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Nucleic Acid Based Pathogen Diagnostics
KTH, School of Biotechnology (BIO).
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 [en]
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: urn:nbn:se:kth:diva-4684ISBN: 978-91-7178-899-3 (print)OAI: oai:DiVA.org:kth-4684DiVA: diva2:13412
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
List of papers
1. Methodological improvements of pyrosequencing technology
Open this publication in new window or tab >>Methodological improvements of pyrosequencing technology
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2006 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 124, no 3, 504-511 p.Article in journal (Refereed) Published
Abstract [en]

Pyrosequencing technology is a rather novel DNA sequencing method based on the sequencing-by-synthesis principle. This bioluminometric, real-time DNA sequencing technique employs a cascade of four enzymatic reactions producing sequence peak signals. The method has been proven highly suitable for single nucleotide polymorphism analysis and sequencing of short stretches of DNA. Although the pyrosequencing procedure is relatively straightforward, users may face challenges due to varying parameters in PCR and sequencing primer design, sample preparation and nucleotide dispensation; such challenges are labor and cost intensive. In this study, these issues have been addressed to increase signal quality and assure sequence accuracy.

Keyword
DNA sequencing; primer-dimers; pyrosequencing technology; sample preparation; single-strand binding protein; SINGLE-NUCLEOTIDE POLYMORPHISM; TUMOR-SUPPRESSOR GENE; STRANDED DNA-BINDING; SEQUENCING PRIMERS; MUTATION; PROTEIN
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-8158 (URN)10.1016/j.jbiotec.2006.01.025 (DOI)000239156700004 ()2-s2.0-33745504382 (Scopus ID)
Note

QC 20100624

Available from: 2008-04-03 Created: 2008-04-03 Last updated: 2013-02-26Bibliographically approved
2. Detection of gyrA mutations associated with ciprofloxacin resistance in Neisseria gonorrhoeae by rapid and reliable pre-programmed short DNA sequencing
Open this publication in new window or tab >>Detection of gyrA mutations associated with ciprofloxacin resistance in Neisseria gonorrhoeae by rapid and reliable pre-programmed short DNA sequencing
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2005 (English)In: International Journal of Antimicrobial Agents, ISSN 0924-8579, E-ISSN 1872-7913, Vol. 26, no 6, 486-490 p.Article in journal (Refereed) Published
Abstract [en]

Quinolone resistance is rapidly increasing in Neisseria gonorrhoeae and is posing a significant public health threat that requires constant surveillance. A rapid and reliable mutation detection assay has been developed. The assay is based on pre-programmed short DNA sequencing and is designed to detect point mutations in the gyrA gene that are highly related to ciprofloxacin resistance, i.e. in codons 91 and 95. By developing an assay based on pyrosequencing and exploiting the pre-programmed nucleotide dispensation capability of this technology, the sequence comprising the mutations will be analysed and promptly reveal whether the N. gonorrhoeae pathogen carries resistance to ciprofloxacin. A panel of 40 N. gonorrhoeae clinical isolates, of which 27 phenotypically displayed decreased susceptibility or resistance to ciprofloxacin, was used in the present study. All point mutations in the short stretch of the N. gonorrhoeae gyrA gene were easily discriminated, and the genotypic results obtained by pre-programmed sequencing were mainly in agreement with the phenotypically identified decreased susceptibility or resistance to ciprofloxacin. The new method used in the present study has the potential for rapid and reliable identification of known as well as previously unknown drug resistance mutations.

Keyword
DNA sequencing; ciprofloxacin resistance; Neisseria gonorrhoeae; pre-programmed DNA sequencing; pyrosequencing technology; ANTIMICROBIAL RESISTANCE; MECHANISMS; FAILURE; ISOLATE; PARC
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-8159 (URN)10.1016/j.ijantimicag.2005.08.017 (DOI)000234043000008 ()2-s2.0-27744514327 (Scopus ID)
Note
QC 20100624Available from: 2008-04-03 Created: 2008-04-03 Last updated: 2010-06-24Bibliographically approved
3. Sentinel-base DNA genotyping using multiple sequencing primers for high-risk human papillomaviruses
Open this publication in new window or tab >>Sentinel-base DNA genotyping using multiple sequencing primers for high-risk human papillomaviruses
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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.

Keyword
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:nbn:se:kth:diva-8160 (URN)10.1016/j.mcp.2006.01.002 (DOI)000238113600011 ()2-s2.0-33646252262 (Scopus ID)
Note
QC 20100624Available from: 2008-04-03 Created: 2008-04-03 Last updated: 2010-06-24Bibliographically approved
4. PathogenMip Assay: A Multiplex Pathogen Detection Assay
Open this publication in new window or tab >>PathogenMip Assay: A Multiplex Pathogen Detection Assay
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2007 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 2, no 2, e223- p.Article in journal (Refereed) Published
Abstract [en]

The Molecular Inversion Probe (MIP) assay has been previously applied to a large-scale human SNP detection. Here we describe the PathogenMip Assay, a complete protocol for probe production and applied approaches to pathogen detection. We have demonstrated the utility of this assay with an initial set of 24 probes targeting the most clinically relevant HPV genotypes associated with cervical cancer progression. Probe construction was based on a novel, cost-effective, ligase-based protocol. The assay was validated by performing pyrosequencing and Microarray chip detection in parallel experiments. HPV plasmids were used to validate sensitivity and selectivity of the assay. In addition, 20 genomic DNA extracts from primary tumors were genotyped with the PathogenMip Assay results and were in 100% agreement with conventional sequencing using an L1-based HPV genotyping protocol. The PathogenMip Assay is a widely accessible protocol for producing and using highly discriminating probes, with experimentally validated results in pathogen genotyping, which could potentially be applied to the detection and characterization of any microbe.

Keyword
Alphapapillomavirus; Automatic Data Processing; Disease Progression; DNA Ligases; DNA Probes, HPV; DNA, Neoplasm; DNA, Viral; Female; Genotype; Humans; Oligonucleotide Array Sequence Analysis; Papillomavirus Infections; Polymerase Chain Reaction; Sensitivity and Specificity; Sequence Analysis, DNA; Uterine Cervical Neoplasms
National Category
Natural Sciences
Identifiers
urn:nbn:se:kth:diva-8161 (URN)10.1371/journal.pone.0000223 (DOI)000207444500004 ()2-s2.0-54949150194 (Scopus ID)
Note

QC 20100624

Available from: 2008-04-03 Created: 2008-04-03 Last updated: 2017-03-23Bibliographically approved
5. Connector Inversion Probe Technology: A Powerful One- Primer Multiplex DNA Amplification System for Numerous Scientific Applications
Open this publication in new window or tab >>Connector Inversion Probe Technology: A Powerful One- Primer Multiplex DNA Amplification System for Numerous Scientific Applications
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2007 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 2, no 9, e915- p.Article in journal (Refereed) Published
Abstract [en]

We combined components of a previous assay referred to as Molecular Inversion Probe (MIP) with a complete gap filling strategy, creating a versatile powerful one-primer multiplex amplification system. As a proof-of-concept, this novel method, which employs a Connector Inversion Probe (CIPer), was tested as a genetic tool for pathogen diagnosis, typing, and antibiotic resistance screening with two distinct systems: i) a conserved sequence primer system for genotyping Human Papillomavirus (HPV), a cancer-associated viral agent and ii) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae. We also discuss future applications and advances of the CIPer technology such as integration with digital amplification and next-generation sequencing methods. Furthermore, we introduce the concept of two-dimension informational barcodes, i. e. "multiplex multiplexing padlocks'' (MMPs). For the readers' convenience, we also provide an on-line tutorial with user-interface software application CIP creator 1.0.1, for custom probe generation from virtually any new or established primer-pairs.

Keyword
DNA; DNA Probes; Drug Resistance, Microbial; Mutation
National Category
Natural Sciences
Identifiers
urn:nbn:se:kth:diva-8162 (URN)10.1371/journal.pone.0000915 (DOI)000207455700023 ()2-s2.0-41249094334 (Scopus ID)
Note
QC 20100624Available from: 2008-04-03 Created: 2008-04-03 Last updated: 2010-06-24Bibliographically approved

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