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Padlock Probe-Based Assays for Molecular Diagnostics
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Treatment success often depends on the availability of accurate and reliable diagnostic assays to guide clinical practitioners in their treatment choices. An optimal test must excel in specificity and sensitivity, and depending on the application area time, low-cost and simplicity are equally important. For instance, time is essential in infectious diagnostics but this is less important in non-invasive prenatal testing (NIPT). In NIPT, specificity and sensitivity are the most important parameters.

In this thesis I describe the development of four different methods, all based on padlock probes and rolling circle amplification, intended for molecular diagnostics. Application areas range from infectious disease diagnostics to NIPT and oncology. The methods described have in common that they overcome certain limitations of currently available assays. This thesis includes two new assays targeting infectious agents: one assay specifically detecting a highly variable double stranded RNA virus and the second assay demonstrating a new format of antibiotic susceptibility testing, which is rapid and generally applicable to different pathogens. Furthermore, I describe the development of a method that uses methylation markers to enrich fetal DNA, accurately quantify chromosome ratios and thus, detecting trisomy 21 and 18. The fourth method described in this thesis uses gap-fill ligation of padlock probes to detect diagnostic relevant point mutations with high specificity in situ.

The assays presented have the potential, after automation and successful validation and verification studies, to be implemented into clinical practice. Furthermore, these assays demonstrate the wide applicability of padlock probes which, due to their properties in regard to specificity and multiplexity, are useful tools for nucleic acid detection in vitro as well as in situ.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2015. , 53 p.
Keyword [en]
Padlock probes; rolling circle amplification; molecular diagnostics; in situ mutation detection
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-116214ISBN: 978-91-7649-155-3 (print)OAI: oai:DiVA.org:su-116214DiVA: diva2:805174
Public defence
2015-06-12, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Available from: 2015-05-21 Created: 2015-04-14 Last updated: 2015-07-08Bibliographically approved
List of papers
1. Detection of Rotavirus Using Padlock Probes and Rolling Circle Amplification
Open this publication in new window or tab >>Detection of Rotavirus Using Padlock Probes and Rolling Circle Amplification
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 11, e111874- p.Article in journal (Refereed) Published
Abstract [en]

Rotavirus infections are one of the most common reasons for hospitalizations due to gastrointestinal diseases. Rotavirus is often diagnosed by latex agglutination assay, chromatography immunoassay, or by electron microscopy, which are all quite insensitive. Reverse transcription polymerase chain reaction, on the other hand, is very sensitive to variations at the genomic level. We developed a novel assay based on a set of 58 different padlock probes with a detection limit of 1,000 copies. Twenty-two patient samples were analyzed and the assay showed high concordance with a PCR-based assay. In summary, we present a new assay for sensitive and variation tolerant detection of rotavirus.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-110753 (URN)10.1371/journal.pone.0111874 (DOI)000344402000115 ()
Note

AuthorCount:5;

Available from: 2014-12-18 Created: 2014-12-17 Last updated: 2017-12-05Bibliographically approved
2. A General Method for Rapid Determination of Antibiotic Susceptibility and Species in Bacterial Infections
Open this publication in new window or tab >>A General Method for Rapid Determination of Antibiotic Susceptibility and Species in Bacterial Infections
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2015 (English)In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 53, no 2, 425-432 p.Article in journal (Refereed) Published
Abstract [en]

To ensure correct antibiotic treatment and reduce the unnecessary use of antibiotics, there is an urgent need for new rapid methods for species identification and determination of antibiotic susceptibility in infectious pathogenic bacteria. We have developed a general method for the rapid identification of the bacterial species causing an infection and the determination of their antibiotic susceptibility profiles. An initial short cultivation step in the absence and presence of different antibiotics was combined with sensitive species-specific padlock probe detection of the bacterial target DNA to allow a determination of growth (i.e., resistance) and no growth (i.e., susceptibility). A proof-of-concept was established for urinary tract infections in which we applied the method to determine the antibiotic susceptibility profiles of Escherichia coli for two drugs with 100% accuracy in 3.5 h. The short assay time from sample to readout enables fast appropriate treatment with effective drugs and minimizes the need to prescribe broad-spectrum antibiotics due to unknown resistance profiles of the treated infection.

National Category
Microbiology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-115289 (URN)10.1128/JCM.02434-14 (DOI)000348568500009 ()25411178 (PubMedID)
Note

AuthorCount:8;

Available from: 2015-03-31 Created: 2015-03-18 Last updated: 2017-12-04Bibliographically approved
3. Elimination of maternal aneuploidy DNA for accurate non-invasive prenatal diagnosis: a pilot study
Open this publication in new window or tab >>Elimination of maternal aneuploidy DNA for accurate non-invasive prenatal diagnosis: a pilot study
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(English)Manuscript (preprint) (Other academic)
National Category
Biomedical Laboratory Science/Technology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-116213 (URN)
Available from: 2015-04-14 Created: 2015-04-14 Last updated: 2016-01-29Bibliographically approved
4. Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ
Open this publication in new window or tab >>Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ
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(English)Manuscript (preprint) (Other academic)
National Category
Biomedical Laboratory Science/Technology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-109696 (URN)
Available from: 2014-11-27 Created: 2014-11-27 Last updated: 2016-01-29Bibliographically approved

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