Magnetic Capture qPCR

The selection of tissue samples to assess parasite load in animals was based on our previous T. gondii IPB LR and/or Gangji strain infection study, in which we demonstrated that the tissues with the highest parasite load were detected in brain and heart followed by skeletal muscle intercostales, longissimus dorsi, psoas major, diaphragm, and gastrocnemius via Magnetic Capture qPCR (21).

The tissue samples from brain, heart and the skeletal muscles gastrocnemius and longissimus dorsi were collected from all pigs. The parasite load was determined by an ISO 17025 validated magnetic capture qPCR as previously described (26), in which the magnetic isolation of T. gondii-specific DNA from large tissue samples (>100 g) was combined with the sensitivity of qPCR. In addition, the developed MCqPCR has a sensitivity of 99% and a better limit of detection (26) than a previous MCqPCR (27).

All the samples with an exponential amplification curve crossing the threshold (Cq) were considered positive for T. gondii, while samples with no amplification curve for the T. gondii target, but amplification of the NCIAC (Not Competitive Internal Amplification Control) were considered negative. The detection limit of this method is 65.4 parasites per 100 g of tissue sample. For each round of samples, a positive control with a known number of parasites (calibrator) was included to correct for possible deviations due to manipulation errors. The number of parasites (n° p) was calculated according to the following formula:

The formula resulted from a standard curve established with known concentrations of parasites ranging from 100 to 10⁵ spiked in 100 g of muscle tissue samples or in 50 g of brain tissue (28). Log₁₀(n° P) represents the log₁₀-transformed parasitic load, while the Cq_value represents the point on the exponential amplification curve crossing the threshold.