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Genotyping male-specific RNA coliphages by hybridization with oligonucleotide probes
F C Hsu1, Y S Shieh, J van Duin
1University of North Carolina, Chapel Hill 27599, USA.
Abstract:
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.
Insights
Genotyping F-specific (F+) RNA coliphages using oligonucleotide probes offers a practical alternative to serotyping. This method reliably distinguishes between human and animal fecal waste sources.
Area of Science:
- Environmental microbiology
- Molecular biology
- Virology
Background:
- F-specific (F+) RNA coliphages are common in human and animal fecal waste.
- Distinct serogroups of these phages predominate in humans versus animals, suggesting potential for waste source tracking.
- Traditional serotyping is labor-intensive and requires specialized reagents.
Purpose of the Study:
- To develop and evaluate a genotyping method using synthetic oligonucleotide probes as an alternative to serotyping F+ RNA coliphages.
- To differentiate between the four antigenically distinct serogroups of F+ RNA coliphages.
- To assess the practicality and reliability of genotyping for classifying phages from environmental samples.
Main Methods:
- Synthetic oligonucleotide probes (Oligoprobes I, II, III, IV, A, B) were designed to detect specific phage groups.
- Phages were transferred to membrane filters, and genomic nucleic acid was fixed.
- DNA-RNA hybridization was performed using digoxigenin-labeled probes, with detection via colorimetric, immunoenzymatic methods.
Main Results:
- Genotyping classified 96.6% of 203 environmental F+ RNA coliphage isolates into their respective groups, comparable to serotyping (99.5%).
- Probes A and B demonstrated 100% accuracy in classifying isolates.
- The optimized method proved practical for analyzing field samples.
Conclusions:
- Genotyping F+ RNA coliphages with oligonucleotide probes is a practical and reliable alternative to serotyping.
- This molecular approach facilitates the distinction of phage groups, aiding in the identification of human versus animal waste sources.
- The method shows promise for routine application in environmental monitoring and public health surveillance.