Molecular detection and genotyping of male-specific coliphages by reverse transcription-PCR and reverse line blot hybridization.
In recent years, there has been increased interest in the use of male-specific or F+ coliphages as indicators of microbial inputs to source waters. Sero- or genotyping of these coliphages can also be used for microbial source tracking (MST). Among the male-specific coliphages, the F+ RNA (FRNA) viruses are well studied, while little is known about the F+ DNA (FDNA) viruses. We have developed a reverse line blot hybridization (RLB) assay which allows for the simultaneous detection and genotyping of both FRNA as well as FDNA coliphages. These assays included a novel generic duplex reverse transcription-PCR (RT-PCR) assay for FRNA viruses as well as a generic PCR for FDNA viruses. The RT-PCR assays were validated by using 190 field and prototype strains. Subsequent DNA sequencing and phylogenetic analyses of RT-PCR products revealed the classification of six different FRNA clusters, including the well-established subgroups I through IV, and three different FDNA clusters, including one (CH) not previously described. Within the leviviruses, a potentially new subgroup (called JS) including strains having more than 40% nucleotide sequence diversity with the known levivirus subgroups (MS2 and GA) was identified. We designed subgroup-specific oligonucleotides that were able to genotype all nine (six FRNA, three FDNA) different clusters. Application of the method to a panel of 351 enriched phage samples from animal feces and wastewater, including known prototype strains (MS2, GA, Q beta, M11, FI, and SP for FRNA and M13, f1, and fd for FDNA), resulted in successful genotyping of 348 (99%) of the samples. In summary, we developed a novel method for standardized genotyping of F+ coliphages as a useful tool for large-scale MST studies.
Vinjé
Jan
J
Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC 27599, USA. janvinje@email.unc.edu.
Oudejans
Sjon J G
SJ
Stewart
Jill R
JR
Sobsey
Mark D
MD
Long
Sharon C
SC
D000818:Animals; D003090:Coliphages / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D005243:Feces / Q000821:virology; D005838:Genotype; D006801:Humans; D017911:Inoviridae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D017907:Leviviridae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014871:Water Microbiology
D004279:DNA, Viral