Development of a routine laboratory direct detection system of staphylococcal enterotoxin genes.
A novel direct detection system has been developed for eight staphylococcal enterotoxin (SE)-encoding genes (sea, seb, sec, sed, see, seg, seh and sei) in milk. Specific detection by real-time PCR was successful for all SE-encoding genes in the reference strains. Furthermore, a novel DNA-preparation method with good reproducibility [coefficients of variation 0.31, 0.99 and 1.21 % at 10(6), 10(4) and 10(2) c.f.u. (ml milk sample)(-1), respectively] was developed to overcome PCR inhibition in the milk samples. The combination of this DNA-preparation method and real-time PCR resulted in high sensitivity [between 1.1 x 10(2) and 1.0 x 10(4) c.f.u. (ml milk sample)(-1)] and allowed the completion of the entire procedure within 4 h. Results of an evaluation of this method for the detection of SE-encoding genes using known outbreak milk samples produced results showing good correspondence with the reversed passive latex agglutination assay. In addition, this newly developed system can be applied to clinical samples such as faeces and vomit. Consequently, the system should be useful in the routine direct detection of SE-encoding genes in food-borne-poisoning samples.
Nara Prefectural Institute for Hygiene and Environment, 57-6 Ohmori-cho, Nara City, Nara 630-8131, Japan.
Akifumi
A
Nakayama
Nara Prefectural Institute for Hygiene and Environment, 57-6 Ohmori-cho, Nara City, Nara 630-8131, Japan.
Akiko
A
Okayama
Nara Prefectural Institute for Hygiene and Environment, 57-6 Ohmori-cho, Nara City, Nara 630-8131, Japan.
Misao
M
Hashida
Nara Prefectural Institute for Hygiene and Environment, 57-6 Ohmori-cho, Nara City, Nara 630-8131, Japan.
Yasuzumi
Y
Yamamoto
Nara Prefectural Institute for Hygiene and Environment, 57-6 Ohmori-cho, Nara City, Nara 630-8131, Japan.
Hisakatsu
H
Takebe
Sakai City Institute of Public Health, 3-2-8 Kaichyo Higashi, Sakai City, Osaka 590-0953, Japan.
Takashi
T
Ohnaka
Sakai City Institute of Public Health, 3-2-8 Kaichyo Higashi, Sakai City, Osaka 590-0953, Japan.
Tomoyuki
T
Tanaka
Nara Prefectural Institute for Hygiene and Environment, 57-6 Ohmori-cho, Nara City, Nara 630-8131, Japan.
Shunsuke
S
Imai
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis; D004768:Enterotoxins / Q000235:genetics* / Q000378:metabolism; D005243:Feces / Q000382:microbiology; D006801:Humans; D008892:Milk / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013202:Staphylococcal Food Poisoning / Q000382:microbiology*; D013211:Staphylococcus aureus / Q000235:genetics* / Q000378:metabolism; D014839:Vomiting
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004768:Enterotoxins