Development of novel isothermal-based DNA amplification assay for detection of pig tissues in adulterated meat
Abstract
For the first time, we describe an innovative polymerase spiral reaction (PSR) assay for the rapid, simple, and accurate detection of pig tissues or pork in adulterated meat including heat-treated and processed ones. The PSR assay specifically targeting the mitochondrial cytochrome b ( cyt-b ) gene of the pig was successfully optimized permitting assay results in 65 min time. The developed detection method was 100% specific amplifying only the cyt-b gene and displaying negative results with all the tested non-pork meats. The sensitivity of the developed PSR (760 fg porcine DNA) was tenfold better than the end-point PCR and able to detect heat-treated (121 °C) and adulterated (0.5% pork in beef) meat and processed pork products such as sausages, salami, meatball, soup, curry, etc. The developed PSR-based method can be used for point-of-care detection with minimum instrumentation and technical expertise to guarantee instant clearance of exported and imported meat products. This is the first time that PSR has been adapted for food authenticity purposes.
Introduction
Globally, meat production and per capita consumption of meat are increasing and are expected to increase further in the next decades [ 1 ]. There is a predicted upsurge in pork consumption (> 36%) followed by chicken (35%), beef (22%), and mutton (4%) [ 2 ]. This ever-expanding globalized and tortuous food value chains contribute to a bourgeoning issue of meat adulteration or fraud [ 3 ]. Consumers’ increased awareness and concerns of zoonotic disease risks, food-related allergy/toxicity, religious (halal) apprehensions, and fraudulent tagging of food products with undeclared ingredients have signified the importance of food authenticity [ 4 , 5 ]. Moreover, after the horsemeat scandal in Europe, there is increased global attention toward meat mislabeling and adulteration activities [ 6 ]. Fraudulent substitution of meat affects the confidence and interests of consumers and thereby poses a negative impact on the meat industry [ 3 ]. This remains an important challenge to the government regulatory agencies and accurate labeling of meat and meat products is the responsibility of all the actors in the food chain which allows the consumers to make informed decisions or choices [ 3 , 7 ]. Pork, the most commonly consumed meat possesses similar color and texture as that of beef (veal) and mutton and, hence, is extensively used as a substitute for these two types of meat [ 8 , 9 ]. The substitution of pork ingredients in other meat foods also jeopardizes religious sentiments among Muslims and Jews and others for whom any amount of pork consumption is not acceptable, even at little levels [ 10 ]. Apart from this, undeclared pork products may contaminate the food with some tissue-dwelling zoonotic parasites such as Toxoplasma gondi and Trichinella and may also introduce some banned veterinary drugs [ 11 , 12 ]. Therefore, rapid and accurate detection methods to specifically identify the pig-derived ingredients in food products would be in the greatest interest and assurance of consumers. Initially, meat adulteration with pork was identified using physicochemical methods such as anatomical structures, glycogen content, refractive index, iodine value, etc. [ 13 ]. Later on, spectroscopic techniques such as near infrared spectroscopy [ 14 ], Fourier transform infrared spectrometry [ 15 ], Raman spectroscopy [ 16 ], etc., were deployed for detection of pork in adulterated meat. Immunological assays such as enzyme-linked immunosorbent assay (ELISA) and lateral-flow assays were frequently used for pork speciation as they have cost-effectiveness, high throughput possibility, high sensitivity, and non-requirement of trained personnel [ 10 , 17 , 18 ]. The major drawbacks of immunoassays are false-negative results due to cross-reactivity, variable affinity, and denaturation of the epitope of target proteins due to heat treatment and processing [ 10 , 19 ]. Nucleic acid (DNA) amplification assays offer many advantages in identification of meat such as rapidity, specificity, and simplicity as DNA is a stable particle that permits detection even after processing (baking, microwaving, spray drying, etc.) and heat treatment [ 10 ]. Among widely used DNA-based meat authentication assays, pork speciation was demonstrated employing conventional end-point PCR [ 20 ], SYBR Green real-time PCR [ 21 ], Taqman probe real-time PCR [ 22 ], random amplification of polymorphic DNA (RAPD) PCR [ 23 ], and droplet digital PCR [ 24 ]. The limitations of PCR-based assays are the requirement for sophisticated instruments and trained laboratory personnel [ 25 ]. Isothermal amplification approaches such as loop-mediated isothermal amplification (LAMP) have obviated the necessity of instruments including thermocycler, gel electrophoresis, and gel documentation system, and also offer additional benefits like low operational cost, high amplification efficiency, easy-to-perform nature, etc. [ 3 , 10 , 25 ]. The main drawback of LAMP assays is the primer–primer interactions owing to the requirement of multiple (4–6) primers for initiating the reaction [ 10 ]. Polymerase spiral reaction (PSR) assay is an innovative isothermal detection system, which is a combination of loop-mediated isothermal amplification (LAMP) and end-point PCR as it operates under isothermal conditions with single pair of primer and permits visual interpretation of results [ 26 ]. PSR has been gaining popularity and it has also been deployed successfully in the detection of foodborne pathogens like Salmonella [ 27 , 28 ], V. parahaemolyticus [ 29 ] , Campylobacter jejuni [ 30 ], and recently the PSR was also successfully adapted for detection of SARS-CoV-2 [ 31 ] . To date, PSR assay for meat authentication or speciation has not been reported. Therefore, in this study, we have developed a PSR assay to detect pork in adulterated meat including heat-treated and processed ones. The detection performance of the established PSR was compared to the end-point PCR assay.
Materials and methods
Instruments, chemicals, reagents, kits, and samples
The laboratory instruments used in the current study are as follows: refrigerated centrifuge (Hettich, United States), homogenizer (Remi, India), vortex (Tarsons, India), microfuge (Tarsons, India), − 20 °C deep freezer (Bluestar, India), micropipettes (Thermo Fischer Scientific, United States), temperature-controlled dry bath (Labnet International, United States), thermocycler (Eppendorf, Germany), horizontal gel electrophoresis unit and power pack (Cleaver Scientific, United Kingdom), gel documentation system (GeNei, India) and nanodrop spectrophotometer (Thermo Fisher Scientific, United States). The reagents and chemicals used are as follows: DreamTaq Green PCR Master Mix, 10 mM dNTP mix, Tris–acetate-EDTA buffer, and GeneRuler 100 bp Plus DNA Ladder procured from ThermoFisher Scientific (USA); Bst 2.0 Warm start polymerase, magnesium sulfate (MgSO 4 ) and 10X thermopol Bst buffer were from New England Biolabs (USA); and betaine and SYBR Green dye were from Sigma-Aldrich (USA). DNeasy blood and tissue kit (Qiagen, Germany) was used for the extraction of DNA from all the meat samples.
Designing of primer
The primer for conventional end-point PCR was designed using NCBI’s primer designing resource ( https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) targeting mitochondrial cytochrome b ( cyt-b ) gene (GenBank Acc.No. NC_000845.1) of the pig. The primer design for PSR was done as previously delineated by Liu et al. [ 26 ]. In the PSR primers, the capital 3′ sequences of the forward and reverse primers are complementary to the cytochrome b gene (target) sequences. The small letter 5′ sequence of the forward primer is reverse to the small letter 5′ sequence of the reverse primer. The lowercase sequences were taken from an exogenous botanic gene. Oligonucleotide primers were custom-synthesized from Imperial Life Sciences, Haryana, India. The oligos for PCR and PSR were Cyt-b F:5′-GCCCATACTACACACATCCA-3; Cyt-b R:5′-TTCTACGGGTTGTCCTCCAA-3′ and Cyt-b PSRF:5′-acgattcgtacatagaagtatagGCCCATACTACACACATCC-3′; Cyt-b PSRR:5′-gatatgaagatacatgcttagcaTTCTACGGGTTGTCCTCCAA-3′, respectively.
Preparation of tissue samples
Fresh and processed meat (pork, beef, carabeef, mutton, chevon, and chicken) samples (50–250 g) were collected from local retail markets. The species of the animals were identified morphologically before collection. Following collection, all meat samples were labeled and transported to the laboratory in a sterile plastic container under chilled conditions (4 °C) and kept at − 20 °C till additional analysis. The genomic DNA was extracted from 25 g of samples (raw/ processed/ heat-treated) using DNeasy Blood and Tissue Kit following the method given by the manufacturer of the kit. The quality and purity of the extracted DNA were confirmed using agarose (2%) gel electrophoresis and nanodrop spectrophotometer, respectively. The genomic DNA samples exhibiting OD260:OD280 ratios of 1.7–1.9 were considered good and were used for further study.
PSR and PCR assay—optimization and result visualization
The PSR assay was optimized for different reagent concentrations, incubation times, and temperature ranges. The optimization was accomplished for primers (8 μM–20 μM), dNTP (1.2–2.0 mM), MgSO 4 (4.0–8.0 mM), betaine (0–1 mM), Bst 2.0 warm start polymerase (8–16 U), time (45–90 min), and amplification temperature (60–70 °C). PSR assay was optimized sequentially as defined earlier by Milton et al. [ 30 ]. The visual interpretation of amplified PSR products was done by adding up 1 μL of SYBR Green I dye (1:10) to the tube after amplification and the results were interpreted in white and UV lights. The results were also interpreted by resolving the amplified products in 2.5% agarose gel electrophoresis. The conventional PCR reaction blend in 25 μL quantity included 10 pmol of each primer (1 μL), 12.5 μL 2 × DreamTaq PCR Master Mix, and 1 μL template. The cycling setting of PCR comprised 95 °C (5 min), 35 cycles of 95 °C (30 s), 64 °C (1 min), 72 °C (30 s), and 72 °C (7 min). The results were interpreted on agarose gel electrophoresis (1.5%).
Specificity, sensitivity, and repeatability
The specificity testing (PCR and PSR) was done to check any cross-amplification using DNA templates obtained from targeted pig meat and non-targeted meat of other food animal species such as cattle, buffalo, sheep, goat, and chicken. The analytical sensitivity testing (PCR and PSR) was done to establish the lowest detectable limit of DNA from pork. This was accomplished using tenfold serial dilutions (with nuclease-free water) of DNA obtained from pork starting from 76 ng to 0.076 fg. The specificity and sensitivity testing were executed thrice to check the repeatability of the detected results.
Detection of raw, heat-treated, and processed pork samples
To check the applicability of the developed technique in the real-world, 30 raw and processed (sausage, meatball, salami, pork curry, and pork soup) pork samples were procured from the retail meat shops and tested. Further, to check the effectiveness of the assay in testing heat-processed or cooked meat samples, raw pork samples were heat treated at 60 °C, 80 °C, 100 °C, and 121 °C for 30 min and genomic DNAs were extracted and PSR assay was deployed in these samples.
Validation of PSR assay in meat admixtures
For testing the detection of pork in binary meat mixtures using PSR assay, various admixture proportions of pork in beef viz ., 20:80, 10:90, 5:95; 1:99, 0.5:99.5 were prepared in 500 mg total quantity. Genomic DNAs were extracted and PSR assay was deployed in these pork–beef mix proportions.
Results and discussion
Optimization of PSR
The PSR assay specifically targeting cyt-b gene of the pig was successfully optimized permitting assay results in 65 min time. The optimized 25 μL PSR reaction mix comprised of “18 µM forward and reverse oligos, 1.4 mM dNTP, 6 mM MgSO 4 , 0.8 M betaine, 12 U of Bst 2.0 warm start DNA polymerase, 3 μL of 10X amplification buffer and 1μL of template”. The standardized incubation temperature was 64 °C (Fig. 1 ). With 2.5% gel electrophoresis, positive (pork) samples showed a ladder-like pattern. The naked eye interpretation was achieved by adding SYBR Green dye which revealed positive (pork) samples as the fluorescent green color and negative (non-pork) samples as orange and below UV light, strong fluorescence was detected. The significant uniqueness of the developed technique is that it necessitates only one enzyme ( Bst polymerase) and one pair of primers contrasting to other isothermal methods (like LAMP, NASBA, etc.) which involve multiple primers and enzymes [ 3 , 32 ]. The primers were designed explicitly to target the cyt-b gene, which is a mitochondrial gene. Many previous studies on pork authentication have recommended cyt-b gene as the target gene [ 33 – 36 ]. The advantages of choosing the mitochondrial gene as the target gene instead of the nuclear gene are that it exists in multiple copies per cell (approx. 1000 copies) and it is relatively tolerant to cooking or processing and other environmental stresses like salt, heat, and pressure [ 7 ]. Furthermore, mitochondrial DNA evolves much quicker than nuclear DNA, and hence encompasses more sequence diversity enabling the identification of phylogenetically related species [ 7 , 10 ]. As far as detection time is concerned, the presently developed PSR technique is much faster compared to end-point PCR and qPCR as it does not involve longer thermal cycling and post-PCR processing [ 28 ]. Fig.1 Optimization of PSR for detection of pork
Specificity and analytical sensitivity
The developed PSR-based detection method to specifically detect the cyt-b gene of pigs exhibited 100% specificity. The characteristic ladder-like patterns (in gel) and fluorescent green color (in the tube) were clearly spotted in samples containing pork meat and products and were absent in other meat (beef, carabeef, chevon, mutton, chicken) (Fig. 2 ). The results advocate that the PSR method has high specificity/ exclusivity for pork and pork products. The analytical sensitivity tested with tenfold serially diluted pork DNA, revealed PSR to be tenfold more sensitive than end-point PCR as the former could detect 760 fg pork DNA per tube, whereas the latter could detect only 7.6 pg pork DNA per tube (Fig. 3 ). The observed sensitivity of 760 fg (0.76 pg) per tube is higher than the formerly reported nucleic acid-based assays for the detection of pork, for example, 1 pg could be detected by a LAMP assay [ 37 ], 100 pg could be detected by an SYBR Green real-time PCR assay [ 38 ], 10 pg could be detected by a PCR assay [ 20 ], 500 pg could be detected by AL-LAMP assay [ 25 ], and 1760 fg could be detected by a real-time LAMP assay [ 3 ]. The repeatability of the developed test was established by repeating the test three times. Fig. 2 Specificity of the PSR assay Fig. 3 Analytical sensitivity A PSR, B end-point PCR
Detection of raw, heat-treated, and processed pork samples
To check the applicability of the developed technique in the real-world, 30 raw and processed (sausage, meatball, salami, pork curry, and pork soup) pork samples and heat-treated (60 °C, 80 °C, 100 °C, and 121 °C for 30 min) samples were tested simultaneously with PSR and PCR methods. The results of the above analysis displayed 100% accuracy as all the raw, processed (Fig. 4 ), and heat-treated pork (Fig. 5 ) samples were detected as positive and non-pork samples were detected as negative. The processed meat generally contains spices, salt, and cooking oil. These ingredients are inhibitors of PCR and for the same reason, immunoassays cannot be used to test adulteration in processed products [ 3 , 39 , 40 ]. Isothermal methods are proved to be less affected by these ingredients and have been successfully deployed in detecting adulteration in processed foods [ 3 , 25 , 41 , 42 ]. Similarly, heat processing or cooking does not affect the efficiency of nucleic acid-based detection assays including isothermal-based techniques, and the heating process also assists in membrane disruption and, thus, has been traditionally used in DNA extraction [ 25 , 43 , 44 ]. Similar to our results, in a previous LAMP-based study, dependable amplification was observed in heat-treated samples heated up to 121 °C for 30 min [ 25 ]. Fig. 4 Detection of processed pork samples by PSR Fig. 5 Detection of heat-treated pork samples by PSR
Validation of PSR assay in meat admixtures
The detection efficiency of PSR in detecting pork in binary meat mixtures was tested using various admixture proportions of pork in beef viz ., 20:80, 10:90, 5:95; 1:99, 0.5:99.5. The nucleic acid was extracted from the pork–beef mixtures and tested using the PSR method. The results revealed that the developed PSR and PCR methods were able to detect up to 0.5 percent of pork admixture in beef (Fig. 6 ). A previously established RPA-LF assay to detect the presence of pig origin tissues has demonstrated detection efficiency of 1% in meat admixtures [ 45 ]. Another LAMP assay was able to detect up to 0.1% pork in beef admixture [ 25 ]. Therefore, the developed PSR-based method can be used for point-of-care detection with minimum instrumentation and technical expertise to guarantee instant clearance of exported and imported meat products. This assay can be deployed to protect consumer rights and prevent unscrupulous competition, and the sale of adulterated and mislabelled products. Fig. 6 Detection of various levels of pork in binary meat mixtures (pork–beef) by A PSR and B end-point PCR
Conclusion
“To the best of our knowledge, this is the first-time PSR has been adopted for food authentication purposes”. The novel PSR-based technique, developed for the detection of pork in adulterated meat, is a promising alternative nucleic acid amplification test (NAAT)-based technique permitting meat authentication in 65 min. The technique possessed 100% specificity and sensitivity (760 fg porcine DNA), able to detect heat-treated (121 °C) and adulterated (0.5% pork in beef) meat and processed pork products such as sausages, pork soup, curry, etc. Further, the results of the current study suggest that it has potential use for the detection of pork adulteration in foods. It can be a simple, robust, rapid, cost-effective, and sophisticated instrument-free method for onsite detection of pork ingredients in other meat foods.
Footnotes
Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Author contributions
AAPM, SWR conceived and designed the experiments. VND and KMM performed the experiments. MK and HVM analyzed the data. The manuscript was written by AAPM and KP, and reviewed by SG. All authors read and approved the final version of the manuscript.
Funding
No funding was received for this study.
Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on request.
Declarations
Conflict of interest
The authors declare that they have no recognized competing financial/personal interests/ relationships that could have seemed to influence the study described in this paper.
Ethical approval
This article contains no studies with human or animal participants conducted by any of the authors.
