Identification of short-length oligonucleotides biomarker for canine species detection using mitochondrial cytochrome b gene

Introduction: Stray dogs are still available in certain countries without any offered price and made it as a potential source for adulteration with costly meats for more benefit. Furthermore, human forensic evidences from crime scenes were often integrated with biomaterial of canine origin. Most of the DNA based assay for canine species detection used longer amplicon size (>150 bp) which are not suitable for highly degraded food or forensic sample analysis. Therefore, in this study for development of short length canine specific biomarker, mitochondrial cytochrome b (cytb) gene was targeted using simple PCR assay. Objective: Detection of canine species using short length DNA biomarker targeting cytb gene. Methods: The assay targeted a 100-bp fragment of cytochrome b gene using a pair of canine specific primers. The primers specificity were tested under Insilco, as well as in real PCR assay using dog and eight other species DNAs. The consensus 100 bp canine specific site along with cytb sequences of 14 species including dog and human were used for analysis of pair wise distances, construct dendogram and primers mismatch calculation. The stability of the biomarker was tested under commonly used cooking condition and extensive autoclaving state which was known for degradation of target DNA. The sensitivity of the assay was tested using binary admixture composed of dog and most consumed chicken DNA pool. Results & Discussion: The biomarker was 100% canine specific and successfully amplified 100 bp region of canine cytb gene specific target. It was highly stable and sensitive enough to detect as low as 0.1% (0.02 ng) of canine specific target from admixed DNAs. Conclusion: The primers provided the shortest DNA biomarker for canine species detection. The shortest amplicon length, high stability and sensitivity offered its potentiality for canine biomaterials determination from food as well as from degraded samples

Gold nanoparticle sensor for the visual detection of pork adulteration in meatball formulation.

We visually identify pork adulteration in beef and chicken meatball preparations using 20 nm gold nanoparticles (GNPs) as colorimetric sensors. Meatball is a popular food in certain Asian and European countries. Verification of pork adulteration in meatball is necessary to meet the Halal and Kosher food standards. Twenty nm GNPs change color from pinkish-red to gray-purple, and their absorption peak at 525 nm is red-shifted by 30–50 nm in 3 mM phosphate buffer saline (PBS). Adsorption of single-stranded DNA protects the particles against salt-induced aggregation. Mixing and annealing of a 25-nucleotide (nt) single-stranded (ss) DNA probe with denatured DNA of different meatballs differentiated well between perfectly matched and mismatch hybridization at a critical annealing temperature. The probes become available in nonpork DNA containing vials due to mismatches and interact with GNPs to protect them from salt-induced aggregation. Whereas, all the pork containing vials, either in pure and mixed forms, consumed the probes totally by perfect hybridization and turned into grey, indicating aggregation. This is clearly reflected by a well-defined red-shift of the absorption peak and significantly increased absorbance in 550–800 nm regimes. This label-free low-cost assay should find applications in food analysis, genetic screening, and homology studies

Duplex real-time PCR assay using SYBR Green to detect and quantify Malayan box turtle (Cuora amboinensis) materials in meatballs, burgers, frankfurters and traditional Chinese herbal jelly powder

The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected species widely used in exotic foods and traditional medicines. Currently available polymerase chain reaction (PCR) assays to identify MBT lack automation and involve long targets which break down in processed or denatured tissue. This SYBR Green duplex real-time PCR assay has addressed this research gap for the first time through the combination of 120- and 141-bp targets from MBT and eukaryotes for the quantitative detection of MBT DNA in food chain and herbal medicinal preparations. This authentication ensures better security through automation, internal control and short targets that were stable under the processing treatments of foods and medicines. A melting curve clearly demonstrated two peaks at 74.63 ± 0.22 and 78.40 ± 0.31°C for the MBT and eukaryotic products, respectively, under pure, admixed and commercial food matrices. Analysis of 125 reference samples reflected a target recovery of 93.25–153.00%, PCR efficiency of 99–100% and limit of detection of 0.001% under various matrices. The quantification limits were 0.00001, 0.00170 ± 0.00012, 0.00228 ± 0.00029, 0.00198 ± 0.00036 and 0.00191 ± 0.00043 ng DNA for the pure meat, binary mixtures, meatball, burger and frankfurter products, respectively. The assay was used to screen 100 commercial samples of traditional Chinese herbal jelly powder from eight different brands; 22% of them were found to be MBT-positive (5.37 ± 0.50–7.00 ± 0.34% w/w), which was reflected through the Ct values (26.37 ± 0.32–28.90 ± 0.42) and melting curves (74.63– 78.65 ± 0.22°C) of the amplified MBT target (120 bp), confirming the speculation that MBT materials are widely used in Chinese herbal desserts, exotic dishes consumed with the hope of prolonging life and youth

A Suitable method to detect potential fraud of bringing Malayan box turtle (Cuora amboinensis) meat into the food chain

Malayan box turtle (Cuora amboinensis) has been a wildlife-protected vulnerable turtle species in Malaysia since 2005. However, because of its purported usage in traditional medicine, tonic foods and feeds, clandestine black market trade is rampant. Several polymerase chain reaction (PCR) assays for the taxonomic detection and classification of turtle species have been proposed. These assays are based on long-length target amplicons which are assumed to break down under compromised states and, hence, might not be suitable for the forensic tracing and tracking of turtle trafficking. For the first time this paper develops a very short-amplicon-length PCR assay (120 bp) for the detection of Malayan box turtle meat in raw, processed and mixed matrices, and experimental evidence is produced that such an assay is not only more stable and reliable but also more sensitive than those previously published. We checked the assay specificity against 20 different species and no cross-species detection was observed. The possibility of any false-negative detection was eliminated by a universal endogenous control for eukaryotes. The assay detection limit was 0.0001 ng of box turtle DNA from pure meat and 0.01% turtle meat in binary and ternary admixtures and commercial meatballs. Superior target stability and sensitivity under extreme treatments of boiling, autoclaving and microwave cooking suggested that this newly developed assay would be suitable for any forensic and/or archaeological identification of Malayan box turtle species, even in severely degraded specimens. Further, in silico studies indicated that the assay has the potential to be used as a universal probe for the detection of nine Cuora species, all of which are critically endangered

A novel catalytic method for the synthesis of spherical aragonite nanoparticles from cockle shells

For the first time, we report here a novel top down catalytic approach for the synthesis of aragonite nanoparticles with spherical morphology from cockleshells. Cockle shell is a natural reservoir of aragonite which is a biogenic polymorph of calcium carbonate. Aragonite polymorph is widely used in the repair of fractured bone, development of advanced drug delivery systems, and tissue scaffolds. The method involves an easily performable and low-cost mechanical stirring of the micron-sized cockle shell powders in presence of a nontoxic biomineralization catalyst, dodecyl dimethyl betaine (BS-12). It produces spherical shaped aragonite nanoparticles of 35 ± 5 nm in diameter with a good reproducibility and without any additional impurities at room temperature. The findings were verified with a variable pressure scanning electron microscopy (VPSEM), energy dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM),Fourier transmission infrared spectroscopy (FT-IR), X-ray diffractometer (XRD), and thermogravimetric analyzer (TGA).The reproducibility, low-cost and simplicity of the method suggested its potential applications in large scale synthesis of aragonite nanoparticles with spherical morphology in an industrial set up

Lab-on-a-chip-based PCR-RFLP assay for the detection of Malayan box turtle (Cuora amboinensis)in the food chain and traditional Chinese medicines

The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected turtle species, but it is a lucrative item in the illegal wildlife trade because of its great appeal as an exotic food item and in traditional medicine. Although several polymerase chain reaction (PCR) assays to identify MBT by various routes have been documented, their applicability for forensic authentication remains inconclusive due to the long length of the amplicon targets, which are easily broken down by natural decomposition, environmental stresses or physiochemical treatments during food processing. To address this research gap, we developed, for the first time, a species-specific PCR-restriction fragment length polymorphism (RFLP) assay with a very short target length (120 bp) to detect MBT in the food chain; this authentication ensured better security and reliability through molecular fingerprints. The PCR-amplified product was digested with Bfa1 endonuclease, and distinctive restriction fingerprints (72, 43 and 5 bp) for MBT were found upon separation in a microfluidic chip-based automated electrophoresis system, which enhances the resolution of short oligos. The chances of any false negative identifications were eliminated through the use of a universal endogenous control for eukaryotes, and the limit of detection was 0.0001 ng DNA or 0.01% of the meat under admixed states. Finally, the optimized PCR-RFLP assay was validated for the screening of raw and processed commercial meatballs, burgers and frankfurters, which are very popular in most countries. The optimized PCR-RFLP assay was further used to screen MBT materials in 153 traditional Chinese medicines of 17 different brands and 62 of them were found MBT positive; wherein the ingredients were not declared in product labels. Overall, the novel assay demonstrated sufficient merit for use in any forensic and/or archaeological authentication of MBT, even under a state of decomposition

Effect of salt stress on morpho-physiology, vegetative growth and yield of rice

Selection of salt tolerant rice varieties has a huge impact on global food supply chain. Five Malaysian rice (Oryza sativa L.) varieties, MR33, MR52, MR211, MR219 and MR232 were tested in pot experiment under different salinity levels for their response in term of vegetative growth, physiological activities, development of yield components and grain yield. Rice varieties, BRRI dhan29 and IR20 were used as a salt-sensitive control and Pokkali was used as a salt-tolerant control. Three different salinity levels viz. 4, 8, and 12 dS m-1 were used in a randomized complete block design with four replications under glass house conditions. Two Malaysia varieties, MR211 and MR232 performed better in terms of vegetative growth (plant height, leaf area plant-1, number of tillers plant-1, dry matter accumulation plant-1), photosynthetic rate, transpiration rate, yield components, grain yield and injury symptoms. While, MR33, MR52 and MR219 verities were able to withstand salinity stress over salt-sensitive control, BRRI dhan29 and IR20

Bambangan (Mangifera pajang) kernel fat: a potential new source of cocoa butter alternative

Bambangan (Mangifera pajang) is one of the underutilised tropical fruits found in the Borneo islands (Malaysia, Indonesia and Brunei). The physicochemical and thermal properties of bambangan kernel fat (BKF) were investigated in an effort to identify an innovative fat that could be exploited in confectionery applications. The fatty acids and triglyceride (TG) contents, melting behaviour and solid fat content (SFC) of the BKF were determined by various chromatographic and thermal techniques. BKF had three main TGs, namely 1-palmitoyl-2-oleoyl-3-stearoyl-glycerol (POS) (11.6%), 1,3-distearoyl-2-oleoyl-glycerol (SOS) (28.7%) and 1-stearoyl-2,3-dioleoyl-glycerol (SOO) (11.2%), with SOS being the major component. Stearic, oleic and palmitic acids were the dominant fatty acids with the area percentage of 36.4%, 44.5% and 8.4%, respectively. The melting behaviour indicated a single curve with only one maximum shoulder. With respect to the physicochemical and thermal properties, BKF is ideal for use in formulations (blending components) as an alternative to CB in food products, especially confectionary products

Characterisation of calcium carbonate and its polymorphs from cockle shells (Anadara granosa)

Calcium carbonate and its polymorphs from cockle shells (Anadara granosa) and commercial calcium carbonate were characterised using a variable pressure scanning electron microscopes (VPSEM), a transmission electron microscope (TEM), an energy dispersive X- ray analyser (EDX), X-ray diffraction (XRD) and Fourier transmission infrared spectroscopy (FT-IR). Cubic-like calcite crystals of commercial calcium carbonate and rod-like aragonite crystals of cockle shell powders were observed by both SEM and TEM. The EDX results showed that the cockle shells contained more calcium and carbon than the commercial calcium carbonate, whereas the commercial calcium carbonate contained more oxygen than the cockle shells. FT-IR analyses revealed the presence of carbonate groups in both the cockle shells and the commercial calcium carbonate. FT-IR analyses also showed the presence of aragonite in the cockle shells and calcite in the commercial calcium carbonate. XRD analyses showed that the cockle shells powder contained aragonite, whereas the commercial calcium carbonate contained calcite. The cockle shells powder was formed with good quality calcium carbonate and contained calcium carbonate in the aragonite phase

Inhibitory effect of chocolate components toward lard detection in chocolate using real time PCR

An identification method of lard in chocolates using real-time polymerase chain reaction was developed to address Halal authentication. However, polymerase chain reaction detection of lard in chocolate has been in vain. In order to investigate the inhibitory effect exerted by each of the chocolate components, four basic chocolate components, sugar, milk powder, cocoa butter, and cocoa powder, were adulterated with lard and examined using porcine-specific real-time polymerase chain reaction assay. The results discovered cocoa powder, as the only component that prevents DNA extraction of lard in chocolate. No substantial polymerase chain reaction inhibition was detected, and thus confirms the cocoa powder’s inhibition on DNA extraction of lard from lard-adulterated chocolate. This finding will expedite new research to develop a method to dissociate the lard from the lard-cocoa powder complex, which will have high potential to be applied as a pre-treatment of the chocolate prior to the DNA extraction and polymerase chain reaction