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

Jun Kawase; Morito Kurosaki; Yuta Kawakami; Takashi Kashimoto; Yoshie Tsunomori; Koji Sato; Tetsuya Ikeda; Keiji Yamaguchi; Masanori Watahiki; Tomoko Shima; Mitsuhiro Kameyama; Yoshiki Etoh; Kazumi Horikawa; Hiroshi Fukushima; Ryoichi Goto; Komei Shirabe

doi:10.7883/yoken.67.441

Original Article

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

Jun Kawase, Morito Kurosaki, Yuta Kawakami, Takashi Kashimoto, Yoshie Tsunomori, Koji Sato, Tetsuya Ikeda, Keiji Yamaguchi, Masanori Watahiki, Tomoko Shima, Mitsuhiro Kameyama, Yoshiki Etoh, Kazumi Horikawa, Hiroshi Fukushima, Ryoichi Goto, Komei Shirabe

Author information

Jun Kawase
Shimane Prefectural Institute of Public Health and Environmental Science
Morito Kurosaki
Shimane Prefectural Institute of Public Health and Environmental Science
Yuta Kawakami
Shimane Prefectural Institute of Public Health and Environmental Science
Takashi Kashimoto
Shimane Prefectural Institute of Public Health and Environmental Science
Yoshie Tsunomori
Shimane Prefectural Institute of Public Health and Environmental Science
Koji Sato
Shimane Prefectural Institute of Public Health and Environmental Science
Tetsuya Ikeda
Hokkaido Institute of Public Health
Keiji Yamaguchi
Hokkaido Institute of Public Health
Masanori Watahiki
Toyama Institute of Health
Tomoko Shima
Toyama Institute of Health
Mitsuhiro Kameyama
Yamaguchi Prefectural Institute of Public Health and Environment
Yoshiki Etoh
Fukuoka Institute of Health and Environmental Sciences
Kazumi Horikawa
Fukuoka Institute of Health and Environmental Sciences
Hiroshi Fukushima
Shimane Prefectural Livestock Technology Center
Ryoichi Goto
Hokkaido Institute of Public Health
Komei Shirabe
Yamaguchi Prefectural Institute of Public Health and Environment

Keywords: DNA extraction, fecal sample, foodborne outbreak, real-time PCR

JOURNAL FREE ACCESS

2014 Volume 67 Issue 6 Pages 441-446

DOI https://doi.org/10.7883/yoken.67.441

Details

Abstract

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 real-time 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.

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