A TaqMan real-time PCR method based on alternative oxidase genes for detection of plant species in animal feed samples

Abstract

Traceability of processed food and feed products has been gaining importance due to the impact that those products can have on human/animal health and to the associated economic and legal concerns, often related to adulterations and frauds as it can be the case for meat and milk. Despite mandatory traceability requirements for the analysis of feed composition, few reliable and accurate methods are presently available to enforce the legislative frame and allow the authentication of animal feeds. In this study, nine sensitive and species-specific real-time PCR TaqMan MGB assays are described for plant species detection in animal feed samples. The method is based on selective real-time qPCR (RT-qPCR) amplification of target genes belonging to the alternative oxidase (AOX) gene family. The plant species selected for detection in feed samples were wheat, maize, barley, soybean, rice and sunflower as common components of feeds, and cotton, flax and peanut as possible undesirable contaminants. The obtained results were compared with end-point PCR methodology. The applicability of the AOX TaqMan assays was evaluated through the screening of commercial feed samples, and by the analysis of plant mixtures with known composition. The RT-qPCR methodology allowed the detection of the most abundant species in feeds but also the identification of contaminant species present in lower amounts, down to 1% w/w. AOX-based methodology provides a suitable molecular marker approach to ascertain plant species composition of animal feed samples, thus supporting feed control and enforcement of the feed sector and animal production.

Establishment of a Sensitive qPCR Methodology for Detection of the Olive-Infecting Viruses in Portuguese and Tunisian Orchards

“…Detection sensitivities of the assays were determined from the Cq of the lowest plasmid dilution that fell within the linear standard curve. The method performed for absolute DNA quantification was based on the determination of the absolute number of target copies (TCN) previously described by Campos et al (2018).…”

DNA-Based Tools to Certify Authenticity of Rice Varieties—An Overview

“…Developed over two decades ago, Real-Time PCR, also known as Quantitative PCR (qPCR), rapidly emerged as an important tool to detect contaminants and adulteration in the food industry. Real-time PCR has several advantages, when compared with classical PCR, namely its higher sensitivity and specificity, also allowing the simultaneous processing of multiple samples with short hands-on time, and the automated, simple and reproducible detection of the amplified products without need for gel electrophoresis for product visualization [108,109]. To explore the qPCR potential, several modifications were introduced including TaqMan-based Real-time PCR, multiplex-SSR, droplet digital PCR, Kompetitive Allele PCR, nanofluid arrays, and padlock probe ligation with multiplex microarray detection, among others.…”

“…Although expensive, the technique is very accurate with 1% detection limit [110]. This technology has been used in the authentication of animal feeds [108] and it also allowed the identification of economically important adulteration of Basmati rice [60].…”

Detection and Quantification of Fusarium spp. (F. oxysporum, F. verticillioides, F. graminearum) and Magnaporthiopsis maydis in Maize Using Real-Time PCR Targeting the ITS Region

“…Amplification efficiencies were calculated through the equation E = (10 (−1/slope) − 1) × 100, as well as slope and linearity (coefficient of determination, R 2 ). The method performed for absolute DNA quantification was based on the determination of the absolute number of target copies, TCN [29].…”

“…For each TaqMan assay, Cq values between 34 and 35 were taken as being indicative of trace amounts, while a Cq = 35 was considered the cut-off limit, defining no detection [29]. The method showed a high sensitivity for both Fusarium spp.…”