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Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
Published on: March 30, 2015
Internal amplification controls have not been employed in fungal PCR hence potential false negative results
1Centro de Engenharia Biológica, Micoteca da Universidade do Minho, Braga, Portugal. russell.paterson@deb.uminho.pt
Abstract:
Polymerase chain reaction (PCR) is subject to false negative results. Samples of fungi with the genes of interest (e.g. a disease or mycotoxin) may be categorized as negative and safe as a consequence. Fungi are eukaryotic organisms that are involved in many fields of human activity such as antibiotic, toxin and food production. Certain taxa are implicated in human, animal and plant diseases. However, fungi are difficult to identify and PCR techniques have been proposed increasingly for this purpose. Internal amplification controls (IACs) will ameliorate the situation and need to become mandatory. These are nucleic acids that posses a sequence which will provide a PCR product (i) using the same primers employed for the target gene, and (ii) that will not coincide on the gel with the product of the target gene. Only one group of workers employed an IAC, to respond to potential inhibition, which was reported in 1995 from this present assessment of numerous reports. Inhibitors in cultures need to be minimized, and secondary metabolites are an obvious source. The fields reviewed herein include medical mycology, mycotoxicology, environmental mycology and plant mycology. The conclusion is that previous reports are compromised because IACs have not been employed in fungal PCR; future research must include this control at an early stage.
Insights
Internal amplification controls (IACs) are crucial for accurate fungal polymerase chain reaction (PCR) testing. Their absence in most studies compromises results, necessitating their mandatory inclusion in future fungal PCR research for reliable disease and toxin detection.
Area of Science:
- Mycology
- Molecular Biology
- Biotechnology
Background:
- Fungi are vital in various industries (food, medicine) but challenging to identify.
- Polymerase chain reaction (PCR) is increasingly used for fungal identification.
- PCR can yield false negatives, misclassifying fungal samples as safe.
Purpose of the Study:
- To highlight the critical need for internal amplification controls (IACs) in fungal PCR.
- To assess the impact of IACs on the reliability of fungal detection methods.
- To advocate for the mandatory implementation of IACs in future fungal research.
Main Methods:
- Review of numerous scientific reports on fungal PCR applications.
- Analysis of the prevalence and impact of internal amplification controls (IACs).
- Identification of potential sources of inhibition in fungal cultures, such as secondary metabolites.
Main Results:
- A significant lack of IAC implementation was observed across reviewed fungal PCR studies.
- Only one study, dating back to 1995, utilized an IAC to address potential inhibition.
- The absence of IACs compromises the accuracy and reliability of fungal detection results.
Conclusions:
- Previous fungal PCR research is compromised due to the non-use of IACs.
- Minimizing inhibitors in fungal cultures is essential for reliable PCR outcomes.
- Future fungal PCR research must incorporate IACs from the outset to ensure data integrity.
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