Abstract
Paper-based DNA biosensors are powerful tools in point-of-care diagnostics since they are affordable, portable, user-friendly, rapid and robust. However, their sensitivity is not always as high as required to enable DNA quantification. To improve the response of standard dot blots, we have applied a new enhancement strategy that increases the sensitivity of assays based on the use of biotinylated silica-nanoparticles (biotin-Si-NPs). After immobilization of a genomic Campylobacter DNA onto a paper membrane, and addition of a biotinylated-DNA detection probe, hybridization was evidenced using streptavidin-conjugated to horseradish peroxidase (HRP) in the presence of luminol and H2O2. Replacement of the single biotin by the biotin-Si-NPs boosted on average a 30 fold chemiluminescent read-out of the biosensor. Characterization of biotin-Si-NPs onto a paper with immobilized DNA was done using a scanning electron microscope. A limit of detection of 3 pg/μL of DNA, similar to the available qPCR kits, is achieved, but it is cheaper, easier and avoids inhibition of DNA polymerase by molecules from the food matrices. We demonstrated that the new dot blot coupled to biotin-Si-NPs successfully detected Campylobacter from naturally contaminated chicken meat, without needing a PCR step. Hence, such an enhanced dot blot paves the path to the development of a portable and multiplex paper based platform for point-of-care screening of chicken carcasses for Campylobacter.
Highlights
Campylobacter is considered the most common bacterial cause of human gastroenteritis in the world
transmission electron microscopy (TEM) observations showed that the NP morphology was stable during synthesis (Fig. 1A)
Our results demonstrated that the biotin-Si-NPs enhanced dot blot platform is sensitive enough to detect the whole DNA extracted from the most frequent Campylobacter spp. and could distinguish between Campylobacter and other bacteria
Summary
Campylobacter is considered the most common bacterial cause of human gastroenteritis in the world. The World Health Organization (WHO) estimates that 550 million people fall ill every year from gastroenteritis, of which 220 million are children (WHO 2020). This zoonosis is transmitted mostly through exposure to under-cooked poultry products (50-80% cases), and in a minor way, to contaminated milk, vegetables and water (ECDC 2018; Hermans et al 2012; Humphrey et al 2007). Campylobacteriosis in humans is usually a self-limiting condition involving bloody diarrhea, abdominal cramping, nausea and fever, which can last up to two weeks. To prevent the entry of Campylobacter contaminated broilers into the market, the European Commission adopted a process hygiene criterion (EU 2017/1495) with the critical limit of 1000 cfu/g of broiler meat or skin and obligatory systematic screening of broiler carcasses for Campylobacter spp.
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