OWML: QA/QC

Quality Assurance/Quality Control Manual: Ohio Water Microbiology Laboratory

Enteric viruses
RT-PCR and cell-culture methods are recommended for the detection of enteric viruses in water. To prepare samples for RT-PCR and cell culture, attached viruses are eluted from a 1MDS filter with beef extract (pH 9.5), concentrated using celite (pH 4.0), and eluted with sodium phosphate (pH 9.5).  These steps are done in the OWML, and a protocol is included as Appendix N.

The RT-PCR method was written and laboratory tested by USEPA and is experimental (G. Shay Fout, U.S. Environmental Protection Agency, written commun., 1997; Fout and others, 2003). Viruses are isolated from the eluate by ultracentrifugation through a sucrose gradient, and trace contaminants are removed by extraction with a sol­vent mixture (Appendix N1). An aliquot of the concentrate is used for RT-PCR, wherein any target viral RNA is converted to DNA (by reverse transcriptase) and amplified by the polymerase chain reaction. The RT-PCR products confirmed by hybridization. The enteric viruses detected by use of this method include enterovirus, hepatitis A virus, rotavirus, reovirus, and calicivirus (including Norwalk-like virus).

The OWML consists of a 1,000 ft² main laboratory and a 300 ft² limited-use laboratory. Virus elutions, inhibitor removal, and reaction preparations for RT-PCR are done in the main lab, along with media preparation, membrane filtration, incubation, and culture maintenance. The limited-use lab is the only area in the building in which PCR products are handled. Gel electrophoresis and hybridization are performed in this room. To avoid contamination of incoming samples, staff that have entered the limited-use lab are not allowed to reenter the main lab unless they have showered and changed their clothes. Equipment and supplies are also not to be transferred into the main lab, unless adequate sterilization and decontamination procedures have been followed.

A list describing QC samples for all stages of sample preparation and analysis by the RT-PCR method are listed in Appendix N2.  A check list to be used when interpreting all results of RT-PCR analyses is also contained in Appendix N2. 

Cell-culture analysis is not done at the OWML; these samples need to be sent to a contract laboratory for analysis.  The recommended cell-culture method is an experimental method and was modified from U.S. Environmental Protection Agency (1996) by USEPA (G. Shay Fout, U.S. Environmental Protection Agency, written commun., 1999).  The sample eluate is added to a monolayer of a continuous cell line derived from African Green monkey kidney cells.   Each cell culture is examined microscopically for the appearance of cytopathic effects (CPE) for a total of 14 days; if CPE is not observed in 14 days, a second passage is done. Results are reported as most probable number of infectious units per volume of water.  The following QC samples are included in cell-culture analysis:

• A negative control, containing cells and sodium phosphate buffer, is incubated in a roller bottle with each batch of samples inoculated for the first passage.
   
• A positive control, containing cells, sodium phosphate buffer, and polio virus vaccine, is incubated in a 75 cm² flask with each batch of samples inoculated for the first passage and the second passage.

Media and reagent preparations are done in the OWML or at the contract laboratory and are prepared and stored per method instructions.  The contract laboratory is required to strictly follow the QA/QC guidelines listed in the method documentation (G. Shay Fout, U.S. Environmental Protection Agency, written commun., 1997 and 1999).