United States
   Environmental Protection
  Acceptability of the EPA qPCR Test at Your Beach
                Office of Science and Technology (4303T)
                          Office of Water
                       Washington, DC 20460

                          December 2013


Neither the United States Government nor any of its employees, contractors, or their employees
make any warranty, expressed or implied, or assumes any legal liability or responsibility for any
third party's use of apparatus, product, or process discussed in this document, or represents that
its use by such party would not infringe on privately owned rights. Mention of trade names or
commercial products does not constitute endorsement or recommendation for use.
Questions concerning this method or its application should be addressed to:

    Robin K. Oshiro
    Engineering and Analysis Division (4303T)
    U.S. EPA Office of Water, Office of Science and Technology
    1200 Pennsylvania Avenue, NW
    Washington, DC 20460
    oshiro.robin@epa.gov or OSTCWAMethods@epa.gov

            Acceptability of the EPA qPCR Test at Your Beach
Section 5.2 of the 2012 Recreational Water Quality Criteria (RWQC) states that "EPA
encourages a site-specific analysis of the method's performance prior to use in a beach
notification program or adoption of WQS [water quality standards] based on the method." [1]
This document provides guidance on how to evaluate the acceptability of the quantitative
polymerase chain reaction (qPCR) test at your beach.

The methods referred to in this document are two qPCR Enterococcus spp. as measured by
qPCR methods, EPA Methods 1611 and 1609 [2-3]. The former method was released
simultaneously with the 2012 RWQC. The latter method is an improved version of the method
that uses a newer formulation of PCR reagent (environmental master mix) that has shown
reduced susceptibility to inhibition compared to the reagent used in Method 1611 (universal
master mix).  In addition, Method 1609 includes a competitive internal amplification control
(IAC) assay to help specifically identify false negative reactions or reduced amplification
efficiency due to Taq DNA polymerase inhibit!on[4]. Although either method is acceptable for
use, EPA recommends the use of Method 1609 due to these enhancements.

This document does not address performance acceptability criteria of the method itself, as these
are stated within the method (see Section 14 in Methods 1609 and 1611) as based on a
nationwide method validation. Additionally, this document assumes that the testing laboratory
has been able to perform the method within the acceptance criteria, and now wishes to ascertain
whether or not the method would be acceptable for use at a particular site.

The demonstration should have the following characteristics:

   1.  At least 10 samples should be taken on different days for site evaluation in advance of
       using the method for beach  action decisions. Among these samples, a maximum of 10%
       can fail the Salmon DNA SPC control assay criterion (see Section 9.12 in Method 1611)
       or the SPC and IAC control assays criteria (see Sections 9.12 and 9.13 in Method 1609).
       For any samples that fail the initial analysis, one or both of the interference mitigation
       approaches: extract dilution (see Section 9.12 in Methods  1609 and 1611); higher Salmon
       DNA (see reference 17.5 in method 1609), can be used to assess for mitigation of the
       interference. If mitigation by one of these approaches is successful (i.e., samples now
       pass the control assay criteria specified above), these samples can be considered as not
       having failed in the site evaluation. As an option, beach managers localities can choose
       to sample for an entire beach season or year for initial site evaluation so that their data is
       more representative, but this is not required.

   2.  Particularly if beach closures are not mandated by local standards after a heavy rain
       event, site evaluation sampling should include a representative number of samples
       collected after such events. In general sampling should represent the conditions when
       recreation is expected or known to occur.

   3.  Sites should be re-evaluated every year, preferably prior to using the method for beach
       action decisions, since water characteristics, including the appearance and disappearance
       of inhibitors to the method, have been shown to change over time.

Some localities may be inclined to compare the frequency of beach advisories based on the
qPCR test versus culture test. EPA neither encourages nor discourages such comparisons;
however, it is noted that the results should be interpreted carefully. Comparisons between
exceedances are not reflective of the respective method performance assuming all the controls in
the methods are performing properly.  Caution should also be exercised because comparing
exceedances does not take into account the uncertainty of the respective health relationships
between the two methods.  Also, it is noted that the same day notification potential for qPCR
more accurately reflects water quality for beach goers compared with methods where results are
not available until the following day because water quality is known to fluctuate day-to-day.

This document was developed by Rich Haugland, Kevin Oshima and Larry Wymer of EPA's
Office of Research and Development (ORD) and Robin K. Oshiro of EPA's Office of Water
(OW). Alfred Dufour and Tim Wade of ORD, and Samantha Fontenelle, Tracy Bone, and
Shamima Akhter of OW are also gratefully acknowledged.

(1) US EPA. Recreational Water Quality Criteria. 2012. EPA-820-F-12-058.
(2) Method 1611: Enterococci in Water by TaqManŽ Quantitative Polymerase Chain Reaction
   (qPCR) Assay.  October 2012. EPA-821-R-12-008.
(3) Method 1609: Enterococci in Water by TaqManŽ Quantitative Polymerase Chain Reaction
   (qPCR) with Internal Amplification Control (IAC) Assay. March 2013.  EPA-820-R-13-005.
(4) Haugland, R.A., Siefring, S., Lavender, J., Varma, M. Influences of sample interference and
   interference controls on quantification of enterococci fecal indicator bacteria in surface water
   by the qPCR method. Water Research 2012, 46(18): 5989-6001.