Rapid Identification of Bio-Molecules Applied for Detection of Biosecurity Agents Using Rolling Circle Amplification

Detection and identification of pathogens in environmental samples for biosecurity applications are challenging due to the strict requirements on specificity, sensitivity and time. We have developed a concept for quick, specific and sensitive pathogen identification in environmental samples. Target identification is realized by padlock- and proximity probing, and reacted probes are amplified by RCA (rolling-circle amplification). The individual RCA products are labeled by fluorescence and enumerated by an instrument, developed for sensitive and rapid digital analysis. The concept is demonstrated by identification of simili biowarfare agents for bacteria (Escherichia coli and Pantoea agglomerans) and spores (Bacillus atrophaeus) released in field

Detection of Rotavirus Using Padlock Probes and Rolling Circle Amplification

Abstract 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

Sensitive detection of Myobacterium avium subsp paratuberculosis in bovine semen by real-time PCR

Aims: To develop a fast and sensitive protocol for detection of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine semen and to make a critical evaluation of the analytical sensitivity. Methods and Results: Processed semen was spiked with known amounts of MAP. Semen from different bulls as well as semen of different dilutions was tested. The samples were treated with lysing agents and beadbeating and the DNA was extracted with phenol and chloroform. Real-time PCR with a fluorescent probe targeting the insertion element IS900 detected as few as 10 organisms per sample of 100 ¿l semen. PCR-inhibition was monitored by inclusion of an internal control. Pre-treatment with immunomagnetic separation was also evaluated, but was not shown to improve the overall sensitivity. Conclusions: Real-time PCR is a sensitive method for detection of MAP in bovine semen. Lysis by mechanical disruption followed by phenol and chloroform extraction efficiently isolated DNA and removed PCR-inhibitors. Significance and Impact of the Study: The high sensitivity of the applied method allows reliable testing of bovine semen used for artificial insemination to prevent the spread of Johne's disease, caused by MAP