Patent Application: US-81898201-A

Abstract:
the l1 / e1 gene region of the hpv virus maybe deleted during integration into the genome of the host cell , but the e6 / e7 gene region is always retained . there is a need to detect hpv infection and cervical cancer in way that provides information about the stage of infection so that the proper treatment can be undertaken .

Description:
there are methods known in the art for detecting dna and rna . a number of methods are particularly suited for detecting viral dna or rna . any method that can be used to detect the presence or absence of the e6 and / or e7 and l1 and / or l2 regions of the hpv genome can be used in the method of this invention . methods for detection of hpv dna or rna include but are not limited to polymerase chain reaction ( pcr ) ( see u . s . pat . nos . 4 , 683 , 195 ; 4 , 683 , 202 ; 4 , 800 , 159 ; 4 , 965 , 188 ; 5 , 008 , 182 5 , 176 , 995 ; 5 , 182 , 377 ; 5 , 283 , 171 , and wo 88 / 06634 ); light cycla , taq man , q - beta replicase , ligase chain reaction , pcr - elisa and nasba dna detection . pcr can be used with elisa ( enzyme - linked immunosorbent assay ) to identify dna sequences in the e6 / e7 and l1 / l2 regions of hpv . the ligase chain reaction is a dna amplification technique which is a cyclic two - step reaction : 1 ) a high - temperature melting step in which double - stranded target dna unwinds to become single - stranded and 2 ) a cooling step in which two sets of adjacent , complementary oligonucleotides anneal to the single - stranded target molecules and ligate together . the products of the ligation from one cycle serve as templates for the next cycle &# 39 ; s ligation reaction . amplification is achieved in a similar manner to pcr . see weiss , ( 1988 ) science 254 , 1292 - 1293 ; landegren , u . et al . ( 1988 ) science 241 : 1077 - 1080 ; barany , f . ( 1991 ) pcr methods and applications 1 : 5 - 16 and marsh , e ., et al . ( 1992 ) strategies 5 : 73 - 76 . q - beta replicase is an isothermal nucleic acid amplification system which uses the enzyme q - beta replicase . see u . s . pat . nos . 5 , 556 , 751 and 6 , 004 , 747 . taq man involves the utilization of an additional oligonucleotide in the standard pcr reaction . the additional oligonucleotide hybridizes to a region between the forward and reverse oligonucleotides . the additional oligonucleotide is conjugated to a fluorescence molecule on the 5 ′ end and a quenching molecule on the 3 ′ end . when these two molecules are in proximity the fluorescence is quenched . when the taq polymerase encounters this oligonucleotide it chews it off releasing the fluor and the quencher causing a change in fluorescence . the oligonucleotide probe that is specific for the target to be amplified is labelled with a fluorescent tag and a quenching molecule . during the extension step of pcr the taq enzyme will disrupt probe bound to the target separating the fluorescent tag from its quencher molecule thus permitting fluorescence . in another approach an oligonucleotide probe containing a reporter molecule - quencher molecule pair that specifically anneals to a region of a target polynucleotide downstream i . e . in the direction of extension of primer binding sites . the reporter molecule and quencher molecule are positioned on the probe sufficiently close to each other such that whenever the reporter molecule is excited , the energy of the excited state nonradiatively transfers to the quencher molecule where it either dissipates nonradiatively or is emitted at a different emission frequency see u . s . pat . nos . 5 , 210 , 015 and 6 , 030 , 787 . nasba ( nucleic acid sequence based amplification ) is an isothermal rna amplification method using reverse transcriptase . nasba is continuous rather than cyclic which means it measures all at once instead of waiting for a series of copies to be made . quantitative detection is achieved by way of internal calibrators which are added at isolation and are co - amplified and subsequently identified along with the wild type of rna using electrochemiluminscence . these methods and others known in the art can be used to detect dna and / or rna and / or expression of genes of hpv . the dna sequences of strains of hpv are known . known hpv include hpv 1a , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 72 , 73 , 74 , 75 , 76 , and 77 . the dna sequences of these viruses can be found in the genbank database . the sequences of hpv 6 , 11 , 16 , 18 , and 33 , are also described in the following references . the sequence of hpv6 is given in schwartz et al ., embo l 2 : 2361 - 8 , 1983 . the sequence of hpv11 is given in dartman et al . virology 151 : 124 - 30 , 1986 . the sequence of hpv16 is given in seedorf et al . virology 145 : 181 - 5 , 1985 . the sequence of hpv18 is given in matlashewski et al ., j . gen . virol . 67 : 1909 - 16 , 1986 . the sequence of hpv33 is given in cole and streeck . j . virol 58 : 991 - 5 , 1986 . the methods described above can be used to detect the presence or absence of the e6 , e7 or e6 / e7 region and the presence or absence of the li , l2 or l1 / l2 region . the presence or absence can be detected by using a method that detects or measures dna , rna or expression of the gene . the methods can also be used to determine whether the dna , rna or gene product is from a high risk or low risk strain of hpv . if it is determined that the dna , rna or gene product is derived from a high risk strain of hpv , the presence or absence of dna , rna or gene product from the l1 or l2 region will provide the clinician with important information about the progression of the infection . if the l1 or l2 regions are present then it is likely that the cell has not completed its transformation to the malignant state . if no l1 or l2 regions are present and only the e6 , e7 or e6 / e7 regions can be detected then the cell has been transformed to a malignant state and clinical intervention and further testing and treatment is warranted . if integration occurs in the precancerous stage and this occurs in the l1 gene region , and if the e6 region is not integrated and remains detectable in the advancing stages of carcinogenesis , then combined l1 / e6 pcr testing provides a diagnostic / prognostic indicator of progression . [ 0061 ] table 2 frequency of hpv16 in cervical cancers by l1 / e6 consensus primers compared to e6 - e7 specific primers reference number of sample l1 / e1 e6 / e7 * van den brule et al . ( 1990 ) 21 84 % ter maulen et al . ( 1992 ) 53 38 % guerrero et al . ( 1992 ) 302 48 % prussia et al . ( 1993 ) 20 65 % eluf - neto et al ( 1994 ) 186 54 % monk et al . ( 1994 ) 218 44 % williamson et al . ( 1994 ) 68 46 % karlsen et al . ( 1995 ) 143 63 % [ 0062 ] table 3 variability in rate of detection of hpv in carcinoma samples by l1 / e1 consensus primers in different studies target and primers l1 l1 e1 reference number of samples my gp cp van den brule et al . ( 1990 ) 21 — 91 % — ter maulen et al . ( 1992 ) 53 — 89 % — guerrero et al . ( 1992 ) 302 69 % — — prussia et al . ( 1993 ) 20 — — 90 % eluf - neto et al . ( 1994 ) 186 — 84 % — monk et al . ( 1994 ) 218 79 % — — williamson et al . ( 1994 ) 68 81 % — — karlsen et al . ( 1995 ) 143 91 % 9 % 89 % herrington et al . 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