATTGTCCCTGTAATAAACCCGA ?(SEQ?ID?NO：108)	46
					????F3	?CCCCAAAGTCAAGGAGTAGT(SEQ?ID?NO：109)	20
	????B3	?TTTGCTGGTCCTTTCCAA(SEQ?ID?NO：110)	18
					????Loop?F	?TGGATGAATACTGCCAT(SEQ?ID?NO：111)	17
	????Loop?B	?AACAGACATACAAACTAGAGAATTAC(SEQ?ID?NO：112)	26
				?3	????FIP	?CCCCAATCCCCCCTTTTCTTAGACAGCAGTACAAATGGCA ?(SEQ?ID?NO：113)	40
	????BIP	?AGTGCAGGGGAAAGAATAGTAGACGCTGTCCCTGTAATAAACCCGA ?A(SEQ?ID?NO：114)	47
					????F3	?GGTAAGAGATCAGGCTGAACATC(SEQ?ID?NO：115)	23
	????B3	?GCTGGTCCTTTCCAAAGTGG(SEQ?ID?NO：116)	20
					????Loop?F	?TTAAAATTGTGGATGAAT(SEQ?ID?NO：117)	18
	????Loop?B	?GCAACAGACATACAAACTAAAG(SEQ?ID?NO：118)	22
				?4	????FIP	?CCCCAATCCCCCCTTTTCTTAGACAGCAGTACAAATGGCA ?(SEQ?ID?NO：119)	40
	????BIP	?AGTGCAGGGGAAAGAATAGTAGACCTGCTGTCCCTGTAATAAACCC ?(SEQ?ID?NO：120)	46
					????F3	?GGTAAGAGATCAGGCTGAACATC(SEQ?ID?NO：121)	23
	????B3	?GCTGGTCCTTTCCAAAGTGG(SEQ?ID?NO：122)	20
					????Loop?F	?TTAAAATTGTGGATGAAT(SEQ?ID?NO：123)	18
	????Loop?B	?GCAACAGACATACAAACTAAAG(SEQ?ID?NO：124)	22
				?5	????FIP	?CGTAACACTAGGCAAAGGTGGCTTCTCTACAATACTTGGCACTAGC	46

		?(SEQ?ID?NO：125)
					????BIP	?CCAGAAGACCAAGGGCCACAACAGCTTCATTCTTAAGCTCCT ?(SEQ?ID?NO：126)	42
	????F3	?GGTGTGAATATCAAGCAGGACAT(SEQ?ID?NO：127)	23
					????B3	?GAGCCAAATCCTAGGAAAATGTC(SEQ?ID?NO：128)	23
	????Loop?B	?CCACACAATGAATGGACACTAGAG(SEQ?ID?NO：129)	24
				?6	????FIP	?CGTAACACTAGGCAAAGGTGGCTCTCTACAATACTTGGCACTAGCA ?(SEQ?ID?NO：130)	46
	????BIP	?AGATGGAACAAGCCCCAGAAGACTCTAGTGTCCATTCATTGTGTGG ?(SEQ?ID?NO：131)	46
					????F3	?AGCAGGACATAACAAGGTAGGA(SEQ?ID?NO：132)	22
	????B3	?CAGCTTCATTCTTAAGCTCCTCT(SEQ?ID?NO：133)	23
				?7	????FIP	?CGTAACACTAGGCAAAGGTGGCTCTCTACAATACTTGGCACTAGCA ?(SEQ?ID?NO：134)	46
	????BIP	?AGATGGAACAAGCCCCAGAAGACCTCCTCTAAAAGCTCTAGTGTCC ?(SEQ?ID?NO：135)	46
					????F3	?GGTGTGAATATCAAGCAGGACA(SEQ?ID?NO：136)	22
	????B3	?GAGCCAAATCCTAGGAAAATGTC(SEQ?ID?NO：137)	23
				?8	????FIP	?CGTAACACTAGGCAAAGGTGGCTTCTACAATACTTGGCACTAGCAG ?(SEQ?ID?NO：138)	46
	????BIP	?AGATGGAACAAGCCCCAGAAGACTCTAGTGTCCATTCATTGTGTGG ?(SEQ?ID?NO：139)	46
					????F3	?GCAGGACATAACAAGGTAGGATC(SEQ?ID?NO：140)	23
	????B3	?CAGCTTCATTCTTAAGCTCCTCT(SEQ?ID?NO：141)	23
				?9	????FIP	?GTCTTCTGGGGCTTGTTCCATCTAAAAGATAAAGCCACCTTTGCC ?(SEQ?ID?NO：142)	45
	????BIP	?GGGAGCCACACAATGAATGGACAGAGCCAAATCCTAGGAAAATGTC ?(SEQ?ID?NO：143)	46
					????F3	?GCACTAGCAGCATTAATAACACC(SEQ?ID?NO：144)	23
	????B3	?AGATATGTTGCCCTAAGCCATG(SEQ?ID?NO：145)	22
					????Loop?F	?ATCCTCTGTCAGTTTCGTAACACTA(SEQ?ID?NO：146)	25

	??Loop?B	?GAGGAGCTTAAGAATGAAGCTGTTA(SEQ?ID?NO：147)	25
				?10	??FIP	?GTCTTCTGGGGCTTGTTCCATCTAGATAAAGCCACCTTTGCCT ?(SEQ?ID?NO：148)	43
	??BIP	?GGGAGCCACACAATGAATGGACAGAGCCAAATCCTAGGAAAATGTC ?(SEQ?ID?NO：149)	46
					??F3	?GCACTAGCAGCATTAATAACACC(SEQ?ID?NO：150)	23
	??B3	?AGATATGTTGCCCTAAGCCATG(SEQ?ID?NO：151)	22
					??Loop?F	?TCCTCTGTCAGTTTCGTAACACT(SEQ?ID?NO：152)	23
	??Loop?B	?GAGGAGCTTAAGAATGAAGCTGTTA(SEQ?ID?NO：153)	25

(4) Res-1 primer, totally 5 covers

Numbering	Title	Sequence	Length
				?1	??FIP	?ACGAGCGGCTTCCATCTTTGATACGACTCTTTTGGCCAAACC ?(SEQ?ID?NO：154)	42
	??BIP	?CACAATCCGTTGATGTCGCTGCAAAGAGCGGTATCCCCATGA (SEQ?ID?NO：155)	42
					??F3	?CGGAGTTGCTTCAATCTGCT(SEQ?ID?NO：156)	20
	??B3	?GCGCATTTCTTTGCACCATT(SEQ?ID?NO：157)	20
					??Loop?F	?AGAAGAAGCTAATAATGCGATGTGG(SEQ?ID?NO：158)	25
?2	??FIP	?ACGAGCGGCTTCCATCTTTGATCTCTTTTGGCCAAACCGTCT ?(SEQ?ID?NO：159)	42
					??BIP	?CACAATCCGTTGATGTCGCTGCCAAAAGAGCGGTATCCCCAT ?(SEQ?ID?NO：160)	42
	??F3	?CGGAGTTGCTTCAATCTGCT(SEQ?ID?NO：161)	20
					??B3	?GCGCATTTCTTTGCACCATT(SEQ?ID?NO：162)	20
	??Loop?F	?AGAAGAAGCTAATAATGCGATGTGG(SEQ?ID?NO：163)	25
				?3	??FIP	?ACGAGCGGCTTCCATCTTTGATTTGGCCAAACCGTCTTTCA ?(SEQ?ID?NO：164)	41
	??BIP	?CACAATCCGTTGATGTCGCTGCCAAAAGAGCGGTATCCC ?(SEQ?ID?NO：165)	42

	??F3	?CGGAGTTGCTTCAATCTGCT(SEQ?ID?NO：166)	20
					??B3	?GCGCATTTCTTTGCACCATT(SEQ?ID?NO：167)	20
	??Loop?F	?AGAAGAAGCTAATAATGCGATGTGG(SEQ?ID?NO：168)	25
				?4	??FIP	?ACGAGCGGCTTCCATCTTTGATTCTTTTGGCCAAACCGTCTT ?(SEQ?ID?NO：169)	42
	??BIP	?CACAATCCGTTGATGTCGCTGCATCAAAAGAGCGGTATCCCC ?(SEQ?ID?NO：170)	42
					??F3	?CGGAGTTGCTTCAATCTGCT(SEQ?ID?NO：171)	20
	??B3	?GCGCATTTCTTTGCACCATT(SEQ?ID?NO：172)	20
					??Loop?F	?AGAAGAAGCTAATAATGCGATGTGG(SEQ?ID?NO：173)	25
?5	??FIP	?TAACGAGCGGCTTCCCCAAACCGTCTTTCACCAC ?(SEQ?ID?NO：174)	20
					??BIP	?GCAACACAATCCGTTGAAAGAGCGGTATCCCCAT ?(SEQ?ID?NO：175)	18
	??F3	?GCTATCTGCATGAACGACTC(SEQ?ID?NO：176)	34
					??B3	?GCGCATTTCTTTGCACCA(SEQ?ID?NO：177)	34
	??Loop?F	?GATTTAGAAGAAGCTAATAATGCG(SEQ?ID?NO：178)	24
					??Loop?B	?GTCGCTGCCCAAATTATAAACC(SEQ?ID?NO：179)	22

Auxiliary reagent in the differential diagnosis of the present invention system comprises:

(1) nucleic acid extraction agent: this reagent can be used for extracting simultaneously viral DNA and the RNA in the blood plasma

Solution 1

Guanidinium isothiocyanate 68%

DTT???????????????????????????????3％

TrisHCl(pH8.0)????????????????????10mM

Carrier

Amylopectin?azure?????????????????10mg/mL

Solution 2

Virahol 100%

Solution 3

Ethanol 70%

The deionization distilled water

The TE damping fluid

TirsHCl(pH8.0)????????????????10mM

EDTA·2Na?????????????????????10mM

(2) LAMP nucleic acid amplification reagent

Master?Mix.(50mM?Tris/pH8.8，25mM?KCl，25mM(NH4)2SO4，20mM?MgSO4，0.25％Tween20，2mM?Betain)

Primer Mix. (0.5～1 * Master Mix.; 1.5～2mM dNTP, the 40pmol primer)

Bst DNA polymerase

The detection method of differential diagnosis of the present invention system, undertaken by following step successively:

(1) nucleic acid extracting,

Use following method can extract HBV DNA

The Eppendorf tube that adds 300 μ l solution 1 to 1.5ml

Add 100 μ l serum or blood plasma to centrifuge tube, and vortex vibrated 5 seconds

Incubated at room (15-25 ℃) was boiled cool to room temperature 10 minutes after 10 minutes

Add 400 μ l solution 2 to centrifuge tube, and vortex vibration 5 seconds 15, centrifugal 15 minutes of 000rpm, the careful suction removed supernatant

Add 500 μ l solution 3 to centrifuge tube, and vortex vibrated 5 seconds; 15, centrifugal 5 minutes of 000rpm, the careful suction removed supernatant

Air drying 10 minutes adds 5 μ l distilled waters to centrifuge tube, complete dissolution precipitation, and 4 ℃ of preservations are stand-by.

Use following method can extract HBV DNA simultaneously, HCV RNA and HIV RNA

The Eppendorf tube that adds 300 μ l solution 1 to 1.5ml

Add 100 μ l serum or blood plasma to centrifuge tube, and vortex vibrated 5 seconds

Incubated at room (15-25 ℃) 10 minutes

Add 400 μ l solution 2 to centrifuge tube, and vortex vibrated 5 seconds; 15, centrifugal 15 minutes of 000rpm, the careful suction removed supernatant

Add 500 μ l solution 3 to centrifuge tube, and vortex vibrated 5 seconds; 15, centrifugal 5 minutes of 000rpm, the careful suction removed supernatant

Air drying 10 minutes adds 5 μ l distilled waters to centrifuge tube, complete dissolution precipitation, and 4 ℃ of preservations are stand-by

(2) LAMP reaction

1. carry out the LAMP DNA cloning according to the following step

Prepare Reaction Mix. (10 person-portion)

Master?Mix.????????????????100.0μl

Primer?Mix.????????????????30.0μl

Bst DNA Polymerase 20.0 μ l (16U/ pipe)

Mending distilled water to final volume is 200.0 μ l

With Reaction Mix. mixing, divide to install to 0.2ml eight PCR tubules, every pipe 20 μ l

Add the template that (1) step extracting goes out in the eight PCR tubules respectively, every pipe 5 μ l, each 5 μ l of positive reference substance and negative control product are used separately as positive control, negative control, mix

Eight PCR tubules are put into real-time turbidimeter LA-200, set reaction conditions: 60 ℃, and 60 minutes

Experiment finishes, and takes out eight PCR tubules, and preserves data

2. carry out LAMP RNA amplification according to the following step

A. prepare Reaction Mix. (10 person-portion)

Master?Mix.????????????????100.0μl

Primer?Mix.????????????????30.0μl

AMV ThermoScript II 12.5U

Bst DNA polymerase 160U

Mending distilled water to final volume is 200.0 μ l

B. with Reaction Mix. mixing, divide to install to eight PCR tubules, every pipe 20 μ l

C. add the template that (1) step extracting goes out in the eight PCR tubules respectively, every pipe 5 μ l, each 5 μ l of positive reference substance and negative control product are used separately as positive control, negative control, mix

D. eight PCR tubules are put into real-time turbidimeter LA-200, set reaction conditions: 60 ℃, and 60 minutes

E. experiment finishes, and takes out eight PCR tubules, and preserves data

3. carry out LAMP HBV, HCV and HIV three amplifications or HCV and the amplification of HIV bigeminy according to the following step

Prepare Reaction Mix. (10 person-portion)

Master?Mix.?????????????100.0μl

Primer?Mix.(HBV)????????30.0μl

Primer?Mix.(HCV)????????30.0μl

Primer?Mix.(HIV)????????30.0μl

AMV ThermoScript II 12.5U

Bst DNA polymerase 160U

Mending distilled water to final volume is 200.0 μ l

When carrying out three joint inspections and surveying, all Primer Mix will join reaction system, but carry out HCV and HIV bigeminy when detecting, and do not add HBV Primer Mix

With Reaction Mix. mixing, divide to install to eight PCR tubules, every pipe 20 μ l

Add the template that (1) step extracting goes out in the eight PCR tubules respectively, every pipe 5 μ l, each 5 μ l of positive reference substance and negative control product are used separately as positive control, negative control, mix

Eight PCR tubules are put into real-time turbidimeter LA-200, set reaction conditions: 60 ℃, and 60 minutes

Experiment finishes, and takes out eight PCR tubules, and preserves data

(3) result judges

Nephelometry is adopted in the detection of amplified production of the present invention.Under the effect of Mg+, LAMP amplification and the long-chain nucleic acid molecule can be cross-linked with each other into flocks, and sedimentary amount increases with the increase of amplified production.Therefore the judgement of experimental result has following two kinds of methods:

Qualitative observation: after reaction finished, experimental result can be by the direct interpretation of visual inspection (accompanying drawing 1), can be with the direct result of determination of this method when not adding the internal reference quality control product in the reaction system.

Detect in real time: can by LA-200 with and supporting LA-200 Program Ver0.18 software, obtains the delta data of turbidity in the real-time reaction solution, the row image of going forward side by side processing, result of determination (accompanying drawing 2,3).When adding the internal reference quality control product in the reaction system, can only be with this method result of determination.

Description of drawings

Fig. 1 has shown when not adding the internal reference quality control product, naked eyes direct viewing turbidity under the UV-light.

When Fig. 2 has shown the inspection of HBV list, after adding the internal reference quality control product, by the real time data that LA-200 Program Ver0.18 software provides, the result who carries out image processing.Wherein, the sample of two dotted line representatives is the HBV DNA positive, and the sample of two solid line representatives is a HBV DNA feminine gender.

Fig. 3 has shown when HBV, HCV and HIV three joint inspections are surveyed, by the real time data that LA-200 Program Ver0.18 software provides, the result who carries out image processing.Wherein, sample Sl is positive, and sample S2 is positive, and all viral nucleic acids are all negative among the sample S3, and sample S4 is suspicious result, needs duplicate detection to confirm, sample S5 needs to detect again for detecting failure.

Embodiment

The application example of embodiment 1 HBV blood screening identification system working method

HBV blood screening identification system (50 person-portion), its composition comprises:

Reagent		The Packing Unit preservation condition
			The nucleic acid extraction agent	Solution 1	4 ℃ of preservations of 300 μ l
Solution 2	4 ℃ of preservations of 400 μ l
		Solution 3		4 ℃ of preservations of 500 μ l
LAMP reagent	??Master?Mix.				5ml-20 ℃ preservation
		??Primer?Mix.	3ml-20 ℃ preservation
	The DNA polymerase			2ml-20 ℃ preservation
		Analysis software			LA-200 Program Ver0.18 software

Concrete operation method is as follows:

(1) nucleic acid extracting

Carry out the extracting of HBV nucleic acid according to the following step

A. the Eppendorf tube that adds 300 μ l solution 1 to 1.5ml

B. add 100 μ l serum or blood plasma to centrifuge tube, and vortex vibrated 5 seconds

C. incubated at room (15-25 ℃) was boiled cool to room temperature 10 minutes after 10 minutes

D. add 400 μ l solution 2 to centrifuge tube, and vortex vibrated 5 seconds; 15, centrifugal 15 minutes of 000rpm, the careful suction removed supernatant

E. add 500 μ l solution 3 to centrifuge tube, and vortex vibrated 5 seconds; 15, centrifugal 5 minutes of 000rpm, the careful suction removed supernatant

F. air drying is 10 minutes, adds 5 μ l distilled waters to centrifuge tube, complete dissolution precipitation, and 4 ℃ of preservations are stand-by

(2) LAMP reaction

1. carry out HBV LAMP nucleic acid amplification (10 person-portion) according to the following step

A. prepare Reaction Mix.

Master?Mix.????????????100.0μl

Primer?Mix.????????????30.0μl

Distilled water 50.0 μ l

Bst?DNA?Polymerase????????20.0μl

Total?????????????????????200.0μl

B. with Reaction Mix. mixing, divide to install to eight PCR tubules, every pipe 20 μ l

C. add the template that (1) step extracting goes out in the eight PCR tubules respectively, every pipe 5 μ l, each 5 μ l of positive reference substance and negative control product are used separately as positive control, negative control, mix

D. eight PCR tubules are put into real-time turbidimeter LA-200, set reaction conditions: 60 ℃, and 60 minutes

Experiment finishes, and takes out eight PCR tubules, and preserves data

The result judges:

When not adding the internal reference quality control product, experimental result can the results are shown in accompanying drawing 1 by the direct interpretation of visual inspection; After adding the internal reference quality control product, also the real time data that can provide by LA-200 Program Ver0.18 software is carried out image processing, result of determination such as accompanying drawing 2.In the accompanying drawing 2, the sample of two red curve representatives is the HBV DNA positive, and the sample of two black curve representatives is a HBV DNA feminine gender.

Embodiment 2 HBV, HCV and HIV three blood screening identification system working method

One, viral blood screening identification system (50 person-portion), its composition comprises:

Reagent		The Packing Unit preservation condition
			The nucleic acid extraction agent	Solution 1	4 ℃ of preservations of 300 μ l
Solution 2	4 ℃ of preservations of 400 μ l
		Solution 3		4 ℃ of preservations of 500 μ l
LAMP reagent	Master?Mix.				5ml-20 ℃ preservation
		Primer?Mix.	3ml-20 ℃ preservation
	The enzyme mixed solution			2ml-20 ℃ preservation
		Analysis software			LA-200 Program Ver0.18 software

Concrete operation method is as follows:

(1) nucleic acid extracting

Carry out HBV according to the following step, HCV and the extracting of HIV nucleic acid

The Eppendorf tube that adds 300 μ l solution 1 to 1.5ml

Add 100 μ l serum or blood plasma to centrifuge tube, and vortex vibrated 5 seconds

Incubated at room (15-25 ℃) 10 minutes

Add 400 μ l solution 2 to centrifuge tube, and vortex vibrated 5 seconds; 15, centrifugal 15 minutes of 000rpm, the careful suction removed supernatant

Add 500 μ l solution 3 to centrifuge tube, and vortex vibrated 5 seconds; 15, centrifugal 5 minutes of 000rpm, the careful suction removed supernatant

Air drying 10 minutes adds 5 μ l distilled waters to centrifuge tube, complete dissolution precipitation, and 4 ℃ of preservations are stand-by

(2) LAMP reaction

1. carry out HBV, HCV, HIV LAMP nucleic acid amplification (10 person-portion) according to the following step

A. prepare Reaction Mix.

Master?Mix.????????????100.0μl

Primer?Mix.????????????30.0μl

Distilled water 50.0 μ l

Enzyme cocktail buffer 20.0 μ l

Total??????????????????200.0μl

With Reaction Mix. mixing, divide to install to eight PCR tubules, every pipe 20 μ l

Add the template that (1) step extracting goes out in the eight PCR tubules respectively, every pipe 5 μ l, each 5 μ l of positive reference substance and negative control product are used separately as positive control, negative control, mix

Eight PCR tubules are put into real-time turbidimeter LA-200, set reaction conditions: 60 ℃, and 60 minutes

Experiment finishes, and takes out eight PCR tubules, and preserves data

The result judges:

Experimental result can be by the direct interpretation of visual inspection; Also the real time data that can provide by LA-200 Program Ver0.18 software is carried out image processing, and result of determination is seen accompanying drawing 3.The result: sample S1 is positive, and sample S2 is positive, and all viral nucleic acids are all negative among the sample S3, and sample S4 is suspicious result, needs duplicate detection to confirm, sample S5 needs to detect again for detecting failure.The introducing of internal reference quality control product has prevented the omission that the false negative result of similar sample S5 causes effectively.

Sequence table

＜110〉Shanghai City Blood Center

＜120〉based on the method for sceening viral nucleic acid of blood through that encircles the isothermal amplification technique that mediates

<160>179

<170>Patentln?version?3.1

<210>1

<211>42

<212>DNA

＜213〉primer

<400>1

aggacaaacg?ggcaacatac?cttcttcatc?ctgctgctat?gc??????????????????42

<210>2

<211>42

<212>DNA

＜213〉primer

<400>2

tccaggaaca?tcaaccacca?gcaggttcct?tgagcaggaa?tc??????????????????42

<210>3

<211>20

<212>DNA

＜213〉primer

<400>3

gctatcgctg?gatgtgtctg???????????????????????????????????????????20

<210>4

<211>20

<212>DNA

＜213〉primer

<400>4

acagcaacaa?gagggaaaca???????????????????????????????????????????20

<210>5

<211>24

<212>DNA

＜213〉primer

<400>5

ccagaagaac?caacaagaag?atga??????????????????????????????????????24

<210>6

<211>42

<212>DNA

＜213〉primer

<400>6

aggacaaacg?ggcaacatac?cttcttcatc?ctgctgctat?gc??????????????????42

<210>7

<211>42

<212>DNA

＜213〉primer

<400>7

tccaggaaca?tcaaccacca?gcaggttcct?tgagcaggaa?tc??????????????????42

<210>8

<211>20

<212>DNA

＜213〉primer

<400>8

gctatcgctg?gatgtgtctg???????????????????????????????????????????20

<210>9

<211>20

<212>DNA

＜213〉primer

<400>9

acagcaacaa?gagggaaaca???????????????????????????????????????????20

<210>10

<211>23

<212>DNA

＜213〉primer

<400>10

ggtagtccag?aagaaccaac?aag???????????????????????????????????????23

<210>11

<211>42

<212>DNA

＜213〉primer

<400>11

gataaaacgc?cgcagacaca?tccccaacct?cttgtcctcc?aa?????????????????42

<210>12

<211>44

<212>DNA

＜213〉primer

<400>12

cctgctgcta?tgcctcatct?tctgacaaac?gggcaacata?cctt???????????????44

<210>13

<211>20

<212>DNA

＜213〉primer

<400>13

caaaattcgc?agtccccaac???????????????????????????????????????????20

<210>14

<211>19

<212>DNA

＜213〉primer

<400>14

ggtggttgat?gttcctgga????????????????????????????????????????????19

<210>15

<211>19

<212>DNA

＜213〉primer

<400>15

cagcgatagc?caggacaaa????????????????????????????????????????????19

<210>16

<211>20

<212>DNA

＜213〉primer

<400>16

gttggttctt?ctggactacc???????????????????????????????????????????20

<210>17

<211>42

<212>DNA

＜213〉primer

<400>17

gataaaacgc?cgcagacaca?tccccaacct?cttgtcctcc?aa??????????????????42

<210>18

<211>44

<212>DNA

＜213〉primer

<400>18

cctgctgcta?tgcctcatct?tctgacaaac?gggcaacata?cctt????????????????44

<210>19

<211>20

<212>DNA

＜213〉primer

<400>19

ccaaaattcg?cagtccccaa???????????????????????????????????????????20

<210>20

<211>18

<212>DNA

＜213〉primer

<400>20

gcaggttttg?catggtcc?????????????????????????????????????????????18

<210>21

<211>18

<212>DNA

＜213〉primer

<400>21

cagcgataac?caggacaa?????????????????????????????????????????????18

<210>22

<211>16

<212>DNA

＜213〉primer

<400>22

tgttggttct?tctgga???????????????????????????????????????????????16

<210>23

<211>41

<212>DNA

＜213〉primer

<400>23

gattggaggt?tggggactgc?gaattttcta?gggggagcac?c???????????????????41

<210>24

<211>42

<212>DNA

＜213〉primer

<400>24

tggctatcgc?tggatgtgtc?tgatgaggca?tagcagcagg?at??????????????????42

<210>25

<211>20

<212>DNA

＜213〉primer

<400>25

agagtctaga?ctcgtggtgg???????????????????????????????????????????20

<210>26

<211>20

<212>DNA

＜213〉primer

<400>26

gggcaacata?ccttggtagt???????????????????????????????????????????20

<210>27

<211>41

<212>DNA

＜213〉primer

<400>27

tgattggagg?ttggggactg?caattttcta?gggggagcac?c???????????????????41

<210>28

<211>42

<212>DNA

＜213〉primer

<400>28

tggctatcgc?tggatgtgtc?tgatgaggca?tagcagcagg?at??????????????????42

<210>29

<211>20

<212>DNA

＜213〉primer

<400>29

agagtctaga?ctcgtggtgg???????????????????????????????????????????20

<210>30

<211>20

<212>DNA

＜213〉primer

<400>30

gggcaacata?ccttggtagt???????????????????????????????????????????20

<210>31

<211>42

<212>DNA

＜213〉primer

<400>31

gtgattggag?gttggggact?gcaattttct?agggggagca?cc??????????????????42

<210>32

<211>42

<212>DNA

＜213〉primer

<400>32

ggctatcgct?ggatgtgtct?gcatgaggca?tagcagcagg?at??????????????????42

<210>33

<211>20

<212>DNA

＜213〉primer

<400>33

agagtctaga?ctcgtggtgg???????????????????????????????????????????20

<210>34

<211>20

<212>DNA

＜213〉primer

<400>34

gggcaacata?ccttggtagt???????????????????????????????????????????20

<210>35

<211>20

<212>DNA

＜213〉primer

<400>35

gaattttggc?caggacacgt???????????????????????????????????????????20

<210>36

<211>42

<212>DNA

＜213〉primer

<400>36

aggacaaacg?ggcaacatac?cttcttcatc?ctgctgctat?gc??????????????????42

<210>37

<211>42

<212>DNA

＜213〉primer

<400>37

tccaggaaca?tcaaccacca?gcaggttcct?tgagcaggaa?tc??????????????????42

<210>38

<211>20

<212>DNA

＜213〉primer

<400>38

gctatcgctg?gatgtgtctg???????????????????????????????????????????20

<210>39

<211>20

<212>DNA

＜213〉primer

<400>39

acagcaacaa?gagggaaaca???????????????????????????????????????????20

<210>40

<211>25

<212>DNA

＜213〉primer

<400>40

gtccagaaga?accaacaaga?agatg?????????????????????????????????????25

<210>41

<211>42

<212>DNA

＜213〉primer

<400>41

aggacaaacg?ggcaacatac?cttcttcatc?ctgctgctat?gc??????????????????42

<210>42

<211>42

<212>DNA

＜213〉primer

<400>42

tccaggaaca?tcaaccacca?gcaggttcct?tgagcaggaa?tc??????????????????42

<210>43

<211>20

<212>DNA

＜213〉primer

<400>43

gctatcgctg?gatgtgtctg???????????????????????????????????????????20

<210>44

<211>20

<212>DNA

＜213〉primer

<400>44

acagcaacaa?gagggaaaca???????????????????????????????????????????20

<210>45

<211>23

<212>DNA

＜213〉primer

<400>45

gtccagaaga?accaacaaga?aga???????????????????????????????????????23

<210>46

<211>42

<212>DNA

＜213〉primer

<400>46

aggacaaacg?ggcaacatac?cttcttcatc?ctgctgctat?gc??????????????????42

<210>47

<211>42

<212>DNA

＜213〉primer

<400>47

tccaggaaca?tcaaccacca?gcaggttcct?tgagcaggaa?tc??????????????????42

<210>48

<211>20

<212>DNA

＜213〉primer

<400>48

gctatcgctg?gatgtgtctg???????????????????????????????????????????20

<210>49

<211>20

<212>DNA

＜213〉primer

<400>49

acagcaacaa?gagggaaaca???????????????????????????????????????????20

<210>50

<211>25

<212>DNA

＜213〉primer

<400>50

ggtagtccag?aagaaccaac?aagaa?????????????????????????????????????25

<210>51

<211>43

<212>DNA

＜213〉primer

<400>51

ggttkatcca?agaaaggacc?cagtcgccat?agtggtctgc?gga?????????????????43

<210>52

<211>38

<212>DNA

＜213〉primer

<400>52

ccgcaagact?gctagccgag?gcaagcaccc?tatcaggc???????????????????????38

<210>53

<211>22

<212>DNA

＜213〉primer

<400>53

ggcgttagta?tgagtgtcgt?ac????????????????????????????????????????22

<210>54

<211>18

<212>DNA

＜213〉primer

<400>54

catggtgcac?ggtctacg?????????????????????????????????????????????18

<210>55

<211>15

<212>DNA

＜213〉primer

<400>55

ttgggttgcg?aaagg????????????????????????????????????????????????15

<210>56

<211>38

<212>DNA

＜213〉primer

<400>56

caccctatca?ggcagtacca?agactgctag?ccgagtag???????????????????????38

<210>57

<211>39

<212>DNA

＜213〉primer

<400>57

gggaggtctc?gtagaccgtc?gtttggtttt?tctttgagg??????????????????????39

<210>58

<211>22

<212>DNA

＜213〉primer

<400>58

ggcgttagta?tgagtgtcgt?ac????????????????????????????????????????22

<210>59

<211>18

<212>DNA

＜213〉primer

<400>59

ctccaccaac?gatctgac?????????????????????????????????????????????18

<210>60

<211>14

<212>DNA

＜213〉primer

<400>60

caaggccttt?cgcg?????????????????????????????????????????????????14

<210>61

<211>19

<212>DNA

＜213〉primer

<400>61

catgagcacg?aatcctaaa????????????????????????????????????????????19

<210>62

<211>38

<212>DNA

＜213〉primer

<400>62

caccctatca?ggcagtacca?agactgctag?ccgagtag???????????????????????38

<210>63

<211>39

<212>DNA

＜213〉primer

<400>63

gggaggtctc?gtagaccgtc?gtttggtttt?tctttgagg??????????????????????39

<210>64

<211>22

<212>DNA

＜213〉primer

<400>64

ggcgttagta?tgagtgtcgt?ac????????????????????????????????????????22

<210>65

<211>18

<212>DNA

＜213〉primer

<400>65

ctccaccaac?gatctgac?????????????????????????????????????????????18

<210>66

<211>13

<212>DNA

＜213〉primer

<400>66

caaggccttt?cgc??????????????????????????????????????????????????13

<210>67

<211>19

<212>DNA

＜213〉primer

<400>67

catgagcacg?aatcctaaa????????????????????????????????????????????19

<210>68

<211>40

<212>DNA

＜213〉primer

<400>68

tggaggctgc?acgacactca?actgtcttca?cgcagaaagc?????????????????????40

<210>69

<211>40

<212>DNA

＜213〉primer

<400>69

ggaaccggtg?agtacaccgg?cccaaatctc?caggcattga?????????????????????40

<210>70

<211>19

<212>DNA

＜213〉primer

<400>70

cactcccctg?tgaggaact????????????????????????????????????????????19

<210>71

<211>18

<212>DNA

＜213〉primer

<400>71

actcggctag?cagtctcg?????????????????????????????????????????????18

<210>72

<211>18

<212>DNA

＜213〉primer

<400>72

aattgccagg?acgaccgg?????????????????????????????????????????????18

<210>73

<211>40

<212>DNA

＜213〉primer

<400>73

tggaggctgc?acgacactca?actgtcttca?cgcagaaagc?????????????????????40

<210>74

<211>40

<212>DNA

＜213〉primer

<400>74

ggaaccggtg?agtacaccgg?cccaaatctc?caggcattga?????????????????????40

<210>75

<211>19

<212>DNA

＜213〉primer

<400>75

cactcccctg?tgaggaact????????????????????????????????????????????19

<210>76

<211>18

<212>DNA

＜213〉primer

<400>76

actcggctag?cagtctcg?????????????????????????????????????????????18

<210>77

<211>20

<212>DNA

＜213〉primer

<400>77

acgaccgggt?cctttcttgg???????????????????????????????????????????20

<210>78

<211>40

<212>DNA

＜213〉primer

<400>78

tggaggctgc?acgacactca?actgtcttca?cgcagaaagc?????????????????????40

<210>79

<211>38

<212>DNA

＜213〉primer

<400>79

cctcccggga?gagccatagt?gtcgtcctgg?caattccg???????????????????????38

<210>80

<211>19

<212>DNA

＜213〉primer

<400>80

cactcccctg?tgaggaact????????????????????????????????????????????19

<210>81

<211>20

<212>DNA

＜213〉primer

<400>81

cgggttgatc?caagaaagga???????????????????????????????????????????20

<210>82

<211>40

<212>DNA

＜213〉primer

<400>82

tggaggctgc?acgacactca?actgtcttca?cgcagaaagc?????????????????????40

<210>83

<211>39

<212>DNA

＜213〉primer

<400>83

ggaaccggtg?agtacaccgg?agcccaaatc?tccaggcat??????????????????????39

<210>84

<211>19

<212>DNA

＜213〉primer

<400>84

cactcccctg?tgaggaact????????????????????????????????????????????19

<210>85

<211>18

<212>DNA

＜213〉primer

<400>85

actcggctag?cagtctcg?????????????????????????????????????????????18

<210>86

<211>18

<212>DNA

＜213〉primer

<400>86

gacgaccggg?tcctttct?????????????????????????????????????????????18

<210>87

<211>38

<212>DNA

＜213〉primer

<400>87

actatggctc?tcccgggagg?acgcagaaag?cgtctagc???????????????????????38

<210>88

<211>40

<212>DNA

＜213〉primer

<400>88

accggtgagt?acaccggaat?tggcacgccc?aaatctccag?????????????????????40

<210>89

<211>20

<212>DNA

＜213〉primer

<400>89

cccctgtgag?gaactactgt???????????????????????????????????????????20

<210>90

<211>20

<212>DNA

＜213〉primer

<400>90

acccaacact?actcggctag???????????????????????????????????????????20

<210>91

<211>21

<212>DNA

＜213〉primer

<400>91

cacgacactc?atactaacgc?c?????????????????????????????????????????21

<210>92

<211>19

<212>DNA

＜213〉primer

<400>92

aggacgaccg?ggtcctttc????????????????????????????????????????????19

<210>93

<211>38

<212>DNA

＜213〉primer

<400>93

actcggctag?cagtctcgcg?accgggtcct?ttcttgga???????????????????????38

<210>94

<211>42

<212>DNA

＜213〉primer

<400>94

aaaggccttg?tggtactgcc?tgggattcgt?actcatggtg?ca??????????????????42

<210>95

<211>19

<212>DNA

＜213〉primer

<400>95

gagtacaccg?gaattgcca????????????????????????????????????????????19

<210>96

<211>20

<212>DNA

＜213〉primer

<400>96

cggttggtgt?tacgtttggt???????????????????????????????????????????20

<210>97

<211>40

<212>DNA

＜213〉primer

<400>97

gcctttcgcg?acccaacact?accctggaga?tttgggcgtg?????????????????????40

<210>98

<211>39

<212>DNA

＜213〉primer

<400>98

cgggaggtct?cgtagaccgt?gcggttggtg?ttacgtttg??????????????????????39

<210>99

<211>19

<212>DNA

＜213〉primer

<400>99

tggatcaacc?cgctcaatg????????????????????????????????????????????19

<210>100

<211>19

<212>DNA

＜213〉primer

<400>100

ggaacttgac?gtcctgtgg????????????????????????????????????????????19

<210>101

<211>25

<212>DNA

＜213〉primer

<400>101

gcaccatgag?tacgaatcct?aaacc?????????????????????????????????????25

<210>102

<211>46

<212>DNA

＜213〉primer

<400>102

cgtaacacta?ggcaaaggtg?gcttctctac?aatacttggc?actagc??????????????46

<210>103

<211>43

<212>DNA

＜213〉primer

<400>103

cccagaagac?caagggccac?cagcttcatt?cttaagctcc?tct?????????????????43

<210>104

<211>22

<212>DNA

＜213〉primer

<400>104

agcaggacat?aacaaggtag?ga????????????????????????????????????????22

<210>105

<211>18

<212>DNA

＜213〉primer

<400>105

gccatggagc?caaatcct?????????????????????????????????????????????18

<210>106

<211>25

<212>DNA

＜213〉primer

<400>106

gccacacaat?gaatggacac?tagag?????????????????????????????????????25

<210>107

<211>47

<212>DNA

＜213〉primer

<400>107

ccccaatccc?cccttttctt?ttaatgaaca?tcttaagaca?gcagtac?????????????47

<210>108

<211>46

<212>DNA

＜213〉primer

<400>108

agtgcagggg?aaagaatagt?agacattgtc?cctgtaataa?acccga??????????????46

<210>109

<211>20

<212>DNA

＜213〉primer

<400>109

ccccaaagtc?aaggagtagt???????????????????????????????????????????20

<210>110

<211>18

<212>DNA

＜213〉primer

<400>110

tttgctggtc?ctttccaa?????????????????????????????????????????????18

<210>111

<211>17

<212>DNA

＜213〉primer

<400>111

tggatgaata?ctgccat??????????????????????????????????????????????17

<210>112

<211>26

<212>DNA

＜213〉primer

<400>112

aacagacata?caaactagag?aat?tac???????????????????????????????????26

<210>113

<211>40

<212>DNA

＜213〉primer

<400>113

ccccaatccc?cccttttctt?agacagcagt?acaaatggca?????????????????????40

<210>114

<211>47

<212>DNA

＜213〉primer

<400>114

agtgcagggg?aaagaatagt?agacgctgtc?cctgtaataa?acccgaa?????????????47

<210>115

<211>23

<212>DNA

＜213〉primer

<400>115

ggtaagagat?caggctgaac?atc??????????????????????????????????????23

<210>116

<211>20

<212>DNA

＜213〉primer

<400>116

gctggtcctt?tccaaagtgg???????????????????????????????????????????20

<210>117

<211>18

<212>DNA

＜213〉primer

<400>117

ttaaaattgt?ggatgaat?????????????????????????????????????????????18

<210>118

<211>22

<212>DNA

＜213〉primer

<400>118

gcaacagaca?tacaaactaa?ag????????????????????????????????????????22

<210>119

<211>40

<212>DNA

＜213〉primer

<400>119

ccccaatccc?cccttttctt?agacagcagt?acaaatggca?????????????????????40

<210>120

<211>46

<212>DNA

＜213〉primer

<400>120

agtgcagggg?aaagaatagt?agacctgctg?tccctgtaat?aaaccc??????????????46

<210>121

<211>23

<212>DNA

＜213〉primer

<400>121

ggtaagagat?caggctgaac?atc???????????????????????????????????????23

<210>122

<211>20

<212>DNA

＜213〉primer

<400>122

gctggtcctt?tccaaagtgg???????????????????????????????????????????20

<210>123

<211>18

<212>DNA

＜213〉primer

<400>123

ttaaaattgt?ggatgaat?????????????????????????????????????????????18

<210>124

<211>22

<212>DNA

＜213〉primer

<400>124

gcaacagaca?tacaaactaa?ag????????????????????????????????????????22

<210>125

<211>46

<212>DNA

＜213〉primer

<400>125

cgtaacacta?ggcaaaggtg?gcttctctac?aatacttggc?actagc??????????????46

<210>126

<211>42

<212>DNA

＜213〉primer

<400>126

ccagaagacc?aagggccaca?acagcttcat?tcttaagctc?ct??????????????????42

<210>127

<211>23

<212>DNA

＜213〉primer

<400>127

ggtgtgaata?tcaagcagga?cat???????????????????????????????????????23

<210>128

<211>23

<212>DNA

＜213〉primer

<400>128

gagccaaatc?ctaggaaaat?gtc???????????????????????????????????????23

<210>129

<211>24

<212>DNA

＜213〉primer

<400>129

ccacacaatg?aatggacact?agag??????????????????????????????????????24

<210>130

<211>46

<212>DNA

＜213〉primer

<400>130

cgtaacacta?ggcaaaggtg?gctctctaca?atacttggca?ctagca??????????????46

<210>131

<211>46

<212>DNA

＜213〉primer

<400>131

agatggaaca?agccccagaa?gactctagtg?tccattcatt?gtgtgg??????????????46

<210>132

<211>22

<212>DNA

＜213〉primer

<400>132

agcaggacat?aacaaggtag?ga????????????????????????????????????????22

<210>133

<211>23

<212>DNA

＜213〉primer

<400>133

cagcttcatt?cttaagctcc?tct???????????????????????????????????????23

<210>134

<211>46

<212>DNA

＜213〉primer

<400>134

cgtaacacta?ggcaaaggtg?gctctctaca?atacttggca?ctagca??????????????46

<210>135

<211>46

<212>DNA

＜213〉primer

<400>135

agatggaaca?agccccagaa?gacctcctct?aaaagctcta?gtgtcc??????????????46

<210>136

<211>22

<212>DNA

＜213〉primer

<400>136

ggtgtgaata?tcaagcagga?ca????????????????????????????????????????22

<210>137

<211>23

<212>DNA

＜213〉primer

<400>137

gagccaaatc?ctaggaaaat?gtc???????????????????????????????????????23

<210>138

<211>46

<212>DNA

＜213〉primer

<400>138

cgtaacacta?ggcaaaggtg?gcttctacaa?tacttggcac?tagcag??????????????46

<210>139

<211>46

<212>DNA

＜213〉primer

<400>139

agatggaaca?agccccagaa?gactctagtg?tccattcatt?gtgtgg??????????????46

<210>140

<211>23

<212>DNA

＜213〉primer

<400>140

gcaggacata?acaaggtagg?atc???????????????????????????????????????23

<210>141

<211>23

<212>DNA

＜213〉primer

<400>141

cagcttcatt?cttaagctcc?tct???????????????????????????????????????23

<210>142

<211>45

<212>DNA

＜213〉primer

<400>142

gtcttctggg?gcttgttcca?tctaaaagat?aaagccacct?ttgcc???????????????45

<210>143

<211>46

<212>DNA

＜213〉primer

<400>143

gggagccaca?caatgaatgg?acagagccaa?atcctaggaa?aatgtc??????????????46

<210>144

<211>23

<212>DNA

＜213〉primer

<400>144

gcactagcag?cat?taataac?acc??????????????????????????????????????23

<210>145

<211>22

<212>DNA

＜213〉primer

<400>145

agatatgttg?ccctaagcca?tg????????????????????????????????????????22

<210>146

<211>25

<212>DNA

＜213〉primer

<400>146

atcctctgtc?agtttcgtaa?cacta?????????????????????????????????????25

<210>147

<211>25

<212>DNA

＜213〉primer

<400>147

gaggagctta?agaatgaagc?tgtta?????????????????????????????????????25

<210>148

<211>43

<212>DNA

＜213〉primer

<400>148

gtcttctggg?gcttgttcca?tctagataaa?gccacctttg?cct?????????????????43

<210>149

<211>46

<212>DNA

＜213〉primer

<400>149

gggagccaca?caatgaatgg?acagagccaa?atcctaggaa?aatgtc??????????????46

<210>150

<211>23

<212>DNA

＜213〉primer

<400>150

gcactagcag?cattaataac?acc???????????????????????????????????????23

<210>151

<211>22

<212>DNA

＜213〉primer

<400>151

agatatgttg?ccctaagcca?tg????????????????????????????????????????22

<210>152

<211>23

<212>DNA

＜213〉primer

<400>152

tcctctgtca?gtttcgtaac?act???????????????????????????????????????23

<210>153

<211>25

<212>DNA

＜213〉primer

<400>153

gaggagctta?agaatgaagc?tgtta?????????????????????????????????????25

<210>154

<211>42

<212>DNA

＜213〉primer

<400>154

acgagcggct?tccatctttg?atacgactct?tttggccaaa?cc??????????????????42

<210>155

<211>42

<212>DNA

＜213〉primer

<400>155

cacaatccgt?tgatgtcgct?gcaaagagcg?gtatccccat?ga??????????????????42

<210>156

<211>20

<212>DNA

＜213〉primer

<400>156

cggagttgct?tcaatctgct???????????????????????????????????????????20

<210>157

<211>20

<212>DNA

＜213〉primer

<400>157

gcgcatttct?ttgcaccatt???????????????????????????????????????????20

<210>158

<211>25

<212>DNA

＜213〉primer

<400>158

agaagaagct?aataatgcga?tgtgg?????????????????????????????????????25

<210>159

<211>42

<212>DNA

＜213〉primer

<400>159

acgagcggct?tccatctttg?atctcttttg?gccaaaccgt?ct??????????????????42

<210>160

<211>42

<212>DNA

＜213〉primer

<400>160

cacaatccgt?tgatgtcgct?gccaaaagag?cggtatcccc?at??????????????????42

<210>161

<211>20

<212>DNA

＜213〉primer

<400>161

cggagttgct?tcaatctgct???????????????????????????????????????????20

<210>162

<211>20

<212>DNA

＜213〉primer

<400>162

gcgcatttct?ttgcaccatt???????????????????????????????????????????20

<210>163

<211>25

<212>DNA

＜213〉primer

<400>163

agaagaagct?aataatgcga?tgtgg?????????????????????????????????????25

<210>164

<211>41

<212>DNA

＜213〉primer

<400>164

acgagcggct?tccatctttg?atttggccaa?accgtctttc?a???????????????????41

<210>165

<211>39

<212>DNA

＜213〉primer

<400>165

cacaatccgt?tgatgtcgct?gccaaaagag?cggtatccc??????????????????????39

<210>166

<211>20

<212>DNA

＜213〉primer

<400>166

cggagttgct?tcaatctgct???????????????????????????????????????????20

<210>167

<211>20

<212>DNA

＜213〉primer

<400>167

gcgcatttct?ttgcaccatt???????????????????????????????????????????20

<210>168

<211>25

<212>DNA

＜213〉primer

<400>168

agaagaagct?aataatgcga?tgtgg?????????????????????????????????????25

<210>169

<211>42

<212>DNA

＜213〉primer

<400>169

acgagcggct?tccatctttg?attcttttgg?ccaaaccgtc?tt??????????????????42

<210>170

<211>42

<212>DNA

＜213〉primer

<400>170

cacaatccgt?tgatgtcgct?gcatcaaaag?agcggtatcc?cc??????????????????42

<210>171

<211>20

<212>DNA

＜213〉primer

<400>171

cggagttgct?tcaatctgct???????????????????????????????????????????20

<210>172

<211>20

<212>DNA

＜213〉primer

<400>172

gcgcatttct?ttgcaccatt???????????????????????????????????????????20

<210>173

<211>25

<212>DNA

＜213〉primer

<400>173

agaagaagct?aataatgcga?tgtgg?????????????????????????????????????25

<210>174

<211>34

<212>DNA

＜213〉primer

<400>174

taacgagcgg?cttccccaaa?ccgtctttca?ccac???????????????????????????34

<210>175

<211>34

<212>DNA

＜213〉primer

<400>175

gcaacacaat?ccgttgaaag?agcggtatcc?ccat???????????????????????????34

<210>176

<211>20

<212>DNA

＜213〉primer

<400>176

gctatctgca?tgaacgactc???????????????????????????????????????????20

<210>177

<211>18

<212>DNA

＜213〉primer

<400>177

gcgcatttct?ttgcacca?????????????????????????????????????????????18

<210>178

<211>24

<212>DNA

＜213〉primer

<400>178

gatttagaag?aagctaataa?tgcg??????????????????????????????????????24

<210>179

<211>22

<212>DNA

＜213〉primer

<400>179

gtcgctgccc?aaattataaa?cc????????????????????????????????????????22

Claims (5)

1. viral nucleic acid detection method that is applied to blood screening, utilize the isothermal amplification technique LAMP of ring mediation to carry out, it is characterized in that, by using Auele Specific Primer, utilize the specific region of LMAP technology platform amplified target gene, assisting down of positive and negative Quality Control and internal reference detection architecture, blood disease is carried out nucleic acid screening or detection from molecular level.

2. the viral nucleic acid detection method that is used for blood screening according to claim 1 is characterized in that utilizing the particular target nucleotide sequence zone of LAMP technology amplification HBV, HCV, HIV and res-1.

3. the viral nucleic acid detection method that is used for blood screening according to claim 2, it is characterized in that in the described internal reference detection architecture, the internal reference quality control product is res-1 DNA or the RNA that is different from the amplicon virus target gene, and differentiates nephelometry duration of service and detect internal reference and viral target gene amplified production simultaneously.

4. according to claim 1,2,3 arbitrary described viral nucleic acid detection methods that are used for blood screening, it is characterized in that described Auele Specific Primer is selected from:

HBV primer: SEQ ID NO:1-SEQ ID NO:50;

HCV primer: SEQ ID NO:51-SEQ ID NO:101;

HIV primer: SEQ ID NO:102-SEQ ID NO:153;

Res-1 primer: SEQ ID NO:154-SEQ ID NO:179.

5. the viral nucleic acid detection method that is used for blood screening according to claim 4 is characterised in that HBV, HCV and duplex is carried out in the HIV merging or three joint inspections are surveyed.

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