Journal Pre-proofs

Rapid detection of porcine DNA in processed food samples using a streamlined

DNA extraction method combined with the SYBR Green real-time PCR assay

Lee Lee Tan, Siti Aminah Ahmed, Siew Kit Ng, Marimuthu Citartan, Carsten
A. Raabe, Timofey S. Rozhdestvensky, Thean Hock Tang

PII: S0308-8146(19)31780-7
DOI: https://doi.org/10.1016/j.foodchem.2019.125654
Reference: FOCH 125654

To appear in: Food Chemistry

Received Date: 4 July 2018

Revised Date: 2 September 2019
Accepted Date: 5 October 2019

Please cite this article as: Lee Tan, L., Aminah Ahmed, S., Kit Ng, S., Citartan, M., Raabe, C.A., Rozhdestvensky,
T.S., Hock Tang, T., Rapid detection of porcine DNA in processed food samples using a streamlined DNA extraction
method combined with the SYBR Green real-time PCR assay, Food Chemistry (2019), doi: https://doi.org/10.1016/
j.foodchem.2019.125654

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Rapid detection of porcine DNA in processed food samples using a streamlined DNA

extraction method combined with the SYBR Green real-time PCR assay.

Lee Lee Tana, Siti Aminah Ahmeda, Siew Kit Nga, Marimuthu Citartana, Carsten A. Raabeb,d,e,

Timofey S. Rozhdestvenskyc, Thean Hock Tanga*

aAdvanced Medical & Dental Institute, Universiti Sains Malaysia, Bertam, 13200 Kepala Batas,

Malaysia
bInstitute of Experimental Pathology, Centre for Molecular Biology of Inflammation (ZMBE),

University of Muenster, Von-Esmarch-Strasse 56, D-48149 Muenster, Germany

cCore Facility Transgenic Animal and Genetic Engineering Models (TRAM), University

Hospital of Muenster, D-48149 Muenster, Germany

dInstitute of Medical Biochemistry, Centre for Molecular Biology of Inflammation (ZMBE),

University of Muenster, Von-Esmarch-Strasse 56, D-48149 Muenster, Germany

eBrandenburg Medical School (MHB), Fehrbelliner Strasse 38, D-16816 Neuruppin, Germany

Address for correspondence*

Thean Hock Tang
Advanced Medical & Dental Institute, Universiti Sains Malaysia, Bertam, 13200 Kepala Batas,
Malaysia
Phone: +604-5622302
Fax: +604-5622349
Email: tangth@usm.my

1
Lee Lee Tan (leelee.tan@aiesec.net)

Siti Aminah Ahmed (asiti2000@usm.my)

Siew Kit Ng (skng@usm.my)

Marimuthu Citartan (citartan@usm.my)

Carsten A. Raabe (raabec@uni-muenster.de)

Timofey S. Rozhdestvensky (rozhdest@uni-muenster.de)

Abstract: A streamlined DNA extraction method and a quantitative SYBR Green quantitative

polymerase chain reaction (SyG-qPCR) assay were combined to generate a ready-to-use kit for

rapid detection of porcine admixtures in processed meat products. Our qPCR assay utilized

repetitive LINE-1 elements specific to the genome of Sus scrofa domesticus (pig) as a target and

incorporated internal controls. We improved the genomic DNA extraction method and reduced

extraction times. The method was validated for specificity, sensitivity (0.001% w/w) and

robustness, and values were compared with those of a commercially available kit. We also tested

our method using 121 processed food products and consistently detected amplification only in

samples containing pork. Due to its efficiency and cost-effectiveness, our method represents a

valuable new method for detecting food adulteration with pork that is superior to existing quality

control approaches.

Keywords: Genomic DNA extraction; food authentication; Sus scrofa; adulteration with pork;

SYBR Green PCR, melting curve analysis.

2
1. Introduction

Incorporation of undeclared or falsely declared ingredients is referred to as food

adulteration. In this publication, we focus specifically on the illegal substitution of expensive

meats with pork and/or derivatives thereof. Adulteration with pork can have various economic

and health effects. It also has cultural implications, particularly among Muslim and orthodox

Jewish populations for which pork consumption is strictly prohibited for religious reasons (Man

et al., 2007, Nakyinsige et al., 2012). Therefore, detection of pork as an adulterant is important

for controlling illegal competitive practices and for protecting consumer well-being.

Detection of pork in processed food products is achievable by assays that utilize either

proteins or genomic DNA as the molecular target (Ali et al., 2014). Protein-based assays are

highly sensitive and specific for the analysis of raw meat, but they are less effective for

processed foods. This is because proteins are prone to denaturation at the high temperatures that

are commonly used in the production of processed food items (e.g. cooking, sterilization, etc.). In

addition, protein expression levels may differ between different tissues or body parts, which

might result in erroneous decision making based on the perceived presence or absence of the

pork adulterant (Ibarguren and Villamarín, 2017). DNA-based strategies do not have the

shortcomings associated with protein-based assays for the following reasons: (1) the genomic

DNA sequence is identical in all the tissues of a given organism and, (2) it remains stable under

the high temperatures used during industrial food processing.

One DNA-based method that is commonly used for pork adulterant detection in food is

the polymerase chain reaction (PCR). PCR involves amplification of a specific gene or genes,

and the amplified product is then detected qualitatively or quantitatively. Compared to the

3
protein-based method, PCR is less compromised by conditions associated with food processing.

The technique is also simple, rapid, specific and sensitive (Lockley and Bardsley, 2000). An

even more sensitive and automated PCR assay is real-time quantitative PCR (qPCR) (Navarro et

al., 2015). Various real-time qPCR assays based on intercalating dyes have been developed to

detect food adulteration with pork (Şakalar and Kaynak, 2016, Amaral et al., 2017, Kang and

Tanaka 2018). Coupled with melting curve analysis (MCA), this format is the most effective and

economically viable assay for the quality control of processed meat products (Varga and James,

2006; Navarro et al., 2015).

The reliability and sensitivity of PCR assays depend on DNA quality (Cseke et al., 2011;

Talley and Cseke, 2011). Preparation of high-quality DNA from processed meat products for

PCR is challenging due to the presence of PCR inhibitors such as fats, glycogen,

polysaccharides, and minerals, which can cause false-negative results (Amagliani et al., 2007,

Schrader et al., 2012; Ahmed et al., 2016). In addition, industrial treatment of meat during food

production causes significant DNA degradation (Gryson, 2010). Commercially available DNA

extraction kits, many of which employ spin column technology and/or mobile solid phase

extraction, ensure high DNA quality, but they are expensive, time-consuming, and cumbersome

(Di Pinto et al., 2007).

The goal of this study was to develop an inexpensive, reliable, and easy-to-use protocol

for DNA extraction from meat-based food and subsequently integrate it with an intercalating

dye-based qPCR assay. The DNA extraction method was optimized from our previous study

(Ahmed et al., 2016) by reducing overnight incubation time to 10 min. The qPCR assay was

based on the simultaneous (i.e., in-tube) amplification of the porcine-specific LINE-1 element

and a subsection of the mitochondrial 16S rRNA gene as the target for the vertebrate-specific

4
internal control amplification. The mitochondrial 16S rRNA gene is well-suited as a control

target due to its high sequence conservation amongst vertebrates and its high copy number (Yang

et al., 2014). To identify the DNA threshold concentrations and evaluate assay sensitivity, raw

and cooked meat mixtures were used. The assay then was evaluated using 121 commercial meat-

based food products, and results were compared with those of a commercially available porcine

DNA detection kit.

2. Materials and Methods

2.1 Preparation of reference meat samples and sample collection

Pig (Sus scrofa domesticus), cow (Bos taurus), goat (Capra hircus), chicken (Gallus

gallus), deer (Rusa unicolor), pomfret fish (Pampus argenteus) and whiteleg shrimp

(Litopaneous vannamei) were purchased from local markets. Reference samples were prepared

by spiking ground beef samples with 0.001 to 10% ground pork (Table S1). The resulting pork-

beef admixtures were divided into two batches. The first batch was subjected to immediate DNA

extraction and qPCR assay analysis. The second batch was autoclaved (sterilized) at 121°C for

20 minutes (to mimic genomic DNA degradation that potentially takes place during industrial

food processing) prior to DNA extraction. To evaluate the effectiveness of the assay, 121

commercial meat products of different brands, including sausages (n = 32), meatballs (n = 17),

nuggets (n = 24), burger patties (n = 15), meatloaf (n = 9), and others (n = 24), such as crab

filament and fish cake, were purchased from supermarkets around the northern region of

Peninsular Malaysia. These commercial meat products are enlisted in Table S2. Out of these 121

samples, 117 products were labeled as pork-free and 4 products contained pork to represent

positive samples.

5
2.2 DNA extraction and purification

To generate a standard curve for the qPCR analysis, the genomic DNA used was isolated

from raw meat using a DNA extraction method for mammalian tissues with minor modifications

(Strauss, 2001). Approximately 200 mg of sample were ground with an Axygen tissue grinder

pestle (Fisher Scientific UK Ltd., Loughborough, UK) and suspended in 1 mL of digestion

buffer containing 100 mM NaCl (Merck Millipore, Darmstadt, Germany), 10 mM Tris-Cl

(Fisher Scientific UK Ltd.), 25 mM EDTA (Sigma-Aldrich, St. Louis, MO, USA), 0.5% SDS,

pH 8.0 (Promega, Madison, WI, USA), and 0.1 mg/mL proteinase K (Merck Millipore). The

suspension was incubated at 55°C for 16 h. One milliliter of phenol:chloroform:isoamyl alcohol,

pH 8.0 (25:24:1 [v/v]) (Sigma-Aldrich) was added to the suspension, which then was mixed and

centrifuged at 12,000 x g for 10 min. The aqueous upper layer was transferred to a fresh tube and

mixed with 100 µL of 3 M sodium acetate, pH 5.2 and 2 mL of pre-chilled (4oC) absolute ethanol

(Merck Millipore). The tubes were centrifuged at 12,000 x g for 12 min. Supernatants were

discarded and the pellet rinsed with 1 mL of pre-chilled (4oC) 70% ethanol. Finally, pellets were

air dried for about 5 min and resuspended in 50 µL of ddH₂O. Purity and DNA concentrations

were determined using a NanoPhotometer (IMPLEN GmbH, Munich, Germany).

Crude DNA extracts were utilized for the specificity and sensitivity analyses. Crude

DNA from both raw autoclaved reference samples and processed meat products was extracted

according to the method described by Ahmed et al. (2016) with some modifications.

Approximately 400 mg of each meat mixture sample were ground in a microcentrifuge tube with

the Axygen tissue grinder pestle. Commercial meat-based products had different densities, so

instead of testing a 400 mg sample, up to one-third of the microcentrifuge tube was filled up with

the sample. Next, 1 mL of digestion buffer (100 mM NaCl, 10 mM Tris-Cl, 25 mM EDTA, 0.5%

6
SDS, pH 8.0 and 0.1 mg/mL proteinase K) was added to each sample, which then was vortexed

for 10 s to eliminate clumps of tissue. The resulting suspension was incubated at 65°C for 10 min

followed by centrifugation at 14,000 x g for 2 min. Each supernatant was transferred to a fresh

tube and stored at -20°C. Two microliters of the supernatant were diluted in 200 µL of ddH₂O

prior to qPCR analysis.

2.3 Design of oligonucleotide primers

Porcine specific primers for amplification of repetitive LINE-1 (L1) retrocopies (L1SsF

and L1SsR) within the genome of Sus scrofa were designed. The design of oligonucleotide

primer sequences (16SrF and 16SrR) targeting the mitochondrial 16S ribosomal DNA gene for

internal control amplification was based on a multiple sequence alignment (MSA)

(http://www.ebi.ac.uk/Tools/msa/muscle/) (Madeira et al., 2019). The MSA helped to identify

regions with high conservation across vertebrate genomes. PCR primers were checked for

specificity using the NCBI basic local alignment search tool

(https://www.ncbi.nlm.nih.gov/tools/primer-blast/) (Ye et al., 2012). Table S3 lists the

oligonucleotide primer sequences and their corresponding locations.

Thermodynamic properties of all primers, including their secondary structures and

potential duplex formations, were determined with the Oligo Analyzer package

(https://sg.idtdna.com/calc/analyzer). Theoretical melting temperatures (Tm) of each PCR

product were first predicted using uMelt software (https://www.dna.utah.edu/umelt/um.php) and

then confirmed by MCA. The temperature with the highest rate of change in fluorescent signal

against temperature (-d(RFU)/dT) corresponds to the temperature at which 50% of the PCR

amplicon duplex is unwound (Pfaffl, 2001). Tm values of PCR amplicons (or DNA duplexes in

7
general) depend on concentration, length, and nucleotide composition; they have to be different

from each other to enable clear discrimination between two or more PCR products by SYBR

Green multiplex real-time quantitative PCR (SyG-qPCR) assays. All primers were synthesized

by Integrated DNA Technologies Pte. Ltd. (Singapore, Republic of Singapore).

2.4 Development of the SyG-qPCR assay

2.4.1 Optimization of annealing temperature (Ta)

The optimal Ta for this PCR assay was identified using gradient PCR. All reactions were

performed with the T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA). Both primer pairs used

for genomic LINE-1 elements and the internal control amplification were combined in a single

PCR reaction. All PCR reagents, with the exception of primers, were purchased from Biotools

B&M Labs (Madrid, Spain). The assay was conducted in a 20 µL total reaction volume

containing 0.5 µM of both the forward and reverse primer, 200 µM of each dNTP, 1.5 mM

MgCl2, 1 U of Taq DNA polymerase, and 1 ng of porcine genomic DNA as template in 1X PCR

buffer (20 mM (NH4)2SO4, 75 mM Tris HCl [pH 9.0], 50 mM KCl,).

The PCR temperature profile was as follows: initial DNA denaturation at 95°C for 5 min,

followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 53-68°C for 30 s, primer

extension at 72°C for 30 s, with a final extension step at 72°C for 5 min. PCR products were

separated by 8% native polyacrylamide gel electrophoresis (PAGE) in 1X TBE [40 mM Tris

borate, 1mM EDTA (pH 8)] (Sigma-Aldrich).

8
2.4.2 Optimization of primer concentrations

Duplex real-time PCR assays were performed with the LightCycler 480 Instrument II

(Roche Applied Science, Mannheim, Germany). To determine the optimal primer concentrations

for internal control amplification, assays were conducted in 10 µL of total reaction volume

including 1X LightCycler 480 SYBR Green I Master Mix (Roche Applied Science), 0.5 µM of

each forward (16SrF) and reverse primers (16SrR), and 1 µL of crude DNA extract. The

concentration for the porcine-specific primer pair (0.5 µM) remained constant throughout the

optimization. Four different concentrations of the internal control primer pair were tested: 0.25,

0.20, 0.15, and 0.10 µM. qPCR amplification was performed as follows: pre-incubation at 95°C

for 10 min, 40 cycles at 95°C for 10 s, 65°C for 20 s, and 72°C for 10 s, followed by MCA (65°C

to 97°C, 2.2°C/s). Quantification Cycle (Cq) values were calculated using Light-cycler 480

software, version 1.5.0.39 (Roche Applied Science), and all raw data were exported to Microsoft

Excel for further analysis.

2.5 Analytical specificity of SyG-qPCR

The specificity of the assay was evaluated with DNA representing seven different types

of raw meat from the following widely consumed species: pig, cow, goat, chicken, deer, pomfret

fish and whiteleg shrimp. Each PCR run included a positive control containing porcine DNA and

a negative “no template control” (NTC) sample to check for any potential amplification derived

from DNA contaminants.

9
2.6 Analytical sensitivity of SyG-qPCR

The sensitivity of the assay was determined by testing (1) porcine DNA, (2) mixtures of

porcine and bovine DNA, (3) crude DNA extracted from raw mixed meat samples, and (4) crude

DNA isolated from autoclaved mixed meat samples. A serial dilution of porcine DNA ranging

from 10 ng to 1 fg was prepared to analyze the sensitivity of this assay. For mixed binary DNA

admixture-containing samples, porcine DNA from (0% w/w) to 100% (w/w) was spiked into

bovine DNA samples (10 ng). The final DNA amount was 25 ng for each sample (Table S4).

The analytical sensitivity of the assay was also determined with DNA extracted from raw

and cooked meat mixture samples. Porcine meat was spiked into beef samples in amounts

ranging from 0% to 100% (w/w) to produce the admixtures (Table S1) before subjected to DNA

extraction. Each run was repeated three times to assess the reproducibility of the assay.

2.7 Application of the assay to processed food

To evaluate the performance and robustness of this assay, 121 commercial processed

meat products (Table S2) were purchased from supermarkets around the northern region of

Peninsular Malaysia by two independent researchers. Genomic DNA samples were extracted and

analyzed using the SyG-qPCR assay. For verification, all samples were evaluated in parallel with

a commercially available porcine detection kit (PorcineTrace Real-time PCR Kit) purchased

from 7FoodPillars Sdn. Bhd. (Subang Jaya, Malaysia).

3. Results and Discussion

10
3.1 Streamlined DNA extraction method

A fast and efficient DNA extraction method can shorten the overall assay time of qPCR-

based detection. For this purpose, we optimized our previously reported DNA extraction method

(Ahmed et al., 2016) by reducing the proteinase K treatment from the original 16 h to as short as

10 min. We also omitted the proteinase K deactivation step and increased the incubation

temperature from 55oC to 65oC (Fig. S1). Following the incubation, an aliquot of the supernatant

was used directly for qPCR analysis. Our newly developed in-house DNA extraction method has

the potential to replace conventional and currently available protocols for DNA extraction, which

often are far more laborious and expensive (Gallup et al., 2010).

3.2 Molecular targets for the design of real-time qPCR assays

This PCR assay was designed to amplify porcine specific L1_SS (L1 Sus scrofa)

elements. L1 repeats are the result of genomic amplification via retroposition. Several LINE-

based PCR assays with significantly improved sensitivities were previously reported (Ballin et

al., 2012, Cai et al., 2012). In general, LINE1 elements are amongst the most active autonomous

non-long terminal repeat retrotransposons in mammals; they cover approximately 15% of the Sus

scrofa genome (Ballin et al., 2009). The high copy number of L1 repeats rendered the elements

feasible for the design of our qPCR assay. We anticipated lower threshold levels for the detection

of pork admixtures due to the significantly higher copy number of the gene (i.e., compared to

single copy genes). For internal control amplification, we selected a highly conserved domain of

the mitochondrial 16S ribosomal RNA gene, which is suitable for the design of control

amplifications due to the high copy number of this extranuclear genome (Yang et al., 2014).

11
Internal control amplifications serve as an indicator of PCR failure and account for false

negatives, which might be the result of poor DNA recovery, degradation, or the presence of PCR

inhibitors within template samples (Schrader et al., 2012). Certain protein components or

complex polysaccharides are known to reduce the activity of Taq DNA polymerase and interfere

with the efficiency of PCR amplification (Kermekchiev et al., 2009; Schrader et al., 2012). In

addition, DNA degradation in processed food items limits the amount of intact template

molecules within samples and might render successful target amplification impossible. These

possibilities reiterate the need for the inclusion of suitable internal controls.

Table S3 lists the sequences of oligonucleotide primers that were utilized in our SyG-

qPCR reaction. Short amplicon sizes, which are less viable to degradation, were selected to

improve the sensitivity of the assay (Murray et al., 2009). In addition, amplified DNA fragments

of less than 150 bp are most suitable for the detection of adulteration in processed foods

(Vijayakumar et al., 2009; Gryson, 2010). Amplicon size selection is vital when SyG-qPCR

assays are coupled to MCA. Binding of SYBR Green I to the DNA minor groove during PCR

reactions results in an over thousand-fold increased intensity of detectable fluorescence signals

(Dragan et al., 2012). In the case of two or more PCR products that coexist in a single reaction,

the difference in Tms for each amplicon is crucial to enable a clear-cut differentiation of these

products by MCA. Amplicons of porcine-specific L1 elements and internal control corresponded

to peaks of 74.95°C ± 0.31 and 81.08°C ± 0.16, respectively, which were well resolved in the

MCA profile.

12
3.3 Optimization of the SyG-qPCR assay

3.3.1 Optimization of Ta

The optimal Ta for the SyG-qPCR was determined by gradient PCR. Resulting PCR

products were resolved by analytical 8% native PAGE. The temperature gradient used ranged

from 53°C to 68°C. Two readily distinguishable PCR products of expected sizes [77 base pairs

(bp) for the porcine-specific amplicon and 119 bp for the internal control] were observed for the

Tas of 65°C and below (Fig. S2, lanes 1-6). However, an additional non-specific amplification of

approximately 200 bp was detected for Ta below 65°C. Therefore, 65°C was chosen as the best

Ta that permitted specific parallel amplification of both targets within a single reaction.

3.3.2 Optimization of primer concentrations

We determined the optimal concentrations of both porcine-specific and internal control

primer sets that yielded the most robust parallel amplifications for this real-time PCR assay

format. Amplifications with equimolar concentrations (0.5 µM) of each primer set resulted in an

unbalanced amplification of the internal control and the porcine-specific L1 element. A strong

signal for the internal control amplification and a very weak signal for porcine-specific L1

element were observed (Fig. S3A). Interestingly, both specific amplification products were

readily identified and resolved via native PAGE, indicating that both porcine-specific and

internal control primers yielded correctly amplified products (Fig. S2). Stronger signals for the

internal control presumably are caused by higher relative binding of SYBR Green to the longer

fragment compared to the shorter porcine-specific amplicons, resulting in a higher melting peak

for the internal control (Marín et al., 2010). It is well known that the concentration of internal

13
control primers should be kept as low as possible to minimize interference with the target-

specific amplification in a duplex qPCR assay (Markoulatos et al., 2002). By lowering the

concentration of internal control primers in the duplex assay, we increased the relative

amplification of the porcine-specific product and in turn enhanced its fluorescent signal

production, leading to better discrimination of the peaks by MCA (Fig. S3B). Optimal

amplification with distinct peaks in MCA were obtained with 0.5 µM of porcine-specific primers

and 0.15 µM of internal control primers (Fig. S3B). This combination of primer concentrations

was utilized for all subsequent analyses.

3.4 The assay specifically detects Sus scrofa DNA

The specificity of this SyG-qPCR assay was determined with pig (Sus scrofa) and 6 non-

target species (cow, goat, chicken, deer, pomfret fish and whiteleg shrimp). Amplification of the

internal control was observed for all species tested except for shrimp DNA (the result was

anticipated because prawns are invertebrates). Porcine-specific L1 amplification was restricted to

samples containing Sus scrofa DNA (Fig. 1, red arrow), whereas no amplification was detected

in the NTC. In summary, the assay specifically detects porcine DNA and does not yield any

notable amplification with DNA templates of other non-target species.

3.5 Sensitivity of the SyG-qPCR assay

The sensitivity of this assay was evaluated with serially diluted porcine genomic DNA

ranging from 10 ng to 1 fg. Cq values are inversely proportional to the actual concentration of

14
template DNA within analyzed samples (Pennington 2014) (Fig. S4A). The LightCycler

software determines the Cq value, which refers to the number of PCR cycles required for the

amplicon’s fluorescence to be significantly higher than the background level (Pfaffl, 2001;

Rasmussen, 2001). These values permit the generation of a standard curve by plotting Cq values

against the logarithm of DNA dilutions (Fig. S4B). The dynamic range of our assay

encompassed seven orders of magnitude with a strong linear regression between Cq values and

the logarithm of starting DNA concentrations. Using the LightCycler software, the slope of the

resulting linear regression was estimated to be -3.379 with a correlation coefficient (r2) of 0.999.

Thus, the efficiency and r2 obtained in this study indicate that this assay complies with

the guidelines established by the Minimum Information for Publication of Quantitative Real-

Time PCR Experiments. The percentage of amplification efficiency (%E) was calculated as % E

= (E-1) * 100%, where E = [10^(-1/slope)] (Rasmussen, 2001). A value of 97.7% was acquired,

which is in agreement with the recommended range (90-110%) for real-time qPCR (Bustin et al.,

2009). Moreover, the r2 obtained was 0.999, which is higher than the accepted criterion value of

≥ 0.98 (ENGL, 2008; Bustin et al., 2009;).

Detection limits/thresholds are defined as the lowest quantity of the porcine-containing

sample that is still distinguishable from that of the non-target-containing sample. The detection

limit of this assay was 10 fg of porcine DNA (Fig. S4A) and 0.001% (w/w) of porcine DNA in

DNA mixture (Fig.2). This threshold was the same for both the raw (Fig. 3A) and autoclaved

meat mixture samples (Fig. 3B) when template DNA was extracted using our streamlined

method. Samples were autoclaved to mimic the potential effects of temperature and pressure

(i.e., causing DNA fragmentation or denaturation) during the food processing step. Internal

control peaks were present in all the test samples (Fig. 3, highlighted in blue). This substantiates

15
the potential applicability of our assay for detecting porcine admixtures, as its sensitivity is not

affected by DNA fragmentation due to food processing.

3.6 Evaluation of the assay against commercially available real-time qPCR kits

Results of our assay were compared with those the commercially available PorcineTrace

real-time PCR kit. The PorcineTrace kit was tested using purified porcine DNA, mixed DNA,

raw mixed meat, and autoclaved mixed meat samples before we screened the more complex

commercial meat products for porcine admixtures. For the PorcineTrace kit, the DNA detection

limits were 1 pg for pure DNA and 0.01% (w/w) for porcine DNA in DNA mixture. These

detection limits were lower than those obtained in our assay, which were 10 fg and 0.001%,

respectively, for pure DNA and porcine DNA in DNA mixture. However, both the commercial

kit and our assay yielded similar sensitivities for raw and autoclaved mixed meat (results not

shown).

Of the 121 commercial meat products tested, 117 were halal-labeled (i.e., declared as not

containing pork) and 4 represented known pork products and served as the positive control.

Figure 4 shows results for 12 representative samples (8 halal-labeled and 4 pork-derived meat

products). The MCA revealed amplification of the 16S ribosomal RNA gene (internal control)

for all 121 meat-based products at 81 ± 0.12°C. As anticipated, additional porcine-specific peaks

were detectable at 75 ± .26°C for all four positive control samples, and no peak was observed in

the NTC reaction. Results for all 121 commercial meat products are summarized in Table S2 and

results were consistent across all three replicates.

16
 We also validated these results using the PorcineTrace Real-time PCR kit. Table 1 shows

the results obtained from our qPCR assay and the commercial kit. A correlation of 100% was

obtained for our method and the commercial kit, which corroborated and emphasized the

diagnostic value of the SyG-qPCR assay for the detection of porcine meat. The data acquired in

this study suggest that halal-labeled Malaysian products are free from pork. Furthermore, our

findings also correlate with a few similar analyses that identified no pork in Malaysian halal-

labeled products (Ali et al., 2012; Hossain et al., 2016).

3.7 The newly developed assay is a promising porcine detection test

In this study, we described the development of a promising porcine detection assay. Our

DNA extraction method coupled with the SyG-qPCR assay permits real-time analysis and

eliminates the post-PCR steps, resulting in an assay that can be completed within 1.5 h. The use

of SYBR Green dye, which is four times less expensive compared to the application of other

assay formats (e.g., TaqMan probe and beacon), also allows high sample throughput (Marín et

al., 2010).

4. Conclusions

The streamlined DNA extraction method coupled with SyG-qPCR assays is a practical

and reliable tool for rapid detection of porcine admixtures in a wide variety of processed meat

products. The combination of cost-effectiveness and high sensitivity provided by this assay

17
method make it a useful option for routine testing for adulteration with pork to control food

quality and safety.

Acknowledgements

THT was supported by the Research University Grant for Individual (RUI, Grant No.:

1001/CIPPT/811319), Fundamental Research Grant Scheme (FRGS, Grant No.:

203/CIPPT/6711510), Trans-Disciplinary Research Grant Scheme (TRGS, Grant No.:

203/CIPPT/6769003) and Long-Term Research Grant Scheme [LRGS, Grant No.: 600-

IRMI/LRGS 5/3 (0003/2016)]. SAA was supported by the eScience Fund (Grant No.:

305/CIPPT/613237) and Short-Term Research Grants (Grant No.: 304/CIPPT/6313232). LLT

was supported by JPA scholarship from Ministry of Higher Education, Malaysia. CAR was

supported by internal funds of the University of Münster and the Brandenburg Medical School

(MHB).

Conflict of interest statement

The authors declare no conflict of interest.

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Figure Legends

Fig. 1. Determination of the specificity of the duplex SyG-qPCR assay. Pure meat samples from
seven commonly consumed animals (i.e., cow, goat, deer, chicken, fish, prawn, and pig) were
utilized as starting materials. Genomic DNA was extracted and 10 ng from each species were
tested to evaluate the assay’s specificity. Amplified DNA of each sample was examined by
MCA. Highlighted in blue are the internal control peaks, which were detected in all samples
except for prawn. Highlighted in red is the porcine-specific peak, which was only present in pig
DNA samples. NTC: no template control. (*) The area under curve for the corresponding peak
was too small to represent the result of DNA amplification.

Fig. 2. Identification of detection thresholds with purified bovine and porcine gDNA admixtures.
Bovine genomic DNA mixed with 10%, 1%, 0.1%, 0.01%, 0.001% and 0.0001% porcine
genomic DNA was tested. A sample containing 100% porcine genomic DNA was used as the
positive control, and 100% bovine genomic DNA served as the negative control. MCA is shown.
Control peaks are highlighted in blue, and red colour indicates the porcine-specific peak. NTC
indicated the no-template control.

Fig. 3. Identification of detection thresholds with crude DNA extracted using our in-house
method from samples of raw meat mixtures (A) and samples of autoclaved meat mixtures (B).
Minced beef samples containing additions of 10%, 1%, 0.1%, 0.01%, 0.001%, and 0.0001% pork
were tested. A sample containing 100% pork was used as the positive control, and 100% beef
served as the negative control. Internal control peaks are highlighted in blue, and red colour
indicates porcine-specific peaks. NTC: no-template control.

Fig. 4. Analysis of commercial meat products using our PCR assay. A total of 121 meat products
were evaluated. Crude lysates from optimized DNA extractions were used as the template.
Twelve commercial meat products are displayed as representative results. B2-B9 are halal
labeled products, and G1-G3 and H2 are pork products. Additionally, 10 ng of porcine DNA and
NTC were included as positive and negative controls, respectively. Internal control peaks are
highlighted in blue, and red colour indicates porcine-specific peaks.

Table 1
Comparison of results from our assay and the PorcineTrace kit for 121 commercial meat-based
food products.

Subject SyG-qPCR positive SyG-qPCR negative Total

PorcineTrace kit positive 4 0 4
PorcineTrace kit negative 0 117 117
Total 4 117 121

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Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☒The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:

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Highlights:

 Quantitative PCR assay to detect food adulterated with pork was established.

 Amplification of porcine-specific LINE-1 and internal control in a single tube was

achieved.

 Rapid and efficient DNA extraction from samples and PCR analysis were completed in

10 minutes.

 Detection threshold of 0.001% pork within meat mixtures was achieved.

 Robustness was demonstrated with analysis of 121 commercial meat-based food products.

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