Skip to main content
NCBI home page
Journal of Food Science and Technology logoLink to Journal of Food Science and Technology
. 2021 Jan 6;58(12):4504–4513. doi: 10.1007/s13197-020-04932-2

Rapid porcine detection in gelatin-based highly processed products using loop mediated isothermal amplification

Nor Asmara Tasrip 1, Mohd Nasir Mohd Desa 1,2,3,✉, Nur Fadhilah Khairil Mokhtar 1,2, Nurhayatie Sajali 1,4, Amalia Mohd Hashim 2,5, Md Eaqub Ali 6,7, Cheah Yoke Kqueen 3
PMCID: PMC8478990  PMID: 34629514

Abstract

Low DNA concentration recovered from highly processed products such as gelatin and gelatin-based products renders difficulty in detecting porcine contamination using conventional PCR techniques. We documented here a porcine-specific loop-mediated isothermal amplification (LAMP) to identify porcine traces in gelatin products. The porcine-specific primers were designed according to mitochondrial DNA of Cytochrome b gene sequence. Here we used two different reaction mixtures for LAMP assay (GENIE and MYRM) against the same DNA samples extracted from gelatin products and porcine-specific primers to detect the presence of porcine DNA. The porcine-specific primers were shown to be specific only to Sus scrofa against 14 DNA of other meat species. The analytical sensitivity of the LAMP assay for porcine DNA detection is 1 pg/µL using both GENIE (within 30 m) and MYRM (within 60 m) reaction mixtures. Analysis against 32 samples of gelatin products showed that five samples were found to contain porcine DNA; two samples out of six gelatin powder samples and three gelatin capsule samples out of nine. Out of these five positive samples, three were not labeled containing porcine gelatin. Overall, LAMP assay in this study showed an excellent specificity, sensitivity and rapidity in detection of porcine DNA in gelatin products.

Supplementary Information

The online version contains supplementary material available at (10.1007/s13197-020-04932-2).

Keywords: Gelatin authentication, Loop isothermal amplification, LAMP, Porcine DNA, Fluorescent dye, Real-time LAMP, Calcein

Introduction

Gelatin is derived from selective hydrolysis of a structural protein called collagen that can be found in many animals, including humans. Gelatin has gelling, thickening and stabilizing properties. Therefore, it is widely used in a variety of confectionery products such as gummies, marshmallow and candies (Cai et al. 2012). In pharmaceutical industries, gelatin is also commonly used to make capsules, dietary supplements, and protective coatings for medicine (Cai et al. 2012). Meanwhile, gelatin may harbour traces of protein, peptide and nucleic acid residues (Malik et al. 2016), which may remain in the processed products containing the gelatin even after undergoing heating process (Buckley et al. 2009). These traces may serve as the favourable target for identification of the gelatin origin. However, large similarities between structures of gelatin from different sources make their differentiation difficult (Nemati et al. 2004).

Numerous applications of gelatin in food and pharmaceutical products have raised a concern among consumers regarding the animal origin from which the gelatin was derived. The raw materials to make gelatin may come from various animal-by-products such as skins, hides, splits and bones which could be of porcine, bovine and poultry origins (Cai et al. 2012). In addition, plant-based gelatin derived from plant hydrocolloids and cellulose have also been developed (Mohamad et al. 2016). Although various sources of gelatin have been made available, gelatin from porcine has been more popular among manufacturers due to the low cost and bulk quantity of the raw materials. However, religions such as Islam and Judaism forbid the consumption of gelatin that is derived from porcine materials.

Meanwhile, the incidences of food fraud create concern on the quality of food products due to potential undeclared substitution or addition of substances that are cheaper or of inferior quality. This is due to poor manufacturing conduct and economic pressure to save cost and increase profit (Sajali et al. 2020). Cases had been reported such as Sultana et al. (2018) study, where they found branded gelatine samples from various Malaysian outlets that were porcine-positive although the product was labelled as halal gelatin. Demirhan et al. (2012) also found porcine DNA traces in samples of gum drops and marshmallows obtained from markets in Turkey and Germany. Similarly, Nikzad et al. (2017) reported that they identified 50% (n = 12) from 24 pharmaceutical capsule shells in Tehran, Iran were positive for porcine DNA. This shows that highly processed gelatin based products are vulnerable to adulteration with undeclared elements such as porcine traces; it could also be due to contamination indicating poor manufacturing practice. Therefore, to assess the quality of the products, screening of porcine element in gelatin products is essential and this requires a reliable method for detection.

The species determination of the animal by-products has been achieved mainly through analysis of proteins and DNA. Protein-based species authentication often relies on assays utilizing HPLC (Zhang et al. 2009), mass spectrometry (Kumazawa et al. 2016; Yang et al. 2018), enzyme-linked immunosorbence (ELISA) (Venien and Levieux 2005; Tukiran et al. 2016), isoelectric focusing (Tepedino et al. 2001), spectroscopic method (Hashim et al. 2010; Hermanto and Fatimah 2013) and electrophoretic analysis (Hermanto and Fatimah 2013; Azira et al. 2014). However, for highly processed samples such as gelatin products, protein-based analysis may be hindered by progressive denaturation of the protein markers caused by pH treatments and also extreme temperature during manufacturing process, resulting in the loss of heterogeneity and antigenicity of the proteins.

Because of the greater stability of DNA, DNA-based analysis such as PCR may offer an advantage. To date, PCR and qPCR analysis with species-specific primers has been extensively used for sensitive and specific detection of various animal species from highly processed products (Cai et al. 2012; Demirhan et al. 2012; Mutalib et al. 2015; Shabani et al. 2015; Sudjadi et al. 2015; Malik et al. 2016). Nevertheless, PCR based methods on determination of porcine DNA from gelatin based products have been limited (Muñoz‐Colmenero et al. 2016; Mohamad et al. 2016). This is due to the lower amount of DNA surrounded with potential high presence of inhibitors leading to difficulties in analysis and confirmation of results. At the same time, high financial demands of setting up a PCR assay, to highly resourced settings and the need for skilful personnel limit its application especially for on-site detection (Erwanto et al. 2011).

Loop-mediated isothermal amplification (LAMP) assay is an extension of the DNA based detection which is potentially promising because of its high sensitivity and specificity. LAMP assay is simple because it amplifies DNA in a single thermal condition using an incubator or water bath from a very minimum target DNA copies per analysis (Roy et al. 2016). This method relies on four specific primers, inner primers (FIP and BIP) and outer primers (F3 and B3), which recognize six distinct regions of target DNA (Notomi et al. 2000) thus increasing the specificity of detection. Many studies have applied LAMP assay in the identification of meat species especially porcine (Ahmed et al. 2009; Kanchanaphum et al. 2014; Yang et al. 2014; Lee et al. 2016; Ran et al. 2016). For examples, Ran et al. (2016) successfully detected porcine in meat samples using LAMP assay (NEB, USA) containing calcein dye to allow observation using naked eye. Meanwhile, Lee et al. (2016) have developed a direct real-time LAMP assay to detect porcine DNA in processed meat products using a portable, real-time fluorescence detector (Genie II, Optigene, UK).

In view of the potential product fraud, and given the rapidity and high sensitivity of LAMP approach, LAMP application on gelatin products is of interest. In this study, we developed a porcine LAMP assay using two different reaction mixtures based on a single set of LAMP-porcine specific primer on gelatin products; (1) Lab prepared ThermoPol reaction buffer (NEB, USA) with calcein dye ‘MYRM’ and (2) Commercial Isothermal Master Mix ISO-001 reaction (Optigene, UK) ‘GENIE’.

Methods

DNA extraction from meat samples and highly processed products

Meat samples from domestic animals were used in this project. Preliminarily, fresh meat samples for pig, cow, chicken, buffalo, sheep, duck, and turkey were purchased from a local supermarket as labeled in the packaging. Meat samples for dog, cat, and frog, were generously donated by Dr. Md. Eaqub Ali from the Nanotechnology & Catalysis Research Centre, University of Malaya. Meat samples for mouse and rat were obtained from the Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia. The fresh meat samples were stored at − 20 °C prior to DNA extraction. Porcine meat-based products such as sausages, meatballs, burger and canned meat were purchased from a local supermarket. A total of 32 gelatin-based products (9 capsules, 8 gummies, 6 marshmallows, 6 gelatin powders and 3 gelatin strings) were obtained from different retailers within and outside of Malaysia. Summary of all samples analyzed were shown in Tables 1 and 2.

Table 1.

Summary of meat-based samples used in this study and detection using GENIE and MYRM methods

Sample Type/species Source Genie MM (m:s)* MYRM
Raw meat Porcine A local supermarket, Malaysia 10:15 +
Dog UM Malaysiaa ND ND
Cat UM Malaysiaa ND ND
Frog UM Malaysiaa ND ND
Mouse UPM Malaysiab ND ND
Rat UPM Malaysiab ND ND
Cow A local supermarket, Malaysia ND ND
Buffalo A local supermarket, Malaysia ND ND
Sheep A local supermarket, Malaysia ND ND
Chicken A local supermarket, Malaysia ND ND
Turkey A local supermarket, Malaysia ND ND
Duck A local supermarket, Malaysia ND ND
Shrimp A local supermarket, Malaysia ND ND
Squid A local supermarket, Malaysia ND ND
Tuna A local supermarket, Malaysia ND ND
Meat-based product Porcine sausage A local supermarket, Malaysia 12:15 +
Porcine burger A local supermarket, Malaysia 15:15 +
Porcine meatball A local supermarket, Malaysia 14:15 +
Porcine canned food A local supermarket, Malaysia 13:15 +
Open in a new tab

aObtained from Nanotechnology and Catalysis Research Center, University of Malaya (UM), 50603, Kuala Lumpur, Malaysia

bObtained from Faculty of Medicine and Health Sciences, Universiti Putra Malaysia (UPM), 43400, Serdang, Selangor, Malaysia

+ = Color change indicating porcine DNA detected, *m minute, s second, ND not detected

Table 2.

Summary of highly processed gelatin products used in this study and detection using GENIE and MYRM methods

Sample ID Brand/source Labeled information DNA (ng) GENIE (m:s)* MYRM
Gelatin powder GM1 Swallow Globe, Singapore Halal 0.135 ND ND
GM2 JELL-O, Heinz, Illinois Cornstarch (artificial) 0.098 ND ND
GM3 Bites, Singapore Halal 0.033 ND ND
GM7 HALAGEL, Malaysia Halal 0.267 ND ND
GE8 SIGMA, USA Bovine 0.421 15:15 +
GE9 FLUKA, UK Porcine skin 0.003 15:30 +
Capsule C1 HALAGEL, Malaysia Halal 0.250 ND ND
C2 NOW Healthy Food, Illinois, USA Natural gelatin 0.049 13:45 +
C3 Nasmir Hard, Malaysia Halal 0.105 ND ND
C4 TORPAC, USA Porcine gelatin 0.106 16:00 +
C5 TORPAC, USA NA 0.197 ND +
C6 A local store, East Malaysia NA 0.093 14:15 ND
C7 A local store, West Malaysia NA 0.054 ND ND
C8 A local brand, West Malaysia NA 0.045 ND ND
C9 Empty Hard Gelatin, India Hard gelatin 0.018 ND ND
Gummies GE1 Paris, France NA (fish-shape) 0.008 ND ND
GE2 Paris, France NA (Teddy-shape) 0.004 ND ND
GE3 HARIBO GOLDBAREN, Germany Fruit flavour 0.004 ND ND
GE4 Paris, France Coke flavour 0.013 ND ND
GE5 Paris, France NA (big circle-shape) 0.003 ND ND
GE6 Paris, France NA (small circle-shape) 0.004 ND ND
GE7 Paris, France NA (cuboid-shape) 0.004 ND ND
GM6 BOURBON FETTUCCINE, Japan Cola flavour 0.003 ND ND
Marshmallow M1 CVmallow, Malaysia Halal 0.050 ND ND
M2 Guangzhou, China NA (flower-shape) 0.035 ND ND
M3 Beijing, China NA (duck-shape) 0.038 ND ND
M4 Beijing, China NA (bunny-shape) 0.017 ND ND
M5 Beijing, China NA (cylinder big white-shape) 0.020 ND ND
M6 Beijing, China NA (cylinder small pink-shape) 0.028 ND ND
Gelatin string GM10 Agar–agar Pilihan, Malaysia NA (red color) 0.038 ND ND
GM11 Agar–agar Pilihan, Malaysia NA (pale yellow color) 0.014 ND ND
GM12 Pilihan Agar–agar, Malaysia NA 0.063 ND ND
Open in a new tab

+ = Color change indicating porcine DNA detected, NA not available, ND not detected, UK United Kingdom, USA United States of America, *m minute, s second

DNA isolation from all meat samples were extracted using a GeneAll® Exgene™ kit for Animal Tissue (GeneAll Biotechnology co., ltd., Seoul, Korea). For gummy products, DNA was extracted from 2 g of each sample using a PorcineTrace Gelatin DNA Extraction Kit (7FoodPillars, Selangor, Malaysia). Meanwhile, remaining gelatin samples were extracted using DNeasy mericon Food Kit (Qiagen, Hilden, Germany). DNA samples were stored at − 20 °C for further analysis.

DNA quantification

DNA concentration was measured using PicoGreen dye kit Quant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies™, Singapore) according to the manufacturer’s protocol. Series of dilution of the lambda DNA standards provided in the kit were freshly prepared prior to the measurement. A total of 200 μL solution containing 100 μL DNA sample or standard, and 100 μL of 1 × PicoGreen dye in 1 × TE buffer were used. After a proper mixing of the solutions, the solution was transferred into 392 μL UV-Star microplate (Greiner Bio-One GmbH, Frickenhausen, Germany) to be measured using a microplate reader (Infinite M200; Tecan Group Ltd., Switzerland). The samples were excited at 480 nm and the fluorescence intensity was measured at 520 nm. Each sample including the standards was run in triplicates. A standard curve was then plotted and was used to calculate the concentration of the DNA samples. DNA samples with a concentration above 0.001 ng/µL were used as template DNA in the subsequent analysis.

Species verification of fresh meat samples

PCR amplification used universal oligonucleotide primers targeting a section of 16S rRNA gene; Forward: 5′-AYAAGACGAGAAGACCC-3′ and Reverse: 5′-GATTGCGCTGTTATTCC-3′, yielding variable amplicon sizes depending on meat species (Sarri et al. 2014). PCR reactions (50 µl) contained 10 ng DNA of meat samples, 25 µl of 10 Taq buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP, 50 pmol of each primer and 1 U Taq DNA polymerase (REDiant 2X PCR Master Mix, Axil Scientific Pte Ltd, Singapore). The optimal annealing temperature was at 54 °C, as determined using a gradient thermocycler. The cycling conditions consisted of an initial denaturation at 95 °C for 5 min followed by 35 cycles of denaturation at 95 °C for 1 min, annealing at 54 °C for 1 min and extension at 72 °C for 1 min, with a final extension at 72 °C for 10 min. PCR amplicons were visualised by agarose gel electrophoresis in UV chamber.

PCR amplicons were sequenced using the same primers for PCR (1st Base Lab, Malaysia). The nucleotide sequences were checked for sequence homology, using the BLASTn software available at the National Center of Biotechnology Information (http:// www.ncbi-nlm-nih.gov). The determined 16S rDNA nucleotide sequences of the 15 animal species were subjected to phylogenetic analysis in comparison to respective related species retrieved from the GenBank sequence databases. In total, the analysis involved 30 nucleotide sequences. All positions containing gaps and missing data were eliminated. Evolutionary analysis was conducted in MEGA X program using UPGMA algorithm with bootstrap sampling at 1000 replicates (Kumar et al. 2018).

Design of porcine-specific LAMP primers

DNA sequences of the porcine mitochondrial genes were retrieved from the GenBank database. Multiple sequences were aligned using an online MultAlin interface calculator (http://multalin.toulouse.inra.fr/multalin/multalin.html), and a set of LAMP primers were designed based on the conserved regions identified using Primer Explorer software, version 4 (Eiken Chemical Co., Tokyo, Japan) as described in Fig. 1. Specific primers for porcine were used, which consisted of five primer sequences covering approximately 190 bp amplicon size; two outer primers (F3:5’-TCATAGGCTACGTCCTGC-3’ and B3:5’-GCGGTAATGATGAATGGCA-3’), two inner primers (FIP:5’-TCCGATATAAGGGATAGCTGATAGT-AGGACAAATATCATTCTGAGGAG-3’ and BIP:5’-ACAGACCTCGTAGAATGAATCTGA-GGATAAAGTGAAAGGCGAAGA-3’) and a loop primer (LB:5’-TTTTCCGTCGACAAAGCAACCCTC-3’).

Fig. 1.

Fig. 1

Open in a new tab

Nucleotide sequences used for designing the primers. a Primer design in Primer Explorer Version 4.0. Arrows indicate the primers sequence used for the porcine-specific LAMP assay. b Schematic diagram showing the positions of the porcine-specific LAMP assay

Development of porcine-specific LAMP assay

Two different assays format were developed in this study. The first format (GENIE) was carried out using 1X Isothermal Master Mix ISO-001 (Optigene, West Sussex, UK), containing 5 pmol of each outer primer (F3 and B3), 20 pmol of loop primer (LF) and 40 pmol of each inner primer (FIP and BIP) and 1 ng/µL of DNA for meat samples in a total volume of 25 µL. LAMP assay was performed in 60 min for meat samples and 80 min for gelatin samples, followed by annealing curve analysis (98–80 °C, 0.05 °C /s) to confirm the annealing temperature of each amplified sample of DNA. Emitted fluorescence released from LAMP products was monitored using Genie II LAMP detector (Optigene, West Sussex, UK). This assay used a closed system tube in which reaction vessels were not opened either during or after the analysis. The purpose was to reduce contamination of subsequent samples by aerosolized products (Niessen et al. 2013). In addition, this machine showed real-time fluorometer results with summary of outputs in table format.

The second format (MYRM) was developed based on modification of method proposed by Tomita et al. (2008). In contrast to the first format, positive detection in the second format was determined by a color change of the reaction mixture from orange to green upon successful amplification of the target species. To confirm the results, all LAMP products were subjected to gel electrophoresis and visualized in a UV chamber (Infinite M200; Tecan Group Ltd, Switzerland). The reaction mixture included 5 pmol of each outer primer (F3 and B3), 20 pmol of loop primer (LF), 40 pmol of each inner primer (FIP and BIP), 1.8 mM dNTPs (Tiangen, China), × 1 ThermoPol reaction buffer (20 mMTris-HCl, 10 mM KCl, 2 mM MgSO4, 10 mM (NH4)2SO4, 0.1% Triton X-100) (New England Biolabs, USA), 6 mM MgSO4, 0.6 M betaine (Sigma-Aldrich, Canada), 0.5 mM MnCl2, 25 μM calcein (Dojindo, Japan), 8 U of Bst DNA polymerase (New England Biolabs, USA), and 1 ng/μL of template DNA. The reaction mixture was incubated for 60 min for meat samples and 80 min for gelatin samples, and then heated to 80 °C for 5 min to terminate the reaction. Each LAMP assay was repeated three times to ensure reproducibility of the method.

Optimization of temperature for porcine-specific LAMP assays was performed for each buffer (GENIE and MYRM). The incubation temperature for each buffer was initially set at 60, 63 and 65 °C (Kaewphinit et al. 2013; de Lira Nunes et al. 2014) within 60 min. The optimum temperature for porcine-specific primer was 63 ˚C and 65 ˚C, respectively for GENIE and MYRM buffer. Specificity of the primers used for porcine detection in each assay format was tested against DNA from 15 different animals. Sensitivity was assessed using serially diluted porcine DNA; 1 ng/µL to 0.01 pg/µL.

Results and discussions

Species verification of animal species

The size of the amplified sequences in this study ranged from 206 to 322 bp. The variation in size was expected as the primers are universal-based, capable of amplifying various species with potential differences in target size. Subsequently, the identity of all animal species used in this study were successfully verified using 16S rRNA gene sequencing with > 95% sequence similarities; porcine (Sus Scrofa), dog (Canis lupus familiaris), cat (Felis catus), mouse (Mus Musculus), rat (Rattus norvegicus), frog (Hoplobatrachus rugulosus), beef (Bos taurus), buffalo (Bubalus bubalis), goat (Capra hircus), chicken (Gallus gallus), duck (Anas platyrhynchos), turkey (Meleagris gallopavo), shrimp (Litopenaeus vannamei), squid (Uroteuthis edulis), and tuna (Euthynnus affinis). Furthermore, phylogenetic analysis showed that the 16S rRNA gene sequence of each meat species was paired together with their respective reference sequence, supported by high bootstrap values ranging from 96 to 100% (see supplementary material). The tree also showed a good clustering according to the taxonomic categories where all mammals and birds are rooted in their own group, while others are branched away in their own lineage respectively. This indicates that the DNA sequences of the respective meat species are in a good quality and reliable to be used in the specificity test.

Performance of porcine-specific LAMP assays

Specificity of the porcine primer set was confirmed as no cross reactivity was detected when tested against meat DNA of 14 non target animal species. In the GENIE assay, annealing curve analysis did not show an unspecific peak confirming thereby the specificity of the design assay. Based on the porcine-specific amplicons, annealing temperature of porcine DNA was 85.12 ± 0.26 °C. Meanwhile, that for DNA derived from gelatin products were very close at 84.86 ± 0.56 °C (Table 3). The slight difference of annealing temperature between meat sources and gelatin products may be due to the condition of the gelatin samples that have gone through many processes of hydrolysis and progressive denaturation to affect the integrity of the end products, causing deformed DNA structure (Cai et al. 2012). In MYRM reaction, in addition to color changes reaction, the presence of DNA bands on agarose gel electrophoresis (Fig. 2) also confirmed that amplification was only detected in tubes containing traces of porcine DNA.

Table 3.

Sensitivity test for porcine-specific primers using DNA extracted from porcine meat by LAMP assay (A), porcine-specific GENIE assay tested on porcine meat-based (B) and porcine-specific GENIE assay tested on gelatin-based products (C)

Test DNA amount (ng) Detection time (m:s)* Annealing temperature (○C)
A
Sensitivity 1 10:15 84.86
0.1 17:30 85.27
0.01 17:30 85.20
0.001 27:45 85.15
0.0001 ND ND
0.00001 ND ND
B
Meat-based products Porcine sausage 12:15 85.01
Porcine meatball 14:15 84.86
Porcine burger 15:15 84.87
Porcine canned Food 13:15 84.77
C
Gelatin-based products C2 13:45 84.67
C4 16:00 84.70
C5 ND ND
C6 14:15 84.76
GE8 15:15 84.76
GE9 15:30 84.61
Negative control ND ND
Open in a new tab

*m minute, s second, ND not detected

Fig. 2.

Fig. 2

Open in a new tab

Determination of the sensitivity of the porcine-specific primers using MYRM assay tested on porcine meat-based products. Lane No. 1–5; 0.1 ng, 0.01 ng, 0.001 ng, 0.0001 ng and 0.00001 ng. Lane No. 6–9; Porcine sausage, Porcine meatball, Porcine burger, and Porcine canned food. a, c Fluorescent LAMP assay in UV light in correspondence to b, d LAMP assay products viewed in gel electrophoresis respectively (a to b, c to d)

In sensitivity test, Table 3 showed porcine-specific LAMP successfully detected as low as 1 pg/µL of porcine DNA samples. An annealing curve analysis showed only one single peak, with average temperature value of 85.12 ± 0.26 °C. Rapidity of the porcine-specific LAMP GENIE assay was indicated by its ability to detect a low amount of porcine DNA within 30 min of reaction time.

Meanwhile, successful amplification and detection as low as 1 pg/µL of porcine DNA in porcine-specific LAMP of MYRM assay as indicated by the color changes from yellow to green was obtained in 60 min incubation time (Fig. 2a). Compared with a previous study, Ran et al. (2016) conducted a similar color change based-assay and detected as low as 0.5 pg/reaction targeting ND1 gene. The lower sensitivity could be due to different target region of primer or different dilution of DNA. In our study, we did tenfold dilution and thus 0.5 was not included in the assay. Nevertheless, agarose gel electrophoresis of the amplicons detected the presence of bands as low as 0.1 pg/µL of porcine DNA (Fig. 2b). The inability of 0.1 pg/µL of porcine DNA sample to cause a color change is probably due to limited capability of calcein to induce color changes upon binding to a low amount of amplicons in the tube. This was also observed in a study by Zhang et al. (2019) in their sensitivity test, with a minimum detectable concentration at 1.0 × 102 CFU/mL. Meanwhile, in other LAMP assay studies using calcein dye, the electrophoretic analysis of LAMP products was in accordance with naked eye visualization (Xie et al. 2014; Ran et al. 2016; Ezzatyhusna et al. 2017).

Application of porcine-specific LAMP assays on meat-based products

Porcine DNA was detected in all porcine meat-based products tested in GENIE assay, although a slightly different detection time was obtained for each product. The results are as expected since ‘pork’ was indicated on the label of the commercial products. Annealing curve analysis consistently showed only one single peak in all positively tested samples, with average peak temperature value of 84.88 ± 0.09 °C as shown in Table 3. Lee et al. (2016), using a similar reaction mixture from Optigene but targeting mitochondrial D-loop gene, also showed slightly different detection times among 32 positive samples of commercial porcine meat-based products. This indicates that amplification of DNA targeting the same target region from products with different sample texture could be differentially affected due to different quality level of the extracted DNA.

In MYRM assay, all porcine meat-based products were able to induce color changes as shown in Fig. 2c. This was further supported by agarose gel electrophoresis of the LAMP products (Fig. 2d) where all bands appeared in positive DNA samples. In another study, Ran et al. (2016) used a similar approach and detected 15 positive samples out of 42 samples with 36% of the selected samples did not conform to the labeling. The study also compared the result with porcine-qPCR kit as a standard and found two discrepancies. The LAMP assay for the remaining 40 samples was in accordance with the standard testing.

Application of porcine-specific LAMP assays on highly processed gelatin-based products

Next, we divided 32 samples of highly processed gelatin-based products into five groups which were (1) gelatin powder (N = 6 with the following distribution; labeled as porcine: n = 1, halal: n = 3, not porcine: n = 2), (2) capsule (N = 9; labeled as porcine: n = 1, halal: n = 2, not available: n = 6), (3) gummies (N = 8; label not available), (4) marshmallow (N = 6; label not available) and (5) gelatin string (N = 3; label not available). Out of the 32 samples, we detected porcine DNA in two samples of gelatin powder group and three of capsule group. As shown in Fig. 3, the two samples, GE8 and GE9 of gelatin powder group showed a positive result using both LAMP reaction mixtures. GE9 (FLUKA, UK) is expected to be positive because it is a porcine labeled-gelatin powder while others were not labeled to have porcine origin. Meanwhile GE8 (Sigma-Aldrich, USA) was labeled as bovine gelatin. Cai et al. (2012) and Mohamad et al. (2016) analyzed products of a similar brand using qPCR assay and also detected a mixture of porcine and bovine gelatin in this product. As far as the efficacy of LAMP assay of our study is concerned, the results were consistent with the previous studies indicating the reliability of our LAMP assay in detecting porcine DNA in mixture of bovine DNA in highly processed gelatin powder.

Fig. 3.

Fig. 3

Open in a new tab

Porcine-specific MYRM assay tested on gelatin-based products. Lane No. 1–7; C2, C4, C6, GE8, GE9, C5, and GE2. Fluorescent LAMP assay in UV light (a) in correspondence to LAMP assay products viewed in gel electrophoresis (b)

On the other hand, a discrepancy was observed in comparing the two LAMP reaction mixtures involving sample C5 and C6 (The labels for source of gelatin of both products were not available) of the capsule group. Using GENIE reaction mixture, LAMP primers successfully amplified porcine DNA in three samples of capsule group (C2, C4, C6) out of 9 samples (C2 and C6; label not available, C4; labeled as porcine gelatin). Meanwhile, the MYRM reaction mixture consistently showed a positive result for C2 and C4. C5 was also positive (negative in GENIE) while the remaining samples including C6 (positive by GENIE) were negative. For clarification, we further analyzed the DNA samples of C5, C6, GE1, GE2, M4 and M5 to check for the possibility that the negative reaction was due to presence of inhibitors. We added 0.1 ng/µL of porcine DNA to each representative of the gelatin sample that gave negative results, and run again the LAMP assay using both reaction mixtures. It turned out that all the samples were amplified for the porcine DNA. Thus, there were most likely no inhibitory factors in the DNA samples that may inhibit amplification in this LAMP assay. Therefore, the discrepancy could be due to the different buffer that may have a different chemistry to affect the results, although we used the same DNA samples and porcine-specific primer set for each assay. In addition, this may also be due to the different DNA polymerase in each buffer. MYRM buffer contained Bst DNA polymerase (New England Biolabs, Ipswich, MA), while GENIE buffer used GspSSD DNA polymerase (Optigene Ltd., UK). These enzymes were reacting perfectly when we tested using meat-based products but slightly different when we tested on gelatin-processed products (capsules group). Another observation was DNA of capsule sample C4 (labeled as porcine gelatin) produced a positive result using both LAMP reaction mixtures. The same capsule product was also previously analyzed by Mohamad et al. (2016) and showed a porcine positive reaction using qPCR. This may suggest that our LAMP assay was as good as qPCR to detect traces of porcine DNA in highly processed gelatin products of capsules.

Conclusion

We successfully developed LAMP specific porcine primers on gelatin samples and tested using two different buffers, GENIE and MYRM. The primers allowed sensitive detection of porcine DNA at a concentration as low as 1 pg/µL with no cross-reactivity occurred in DNA samples from 14 other animal species, indicating a high specificity of these primers. In addition, porcine detection was possible in gelatin products despite the commonly low DNA concentration recovered from this highly processed product due to extensive DNA degradation during extreme processing. Therefore, this LAMP assay may represent a simple, rapid, reliable and sensitive DNA-based test for determining the species origin of highly processed products. Although we found a contradicting result when using different buffers in GENIE and MYRM assays against the capsule samples, it involved only two samples. The two methods may not be readily compared as each is utilizing different buffers and DNA polymerase enzymes. Nevertheless, GENIE assay could be better as it is using a real time-based reading device to allow a better sensitivity in detection while MYRM assay is relying on visual by eye-observation and gel image. Due to a low number of positive samples in this study and limited information on the status of the gelatin origin, as well as the nature of the manufacturing process, more assessments using a larger number of samples of known characteristics, with robust validation measures are highly recommended to further establish the method.

Supplementary Information

Supplementary material 1 (DOCX 18 kb) (18.4KB, docx)

Acknowledgements

The authors would like to acknowledge the financial support provided by Universiti Putra Malaysia and Konsortium Institut Halal IPT Malaysia. Nor Asmara Tasrip wishes to acknowledge the financial support from MyBrain15 under the Ministry of Higher Education, Malaysia for her PhD fellowship.

Funding

This study was funded by grants of Universiti Putra Malaysia (GP-IPS/2018/9625800 and GP-B/2018/9658200) and Konsortium Institut Halal IPT Malaysia, Ministry of Higher Education, Malaysia (6300911).

Compliance with ethical standards

Conflict of interest

Nor Asmara Tasrip declares that she has no conflict of interest. Mohd Nasir Mohd Desa declares that he has no conflict of interest. Nur Fadhilah Khairil Mokhtar declares that she has no conflict of interest. Nurhayatie Sajali declares that she has no conflict of interest. Amalia Mohd Hashim declares that she has no conflict of interest. Md. Eaqub Ali declares that he has no conflict of interest. Cheah Yoke Kqueen declares that he has no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or live animal subjects by any of the authors.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. Ahmed MU, Saito M, Hossain MM, Rao SR, Furui S, Hino A, Takamura Y, Takagi M, Tamiya E. Electrochemical genosensor for the rapid detection of GMO using loop-mediated isothermal amplification. Analyst. 2009;134(5):966–972. doi: 10.1039/b812569d. [DOI] [PubMed] [Google Scholar]
  2. Azira TN, Man YBC, Hafidz RNRM, Aina MA, Amin I. Use of principal component analysis for differentiation of gelatine sources based on polypeptide molecular weights. Food Chem. 2014;151:286–292. doi: 10.1016/j.foodchem.2013.11.066. [DOI] [PubMed] [Google Scholar]
  3. Buckley M, Collins M, Thomas-Oates J, Wilson JC. Species identification by analysis of bone collagen using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Rapid Commun Mass Spectrom. 2009;23:3843–3854. doi: 10.1002/rcm.4316. [DOI] [PubMed] [Google Scholar]
  4. Cai H, Gu X, Scanlan MS, Ramatlapeng DH, Lively CR. Real-time PCR assays for detection and quantification of porcine and bovine DNA in gelatin mixtures and gelatin capsules. J Food Compos Anal. 2012;25:83–87. doi: 10.1016/j.jfca.2011.06.008. [DOI] [Google Scholar]
  5. de Lira NM, Mendes-Marques CL, de Almeida AMP, Leal NC. The development of a loop-mediated isothermal amplification (LAMP) procedure for plague diagnostic. Am J Anal Chem. 2014;5:1069–1077. doi: 10.4236/ajac.2014.516114. [DOI] [Google Scholar]
  6. Demirhan Y, Ulca P, Senyuva HZ. Detection of porcine DNA in gelatin and gelatin-containing processed food products—Halal/Kosher authentication. Meat Sci. 2012;90:686–689. doi: 10.1016/j.meatsci.2011.10.014. [DOI] [PubMed] [Google Scholar]
  7. Erwanto Y, Abidin MZ, Rohman A, Sismindari S. PCR-RFLP using BseDI enzyme for pork authentication in sausage and nugget products. Trop Anim Sci J. 2011;34(1):14–18. doi: 10.5398/medpet.2011.34.1.14. [DOI] [Google Scholar]
  8. Ezzatyhusna NMK, Izzati NH, Suraiya SMN, Suharni M. Sensitivity evaluation of SYBR Green I, SYBR safe and calcein dyes for detection of human papillomavirus 16 by loop-mediated isothermal amplification. Med Health. 2017;12(2):220–229. doi: 10.17576/MH.2017.1202.07. [DOI] [Google Scholar]
  9. Hashim DM, Man YBC, Norakasha R, Suhaimi M, Salmah Y, Syahariza ZA. Potential use of fourier transform infrared spectroscopy for differentiation of bovine and porcine gelatins. Food Chem. 2010;118(3):856–860. doi: 10.1016/j.foodchem.2009.05.049. [DOI] [Google Scholar]
  10. Hermanto S, Fatimah W. Differentiation of bovine and porcine gelatin based on spectroscopic and electrophoretic analysis. J Food Pharm Sci. 2013;1(3):68–73. [Google Scholar]
  11. Kaewphinit T, Arunrut N, Kiatpathomchai W, Santiwatanakul S, Jaratsing P, Chansiri K. Detection of mycobacterium tuberculosis by using loop-mediated isothermal amplification combined with a lateral flow dipstick in clinical samples. Biomed Res Int. 2013;2013:926230. doi: 10.1155/2013/926230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kanchanaphum P, Maneenin S, Chaiyana W. Analysis of pork meat using loop mediated isothermal amplification (LAMP) to confirm halal status. Int J Biosci. 2014;4(9):62–68. [Google Scholar]
  13. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018 doi: 10.1093/molbev/msy096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kumazawa Y, Taga Y, Iwai K, Koyama Y. A Rapid and simple LC-MS method using collagen marker peptides for identification of the animal source of leather. J Agric Food Chem. 2016;64(30):6051–6057. doi: 10.1021/acs.jafc.6b02132. [DOI] [PubMed] [Google Scholar]
  15. Lee S, Kim M, Hong Y, Kim H. Development of a rapid on-site detection method for pork in processed meat products using real-time loop mediated isothermal amplification. Food Control. 2016;66:53–61. doi: 10.1016/j.foodcont.2016.01.041. [DOI] [Google Scholar]
  16. Malik A, Sutantyo ML, Hapsari I, Sinurat AV, Purwati EM, Jufri M, Suryadi H. Simultaneous identification and verification of gelatin type in capsule shells by electrophoresis and polymerase chain reaction. J Pharm Investig. 2016;46(5):475–485. doi: 10.1007/s40005-016-0245-0. [DOI] [Google Scholar]
  17. Mohamad NA, Mustafa S, El Sheikha AF, KhairilMokhtar NF, Ismail A, Eaqub MA. Modification of gelatin-DNA interaction for optimised DNA extraction from gelatin and gelatin capsule. J Sci Food Agric. 2016;96(7):2344–2351. doi: 10.1002/jsfa.7482. [DOI] [PubMed] [Google Scholar]
  18. Muñoz-Colmenero M, Martínez JL, Roca A, Garcia-Vazquez E. Authentication of commercial candy ingredients using DNA PCR-cloning methodology. J Sci Food Agric. 2016;96(3):859–867. doi: 10.1002/jsfa.7158. [DOI] [PubMed] [Google Scholar]
  19. Mutalib SA, Muin NM, Abdullah A, Hassan O, Mustapha WAW, Sani NA, Maskat MY. Sensitivity of polymerase chain reaction (PCR)-southern hybridization and conventional PCR analysis for Halal authentication of gelatin capsules. LWT Food Sci Technol. 2015;63(1):714–719. doi: 10.1016/j.lwt.2015.03.006. [DOI] [Google Scholar]
  20. Nemati M, Oveisi M, Abdollahi H, Sabzevari O. Differentiation of bovine and porcine gelatins using principal component analysis. J Pharm Biomed Anal. 2004;34:485–492. doi: 10.1016/S0731-7085(03)00574-0. [DOI] [PubMed] [Google Scholar]
  21. Niessen L, Luo J, Denschlag C, Vogel RF. The application of loop-mediated isothermal amplification (LAMP) in food testing for bacterial pathogens and fungal contaminants. Food Microbiol. 2013;36(2):191–206. doi: 10.1016/j.fm.2013.04.017. [DOI] [PubMed] [Google Scholar]
  22. Nikzad J, Shahhosseini S, Tabarzad M, Nafissi-Varcheh N, Torshabi M. Simultaneous detection of bovine and porcine DNA in pharmaceutical gelatin capsules by duplex PCR assay for Halal authentication. DARU J Pharm Sci. 2017;25(1):3. doi: 10.1186/s40199-017-0171-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. Loop mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28(12):E63. doi: 10.1093/nar/28.12.e63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ran G, Ren L, Han X, Liu X, Li Z. Development of a rapid method for the visible detection of pork DNA in halal products by loop mediated isothermal amplification. Food Anal Methods. 2016;9:565–570. doi: 10.1007/s12161-015-0246-z. [DOI] [Google Scholar]
  25. Roy S, Rahman IA, Ahmed MU. Paper based rapid detection of pork and chicken using LAMP-magnetic bead aggregates. Anal Methods. 2016;8:2391–2399. doi: 10.1039/C6AY00274A. [DOI] [Google Scholar]
  26. Sajali N, Wong SC, Abu Bakar S, KhairilMokhtar NF, Manaf YN, Yuswan MH, MohdDesa MN. Analytical approaches of meat authentication in food. Int J Food SciTechnol. 2020 doi: 10.1111/ijfs.14797. [DOI] [Google Scholar]
  27. Sarri C, Stamatis C, Sarafidou T, Galara I, Godosopoulo V, Kolovos M, Liakou C, Tastsoglou S, Mamuris Z. A new set of 16S rRNA universal primers for identification of animal species. Food Control. 2014;43(9):35–41. doi: 10.1016/j.foodcont.2014.02.036. [DOI] [Google Scholar]
  28. Shabani H, Mehdizadeh M, Mousavi SM, Dezfouli EA, Solgi T, Khodaverdi M, Rabiei M, Rastegar H, Alebouyeh M. Halal authenticity of gelatin using species-specific PCR. Food Chem. 2015;184:203–206. doi: 10.1016/j.foodchem.2015.02.140. [DOI] [PubMed] [Google Scholar]
  29. Sudjadi WHS, Sepminarti T, Rohman A. Analysis of porcine gelatin DNA in commercial capsule shell using real-time polymerase chain reaction for halal authentication. Int J Food Prop. 2015;19(9):2127–2134. doi: 10.1080/10942912.2015.1110164. [DOI] [Google Scholar]
  30. Sultana S, Hossain MAM, Zaidul ISM, Ali ME. Multiplex PCR to discriminate bovine, porcine, and fish DNA in gelatin and confectionery products. LWT Food Sci Technol. 2018;92(6):169–176. doi: 10.1016/j.lwt.2018.02.019. [DOI] [Google Scholar]
  31. Tepedino V, Berrini A, Borromeo V, Gaggioli D, Cantoni C, Manzoni P, Secchi C. Identification of commercial fish species belonging to the orders pleuronectiformes and gadiformes: library of isoelectric focusing patterns. J AOAC Int. 2001;84(5):1600–1607. doi: 10.1093/jaoac/84.5.1600. [DOI] [PubMed] [Google Scholar]
  32. Tomita N, Mori Y, Kanda H, Notomi T. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc. 2008;3(5):877–882. doi: 10.1038/nprot.2008.57. [DOI] [PubMed] [Google Scholar]
  33. Tukiran NA, Ismail A, Mustafa S, Hamid M. Determination of porcine gelatin in edible bird's nest by competitive indirect ELISA based on anti-peptide polyclonal antibody. Food Control. 2016;59:561–566. doi: 10.1016/j.foodcont.2015.06.039. [DOI] [Google Scholar]
  34. Venien A, Levieux A. Differentiation of bovine from porcine gelatins using polyclonal anti-peptide antibodies in indirect and competitive indirect ELISA. J Pharm Biomed Anal. 2005;39:418–424. doi: 10.1016/j.jpba.2005.04.013. [DOI] [PubMed] [Google Scholar]
  35. Xie L, Xie Z, Zhao G. A loop-mediated isothermal amplification assay for the visual detection of duck circovirus. Virol J. 2014;11:76. doi: 10.1186/1743-422X-11-76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yang L, Fu S, Peng X, Li L, Song T, Li L. Identification of pork in meat products using real-time loop-mediated isothermal amplification. Biotechnol Biotechnol Equip. 2014;28(5):882–888. doi: 10.1080/13102818.2014.963789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yang L, Wu T, Liu Y, Zou J, Huang Y, Babu SV, Lin L. Rapid identification of pork adulterated in the beef and mutton by infrared spectroscopy. J Spectrosc. 2018;2018:1–10. doi: 10.1155/2018/2413874. [DOI] [Google Scholar]
  38. Zhang G, Liu T, Wang Q, Chen L, Lei J, Luo J, Ma G, Su Z. Mass spectrometric detection of marker peptides in tryptic digests of gelatin: a new method to differentiate between bovine and porcine gelatin. Food Hydrocoll. 2009;23:2001–2007. doi: 10.1016/j.foodhyd.2009.03.010. [DOI] [Google Scholar]
  39. Zhang J, Cao J, Zhu M, Xu M, Shi F. Loop-mediated isothermal amplification-lateral-flow dipstick (LAMP-LFD) to detect Mycoplasma ovipneumoniae. World J Microbiol Biotechnol. 2019;35:31. doi: 10.1007/s11274-019-2601-5. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material 1 (DOCX 18 kb) (18.4KB, docx)

Articles from Journal of Food Science and Technology are provided here courtesy of Springer

RESOURCES