Development and validation of a real-time PCR assay for the detection of Toxoplasma gondii DNA in animal and meat samples.
We report a rapid and reliable method for the detection of Toxoplasma gondii in meat and animal tissues based on real-time polymerase chain reaction (PCR). Samples were collected from cattle, small ruminants, horses, and pigs raised or imported into Sicily, Italy. All DNA preparations were assayed by real-time PCR tests targeted to a 98-bp long fragment in the AF 529-bp repeat element and to the B1 gene using specific primers. Diagnostic sensitivity (100%), diagnostic specificity (100%), limit of detection (0.01 pg), efficiency (92-109%), and precision (mean coefficient of variation = 0.60%), repeatability (100%), reproducibility (100%), and robustness were evaluated using 240 DNA extracted samples (120 positives and 120 negative as per the OIE nested PCR method) from different matrices. Positive results were confirmed by the repetition of both real-time and nested PCR assays. Our study demonstrates the viability of a reliable, rapid, and specific real-time PCR on a large scale to monitor contamination with Toxoplasma cysts in meat and animal specimens. This validated method can be used for postmortem detection in domestic and wild animals and for food safety purposes.
National Reference Centre for Toxoplasmosis, Istituto Zooprofilattico Sperimentale della Sicilia, Catania, Italy (Marino, Giunta, Salvaggio, Percipalle, Caracappa, Alfonzetti, Aparo).|Molecular Biology Department, Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy (Reale).
Anna Maria Fausta
AM
Marino
National Reference Centre for Toxoplasmosis, Istituto Zooprofilattico Sperimentale della Sicilia, Catania, Italy (Marino, Giunta, Salvaggio, Percipalle, Caracappa, Alfonzetti, Aparo).|Molecular Biology Department, Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy (Reale).
Maurizio
M
Percipalle
National Reference Centre for Toxoplasmosis, Istituto Zooprofilattico Sperimentale della Sicilia, Catania, Italy (Marino, Giunta, Salvaggio, Percipalle, Caracappa, Alfonzetti, Aparo).|Molecular Biology Department, Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy (Reale).
Renato Paolo
RP
Giunta
National Reference Centre for Toxoplasmosis, Istituto Zooprofilattico Sperimentale della Sicilia, Catania, Italy (Marino, Giunta, Salvaggio, Percipalle, Caracappa, Alfonzetti, Aparo).|Molecular Biology Department, Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy (Reale).
Antonio
A
Salvaggio
National Reference Centre for Toxoplasmosis, Istituto Zooprofilattico Sperimentale della Sicilia, Catania, Italy (Marino, Giunta, Salvaggio, Percipalle, Caracappa, Alfonzetti, Aparo).|Molecular Biology Department, Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy (Reale).
Giulia
G
Caracappa
National Reference Centre for Toxoplasmosis, Istituto Zooprofilattico Sperimentale della Sicilia, Catania, Italy (Marino, Giunta, Salvaggio, Percipalle, Caracappa, Alfonzetti, Aparo).|Molecular Biology Department, Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy (Reale).
Tiziana
T
Alfonzetti
National Reference Centre for Toxoplasmosis, Istituto Zooprofilattico Sperimentale della Sicilia, Catania, Italy (Marino, Giunta, Salvaggio, Percipalle, Caracappa, Alfonzetti, Aparo).|Molecular Biology Department, Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy (Reale).
Alessandra
A
Aparo
National Reference Centre for Toxoplasmosis, Istituto Zooprofilattico Sperimentale della Sicilia, Catania, Italy (Marino, Giunta, Salvaggio, Percipalle, Caracappa, Alfonzetti, Aparo).|Molecular Biology Department, Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy (Reale).
Stefano
S
Reale
D000818:Animals; D002417:Cattle; D017931:DNA Primers; D016054:DNA, Protozoan / Q000235:genetics; D006736:Horses; D007558:Italy; D008460:Meat; D016133:Polymerase Chain Reaction / Q000662:veterinary; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013552:Swine; D014122:Toxoplasma / Q000235:genetics / Q000302:isolation & purification*; D014123:Toxoplasmosis / Q000175:diagnosis*
D017931:DNA Primers; D016054:DNA, Protozoan
AF repeat element; Toxoplasma gondii; animal tissues; meat; real-time PCR