Sumyya Waliullah, PhD

Development of a portable rapid molecular detection and quantification tools for aflatoxin-producing fungi in peanuts and peanut products

Aflatoxin, a toxic secondary metabolite primarily produced by Aspergillus flavus (A. flavus), has been classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC) Aflatoxin, a toxic secondary metabolite primarily produced by Aspergillus flavus (A. flavus), has been classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC) (Mamo et al. 2021). Aflatoxin contamination not only compromises the safety of peanuts but also presents grave health risks upon consumption like immunity suppression, stunt growth in children, hepatotoxicity (Bbosa et al., 2013). Mississippi with its warm and humid climate provides optimal conditions for the growth of A. flavus and aflatoxin production in peanuts and peanut products. A primary limitation to controlling ingestion of contaminated peanuts is the lack of rapid and accurate detection and quantification methods to screen of aflatoxin-producing fungi in foods contamination. The conventional methods for identifying and detecting aflatoxigenic fungi in foods and feedstuffs rely on morphological characteristics on plate assay or blotter assay and the outcomes can be highly variable depending on the media and culture conditions. Furthermore, they are time consuming (~2 weeks to complete), labor intensive and require the expertise of mycologists. Molecular diagnostic based on polymerase chain reaction (PCR) also exists to diagnosis this pathogen. However, this method is less sensitive for the screening of asymptomatic or early infected peanuts that harbor relatively low pathogen populations. In particular, the conventional PCR assay cannot detect under 500 copies (1 pg/μl) of a pathogen. In contrast, previous studies on a portable real-time PCR with other microbes easily detect below 1fg/μl or lower concentration (Waliullah et al., 2019). Therefore, the development of a rapid and portable method for detection and identification of aflatoxigenic fungi in foods and feedstuffs is needed for the estimation and neutralization of the associated potential health risks. 

Rapid detection of contamination of peanuts and peanut products by aflatoxigenic fungi is extremely useful because aflatoxins exert adverse health effects (Konietzny et al., 2003) To tackle the limitation of traditional detection issues effectively, we propose the development of a portable real-time PCR method for quantifying of aflatoxin-producing fungi A. flavus in peanuts and peanut products. This method combines high sensitivity with the convenience of portability, allowing for on-site testing and rapid results. Currently, sequencing of barcoding genes such as ITS (internal transcribed spacer), beta-tubulin gene, RNA polymerase II gene (RPB2) or calmodulin gene can identify almost all the species of Aspergillus section Flavi, however, they cannot distinguish between aflatoxigenic and non-aflatoxigenic species A. flavus (Makhlouf et al., 2019). Aflatoxins are the end product of a biosynthetic pathway comprising many enzymatic steps. Several studies have measured the expression of genes involved in the aflatoxin biosynthesis pathway to distinguish between aflatoxin producers and non-producers (Rodrigues et al., 2009). Therefore, the proposed detection method will be designed by targeting specific gene’s coding regions of the DNA sequences of A. flavus involved in the aflatoxin biosynthesis pathway such as the nor-1, omt A, aflR etc. This method will be helpful to provide rapid and early detection and quantification tools of aflatoxigenic fungi Aspergillus flavus in peanuts and peanut products which is critical for preventing spread aflatoxin contamination at various stages of the supply chain. Moreover, this fast and less tedious detection technique will be useful for routine diagnostics, surveillance, biosecurity, and epidemiological studies. For both aims, the experimental preparation will take place in the laboratory space of the PI. The students will engage in sample collection, preparation and pathogen identification using various molecular techniques in a laboratory classroom. The learning objectives include the concepts of food safety and mycotoxins, tools and techniques of molecular diagnosis, regulation of gene expression, technical and biological variability, and data analysis/result interpretation. 

Aim 1 will focus on marker/primer development of a portable real time PCR assay for the detection and quantification of aflatoxigenic A. flavus in peanuts. Aim 2a will focus on the optimization of a portable real time PCR assay for the detection and quantification of A. flavus in peanuts. Aim 2b will compare them with other existing techniques like PCR with a comprehensive analysis of which factors in portability, sensitivity, cost-effectivity, and robustness between detection techniques is needed.