Development and Evaluation of a Multiplexed Real-Time TaqMan RT-PCR Assay with a Sample Process Control for Detection of F-specific RNA Coliphage Genogroups I and IV
- 170 Downloads
There are increasing concerns of zoonotic transmission of some animal enteric viruses, such as calicivirus, hepatitis E virus, and rotavirus, which are closely related to human pathogenic strains. Most enteric viruses are detected by molecular techniques because they cannot be cultured. Surrogates such as F-RNA coliphages are cultivable but few molecular methods exist. Individual real-time TaqMan RT-PCR assays for the replicase gene of F-RNA coliphage genogroups I and IV were developed and multiplexed with a real-time TaqMan RT-PCR assay for feline calicivirus as a sample process control for the simultaneous detection and enumeration of genogroup I and IV F-RNA coliphages. Genogroup IV were successfully detected with the multiplexed assay in 80% of fecal samples that contained F-RNA coliphage levels ≥3.2 log plaque forming units (pfu). F-RNA coliphage were at or below the limit of detection in most fecal samples when levels were ≤4 log pfu/g.
KeywordsF+ RNA bacteriophages Virus surrogate Real-time RT-PCR Feline calicivirus Sample process control
The authors thank Danielle Leblanc at AAFC, Food Research and Development Centre, St-Hyacinthe, QC, Canada, for her valuable discussion and FCV stock production and titration. This research was supported by Agriculture and Agri-Food Canada Research Branch Peer Reviewed Research Projects 75 and 162.
- Anon. (1995). ISO 10705-1. Water quality-detection and enumeration of bacteriophages. Part 1. Enumeration of F-specific RNA bacteriophages. Geneva: International Organization for Standardization.Google Scholar
- Anon. (2001). USEPA Method 1602. Male specific and somatic coliphage in water by single agar layer (SAL) procedure. Washington, DC: United States Environmental Protection Agency, Office of Water.Google Scholar
- Cirino, N. M., Tavakoli, N. P., Madison-Antenucci, S., & Egan, C. (2007). Multiplex rtPCR in microbiology. In I. M. Mackay (Ed.), Real-time PCR in microbiology: From diagnosis to characterization (pp. 183–229). Norfolk: Caister Academic Press.Google Scholar
- Formiga-Cruz, M., Allard, A. K., Conden-Hansson, A. C., Henshilwood, K., Hernroth, B. E., Jofre, J., et al. (2003). Evaluation of potential indicators of viral contamination in shellfish and their applicability to diverse geographical areas. Applied and Environmental Microbiology, 69(3), 1556–1563.PubMedCrossRefGoogle Scholar
- Furuse, K. (1987). Distribution of coliphages in the environment: General considerations. In S. G. Goyal, C. P. Gerba, & G. Bitton (Eds.), Phage ecology (pp. 87–124). New York: Wiley.Google Scholar
- Havelaar, A. H., Hogeboom, W. M., & Pot, R. (1984). F-specific RNA bacteriophages in sewage: Methodology and occurrence. Water Science and Technology, 17(4/5), 645–655.Google Scholar
- Mattison, K., Brassard, J., Houde, A., Simard, C., Pagotto, F., Jones, T., et al. (2007a). The feline calicivirus (FCV) as an internal control for the detection of RNA viruses from foods. Federal food safety and nutrition research meeting. Winnipeg, Manitoba, Canada.Google Scholar
- Sundram, A., Jumanlal, N., & Ehlers, M. M. (2006). Genotyping of F-RNA coliphages isolated from wastewater and river water samples. Water SA, 32(1), 65–70.Google Scholar