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AN ENVIRONMENTAL TECHNOLOGY VERIFICATION (ETV) PERFORMANCE TESTING OF THREE RAPID PCR TECHNOLOGIES FOR IDAHO TECHNOLOGY R.A.I.D.® SYSTEM, APPLIED BIOSYSTEMS TAQMAN® E. COLI 0157:H7 DETECTION SYSTEM, AND INVITROGEN CORPORATION PATHALERTTM DETECTION KITS
Citation:
RIGGS, K. AN ENVIRONMENTAL TECHNOLOGY VERIFICATION (ETV) PERFORMANCE TESTING OF THREE RAPID PCR TECHNOLOGIES FOR IDAHO TECHNOLOGY R.A.I.D.® SYSTEM, APPLIED BIOSYSTEMS TAQMAN® E. COLI 0157:H7 DETECTION SYSTEM, AND INVITROGEN CORPORATION PATHALERTTM DETECTION KITS. U.S. Environmental Protection Agency, Washington, DC, EPA/600/R-05/026, 2004.
Impact/Purpose:
The overall objective of the ETV Program is to accelerate the entrance of new environmental technologies into the domestic and international marketplace by identifying the environmental performance characteristics of commercial-ready technology through the evaluation of objective and quality assured data. This provides the potential purchasers and permitters with an independent and credible assessment of what they are buying and/or permitting. The AMS Center has received funding to performance verify monitoring technologies relevant for homeland security.
Description:
The Environmental Technology Verification (ETV) Program, beginning as an initiative of the U.S. Environmental Protection Agency (EPA) in 1995, verifies the performance of commercially available, innovative technologies that can be used to measure environmental quality. The ETV provides, through a third-party, quality-assured performance data so buyers and users of environmental technologies can make informed purchase and application decisions, thus providing one path to reducing emissions and improving human health. To provide cost-effective testing, Stakeholder committees, made up of members with diverse backgrounds, provide guidance to the ETV by identifying and prioritizing environmental technologies to address present day environmental quality challenges.
The ETV Advanced Monitoring Systems (AMS) Center, one of six ETV Centers, is actively involved in verifying the performance of advanced monitoring systems available to the public for purchase. The AMS Center participates in the mission of the ETV by providing Test Plans, Protocols, conducting independent performance tests of technologies, and preparing Verification Reports and Statements describing the results of the testing. Vendors of tested technologies can use the Verification Reports and Statements for marketing purposes. All approved Verification Reports are posted on the ETV Web Site as a form of distribution. The individual verification reports and statements describe by this abstract are for three Rapid Polymerase Chain Reaction (PCR) technologies: Idaho Technology R.A.P.I.D®. System, Applied Biosystems Taqman® E. Coli 0157:H7 Detection System, and Invitrogen Corporation PathAlert Detection Kits.
The purpose of this verification test of rapid PCR technologies was to evaluate the ability of these technologies to detect the presence of specific bacteria in water and to determine the technologies susceptibility to specific interferents added to pure water and to those interferents inherently present in several drinking water matrices. The technologies for this verification test operate based on the PCR process, which involves enzyme-mediated reactions that allow for target DNA (that from the bacteria of interest) replication and amplification through a series of temperature cycles. Before the target DNA can be amplified, however, it must first be extracted from the bacteria and then purified.
Because rapid PCR technologies are anticipated to serve mostly as screening tools in water monitoring scenarios, providing rapid results as to whether or not a pathogen or biological agent is present in the water, this verification test involved only qualitative results. This verification test of these technology systems were conducted according to procedures specified in the Test/QA Plan for Verification of Rapid PCR Technologies. The performance of these technologies system were verified in terms of the following parameters: accuracy, specificity, false positive/negative responses, precision, and interferences.
The performance of these technology systems were verified by subjecting them to various concentration levels of Bacillus anthracis (B. anthracis) Ames strain, Francisella tularensis (F. tularensis) LVS (ATCC# 29684), Yersinia pestis (Y. pestis) CO92, Brucella suis (ATCC#23444), and Escherichia coli O157:H7 (E. coli) in American Society of Testing and Materials (ASTM) Type II deionized (DI) water; ASTM Type II DI water spiked with various interferents; and concentrated drinking water (DW) samples obtained from four water utilities from different geographical locations in the United States. Each source of DW represented a unique water treatment process. In addition, the interferent and DW samples were analyzed without adding any contaminant to evaluate the potential for false positive results. These system were tested for one bacteria at a time and only for the bacteria it was capable of detecting.
Contaminant concentrations included the infective/lethal dose concentrations for each contaminant and approximately 2, 5, 10, and 50 times the vendor-reported limit of detection (LOD). The infective/lethal dose of each contaminant was determined by calculating the concentration at which 250 mL of water is likely to cause the death of a 70-kilogram person based on human LD50 or ID50 data.
For the purposes of this verification test, the LOD is anticipated to be the level at which quantifiably reproducible positive results are obtained. It represents the level for the entire system at which reproducible positive results are obtained from a raw water sample. As such, it is more of a method or experimental detection limit than a true instrument detection limit. This detection level, then, incorporates the sensitivities and uncertainties of the entire technology system, in particular the DNA purification step as well as additional reagents. Detection limits for individual components of these technology systems may differ and were not verified in this test.
The verification test was conducted at Battelle's Medical Research and Evaluation Facility in West Jefferson, Ohio, as well as Battelle headquarters in Columbus, Ohio, from May 27, 2004, through July 8, 2004. Aqua Tech Environmental Laboratories, Inc. (ATEL) of Marion, Ohio, performed physicochemical characterization for each DW sample, including turbidity, dissolved and total organic carbon, specific conductivity, alkalinity, pH, magnesium (Mg), calcium (Ca), hardness, total organic halides, trihalomethanes, and haloacetic acids.
QA oversight of the verification testing was provided by the EPA and Battelle. Battelle QA staff conducted a technical systems audit and a data quality audit of 10% of the test data. The verification statement, the full report on which it is based, and the test/QA plan for this verification are all available at http://www.epa.gov/etv/centers/center1.html.