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Sentinel-base DNA genotyping using multiple sequencing primers for high-risk human papillomaviruses
Stanford Univ, Stanford Genome Technol Ctr.
Karolinska Inst, Dept Mol Med.
KTH, School of Biotechnology (BIO).
Karolinska Univ Hosp, Clinical Pathology/Cytology.
Show others and affiliations
2006 (English)In: Molecular and Cellular Probes, ISSN 0890-8508, E-ISSN 1096-1194, Vol. 20, no 3-4, 230-238 p.Article in journal (Refereed) Published
Abstract [en]

Despite the various technologies in place for genotyping human papillomaviruses (HPV), clinical use and clinical research demand a method that is fast, more reliable and cost-effective. The technology described here represents a breakthrough development in that direction. By combining the method of multiple sequencing primers with DNA sequencing, we have developed a rapid assay for genotyping HPV that relies on the identification of a single, type-specific 'sentinel' base. As described here, the prototype assay has been developed to recognize the 12 most high-risk HPV types (HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59) and is capable of recognizing and simultaneously genotyping multiple HPV co-infections. By providing sequence information on multiple HPV infections, this method eliminates the need for labor- and cost-intensive PCR cloning. These proof-of-concept studies establish the assay to be accurate, reliable, rapid, flexible, and cost-effective, providing evidence of the feasibility this technique for use in clinical settings.

Place, publisher, year, edition, pages
2006. Vol. 20, no 3-4, 230-238 p.
Keyword [en]
human papillomaviruses (HPV); DNA sequencing; multiple infections; multiple sequencing primers; sentinel-base DNA sequencing; pyrosequencing technology; POLYMERASE-CHAIN-REACTION; GENITAL HUMAN PAPILLOMAVIRUSES; CERVICAL-CANCER; HYBRID CAPTURE; GENERAL PRIMERS; NESTED PCR; SPECIMENS; SAMPLES; HPV; IDENTIFICATION
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-8160DOI: 10.1016/j.mcp.2006.01.002ISI: 000238113600011Scopus ID: 2-s2.0-33646252262OAI: oai:DiVA.org:kth-8160DiVA: diva2:13409
Note
QC 20100624Available from: 2008-04-03 Created: 2008-04-03 Last updated: 2010-06-24Bibliographically approved
In thesis
1. Nucleic Acid Based Pathogen Diagnostics
Open this publication in new window or tab >>Nucleic Acid Based Pathogen Diagnostics
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Pathogenic organisms are transmitted to the host organism through all possible connected pathways, and cause a myriad of diseases states. Commonly occurring curable infectious diseases still impose the greatest health impacts on a worldwide perspective. The Bill & Melinda Gates Foundation partnered with RAND Corporation to form the Global Health Diagnostics Forum, with the goal of establishing and interpreting mathematical models for what effects a newly introduced point-of-care pathogen diagnostic would have in developing countries. The results were astonishing, with potentially millions of lives to be saved on an annual basis.

Golden standard for diagnostics of pathogenic bacteria has long been cultureable medias. Environmental biologists have estimated that less than 1% of all bacteria are cultureable. Genomic-based approaches offer the potential to identify all microbes from all the biological kingdoms. Nucleic acid based pathogen diagnostics has evolved significantly over the past decades. Novel technologies offer increased potential in sensitivity, specificity, decreased costs and parallel sample management. However, most methods are confined to core laboratory facilities. To construct an ultimate nucleic acid based diagnostic for use in areas of need, potential frontline techniques need to be identified and combined.

The research focus of this doctoral thesis work has been to develop and apply nucleic acid based methods for pathogen diagnostics. Methods and assays were applied to the two distinct systems i) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae, and ii) genotype determination of the cancer causative Human Papillomavirus (HPV). The first part of the study included development of rapid, direct and multiplex Pyrosequencing nucleic acid screenings. With improved methodology in the sample preparation process, we could detect an existence of multiple co-infecting HPV genotypes at greater sensitivities than previously described, when using the same type of methodology. The second part of the study focused on multiplex nucleic acid amplification strategies using Molecular Inversion Probes with end-step Pyrosequencing screening. The PathogenMip assay presents a complete detection schematic for virtually any known pathogenic organism. We also introduce the novel Connector Inversion Probe, a padlock probe capable of complete gap-fill reactions for multiplex nucleic acid amplifications.

Abstract [sv]

Patogena organismer smittas till värd organismen genom alla möjliga kontaktnätverk och skapar en mångfald olika sjukdomstillstånd. Dock är det fortfarande vanligt förekommande behandlingsbara infektiösa sjukdomar som orsakar den största hälsoförlusten, sett från ett globalt perspektiv. Bill och Melinda Gates Stiftelsen samarbetade med RAND kooperation för att forma “The Global Health Diagnostics Forum”. Deras mål var att etablera och analysera matematiska modeller för vilka effekter en ny diagnostisk metod utrustat för fältarbete skulle ha i utvecklingsländer. Resultaten var häpnadsveckande, med potentiellt miljoner av liv som skulle kunna räddas på en årlig basis.

Den etablerade standarden för diagnostik av patogena bakterier har länge varit kultiveringsmedia baserad. Miljö specialiserade biologer har estimerat att mindre än 1 % av alla bakterie arter går att kultivera. Dock erbjuder genetiska analyser potentialen att kunna identifiera alla mikrober från alla de biologiska rikena. Nukleinsyrebaserade diagnostiska metoder har märkbart förbättrats över de senaste årtionden. Nya tekniker erbjuder utökad sensitivitet, selektivitet, sänkta kostnader och parallella analyser av patient prover. Dock är de flesta metoderna begränsade till standardiserade laboratoriemiljöer. För att konstruera en väl fungerande diagnostisk fältutrustning för användning i problem områden, behöver världsledande tekniker identifieras och kombineras.

Fokuseringsområdet för denna doktorsavhandling har varit att utveckla och utföra nukleinsyrebaserade metoder för patogen diagnostik. Metoder och experimentella utförande applicerades på två distinkta system i) sökning av antibiotika resistens relaterade mutationer i den patogena bakterien Neisseria gonorrhoeae och ii) genotypning av det cancer orsakande Humana Papillomaviruset (HPV). Den första delen av studien inriktade sig mot utveckling av snabba, direkta och multiplexa Pyrosekvenserings baserade nukleinsyreanalyser. Med förbättrad provprepareringsmetodologi kunde vi detektera multipla HPV infektioner med högre sensitivitet än vad tidigare beskrivits med liknande metodologi. Den andra delen av studien fokuserades på multiplexa nukleinsyre amplifikationer med “Molecular Inversion Probe” tekniken med sista steg Pyrosekvenserings analys. “PathogenMip assay” erbjuder ett komplett detektionsprotokoll för alla kända patogena organismer. Vi introducerar även den nya “Connector Inversion Probe”, en “Padlock Probe” kapabel att genomföra kompletta gap fyllningar för multiplex nukleinsyre amplifiering.

Place, publisher, year, edition, pages
Stockholm: KTH, 2008. [8], 51 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2008:4
Keyword
pathogen diagnostics, antibiotic resistance, connector inversion probes (CIPer), human papillomavirus (HPV), genotyping, global health diagnostic forum, molecular inversion probe (MIP), neisseria gonorrhoeae, padlock probes, pyrosequencing
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-4684 (URN)978-91-7178-899-3 (ISBN)
Public defence
2008-04-18, FR4, AlbaNova University Center, Roslagstullsbacken 21, Stockholm, 13:00
Opponent
Supervisors
Note
QC 20100624Available from: 2008-04-03 Created: 2008-04-03 Last updated: 2010-06-30Bibliographically approved

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