TaqMan-based duplex real-time PCR approach for analysis of grain composition (Zea mays - Sorghum bicolor) in feedstock flour mixes for bioethanol production

Authors: Kessler-Mathieu, Melanie; Tilley, Michael - Mike; Prakash, Sushma; Bean, Scott; Peiris, Kamaranga; Aramouni, Fadi

Submitted to: ACS Agricultural Science and Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/25/2023
Publication Date: 2/2/2023
Citation: Kessler-Mathieu, M.S., Tilley, M., Prakash, S.R., Bean, S.R., Peiris, K.H., Aramouni, F.M. 2023. TaqMan-based duplex real-time PCR approach for analysis of grain composition (Zea mays - Sorghum bicolor) in feedstock flour mixes for bioethanol production. ACS Agricultural Science and Technology. 3(2):232–240. https://doi.org/10.1021/acsagscitech.2c00314.
DOI: https://doi.org/10.1021/acsagscitech.2c00314

Interpretive Summary: Bioethanol is a domestically produced alternative fuel made mainly from maize. It can be also made from other plant-based starch/sugar feedstocks (sorghum, wheat, sugar beet) or cellulosic feedstocks (wood, crop residues). In the sorghum-growing region of the United States (Midwest), some bioethanol plants use mixtures of corn and sorghum grains as feedstocks depending on price and availability. For regulatory purposes and for optimizing the ethanol manufacturing process, knowledge of the grain composition of the milled feedstock is important. Thus, a highly sensitive real-time polymerase chain reaction (PCR) method was developed to simultaneously analyze the content of sorghum and corn in corn-sorghum flour mixtures. Real-time PCR is a molecular biology laboratory technique that monitors the amplification of a targeted DNA molecule during the PCR in real time. The real-time PCR method was able to predict the percentage of sorghum in the binary mixture within 2.23% in average with a bias of 9.73%. The method developed in this study offers a reliable quantification strategy for sorghum flour quantification in a binary sorghum/maize flour blend.

Technical Abstract: Some US bioethanol plants use a mixture of maize and sorghum grains as feedstocks depending on availability and price. For bioethanol marketing and regulatory purposes, knowledge of the grain composition of the milled feedstock is useful and may be required in the future. We developed a duplex real-time PCR assay based on Taqman™ probes for the simultaneous analysis of Sorghum and Maize flour blends. For sorghum, the method amplified a sequence within the F-Box domain-containing protein and for maize, the method amplified a sequence within the high mobility group of genes (HMGa). To obtain simultaneous fluorescent signals, FAM and TAMRA fluorescent probes were used to label the sorghum- and maize- specific probes respectively. Results showed that the primers and probes were highly specific either for maize or sorghum. The real-time duplex PCR assay was validated on simulated flour binary mixtures and high in the precision and accuracy for the Sorghum PCR system. Results with the established method showed specificity and sensitivity in quantifying sorghum composition in bioethanol feedstock mixtures.