Comparison of Real-Time PCR Quantification Methods in the Identification of Poultry Species in Meat Products

Abstract

Poultry meat is consumed worldwide and is prone to food fraud because of large price differences among meat from different poultry species. Precise and sensitive analytical methods are necessary to control poultry meat products. We chose species–specific sequences of the cytochrome b gene to develop two multiplex real-time polymerase chain reaction (real-time PCR) systems: one for chicken (Gallus gallus), guinea fowl (Numida meleagris), and pheasant (Phasianus colchicus), and one for quail (Coturnix japonica) and turkey (Meleagris gallopavo). For each species, added meat could be detected down to 0.5 % w/w. No cross reactions were seen. For these two real-time PCR systems, we applied three different quantification methods: (A) with relative standard curves, (B) with matrix-specific multiplication factors, and (C) with an internal DNA reference sequence to normalize and to control inhibition. All three quantification methods had reasonable recovery rates from 43% to 173%. Method B had more accepted recovery rates, i.e., in the range 70–130%, namely 83% compared to 75% for method A or C.

Target and Non-Target Approaches for Food Authenticity and Traceability

“…In this regard, this Special Issue aimed at gathering original research and review papers focusing on the development and application of both targeted and non-targeted methodologies to verify food authenticity and traceability. This Special Issue includes eighteen notable contributions, comprising one review paper and seventeen original research papers, these last dealing with the authentication of different foods, including some considered as highly prone to food fraud such as olive oil [ 14 , 15 ], honey [ 16 , 17 ], fish [ 18 , 19 , 20 ] and meat [ 21 , 22 , 23 , 24 ].…”

“…These techniques are highly specific and sensitive and are frequently considered as the most suitable tools for the identification of species. Various research papers on the use of DNA-based approaches are also included in this Special Issue, from the comparison of different DNA extraction methods [ 28 ] to the use of multiplex polymerase chain reaction (PCR) [ 23 ], real-time PCR [ 19 , 22 , 24 , 29 , 30 ], or more advanced techniques such as Digital PCR [ 31 ]. Kim et al [ 23 ] proposed the use of a simple qualitative assay based on the use of multiplex PCR to identify three deer species, namely red deer ( Cervus elaphus ), roe deer ( Capreolus capreolus ), and water deer ( Hydropotes inermis ).…”

“…Kyriakopoulou and Kalogianni [ 15 ] described the development of a new allele-specific real-time PCR to specifically differentiate olive oil from the valuable wild-type Olea europaea var Sylvestris from the commonly cultivated type Olea europaea L. var Europaea . Besides being used for species-specific identification, real-time PCR is also reported for quantification purposes [ 22 , 29 , 30 ]. While Oh et al [ 29 ] estimate the percentage of corn ( Zea mays ) as an added adulterant in turmeric powder ( Curcuma longa ) by using the fluorescent dye SYBRGreen, others propose the use of specific probes [ 22 , 30 ].…”

“…Besides being used for species-specific identification, real-time PCR is also reported for quantification purposes [ 22 , 29 , 30 ]. While Oh et al [ 29 ] estimate the percentage of corn ( Zea mays ) as an added adulterant in turmeric powder ( Curcuma longa ) by using the fluorescent dye SYBRGreen, others propose the use of specific probes [ 22 , 30 ]. Dolch et al [ 22 ] developed two multiplex real-time PCR assays using specific primers and probes, one for the detection and quantification of chicken ( Gallus gallus ), guinea fowl ( Numida meleagris ) and pheasant ( Phasianus colchicus ), and other for quail ( Coturnix japonica ) and turkey ( Meleagris gallopavo ).…”

“…While Oh et al [ 29 ] estimate the percentage of corn ( Zea mays ) as an added adulterant in turmeric powder ( Curcuma longa ) by using the fluorescent dye SYBRGreen, others propose the use of specific probes [ 22 , 30 ]. Dolch et al [ 22 ] developed two multiplex real-time PCR assays using specific primers and probes, one for the detection and quantification of chicken ( Gallus gallus ), guinea fowl ( Numida meleagris ) and pheasant ( Phasianus colchicus ), and other for quail ( Coturnix japonica ) and turkey ( Meleagris gallopavo ). For each system, three different quantification methods were compared for estimating the relative meat content of these poultry species in meat mixtures.…”