Patent Application: US-9211093-A

Abstract:
the ability to rapidly detect wild polioviruses in clinical specimens is a major concern for the world - wide eradication of polioviruses . this report describes a method of detecting polioviruses of all three serotypes from viral isolates of clinical specimens using a pair of degenerate pcr primers . this primer set , which uses deoxyinosine residues to compensate for third position mismatches , recognizes nucleotide sequences near the receptor binding site of polioviruses . these sequences are unique to polioviruses and are absolutely conserved at the amino acid level . as a result , these pcr primers do not recognize nonpoliovirus enteroviruses . all poliovirus serotypes tested positive . all 14 prototype strains of nonpoliovirus enteroviruses tested negative . this &# 34 ; pan - poliovirus &# 34 ; degenerate pcr primer set will be useful in rapidly diagnosing poliovirus infections from world - wide clinical specimens .

Description:
the present invention may be understood more readily by reference to the following detailed description of specific embodiments and the examples , tables and sequence listing included therein . as used in the claims , &# 34 ; a &# 34 ; can mean one or more . the acronym &# 34 ; pcr &# 34 ; is used interchangeably with &# 34 ; polymerase chain reaction .&# 34 ; the acronym &# 34 ; rt / pcr &# 34 ; is used interchangeably with &# 34 ; reverse transcriptase - polymerase chain reaction .&# 34 ; the present invention provides an isolated nucleic acid comprising the nucleotide sequence set forth in the sequence listing as seq id no : 1 . the consensus sequence set forth in seq id no : 1 denotes the possible combinations of nucleotides that are found in seq id nos : 5 - 12 . the present invention also provides an isolated nucleic acid complementary to the nucleotide sequence set forth in the sequence listing as seq id no : 1 . also provided is a composition comprising a nucleic acid comprised of the nucleotide sequence set forth in the sequence listing as seq id no : 1 and a nucleic acid that is capable of selectively hybridizing with that nucleic acid . by &# 34 ; selectively hybridizing &# 34 ; is meant that the nucleic acid does not hybridize with sequences from other enteroviruses to prevent adequate positive hybridization with nucleic acids from a poliovirus . the present invention further provides an isolated nucleic acid comprising the nucleotide sequence set forth in the sequence listing as seq id no : 2 . the consensus sequence set forth in seq id no : 2 denotes the possible combinations of nucleotides that are found in seq id nos : 13 - 20 . the present invention also provides an isolated nucleic acid complementary to the nucleotide sequence set forth in the sequence listing as seq id no : 2 . also provided is a composition comprising a nucleic acid comprised of the nucleotide sequence set forth in the sequence listing as seq id no : 2 and a nucleic acid that is capable of selectively hybridizing with that nucleic acid . in a further embodiment , the present invention provides a primer for the detection of a poliovirus in a sample utilizing a nucleic acid amplification technique , comprising the nucleotide sequence set forth in the sequence listing as seq id no : 1 . the primer of the present invention can be utilized as a degenerate primer comprised of one or more of the possible combinations of nucleotide sequences set forth in the sequence listing as seq id nos : 5 - 12 . it is contemplated by the present invention that the nucleic acids described herein can be utilized in any of a number of nucleic acid detection techniques including , but not limited to polymerase chain reaction , isothermal dna amplification , etc . likewise , the nucleic acid set forth in seq id no : 1 can be used as a probe for detecting or capturing a nucleic acid which hybridizes with the nucleic acid of seq id no : 1 . the present invention also provides a primer for the detection of a poliovirus in a sample utilizing a nucleic acid amplification technique , comprising the nucleotide sequence set forth in the sequence listing as seq id no : 2 . the primer set forth in seq id no : 2 can be utilized as a degenerate primer comprised of one or more of the possible combinations of nucleotide sequences set forth in the sequence listing as seq id nos : 13 - 20 . similarly , the nucleic acid set forth in seq id no : 2 can be used as a probe for detecting or capturing a nucleic acid which hybridizes with the nucleic acid of seq id no : 2 . it is also contemplated by the present invention that any of the primers or probes described herein can be labeled or tagged for use in e . g ., chemiluminescent or fluorescent detection systems . in a further embodiment , the present invention provides a method for detecting the presence or absence of a poliovirus in a sample containing nucleic acids comprising the steps of : a ) amplifying the nucleic acids from the sample with a primer pair comprised of a primer comprising the nucleotide sequence set forth in the sequence listing as seq id no : 1 and a suitable upstream primer ; b ) determining the presence or absence of a nucleic acid from poliovirus , thereby detecting the presence or absence of poliovirus in the sample . as used herein , a &# 34 ; suitable upstream primer &# 34 ; is any of the possible primers which can be designed from known sequences for the vp1 gene located upstream ( i . e . 5 &# 39 ;) of position number 2914 following the numbering system of kew et al ., ( 1990a ). examples of a suitable upstream primer include , but are not limited to the panpv 2s and panpv 13s primers described herein . in one embodiment , the invention provides a method for detecting the presence or absence of a poliovirus in a sample containing nucleic acids comprising the steps of : a ) amplifying the nucleic acids from the sample with a primer pair comprised of a primer comprising the nucleotide sequence set forth in the sequence listing as seq id no : 1 and a primer comprising the nucleotide sequence set forth in the sequence listing as seq id no : 2 ; b ) determining the presence or absence of a nucleic acid from poliovirus , thereby detecting the presence or absence of poliovirus in the sample . also provided by the present invention is a kit for detecting a nucleic acid of a poliovirus by nucleic acid amplification comprising a primer comprised of the nucleotide sequence set forth in the sequence listing as seq id no : 1 and a suitable upstream primer . in one embodiment the invention provides a kit for detecting a nucleic acid of a poliovirus by nucleic acid amplification comprising a primer comprised of the nucleotide sequence set forth in the sequence listing as seq id no : 1 and a primer comprising the nucleotide sequence set forth in the sequence listing as seq id no : 2 . the nucleic acid specific for each genotype of poliovirus can be detected utilizing a nucleic acid amplification technique , such as polymerase chain reaction ( pcr ) as taught in the examples described herein . alternatively , the nucleic acid is detected utilizing direct hybridization or by utilizing a restriction fragment length polymorphism . for example , the present invention contemplates a : method of detecting the presence of all poliovirus genotypes to the exclusion of nonpolio enteroviruses . pcr primers which hybridize only with nucleic acids specific for a target sequence ( e . g ., seq id no : 3 ) of the poliovirus can be utilized . the presence of amplification indicates the presence of the virus . alternatively , the poliovirus can be detected by directly hybridizing the target sequence with a nucleic acid probe selective for the specific target sequence of the poliovirus . polymerase chain reaction ( pcr ) and rt / pcr are examples of techniques that amplify specific nucleic acid sequences with remarkable efficiency . repeated cycles of denaturation , primer annealing and extension carried out with a polymerase , e . g ., the heat stable enzyme taq polymerase , leads to exponential increases in the concentration of desired nucleic acid sequences . given a knowledge of the appropriate target nucleic acid sequence of the poliovirus as provided by the present invention , synthetic oligonucleotides can be prepared which are complementary to all of the possible sequences in the poliovirus of interest . each oligonucleotide primer species is complementary to one of the possible poliovirus specific degenerate sequences of interest . the nucleic acid can be denatured at high temperatures ( e . g ., 95 ° c .) and then reannealed in the presence of a large molar excess of the oligonucleotides . the oligonucleotides , oriented with their 3 &# 39 ; ends pointing towards each other , hybridize to opposite strands of the target sequence and prime enzymatic extension along the nucleic acid template in the presence of the four deoxyribonucleotide triphosphates . the end product is then denatured again for another cycle . after this three - step cycle has been repeated several times , amplification of a nucleic acid segment by more than one million - fold can be achieved . the resulting nucleic acid may then be directly detected by any of a number of methods well known in the art ( for example , southern blotting using poliovirus specific probes as descried above ). in general , primers for pcr are usually about 20 bp in length and the preferable range is from 15 - 25 bp . better amplification is obtained when both primers are the same length and with roughly the same nucleotide composition . denaturation of strands usually takes place at about 94 ° c . and extension from the primers is usually at about 72 ° c . the annealing temperature varies according to the sequence under investigation . examples of reaction times are : 20 mins denaturing ; 35 cycles of 2 min , and 1 min , 1 min for annealing , extension and denaturation ; respectively and finally , a 5 min extension step . the present invention is more particularly described in the following examples which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art . poliovirus isolates ( tables 1 and 2 ) have been previously characterized by neutralization with hyperimmune equine sera and partial genomic sequencing ( rico - hesse et al ., 1987 ; kew et al 1990a ; de et al ., in preparation ). vaccine - related strains were also positively identified by pcr using the sabin strain - specific primer pairs ( yang et al ., 1991 ). fourteen human nonpolio enteroviruses were identified by confirmation of serotype with monotypic neutralizing polyclonal antibodies . viruses were propagated in hela or rd monolayers to produce high - titer inoculation stocks . table 1______________________________________vaccine - related poliovirus genotypes detected by pan - poliopcrtype 10584 / gut91 0246 / gut90 9825 / usa89 9703 / els899360 / ven89 9240 / hon89 2800 / h0n91 8315 / mex886258 / mor85 5498 / usa84type 20636 / els91 0042 / els90 9897 / gut90 0078 / per899818 / per89 9519 / usa89 8370 / per88 8018 / gut877653 / soa86 7170 / mex86 6700 / hon86 7837 / per846886 / gut83type 31063 / usa91 0644 / hon91 0642 / els91 0405 / gut900040 / els90 0131 / mex89 0044 / gut89 9896 / gut899442 / nic89 9441 / gut89 8774 / trt88 1339 / chn898239 / gut87 6880 / col86______________________________________ table 2______________________________________wild poliovirus genotypes detected by pan - polio pcrtype 10006 / chn89 0109 / chn86 0032 / chn91 0124 / chn910285 / ino86 0289 / por87 0427 / ssr91 0440 / ssr900467 / col89 0941 / srl87 0955 / srl88 1184 / rom911187 / rom91 1338 / chn89 1607 / soa88 2609 / eth912611 / pak90 2662 / col87 2758 / svn89 2786 / vtn902854 / hon91 3638 / chn85 3643 / chn91 3647 / chn913677 / cyp92 3706 / maa92 3907 / phl91 3940 / tha926224 / zim85 6536 / nep86 6700 / tur90 6701 / tur906750 / sen86 7054 / ind86 7169 / bul91 7362 / pak917377 / bol86 8223 / gut87 8425 / isr88 8644 / ind918645 / ind92 8649 / ind91 8771 / oma88 9366 / saa899475 / zai89 05145 / uzb88 07470 / tog92 09323 / mog9111231 / 11236 / egy91 11267 / egy91 11270 / egy91egy9115949 / fra89 16834 / tur90 16838 / tur90 18641 / pak9118655 / pak91type 20290 / tur73 0291 / tur73 0295 / isr78 0297 / kuw780298 / egy79 0302 / yug81 0305 / ira71 1155 / alb911534 / ind82 2613 / pak89 2710 / ken71 6876 / col867079 / ind82 7354 / pak91 8650 / ind91 8654 / ind9105144 / uzb88 11263 / egy91 18637 / pak91 18638 / pak91type 30314 / rom80 0380 / mex90 0426 / ssr90 0672 / oma912615 / mol90 2619 / mol90 2723 / tur90 2728 / arm902731 / urz89 4075 / arm90 6184 / fin84 7095 / ind867350 / pak91 7377 / bol86 8178 / ven87 8668 / ind918670 / ind91 9035 / bra88 9259 / tun88 05141 / uzb8805142 / uzb88 11246 / egy91 11252 / egy91 11257 / egy9115952 / fra90 16837 / tur90 18643 / pak91 18653 / pak91______________________________________ synthetic oligodeoxynucleotides were prepared , purified , and analyzed as described ( yang et al ., 1991 ). the degenerate primers used for amplifying poliovirus are : the numbers in parentheses indicate the genomic intervals matching the primers ( a = antigenome polarity primer ; s = sense or genome polarity primer ; following the numbering system of kew et al . ( 1990a ). primer panpv 1a as used herein refers to the consensus sequence set forth in the sequence listing as seq id no : 1 . the eight possible primer species for the consensus sequence seq id no : 1 are set forth in the sequence listing as seq id nos : 5 - 12 . primer panpv 13s as used herein refers to the consensus sequence set forth in the sequence listing as se0 id no : 2 . the eight possible primer species for the consensus sequence seq id no : 2 are set forth in the sequence listing as seq id nos : 13 - 20 . primer panpv 2s as used herein refers to the consensus sequence set forth in the sequence listing as seq id no : 21 . in vitro amplification by pcr was performed as described previously ( yang et al ., 1992 ). amplification reactions were carried out in 50 μl reaction mixtures containing 1 μl of each individual virus tissue culture lysate in 50 mm tris - hcl ( ph 8 . 3 ), 70 mm kcl , 5 mm mgcl 2 , 10 mm dithiothreitol , 10 pmol of each primer , 200 μm each of datp , dctp , dctp , dttp ( pharmacia ), 0 . 5 % np - 40 , 10 u placenta ribonuclease inhibitor ( boehringer mannheim biochemicals , indianapolis , ind . ), 2 . 5 u amv reverse transcriptase ( boehringer mannheim ), and 2 . 5 u of taq dna polymerase ( perkin elmer - cetus , norwalk , conn .). the reaction mixtures were prepared , excluding the ribonuclease inhibitor , amv reverse transcriptase , and taq dna polymerase , overlaid with mineral oil , heated for 5 min at 95 ° c . to release the virion rna and chilled on ice . the enzymes were then added and the samples incubated at 42 ° c . for 30 min before 30 cycles of programmed amplification ( denaturation : 94 ° c ., 1 min ; annealing : 42 ° c ., 1 min ; extension : 60 ° c ., 1 min ) in a dna thermal cycler ( perkin elmer - cetus ). conditions for polyacrylamide gel electrophoresis , and detection of amplified products by ethidium bromide staining were as described ( yang et al ., 1991 ). the amino acid alignment in the capsid protein region ( palmenberg , 1989 ) of a wide variety of picornaviruses was used to find poliovirus amino acid residues that were near residues suspected to be involved in receptor attachment / recognition and conserved among only picornaviruses . a 7 amino acid sequence in vp1 ( nnghaln , as set forth in the sequence listing as seq id no : 3 ) that was unique to only polioviruses was chosen as a possible pcr primer site . a degenerate pcr primer ( anti - sense ; designated as panpv 1a ) was designed using this sequence information as well as possible nucleotide incorporation at the first and third codon positions due to codon degeneracy . deoxyinosine residues were used in those positions where 3 or 4 different nucleotides were possible . this was done to keep the number of possible primer species at a minimum . since there are 8 possible species of panpv 1a ( seq id nos : 5 - 12 ), a concentration of 80 picomoles was used per reaction ( 10 pm / primer species ). similarly , another 7 amino acid ( ftysrfd , as set forth in the sequence listing as seq id no : 4 ) sequence was located upstream from panpv 1a and chosen as the sense pcr primer site ( designated as panpv 2s ). this pcr primer set yields an 83 bp pcr product . we generally use primer pairs that are closely spaced (& lt ; 250 nucleotides ) along the template because amv reverse transcriptase has relatively low processivity ( berger et al ., 1983 ). diagnostic sensitivities are generally improved by reducing the lengths of the cdna transcripts required to initiate the chain reactions . the panpv 1a / 2s primer pair was first tested against different vaccine - related poliovirus genotypes since they would have the least amount of nucleotide sequence heterogeneity . one microliter of each infected tissue culture lysate was amplified in an rt / pcr reaction mixture . after 30 amplification cycles , dna products were separated by electrophoresis on 12 % polyacrylamide gels and visualized the ethidium bromide staining . a single 83 bp product was seen from all samples . the remaining vaccine - related isolates also yielded this same 83 bp product . a wide range of genotypes from around the world and representing all three serotypes was also tested . all of the isolates tested positive ( table 1 ). poliovirus genomes evolve rapidly during replication in humans . however , the 7 amino acid sequences set forth in seq id no : 3 were found to be absolutely conserved in the 23 complete vp1 nucleotide sequences presently in the centers for disease control and prevention data base . an 83 bp pcr product was found when 13 wild type 1 poliovirus isolates were tested with the panpv 1a / 2s primer set . subsequently , all 120 poliovirus isolates ( table 2 ) were found to be positive . this : suggests that the nnghaln amino acid sequence is conserved among all polioviruses . however , in six isolates a weak pcr product was detected . this was thought to be a result of poor primer homology due to the upstream panpv 2s primer . further analysis found that in some instances the minus 3 and minus 8 positions from the 3 &# 39 ;- terminus of the 2s primer do not correctly match the virus sequence ( for example isolate 9288 / mexvp1 has a c at positions minus 3 and minus 8 ). proper annealing at the 3 &# 39 ; end of the primer is known to be very important to the fidelity of taq polymerase in extending the sequence . panpv 2s was re - designed to contain a t or c at the minus 3 position and an a or c at the minus 8 position to see if this would increase the yield of the pcr product ( since the nucleotide sequences for these isolates was unknown ). a deoxyinosine residue was also introduced at the minus 6 position to reduce the number of primer species . this new primer , panpv 13s was used along with panpv 1a to amplify the isolates which gave the weakest priming . the results showed a stronger pcr product when this new primer was used , as compared to the original panpv 2s primer . this indicates that the weaker pcr product found with a few virus isolates is due to poor annealing of the panpv 2s primer and noir to weak annealing of panpv 1a . the primary need for developing poliovirus specific pcr is to rapidly distinguish poliovirus cases of acute flaccid paralysis ( afp ) from npev cases of afp . this is becoming increasingly important in the surveillance of afp cases in those areas of the world that have essentially eliminated wild poliovirus . when the panpv 1a / 2s primer pair was tested against a wide range of nonpoliovirus enteroviruses , no amplification products were detected . these data supported our early hypothesis that the nnghaln amino acid sequence in vp1 is unique among all polioviruses . to prove that each isolate tested did indeed contain viable virus , these same isolates were tested with an enterovirus specific primer pair ( ev / pcr - 1 & amp ; ev pcr - 2 ). this primer pair recognizes highly conserved nucleotide sequences in the 5 &# 39 ; noncoding region in a wide range of enteroviruses ( yang et al ., 1992 ). the expected 114 bp pcr product of the enterovirus primer pair was identified in all of the isolates tested . this indicates that the panpv 1a / 2s primer pair is specific for polioviruses and does not recognize other enteroviruses . virus isolates are presently typed as npev by their ability to replicate in the presence of neutralizing antisera specific to polioviruses . however , low titers of poliovirus can be masked by the presence of higher npev titers . such a case was suspected due to uncharacteristic growth in tissue culture during typing . two suspected poliovirus cases originally typed as npev were tested with the panpv 1a / 2s primer set . the 83 bp pcr product characteristic of the primer pair was detected and clearly indicated the presence of poliovirus . a serotype 1 poliovirus was eventually isolated from this sample . this shows that the pan - poliovirus pcr primer set would be very useful in rapidly distinguishing poliovirus from npev in samples containing both virus types . throughout this application , various publications are referenced by author and year . the disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains . a complete reference citation is provided below . although the present process has been described with reference to specific details of certain embodiments thereof , it is not intended that such details should be regarded as limitations upon the scope of the invention except as and to the extent that they are included in the accompanying claims . abraham , r ., chonmaitree , t ., mccombs , j ., prabhakar , b ., lo verde , p . t . and ogra , p . l . 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( 1992 ). genotype - specific in vitro amplification of sequences of the wild type 3 polioviruses from mexico and guatemala . virus research 24 , 277 - 296 . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 21 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( iv ) anti - sense : yes ( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 1 .. 20 ( d ) other information : / product = &# 34 ; synthetic dna &# 34 ;/ note = &# 34 ; in the primer sequence submitted n = inosine ; r = aor g ; and nucleotide # for the entire sequence is2915 - 2934 .&# 34 ;( xi ) sequence description : seq id no : 1 : ttnanngcrtgnccrttrtt20 ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( iv ) anti - sense : no ( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 1 .. 20 ( d ) other information : / product = &# 34 ; synthetic dna &# 34 ;/ note = &# 34 ; in the primer sequence submitted n = inosine ; m = aor c ; y = t or c ; and nucleotide # for the entire sequenceis 2852 - 2871 .&# 34 ;( xi ) sequence description : seq id no : 2 : ttcacmtantcnmgnttyga20 ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 7 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 3 : asnasnglyhisalaleuasn15 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 7 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 4 : phethrtyrserargpheasp15 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( iv ) anti - sense : yes ( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 1 .. 20 ( d ) other information : / product = &# 34 ; synthetic dna &# 34 ;/ note = &# 34 ; in the primer sequence submitted n = inosine ; andnucleotide # for the entire sequence is 2915 - 2934 .&# 34 ;( xi ) sequence description : seq id no : 5 : ttnanngcgtgnccgttgtt20 ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( iv ) anti - sense : yes ( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 1 .. 20 ( d ) other information : / product = &# 34 ; synthetic dna &# 34 ;/ note = &# 34 ; in the primer sequence submitted n = insoine ; andnucleotide # for the entire sequence is 2915 - 2934 .&# 34 ;( xi ) sequence description : seq id no : 6 : ttnanngcatgnccgttgtt20 ( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( iv ) anti - sense : yes ( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 1 .. 20 ( d ) other information : / product = &# 34 ; synthetic dna &# 34 ;/ note = &# 34 ; in the primer sequence submitted n = inosine ; andnucleotide # for the entire sequence is 2915 - 2934 .&# 34 ;( xi ) sequence description : seq id no : 7 : ttnanngcatgnccattgtt20 ( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( iv ) anti - sense : yes ( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 1 .. 20 ( d ) other information : / product = &# 34 ; synthetic dna &# 34 ;/ note = &# 34 ; in the primer sequence submitted n = inosine ; andnucleotide # for the entire sequence is 2915 - 2934 .&# 34 ;( xi ) sequence description : seq id no : 8 : ttnanngcatgnccattatt20 ( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( iv ) anti - sense : yes ( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 1 .. 20 ( d ) other information : / product = &# 34 ; synthetic dna &# 34 ;/ note = &# 34 ; in the primer sequence submitted n = inosine ; andnucleotide # for the entire sequence is 2915 - 2934 .&# 34 ;( xi ) sequence description : seq id no : 9 : ttnanngcgtgnccattgtt20 ( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( iv ) anti - sense : yes ( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 1 .. 20 ( d ) other information : / product = &# 34 ; synthetic dna &# 34 ;/ note = &# 34 ; in the primer sequence submitted n = inosine ; andnucleotide # for the entire sequence is 2915 - 2934 .&# 34 ;( xi ) sequence description : seq id no : 10 : ttnanngcgtgnccattatt20 ( 2 ) information for seq id no : 11 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( iv ) anti - sense : yes ( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 1 .. 20 ( d ) other information : / product = &# 34 ; synthetic dna &# 34 ;/ note = &# 34 ; in the primer sequence submitted n = inosine ; andnucleotide # for the entire sequence is 2915 - 2934 .&# 34 ;( xi ) sequence description : seq id no : 11 : ttnanngcgtgnccgttatt20 ( 2 ) information for seq id no : 12 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( iv ) anti - sense : yes ( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 1 .. 20 ( d ) other information : / product = &# 34 ; synthetic dna &# 34 ;/ note = &# 34 ; in the primer sequence submitted n = inosine ; andnucleotide # for the entire sequence is 2915 - 2934 .&# 34 ;( xi ) sequence description : seq id no : 12 : ttnanngcatgnccgttatt20 ( 2 ) information for seq id no : 13 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( iv ) anti - sense : no ( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 1 .. 20 ( d ) other information : / product = &# 34 ; synthetic dna &# 34 ;/ note = &# 34 ; in the primer sequence submitted n = inosine ; andnucleotide # for the entire sequence is 2852 - 2871 .&# 34 ;( xi ) sequence description : seq id no : 13 : ttcacatantcnagntttga20 ( 2 ) information for seq id no : 14 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( iv ) anti - sense : no ( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 1 .. 20 ( d ) other information : / product = &# 34 ; synthetic dna &# 34 ;/ note = &# 34 ; in the primer sequence submitted n = inosine ; andnucleotide # for the entire sequence is 2852 - 2871 .&# 34 ;( xi ) sequence description : seq id no : 14 : ttcacctantcnagntttga20 ( 2 ) information for seq id no : 15 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( iv ) anti - sense : no ( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 1 .. 20 ( d ) other information : / product = &# 34 ; synthetic dna &# 34 ;/ note = &# 34 ; in the primer sequence submitted n = inosine ; andnucleotide # for the entire sequence is 2852 - 2871 .&# 34 ;( xi ) sequence description : seq id no : 15 : ttcacatantcncgntttga20 ( 2 ) information for seq id no : 16 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( iv ) anti - sense : no ( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 1 .. 20 ( d ) other information : / product = &# 34 ; synthetic dna &# 34 ;/ note = &# 34 ; in the primer sequence submitted n = inosine ; andnucleotide # for the entire sequence is 2852 - 2871 .&# 34 ;( xi ) sequence description : seq id no : 16 : ttcacatantcncgnttcga20 ( 2 ) information for seq id no : 17 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 1 .. 20 ( d ) other information : / product = &# 34 ; synthetic dna &# 34 ;/ note = &# 34 ; in the primer sequence submitted n = inosine ; andnucleotide # for the entire sequence is 2852 - 2871 .&# 34 ;( xi ) sequence description : seq id no : 17 : ttcacatantcnagnttcga20 ( 2 ) information for seq id no : 18 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( iv ) anti - sense : no ( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 1 .. 20 ( d ) other information : / product = &# 34 ; synthetic dna &# 34 ;/ note = &# 34 ; in the primer sequence submitted n = inosine ; andnucleotide # for the entire sequence is 2852 - 2871 .&# 34 ;( xi ) sequence description : seq id no : 18 : ttcacctantcncgntttga20 ( 2 ) information for seq id no : 19 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( iv ) anti - sense : no ( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 1 .. 20 ( d ) other information : / product = &# 34 ; synthetic dna &# 34 ;/ note = &# 34 ; in the primer sequence submitted n = inosineresidues ; and nucleotide # for the entire sequence is2852 - 2871 .&# 34 ;( xi ) sequence description : seq id no : 19 : ttcacctantcncgnttcga20 ( 2 ) information for seq id no : 20 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( iv ) anti - sense : no ( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 1 .. 20 ( d ) other information : / product = &# 34 ; synthetic dna &# 34 ;/ note = &# 34 ; in the primer sequence submitted n = inosineresidues ; and nucleotide # for the entire sequence is2852 - 2871 .&# 34 ;( xi ) sequence description : seq id no : 20 : ttcacctantcnagnttcga20 ( 2 ) information for seq id no : 21 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( iv ) anti - sense : no ( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 1 .. 20 ( d ) other information : / product = &# 34 ; synthetic dna &# 34 ;/ note = &# 34 ; at position # 2860 and position # 2863 i = inosineresidues ; at position # 2866 n = a or c or g or t ; atposition # 2857 m = a or c ; and nucleotide # for the entiresequence is 2852 - 2871 .&# 34 ;( xi ) sequence description : seq id no : 21 : ttcacmtantcnagntttga20__________________________________________________________________________