This IMS method was subsequently combined with a qPCR method to achieve rapid detection and quantitation of monitoring purposes, subject to some further optimization. In order to produce novel recombinant scFv antibodies specific for cells, two rabbits were immunized with whole cells that had been subjected to gamma radiation rather than heat-inactivation before use. method to rapidly, sensitively, and specifically detect low numbers of cells. This AR234960 library was screened by surface biopanning against whole cells. Enriched clones were analyzed by enzyme-linked immunosorbent assay (ELISA). Two scFv antibodies that strongly and specifically acknowledged cell were expressed in cells. This recombinant monoclonal scFv antibody was purified and used to covalently coat paramagnetic beads to be used for IMS-qPCR. The IMS-qPCR method was able to specifically and sensitively detect in mixed cultures within 3?h. Electronic supplementary material The online version of this article (10.1007/s00253-018-8949-x) contains AR234960 supplementary material, which is available to authorized users. Keywords: spp. are the most common cause of foodborne illness in humans, ahead of (Food Standards Agency 2009; Dominguez 2012). Data on Campylobacteriosis from developing countries is usually scarce, but there is a suggestion that this human burden due to infection is considerable (World Health Business 2012; Platts-Mills and Kosek 2014). Mishandling of contaminated raw poultry and consumption of undercooked poultry have been incriminated as the most significant source of infections for humans (Tam et al. 2009; Sheppard et al. 2009; Dominguez 2012). is usually a harmless commensal intestinal inhabitant of chicken (Doyle 1994), which makes it difficult to spot early contamination of in a flock. It spreads quickly and 95% of a flock of 20,000 chickens can be infected by within 4 to 7?days after colonization of the first bird (Van Gerwe et al. 2009). AR234960 Cross contamination of broiler carcasses can also occur in the slaughterhouse (Hermani et al. 2003; Tram et al. 2012). A significant correlation between the contamination of the broilers during rearing and the carcasses after processing has been observed (Hermani et al. 2003). There is a need to monitor during the life of the chickens in broiler houses, to spot the infection as early as possible and to determine effectiveness of interventions. In the slaughter house, infection status needs to be checked in order to routine flocks for slaughtering and prevent cross contamination (Tram et al. 2012). However, such monitoring has been hampered by the current gold standard culture-based detection method, ISO/TS/EN 10272-2:2006 (International Requirements Organization 2006). This method generally require 24C48?h enrichment culture before isolation of on selective culture media. Enrichment is usually followed by confirmatory methods such as biochemical and serological assessments (Goossens and Butzler 1992), DNA/RNA hybridizations (Cudjoe et al. 1991; Ransom et al. 1994), enzyme-linked immunosorbent (ELISA) assay (Haas 1997), randomly amplified polymorphic DNA (RAPD) analysis (Mazurier et al. 1992; Hilton et al. 1997), polymerase chain reaction (PCR) or quantitative AR234960 PCR (qPCR) assays (Giesendorf et al. 1992; Sails et al. 2003), and loop-mediated isothermal amplification (LAMP) of DNA (Yamazaki et al. 2009). Combination of culture enrichment process with these confirmatory methods prospects to 3C5?day detection time, which is too long, bearing in mind the 4C7?day flock infection rate. By the time the results become available, the status of the flock may have changed, AR234960 which may result in a false negative flock status. Another limitation of culture enrichment-dependent methods is their failure to detect viable but non-culturable (VBNC) cells that may be able to regenerate and become infectious again (Rollins and Colwell 1986; Silva et al. 2011). Direct detection of by PCR and qPCR methods without culture enrichment has been published (Rasmussen et al. 1996; Bang et al. 2001; Vondrakova et al. 2014). However, sensitive, specific, and quick PCR detection can be hampered by inhibitors present in the crude sample matrix leading to false negative results (Tram et al. 2012). The other limitation of all the described culture-based methods is that only a very small portion (10C500?l) of the considerable volume of biological samples (50C250?ml) is actually tested; this may also lead to false unfavorable results, given that the large quantity of spp. in biological samples may be low. Detection sensitivity of assessments for spp. could be substantially improved if most of the cells in Rabbit polyclonal to PIWIL3 the large initial volume of.