Browsing by Author "Blomberg, Jonas"

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    Development of single-tube nested real-time PCR assays with long internally quenched probes for detection of norovirus genogroup II
    (2016-01) Xia, Hongyan; Gravelsina, Sabine; Öhrmalm, Christina; Ottoson, Jakob; Blomberg, Jonas; Institute of Microbiology and Virology
    The high sequence variation of RNA viruses necessitates use of degenerate primers and probes or multiple primers and probes in molecular diagnostic assays. We showed previously that PCR amplification in two rounds, first with long target-specific primers and then with short generic primers, followed by detection using long probes, can tolerate sequence variation. Here we demonstrate that long primers and probes of up to 56 nucleotides can also be applied in real-time PCR for the detection of norovirus genogroup II with improved sensitivity. Probe design (method of incorporating quenchers, use of Zen internal quencher or traditional quenchers) greatly affects the sensitivity of the real-time PCR assays.
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    No Definite Evidence for Human Endogenous Retroviral HERV-W and HERV-H RNAS in Plasma of Latvian Patients Suffering from Multiple Sclerosis and Other Neurological Diseases
    (2016-08-01) Užameckis, Dmitrijs; Čapenko, Svetlana; Logina, Ināra; Murovska, Modra; Blomberg, Jonas; Department of Neurology and Neurosurgery; Institute of Microbiology and Virology
    Multiple sclerosis (MS) is a neurological disease of unknown aetiology. Several research groups reported an increased level of human endogenous retroviruses HERV-W and HERV-H RNAs in cerebrospinal fluid, plasma and supernatants of cell cultures from MS individuals. To quantify the abundance of extracellular virion-associated HERV, RNAs in blood, plasma samples from Latvian MS patients, patients with other neurological diseases (OND), as well as blood donors (BD), were retrospectively studied by using both our previously published and newly developed quantitative Real-time reverse transcription PCR assays (QPCRs) targeting different polymerase (pol) gene regions of HERV-W and HERV-H. Unspecific signals due to incomplete removal of DNA were monitored by running the assays with and without reverse transcription (RT±) in parallel. According to our score, a few MS, OND and healthy controls gave borderline signals simultaneously with both newly developed HERV-H and HERV-W QPCRs, but the rest were negative. All borderline positive samples also had small amounts of non-retroviral cellular mRNA with possible origin from cell-free circulating RNA fragments, apoptotic bodies or exosomes, which can mimic the previously described virus-like particles. The results do not confirm the previous reports on prevalence of HERV-H or-W virion-associated RNA in plasma of MS patients.
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    Serology in the Digital Age : Using Long Synthetic Peptides Created from Nucleic Acid Sequences as Antigens in Microarrays
    (2016-08-10) Rizwan, Muhammad; Rönnberg, Bengt; Cistjakovs, Maksims; Lundkvist, Åke; Pipkorn, Rudiger; Blomberg, Jonas; Institute of Microbiology and Virology
    BACKGROUND: Antibodies to microbes, or to autoantigens, are important markers of disease. Antibody detection (serology) can reveal both past and recent infections. There is a great need for development of rational ways of detecting and quantifying antibodies, both for humans and animals. Traditionally, serology using synthetic antigens covers linear epitopes using up to 30 amino acid peptides. METHODS: We here report that peptides of 100 amino acids or longer ("megapeptides"), designed and synthesized for optimal serological performance, can successfully be used as detection antigens in a suspension multiplex immunoassay (SMIA). Megapeptides can quickly be created just from pathogen sequences. A combination of rational sequencing and bioinformatic routines for definition of diagnostically-relevant antigens can, thus, rapidly yield efficient serological diagnostic tools for an emerging infectious pathogen. RESULTS: We designed megapeptides using bioinformatics and viral genome sequences. These long peptides were tested as antigens for the presence of antibodies in human serum to the filo-, herpes-, and polyoma virus families in a multiplex microarray system. All of these virus families contain recently discovered or emerging infectious viruses. CONCLUSION: Long synthetic peptides can be useful as serological diagnostic antigens, serving as biomarkers, in suspension microarrays.