Abstract
Toxoplasmosis is a zoonotic disease caused by the protozoan parasite Toxoplasma gondii. Infection in humans has usually been related to the consumption of raw, undercooked or cured meat. The aim of this study was to develop a droplet digital polymerase chain reaction (ddPCR)-based assay for the detection and quantification of T. gondii in meat samples. To optimize the ddPCR, T.gondii reference DNA aliquots at five known concentrations: 8000 cg/µl, 800 cg/µl, 80 cg/µl, 8 cg/µl were used. Moreover, results obtained by ddPCR and quantitative PCR (qPCR) were compared using 80 known samples (40 positive and 40 negative), as well as 171 unknown diaphragm tissue samples collected at slaughterhouses. The ddPCR showed a sensitivity of 97.5% and a specificity of 100%, with a detection limit of 8 genomic copy/µl of T. gondii. A nearly perfect agreement (κ = 0.85) was found between results obtained by ddPCR and qPCR for both positive and negative known samples analysed. On the 171 diaphragm tissue samples from field, 7.6% resulted positive by ddPCR and only 1.2% by qPCR. Therefore, this innovative method could be very useful for the detection of T. gondii in meat samples, aiming to prevent human infections.
Highlights
Toxoplasmosis, caused by the intracellular protozoan Toxoplasma gondii, is one of the most common parasitic infection in animals and humans worldwide (Almeira and Dubey, 2021)
The aim of this paper is to develop and validate a new droplet digital polymerase chain reaction (ddPCR) assay for detection and quantification of T. gondii DNA in meat of intermediate hosts
DNA concentration was determined by a Biophotometer (Eppendorf, Hamburg, Germany), samples were diluted to be analysed by ddPCR at five concentration levels: 8000 cg/μl, 800 cg/μl, 80 cg/μl, 8 cg/μl, to evaluate the limit of detection at 95% of probability (LOD95)
Summary
Toxoplasmosis, caused by the intracellular protozoan Toxoplasma gondii, is one of the most common parasitic infection in animals and humans worldwide (Almeira and Dubey, 2021). As reported by FAO/WHO (2014), toxoplasmosis is considered one of the most important food-, water- and soilborne diseases. It is estimated that approximately two billion of people are infected with T. gondii (Almeria and Dubey, 2021). The main routes of infection are ingestion of: (i) food or water contaminated with sporulated oocysts (e.g. vegetables, fruit and molluscan shellfish); (ii) uncooked or undercooked meat containing tissue cysts (Ghozzi et al, 2017; Caradonna et al, 2017; EFSA, 2018). Tachyzoites excreted in milk could be a source of infection. Outbreaks of toxoplasmosis associated with consumption of unpasteurized goats’ milk have been reported (FAO/WHO, 2014; EFSA, 2018; Almeria and Dubey, 2021). Tachyzoites can be transmitted vertically from mother to foetus or via organ transplants (Smith et al, 2021)
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