Direct amplification of Escherichia coli O157 vero toxin genes from human faeces by the polymerase chain reaction.
Direct amplification of DNA from clinical specimens, such as blood and faeces, by polymerase chain reaction (PCR) is most often hindered by endogenous inhibitory substances, including haemoglobin and bile acids. We tested whether Ampdirect A (Shimadzu), a novel reagent cocktail that has been shown to suppress the inhibitors in blood, is also useful for faecal samples, and found that the vero toxin genes (VT1 and VT2) of Escherichia coli O157 could be efficiently amplified from the supernatant of boiled faeces by PCR in the presence of this cocktail without prior extraction of DNA. We compared the efficiency of amplification with and without the cocktail, using the supernatant of boiled normal faeces supplemented with E. coli O157. PCR without the cocktail failed to amplify the vero toxin genes from the supernatant diluted < 6400-fold or containing > 0.02% (final concentration) of boiled faeces. By contrast, PCR with Ampdirect A amplified the toxin genes in the mixture containing as much boiled faeces as 0.5% and as few E. coli as 4 to 8 colony-forming units (CFU). The minimum limit for E. coli O157 detection by this method was estimated to be about 10(4) CFU/g faeces. The results obtained by this direct method agreed well with those obtained by the indirect method using DNA pre-extracted from patients' faeces (the detection limit being 10(3) CFU/g faeces).
Okamoto
H
H
Department of Clinical Laboratory, Kyoto National Hospital, Japan.
Takano
E
E
Sugao
T
T
Kage
K
K
Okamoto
E
E
Nishimura
N
N
Ueda
K
K
D001427:Bacterial Toxins / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D019453:Escherichia coli O157 / Q000235:genetics*; D005243:Feces / Q000382:microbiology*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D022622:Shiga Toxin 1
D001427:Bacterial Toxins; D022622:Shiga Toxin 1