Internal amplification controls have not been employed in fungal PCR hence potential false negative results

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.

Advances and Prospects for Molecular Diagnostics of Fungal Infections

“…Although some PCR inhibitors (eg, hemoglobin and heparin) are known or suspected, most are not. However, their impact on the PCR yield can be shown by using an internal control of amplification [17]. An internal control specific for every primer set is one possibility [8].…”

PCR to detect patulin producing moulds validated in foods

“…These findings are very important to further studies such as that reported by Luque et al (2011). Thus, in the work of Luque et al (2011) the authors used four representative works of Dr. Paterson as references, which have been cited in the manuscript (Paterson, 2004(Paterson, , 2006(Paterson, , 2007Paterson et al, 2000), showing that for these authors, findings of Dr. Paterson are essentials for the development of its work. Luque et al (2011) designed specific primers based on the idh gene involved in patulin biosynthesis in order to detect a PCR product of 496 bp in all patulin producing moulds from different genera and species that may contaminate foods.…”

Differential expression of oil palm pathology genes during interactions with Ganoderma boninense and Trichoderma harzianum

“…To further validate the accuracy of the PCR amplification, internal amplification control (IAC) was used to ensure the absence of inhibitory substances in down-regulated samples. The IAC was performed using the actin according to Paterson (2007) and following QIAGEN One Step RT-PCR Kit instructions. The amplified products were purified using the QIAquick Gel Extraction Kit (QIAGEN) according to manufacturer's instructions and sequenced using outsourced commercial sequencing service provider (First BASE Laboratories Sdn Bhd, Malaysia).…”