WO2003083142A2 - Detection universelle de l'arnm du papillomavirus humain - Google Patents

Detection universelle de l'arnm du papillomavirus humain Download PDF

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Publication number
WO2003083142A2
WO2003083142A2 PCT/GB2003/001317 GB0301317W WO03083142A2 WO 2003083142 A2 WO2003083142 A2 WO 2003083142A2 GB 0301317 W GB0301317 W GB 0301317W WO 03083142 A2 WO03083142 A2 WO 03083142A2
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WIPO (PCT)
Prior art keywords
arm
oligonucleotide
hpv
sequence
primer set
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PCT/GB2003/001317
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WO2003083142A3 (fr
Inventor
Frank Karlsen
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ALLARD SUSAN JOYCE
Norchip AS
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ALLARD SUSAN JOYCE
Norchip AS
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Priority to AU2003212544A priority Critical patent/AU2003212544A1/en
Publication of WO2003083142A2 publication Critical patent/WO2003083142A2/fr
Publication of WO2003083142A3 publication Critical patent/WO2003083142A3/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/708—Specific hybridization probes for papilloma

Definitions

  • the present invention is concerned with methods of detecting the presence of human papillomavirus mRNA in clinical samples using nucleic acid sequence-based amplification and in particular with sets of degenerate oligonucleotide primers for use in such methods .
  • Cervical cancer is closely associated with human papillomavirus (HPV) . More than 100 HPV genotypes have been considered to date; types 16, 18, 31, 33, 35, 45, 52, 58 and 67 are considered to belong to the group that puts patients infected with these types at high risk for cervical carcinogenesis.
  • HPV human papillomavirus
  • PCR polymerase chain reaction
  • NASBA Nucleic acid sequence-based amplification
  • RNA Compton, Nature. 350: 91-92 (1991)
  • NASBA is well known by persons of ordinary skill in the art and is described, for example, in US-A-5, 409, 818.
  • NASBA is an effective procedure for generating large quantities of a target RNA sequence in vitro, allowing detection of target RNA sequences that are present in very low concentrations in the original test sample.
  • the NASBA method is based on the same type of primer-sets as for PCR but one primer is modified with a promoter sequence, for example a T7 promoter.
  • NASBA sensitivity and specificity of the NASBA amplification has been shown to be the same as for PCR and better then most RT-PCR protocols. Since the NASBA method is an isothermal assay and it is dependent on RNase H it cannot amplify DNA. This is important in connection with detection of mRNA expression from the HPV genome where the RT-PCR method would amplify both the RNA and DNA. There is extensive literature on the use of NASBA for the detection of HIV-1 in clinical samples (see for example Kievits et al . , Journal of Virological Methods. 35: 273-286 (1991)).
  • an oligonucleotide primer set for use in the detection of HPV in a test sample comprising at least one oligonucleotide primer which is an oligonucleotide selected from group (i) as defined below and at least one oligonucleotide primer which is an oligonucleotide selected from group (ii) as defined below:
  • oligonucleotides comprising one of the following nucleotide sequences:
  • X x represents a nucleotide sequence comprising a promoter
  • the invention also provides a oligonucleotide probe/primer set for use in the detection of HPV in a test sample comprising an oligonucleotide primer set according to the first aspect of the invention and at least one oligonucleotide probe which is an oligonucleotide selected from group (iii) or group (iv) , as defined below:
  • probe oligonucleotides comprising one of the following nucleotide sequences: TTGTTACTGTGGTAGATACTA; or TTTGTTACTGTGGTAGATACTAC
  • X 2 and X 3 represent a fluorescent moiety and a quencher moiety capable of substantially or completely quenching the fluorescence from the fluorescent moiety when the two are held together in close proximity and wherein arm- L and arm 2 represent complementary sequences capable of forming a stem duplex.
  • Suitable pairs of ar- and arm 2 sequences include, but not exclusively, the following:
  • the invention provides the following additional primer sets for use in the detection of HPV in a test sample:
  • a primer set comprising a first oligonucleotide primer having the sequence:
  • X ⁇ represents a nucleotide sequence comprising a promoter; and a second oligonucleotide primer having the sequence: ACAATAATGGCATTTGTT;
  • a primer set comprising a first oligonucleotide primer having the sequence:
  • X ⁇ represents a nucleotide sequence comprising a promoter; and a second oligonucleotide primer having the sequence: AATGGCATTTGTTGGGGTA;
  • a primer set comprising a first oligonucleotide primer having the sequence:
  • X x represents a nucleotide sequence comprising a promoter; and a second oligonucleotide primer having the sequence: ACAATAATGGCATTTGTTG;
  • a primer set comprising a first oligonucleotide primer having the sequence:
  • Xi-ACACCTAAAGGCTGACCA wherein X 1 represents a nucleotide sequence comprising a promoter; and a second oligonucleotide primer having the sequence: AAACTGTAAATCATATTCC .
  • the invention also provides an oligonucleotide probe/primer set for use in the detection of HPV in a test sample comprising one of the additional oligonucleotide primer sets listed above and at least one oligonucleotide probe which is a probe oligonucleotide comprising one of the following sequences:
  • oligonucleotide selected from oligonucleotides comprising one of the following structures :
  • X 2 -arm 1 -GCATACCAATATAGAGTATTTAG-arm 2 -X 3 wherein X 2 and X 3 represent a fluorescent moiety and a quencher moiety capable of substantially or completely quenching the fluorescence from the fluorescent moiety when the two are held together in close proximity and wherein arm x and arm 2 represent complementary sequences capable of forming a stem duplex.
  • X 2 -arm 1 -TTACCTGACCCAAATAAATT-arm 2 -X 3 , arm-* . and arm 2 are preferably selected from:
  • arm x and arm 2 are preferably selected from: CGCAT and ATGCG; or
  • CGTCGT and ACGACG or CCATC and GATGG; or CCAGC and GCTGG.
  • X 2 -arm 1 -GCATACCAATATAGAGTATTTAG-arm 2 -X 3f arm-j . and arm 2 are preferably selected from:
  • CGTCGT and ACGACG CTCGCG and CGCGAG.
  • the oligonucleotide primer sets provided by the invention include at least one NASBA pi primer oligonucleotide and at least one NASBA p2 primer oligonucleotide, which together are suitable for use in amplifying a region of the HPV genome by NASBA.
  • the NASBA primer 1 (PI) oligonucleotide molecules are preferably approximately 50 bases in length, with an average of about 20 bases at the 3' end that are complementary to a region of the target HPV mRNA.
  • the 5' ends of the primer 1 oligonucleotides (represented herein in general terms as X- comprise a promoter sequence that is recognized by a specific RNA polymerase.
  • Bacteriophage promoters for example the T7, T3 and SP6 promoters, are preferred for use in the oligonucleotides of the invention, since they provide advantages of high level transcription which is dependent only on binding of the appropriate RNA polymerase.
  • the 5' terminal sequence of the primer 1 oligonucleotides may comprise the sequence AATTCTAATACGACTCACTATAGGG. This sequence contains a T7 promoter, including the transcription initiation site for T7 RNA polymerase.
  • the primer 1 oligonucleotides are used in combination with NASBA primer 2 (P2) oligonucleotides which preferably comprise a sequence of approximately 20 bases substantially identical to a region of the target HPV mRNA. Several degenerate bases are included in this sequence in order to provide specificity for HPV types.
  • the primer 2 oligonucleotides may, in a particular but non-limiting embodiment, further comprise a sequence of nucleotides at the 5' end which is unrelated to HPV but which is capable of hybridising to a generic detection probe.
  • the detection probe will preferably be labelled, for example with a fluorescent, luminescent or enzymatic label. In one embodiment the detection probe is labelled with a label that permits detection using ECLTM technology, although it will be appreciated that the invention is in no way limited to this particular method of detection.
  • the 5 ' end of the primer 2 oligonucleotides may comprise the sequence GATGCAAGGTCGCATATGAG. This sequence is capable of hybridising to a generic ECLTM probe commercially available from Organon Teknika having the following structure:
  • the primer 2 oligonucleotide may incorporate ⁇ molecular beacons' ' technology, which is known in the art and described, for example, in WO 95/13399 by Tyagi and Kramer, Nature Biotechnology. 14: 303-308, 1996, to allow for real-time monitoring of the NASBA reaction.
  • the sets of primer pairs provided by the invention may be used to drive a NASBA amplification reaction to amplify a region of the HPV genome.
  • the primer 1 and primer 2 oligonucleotides must be capable of priming synthesis of a double-stranded DNA from a target region of mRNA, specifically HPV Ll mRNA.
  • the primer 1 and primer 2 oligonucleotides must comprise HPV-specific sequences which are complementary to regions of the sense and the antisense strand of the target mRNA, respectively.
  • the primer 1 oligonucleotide anneals to a complementary sequence in the target mRNA and it's 3' end is extended by the action of an RNA-dependent DNA polymerase (e.g. reverse transcriptase) to form a first-strand cDNA synthesis.
  • the RNA strand of the resulting RNA: DNA hybrid is then digested, e.g. by the action of RNaseH, to leave a single stranded DNA.
  • the primer 2 oligonucleotide anneals to a complementary sequence towards the 3' end of this single stranded DNA and it's 3' end is extended (by the action of reverse transcriptase) , forming a double stranded DNA.
  • RNA polymerase is then able to transcribe multiple RNA copies from the now transcriptionally active promoter sequence within the double-stranded DNA.
  • This RNA transcript which is antisense to the original target mRNA, can act as a template for a further round of NASBA reactions, with primer 2 annealing to the RNA and priming synthesis of the first cDNA strand and primer 1 priming synthesis of the second cDNA strand.
  • the probe oligonucleotide molecules preferably comprise a sequence of approximately 20-25 bases substantially identical to a region of the target HPV mRNA. These oligonucleotides may be used as HPV-specific hybridisation probes for detection of the products of a NASBA reaction.
  • the probe oligonucleotides may be coupled to a solid support, such as paramagnetic beads, to form a capture probe to immobilise the product of the NASBA amplification reaction (a single stranded RNA) .
  • the 5' end of the probe oligonucleotide may be labelled with biotin. The addition of a biotin label facilitates attachment of the probe to a solid support via a biotin/streptavidin or biotin/avidin linkage.
  • the molecular beacons probe oligonucleotides including those listed as 'group (iv) ' oligonucleotides, are HPV-specific probes incorporating ⁇ molecular beacons' technology which is known in the art and described, for example, by Tyagi and Kramer, Nature Biotechnology. 14: 303-308, 1996 and in WO 95/13399.
  • the use of molecular beacons technology allows for real-time monitoring of the NASBA reaction (Leone et al . , Nucleic Acids Research., 1998, vol: 26, pp 2150-2155) .
  • the molecular beacons probes generally include complementary sequences flanking the HPV-specific sequence, represented herein by the notation arm x and arm 2 , which are capable of hybridising to each other form a stem duplex structure.
  • the precise sequences of arm x and arm 2 are not material to the invention, except for the requirement that these sequences must be capable of forming a stem duplex when the probe is not bound to a target HPV sequence.
  • Molecular beacons probes also include a fluorescent moiety and a quencher moiety, the fluorescent and the quencher moieties being represented herein by the notation X 2 and X 3 .
  • the fluorescer and quencher moieties are selected such that the quencher moiety is capable of substantially or completely quenching the fluorescence from the fluorescent moiety when the two moieties are in close proximity, e.g. when the probe is in the hairpin closed' conformation in the absence of the target sequence.
  • the fluorescent and quencher moieties Upon binding to the target sequence, the fluorescent and quencher moieties are held apart such that the fluorescence of the fluorescent moiety is no longer quenched.
  • Suitable pairs of quencher/fluorescer moieties which may be used in accordance with the invention are known in the art (see WO 95/13399, Tyagi and Kramer, ibid) .
  • Preferred combinations include the fluorophore 5-(2'- aminoethyl) aminonaphthalene-1-sulphonic acid (EDANS) and the quencher 4- (4 ' -dimethylaminophenylazo) benzoic acid (DABCYL) , although it will be appreciated that the invention is not limited to this particular combination.
  • the fluorescer and quencher moieties may be covalently attached to the probe in either orientation, either with the fluorescer at or near the 5 ' end and the quencher at or near the 3 ' end or vice versa.
  • the invention further provides a primer set for use in detection of HPV which is specific for the El region of the HPV genome.
  • a primer set for use in detection of HPV which is specific for the El region of the HPV genome.
  • the invention provides an oligonucleotide primer set for use in the detection of HPV in a test sample comprising a first oligonucleotide primer having the sequence: Xi-ATATTCCTCCCCATGTCGT, wherein X x represents a nucleotide sequence comprising a promoter; and a second oligonucleotide primer having the sequence: TCAGAAATGGTACAATGG.
  • This primer set also comprises a NASBA PI primer and a NASBA P2 primer, as described above.
  • the invention provides a method for the detection of HPV in a test sample using the NASBA technique.
  • This method generally comprise the following steps:
  • the ⁇ test sample suspected of containing HPV will most commonly be a clinical sample, for example a cervical scraping in the cervical screening field.
  • the NASBA reaction will preferably be carried out on a preparation of nucleic acid isolated from the test sample.
  • This preparation of nucleic acid must include mRNA, however it need not be a preparation of purified poly A+ mRNA and preparations of total RNA or even preparations of total nucleic acid containing both RNA and genomic DNA are also suitable as starting material for a NASBA reaction.
  • any technique known in the art for the isolation of a preparation of nucleic acid including mRNA may be used to isolate nucleic acid from the test sample.
  • a preferred technique is the "Boom" isolation method described in US-A-5, 234, 809 and EP-B-0389, 063.
  • This method which can be used to isolate a nucleic acid preparation containing both RNA and DNA, is based on the nucleic acid binding properties of silicon dioxide particles in the presence of the chaotropic agent guanidine thiocyanate (GuSCN) .
  • GuSCN guanidine thiocyanate
  • Detection of the specific product (s) of the NASBA reaction may be carried out in a number of different ways.
  • the NASBA product (s) may be detected with the use of an HPV-specific hybridisation probe capable of specifically annealing to the NASBA product.
  • the hybridisation probe may be attached to a revealing label, for example a fluorescent, luminescent, radioactive or chemiluminescent compound or an enzyme label or any other type of label known to those of ordinary skill in the art.
  • the precise nature of the label is not critical, but it should be capable of producing a signal detectable by external means, either by itself or in conjunction with one or more additional substances (e.g. the substrate for an enzyme).
  • NASBA reaction over the course of the reaction.
  • This may be achieved using a 'molecular beacons' probe comprising an HPV-specific sequence capable of annealing to the NASBA product, a stem-duplex forming oligonucleotide sequence and a pair of fluorescer/quencher moieties, as described in WO 95/13399. If the molecular beacons probe is added to the reaction mixture prior to amplification it may be possible to monitor the formation of the NASBA product in real-time (Leone et al . , Nucleic Acids Research, 1998, Vol 26, 2150-2155) .
  • the molecular beacons technology may be incorporated into the primer 2 oligonucleotide allowing real-time monitoring of the NASBA reaction without the need for a separate hybridisation probe.
  • the products of the NASBA reaction may be monitored using a generic labelled detection probe which hybridises to a nucleotide sequence in the 5 1 terminus of primer 2.
  • a generic labelled detection probe which hybridises to a nucleotide sequence in the 5 1 terminus of primer 2.
  • This is equivalent to the NucliSensTM' detection system supplied by Organon Teknika.
  • specificity for NASBA products derived from the target HPV mRNA may be conferred by using HPV-specific capture probes comprising probe oligonucleotides as described herein attached to a solid support such as a magnetic microbead.
  • the generic labelled detection probe is the ECLTM detection probe supplied by Organon Teknika.
  • NASBA amplicons are hybridized to the HPV-specific capture probes and the generic ECL probe (via a complementary sequence on primer 2) . Following hybridization the bead/amplicon/ECL probe complexes may be captured at the magnet electrode of an automatic ECL reader (e.g. the NucliSensTM reader supplied by Organon Teknika. Subsequently, a voltage pulse triggers the ECLTM reaction.
  • an automatic ECL reader e.g. the NucliSensTM reader supplied by Organon Teknika.
  • kits for use in the detection of HPV by NASBA comprising a primer set or probe/primer set according to the invention.
  • the reagent kits may further comprise a mixture of enzymes required for the NASBA reaction, specifically an enzyme mixture containing an RNA directed DNA polymerase (e.g. a reverse transcriptase) , a ribonuclease that hydrolyses the RNA strand of an RNA-DNA hybrid without hydrolysing single or double stranded RNA or DNA (e.g. RNaseH) and an RNA polymerase.
  • an enzyme mixture containing an RNA directed DNA polymerase (e.g. a reverse transcriptase) , a ribonuclease that hydrolyses the RNA strand of an RNA-DNA hybrid without hydrolysing single or double stranded RNA or DNA (e.g. RNaseH) and an RNA polymerase.
  • an enzyme mixture containing an RNA directed DNA polymerase (e.g. a reverse
  • the RNA polymerase should be one which recognises the promoter sequence present in the 5' terminal region of the primer 1 oligonucleotides in the oligonucleotide primer cocktails or primer sets supplied in the reagent kit.
  • the kit may also comprise a supply of NASBA buffer containing the ribonucleosides and deoxyribonucleosides required for RNA and DNA synthesis. Details of the composition of a standard NASBA reaction buffer are well known in the art.
  • the kit may further contain one or more capture probes, comprising a probe oligonucleotide attached to a solid support as described above, for immobilising the products of a specific NASBA reaction.
  • the kit may still further contain labelled generic detection probes.
  • the detection probes may comprise a sequence of nucleotides complementary to a non-HPV sequence present at the 5' terminal end of the primer 2 oligonucleotides present in the reagent kit.
  • the kit may further contain one or more molecular beacon probes according to the invention.
  • the molecular beacon probes may be supplied as a separate reagent within the kit or may be supplied in a primer cocktail/probe or primer set/probe mixture.
  • the invention also provides oligonucleotide molecules for use as primers or probes in determining the presence of human papilloma virus type in a test sample, these being oligonucleotides selected from any of groups (a) to (c) as follows:
  • X x represents a nucleotide sequence comprising a promoter
  • X 2 and X 3 represent a fluorescent moiety and a quencher moiety capable of substantially or completely quenching the fluorescence from the fluorescent moiety when the two are held together in close proximity and wherein arm x and arm 2 represent complementary sequences capable of forming a stem duplex
  • the oligonucleotide molecules of the invention are selected to be specific for mRNA transcribed from the HPV LI or El genes.
  • the oligonucleotide molecules are preferably single stranded DNA molecules.
  • Non-natural synthetic polynucleotides which retain the ability to base-pair with a complementary nucleic acid molecule and are able to function in the NASBA reaction are also within the scope of the invention, including synthetic oligonucleotides which incorporate modified bases and synthetic oligonucleotides wherein the links between individual nucleosides include bonds other than phosphodiester bonds.
  • oligonucleotide molecules of the invention may be produced according to techniques well known in the art, such as by chemical synthesis using standard apparatus and protocols for oligonucleotide synthesis. Representations of the sequences of degenerate oligonucleotide molecules provided herein use the standard IUB code for mixed base sites:
  • sequence X ⁇ is a sequence comprising a T7 promoter. Most preferably X ⁇ comprises the sequence AATTCTAATACGACTCACTATAGGG.
  • the a ⁇ rij and arm 2 sequences of the group (c) oligonucleotides are as defined above.
  • Oligonucleotides suitable for use as NASBA pi primers HPV-Apl AATTCTAATACGACTCACTATAGGGAGAAGGGAAAAATAAACTGTAAATCATA
  • Oligonucleotides suitable for use as NASBA p2 primers HPV-Ap2 GATGCAAGGTCGCATATGAGGCMCAGGGWCATAAYAAT H PV- Bp 2 GATGCAAGGT CGCAT ATGAGGCMCAGGGWCATAAYAAT GG ONC1P2 GATGCAAGGTCGCATATGAGACAATAATGGCATTTGTTG

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Abstract

L'invention concerne un ensemble d'amorces oligonucléotidiques utilisées dans la détection du papillomavirus dans un échantillon d'essai. Cet ensemble d'amorces comprend au moins une amorce nucléotidique sélectionnée dans le groupe (i), tel que défini ci-après, et au moins une amorce oligonucléotidique sélectionnée dans le groupe (ii), tel que défini ci-après : (i) des oligonucléotides comprenant une des séquences nucléotidiques suivantes: X1 GAAAAATAAACTGTAAATCATA; X1-GAAAAATAAACTGTAAATCATATTC; ou X1-GATAAATAAACTGTAAATCATATTC, où X1 représente une séquence oligonucléotidique qui comprend un promoteur ; (ii) des oligonucléotides comprenant une des séquences nucléotidiques suivantes: GCMCAGGGWCATAAYAAT; ou GCMCAGGGWCATAAYAATGG.
PCT/GB2003/001317 2002-03-27 2003-03-26 Detection universelle de l'arnm du papillomavirus humain Ceased WO2003083142A2 (fr)

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AU2003212544A AU2003212544A1 (en) 2002-03-27 2003-03-26 UNIVERSAL DETECTION OF HUMAN PAPILLOMAVIRUS mRNA

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GB0207242A GB0207242D0 (en) 2002-03-27 2002-03-27 Universal detection of human papillomavirus mRNA
GB0207242.9 2002-03-27

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005100598A1 (fr) * 2004-04-19 2005-10-27 Genomictree Inc. Procede de preparation d'une puce a adn et utilisation de cette puce
WO2006077102A3 (fr) * 2005-01-18 2007-07-19 Delft Diagnostic Lab Bv Methode de detection et matieres associees
WO2008096177A3 (fr) * 2007-02-09 2008-10-16 Health Prot Agency Détection de papillomavirus humain
CN117126966A (zh) * 2023-09-01 2023-11-28 弗雷米德生物医药技术（天津）有限公司 基于HPV mRNA表达量的试剂盒及应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NL9000134A (nl) * 1990-01-19 1991-08-16 Stichting Res Fonds Pathologie Primers en werkwijze voor het detecteren van humaan papilloma virus genotypen m.b.v. pcr.
ES2181765T3 (es) * 1994-02-21 2003-03-01 Stichting Res Fonds Pathologie Deteccion de papiloma virus humano en un proceso de amplificacion de acido nucleico utilizando cebadores generales.
SE515668C2 (sv) * 1999-02-22 2001-09-17 Quantovir Ab Metod och kit för tidig förutsägelse av cancer
DE60141205D1 (de) * 2000-04-03 2010-03-18 Cytyc Corp Nachweis und typisierung des papillomavirus mittels pna-sonden

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005100598A1 (fr) * 2004-04-19 2005-10-27 Genomictree Inc. Procede de preparation d'une puce a adn et utilisation de cette puce
US7741042B2 (en) 2004-04-19 2010-06-22 Genomictree, Inc. Method for preparing a DNA chip and use thereof
WO2006077102A3 (fr) * 2005-01-18 2007-07-19 Delft Diagnostic Lab Bv Methode de detection et matieres associees
WO2008096177A3 (fr) * 2007-02-09 2008-10-16 Health Prot Agency Détection de papillomavirus humain
JP2010517556A (ja) * 2007-02-09 2010-05-27 ヘルス プロテクション エージェンシー ヒトパピローマウイルスの検出
US8741568B2 (en) 2007-02-09 2014-06-03 Health Protection Agency Detection of human papillomavirus
CN117126966A (zh) * 2023-09-01 2023-11-28 弗雷米德生物医药技术（天津）有限公司 基于HPV mRNA表达量的试剂盒及应用

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AU2003212544A8 (en) 2003-10-13
AU2003212544A1 (en) 2003-10-13
GB0207242D0 (en) 2002-05-08

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