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Molecularly imprinted polymers-based DNA biosensors

Nawaz N, Abu Bakar NK, Muhammad Ekramul Mahmud HN, Jamaludin NS

Anal Biochem, 2021 10 01;630:114328.
PMID: 34363786 DOI: 10.1016/j.ab.2021.114328

In multiple biological processes, molecular recognition performs an integral role in detecting bio analytes. Molecular imprinted polymers (MIPs) are tailored sensing materials that can biomimic the biologic ligands and can detect specific target molecules selectively and sensitively. The formulation of molecularly imprinted polymers is followed by the formulation of a control termed as non-imprinted polymer (NIP), which, in the absence of a template, is commonly formulated to evaluate whether distinctive imprints have been produced for the template. Given the difficulties confronting bioanalytical researchers, it is inevitable that this strategy would come out as a central route of multidisciplinary studies to create extremely promising stable artificial receptors as a replacement or accelerate biological matrices. The ease of synthesis, low cost, capability to 'tailor' recognition element for analyte molecules, and stability under harsh environments make MIPs promising candidates as a recognition tool for biosensing. Compared to biological systems, molecular imprinting techniques have several advantages, including high recognition ability, long-term durability, low cost, and robustness, allowing molecularly imprinted polymers to be employed in drug delivery, biosensor technology, and nanotechnology. Molecular imprinted polymer-based sensors still have certain shortcomings in determining biomacromolecules (nucleic acid, protein, lipids, and carbohydrates), considering the vast volume of the latest literature on biomicromolecules. These potential materials are still required to address a few weaknesses until gaining their position in recognition of biomacromolecules. This review aims to highlight the current progress in molecularly imprinted polymers (MIPs)-based sensors for the determination of deoxyribonucleic acid (DNA) or nucleobases.

Matched MeSH terms: DNA/analysis*
IQPA: isothermal nucleic acid amplification-based immunoassay using DNAzyme as the reporter system

Loh Q, Omar N, Glökler J, Lim TS

Anal Biochem, 2014 Oct 15;463:67-9.
PMID: 24972268 DOI: 10.1016/j.ab.2014.06.012

Immunoassays are often coupled to peroxidase activity for antigen detection. Sensitivity and speed of detection has been increased by the advent of hybrid methods such as immuno-PCR (polymerase chain reaction). However, a more simplified immunoassay that retains both colorimetric peroxidase detection and effective DNA amplification in a setting closer to field application conditions has been nonexistent. Here we describe a method that successfully combines a competitive immunoassay with the new isothermal quadruplex-primed amplification (QPA) to generate excess quadruplex reporter molecules with intrinsic peroxidase DNAzyme activity.

Matched MeSH terms: DNA/analysis*
Integrating amperometric detection with electrophoresis microchip devices for biochemical assays: recent developments

Ghanim MH, Abdullah MZ

Talanta, 2011 Jul 15;85(1):28-34.
PMID: 21645665 DOI: 10.1016/j.talanta.2011.04.069

Recent advances in microfluidic systems, particularly in the Micro Total Analysis System (μTAS) or Lab On a Chip (LOC), drive the current analysis tools and equipment towards miniaturization, rapid at-line testing and mobility. The state-of-the-art microfluidic technology targets a wider range but smaller volumes of analytes, making the analytical procedure relatively easier and faster. This trend together with faster electronics and modern instrumentation systems will make real-time and in situ analysis a definite possibility. This review focuses on microchip capillary electrophoresis with amperometric detection (MCE-AD) for the detection of DNA and other electroactive analytes. The problems associated with the microchip design, in particular the choice of materials and the configuration of electrodes are discussed thoroughly and solutions are proposed. Significant developments in the related areas are also covered and reviewed critically.

Matched MeSH terms: DNA/analysis
Fulltext First trimester prenatal diagnosis of beta-thalassaemia following chorionic villus sampling

Chandran R, Ainoon O, Anson I, Anne J, Cheong SK

Med J Malaysia, 1993 Sep;48(3):341-4.
PMID: 8183149

DNA analysis for the diagnosis of beta-thalassaemia is a relatively new technique in Malaysia. This, combined with chorionic villus sampling, has enabled us to offer prenatal diagnosis in the first trimester for this common condition. To the best of our knowledge, this has not hitherto been reported in Malaysia.

Matched MeSH terms: DNA/analysis
A comparative study of two methods for the isolation of human leucocytes for DNA extraction

Lim LH, Ton SH, Cheong SK

Malays J Pathol, 1990 Jun;12(1):39-41.
PMID: 2090888

The 'Dextran' and the 'Buffy-coat' methods for isolation of human leucocytes for DNA extraction were compared on the basis of DNA yield from the same amounts (10 ml) of blood. Human leucocytes from a total of 11 samples were isolated using both methods for each sample after which DNA was extracted. Extracted DNA samples were treated with ribonucleases and proteinase K after which the yields were quantitated by measuring absorbance at 260 nm. The 'Buffy-coat' method yielded a mean concentration of DNA of 476.7 micrograms/ml (range: 212 to 700 micrograms/ml) while the 'Dextran' method yielded 188.4 micrograms/ml (range: 64 to 340 micrograms/ml). The difference was confirmed by subjecting the extracted DNA samples to agarose gel electrophoresis.

Matched MeSH terms: DNA/analysis*
Ant-termite interactions: an important but under-explored ecological linkage

Tuma J, Eggleton P, Fayle TM

Biol Rev Camb Philos Soc, 2020 06;95(3):555-572.
PMID: 31876057 DOI: 10.1111/brv.12577

Animal interactions play an important role in understanding ecological processes. The nature and intensity of these interactions can shape the impacts of organisms on their environment. Because ants and termites, with their high biomass and range of ecological functions, have considerable effects on their environment, the interaction between them is important for ecosystem processes. Although the manner in which ants and termites interact is becoming increasingly well studied, there has been no synthesis to date of the available literature. Here we review and synthesise all existing literature on ant-termite interactions. We infer that ant predation on termites is the most important, most widespread, and most studied type of interaction. Predatory ant species can regulate termite populations and subsequently slow down the decomposition of wood, litter and soil organic matter. As a consequence they also affect plant growth and distribution, nutrient cycling and nutrient availability. Although some ant species are specialised termite predators, there is probably a high level of opportunistic predation by generalist ant species, and hence their impact on ecosystem processes that termites are known to provide varies at the species level. The most fruitful future research direction will be to evaluate the impact of ant-termite predation on broader ecosystem processes. To do this it will be necessary to quantify the efficacy both of particular ant species and of ant communities as a whole in regulating termite populations in different biomes. We envisage that this work will require a combination of methods, including DNA barcoding of ant gut contents along with field observations and exclusion experiments. Such a combined approach is necessary for assessing how this interaction influences entire ecosystems.

Matched MeSH terms: DNA/analysis
Synthesis, Characterization and DNA-Binding Studies of Hydroxyl Functionalized Platinum(II) Salphen Complexes

Mohd Sukri SA, Heng LY, Abd Karim NH

J Fluoresc, 2017 May;27(3):1009-1023.
PMID: 28224358 DOI: 10.1007/s10895-017-2035-0

The platinum(II) salphen complex N,N'-Bis-4-(hydroxysalicylidene)-phenylenediamine-platinum(II); (1) and its two derivatives containing hydroxyl functionalized side chains N,N'-bis-[4-[[1-(2-hydroxyethoxy)] salicylidene] phenylenediamine-platinum(II); (2) and N,N'-bis-[4-[[1-(3-hydroxypropoxy)] salicylidene] phenylenediamine-platinum(II); (3) were synthesized and characterized. The structures of the complexes were confirmed by 1H and 13C NMR spectroscopy, FTIR, ESI-MS and CHN elemental analyses. The effects of the hydroxyl substituent on the spectral properties and the DNA binding behaviors of the Pt(II) complexes were explored. The binding mode and interactions of these complexes with duplex DNA (calf thymus DNA and porcine DNA) and also single-stranded DNA were studied by UV-Vis and emission DNA titration. The complexes interact with DNA by intercalation binding mode with the binding constants in the order of magnitude (Kb = 104 M-1, CT-DNA) and (Kb = 105 M-1, porcine DNA). The intercalation of the complex in the DNA structure was proposed to happen by π-π stacking due to its square-planar geometry and aromatic rings structure. The phosphorescence emission spectral characteristics of Pt(II) complexes when interacted with DNA have been studied. Also, the application of the chosen hydroxypropoxy side chains complex (3) as an optical DNA biosensor, specifically for porcine DNA was investigated. These findings will be valuable for the potential use of the platinum(II) salphen complex as an optical DNA biosensor for the detection of porcine DNA in food products.

Matched MeSH terms: DNA/analysis*
Fulltext DNA barcoding for the identification and authentication of medicinal deer (Cervus sp.) products in China

Li W, Ren Q, Feng J, Lee SY, Liu Y

PLoS One, 2024;19(1):e0297164.
PMID: 38241246 DOI: 10.1371/journal.pone.0297164

Deer products from sika deer (Cervus nippon) and red deer (C. elaphus) are considered genuine and used for Traditional Chinese Medicine (TCM) materials in China. Deer has a very high economic and ornamental value, resulting in the formation of a characteristic deer industry in the prescription preparation of traditional Chinese medicine, health food, cosmetics, and other areas of development and utilization. Due to the high demand for deer products, the products are expensive and have limited production, but the legal use of deer is limited to only two species of sika deer and red deer; other wild deer are prohibited from hunting, so there are numerous cases of mixing and adulteration of counterfeit products and so on. There have been many reports that other animal (pig, cow, sheep, etc.) tissues or organs are often used for adulteration and confusion, resulting in poor efficacy of deer traditional medicine and trade fraud in deer products. To authenticate the deer products in a rapid and effective manner, the analysis used 22 deer products (antler, meat, bone, fetus, penis, tail, skin, and wool) that were in the form of blind samples. Total DNA extraction using a modified protocol successfully yielded DNA from the blind samples that was useful for PCR. Three candidate DNA barcoding loci, cox1, Cyt b, and rrn12, were evaluated for their discrimination strength through BLAST and phylogenetic clustering analyses. For the BLAST analysis, the 22 blind samples obtained 100% match identity across the three gene loci tested. It was revealed that 12 blind samples were correctly labeled for their species of origin, while three blind samples that were thought to originate from red deer were identified as C. nippon, and seven blind samples that were thought to originate from sika deer were identified as C. elaphus, Dama dama, and Rangifer tarandus. DNA barcoding analysis showed that all three gene loci were able to distinguish the two Cervus species and to identify the presence of adulterant species. The DNA barcoding technique was able to provide a useful and sensitive approach in identifying the species of origin in deer products.

Matched MeSH terms: DNA/analysis
Fulltext Hexaferrocenium tri[hexa(isothiocyanato)iron(III)] trihydroxonium complex as a new DNA intercalator for electrochemical DNA biosensor

Ariffin EY, Zakariah EI, Ruslin F, Kassim M, Yamin BM, Heng LY, et al.

Sci Rep, 2021 Apr 12;11(1):7883.
PMID: 33846405 DOI: 10.1038/s41598-021-86939-z

Ferrocene or ferrocenium has been widely studied in the field of organometallic complexes because of its stable thermodynamic, kinetic and redox properties. Novel hexaferrocenium tri[hexa(isothiocyanato)iron(III)]trihydroxonium (HexaFc) complex was the product from the reaction of ferrocene, maleic acid and ammonium thiocyanate and was confirmed by elemental analysis CHNS, FTIR and single crystal X-ray crystallography. In this study, HexaFc was used for the first time as an electroactive indicator for porcine DNA biosensor. The UV-Vis DNA titrations with this compound showed hypochromism and redshift at 250 nm with increasing DNA concentrations. The binding constant (Kb) for HexaFc complex towards CT-DNA (calf-thymus DNA) was 3.1 × 104 M-1, indicated intercalator behaviour of the complex. To test the usefulness of this complex for DNA biosensor application, a porcine DNA biosensor was constructed. The recognition probes were covalently immobilised onto silica nanospheres (SiNSs) via glutaraldehyde linker on a screen-printed electrode (SPE). After intercalation with the HexaFc complex, the response of the biosensor to the complementary porcine DNA was measured using differential pulse voltammetry. The DNA biosensor demonstrated a linear response range to the complementary porcine DNA from 1 × 10-6 to 1 × 10-3 µM (R2 = 0.9642) with a limit detection of 4.83 × 10-8 µM and the response was stable up to 23 days of storage at 4 °C with 86% of its initial response. The results indicated that HexaFc complex is a feasible indicator for the DNA hybridisation without the use of a chemical label for the detection of porcine DNA.

Matched MeSH terms: DNA/analysis*
Fulltext Experimental and theoretical investigation of sensing parameters in carbon nanotube-based DNA sensor

Nouri M, Meshginqalam B, Sahihazar MM, Sheydaie Pour Dizaji R, Ahmadi MT, Ismail R

IET Nanobiotechnol, 2018 Dec;12(8):1125-1129.
PMID: 30964025 DOI: 10.1049/iet-nbt.2018.5068

Nowadays, sensitive biosensors with high selectivity, lower costs and short response time are required for detection of DNA. The most preferred materials in DNA sensor designing are nanomaterials such as carbon and Au nanoparticles, because of their very high surface area and biocompatibility which lead to performance and sensitivity improvements in DNA sensors. Carbon nanomaterials such as carbon nanotubes (CNTs) can be considered as a suitable DNA sensor platform due to their high surface-to-volume ratio, favourable electronic properties and fast electron transfer rate. Therefore, in this study, the CNTs which are synthesised by pulsed AC arc discharge method on a high-density polyethylene substrate are used as conducting channels in a chemiresistor for the electrochemical detection of double stranded DNA. Moreover, the response of the proposed sensor is investigated experimentally and analytically in different temperatures, which confirm good agreement between the presented model and experimental data.

Matched MeSH terms: DNA/analysis*
Multiplex PCR assay for the detection of five meat species forbidden in Islamic foods

Ali ME, Razzak MA, Hamid SB, Rahman MM, Amin MA, Rashid NR, et al.

Food Chem, 2015 Jun 15;177:214-24.
PMID: 25660879 DOI: 10.1016/j.foodchem.2014.12.098

Food falsification has direct impact on public health, religious faith, fair-trades and wildlife. For the first time, here we described a multiplex polymerase chain reaction assay for the accurate identification of five meat species forbidden in Islamic foods in a single assay platform. Five pairs of species-specific primers were designed targeting mitochondrial ND5, ATPase 6, and cytochrome b genes to amplify 172, 163, 141, 129 and 108 bp DNA fragments from cat, dog, pig, monkey and rat meats, respectively. All PCR products were identified in gel-images and electrochromatograms obtained from Experion Bioanalyzer. Species-specificity checking against 15 important meat and fish and 5 plant species detected no cross-species amplification. Screening of target species in model and commercial meatballs reflected its application to detect target species in process foods. The assay was tested to detect 0.01-0.02 ng DNA under raw states and 1% suspected meats in meatball formulation.

Matched MeSH terms: DNA/analysis*
Enhancing the efficiency of polymerase chain reaction using graphene nanoflakes

Abdul Khaliq R, Kafafy R, Salleh HM, Faris WF

Nanotechnology, 2012 Nov 16;23(45):455106.
PMID: 23085573 DOI: 10.1088/0957-4484/23/45/455106

The effect of the recently developed graphene nanoflakes (GNFs) on the polymerase chain reaction (PCR) has been investigated in this paper. The rationale behind the use of GNFs is their unique physical and thermal properties. Experiments show that GNFs can enhance the thermal conductivity of base fluids and results also revealed that GNFs are a potential enhancer of PCR efficiency; moreover, the PCR enhancements are strongly dependent on GNF concentration. It was found that GNFs yield DNA product equivalent to positive control with up to 65% reduction in the PCR cycles. It was also observed that the PCR yield is dependent on the GNF size, wherein the surface area increases and augments thermal conductivity. Computational fluid dynamics (CFD) simulations were performed to analyze the heat transfer through the PCR tube model in the presence and absence of GNFs. The results suggest that the superior thermal conductivity effect of GNFs may be the main cause of the PCR enhancement.

Matched MeSH terms: DNA/analysis
Fulltext An electrochemical DNA microbiosensor based on succinimide-modified acrylic microspheres

Ulianas A, Heng LY, Abu Hanifah S, Ling TL

Sensors (Basel), 2012;12(5):5445-60.
PMID: 22778594 DOI: 10.3390/s120505445

An electrochemical microbiosensor for DNA has been fabricated based on new acrylic microspheres modified with reactive N-acryloxysuccinimide (NAS) functional groups. Hydrophobic poly(n-butylacrylate-N-acryloxysuccinimide) microspheres were synthesized in an emulsion form with a simple one-step photopolymerization technique. Aminated DNA probe was attached to the succinimde functional group of the acrylic microspheres via covalent bonding. The hybridization of the immobilized DNA probe with the complementary DNA was studied by differential pulse voltametry using anthraquninone-2-sulfonic acid monohydrate sodium salt (AQMS) as the electroactive hybridization label. The influences of many factors such as duration of DNA probe immobilization and hybridization, pH, type of ions, buffer concentrations, ionic strength, operational temperature and non-complementary DNA on the biosensor performance were evaluated. Under optimized conditions, the DNA microbiosensor demonstrated a linear response range to target DNA over a wide concentration range of 1.0 × 10(-16) and 1.0 × 10(-8) M with a lower limit of detection (LOD) of 9.46 × 10(-17) M (R(2) = 0.97). This DNA microbiosensor showed good reproducibility with 2.84% RSD (relative standard deviation) (n = 3). Application of the NAS-modified acrylic microspheres in the construction of DNA microbiosensor had improved the overall analytical performance of the resultant DNA microbiosensor when compared with other reported DNA biosensors using other nano-materials for membranes and microspheres as DNA immobilization matrices.

Matched MeSH terms: DNA/analysis*
Quantity and quality assessment of DNA extracted from saliva and blood

Looi ML, Zakaria H, Osman J, Jamal R

Clin. Lab., 2012;58(3-4):307-12.
PMID: 22582505

Saliva has been suggested as an attractive resource for evaluating physiological and pathological conditions in humans. This study aims to evaluate saliva sampling as an alternative to blood sampling for molecular testing.

Matched MeSH terms: DNA/analysis*
Hybridization-ligation versus parallel overlap assembly: an experimental comparison of initial pool generation for direct-proportional length-based DNA computing

Ibrahim Z, Tsuboi Y, Ono O

IEEE Trans Nanobioscience, 2006 Jun;5(2):103-9.
PMID: 16805106

Previously, direct-proportional length-based DNA computing (DPLB-DNAC) for solving weighted graph problems has been reported. The proposed DPLB-DNAC has been successfully applied to solve the shortest path problem, which is an instance of weighted graph problems. The design and development of DPLB-DNAC is important in order to extend the capability of DNA computing for solving numerical optimization problem. According to DPLB-DNAC, after the initial pool generation, the initial solution is subjected to amplification by polymerase chain reaction and, finally, the output of the computation is visualized by gel electrophoresis. In this paper, however, we give more attention to the initial pool generation of DPLB-DNAC. For this purpose, two kinds of initial pool generation methods, which are generally used for solving weighted graph problems, are evaluated. Those methods are hybridization-ligation and parallel overlap assembly (POA). It is found that for DPLB-DNAC, POA is better than that of the hybridization-ligation method, in terms of population size, generation time, material usage, and efficiency, as supported by the results of actual experiments.

Matched MeSH terms: DNA/analysis*
TaqMan probe real-time polymerase chain reaction assay for the quantification of canine DNA in chicken nugget

Rahman MM, Hamid SB, Basirun WJ, Bhassu S, Rashid NR, Mustafa S, et al.

Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2016;33(1):10-8.
PMID: 26458055 DOI: 10.1080/19440049.2015.1104558

This paper describes a short-amplicon-based TaqMan probe quantitative real-time PCR (qPCR) assay for the quantitative detection of canine meat in chicken nuggets, which are very popular across the world, including Malaysia. The assay targeted a 100-bp fragment of canine cytb gene using a canine-specific primer and TaqMan probe. Specificity against 10 different animals and plants species demonstrated threshold cycles (Ct) of 16.13 ± 0.12 to 16.25 ± 0.23 for canine DNA and negative results for the others in a 40-cycle reaction. The assay was tested for the quantification of up to 0.01% canine meat in deliberately spiked chicken nuggets with 99.7% PCR efficiency and 0.995 correlation coefficient. The analysis of the actual and qPCR predicted values showed a high recovery rate (from 87% ± 28% to 112% ± 19%) with a linear regression close to unity (R(2) = 0.999). Finally, samples of three halal-branded commercial chicken nuggets collected from different Malaysian outlets were screened for canine meat, but no contamination was demonstrated.

Matched MeSH terms: DNA/analysis*
Fulltext Non-invasive prenatal diagnosis using fetal DNA in maternal plasma: a preliminary study for identification of paternally-inherited alleles using single nucleotide polymorphisms

Chen JJ, Tan JA, Chua KH, Tan PC, George E

BMJ Open, 2015 Jul 22;5(7):e007648.
PMID: 26201722 DOI: 10.1136/bmjopen-2015-007648

OBJECTIVES: Single nucleotide polymorphism (SNP) with a mutation can be used to identify the presence of the paternally-inherited wild-type or mutant allele as result of the inheritance of either allele in the fetus and allows the prediction of the fetal genotype. This study aims to identify paternal SNPs located at the flanking regions upstream or downstream from the β-globin gene mutations at CD41/42 (HBB:c.127_130delCTTT), IVS1-5 (HBB:c.92+5G>C) and IVS2-654 (HBB:c.316-197C>T) using free-circulating fetal DNA.
SETTING: Haematology Lab, Department of Biomedical Science, University of Malaya.
PARTICIPANTS: Eight couples characterised as β-thalassaemia carriers where both partners posed the same β-globin gene mutations at CD41/42, IVS1-5 and IVS2-654, were recruited in this study.
OUTCOME MEASURES: Genotyping was performed by allele specific-PCR and the locations of SNPs were identified after sequencing alignment.
RESULTS: Genotype analysis revealed that at least one paternal SNP was present for each of the couples. Amplification on free-circulating DNA revealed that the paternal mutant allele of SNP was present in three fcDNA. Thus, the fetuses may be β-thalassaemia carriers or β-thalassaemia major. Paternal wild-type alleles of SNP were present in the remaining five fcDNA samples, thus indicating that the fetal genotypes would not be homozygous mutants.
CONCLUSIONS: This preliminary research demonstrates that paternal allele of SNP can be used as a non-invasive prenatal diagnosis approach for at-risk couples to determine the β-thalassaemia status of the fetus.

Matched MeSH terms: DNA/analysis*
A suitable method to detect potential fraud of bringing Malayan box turtle (Cuora amboinensis) meat into the food chain

Ali ME, Asing, Hamid SB, Razzak MA, Rashid NR, Al Amin M, et al.

Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2015;32(8):1223-33.
PMID: 26062948 DOI: 10.1080/19440049.2015.1058535

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.

Matched MeSH terms: DNA/analysis
Lab-on-a-chip-based PCR-RFLP assay for the confirmed detection of short-length feline DNA in food

Ali ME, Al Amin M, Hamid SB, Hossain MA, Mustafa S

Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2015;32(9):1373-83.
PMID: 26208950 DOI: 10.1080/19440049.2015.1075068

Wider availability but lack of legal market trades has given feline meat a high potential for use as an adulterant in common meat and meat products. However, mixing of feline meat or its derivatives in food is a sensitive issue, since it is a taboo in most countries and prohibited in certain religions such as Islam and Judaism. Cat meat also has potential for contamination with of severe acute respiratory syndrome, anthrax and hepatitis, and its consumption might lead to an allergic reaction. We developed a very short-amplicon-length (69 bp) PCR assay, authenticated the amplified PCR products by AluI-restriction digestion followed by its separation and detection on a lab-on-a-chip-based automated electrophoretic system, and proved its superiority over the existing long-amplicon-based assays. Although it has been assumed that longer DNA targets are susceptible to breakdown under compromised states, scientific evidence for this hypothesis has been rarely documented. Strong evidence showed that shorter targets are more stable than the longer ones. We confirmed feline-specificity by cross-challenging the primers against 10 different species of terrestrial, aquatic and plant origins in the presence of a 141-bp site of an 18S rRNA gene as a universal eukaryotic control. RFLP analysis separated 43- and 26-bp fragments of AluI-digest in both the gel-image and electropherograms, confirming the original products. The tested detection limit was 0.01% (w/w) feline meat in binary and ternary admixed as well as meatball matrices. Shorter target, better stability and higher sensitivity mean such an assay would be valid for feline identification even in degraded specimens.

Matched MeSH terms: DNA/analysis*
Fulltext Colorimetric biosensing of targeted gene sequence using dual nanoparticle platforms

Thavanathan J, Huang NM, Thong KL

Int J Nanomedicine, 2015;10:2711-22.
PMID: 25897217 DOI: 10.2147/IJN.S74753

We have developed a colorimetric biosensor using a dual platform of gold nanoparticles and graphene oxide sheets for the detection of Salmonella enterica. The presence of the invA gene in S. enterica causes a change in color of the biosensor from its original pinkish-red to a light purplish solution. This occurs through the aggregation of the primary gold nanoparticles-conjugated DNA probe onto the surface of the secondary graphene oxide-conjugated DNA probe through DNA hybridization with the targeted DNA sequence. Spectrophotometry analysis showed a shift in wavelength from 525 nm to 600 nm with 1 μM of DNA target. Specificity testing revealed that the biosensor was able to detect various serovars of the S. enterica while no color change was observed with the other bacterial species. Sensitivity testing revealed the limit of detection was at 1 nM of DNA target. This proves the effectiveness of the biosensor in the detection of S. enterica through DNA hybridization.

Matched MeSH terms: DNA/analysis

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