| 1. |
- Gyarmati, Péter, et al.
(författare)
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Simultaneous genotyping of all hemagglutinin and neuraminidase subtypes of avian influenza viruses by use of padlock probes
- 2008
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 46:5, s. 1747-1751
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Tidskriftsartikel (refereegranskat)abstract
- A subtyping assay for both the hemagglutinin (HA) and neuraminidase (NA) surface antigens of the avian influenza virus (AIV) has been developed. The method uses padlock probe chemistry combined with a microarray output for detection. The outstanding feature of this assay is its capability to designate both the HA and the NA of an AIV sample from a single reaction mixture. A panel of 77 influenza virus strains was tested representing the entire assortment of the two antigens. One hundred percent (77/77) of the samples tested were identified as AIV, and 97% (75/77) were subtyped correctly in accordance with previous examinations performed by classical diagnostic methods. Testing of heterologous pathogens verified the specificity of the assay. This assay is a convenient and practical tool for the study of AIVs, providing important HA and NA data more rapidly than conventional methods.
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| 2. |
- Ke, Rongqin, et al.
(författare)
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Colorimetric Nucleic Acid Testing Assay for RNA Virus Detection Based on Circle-to-Circle Amplification of Padlock Probes
- 2011
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 49:12, s. 4279-4285
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Tidskriftsartikel (refereegranskat)abstract
- We developed a molecular diagnostic method for detection of RNA virus based on padlock probes and colorimetric readout. The feasibility of our approach was demonstrated by using detection of Crimean-Congo hemorrhagic fever (CCHF) virus as a model. Compared with conventional PCR-based methods, our approach does not require advanced equipment, involves easier assay design, and has a sensitivity of 103 viral copies/ml. By using a cocktail of padlock probes, synthetic templates representing different viral strain variants could be detected. We analyzed 34 CCHF patient samples, and all patients were correctly diagnosed when the results were compared to those of the current real-time PCR method. This is the first time that highly specific padlock probes have been applied to detection of a highly variable target sequence typical of RNA viruses.
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| 3. |
- Kurt, Kevin, et al.
(författare)
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Multiplexed genotyping of methicillin-resistant Staphylococcus aureus isolates by use of padlock probes and tag microarrays
- 2009
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 47:3, s. 577-585
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Tidskriftsartikel (refereegranskat)abstract
- We developed and tested a ligase-based assay for simultaneous probing of core genome diversity and typing of methicillin resistance determinants in Staphylococcus aureus isolates. This assay uses oligonucleotide padlock probes whose two ends are joined through ligation when they hybridize to matching target DNA. Circularized probes are subsequently amplified by PCR with common primers and analyzed by using a microarray equipped with universal tag probes. Our set of padlock probes includes oligonucleotides targeting diagnostic regions in the mecA, ccrB, and ccrC genes of the SCCmec cassette in methicillin-resistant S. aureus (MRSA). These probes determine the presence and type of SCCmec cassettes (i.e., SCCmec types I to VI). Additional oligonucleotides interrogate a number of highly informative single nucleotide polymorphisms retrieved from a multilocus sequence typing (MLST) database. These latter probes enable the exploration of isolates' phylogenetic affiliation with clonal lineages of MRSA as revealed by MLST. The described assay enables multiplexed genotyping of MRSA based on a single-tube reaction. With a set of clinical isolates of MRSA and methicillin-susceptible S. aureus (n=66), 100% typeability and 100% accuracy were achieved. The assay described here provides valuable genotypic information that may usefully complement existing genotyping procedures. Moreover, the assay is easily extendable by incorporating additional padlock probes and will be valuable for the quick and cost-effective probing of large numbers of polymorphisms at different genomic locations, such as those ascertained through currently ongoing mutation discovery and genome resequencing projects.
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| 4. |
- Mezger, Anja, et al.
(författare)
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A General Method for Rapid Determination of Antibiotic Susceptibility and Species in Bacterial Infections
- 2015
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 53:2, s. 425-432
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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