New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis

BACKGROUND: Johne's disease, a serious chronic form of enteritis in ruminants, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). As the organism is very slow-growing and fastidious, several PCR-based methods for detection have been developed, based mainly on the MAP-specific gene IS900. However, because this gene is similar to genes in other mycobacteria, there is a need for sensitive and reliable methods to confirm the presence of MAP. As described here, two new real-time PCR systems on the IS900 gene and one on the F57 gene were developed and carefully validated on 267 strains and 56 positive clinical faecal samples. RESULTS: Our confirmatory PCR systems on IS900 were found sensitive and specific, only yielding weak false positive reactions in one strain for each system. The PCR system on F57 did not elicit any false positives and was only slightly less sensitive than our primary IS900-system. DNA from both naturally infected and spiked faeces that tested positive with our primary system could be confirmed with all new systems, except one low-level infected sample that tested negative with the F57 system. CONCLUSION: We recommend using the newly constructed DH3 PCR system on the F57 gene as the primary confirmatory test for PCR positives, but should it fail due to its lower sensitivity, the DH1 and DH2 PCR systems should be used

A biosensor assay for the detection of Mycobacterium avium subsp. paratuberculosis in fecal samples

A simple, membrane-strip-based lateral-flow (LF) biosensor assay and a high-throughput microtiter plate assay have been combined with a reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of a small number (ten) of viable Mycobacterium (M.) avium subsp. paratuberculosis (MAP) cells in fecal samples. The assays are based on the identification of the RNA of the IS900 element of MAP. For the assay, RNA was extracted from fecal samples spiked with a known quantity of (101 to 106) MAP cells and amplified using RT-PCR and identified by the LF biosensor and the microtiter plate assay. While the LF biosensor assay requires only 30 min of assay time, the overall process took 10 h for the detection of 10 viable cells. The assays are based on an oligonucleotide sandwich hybridization assay format and use either a membrane flow through system with an immobilized DNA probe that hybridizes with the target sequence or a microtiter plate well. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye, sulforhodamine B. The dye in the liposomes provides a signal that can be read visually, quantified with a hand-held reflectometer, or with a fluorescence reader. Specificity analysis of the assays revealed no cross reactivity with other mycobacteria, such as M. avium complex, M. ulcerans, M. marium, M. kansasii, M. abscessus, M. asiaticum, M. phlei, M. fortuitum, M. scrofulaceum, M. intracellulare, M. smegmatis, and M. bovis. The overall assay for the detection of live MAP organisms is comparatively less expensive and quick, especially in comparison to standard MAP detection using a culture method requiring 6-8 weeks of incubation time, and is significantly less expensive than real-time PCR

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 and confirmation of Mycobacterium avium subsp. paratuberculosis in clinical samples

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of the serious disease paratuberculosis in ruminants. It is a chronic enteric disease that causes considerable economic losses worldwide. Paratuberculosis leads to reduced milk production and eventually, diarrhoea, weight loss and death. Its slow development, the inappropriate immune response of the host and the fastidiousness of the bacteria all contribute to the difficulty of early diagnosis, necessary to restrict spread of the disease. Thanks to rigorous control measures, paratuberculosis is rare or absent in Sweden. However, occasional import-related outbreaks have occurred, during which all animals in the infected herds were culled. Freedom from paratuberculosis is mainly monitored by slow culture methods. In some situations, fast and reliable alternative methods are needed, for instance, when semen is imported for breeding purposes. Donor bulls may be asymptomatic carriers of MAP and the risk for venereal transmission of the disease is insufficiently investigated. In this thesis, the development and sensitivity assessment of a protocol for detection of MAP in bovine semen by real-time PCR is described. Beadbeating with zirconia/silica beads and phenol/chloroform extraction was used to purify the bacterial DNA and it was shown to successfully remove PCR inhibiting substances. A method for accurately evaluating the analytical sensitivity was also developed. By analysis of artificially infected samples, a sensitivity of 10 organisms per 100 µl sample was assessed. The target gene, for the method described here and for many other PCR methods for detection of MAP, is the insertion element IS900. Although specific for MAP, it has been shown to share similarities with genes in other mycobacteria. Positive PCR results must, therefore, be confirmed by an alternative method. Three novel real-time PCR systems were designed and validated on 267 strains and 58 positive clinical faecal and tissue samples. Two systems were based on IS900 and one system on the MAP-specific gene F57. The latter was the most specific for MAP and is therefore the recommended system for confirmation, but as it was slightly less sensitive when tested on pure DNA, the other systems may be applied when necessary

Molecular diagnostic methods for Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of the chronic enteric disease paratuberculosis in ruminants that causes considerable economic losses worldwide. Due to rigorous control measures, paratuberculosis is rare or absent in Sweden. However, import-related outbreaks have occurred. Diagnostic surveillance and outbreak investigations are mainly carried out by very slow culture methods. Faster and equally reliable molecular methods are needed for detection of MAP in several clinical matrixes. MAP is primarily shed in faeces, the most important testing material. The abundance of PCR inhibitory substances in faeces constitutes a diagnostic challenge. Semen, imported for breeding purposes, may contain MAP if the donor bulls are asymptomatic carriers. MAP has been suggested as the causative agent of Crohn's disease and its presence in consumers' milk might be of concern. In this thesis, the development and sensitivity assessment of protocols for detection of MAP in ruminant faeces, semen and milk by real-time PCR are described. The analytical sensitivities were assessed to 104 MAP/g faeces, 10 MAP/100 µl semen and 100 MAP/ml milk. The faeces direct PCR was validated on 202 proficiency test samples. MAP was detected in 97% of previously frozen positive samples - better than culture. Pellet and cream fractions of milk were pooled before cell lysis and DNA extraction by automated magnetic bead separation. In a study of 56 dairy herds, tank milk PCR was compared to culture of environmental faecal samples for herd prevalence testing. By the latter, 68% of the herds were positive, while 30% were positive by PCR. Due to the concluded low abundance of MAP in milk tanks, milk PCR would be more useful for testing of MAP in consumers’ milk, than for herd prevalence testing. Three real-time PCR systems were designed for confirmation of PCR positives and validated on 267 strains and 58 positive faecal and tissue samples. The system based on the gene F57 was the most specific. A faecal culture screening of 501 wild guanacos in Chile yielded MAP colonies from 21 guanacos (4.2%), representing the first isolation of MAP from wild animals in the Chilean Patagonia. Confirmation was done by PCR and typing was performed by PCR-REA. All strains proved to be of C type

Employer branding and motivation in municipalities : A qualitative study on employees’ perceptions of employer brand and its impact on motivation

Bakgrund: En ökad konkurrens på arbetsmarknaden och ett ökat behov av personal har gjort det viktigare för kommuner att fokusera på att skapa en unik organisationsidentitet på arbetsmarknaden. Detta koncept, känt som employer branding, kan bidra till en rad fördelar såsom ökad konkurrenskraft och ökad motivation bland medarbetarna, där framför allt det sistnämnda kan underlätta för organisationer gällande att rekrytera och behålla medarbetare. Med employer branding följer även utmaningar, vilket understryker vikten av att förstå hur det bör hanteras för att dra nytta av dess potentiella fördelar. Syfte: Att undersöka medarbetares uppfattningar av kommuners employer branding och dess påverkan på motivation. Metod: Studien har utgått från en kvalitativ forskningsansats där tio semistrukturerade intervjuer genomfördes på ekonomiavdelningarna inom fyra av Skaraborgs kommuner. Resultat och slutsats: Studiens resultat visade att employer branding-strategierna inte diskuterades öppet inom kommunerna och uppfattades främst undermedvetet av medarbetarna. Inom kommunerna låg också ett fokus på att främja den inre motivationen genom faktorer som organisationskulturen. Det var även den inre motivationen som ansågs vara mest attraktiv hos medarbetarna och motiverade dem mest, så länge de hade en grundläggande nivå av yttre motivation där deras löner låg på en acceptabel nivå, då lönen betraktades som en hygienfaktor för medarbetarna.Background: Increased competition in the labour market and a growing need for personnel have made it more important for municipalities to focus on creating a unique organisational identity in the labour market. This concept, known as employer branding, can contribute to a range of benefits such as increased competitiveness and enhanced motivation among employees, where especially the latter can facilitate organisations in recruiting and retaining employees. However, employer branding also comes with challenges, highlighting the importance of understanding how to manage it to capitalize on its potential advantages. Purpose: To examine employees’ perceptions of municipalities’ employer branding and its impact on motivation. Method: The study has used a qualitative research approach, where ten semi-structured interviews were conducted within the finance departments of four municipalities in Skaraborg. Findings and conclusion: The study’s findings revealed that employer branding strategies were not openly discussed within the municipalities and were primarily perceived subconsciously by the employees. There was also a focus within the municipalities on promoting intrinsic motivation through factors such as organisational culture. Intrinsic motivation was also considered most attractive to the employees and motivated them the most, as long as they had a basic level of extrinsic motivation where their salaries were at an acceptable level, as salary was viewed as a hygiene factor for the employees

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