Development of a real-time PCR assay for the identification and quantification of bovine ingredient in processed meat products

In order to find fraudulent species substitution in meat products, a highly sensitive and rapid assay for meat species identification and quantification is urgently needed. In this study, species-specific primers and probes were designed from the mitochondrial cytb (cytochrome b) fragment for identification and quantification of bovine ingredient in commercial meat products. Bovine samples and non-bovine ones were used to identify the specificity, sensitivity, and applicability of established assay. Results showed that the primers and probes were highly specific for bovine ingredient in meat products. The absolute detection limit of the real-time PCR method was 0.025 ng DNA, and the relative detection limit was 0.002% (w/w) of positive samples. The quantitative real-time PCR assay was validated on simulated meat samples and high in the precision and accuracy. In order to demonstrate the applicability and reliability of the proposed assay in practical products, the 22 commercial meat products including salted, jerkies, and meatball were used. The results indicated the established method has a good stability in detection of bovine ingredient in real food. The established method in this study showed specificity and sensitivity in identification and quantification of beef meat in processed meat products.

fatless muscle, connective tissue) different to those present in the meat products 22 . To address this problem, three mixed matrices (pork, donkey and sheep with known proportions of target meat species, respectively) were prepared to be as the production of food calibrators in this study to render accurate and reproducible quantitative results.
In this study, a probe-based TaqMan ® real-time PCR assay for identification and quantification of bovine ingredient was developed and assessed in terms of the specificity, sensitivity and repeatability. Finally, the assay was applied on different kinds of commercial meat products.
For method validation, 22 commercial beef products were randomly collected from local supermarket, including salted (n = 11), jerkies (n = 10), meatball (n = 1). When arrived in the laboratory, each package was labeled with an internal code, and a visual inspection of the product content was performed by morphological analysis. All specimens were stored at −20 °C for further molecular analysis.
Binary mixture preparation. In order to avoid contamination, the surface tissue, skin, ribs, and fat layers were removed from fresh meat sample using a pair of sterile and clean scissors. The remaining tissues were ground by a high-speed crusher (XJA-100A, Specimen Mould Factory of Shanghai China), and the tissue were ground into a uniform powder from plant samples. All samples were stored at −20 °C for use.
Simulation experiment for mixed sample preparation: 100 g of ground samples (added non-toxic blue dye) were mixed by a high-speed crusher (XJA-100A, Specimen Mould Factory of Shanghai, China) for 5 min intermittently, the color of mixture was observed to be uniform, as well as the time of mixing and crushing in a high-speed crusher was determined to be 6 min intermittently.
Moreover, in order to simulate the cooking and boiling effects that is frequently applied to industrially processed meat products 11 , the binary mixtures were also sent for autoclavation at 121 °C for 20 min. DNA extraction. Total DNA was extracted using the MiniBEST Universal Genomic DNA Extraction Kit (TaKaRa, Japan) for animal and fish tissue, and MiniBEST Plant Genomic DNA Extraction Kit (TaKaRa, Japan) for plants, following manufacturer's instruction.
DNA quality and concentration were determined using a BioPhotometer D30 (Eppendorf, Hamburg, Germany) spectrophotometer and reading samples at 260 nm (A 260 ) and 280 nm (A 280 ). For each sample, a standard working concentration of 100 ng/µL was prepared for further analysis.
The primer and probe used for positive amplification control (PAC) ( Table 1) were previously reported targeting the conserved fragment in the 18S rRNA gene 24 .

Construction of calibration curve and data correction and analysis.
Three mixed matrices (beef mixed with pork, donkey and sheep respectively, raw and autoclaved) were used to construct a calibration curve, (the curves were shown in the Supplementary Figs. S1-S3) The results indicate that it is available for the mixed matrices (beef with pork, autoclaved) to be as production of food calibrators in this study, and that the results agree with the ENGL criteria (the method could be accepted that the R 2 value should be above 0.98, the slope of standard curve was between 3.1 and 3.6, and the PCR efficiency should range between 90% and 110% 26 ).
To apply the quantitative assay to heavily-processed meat products, PCR data were normalized with threshold cycle (Cq) values received by the bovine-specific system and eukaryotic system to construct a calibration curve 17 . The calibration curve was created with reference mixtures containing known beef content (0.01, 0.05, 0.1, 0.5, 1, 5, 100%,w/w) in heated pork.
As historically reported by Fajardo 27 , the Cq values received from the blind sample using the bos-specific system (CqB) were determined by Eq. (1) Where CqS is the normalized Cq value of the sample in the Eq. (1), CqEU is the average Cq value of the binary model mixtures with the endogenous PCR system, and CqEUB is the Cq value of the sample detected with the endogenous PCR system 27 .
The target species in blind samples can be estimated by linear interpolation with the calibration curve of CqS values generated from samples. The correlation between CqS values and content (C) is defined by Eq.
Where a is the slope and b is the intercept in the Eq. (2).

Limit of quantifification (LOQ).
The LOQ is the lowest amount of analyte in a sample that can be reliably quantified at an acceptable level of precision, accuracy, and repeatability 28 . Besides, the LOQ should be ≤ the minimum value included in the dynamic range, and its assessment should be obtained from a abundant number of detection data, at least 15, by analogy with the requirement set for the estimates of Relative Repeatability Standard Deviation (RSDr) (RSDr should be ≤25% over the whole dynamic range of the PCR modules individually) 26 .
Compliance with ethical standards. Ethical Approval this article does not contain any studies with human participants or animals performed by any of the authors.

Results and Discussion
Sequence analysis and primer design. The primer and probe design is a critical step in TaqMan probe assay and it must contain adequate intra-species conserved sequences and inter-species polymorphism 29 . Several sequences of mitochondrial and nuclear genes were aligned using DNAMAN Multiple Alignment to obtain a short-length and intra-species conserved and inter-species fragment. Finally, a short fragment on mitochondrial cytb region was determined to meet the requirements. www.nature.com/scientificreports www.nature.com/scientificreports/ Following absolute intra-species conservation (the 3′ sequences of primer and the whole sequences of probe), the bovine-specific primer and probe were designed by Primer express program version 3.0, and screened for specificity to cross-species binding with other animals, fish or plant species using online BLAST tool in NCBI database (https://www.ncbi.nlm.nih.gov/Blast.cgi). The amplified target sequence is short-length of 119 bp in mitochondrial cytb region, since the short-length nucleic acid sequences are extraordinarily stable under harsh conditions and mitochondrial genes are present in multiple copies 1,30 . Specificity test. Exclusivity was 100% (false positive rate 0%) for all employed primer and probe systems while tested the plant species, with no cross reactivity observed. The Table 2 (the real-time PCR of specific amplification curve are shown in Fig. 2) showed that the Cq values of bovine (cattle, water buffalo and yak) as follow: 9.00 ± 0.02, 9.20 ± 0.01, 9.60 ± 0.02, respectively, were considered as the positive amplification, whereas there was no amplification achieved with DNA from ten non-target animal species and two plant species. The eukaryotic system was used as positive amplification control (PAC) to avoid false negative amplification, and positive amplification by enkaryotic system was obtained for all species ( Table 2).

Sensitivity of the method. Considering the European Network of Genetically Modified Organism (GMO)
Laboratories guidelines 26 , the limit of detection (LOD) of the real-time PCR assay was determined using serially diluted DNA from autoclaved beef to be as a calibrator. The LOD is when there are 26 or more positive amplification times out of 27 reactions, according to the ≥95% confidence level rule.
PCR amplification of DNA from binary model mixtures were used to evaluate the relative LOD. The run was performed under repeatable conditions for reliable generation of absolute and relative LOD 28 .
For the absolute LOD test (Table 3, Cq values and respective SD were shown in the Supplementary Table S2), results showed that positive signals appeared 26 or more out of 27 reactions from 0.025 ng cattle meat DNA. In conclusion, detected the lowest level was 0.025 ng of DNA at a 95% confidence level. (The real-time PCR data of amplification curves obtained directly with the instrument for serially diluted DNA are shown in Fig. 3) For the relative LOD test (Table 4, Cq values and respective SD were shown in the Supplementary Table S3), the positive amplification appeared in 26 or more out of 27 reactions except 0.001% (w/w) cattle meat. In conclusion, the lowest cattle meat content could be test was 0.002% (w/w) at a 95% confidence level. Similar result has also been reported in previous study which concluded that cooking and sterilization processed do not affect significantly the response of PCR to different concentrations of DNA, as well as non-target DNA in the meat admixtures do not affect the PCR efficiency 22 . (The real-time PCR data of amplification curves obtained directly with the instrument for serially diluted mixtures are shown in Fig. 4) The LOD was determined to be 0.005 ng/μL pure bovine DNA, which is equivalent to 0.025 ng DNA. The relative sensitivity of the real-time PCR assay using the reference mixtures was 0.002% (w/w). While previous studies reported an absolute LOD of 0.1 ng using multiplex PCR for horse, dog and pork 31 and 0.01 ng using real time PCR for horse 8,32 . (Safdar and Junejo 2016) 14 reported similar analytical sensitivities, namely 0.01% sensitivity for horse, soybean, sheep, poultry, pork, and cow, respectively. Therefore, the detection limit of the real-time PCR assay established in this study was equal or lower to previously reported values.       Table 6). According to the ENGL criteria, the method could be accepted that the R 2 value should be above 0.98, the slope of standard curve was between 3.1 and 3.6, and the PCR efficiency should range between 90% and 110% 26 .The GMO criteria were used to estimate PCR efficiency in this study because there is no appropriate quantification criterion for detecting of meat products at present 17 . The qPCR (Quantitative PCR) assay constructed in this study meets above criteria, and showed good linearity and PCR reaction efficiency, thus was regarded as a available tool of quantify beef content. The study data showed that there were no significant difference on the standard curve of R 2 values and slopes using raw and autoclaved reference mixture meat. The Linear dynamic range over which a reaction is linear (the maximun to the minimun quantifiable amount created by means of a calibration curve) 34 . The results incidated that the dynamic range is between 0.01% and 100% (w/w) in this study.
Precision, accuracy and trueness. Specific group of food matrix-cattle samples (at five different percentages) were prepared. Cattle tissues (75 g) were mixed with 25 g of pork tissues using a high-speed crusher for 6 min, resulting in a 75% (w/w) sample (coded as Ba1). 80 g of Ba1 were mixed with 40 g of pork tissues for 6 min, resulting in a 50% (w/w) sample (coded as Ba2). 50 g of Ba2 were mixed with 50 g of pork tissues for 6 min, resulting in a 25% (w/w) sample (coded as Ba3). 20 g of Ba3 were mixed with 80 g of pork tissue for 6 min, resulting in a 5% sample (coded as Ba4). The pure cattle tissue was coded as Ba5 (100%,w/w). The mixtures were sent for autoclavation at 121 °C for 20 min. The DNA was extracted from mixtures above, and store at −20 °C for further analysis.
In order to assess the precision (relative standard deviation, RSD) and accuracy (relative mean deviation) of the method, various meat fractions with defined proportions of beef, covering the dynamic range of the assay, were analyzed under repeatability conditions. The results indicated ( Table 7) that the developed assay has a good performance, with the precision, accuracy and trueness of the method lying well the acceptance criterion of ≤25% 26 .   www.nature.com/scientificreports www.nature.com/scientificreports/ Application of method on a reference gene for beef meat species quantification in commercial meat products. At present, many methods for identification of meat species have been published. While the majority of these methods are of a qualitative nature, more recent scandals in the food industry require sensitive quantitative methods to distinguish more accurately between deliberate adulteration and inadvertent  Table 5. The values of normalized Cq (a) and mean (b) obtained from quantitative PCR using pork-specific and eukaryotic systems to create the calibration curve. www.nature.com/scientificreports www.nature.com/scientificreports/ contamination 8 . In this study, 22 commercial processed meat products were selected from different manufacture processes, which could affect DNA degradation and complex food matrices. The quantitative analysis of the meat products was carried out on three different weeks, the results (Table 8) were obtained from repeated data.
There are differences of beef meat contents between these detected by the assay and on the product labels (Table 8), as the processing degree affects DNA degradation result in differences of data tested from different types of commercial processed meat products. As for salted samples as follow 1, 3, 4,10 and 11, the detected beef meat contents are far lower than the contents on labels (Table 8). It is a reasonable explanation that high salt content affects DNA extraction efficiency and PCR amplification, or it might be possibly that there is serious adulteration during processing of meat products. And the beef meat contents estimated for the other salted samples    Table 8. Results of Real-time PCR assay performed on commercial meat products. In this work, positive amplification control (PAC) and negative amplification control (NAC) were set up, all PAC appeared amplification signals, and all NAC is no amplification signal. Extraction DNA concentration from all samples were within the 10 2 range.
are consistent with the ones given on the label. The beef meat contents for jerky samples (2, 3, 7, 9 and 10) are quantified between 5.20% and 38.73% (Table 8), which are extremely lower than the other ones. The detected beef meat content of meatball for sample 1 is 23.92% (as shown in Table 8).
Due to the diversity and the easier changes of composition of meat products during processing, the limit of relative quantitative method of fluorescence PCR for precision lies in standard products, copy number ratio and mass content conversion. Some studies indeed show that the accuracy of real-time PCR assays is significantly increased while calibration sausages on commercial meat products are similar to the detected meat samples, are employed 12 . Besides, the challenge lies in the production of calibration sausages that can contain the various commercial meat products. Clearly, it is difficult on an analytical viewpoint, where food control laboratories 8 . Based on the previous studies for detection of a single type of processed meat product, this study simulated commercial meat product matrixes and developed the PCR assay, which can be applied to various common types of processed meat products.

Conclusion
The assay developed in this study offers a reliable quantification strategy for beef meat products, which was successfully extended to different type beef meat products with varying matrix and composition. The real-time PCR method developed in this work can be used for identification and quantification of bovine ingredient in commercial processed meat products.

Data availability
All data generated or analysed during this study are included in this published article (and its Supplementary information files).