EVALUATION AND COMPARISON OF DNA EXTRACTION KITS FOR THE DETECTION OF Clostridium difficile IN SPIKED AND FIELD FAECES FROM PIGLETS BY USING REAL-TIME PCR

Abstract

In complex samples (faeces, soil, food, etc.), Clostridium difficile is often present in the form of dormant spores that cause reduced effectiveness of DNA extraction. With the aim of determining an optimal DNA extraction procedure from spores, DNA extraction from faecal samples spiked with a known number of C. difficile spores and faecal samples from piglets was performed with three manual protocols, using two commercial kits and subsequent real-time PCR (rtPCR) DNA amplification. DNA extraction protocols, including mechanical disruption by bead beating, gave better results with rtPCR. The SmartHelix DNAid Complex Kit proved to be more efficient than the QIAamp DNA Stool Mini Kit, suggesting that an optimal combination of mechanical, enzymatic, and chemical lysis seems to be required for the best results. Key words: Clostridium difficile; DNA extraction; real-time PCR; piglets; faeces; spores OVREDNOTENJE IN PRIMERJAVA KOMERCIALNIH KOMPLETOV ZA IZOLACIJO DNK ZA DOKAZOVANJE BAKTERIJE Clostridium difficile V BLATU PRAŠIČKOV Z METODO PCR V REALNEM ČASU V kompleksnih vzorcih (blato živali, zemlja, hrana, itd.) je bakterija Clostridium difficile pogosto prisotna v obliki spor, ki so vzrok za slabšo učinkovitost izolacije DNK. Da bi določili optimalni postopek izolacije DNK iz spor, smo s tremi različnimi protokoli in uporabo dveh komercialnih kompletov za izolacijo DNK izolirali DNK iz vzorcev blata z znanim številom C. difficile spor in vzorcev blata prašičkov. DNK smo pomnoževali z metodo PCR v realnem času (rtPCR). Boljše rtPCR rezultate smo dobili pri vzorcih, kjer smo pri izolaciji DNK uporabili tudi mehansko razbitje celic s kroglicami. SmartHelix DNAid Complex Kit je bil učinkovitejši komercialni komplet kot QIAamp DNA Stool Mini Kit, kar nakazuje na optimalno kombinacijo mehanske, encimske in kemične lize za najboljši izplen DNK. Ključne besede: Clostridium difficile; izolacija DNK; spore; PCR v realnem času; prašički; blato