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 ofin faecal samples from cats. MATERIAL AND METHODS: In order to compare the influence of the flotation fluids on microscopy and PCR detection of, saturated solutions of saccharose, MgSO, ZnSOand NaNOwere used. To determine the sensitivity of PCR tests used: Real time PCR (RT) and nested PCR, water samples spiked withtachyzoites 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 ofDNA 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 NaNOwas 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 ofin cat faeces seem to be centrifugal flotation with NaNO, followed by DNA extraction with removing of inhibitors (K2 set) and Real Time PCR targeting B1 gene.