EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay.
A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. The U.S Environmental Protection Agency's (USEPA) Method 1615 was developed with the goal of providing such a standard for measuring Enterovirus and Norovirus in these waters. Virus is concentrated from water using an electropositive filter, eluted from the filter surface with beef extract, and then concentrated further using organic flocculation. Herein we present the protocol from Method 1615 for filter elution, secondary concentration, and measurement of total culturable viruses. A portion of the concentrated eluate from each sample is inoculated onto ten replicate flasks of Buffalo Green Monkey kidney cells. The number of flasks demonstrating cytopathic effects is used to quantify the most probable number (MPN) of infectious units per liter. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. Laboratories must meet defined performance standards. Method 1615 was evaluated by examining virus recovery from reagent-grade and ground waters seeded with Sabin poliovirus type 3. Mean poliovirus recoveries with the total culturable assay were 111% in reagent grade water and 58% in groundwaters.
National Exposure Research Laboratory, U.S. Environmental Protection Agency; fout.shay@epa.gov.
G Shay
GS
Fout
National Exposure Research Laboratory, U.S. Environmental Protection Agency.
Jennifer L
JL
Cashdollar
D000818:Animals; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D060766:Drinking Water / Q000821:virology*; D004770:Enterovirus / Q000235:genetics / Q000302:isolation & purification*; D060587:Groundwater / Q000821:virology*; D029322:Norovirus / Q000235:genetics / Q000302:isolation & purification*; D017955:Poliovirus / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D014481:United States; D014484:United States Environmental Protection Agency; D014871:Water Microbiology
D060766:Drinking Water
J Appl Microbiol. 2013 Jul;115(1):1-11
23360578
Appl Environ Microbiol. 2011 May;77(10):3500-6
21441329
Appl Environ Microbiol. 2008 Jan;74(2):477-84
18065626
N Engl J Med. 1999 Sep 23;341(13):929-35
10498487
MMWR Surveill Summ. 2006 Sep 15;55(8):1-20
16971890
Appl Environ Microbiol. 2000 Jul;66(7):2914-20
10877786
Appl Environ Microbiol. 2009 Apr;75(8):2393-9
19218410
Environ Sci Technol. 2013 May 7;47(9):4096-103
23570447
Clin Infect Dis. 2008 Sep 1;47(5):616-23
18637756
Semin Pediatr Infect Dis. 2002 Jan;13(1):40-7
12118843
Appl Environ Microbiol. 2003 Dec;69(12):7181-7
14660364
Appl Environ Microbiol. 2013 Jan;79(1):215-23
23087037
Appl Environ Microbiol. 1986 Sep;52(3):450-9
3021056
Appl Environ Microbiol. 1986 Apr;51(4):790-812
3010860
J Appl Microbiol. 2011 Nov;111(5):1250-61
21854513
Emerg Infect Dis. 2008 Oct;14(10):1546-52
18826817
N Engl J Med. 2009 Oct 29;361(18):1776-85
19864676
Appl Environ Microbiol. 2010 Aug;76(15):5159-64
20562285
J Infect Dis. 2003 Jan 15;187(2):303-6
12552455