Comparison of Two Methods of Bacterial DNA Extraction from Human Fecal Samples Contaminated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni

Abstract

SUMMARY:In this study, 2 methods of DNA extraction were evaluated for use in conjunction with the screening system Rapid Foodborne Bacterial Screening 24 (RFBS24), which employs multiplex realtime SYBR Green polymerase chain reaction (SG-PCR) and can simultaneously detect 24 target genes of foodborne pathogens in fecal DNA samples. The QIAamp DNA Stool mini kit (Qkit) and Ultra Clean Fecal DNA Isolation Kit (Ukit) were used for bacterial DNA extraction from fecal samples artificially inoculated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni. SG-PCR and simplex real-time quantitative PCR (S-qPCR) analyses revealed higher copy numbers (8-234 times) of DNA in samples obtained using Ukit compared with those obtained using Qkit, resulting in lower cycle threshold values for the Ukit samples of the 4 bacteria on SG-PCR analysis. Fecal DNA samples from patients infected during foodborne outbreaks of Salmonella and Campylobacter were also prepared by Qkit and Ukit methods and subjected to RFBS24 analyses. Higher numbers of RFBS24 bacterial target genes were detected in DNA samples obtained using Ukit compared with those obtained using Qkit. Thus, the higher DNA extraction efficiency of the Ukit method compared with Qkit renders the former more useful in achieving improved detection rates of these 4 bacteria in fecal samples using SG-PCR.

An Improved Multiplex Real-Time SYBR Green PCR Assay for Analysis of 24 Target Genes from 16 Bacterial Species in Fecal DNA Samples from Patients with Foodborne Illnesses

“…The improvement of analytical characteristics of RFBS24 ver. 5 by Ukit may be attributed to highly efficient DNA extraction by disruption of bacterial cells via mechanical shearing, as reported previously (24). For Salmonella and Campylobacter infections, the results of the culture method and of our PCR assay for bacterial pathogens, except for the causative pathogens detected with RFBS24 ver.…”

Developments in Rapid Detection Methods for the Detection of Foodborne Campylobacter in the United States

“…This consideration is true even for commercial DNA extraction kits. For example, when Kawase et al. (2014) compared two commercial fecal DNA extraction kits on human feces to pathogen DNA, they detected higher recovery of target genes and improved detection consistently with one kit versus the other.…”

An electrochemiluminescence sensor with dual signal amplification of Ru(bpy)32+based on PtNPs and glucose dehydrogenase for diagnosis of gas gangrene

“…Compared with the previous reports, the sensitivity of this SPE ECL assay is not the highest. 24 However, the signal amplication of ECL has been improved (dual signal amplication) and the assay process has been simplied. Thus, the adaptability for application in POCT is increased.…”