Description of a DNA amplification procedure for the detection of bacteriophages of Bacteroides fragilis HSP40 in environmental samples.
A molecular test based on DNA amplification by PCR was developed for the detection of bacteriophages of Bacteroides fragilis strain HSP40 in the environment. These specific phages are associated with faecal contamination of human origin. A homologous DNA region of 1.5 kb, identified previously by hybridisation, was used to design primers for the detection of B. fragilis HSP40 phages. A nested-PCR procedure for the DNA amplification of those phages was developed. The sensitivity of the nested-PCR was between 10(-1) and 10(-2) PFU for purified HSP40 phage solutions, sewage and seawater samples, and between 1 and 10 PFU for river water samples. Specific amplification of HSP40 phages was observed when viral suspensions of 10(3) PFU/ml or lower were used. Common levels of B. fragilis phages found in sewage are 10(1)-10(2) PFU/ml. A total of 24 water samples (sewage, river water and seawater) were tested both by PCR and by plaque assay, to evaluate the efficiency of the molecular method in field samples. The data obtained by PCR in environmental samples showed good concordance with the PFU counts and a higher sensitivity.
Puig
M
M
Department of Microbiology, Faculty of Biology, University of Barcelona, Avd. Diagonal 645, 08028, Barcelona, Spain. rosina@bio.ub.es
Pina
S
S
Lucena
F
F
Jofre
J
J
Girones
R
R
D001435:Bacteriophages / Q000235:genetics* / Q000302:isolation & purification; D001441:Bacteroides fragilis / Q000821:virology*; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis* / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D005618:Fresh Water / Q000821:virology*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D012623:Seawater / Q000821:virology*; D012680:Sensitivity and Specificity; D012722:Sewage / Q000821:virology*; D010948:Viral Plaque Assay
D017931:DNA Primers; D004279:DNA, Viral; D012722:Sewage