Genotyping male-specific RNA coliphages by hybridization with oligonucleotide probes.
F-specific (F+) RNA coliphages are prevalent in sewage and other fecal wastes of humans and animals. There are four antigenically distinct serogroups of F+ RNA coliphages, and those predominating in humans (groups II and III) differ from those predominating in animals (groups I and IV). Hence, it may be possible to distinguish between human and animal wastes by serotyping F+ RNA coliphage isolates. Because serotyping is laborious and requires scarce antiserum reagents, we investigated genotyping using synthetic oligonucleotide probes as an alternative approach to distinguishing the four groups of F+ RNA coliphages. Oligoprobes I, II, III, IV, A, and B were selected to detect group I, II, III, IV, I plus II, and III plus IV phages, respectively. Methods for phage transfer from zones of lysis on a host cell lawn to candidate membrane filters and fixation of genomic nucleic acid on the membranes were optimized. The oligoprobes, which were end labeled with digoxigenin, were applied in DNA-RNA hybridization, and hybrids were observed by colorimetric, immunoenzymatic detection. Of 203 isolates of F+ RNA coliphages from environmental samples of water, wastes, and shellfish, 99.5 and 96.6% could be classified into each group by serotyping and genotyping, respectively. Probes A and B correctly identified 100% of the isolates. On the basis of these results, this method for genotyping F+ RNA coliphages appears to be practical and reliable for typing isolates in field samples.
Hsu
F C
FC
University of North Carolina, Chapel Hill 27599, USA.
Shieh
Y S
YS
van Duin
J
J
Beekwilder
M J
MJ
Sobsey
M D
MD
D000818:Animals; D001483:Base Sequence; D003090:Coliphages / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D004783:Environmental Microbiology; D005243:Feces / Q000821:virology; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics*; D012703:Serotyping
D015345:Oligonucleotide Probes; D012367:RNA, Viral