CN1450173A

CN1450173A - SARS related coronal virus total genom chip and use thereof

Info

Publication number: CN1450173A
Application number: CN03122953A
Authority: CN
Inventors: 吴小岳; 马鑫; 任鲁风; 董小岩
Original assignee: AGTC Gene Technology Co Ltd
Current assignee: AGTC Gene Technology Co Ltd
Priority date: 2003-04-25
Filing date: 2003-04-25
Publication date: 2003-10-22

Abstract

The present invention relates to a total genom gene chip for diagnosing coronary virus related to SARS disease, and provides a new primer for obtaining high detection rate. Said total genom gene chip includes detection monitoring system (A) and disease diagnosis system, and can accurately and quickly detect the coronary virus related to SARS, and can shorten the diagnosis time. Said invention is special applicable to living body detection of quarantine office, hospital clinical sample, biological environment virus pollution monitoring, controlling propagation of the disease after patient discharge, and establishment of said virus gene bank and tracking animal source of said SARS coronary virus, and has the important action.

Description

Coronavirus full genome chip relevant and uses thereof with SARS

Technical field the invention belongs to biological technical field, is specifically related to the full genome chip of the coronavirus relevant with SARS and construction strategy, method and purposes.

Background technology virus detection gene chip background.Gene chip, briefly be exactly on the sheet glass or other solid support (as silicon chip, polypropylene screen, nitrocellulose filter, nylon membrane etc.) of a special processing, gene probe is arranged with the form of large scale array, formation can with the interactional solid phase surface of molecules of interest (as viral genome).Probe array behind the molecular hybridization presents different fluorescence emission spectrums because of the difference of binding purposes molecule on the probe respectively under the order of laser excites levies, and according to its wavelength and wave amplitude feature collection signal, analyzes the desired information that obtains thereby make comparisons.

Gene chip application now mainly contains following aspect: the genetic expression space-time characteristic is analyzed; Gene differential expression detects; The discovery of new gene; Extensive dna sequencing; Genotype, transgenation and polymorphism analysis; The location of inherited disease genes involved; The detection of tumor-related gene; The diagnosis of single or a small amount of infectious diseases and drug research etc.

The virus detection chip has following characteristics:

1, high-throughput: can that is to say and to carry out up to ten thousand molecular hybridization reactions simultaneously abreast in conjunction with 5000～10000 nucleic acid probes on every square centimeter.The known thousands of kinds of viruses of gene order only just can be held fully by a chip now.

2, pinpoint accuracy, sensitivity: the existence that detects virus by molecular hybridization is that accuracy and specificity are the highest in the technology of current application, shows in it is reported that the applying gene chip technology carries out clinical diagnosis, and accuracy can reach 98%-100%.

3, associativity: the gene chip product of developing now that can detect virus all can only unicity detects a kind of or a small amount of several viruses, and is like this then can not bring into play the technical superiority of gene chip maximum.Our viral combine detection chip will be almost all the test set of known viruses be combined on the chip, farthest utilize the potentiality of chip technology, improve and reduce cost ageing the time.The combine detection of virus then has epoch making significance for aspects such as environmental monitoring and quality surveillances.

4, short, low-cost, low workload of cycle: by ordinary method virus being carried out Molecular Detection needs week age usually at least, and then needs identical work to repeat for multiple virus.Virus combine detection chip can be in 4～5 hours automation process all viruses of disposable detection, consumption, intensity and experimentation cost when reducing work greatly.

The background technology of sars coronavirus background context and the full genome chip of sars coronavirus:

From in November, 2002 till now, worldwide acute respiratory distress syndrome (severe acute respiratorysyndrome, SARS) outburst is proved to be relevant with a kind of coronavirus of new form variation with popular, in the world this virus is called SARS associated coronavirus (SARS related virus) or SARS virus (SARS virus) at present, we are referred to as " coronavirus relevant with SARS " in the present invention.In China this acute respiratory distress syndrome also is called severe acute respiratory syndrome.March 23, the sample that the U.S. disease prevention and control center of Atlanta mails UCSF Dr.Deresi professor to passes through it to comprising the chip testing of 1000 kinds of viruses, confirms that first causing this extensive popular cause of disease is a kind of coronavirus.Continue it, Canadian BCCA genome scientific center April 13 issued first sars coronavirus (the Genbank sequence number: AY274119) complete sequence, April 15 Genbank in addition note issue (Genbank sequence number: NC_004718) once more.Inoculation is this from the isolating on one's body sars coronavirus of clinical definite patient separately to give monkey on April 16, finds that the symptom that causes monkey is extremely similar to patient SARS on one's body.The SARS multicenter cooperating research member of WHO is basic on the multinational cooperating research of WHO basis to determine that a kind of novel coronavirus is the cause of disease of this acute respiratory symptom SARS, and other suspicious cause of disease such as chlamydozoan still needs further assessment.

The coronavirus of the pathogenic factor of being reported of thinking SARS at present is the very large coronavirus of a kind of degree of variation.It belongs to coronaviridae (from Latin corona, resemble imperial crown), it is a kind of RNA viruses, virosome contains strand normal chain linear rna molecule, infect vertebrates, especially warm-blooded animal comprises some mammalss (such as people, ox, cat, pig and mouse), and several bird (such as turkey and chicken).This coe virus genome is made up of RNA, has 29,000 to 31,000 bases.

The rarest 6 of human SARS virus member is infected in Hong Kong University's latest find.This discovery can be invalid, the state of an illness mystery repeatedly of part patient medicine recently, and the clue that cracks is provided.Yet from the health and epidemic prevention angle, the SARS virus numbers is various, breaks up one by one in a short time, very difficulty.The big medical college in port department of microbiology is made comparisons from SARS Virus Sample all over the world a plurality of, unexpectedly find central 6 Virus Samples, combination inequality appears in a certain fragment gene, comprise two from Hong Kong, two from Guangzhou, one from Canada and one from the U.S., 6 viruses are members different in the family in other words.But, do not know that still the virus of which sample is " parent " at present, the researchist tends to from similar to from the Canada and the U.S. of sample in Hong Kong; And from another sample in Hong Kong, then similar to two samples in Guangzhou.Temporary transient unknown these atypical pneumonia viruses family member is that this exists, is still derived out by a kind of atypical pneumonia virus mutation wherein.And the relation between each family member, so that the similarities and differences on the symptom are all unclear as yet.But, this discovery may help the mystery that the very big difference of the state of an illness appears behind a series of patient infections in answer.

The result of study of Hong Kong University confirms to be correct always with not only a kind of worry of the many people's SARSs of rice virus.Though the relation between unknown these six viral members, because the member has six more than, the relevant expert infers thus, the rehabilitation clients of atypical pneumonia might be after rehabilitation, attacked by another kind of viral member, health does not have under the corresponding antibodies and is infected, causes the phenomenon of superinfection.On the other hand,, in part patient's body two above different virus members may be arranged simultaneously, make the effective treatment plan of the more difficult formulation of medical personnel if virus has 6 members.Medical personnel must know which member is patient's virus on one's body be, just can take the most effective treatment plan, and therefore testing method also is restricted.This adds difficulty doubly to work such as control and rehabilitations undoubtedly.

In history, the standard virus detection technique depends on virus separation and external virus culture or immunodetection.Antibody of current SAR S coronavirus and antigen standard are also set up, and immunodetection mainly depends on the quality and the availability of antibody, and the IgM antibody research of sars coronavirus monoclonal antibody and ELISA is also carried out.SARS stdn cell strain is not set up yet, the sars coronavirus vitro culture is difficult, sars coronavirus metabolic patterns and antibody dynamics research are still unclear, and the susceptibility and the specificity of the sars coronavirus diagnosis ELISA test kit of having developed are still waiting further affirmation.Or even the defective of the best way at present also becomes obvious.The full genome chip of sars coronavirus of our development is except have absolute predominance in diagnosis, to the foundation of gene diversity database; The monitoring that sars coronavirus pollutes in the monitoring of sars coronavirus whole genome sequence variation and the environment among the crowd has the meaning of particularly important.Detect at the laboratory of coronavirus at present and exist limitation, so the application of the full genome chip of sars coronavirus is very necessary.Put up with three aspects below and set forth the significance of the full genome chip of sars coronavirus:

(1) the full genome chip of sars coronavirus is significant for the monitoring of the early discovery of " superspreader " and the back patient's environmental pollution of healing.PCR detects has excellent specificity, but can't detect the patient of all secretion coronavirus.Detecting sars coronavirus as early as possible and reliably can help the doctor to discern as patient fever to occur and whether infected SARS during other suspect, simultaneously it is in time isolated and the strict measure of implementing control infection.This will reduce patient SARS greatly disease will be continued transmission danger.

Superspreader " speech gives a lot of people in order to describe a patient who infects SARS with disease propagation.The phenomenon of " superspreader " appears at the early stage of outbreak of disease, at that time this phenomenon is not had enough knowledge and understands.Initial people only know that SARS is a kind of novel disease, think that the patient who much suffers from severe acute respiratory syndrome is caused by other reasons, therefore the infected are not implemented to isolate and strict measure of control.Therefore the early stage infection control measure of carrying out strictness of outbreak of disease.Lacking under the situation of sfgd., a lot of medical personnels, patient relatives and go to the visiting person of hospital to be exposed under the sars coronavirus also develop into the SARS sufferer subsequently.There is the expert to point out in for some time, all might cause the potential sars coronavirus to threaten behind the SARS patient discharge to environment.

(2) the full genome chip of sars coronavirus is significant for the animal-origin of tracking and definite sars coronavirus.A people is dead because of infecting behind the SARS among the owner of existing report dog, and other members are also isolated according to the related measure of control SARS in the family.This pet dog that the household supports, dead during this period, relevant department does not determine also whether it is dead because infect SARS yet.The Peter Rottier research group of Holland Utrecht university passes through infectivity cat abdomen encephalitis (Feline Infectious Peritonitis Virus, FIPV) gene fragment of importing mouse coronavirus, cat FIPV just can the infecting mouse cell, this studies have shown that coronavirus can change the host easily by gene reorganization, and then infers that sars coronavirus may be that existing animal and human coronary virus are mutated into a kind of more fatal form and cause.If other evidence is supported this zoogenetic infection, carry out full genome chip analysis and can analyze easily and confirm the molecule foundation propagated between this all system by animal being separated the sars coronavirus that obtains on one's body.

(3) the full genome chip of sars coronavirus is for the significance that establishes of sars coronavirus gene variation database.Report from Taiwan two people to be separated to sars coronavirus it SARS relevant clinical symptom do not occur, this sars coronavirus that is separated to is carried out full genome chip analysis help understanding the sars coronavirus genetic resistance.The natural genetic resistance that certain sars coronavirus may exist is not determined by single factors, but the method that only relies on serology and PCR be can not determine the diversity and the contingent minor alteration of the relevant concrete gene of sars coronavirus, when RNA viruses (such as HIV virus and influenza virus) was bred (genome duplication and virosome assembling) rapidly in viable cell, they can more easily change its genetic construction in a short time in different tissues.Therefore, the expert thinks that the mutational range of research sars coronavirus is very important to the diagnosis and the treatment of virus infection.The full genome chip of sars coronavirus can reach this purpose quickly and easily, and is significant to the variation and the Molecule Epidemiology Investigation of virus stain.

The SARS associated coronavirus whole genome sequence that the full genome chip of sars coronavirus of our design is mainly announced according to GenBank (the GenBank sequence number: NC_004718) and so far all sars coronavirus whole genome sequences of announcing of GenBank (comprise 1, sars coronavirus HKU-39849, GenBank sequence number: AY278491; 2, sars coronavirus Urbani, GenBank sequence number: AY278741; 3, sars coronavirus CUHK-W1, GenBank sequence number: AY278554; And GenBank has announced sars coronavirus 1, the sars coronavirus BJ04 of portion gene group sequence, GenBank sequence number: AY279354; 2, sars coronavirus BJ03, GenBank sequence number: AY278490; 3, sars coronavirus GZ01, GenBank sequence number: AY278489; 4, sars coronavirus BJ01, GenBank sequence number: AY278488; 5, sars coronavirus BJ02, GerBank sequence number: AY278487; 6, sars coronavirus Taiwan RNA-directed RNA polymerase (pol) Gene Partial coding region, GenBank sequence number: AY268049; 7, sars coronavirus Vietnam200300592 pol gene part coding region, GenBank sequence number: AY269391).It and present existing coronavirus comparative result show the similarity extreme difference of its nucleotide level.Wherein China's five strain SARS associated coronavirus of having surveyed the sars coronavirus whole genome sequence separate respectively in the patient and death of Guangzhou and Beijing area.Wherein two strains derive from the lung tissue sample that becomes celestial of different areas death respectively; One strain derives from Beijing become celestial liver and lymph node tissue mixture; Also have a strain then to derive from Beijing patient's Nasopharyngeal swabs sample.

With relatively demonstration Canadian and the coronavirus sequence that the U.S. issues, the variation of this virus is very competent, and the many places base mutation is arranged in very short several generations.Comprise into all sample whole genome sequences of these different sourcess, the chip that makes us has been covered all known variations and will determine whether sample is infected by sars coronavirus very exactly, and help to understand the sudden change mechanism of virus, in time grasp the distribution situation of virus, improve the accuracy that detects in each department.

Our selection strategy is, from 5 ' end of genome sequence to be selected, is the probe sl. with 40-80 base, the overlapping 5-30 of an adjacent probe base, and the probe sequence of selecting has covered the whole genome sequence fully.Different with the pcr amplification gene strategy of depositing in a small amount, this makes the maximum unique zone that we can target gene, is able to eliminate when keeping susceptibility the problem of cross hybridization.And it does not need expensive and time-consuming enzymatic amplification and the process that confirms thousands of dna profilings.This is to the coronavirus particularly important, and is about 60% because its genome A/T content is than higher, uses long synthetic oligonucleotide to solve this problem.

According to same selection strategy, we have designed at transmissible gastro-enteritis virus (Porcine transmissiblegastroenteritis virus, TGEV) and the specific oligonucleotide probe of the conserved sequence of the long virus of the 40-80 base pair of birds infectious bronchitis virus (Avian infectious bronchitisvirus), on the basis of investigation sars coronavirus fast, can further investigate the infection that whether also has other type of animal coronavirus like this.The numerous disease that coronavirus causes on people and domestic animal and laboratory animal all has important economic worth, often causes serious enteron aisle of animal or respiratory tract infection.Transmissible gastroenteritis of swine is the transmissible disease of a kind of acute, the height contact that causes of the transmissible gastro-enteritis virus by coronavirus genus.Sick pig and be the main source of infection of this disease with malicious pig.Still not having the specific medicine at present can be for treatment, and this sick sickness rate is very high, propagate fast, in case morbidity is taked to isolate, the disinfectant measure effect is little.Infectious bronchitis of fowl can be dissected finding and make diagnosis according to incidence usually.Still there is not specific medicament at present.

This chip also comprises from tobacco mosaic virus (TMV) whole genome sequence and people's gene group the negative probe of the long oligonucleotide of 40-80 base pair that 10000 knowns and more than 7000 EST fragment select, and the negative control point of hundred chip scannings of dozens of to is arranged altogether.The chip that hybridization is finished places the laser co-focusing chip scanner to carry out the conversion of view data, scan as excitation light source with laser, gather information such as each position of hybridizing the some fluorescent signal, fluorescence intensity, utilization specialty analysis software for example AMAD (abbreviation of Analysis of Microarray Data) carries out data-switching to the data of being gathered, main compositional analysis, various bunches analysis, and then whether definite sample has SARS to infect.Virus sequence that this chip will carefully be selected and the combination of random amplification step have overcome the limitation that in the past detects strategy based on RT-PCR, make sensing range wider, and detected result is more just.

The numerous disease that coronavirus causes on people and domestic animal and laboratory animal all has important economic worth, often causes serious enteron aisle of animal or respiratory tract infection.

Transmissible gastroenteritis of swine is the transmissible disease of a kind of acute, the height contact that causes of the transmissible gastro-enteritis virus by coronavirus genus.Sick pig and be the main source of infection of this disease with malicious pig.Still not having the specific medicine at present can be for treatment, and this sick sickness rate is very high, propagate fast, in case morbidity is taked to isolate, the disinfectant measure effect is little.Infectious bronchitis of fowl can be dissected finding and make diagnosis according to incidence usually.Still there is not specific medicament at present.

We discover and seize on Internet, have only Combimatrix company to announce to issue the chip of having developed diagnosis SARS associated coronavirus within 48 hours at the SARS virus complete sequence in the world.The coronavirus full genome chip relevant with SARS of the present invention comprises the whole genome sequence information of into just having surveyed preface, and be based on existing chip exploitation and the resourceful advantage of viral background, other SARS virus series of variation of constantly finishing is included effectively.Be present known all coronavirus strain isolateds below :+--Corona_S2 (130)+---Viruses (130)

+---ssrna?positive-strand?viruses-no?dna?stage(130)

+---nidovirales(130)

+---coronaviridae??(130)

+---coronavirus?transmissible??(5)

+---coronavirus?enteric??(2)

+---coronavirus?epidemic??(4)

+---coronavirus?coronavirus??(37)

+---coronavirus?sialodacryoadenitis??(1)

+---coronavirus?gastroenteritis??(5)

+---coronavirus?hepatitis??(9)

+---coronavirus?infectious??(61)

+---coronavirushemagglutinating??(1)

+--be the coronavirus that has obtained whole genome sequence at present below the-coronavirus respiratory (5):

·Avian?infectious?bronchitis?virus

.Avian?infectious?bronchitis?virus(strain?6/82)

.Avian?infectious?bronchitis?virus(strain?Beaudette?CK)

.Avian?infectious?bronchitis?virus(strain?Beaudette?M42)

.Avian?infectious?bronchitis?virus(strain?Beaudette?US)

.Avian?infectious?bronchitis?virus(strain?Beaudette)

.Avian?infectious?bronchitis?virus(strain?D274)

.Avian?infectious?bronchitis?virus(strain?D3896)

.Avian?infectious?bronchitis?virus(strain?Gray)

.Avian?infectious?bronchitis?virus(strain?KB8523)

.Avian?infectious?bronchitis?virus(strain?M41)

.Avian?infectious?bronchitis?virus(strain?Portugal/322/82)

.Avian?infectious?bronchitis?virus(strain?UK/123/82)

.Avian?infectious?bronchitis?virus(strain?UK/142/86)

.Avian?infectious?bronchitis?virus(strain?UK/167/84)

.Avian?infectious?bronchitis?virus(strain?UK/183/66)

.Avian?infectious?bronchitis?virus(strain?UK/68/84)·Avian?infectious?laryngotracheitis?virus·Enteric?coronavirus·Equine?coronavirus·Group?1?species

Canine?coronavirus

Feline?coronavirus

Human?coronavirus(strain?229E)

Porcine?epidemic?diarrhea?virus

Transmissible?gastroenteritis?virus·Group?2?species

Bovine?coronavirus

Human?coronavirus(strain?OC43)

Murine?hepatitis?virus

Porcine?hemagglutinating?encephalomyelitis?virus

Rat?coronavirus·Group?3?species

Turkey?enteric?coronavirus·Human?enteric?coronavirus?4408

Among summary of the invention the present invention, the strategy of our chip design is to be detected object with sars coronavirus TOR2 strain whole genome sequence (NC_004718), designs corresponding probe and chip.We with every 40-80 base sequence in the full genome of sars coronavirus as a probe sequence, the adjacent probe sequence repeats 5-30 base, particularly with 70 base sequences as a probe sequence, the adjacent probe sequence repeats the preference pattern of 25 bases as first-selection, thereby covers the genome of whole sars coronavirus.Synthetic good probe utilizes gene chip sample applying instrument point to be formed on surface of solid phase carriers, and solid phase carrier can comprise the sheet base that materials such as sheet glass, silicon chip, polypropylene screen, nitrocellulose filter, nylon membrane are made.

Utilize reverse transcription and polymerase chain reaction technique, we will carry out reverse transcription and amplification from the general RNA that extracts in the standard model of clinical sample such as the blood of human body and secretory product and correlated virus etc., mix fluorescent mark in the process of amplification.After amplification label product and chip carried out hybridization, fluorescent signal was handled by the gene chip scanning instrument scanning analysis, finally obtained the yin and yang attribute result of full genomic probe.Can obtain the band poison situation of test sample through contrast with the standard strain results of hybridization.

Technological line of the present invention is: probe design, probe are synthetic, chip point system, sample process and mark, chip hybridization, interpretation of result.

The invention describes a kind of gene chip, it is characterized in that being fixed on the whole genome sequence that on-chip probe matrix has covered sars coronavirus, the whole genome sequence of described sars coronavirus is meant the sars coronavirus strain that GenBank has announced or the whole genome sequence of hypotype, described sars coronavirus strain or hypotype comprise SARScoronavirus TOR2 (GenBank sequence number: NC 004718/AY274119), SARS coronavirus HKU-39849 (GenBank sequence number: AY278491), SARS coronavirus CUHK-W1 (GenBank sequence number: AY278554), SARS coronavirus Urbani (GenBank sequence number: AY278741), SARS coronavirns BJ01 (GenBank sequence number: AY278488), SARS coronavirns BJ02 (GenBank sequence number: AY278487), SARS coronavirusBJ03 (GenBank sequence number: AY278490), SARS coronavirus BJ04 (the GenBank sequence number: AY279354), SARS coronavirus GZ01 (GenBank sequence number: AY278489).

Probe feature on the gene chip described in the invention is that every 40-80 base sequence from the full genome of sars coronavirus is as a probe sequence, the adjacent probe sequence repeats 5-30 base, probe feature on the described gene chip is meant especially with 70 base sequences that as a probe sequence adjacent probe sequence repeats the pattern of 25 bases.

According to same selection strategy, we also designed at transmissible gastro-enteritis virus (Porcine transmissiblegastroenteritis virus, TGEV) and the long virus-specific oligonucleotide probe of the 40-100 base pair of birds infectious bronchitis virus (Avian infectious bronchitisvirus).The purpose of such layout strategy is can further investigate the infection that whether also has other type of animal coronavirus on the basis of investigating sars coronavirus fast.

One of purposes of gene chip described in the invention is to be used to detect sars coronavirus, described detected object can comprise from clinical sample, animals and plants sample, food water source sample and water surroundings such as the blood of human body and secretory product and atmospheric environment sample or the like, two of the purposes of described gene chip is variation and the hypotypes that are used to monitor sars coronavirus, and described monitoring target can comprise from clinical sample, animals and plants sample, food water source sample and water surroundings such as the blood of human body and secretory product and atmospheric environment sample or the like.

Three of the purposes of gene chip described in the invention is to be used to differentiate sars coronavirus and other coronavirus, and described discriminating object can comprise from clinical sample, animals and plants sample, food water source sample and water surrounding such as the blood of human body and secretory product and atmospheric environment sample or the like.

Figure 1 shows that the full genome chip point sample of the coronavirus relevant synoptic diagram in the description of drawings Figure of description with SARS, point system rule is that every probe repeats three points, be divided into 5 matrixes, 136 probes of each matrix, each matrix comprises 38 positive coordinate point, forms the dot matrix of 25 row * 14 row.Every group of triple junction is a probe.The positive coordinate point of grey color dot shown in the A among the figure, the black triple junction shown in the B is a detection probes.

Embodiment following examples have been done detailed description to the preparation and the purposes of the full genome chip of coronavirus relevant with SARS of the present invention, but and do not mean that restriction content of the present invention.The preparation of embodiment 1 chip

Choosing of probe:

We choose sars coronavirus TOR2 strain whole genome sequence (NC_004718) is detected object, designs corresponding probe and chip.TOR2 strain whole genome sequence (NC_004718) genome total length 29736 bases since 5 ' end, are the probe sl. with 70 bases, overlapping 25 bases of adjacent probe.Head and the tail position as probe 1 is 1-70 base in the genome sequence, the head and the tail position of probe 2 is 46-115 base in the genome sequence, the head and the tail position of probe 3 is 91-160 base in the genome sequence, the head and the tail position of probe 4 is 136-205 base in the genome sequence, by that analogy, obtain 660 probes altogether, as shown in table 1, these 660 probes have covered this genomic full sequence.5 ' end of every probe carries out amido modified.

Table 1 SARS coronavirus TOR2 strain genome probe sequence Serial sequence start site sars001 CTACCCAGGAAAAGCCAACCAACCTCGATCTCTTGTAGATCTGTTCTCTAAACGAACTTTAAAATCTGTG 1 sars002 CTCTAAACGAACTTTAAAATCTGTGTAGCTGTCGCTCGGCTGCATGCCTAGTGCACCTACGCAGTATAAA 46 sars003 GCCTAGTGCACCTACGCAGTATAAACAATAATAAATTTTACTGTCGTTGACAAGAAACGAGTAACTCGTC 91 sars004 GTTGACAAGAAACGAGTAACTCGTCCCTCTTCTGCAGACTGCTTACGGTTTCGTCCGTGTTGCAGTCGAT 136 sars005 CGGTTTCGTCCGTGTTGCAGTCGATCATCAGCATACCTAGGTTTCGTCCGGGTGTGACCGAAAGGTAAGA 181 sars006 GTCCGGGTGTGACCGAAAGGTAAGATGGAGAGCCTTGTTCTTGGTGTCAACGAGAAAACACACGTCCAAC 226 sars007 GTCAACGAGAAAACACACGTCCAACTCAGTTTGCCTGTCCTTCAGGTTAGAGACGTGCTAGTGCGTGGCT 271 sars008 GTTAGAGACGTGCTAGTGCGTGGCTTCGGGGACTCTGTGGAAGAGGCCCTATCGGAGGCACGTGAACACC 316 sars009 GCCCTATCGGAGGCACGTGAACACCTCAAAAATGGCACTTGTGGTCTAGTAGAGCTGGAAAAAGGCGTAC 361 sars010 CTAGTAGAGCTGGAAAAAGGCGTACTGCCCCAGCTTGAACAGCCCTATGTGTTCATTAAACGTTCTGATG 406 sars011 TATGTGTTCATTAAACGTTCTGATGCCTTAAGCACCAATCACGGCCACAAGGTCGTTGAGCTGGTTGCAG 451 sars012 CACAAGGTCGTTGAGCTGGTTGCAGAAATGGACGGCATTCAGTACGGTCGTAGCGGTATAACACTGGGAG 496 sars013 GGTCGTAGCGGTATAACACTGGGAGTACTCGTGCCACATGTGGGCGAAACCCCAATTGCATACCGCAATG 541 sars014 GAAACCCCAATTGCATACCGCAATGTTCTTCTTCGTAAGAACGGTAATAAGGGAGCCGGTGGTCATAGCT 586 sars015 AATAAGGGAGCCGGTGGTCATAGCTATGGCATCGATCTAAAGTCTTATGACTTAGGTGACGAGCTTGGCA 631 sars016 TATGACTTAGGTGACGAGCTTGGCACTGATCCCATTGAAGATTATGAACAAAACTGGAACACTAAGCATG 676 sars017 GAACAAAACTGGAACACTAAGCATGGCAGTGGTGCACTCCGTGAACTCACTCGTGAGCTCAATGGAGGTG 721 sars018 CTCACTCGTGAGCTCAATGGAGGTGCAGTCACTCGCTATGTCGACAACAATTTCTGTGGCCCAGATGGGT 766 sars019 AACAATTTCTGTGGCCCAGATGGGTACCCTCTTGATTGCATCAAAGATTTTCTCGCACGCGCGGGCAAGT 811 sars020 GATTTTCTCGCACGCGCGGGCAAGTCAATGTGCACTCTTTCCGAACAACTTGATTACATCGAGTCGAAGA 856 sars021 CAACTTGATTACATCGAGTCGAAGAGAGGTGTCTACTGCTGCCGTGACCATGAGCATGAAATTGCCTGGT 901 sars022 GACCATGAGCATGAAATTGCCTGGTTCACTGAGCGCTCTGATAAGAGCTACGAGCACCAGACACCCTTCG 946 sars023 AGCTACGAGCACCAGACACCCTTCGAAATTAAGAGTGCCAAGAAATTTGACACTTTCAAAGGGGAATGCC 991 sars024 TTTGACACTTTCAAAGGGGAATGCCCAAAGTTTGTGTTTCCTCTTAACTCAAAAGTCAAAGTCATTCAAC 1036 sars025 AACTCAAAAGTCAAAGTCATTCAACCACGTGTTGAAAAGAAAAAGACTGAGGGTTTCATGGGGCGTATAC 1081 sars026 ACTGAGGGTTTCATGGGGCGTATACGCTCTGTGTACCCTGTTGCATCTCCACAGGAGTGTAACAATATGC 1126 sars027 TCTCCACAGGAGTGTAACAATATGCACTTGTCTACCTTGATGAAATGTAATCATTGCGATGAAGTTTCAT 1171 sars028 TGTAATCATTGCGATGAAGTTTCATGGCAGACGTGCGACTTTCTGAAAGCCACTTGTGAACATTGTGGCA 1216 sars029 AAAGCCACTTGTGAACATTGTGGCACTGAAAATTTAGTTATTGAAGGACCTACTACATGTGGGTACCTAC 1261 sars030 GGACCTACTACATGTGGGTACCTACCTACTAATGCTGTAGTGAAAATGCCATGTCCTGCCTGTCAAGACC 1306 sars031 ATGCCATGTCCTGCCTGTCAAGACCCAGAGATTGGACCTGAGCATAGTGTTGCAGATTATCACAACCACT 1351 sars032 AGTGTTGCAGATTATCACAACCACTCAAACATTGAAACTCGACTCCGCAAGGGAGGTAGGACTAGATGTT 1396 sars033 CGCAAGGGAGGTAGGACTAGATGTTTTGGAGGCTGTGTGTTTGCCTATGTTGGCTGCTATAATAAGCGTG 1441 sars034 TATGTTGGCTGCTATAATAAGCGTGCCTACTGGGTTCCTCGTGCTAGTGCTGATATTGGCTCAGGCCATA 1486 sars035 AGTGCTGATATTGGCTCAGGCCATACTGGCATTACTGGTGACAATGTGGAGACCTTGAATGAGGATCTCC 1531 sars036 GTGGAGACCTTGAATGAGGATCTCCTTGAGATACTGAGTCGTGAACGTGTTAACATTAACATTGTTGGCG 1576 sars037 CGTGTTAACATTAACATTGTTGGCGATTTTCATTTGAATGAAGAGGTTGCCATCATTTTGGCATCTTTCT 1621 sars038 GTTGCCATCATTTTGGCATCTTTCTCTGCTTCTACAAGTGCCTTTATTGACACTATAAAGAGTCTTGATT 1666 sars039 ATTGACACTATAAAGAGTCTTGATTACAAGTCTTTCAAAACCATTGTTGAGTCCTGCGGTAACTATAAAG 1711 sars040 GTTGAGTCCTGCGGTAACTATAAAGTTACCAAGGGAAAGCCCGTAAAAGGTGCTTGGAACATTGGACAAC 1756 sars041 AAAGGTGCTTGGAACATTGGACAACAGAGATCAGTTTTAACACCACTGTGTGGTTTTCCCTCACAGGCTG 1801 sars042 CTGTGTGGTTTTCCCTCACAGGCTGCTGGTGTTATCAGATCAATTTTTGCGCGCACACTTGATGCAGCAA 1846 sars043 TTTGCGCGCACACTTGATGCAGCAAACCACTCAATTCCTGATTTGCAAAGAGCAGCTGTCACCATACTTG 1891 sars044 CAAAGAGCAGCTGTCACCATACTTGATGGTATTTCTGAACAGTCATTACGTCTTGTCGACGCCATGGTTT 1936 sars045 TTACGTCTTGTCGACGCCATGGTTTATACTTCAGACCTGCTCACCAACAGTGTCATTATTATGGCATATG 1981 sars046 AACAGTGTCATTATTATGGCATATGTAACTGGTGGTCTTGTACAACAGACTTCTCAGTGGTTGTCTAATC 2026 sars047 CAGACTTCTCAGTGGTTGTCTAATCTTTTGGGCACTACTGTTGAAAAACTCAGGCCTATCTTTGAATGGA 2071 sars048 AAACTCAGGCCTATCTTTGAATGGATTGAGGCGAAACTTAGTGCAGGAGTTGAATTTCTCAAGGATGCTT 2116 sars049 GGAGTTGAATTTCTCAAGGATGCTTGGGAGATTCTCAAATTTCTCATTACAGGTGTTTTTGACATCGTCA 2161 sars050 ATTACAGGTGTTTTTGACATCGTCAAGGGTCAAATACAGGTTGCTTCAGATAACATCAAGGATTGTGTAA 2206 sars051 TCAGATAACATCAAGGATTGTGTAAAATGCTTCATTGATGTTGTTAACAAGGCACTCGAAATGTGCATTG 2251 sars052 AACAAGGCACTCGAAATGTGCATTGATCAAGTCACTATCGCTGGCGCAAAGTTGCGATCACTCAACTTAG 2296 sars053 GCAAAGTTGCGATCACTCAACTTAGGTGAAGTCTTCATCGCTCAAAGCAAGGGACTTTACCGTCAGTGTA 2341 sars054 AGCAAGGGACTTTACCGTCAGTGTATACGTGGCAAGGAGCAGCTGCAACTACTCATGCCTCTTAAGGCAC 2386 sars055 CAACTACTCATGCCTCTTAAGGCACCAAAAGAAGTAACCTTTCTTGAAGGTGATTCACATGACACAGTAC 2431 sars056 GAAGGTGATTCACATGACACAGTACTTACCTCTGAGGAGGTTGTTCTCAAGAACGGTGAACTCGAAGCAC 2476 sars057 CTCAAGAACGGTGAACTCGAAGCACTCGAGACGCCCGTTGATAGCTTCACAAATGGAGCTATCCTCGGCA 2521 sars058 TTCACAAATGGAGCTATCGTCGGCACACCAGTCTGTGTAAATGGCCTCATGCTCTTAGAGATTAAGGACA 2566 sars059 CTCATGCTCTTAGAGATTAAGGACAAAGAACAATACTGCGCATTGTCTCCTGGTTTACTGGCTACAAACA 2611 sars060 TCTCCTGGTTTACTGGCTACAAACAATGTCTTTCGCTTAAAAGGGGGTGCACCAATTAAAGGTGTAACCT 2656 sars061 GGTGCACCAATTAAAGGTGTAACCTTTGGAGAAGATACTGTTTGGGAAGTTCAAGGTTACAAGAATGTGA 2701 sars062 GAAGTTCAAGGTTACAAGAATGTGAGAATCACATTTGAGCTTGATGAACGTGTTGACAAAGTGCTTAATG 2746 sars063 GAACGTGTTGACAAAGTGCTTAATGAAAAGTGCTCTGTCTACACTGTTGAATCCGGTACCGAAGTTACTG 2791 sars064 GTTGAATCCGGTACCGAAGTTACTGAGTTTGCATGTGTTGTAGCAGAGGCTGTTGTGAAGACTTTACAAC 2836 sars065 GAGGCTGTTGTGAAGACTTTACAACCAGTTTCTGATCTCCTTACCAACATGGGTATTGATCTTGATGAGT 2881 sars066 AACATGGGTATTGATCTTGATGAGTGGAGTGTAGCTACATTCTACTTATTTGATGATGCTGGTGAAGAAA 2926 sars067 TTATTTGATGATGCTGGTGAAGAAAACTTTTCATCACGTATGTATTGTTCCTTTTACCCTCCAGATGAGG 2971 sars068 TGTTCCTTTTACCCTCCAGATGAGGAAGAAGAGGACGATGCAGAGTGTGAGGAAGAAGAAATTGATGAAA 3016 sars069 TGTGAGGAAGAAGAAATTGATGAAACCTGTGAACATGAGTACGGTACAGAGGATGATTATCAAGGTCTCC 3061 sars070 ACAGAGGATGATTATCAAGGTCTCCCTCTGGAATTTGGTGCCTCAGCTGAAACAGTTCGAGTTGAGGAAG 3106 sars071 GCTGAAACAGTTCGAGTTGAGGAAGAAGAAGAGGAAGACTGGCTGGATGATACTACTGAGCAATCAGAGA 3151 sars072 GATGATACTACTGAGCAATCAGAGATTGAGCCAGAACCAGAACCTACACCTGAAGAACCAGTTAATCAGT 3196 sars073 ACACCTGAAGAACCAGTTAATCAGTTTACTGGTTATTTAAAACTTACTGACAATGTTGCCATTAAATGTG 3241 sars074 ACTGACAATGTTGCCATTAAATGTGTTGACATCGTTAAGGAGGCACAAAGTGCTAATCCTATGGTGATTG 3286 sars075 CAAAGTGCTAATCCTATGGTGATTGTAAATGCTGCTAACATACACCTGAAACATGGTGGTGGTGTAGCAG 3331 sars076 CTGAAACATGGTGGTGGTGTAGCAGGTGCACTCAACAAGGCAACCAATGGTGCCATGCAAAAGGAGAGTG 3376 sars077 AATGGTGCCATGCAAAAGGAGAGTGATGATTACATTAAGCTAAATGGCCCTCTTACAGTAGGAGGGTCTT 3421 sars078 GGCCCTCTTACAGTAGGAGGGTCTTGTTTGCTTTCTGGACATAATCTTGCTAAGAAGTGTCTGCATGTTG 3466 sars079 CTTGCTAAGAAGTGTCTGCATGTTGTTGGACCTAACCTAAATGCAGGTGAGGACATCCAGCTTCTTAAGG 3511 sars080 GGTGAGGACATCCAGCTTCTTAAGGCAGCATATGAAAATTTCAATTCACAGGACATCTTACTTGCACCAT 3556 sars081 TCACAGGACATCTTACTTGCACCATTGTTGTCAGCAGGCATATTTGGTGCTAAACCACTTCAGTCTTTAC 3601 sars082 GGTGCTAAACCACTTCAGTCTTTACAAGTGTGCGTGCAGACGGTTCGTACACAGGTTTATATTGCAGTCA 3646 sars083 CGTACACAGGTTTATATTGCAGTCAATGACAAAGCTCTTTATGAGCAGGTTGTCATGGATTATCTTGATA 3691 sars084 CAGGTTGTCATGGATTATCTTGATAACCTGAAGCCTAGAGTGGAAGCACCTAAACAAGAGGAGCCACCAA 3736 sars085 GCACCTAAACAAGAGGAGCCACCAAACACAGAAGATTCCAAAACTGAGGAGAAATCTGTCGTACAGAAGC 3781 sars086 GAGGAGAAATCTGTCGTACAGAAGCCTGTCGATGTGAAGCCAAAAATTAAGGCCTGCATTGATGAGGTTA 3826 sars087 ATTAAGGCCTGCATTGATGAGGTTACCACAACACTGGAAGAAACTAAGTTTCTTACCAATAAGTTACTCT 3871 sars088 AAGTTTCTTACCAATAAGTTACTCTTGTTTGCTGATATCAATGGTAAGCTTTACCATGATTCTCAGAACA 3916 sars089 AAGCTTTACCATGATTCTCAGAACATGCTTAGAGGTGAAGATATGTCTTTCCTTGAGAAGGATGCACCTT 3961 sars090 TCTTTCCTTGAGAAGGATGCACCTTACATGGTAGGTGATGTTATCACTAGTGGTGATATCACTTGTGTTG 4006 sars091 ACTAGTGGTGATATCACTTGTGTTGTAATACCCTCCAAAAAGGCTGGTGGCACTACTGAGATGCTCTCAA 4051 sars092 GGTGGCACTACTGAGATGCTCTCAAGAGCTTTGAAGAAAGTGCCAGTTGATGAGTATATAACCACGTACC 4096 sars093 GTTGATGAGTATATAACCACGTACCCTGGACAAGGATGTGCTGGTTATACACTTGAGGAAGCTAAGACTG 4141 sars094 TATACACTTGAGGAAGCTAAGACTGCTCTTAAGAAATGCAAATCTGCATTTTATGTACTACCTTCAGAAG 4186 sars095 GCATTTTATGTACTACCTTCAGAAGCACCTAATGCTAAGGAAGAGATTCTAGGAACTGTATCCTGGAATT 4231 sars096 ATTCTAGGAACTGTATCCTGGAATTTGAGAGAAATGCTTGCTCATGCTGAAGAGACAAGAAAATTAATGC 4276 sars097 GCTGAAGAGACAAGAAAATTAATGCCTATATGCATGGATGTTAGAGCCATAATGGCAACCATCCAACGTA 4321 sars098 GCCATAATGGCAACCATCCAACGTAAGTATAAAGGAATTAAAATTCAAGAGGGCATCGTTGACTATGGTG 4366 sars099 CAAGAGGGCATCGTTGACTATGGTGTCCGATTCTTCTTTTATACTAGTAAAGAGCCTGTAGCTTCTATTA 4411 sars100 AGTAAAGAGCCTGTAGCTTCTATTATTACGAAGCTGAACTCTCTAAATGAGCCGCTTGTCACAATGCCAA 4456 sars101 AATGAGCCGCTTGTCACAATGCCAATTGGTTATGTGACACATGGTTTTAATCTTGAAGAGGCTGCGCGCT 4501 sars102 TTTAATCTTGAAGAGGCTGCGCGCTGTATGCGTTCTCTTAAAGCTCCTGCCGTAGTGTCAGTATCATCAC 4546 sars103 CCTGCCGTAGTGTCAGTATCATCACCAGATGCTGTTACTACATATAATGGATACCTCACTTCGTCATCAA 4591 sars104 AATGGATACCTCACTTCGTCATCAAAGACATCTGAGGAGCACTTTGTAGAAACAGTTTCTTTGGCTGGCT 4636 sars105 GTAGAAACAGTTTCTTTGGCTGGCTCTTACAGAGATTGGTCCTATTCAGGACAGCGTACAGAGTTAGGTG 4681 sars106 TCAGGACAGCGTACAGAGTTAGGTGTTGAATTTCTTAAGCGTGGTGACAAAATTGTGTACCACACTCTGG 4726 sars107 GACAAAATTGTGTACCACACTCTGGAGAGCCCCGTCGAGTTTCATCTTGACGGTGAGGTTCTTTCACTTG 4771 sars108 CTTGACGGTGAGGTTCTTTCACTTGACAAACTAAAGAGTCTCTTATCCCTGCGGGAGGTTAAGACTATAA 4816 sars109 TCCCTGCGGGAGGTTAAGACTATAAAAGTGTTCACAACTGTGGACAACACTAATCTCCACACACAGCTTG 4861 sars110 AACACTAATCTCCACACACAGCTTGTGGATATGTCTATGACATATGGACAGCAGTTTGGTCCAACATACT 4906 sars111 GGACAGCAGTTTGGTCCAACATACTTGGATGGTGCTGATGTTACAAAAATTAAACCTCATGTAAATCATG 4951 sars112 AAAATTAAACCTCATGTAAATCATGAGGGTAAGACTTTCTTTGTACTACCTAGTGATGACACACTACGTA 4996 sars113 CTACCTAGTGATGACACACTACGTAGTGAAGCTTTCGAGTACTACCATACTCTTGATGAGAGTTTTCTTG 5041 sars114 CATACTCTTGATGAGAGTTTTCTTGGTAGGTACATGTCTGCTTTAAACCACACAAAGAAATGGAAATTTC 5086 sars115 AACCACACAAAGAAATGGAAATTTCCTCAAGTTGGTGGTTTAACTTCAATTAAATGGGCTGATAACAATT 5131 sars116 TCAATTAAATGGGCTGATAACAATTGTTATTTGTCTAGTGTTTTATTAGCACTTCAACAGCTTGAAGTCA 5176 sars117 TTAGCACTTCAACAGCTTGAAGTCAAATTCAATGCACCAGCACTTCAAGAGGCTTATTATAGAGCCCGTG 5221 sars118 CAAGAGGCTTATTATAGAGCCCGTGCTGGTGATGCTGCTAACTTTTGTGCACTCATACTCGCTTACAGTA 5266 sars119 TGTGCACTCATACTCGCTTACAGTAATAAAACTGTTGGCGAGCTTGGTGATGTCAGAGAAACTATGACCC 5311 sars120 GGTGATGTCAGAGAAACTATGACCCATCTTCTACAGCATGCTAATTTGGAATCTGCAAAGCGAGTTCTTA 5356 sars121 TTGGAATCTGCAAAGCGAGTTCTTAATGTGGTGTGTAAACATTGTGGTCAGAAAACTACTACCTTAACGG 5401 sars122 GGTCAGAAAACTACTACCTTAACGGGTGTAGAAGCTGTGATGTATATGGGTACTCTATCTTATGATAATC 5446 sars123 ATGGGTACTCTATCTTATGATAATCTTAAGACAGGTGTTTCCATTCCATGTGTGTGTGGTCGTGATGCTA 5491 sars124 CCATGTGTGTGTGGTCGTGATGCTACACAATATCTAGTACAACAAGAGTCTTCTTTTGTTATGATGTCTG 5536 sars125 GAGTCTTCTTTTGTTATGATGTCTGCACCACCTGCTGAGTATAAATTACAGCAAGGTACATTCTTATGTG 5581 sars126 TTACAGCAAGGTACATTCTTATGTGCGAATGAGTACACTGGTAACTATCAGTGTGGTCATTACACTCATA 5626 sars127 TATCAGTGTGGTCATTACACTCATATAACTGCTAAGGAGACCCTCTATCGTATTGACGGAGCTCACCTTA 5671 sars128 TATCGTATTGACGGAGCTCACCTTACAAAGATGTCAGAGTACAAAGGACCAGTGACTGATGTTTTCTACA 5716 sars129 GGACCAGTGACTGATGTTTTCTACAAGGAAACATCTTACACTACAACCATCAAGCCTGTGTCGTATAAAC 5761 sars130 ACCATCAAGCCTGTGTCGTATAAACTCGATGGAGTTACTTACACAGAGATTGAACCAAAATTGGATGGGT 5806 sars131 GAGATTGAACCAAAATTGGATGGGTATTATAAAAAGGATAATGCTTACTATACAGAGCAGCCTATAGACC 5851 sars132 TACTATACAGAGCAGCCTATAGACCTTGTACCAACTCAACCATTACCAAATGCGAGTTTTGATAATTTCA 5896 sars133 CCAAATGCGAGTTTTGATAATTTCAAACTCACATGTTCTAACACAAAATTTGCTGATGATTTAAATCAAA 5941 sars134 AAATTTGCTGATGATTTAAATCAAATGACAGGCTTCACAAAGCCAGCTTCACGAGAGCTATCTGTCACAT 5986 sars135 GCTTCACGAGAGCTATCTGTCACATTCTTCCCAGACTTGAATGGCGATGTAGTGGCTATTGACTATAGAC 6031 sars136 GATGTAGTGGCTATTGACTATAGACACTATTCAGCGAGTTTCAAGAAAGGTGCTAAATTACTGCATAAGC 6076 sars137 AAAGGTGCTAAATTACTGCATAAGCCAATTGTTTGGCACATTAACCAGGCTACAACCAAGACAACGTTCA 6121 sars138 CAGGCTACAACCAAGACAACGTTCAAACCAAACACTTGGTGTTTACGTTGTCTTTGGAGTACAAAGCCAG 6166 sars139 CGTTGTCTTTGGAGTACAAAGCCAGTAGATACTTCAAATTCATTTGAAGTTCTGGCAGTAGAAGACACAC 6211 sars140 GAAGTTCTGGCAGTAGAAGACACACAAGGAATGGACAATCTTGCTTGTGAAAGTCAACAACCCACCTCTG 6256 sars141 TGTGAAAGTCAACAACCCACCTCTGAAGAAGTAGTGGAAAATCCTACCATACAGAAGGAAGTCATAGAGT 6301 sars142 ACCATACAGAAGGAAGTCATAGAGTGTGACGTGAAAACTACCGAAGTTGTAGGCAATGTCATACTTAAAC 6346 sars143 GTTGTAGGCAATGTCATACTTAAACCATCAGATGAAGGTGTTAAAGTAACACAAGAGTTAGGTCATGAGG 6391 sars144 GTAACACAAGAGTTAGGTCATGAGGATCTTATGGCTGCTTATGTGGAAAACACAAGCATTACCATTAAGA 6436 sars145 GAAAACACAAGCATTACCATTAAGAAACCTAATGAGCTTTCACTAGCCTTAGGTTTAAAAACAATTGCCA 6481 sars146 GCCTTAGGTTTAAAAACAATTGCCACTCATGGTATTGCTGCAATTAATAGTGTTCCTTGGAGTAAAATTT 6526 sars147 AATAGTGTTCCTTGGAGTAAAATTTTGGCTTATGTCAAACCATTCTTAGGACAAGCAGCAATTACAACAT 6571 sars148 TTAGGACAAGCAGCAATTACAACATCAAATTGCGCTAAGAGATTAGCACAACGTGTGTTTAACAATTATA 6616 sars149 GCACAACGTGTGTTTAACAATTATATGCCTTATGTGTTTACATTATTGTTCCAATTGTGTACTTTTACTA 6661 sars150 TTGTTCCAATTGTGTACTTTTACTAAAAGTACCAATTCTAGAATTAGAGCTTCACTACCTACAACTATTG 6706 sars151 AGAGCTTCACTACCTACAACTATTGCTAAAAATAGTGTTAAGAGTGTTGCTAAATTATGTTTGGATGCCG 6751 sars152 GTTGCTAAATTATGTTTGGATGCCGGCATTAATTATGTGAAGTCACCCAAATTTTCTAAATTGTTCACAA 6796 sars153 CCCAAATTTTCTAAATTGTTCACAATCGCTATGTGGCTATTGTTGTTAAGTATTTGCTTAGGTTCTCTAA 6841 sars154 TTAAGTATTTGCTTAGGTTCTCTAATCTGTGTAACTGCTGCTTTTGGTGTACTCTTATCTAATTTTGGTG 6886 sars155 GGTGTACTCTTATCTAATTTTGGTGCTCCTTCTTATTGTAATGGCGTTAGAGAATTGTATCTTAATTCGT 6931 sars156 GTTAGAGAATTGTATCTTAATTCGTCTAACGTTACTACTATGGATTTCTGTGAAGGTTCTTTTCCTTGCA 6976 sars157 TTCTGTGAAGGTTCTTTTCCTTGCAGCATTTGTTTAAGTGGATTAGACTCCCTTGATTCTTATCCAGCTC 7021 sars158 GACTCCCTTGATTCTTATCCAGCTCTTGAAACCATTCAGGTGACGATTTCATCGTACAAGCTAGACTTGA 7066 sars159 ATTTCATCGTACAAGCTAGACTTGACAATTTTAGGTCTGGCCGCTGAGTGGGTTTTGGCATATATGTTGT 7111 sars160 GAGTGGGTTTTGGCATATATGTTGTTCACAAAATTCTTTTATTTATTAGGTCTTTCAGCTATAATGCAGG 7156 sars161 TTAGGTCTTTCAGCTATAATGCAGGTGTTCTTTGGCTATTTTGCTAGTCATTTCATCAGCAATTCTTGGC 7201 sars162 AGTCATTTCATCAGCAATTCTTGGCTCATGTGGTTTATCATTAGTATTGTACAAATGGCACCCGTTTCTG 7246 sars163 ATTGTACAAATGGCACCCGTTTCTGCAATGGTTAGGATGTACATCTTCTTTGCTTCTTTCTACTACATAT 7291 sars164 TTCTTTGCTTCTTTCTACTACATATGGAAGAGCTATGTTCATATCATGGATGGTTGCACCTCTTCGACTT 7336 sars165 ATGGATGGTTGCACCTCTTCGACTTGCATGATGTGCTATAAGCGCAATCGTGCCACACGCGTTGAGTGTA 7381 sars166 AATCGTGCCACACGCGTTGAGTGTACAACTATTGTTAATGGCATGAAGAGATCTTTCTATGTCTATGCAA 7426 sars167 AAGAGATCTTTCTATGTCTATGCAAATGGAGGCCGTGGCTTCTGCAAGACTCACAATTGGAATTGTCTCA 7471 sars168 AAGACTCACAATTGGAATTGTCTCAATTGTGACACATTTTGCACTGGTAGTACATTCATTAGTGATGAAG 7516 sars169 GGTAGTACATTCATTAGTGATGAAGTTGCTCGTGATTTGTCACTCCAGTTTAAAAGACCAATCAACCCTA 7561 sars170 CAGTTTAAAAGACCAATCAACCCTACTGACCAGTCATCGTATATTGTTGATAGTGTTGCTGTGAAAAATG 7606 sars171 GTTGATAGTGTTGCTGTGAAAAATGGCGCGCTTCACCTCTACTTTGACAAGGCTGGTCAAAAGACCTATG 7651 sars172 GACAAGGCTGGTCAAAAGACCTATGAGAGACATCCGCTCTCCCATTTTGTCAATTTAGACAATTTGAGAG 7696 sars173 TTTGTCAATTTAGACAATTTGAGAGCTAACAACACTAAAGGTTCACTGCCTATTAATGTCATAGTTTTTG 7741 sars174 CTGCCTATTAATGTCATAGTTTTTGATGGCAAGTCCAAATGCGACGAGTCTGCTTCTAAGTCTGCTTCTG 7786 sars175 GAGTCTGCTTCTAAGTCTGCTTCTGTGTACTACAGTCAGCTGATGTGCCAACCTATTCTGTTGCTTGACC 7831 sars176 TGCCAACCTATTCTGTTGCTTGACCAAGCTCTTGTATCAGACGTTGGAGATAGTACTGAAGTTTCCGTTA 7876 sars177 GGAGATAGTACTGAAGTTTCCGTTAAGATGTTTGATGCTTATGTCGACACCTTTTCAGCAACTTTTAGTG 7921 sars178 GACACCTTTTCAGCAACTTTTAGTGTTCCTATGGAAAAACTTAAGGCACTTGTTGCTACAGCTCACAGCG 7966 sars179 GCACTTGTTGCTACAGCTCACAGCGAGTTAGCAAAGGGTGTAGCTTTAGATGGTGTCCTTTCTACATTCG 8011 sars180 TTAGATGGTGTCCTTTCTACATTCGTGTCAGCTGCCCGACAAGGTGTTGTTGATACCGATGTTGACACAA 8056 sars181 GTTGTTGATACCGATGTTGACACAAAGGATGTTATTGAATGTCTCAAACTTTCACATCACTCTGACTTAG 8101 sars182 AAACTTTCACATCACTCTGACTTAGAAGTGACAGGTGACAGTTGTAACAATTTCATGCTCACCTATAATA 8146 sars183 AACAATTTCATGCTCACCTATAATAAGGTTGAAAACATGACGCCCAGAGATCTTGGCGCATGTATTGACT 8191 sars184 AGAGATCTTGGCGCATGTATTGACTGTAATGCAAGGCATATCAATGCCCAAGTAGCAAAAAGTCACAATG 8236 sars185 GCCCAAGTAGCAAAAAGTCACAATGTTTCACTCATCTGGAATGTAAAAGACTACATGTCTTTATCTGAAC 8281 sars186 AAAGACTACATGTCTTTATCTGAACAGCTGCGTAAACAAATTCGTAGTGCTGCCAAGAAGAACAACATAC 8326 sars187 AGTGCTGCCAAGAAGAACAACATACCTTTTAGACTAACTTGTGCTACAACTAGACAGGTTGTCAATGTCA 8371 sars188 ACAACTAGACAGGTTGTCAATGTCATAACTACTAAAATCTCACTCAAGGGTGGTAAGATTGTTAGTACTT 8416 sars189 AAGGGTGGTAAGATTGTTAGTACTTGTTTTAAACTTATGCTTAAGGCCACATTATTGTGCGTTCTTGCTG 8461 sars190 GCCACATTATTGTGCGTTCTTGCTGCATTGGTTTGTTATATCGTTATGCCAGTACATACATTGTCAATCC 8506 sars1g1 ATGCCAGTACATACATTGTCAATCCATGATGGTTACACAAATGAAATCATTGGTTACAAAGCCATTCAGG 8551 sars192 ATCATTGGTTACAAAGCCATTCAGGATGGTGTCACTCGTGACATCATTTCTACTGATGATTGTTTTGCAA 8596 sars193 ATTTCTACTGATGATTGTTTTGCAAATAAACATGCTGGTTTTGACGCATGGTTTAGCCAGCGTGGTGGTT 8641 sars194 GCATGGTTTAGCCAGCGTGGTGGTTCATACAAAAATGACAAAAGCTGCCCTGTAGTAGCTGCTATCATTA 8686 sars195 TGCCCTGTAGTAGCTGCTATCATTACAAGAGAGATTGGTTTCATAGTGCCTGGCTTACCGGGTACTGTGC 8731 sars196 GTGCCTGGCTTACCGGGTACTGTGCTGAGAGCAATCAATGGTGACTTCTTGCATTTTCTACCTCGTGTTT 8776 sars197 TTCTTGCATTTTCTACCTCGTGTTTTTAGTGCTGTTGGCAACATTTGCTACACACCTTCCAAACTCATTG 8821 sars198 TGCTACACACCTTCCAAACTCATTGAGTATAGTGATTTTGCTACCTCTGCTTGCGTTCTTGCTGCTGAGT 8866 sars199 TCTGCTTGCGTTCTTGCTGCTGAGTGTACAATTTTTAAGGATGCTATGGGCAAACCTGTGCCATATTGTT 8911 sars200 ATGGGCAAACCTGTGCCATATTGTTATGACACTAATTTGCTAGAGGGTTCTATTTCTTATAGTGAGCTTC 8956 sars201 GGTTCTATTTCTTATAGTGAGCTTCGTCCAGACACTCGTTATGTGCTTATGGATGGTTCCATCATACAGT 9001 sars202 CTTATGGATGGTTCCATCATACAGTTTCCTAACACTTACCTGGAGGGTTCTGTTAGAGTAGTAACAACTT 9046 sars203 GGTTCTGTTAGAGTAGTAACAACTTTTGATGCTGAGTACTGTAGACATGGTACATGCGAAAGGTCAGAAG 9091 sars204 CATGGTACATGCGAAAGGTCAGAAGTAGGTATTTGCCTATCTACCAGTGGTAGATGGGTTCTTAATAATG 9136 sars205 AGTGGTAGATGGGTTCTTAATAATGAGCATTACAGAGCTCTATCAGGAGTTTTCTGTGGTGTTGATGCGA 9181 sars206 GGAGTTTTCTGTGGTGTTGATGCGATGAATCTCATAGCTAACATCTTTACTCCTCTTGTGCAACCTGTGG 9226 sars207 TTTACTCCTCTTGTGCAACCTGTGGGTGCTTTAGATGTGTCTGCTTCAGTAGTGGCTGGTGGTATTATTG 9271 sars208 TCAGTAGTGGCTGGTGGTATTATTGCCATATTGGTGACTTGTGCTGCCTACTACTTTATGAAATTCAGAC 9316 sars209 GCCTACTACTTTATGAAATTCAGACGTGTTTTTGGTGAGTACAACCATGTTGTTGCTGCTAATGCACTTT 9361 sars210 CATGTTGTTGCTGCTAATGCACTTTTGTTTTTGATGTCTTTCACTATACTCTGTCTGGTACCAGCTTACA 9406 sars211 ATACTCTGTCTGGTACCAGCTTACAGCTTTCTGCCGGGAGTCTACTCAGTCTTTTACTTGTACTTGACAT 9451 sars212 TCAGTCTTTTACTTGTACTTGACATTCTATTTCACCAATGATGTTTCATTCTTGGCTCACCTTCAATGGT 9496 sars213 TCATTCTTGGCTCACCTTCAATGGTTTGCCATGTTTTCTCCTATTGTGCCTTTTTGGATAACAGCAATCT 9541 sars214 GTGCCTTTTTGGATAACAGCAATCTATGTATTCTGTATTTCTCTGAAGCACTGCCATTGGTTCTTTAACA 9586 sars215 AAGCACTGCCATTGGTTCTTTAACAACTATCTTAGGAAAAGAGTCATGTTTAATGGAGTTACATTTAGTA 9631 sars216 ATGTTTAATGGAGTTACATTTAGTACCTTCGAGGAGGCTGCTTTGTGTACCTTTTTGCTCAACAAGGAAA 9676 sars217 TGTACCTTTTTGCTCAACAAGGAAATGTACCTAAAATTGCGTAGCGAGACACTGTTGCCACTTACACAGT 9721 sars218 GAGACACTGTTGCCACTTACACAGTATAACAGGTATCTTGCTCTATATAACAAGTACAAGTATTTCAGTG 9766 sars219 TATAACAAGTACAAGTATTTCAGTGGAGCCTTAGATACTACCAGCTATCGTGAAGCAGCTTGCTGCCACT 9811 sars220 TATCGTGAAGCAGCTTGCTGCCACTTAGCAAAGGCTCTAAATGACTTTAGCAACTCAGGTGCTGATGTTC 9856 sars221 TTTAGCAACTCAGGTGCTGATGTTCTCTACCAACCACCACAGACATCAATCACTTCTGCTGTTCTGCAGA 9901 sars222 TCAATCACTTCTGCTGTTCTGCAGAGTGGTTTTAGGAAAATGGCATTCCCGTCAGGCAAAGTTGAAGGGT 9946 sars223 TTCCCGTCAGGCAAAGTTGAAGGGTGCATGGTACAAGTAACCTGTGGAACTACAACTCTTAATGGATTGT 9991 sars224 GGAACTACAACTCTTAATGGATTGTGGTTGGATGACACAGTATACTGTCCAAGACATGTCATTTGCACAG 10036 sars225 TGTCCAAGACATGTCATTTGCACAGCAGAAGACATGCTTAATCCTAACTATGAAGATCTGCTCATTCGCA 10081 sars226 AACTATGAAGATCTGCTCATTCGCAAATCCAACCATAGCTTTCTTGTTCAGGCTGGCAATGTTCAACTTC 10126 sars227 GTTCAGGCTGGCAATGTTCAACTTCGTGTTATTGGCCATTCTATGCAAAATTGTCTGCTTAGGCTTAAAG 10171 sars228 CAAAATTGTCTGCTTAGGCTTAAAGTTGATACTTCTAACCCTAAGACACCCAAGTATAAATTTGTCCGTA 10216 sars229 ACACCCAAGTATAAATTTGTCCGTATCCAACCTGGTCAAACATTTTCAGTTCTAGCATGCTACAATGGTT 10261 sars230 TCAGTTCTAGCATGCTACAATGGTTCACCATCTGGTGTTTATCAGTGTGCCATGAGACCTAATCATACCA 10306 sars231 TGTGCCATGAGACCTAATCATACCATTAAAGGTTCTTTCCTTAATGGATCATGTGGTAGTGTTGGTTTTA 10351 sars232 GGATCATGTGGTAGTGTTGGTTTTAACATTGATTATGATTGCGTGTCTTTCTGCTATATGCATCATATGG 10396 sars233 TCTTTCTGCTATATGCATCATATGGAGCTTCCAACAGGAGTACACGCTGGTACTGACTTAGAAGGTAAAT 10441 sars234 GCTGGTACTGACTTAGAAGGTAAATTCTATGGTCCATTTGTTGACAGACAAACTGCACAGGCTGCAGGTA 10486 sars235 AGACAAACTGCACAGGCTGCAGGTACAGACACAACCATAACATTAAATGTTTTGGCATGGCTGTATGCTG 10531 sars236 AATGTTTTGGCATGGCTGTATGCTGCTGTTATCAATGGTGATAGGTGGTTTCTTAATAGATTCACCACTA 10576 sars237 TGGTTTCTTAATAGATTCACCACTACTTTGAATGACTTTAACCTTGTGGCAATGAAGTACAACTATGAAC 10621 sars238 GTGGCAATGAAGTACAACTATGAACCTTTGACACAAGATCATGTTGACATATTGGGACCTCTTTCTGCTC 10666 sars239 GACATATTGGGACCTCTTTCTGCTCAAACAGGAATTGCCGTCTTAGATATGTGTGCTGCTTTGAAAGAGC 10711 sars240 GATATGTGTGCTGCTTTGAAAGAGCTGCTGCAGAATGGTATGAATGGTCGTACTATCCTTGGTAGCACTA 10756 sars241 GGTCGTACTATCCTTGGTAGCACTATTTTAGAAGATGAGTTTACACCATTTGATGTTGTTAGACAATGCT 10801 sars242 CCATTTGATGTTGTTAGACAATGCTCTGGTGTTACCTTCCAAGGTAAGTTCAAGAAAATTGTTAAGGGCA 10846 sars243 AAGTTCAAGAAAATTGTTAAGGGCACTCATCATTGGATGCTTTTAACTTTCTTGACATCACTATTGATTC 10891 sars244 ACTTTCTTGACATCACTATTGATTCTTGTTCAAAGTACACAGTGGTCACTGTTTTTCTTTGTTTACGAGA 10936 sars245 TCACTGTTTTTCTTTGTTTACGAGAATGCTTTCTTGCCATTTACTCTTGGTATTATGGCAATTGCTGCAT 10981 sars246 CTTGGTATTATGGCAATTGCTGCATGTGCTATGCTGCTTGTTAAGCATAAGCACGCATTCTTGTGCTTGT 11026 sars247 CATAAGCACGCATTCTTGTGCTTGTTTCTGTTACCTTCTCTTGCAACAGTTGCTTACTTTAATATGGTCT 11071 sars248 ACAGTTGCTTACTTTAATATGGTCTACATGCCTGCTAGCTGGGTGATGCGTATCATGACATGGCTTGAAT 11116 sars249 ATGCGTATCATGACATGGCTTGAATTGGCTGACACTAGCTTGTCTGGTTATAGGCTTAAGGATTGTGTTA 11161 sars250 GGTTATAGGCTTAAGGATTGTGTTATGTATGCTTCAGCTTTAGTTTTGCTTATTCTCATGACAGCTCGCA 11206 sars251 TTGCTTATTCTCATGACAGCTCGCACTGTTTATGATGATGCTGCTAGACGTGTTTGGACACTGATGAATG 11251 sars252 AGACGTGTTTGGACACTGATGAATGTCATTACACTTGTTTACAAAGTCTACTATGGTAATGCTTTAGATC 11296 sars253 GTCTACTATGGTAATGCTTTAGATCAAGCTATTTCCATGTGGGCCTTAGTTATTTCTGTAACCTCTAACT 11341 sars254 TTAGTTATTTCTGTAACCTCTAACTATTCTGGTGTCGTTACGACTATCATGTTTTTAGCTAGAGCTATAG 11386 sars255 ATCATGTTTTTAGCTAGAGCTATAGTGTTTGTGTGTGTTGAGTATTACCCATTGTTATTTATTACTGGCA 11431 sars256 TACCCATTGTTATTTATTACTGGCAACACCTTACAGTGTATCATGCTTGTTTATTGTTTCTTAGGCTATT 11476 sars257 CTTGTTTATTGTTTCTTAGGCTATTGTTGCTGCTGCTACTTTGGCCTTTTCTGTTTACTCAACCGTTACT 11521 sars258 CTTTTCTGTTTACTCAACCGTTACTTCAGGCTTACTCTTGGTGTTTATGACTACTTGGTCTCTACACAAG 11566 sars259 TATGACTACTTGGTCTCTACACAAGAATTTAGGTATATGAACTCCCAGGGGCTTTTGCCTCCTAAGAGTA 11611 sars260 CAGGGGCTTTTGCCTCCTAAGAGTAGTATTGATGCTTTCAAGCTTAACATTAAGTTGTTGGGTATTGGAG 11656 sars261 AACATTAAGTTGTTGGGTATTGGAGGTAaACCATGTATCAAGGTTGCTACTGTACAGTCTAAAATGTCTG 11701 sars262 GCTACTGTACAGTCTAAAATGTCTGACGTAAAGTGCACATCTGTGGTACTGCTCTCGGTTCTTCAACAAC 11746 sars263 GTACTGCTCTCGGTTCTTCAACAACTTAGAGTAGAGTCATCTTCTAAATTGTGGGCACAATGTGTACAAC 11791 sars264 AAATTGTGGGCACAATGTGTACAACTCCACAATGATATTCTTCTTGCAAAAGACACAACTGAAGCTTTCG 11836 sars265 GCAAAAGACACAACTGAAGCTTTCGAGAAGATGGTTTCTCTTTTGTCTGTTTTGCTATCCATGCAGGGTG 11881 sars266 TCTGTTTTGCTATCCATGCAGGGTGCTGTAGACATTAATAGGTTGTGCGAGGAAATGCTCGATAACCGTG 11926 sars267 TGCGAGGAAATGCTCGATAACCGTGCTACTCTTCAGGCTATTGCTTCAGAATTTAGTTCTTTACCATCAT 11971 sars268 TCAGAATTTAGTTCTTTACCATCATATGCCGCTTATGCCACTGCCCAGGAGGCCTATGAGCAGGCTGTAG 12016 sars269 CAGGAGGCCTATGAGCAGGCTGTAGCTAATGGTGATTCTGAAGTCGTTCTCAAAAAGTTAAAGAAATCTT 12061 sars270 GTTCTCAAAAAGTTAAAGAAATCTTTGAATGTGGCTAAATCTGAGTTTGACCGTGATGCTGCCATGCAAC 12106 sars271 TTTGACCGTGATGCTGCCATGCAACGCAAGTTGGAAAAGATGGCAGATCAGGCTATGACCCAAATGTACA 12151 sars272 GATCAGGCTATGACCCAAATGTACAAACAGGCAAGATCTGAGGACAAGAGGGCAAAAGTAACTAGTGCTA 12196 sars273 AAGAGGGCAAAAGTAACTAGTGCTATGCAAACAATGCTCTTCACTATGCTTAGGAAGCTTGATAATGATG 12241 sars274 ATGCTTAGGAAGCTTGATAATGATGCACTTAACAACATTATCAACAATGCGCGTGATGGTTGTGTTCCAC 12286 sars275 AATGCGCGTGATGGTTGTGTTCCACTCAACATCATACCATTGACTACAGCAGCCAAACTCATGGTTGTTG 12331 sars276 ACAGCAGCCAAACTCATGGTTGTTGTCCCTGATTATGGTACCTACAAGAACACTTGTGATGGTAACACCT 12376 sars277 AAGAACACTTGTGATGGTAACACCTTTACATATGCATCTGCACTCTGGGAAATCCAGCAAGTTGTTGATG 12421 sars278 TGGGAAATCCAGCAAGTTGTTGATGCGGATAGCAAGATTGTTCAACTTAGTGAAATTAACATGGACAATT 12466 sars279 CTTAGTGAAATTAACATGGACAATTCACCAAATTTGGCTTGGCCTCTTATTGTTACAGCTCTAAGAGCCA 12511 sars280 CTTATTGTTACAGCTCTAAGAGCCAACTCAGCTGTTAAACTACAGAATAATGAACTGAGTCCAGTAGCAC 12556 sars281 AATAATGAACTGAGTCCAGTAGCACTACGACAGATGTCCTGTGCGGCTGGTACCACACAAACAGCTTGTA 12601 sars282 GCTGGTACCACACAAACAGCTTGTACTGATGACAATGCACTTGCCTACTATAACAATTCGAAGGGAGGTA 12646 sars283 TACTATAACAATTCGAAGGGAGGTAGGTTTGTGCTGGCATTACTATCAGACCACCAAGATCTCAAATGGG 12691 sars284 TCAGACCACCAAGATCTCAAATGGGCTAGATTCCCTAAGAGTGATGGTACAGGTACAATTTACACAGAAC 12736 sars285 GGTACAGGTACAATTTACACAGAACTGGAACCACCTTGTAGGTTTGTTACAGACACACCAAAAGGGCCTA 12781 sars286 GTTACAGACACACCAAAAGGGCCTAAAGTGAAATACTTGTACTTCATCAAAGGCTTAAACAACCTAAATA 12826 sars287 ATCAAAGGCTTAAACAACCTAAATAGAGGTATGGTGCTGGGCAGTTTAGCTGCTACAGTACGTCTTCAGG 12871 sars288 TTAGCTGCTACAGTACGTCTTCAGGCTGGAAATGCTACAGAAGTACCTGCCAATTCAACTGTGCTTTCCT 12916 sars289 CCTGCCAATTCAACTGTGCTTTCCTTCTGTGCTTTTGCAGTAGACCCTGCTAAAGCATATAAGGATTACC 12961 sars290 CCTGCTAAAGCATATAAGGATTACCTAGCAAGTGGAGGACAACCAATCACCAACTGTGTGAAGATGTTGT 13006 sars291 ATCACCAACTGTGTGAAGATGTTGTGTACACACACTGGTACAGGACAGGCAATTACTGTAACACCAGAAG 13051 sars292 CAGGCAATTACTGTAACACCAGAAGCTAACATGGACCAAGAGTCCTTTGGTGGTGCTTCATGTTGTCTGT 13096 sars293 TTTGGTGGTGCTTCATGTTGTCTGTATTGTAGATGCCACATTGACCATCCAAATCCTAAAGGATTCTGTG 13141 sars294 CATCCAAATCCTAAAGGATTCTGTGACTTGAAAGGTAAGTACGTCCAAATACCTACCACTTGTGCTAATG 13186 sars295 CAAATACCTACCACTTGTGCTAATGACCCAGTGGGTTTTACACTTAGAAACACAGTCTGTACCGTCTGCG 13231 sars296 AGAAACACAGTCTGTACCGTCTGCGGAATGTGGAAAGGTTATGGCTGTAGTTGTGACCAACTCCGCGAAC 13276 sars297 TGTAGTTGTGACCAACTCCGCGAACCCTTGATGCAGTCTGCGGATGCATCAACGTTTTTAAACGGGTTTG 13321 sars298 GCATCAACGTTTTTAAACGGGTTTGCGGTGTAAGTGCAGCCCGTCTTACACCGTGCGGCACAGGCACTAG 13366 sars299 TTACACCGTGCGGCACAGGCACTAGTACTGATGTCGTCTACAGGGCTTTTGATATTTACAACGAAAAAGT 13411 sars300 CTTTTGATATTTACAACGAAAAAGTTGCTGGTTTTGCAAAGTTCCTAAAAACTAATTGCTGTCGCTTCCA 13456 sars301 TAAAAACTAATTGCTGTCGCTTCCAGGAGAAGGATGAGGAAGGCAATTTATTAGACTCTTACTTTGTAGT 13501 sars302 ATTTATTAGACTCTTACTTTGTAGTTAAGAGGCATACTATGTCTAACTACCAACATGAAGAGACTATTTA 13546 sars303 ACTACCAACATGAAGAGACTATTTATAACTTGGTTAAAGATTGTCCAGCGGTTGCTGTCCATGACTTTTT 13591 sars304 CAGCGGTTGCTGTCCATGACTTTTTCAAGTTTAGAGTAGATGGTGACATGGTACCACATATATCACGTCA 13636 sars305 ACATGGTACCACATATATCACGTCAGCGTCTAACTAAATACACAATGGCTGATTTAGTCTATGCTCTACG 13681 sars306 TGGCTGATTTAGTCTATGCTCTACGTCATTTTGATGAGGGTAATTGTGATACATTAAAAGAAATACTCGT 13726 sars307 GTGATACATTAAAAGAAATACTCGTCACATACAATTGCTGTGATGATGATTATTTCAATAAGAAGGATTG 13771 sars308 ATGATTATTTCAATAAGAAGGATTGGTATGACTTCGTAGAGAATCCTGACATCTTACGCGTATATGCTAA 13816 sars309 CTGACATCTTACGCGTATATGCTAACTTAGGTGAGCGTGTACGCCAATCATTATTAAAGACTGTACAATT 13861 sars310 AATCATTATTAAAGACTGTACAATTCTGCGATGCTATGCGTGATGCAGGCATTGTAGGCGTACTGACATT 13906 sars311 CAGGCATTGTAGGCGTACTGACATTAGATAATCAGGATCTTAATGGGAACTGGTACGATTTCGGTGATTT 13951 sars312 GGAACTGGTACGATTTCGGTGATTTCGTACAAGTAGCACCAGGCTGCGGAGTTCCTATTGTGGATTCATA 13996 sars313 GCGGAGTTCCTATTGTGGATTCATATTACTCATTGCTGATGCCCATCCTCACTTTGACTAGGGCATTGGC 14041 sars314 TCCTCACTTTGACTAGGGCATTGGCTGCTGAGTCCCATATGGATGCTGATCTCGCAAAACCACTTATTAA 14086 sars315 CTGATCTCGCAAAACCACTTATTAAGTGGGATTTGCTGAAATATGATTTTACGGAAGAGAGACTTTGTCT 14131 sars316 ATTTTACGGAAGAGAGACTTTGTCTCTTCGACCGTTATTTTAAATATTGGGACCAGACATACCATCCCAA 14176 sars317 ATTGGGACCAGACATACCATCCCAATTGTATTAACTGTTTGGATGATAGGTGTATCCTTCATTGTGCAAA 14221 sars318 ATAGGTGTATCCTTCATTGTGCAAACTTTAATGTGTTATTTTCTACTGTGTTTCCACCTACAAGTTTTGG 14266 sars319 CTGTGTTTCCACCTACAAGTTTTGGACCACTAGTAAGAAAAATATTTGTAGATGGTGTTCCTTTTGTTGT 14311 sars320 TTGTAGATGGTGTTCCTTTTGTTGTTTCAACTGGATACCATTTTCGTGAGTTAGGAGTCGTACATAATCA 14356 sars321 GTGAGTTAGGAGTCGTACATAATCAGGATGTAAACTTACATAGCTCGCGTCTCAGTTTCAAGGAACTTTT 14401 sars322 CGCGTCTCAGTTTCAAGGAACTTTTAGTGTATGCTGCTGATCCAGCTATGCATGCAGCTTCTGGCAATTT 14446 sars323 CTATGCATGCAGCTTCTGGCAATTTATTGCTAGATAAACGCACTACATGCTTTTCAGTAGCTGCACTAAC 14491 sars324 CATGCTTTTCAGTAGCTGCACTAACAAACAATGTTGCTTTTCAAACTGTCAAACCCGGTAATTTTAATAA 14536 sars325 CTGTCAAACCCGGTAATTTTAATAAAGACTTTTATGACTTTGCTGTGTCTAAAGGTTTCTTTAAGGAAGG 14581 sars326 TGTCTAAAGGTTTCTTTAAGGAAGGAAGTTCTGTTGAACTAAAACACTTCTTCTTTGCTCAGGATGGCAA 14626 sars327 ACTTCTTCTTTGCTCAGGATGGCAACGCTGCTATCAGTGATTATGACTATTATCGTTATAATCTGCCAAC 14671 sars328 ACTATTATCGTTATAATCTGCCAACAATGTGTGATATCAGACAACTCCTATTCGTAGTTGAAGTTGTTGA 14716 sars329 TCCTATTCGTAGTTGAAGTTGTTGATAAATACTTTGATTGTTACGATGGTGGCTGTATTAATGCCAACCA 14761 sars330 ATGGTGGCTGTATTAATGCCAACCAAGTAATCGTTAACAATCTGGATAAATCAGCTGGTTTCCCATTTAA 14806 sars331 ATAAATCAGCTGGTTTCCCATTTAATAAATGGGGTAAGGCTAGACTTTATTATGACTCAATGAGTTATGA 14851 sars332 TTTATTATGACTCAATGAGTTATGAGGATCAAGATGCACTTTTCGCGTATACTAAGCGTAATGTCATCCC 14896 sars333 CGTATACTAAGCGTAATGTCATCCCTACTATAACTCAAATGAATCTTAAGTATGCCATTAGTGCAAAGAA 14941 sars334 TTAAGTATGCCATTAGTGCAAAGAATAGAGCTCGCACCGTAGCTGGTGTCTCTATCTGTAGTACTATGAC 14986

In addition, choose four tobacco mosaic virus (TMV) specific probes as negative control probe, sequence derives from tmv cdna group (GenBank sequence number: NC_001367).As shown in table 2

Table 2 tobacco mosaic virus (TMV) Te Yi Tan Zhen Xu row Xu Xu row initiation site tmv1 ATATTCTTAAGTATGTGTGCAAAACTTACTTCCCGGCCTCTAATAGAGAGGTTTAC ATGAAGGAGTTTTT 895tmv2 TCTGCTGCGGTGTCGAATCTCGTCAAGATCCTCAAAGATACAGCTGCTATTGACCT TGAAACCCGTCAAA 2220tmv3 CTTCTTAAAGGAGTTAAGCTTATTGATAGTGGATACGTCTGTTTAGCCGGTTTGGT CGTCACGGGCGAGT 5065tmv4 TGACAATTGCAGAGGAGGTGTGAGCGTGTGTCTGGTGGACAAAAGGATGGAAAGAG CCGACGAGGCCACT 5145

We have also chosen transmissible gastro-enteritis virus and birds infectious bronchitis virus specific probe sequence totally 12 probes, and sequence sees Table 3:

Table 3 porcine transmissible gastroenteritis virus and avian infectious bronchitis virus-specific probe sequences TGEV1 CTCTTATGGTGGTGCTTCAGTTTGTATTTATTGCAGATGCCATGTTGAACATCCTGCTATTGATGGATTATGEV1_rc TAATCCATCAATAGCAGGATGTTCAACATGGCATCTGCAATAAATACAAACTGAAGCACCACCATAAGAGTGEV2 AGGTGTTATAACACTTGACAACCAAGATCTTAATGGCAATTTCTACGATTTCGGCGATTTCGTGAAGACTTGEV2_rc AGTCTTCACGAAATCGCCGAAATCGTAGAAATTGCCATTAAGATCTTGGTTGTCAAGTGTTATAACACCTTGEV3 GATAATGGTTGTTTGATGGGATGGGACTATCCTAAGTGTGACCGTGCTTTACCTAATATGATTAGAATGGTGEV3_rc CCATTCTAATCATATTAGGTAAAGCACGGTCACACTTAGGATAGTCCCATCCCATCAAACAACCATTATCAIBV1 AACACTAGAAATGCTTCTGTAGTTATTGGAACAACCAAGTTTTATGGCGGTTGGGACAACATGTTGAGAAAIBV1_rc TTCTCAACATGTTGTCCCAACCGCCATAAAACTTGGTTGTTCCAATAACTACAGAAGCATTTCTAGTGTTAIBV2 TTATGGGTTGGGATTATCCTAAGTGTGATAGAGCAATGCCTAATTTGTTGCGTATAGCAGCATCCTTAGTAIBV2_rc ACTAAGGATGCTGCTATACGCAACAAATTAGGCATTGCTCTATCACACTTAGGATAATCCCAACCCATAAAIBV3 TATTTATGTTAAACCTGGTGGCACTAGCAGTGGTGATGCTACTACTGCTTATGCAAACAGTGTTTTTAACAIBV3_rc GTTAAAAACACTGTTTGCATAAGCAGTAGTAGCATCACCACTGCTAGTGCCACCAGGTTTAACATAAATA

We have also designed specific probe sequence totally 3 probes of the SARS complete sequence of the CUHK-W1-S that announces at Hong Kong, and sequence sees Table 4:

The specific probe sequence CUHK-W1-S-210 TACTATTAATCATACGTTTGGCAACCCTGTCATACCTTTTAAGGATGGTATTTATT TTGCTGCCACAGAGCUHK-W1-S-710 CCTTTTCACCTGCTCAAGACATTTGGGGCACGTCAGCTGCAGCCTATTTTGTTGGC TATTTAAAGCCAACCUHK-W1-S-1710 ATTAGACATTTCACCTTGCTCTTTTGGGGGTGTAAGTGTAATTACACCTGGAACAA ATGCTTCATCTGAA of the SARS of table 4 CUHK-W1-S

Certainly the SARS that is not limited to CUHK-W1-S has also comprised the specific probe designed according to the SARS sequence of other up-to-date announcement.

Choose simultaneously from the contrast as chip scanning single or arranged evenly in groups of the 40-100 bar oligonucleotide of people's gene group sequence.

660 probes of sars coronavirus TOR2 strain, 4 probes of tobacco mosaic virus (TMV), 12 probes of transmissible gastro-enteritis virus and birds infectious bronchitis virus, the SARS complete sequence probe sequence of CUHK-W1-S is totally 3 probes, at last, above-mentioned 679 probes altogether.

Embodiment 2 chip points system

Above-mentioned 679 probes are counted 680 probes approximately carry out matrix design.Synthesize by 679 probes, synthetic product is dissolved in the point sample damping fluid, and (3 * SSC), final concentration is 800ng/ul.By the gene chip sample applying instrument probe solution is put on aldehyde group modified glass chip, in humidity is 60% environment, carried out hydration 30min subsequently, so that the aldehyde radical generation ligation of the amino of probe and sheet base, thereby make probe stationary on the sheet base.

Point system rule is that every probe repeats three points, is divided into 5 matrixes, and 136 probes of each matrix, each matrix comprise 38 positive coordinate point, forms the dot matrix of 25 row * 14 row.As shown in Figure 1 in the Figure of description, Fig. 1 is the full genome chip point sample of a sars coronavirus synoptic diagram, and every group of triple junction is a probe.The positive coordinate point of grey color dot shown in the A among the figure, the black triple junction shown in the B is a detection probes.Embodiment 3 sample process and mark

For the sample to be tested that may contain sars coronavirus, use ordinary method by Trizol reagent and extract general RNA, the nine base random primers (GTTTCCCAGTCACGATCNNNNNNNNN) that have the specificity end with 5 ' end carry out reverse transcription at random, utilize specificity end (GTTTCCCAGTCACGATC) to carry out specific amplification then, in the process of amplification, mix the dCTP of Cy3 or Cy5 mark so that amplified production has fluorescent signal.

Operating process is as follows:

The RT:(20ul system),

The 1ul random primer (GTTTCCCAGTCACGATCNNNNNNNNN, 100pmol/ul)

2ulRNA template (1-2ug general RNA)

1ul?dNTP(10mM)

8ul?DEPC-ddH ₂O

Mixing, 70 ℃ of 5min, ice bath is centrifugal fast rapidly

Add the synthetic Buffer of 4ul5* first chain

2ul0.1M?DTT

1ul RNA enzyme inhibitors (40U/ul)

25 ℃ of 5min add the 1ul ThermoScript II, mixing, and 25 ℃ of 10min, 42 ℃ of 1h, 70 ℃ of 15min stop.

The PCR:A:(20ul system)

RT product 1ul

10*PCR?Buffer?2ul

Random primer 1ul (GTTTCCCAGTCACGATCNNNNNNNNN, 100pmol/ul)

dNTP(10mM)0.6ul

ddH ₂O?14.4ul

Taq(5U/ul)1ul

95?5min-94?1min-30?5min-72?3min-5cycles-72?5min

The B:(50ul system)

PCR-A product 5ul

10*PCR?Buffer?5ul

Special primer 2ul (GTTTCCCAGTCACGATC, 100pmol/ul)

dNTP(10mM)1ul

Cy3/5-dCTP(1mM)1ul

ddH ₂O?34ul

Taq(5U/ul)2ul

95 5min-94 50s-40 1min-50 1min-72 1min-30cycles-72 5min embodiment, 4 chip hybridizations:

Chip is carried out pre-treatment, promptly use 0.5%SDS and rinsed with deionized water chip respectively, dry standby.

The 50ul testing sample amplified production that mark is good carries out vacuum freezedrying, 20ul dissolves again with 1 * hybridization buffer, 95 ℃ of sex change 5min, ice bath is got the 20ul hybridization solution and is dripped in chip surface, and covers with cover glass rapidly, 37 ℃ of temperature were bathed after 1 hour, use 0.5%SDS and rinsed with deionized water chip 5min respectively, dry the back, and utilize analysis software to calculate the signal value and analysis yin and yang attribute result of each bar probe by gene chip scanning instrument scanning chip.Embodiment 5 interpretations of result:

The coronavirus that has homology through the standard strain (serum of clinical patient and respiratory secretions sample) of gathering sars coronavirus and other and sars coronavirus, comprise infectious bronchitis virus (Infectious bronchitis virus), bovine coronavirus (viral standard substance) such as (Bovine coronavirus), adopt normal human serum and respiratory secretions sample as negative experimental subjects, repeatedly test according to above technological line, obtain experimental result and be:

1, negative control probe all presents negative signal in the results of hybridization;

2, in the standard strain results of hybridization in the sars coronavirus detection probes positive rate be 83%-92%;

3, in other coronavirus results of hybridization relevant with sars coronavirus in the sars coronavirus detection probes positive rate be 37%-58%;

4, in the negative experimental subjects results of hybridization in the sars coronavirus detection probes positive rate be 1%-16%.

Obtain hybrid experiment criterion as a result by above experiment, promptly under the prerequisite that negative probe reaction is negative, 80% above detection probes positive as a result the time decidable testing sample have SARS virus; The 30%-80% detection probes positive as a result the time decidable testing sample have the virus relevant with SARS virus; 30% following detection probes positive as a result the time decidable testing sample do not have the virus relevant with SARS virus.

Claims

1. the invention describes a kind of gene chip, it is characterized in that being fixed on the whole genome sequence that on-chip probe matrix has covered sars coronavirus.

2. the whole genome sequence of the described sars coronavirus of claim 1 is meant the sars coronavirus strain that GenBank has announced or the whole genome sequence of hypotype.

3. sars coronavirus strain described in the claim 2 or hypotype comprise SARS coronavirus TOR2 (GenBank sequence number: NC 004718/AY274119), SARS coronavirus HKU-39849 (GenBank sequence number: AY278491), SARS coronavirus CUHK-WI (GenBank sequence number: AY278554), SARScoronavirus Urbani (GenBank sequence number: AY278741), SARS coronavirus BJ01 (GenBank sequence number: AY278488), SARS coronavirus BJ02 (GenBank sequence number: AY278487), SARScoronavirus BJ03 (GenBank sequence number: AY278490), SARS coronavirus BJ04 (the GenBank sequence number: AY279354), SARS coronavirus GZ01 (GenBank sequence number: AY278489).

Probe feature on the described gene chip of claim 1 be every 40-80 base sequence from the full genome of sars coronavirus as a probe sequence, the adjacent probe sequence repeats 5-30 base.

5. the probe feature on the described gene chip of claim 1 is meant with 70 base sequences especially as a probe sequence, and the adjacent probe sequence repeats the pattern of 25 bases.

6. one of purposes of the described gene chip of claim 1 is to be used to detect sars coronavirus.

7. the described detected object of claim 6 can comprise from clinical sample, animals and plants sample, food water source sample and water surroundings such as the blood of human body and secretory product and atmospheric environment sample or the like.

8. two of the purposes of the described gene chip of claim 1 is variation and the hypotypes that are used to monitor sars coronavirus.

9. the described monitoring target of claim 8 can comprise from clinical sample, animals and plants sample, food water source sample and water surroundings such as the blood of human body and secretory product and atmospheric environment sample or the like.

10. three of the purposes of the described gene chip of claim 1 is to be used to differentiate sars coronavirus and other coronavirus.

11. the described discriminating object of claim 10 can comprise from clinical sample, animals and plants sample, food water source sample and water surroundings such as the blood of human body and secretory product and atmospheric environment sample or the like.