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Laboratory experience and guidelines for avoiding false positive polymerase chain reaction results
T Victor1, A Jordaan, R du Toit
1Department of Medical Physiology and Biochemistry, Faculty of Medicine, University of Stellenbosch, Tygerberg, Republic of South Africa.
Abstract:
Despite the widespread use of polymerase chain reaction (PCR) for diagnosis of infectious diseases, the technology has not been generally introduced into routine diagnostic laboratories. One of the most serious problems which has influenced the acceptance of this technology is the occurrence of false positive PCR results. This study describes the experience, in a hospital laboratory setting, of using PCR for the diagnosis of heat-labile enterotoxin-producing E. coli, M. tuberculosis, M. paratuberculosis and human papillomavirus. Results indicate that a build-up of amplicons, generated during the amplification process in the laboratory, is the main source of PCR-contamination. Protocols are described that include both physical and chemical procedures to prevent contamination. The use of photo-induced psoralen is recommended for those laboratories already involved in PCR work where amplicons are likely to be present. An enzymatic system (uracil-N-glycosylase) was evaluated and is recommended for workers intending to start diagnostic PCR. Attention was given to simple control measures which are easily implemented in a routine diagnostic laboratory. Protocols such as these are likely to have a major impact on the introduction of PCR-based methods into routine laboratories.
Insights
False positive polymerase chain reaction (PCR) results are a major hurdle for diagnostic labs. This study identifies amplicon buildup as the main contamination source and offers protocols to prevent it, aiding routine PCR adoption.
Area of Science:
- Clinical Microbiology
- Molecular Diagnostics
- Infectious Disease Detection
Background:
- Polymerase chain reaction (PCR) is widely used for infectious disease diagnosis but not routinely implemented in diagnostic laboratories.
- A significant barrier to PCR adoption is the frequent occurrence of false-positive results, often due to contamination.
- Contamination issues hinder the reliable application of PCR in clinical settings.
Purpose of the Study:
- To investigate the primary sources of PCR contamination in a hospital laboratory setting.
- To evaluate and recommend effective protocols for preventing false-positive PCR results.
- To facilitate the routine implementation of PCR-based diagnostic methods.
Main Methods:
- The study focused on PCR diagnostics for heat-labile enterotoxin-producing E. coli, M. tuberculosis, M. paratuberculosis, and human papillomavirus.
- Identified amplicon buildup during amplification as the main source of contamination.
- Evaluated physical and chemical decontamination protocols, including photo-induced psoralen and uracil-N-glycosylase (UNG) enzymatic systems.
Main Results:
- Amplicon accumulation during the PCR process was confirmed as the principal cause of laboratory contamination.
- Physical and chemical control measures were detailed, with specific recommendations for different laboratory scenarios.
- The uracil-N-glycosylase system proved effective for laboratories initiating diagnostic PCR work.
Conclusions:
- Implementing robust contamination control protocols is crucial for the successful integration of PCR into routine diagnostics.
- Photo-induced psoralen and uracil-N-glycosylase are effective strategies for preventing PCR contamination.
- Simple, implementable control measures can significantly improve the reliability and adoption of PCR-based diagnostic tests.