What is a PCR (Polymerase Chain Reaction) inhibitor?

From the Guidelines

PCR inhibitors can significantly impact the accuracy of molecular diagnostics, and their presence should be carefully considered when interpreting results, particularly in the context of infective endocarditis diagnosis. When dealing with PCR inhibitors, it is essential to understand the potential limitations of the PCR technique, as highlighted in the guidelines on the prevention, diagnosis, and treatment of infective endocarditis by the European Society of Cardiology (ESC) 1. Some key points to consider include:

• The presence of PCR inhibitors in clinical samples can lead to false negatives
• The technique has inherent limitations, including the lack of reliable application to whole blood samples and the risk of contamination
• Improvements in PCR technology, such as real-time PCR, and the availability of other emerging technologies may address some of these deficiencies
• Results still require careful specialist interpretation, particularly in the context of infective endocarditis diagnosis, where PCR positivity has been proposed as a major diagnostic criterion, but is unlikely to supersede blood cultures as a prime diagnostic tool 1. To overcome PCR inhibition, several strategies can be employed, including:
• Using specialized DNA extraction methods that remove inhibitors
• Diluting samples to reduce inhibitor concentration
• Adding PCR enhancers like bovine serum albumin (BSA) or betaine
• Using inhibitor-resistant DNA polymerases such as Hot Start polymerases
• Incorporating internal amplification controls to detect inhibition. In the context of real-life clinical medicine, it is crucial to prioritize the accurate diagnosis and treatment of infective endocarditis, and to carefully consider the potential impact of PCR inhibitors on molecular diagnostics, as highlighted in the ESC guidelines 1.

From the Research

PCR Inhibitors

• PCR inhibitors can originate from the sample itself or as a result of the method used to collect or prepare the sample 2
• These inhibitors can dramatically reduce the sensitivity and amplification efficiency of PCR 2
• PCR inhibitors represent a diverse group of substances with different properties and mechanisms of action 3

Occurrence of PCR Inhibitors

• PCR inhibitors can be found in various types of samples, including clinical, environmental, and food samples 2, 3
• Some PCR inhibitors are predominantly found in specific types of samples, necessitating matrix-specific protocols for preparation of nucleic acids before PCR 3

Removal of PCR Inhibitors

• Various protocols have been developed to remove PCR inhibitors from samples, including the use of PowerClean® DNA Clean-Up kit, DNA IQ™ System, Phenol-Chloroform extraction, and Chelex®-100 methods 4
• The PowerClean® DNA Clean-Up kit and DNA IQ™ System have been shown to be effective in removing commonly encountered PCR inhibitors 4
• Applying inhibitor-tolerant DNA polymerases can also provide a solution to the inhibition problem 5

Solutions to PCR Inhibition

• Purifying or diluting DNA extracts can help to reduce PCR inhibition, but this can lead to DNA loss 5
• Adding bovine serum albumin (BSA) to PCR reactions can also help to alleviate PCR inhibition, particularly in the detection of foodborne viruses 6