Optimization of flotation, DNA extraction and PCR methods for detection of Toxoplasma gondii oocysts in cat faeces

Abstract

Introduction and objective The aim of the study was to compare the effectiveness of selected oocysts concentration methods, DNA extraction protocols and PCR assays targeting the B1 gene, for the development of procedures which would be effective and useful in laboratory practice for the detection of T. gondii in faecal samples from cats. Material and methods In order to compare the influence of the flotation fluids on microscopy and PCR detection of T. gondii , saturated solutions of saccharose, MgSO 4 , ZnSO 4 and NaNO 3 were used. To determine the sensitivity of PCR tests used: Real time PCR (RT) and nested PCR, water samples spiked with T. gondii tachyzoites and oocysts were tested. DNA was extracted using DNeasy Blood and Tissue Kit (Qiagen) (K1). The same PCR tests were used to assess the efficacy of T. gondii DNA detection in samples of cat faeces spiked with oocysts, using DNA extraction by a K1 set and a K2 set (QIamp DNA Stool Mini Kit, (Qiagen). Results The initial results showed that NaNO 3 was most useful as a flotation fluid due to the lack negative effect on the oocysts and amplification efficacy in PCR. The level of detection for water samples (100 µl) was determined as 100 tachyzoites and 1–50 oocysts in RT, and 2–20 oocysts in nested PCR. The limit of detection (LD) for stool samples (250 mg) spiked with oocysts, where the K1 set was used, determined as 250 and 5 oocysts in RT and nested PCR, respectively. For samples extracted with the K2 set, LD in RT was determined as 1–50 oocysts (depending on the variant) and 50 oocysts in nested PCR. Conclusions The most effective methods for detection of T. gondii in cat faeces seem to be centrifugal flotation with NaNO 3 , followed by DNA extraction with removing of inhibitors (K2 set) and Real Time PCR targeting B1 gene.

Keywords

• flotation
• Toxoplasma gondii
• faeces
• Real time PCR
• nested PCR
• cats
• oocysts detection