Displaying publications 1 - 20 of 36 in total

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  1. Fulltext Evaluation of antioxidant properties and mineral composition of Purslane (Portulaca oleracea L.) at different growth stages
    Uddin MK, Juraimi AS, Ali ME, Ismail MR
    Int J Mol Sci, 2012;13(8):10257-67.
    PMID: 22949859 DOI: 10.3390/ijms130810257
    The main objective of this research was to appraise the changes in mineral content and antioxidant attributes of Portulaca oleracea over different growth stages. The antioxidant activity was measured using 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric-reducing antioxidant power (FRAP) assays. The iodine titration method was used to determine the ascorbic acid content (AAC). DPPH scavenging (IC(50)) capacity ranged from 1.30 ± 0.04 to 1.71 ± 0.04 mg/mL, while the ascorbic acid equivalent antioxidant activity (AEAC) values were 229.5 ± 7.9 to 319.3 ± 8.7 mg AA/100 g, total phenol content (TPC) varied from 174.5 ± 8.5 to 348.5 ± 7.9 mg GAE/100 g. AAC 60.5 ± 2.1 to 86.5 ± 3.9 mg/100 g and FRAP 1.8 ± 0.1 to 4.3 ± 0.1 mg GAE/g. There was good correlation between the results of TPC and AEAC, and between IC(50) and FRAP assays (r(2) > 0.9). The concentrations of Ca, Mg, K, Fe and Zn increased with plant maturity. Calcium (Ca) was negatively correlated with sodium (Na) and chloride (Cl), but positively correlated with magnesium (Mg), potassium (K), iron (Fe) and zinc (Zn). Portulaca olerecea cultivars could be used as a source of minerals and antioxidants, especially for functional food and nutraceutical applications.
  2. A lab-on-a-chip-based multiplex platform to detect potential fraud of introducing pig, dog, cat, rat and monkey meat into the food chain
    Razzak MA, Hamid SB, Ali ME
    Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2015;32(11):1902-13.
    PMID: 26437367 DOI: 10.1080/19440049.2015.1087060
    Food forgery has posed considerable risk to public health, religious rituals, personal budget and wildlife. Pig, dog, cat, rat and monkey meat are restricted in most religions, but their sporadic adulteration are rampant. Market controllers need a low-cost but reliable technique to track and trace suspected species in the food chain. Considering the need, here we documented a lab-on-a-chip-based multiplex polymerase chain reaction (PCR) assay for the authentication of five non-halal meat species in foods. Using species-specific primers, 172, 163, 141, 129 and 108-bp sites of mitochondrial ND5, ATPase 6 and cytochrome b genes were amplified to detect cat, dog, pig, monkey and rat species under complex matrices. Species-specificity was authenticated against 20 different species with the potential to be used in food. The targets were stable under extreme sterilisation (121°C at 45 psi for 2.5 h) which severely degrades DNA. The assay was optimised under the backgrounds of various commercial meat products and validated for the analysis of meatballs, burgers and frankfurters, which are popular fast food items across the globe. The assay was tested to detect 0.1% suspected meats under commercial backgrounds of marketed foods. Instead of simplex PCR which detects only one species at a time, such a multiplex platform can reduce cost by at least fivefolds by detecting five different species in a single assay platform.
  3. Novel multiplex PCR-RFLP assay discriminates bovine, porcine and fish gelatin substitution in Asian pharmaceuticals capsule shells
    Sultana S, Hossain MAM, Naquiah NNA, Ali ME
    Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2018 Sep;35(9):1662-1673.
    PMID: 30028648 DOI: 10.1080/19440049.2018.1500719
    Gelatin is widely used in pharmaceuticals as a protective coating, such as soft and hard capsule shells. However, the animal source of gelatin is a sensitive issue because certain gelatins such as porcine and bovine gelatins are not welcome in Halal, Kosher and Hindus' consumer goods. Recently, we have documented DNA barcoding and multiplex PCR platforms for discriminating porcine, bovine and fish gelatins in various fish and confectionary products; but those assays were not self-authenticating and also not tested in highly refined pharmaceutical products. To address this knowledge gap, here we report a self-authenticating multiplex PCR-restriction fragment length polymorphism (RFLP) assay to identify animal sources of various gelatin in pharmaceutical capsules. Three different restriction enzymes, BsaAI, Hpy188I and BcoDI were used to yield distinctive RFLP patterns for gelatin-based bovine (26, 94 bp), fish (97, 198 bp) and porcine (17, 70 bp) DNA in control experiments. The specificity was cross-tested against 16 non-target species and the optimised assay was used to screen gelatin sources in 30 halal-branded pharmaceuticals capsule shells. Bovine and porcine DNA was found in 27 and 3 of the 30 different capsules products. The assay was suitable for detecting 0.1 to 0.01 ng total DNA extracted from pure and mixed gelatins. The study might be useful to authenticate and monitor halal, kosher, vegetarian and Hindu compliant pharmaceuticals, foods and cosmetics.
  4. Fulltext Statistical Optimization for Acid Hydrolysis of Microcrystalline Cellulose and Its Physiochemical Characterization by Using Metal Ion Catalyst
    Karim MZ, Chowdhury ZZ, Hamid SBA, Ali ME
    Materials (Basel), 2014 Oct 13;7(10):6982-6999.
    PMID: 28788226 DOI: 10.3390/ma7106982
    Hydrolyzing the amorphous region while keeping the crystalline region unaltered is the key technology for producing nanocellulose. This study investigated if the dissolution properties of the amorphous region of microcrystalline cellulose can be enhanced in the presence of Fe(3+) salt in acidic medium. The process parameters, including temperature, time and the concentration of metal chloride catalyst (FeCl₃), were optimized by using the response surface methodology (RSM). The experimental observation demonstrated that temperature and time play vital roles in hydrolyzing the amorphous sections of cellulose. This would yield hydrocellulose with higher crystallinity. The factors that were varied for the production of hydrocellulose were the temperature (x₁), time (x₂) and FeCl₃ catalyst concentration (x₃). Responses were measured in terms of percentage of crystallinity (y₁) and the yield (y₂) of the prepared hydrocellulose. Relevant mathematical models were developed. Analysis of variance (ANOVA) was carried out to obtain the most significant factors influencing the responses of the percentage of crystallinity and yield. Under optimum conditions, the percentage of crystallinity and yield were 83.46% and 86.98% respectively, at 90.95 °C, 6 h, with a catalyst concentration of 1 M. The physiochemical characteristics of the prepared hydrocellulose were determined in terms of XRD, SEM, TGA and FTIR analyses. The addition of FeCl₃ salt in acid hydrolyzing medium is a novel technique for substantially increasing crystallinity with a significant morphological change.
  5. 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.
  6. Fulltext Purslane weed (Portulaca oleracea): a prospective plant source of nutrition, omega-3 fatty acid, and antioxidant attributes
    Uddin MK, Juraimi AS, Hossain MS, Nahar MA, Ali ME, Rahman MM
    ScientificWorldJournal, 2014;2014:951019.
    PMID: 24683365 DOI: 10.1155/2014/951019
    Purslane (Portulaca oleracea L.) is an important plant naturally found as a weed in field crops and lawns. Purslane is widely distributed around the globe and is popular as a potherb in many areas of Europe, Asia, and the Mediterranean region. This plant possesses mucilaginous substances which are of medicinal importance. It is a rich source of potassium (494 mg/100 g) followed by magnesium (68 mg/100 g) and calcium (65 mg/100 g) and possesses the potential to be used as vegetable source of omega-3 fatty acid. It is very good source of alpha-linolenic acid (ALA) and gamma-linolenic acid (LNA, 18 : 3 w3) (4 mg/g fresh weight) of any green leafy vegetable. It contained the highest amount (22.2 mg and 130 mg per 100 g of fresh and dry weight, resp.) of alpha-tocopherol and ascorbic acid (26.6 mg and 506 mg per 100 g of fresh and dry weight, resp.). The oxalate content of purslane leaves was reported as 671-869 mg/100 g fresh weight. The antioxidant content and nutritional value of purslane are important for human consumption. It revealed tremendous nutritional potential and has indicated the potential use of this herb for the future.
  7. Inheritance studies of SSR and ISSR molecular markers and phylogenetic relationship of rice genotypes resistant to tungro virus
    Latif MA, Rahman MM, Ali ME, Ashkani S, Rafii MY
    C. R. Biol., 2013 Mar;336(3):125-33.
    PMID: 23643394 DOI: 10.1016/j.crvi.2012.12.002
    Multivariate analyses were performed using 13 morphological traits and 13 molecular markers (10 SSRs and three ISSRs) to assess the phylogenetic relationship among tungro resistant genotypes. For morphological traits, the genotypes were grouped into six clusters, according to D(2) statistic and Canonical vector analysis. Plant height, days to flowering, days to maturity, panicle length, number of spikelet per panicle, number of unfilled grain per panicle and yield were important contributors to genetic divergence in 14 rice genotypes. Based on Nei's genetic distance for molecular studies, seven clusters were formed among the tungro resistant and susceptible genotypes. Mantel's test revealed a significant correlation (r = 0.834*) between the morphological and molecular data. To develop high yielding tungro resistant varieties based on both morphological and molecular analyses, crosses could be made with susceptible (BR10 and BR11) genotypes with low yielding but highly resistant genotypes, Sonahidemota, Kumragoir, Nakuchimota, Khaiyamota, Khairymota and Kachamota. The chi-square analysis for seven alleles (RM11, RM17, RM20, RM23, RM80, RM108 and RM531) of SSR and five loci (RY1, MR1, MR2, MR4 and GF5) of three ISSR markers in F2 population of cross, BR11×Sonahidemota, showed a good fit to the expected segregation ratio (1:2:1) for a single gene model.
  8. Fulltext Genetic dissection of sympatric populations of brown planthopper, Nilaparvata lugens (Stål), using DALP-PCR molecular markers
    Latif MA, Rafii MY, Mazid MS, Ali ME, Ahmed F, Omar MY, et al.
    ScientificWorldJournal, 2012;2012:586831.
    PMID: 22593700 DOI: 10.1100/2012/586831
    Direct amplified length polymorphism (DALP) combines the advantages of a high-resolution fingerprint method and also characterizing the genetic polymorphisms. This molecular method was also found to be useful in brown planthopper, Nilaparvata lugens species complex for the analysis of genetic polymorphisms. A total of 11 populations of Nilaparvata spp. were collected from 6 locations from Malaysia. Two sympatric populations of brown planthopper, N. lugens, one from rice and the other from a weed grass (Leersia hexandra), were collected from each of five locations. N. bakeri was used as an out group. Three oligonucleotide primer pairs, DALP231/DALPR'5, DALP234/DALPR'5, and DALP235/DALPR'5 were applied in this study. The unweighted pair group method with arithmetic mean (UPGMA) dendrogram based on genetic distances for the 11 populations of Nilaparvata spp. revealed that populations belonging to the same species and the same host type clustered together irrespective of their geographical localities of capture. The populations of N. lugens formed into two distinct clusters, one was insects with high esterase activities usually captured from rice and the other was with low esterase activities usually captured from L. hexandra. N. bakeri, an out group, was the most isolated group. Analyses of principal components, molecular variance, and robustness also supported greatly to the findings of cluster analysis.
  9. Fulltext Development of swine-specific DNA markers for biosensor-based halal authentication
    Ali ME, Hashim U, Kashif M, Mustafa S, Che Man YB, Abd Hamid SB
    Genet. Mol. Res., 2012;11(2):1762-72.
    PMID: 22843053 DOI: 10.4238/2012.June.29.9
    The pig (Sus scrofa) mitochondrial genome was targeted to design short (15-30 nucleotides) DNA markers that would be suitable for biosensor-based hybridization detection of target DNA. Short DNA markers are reported to survive harsh conditions in which longer ones are degraded into smaller fragments. The whole swine mitochondrial-genome was in silico digested with AluI restriction enzyme. Among 66 AluI fragments, five were selected as potential markers because of their convenient lengths, high degree of interspecies polymorphism and intraspecies conservatism. These were confirmed by NCBI blast analysis and ClustalW alignment analysis with 11 different meat-providing animal and fish species. Finally, we integrated a tetramethyl rhodamine-labeled 18-nucleotide AluI fragment into a 3-nm diameter citrate-tannate coated gold nanoparticle to develop a swine-specific hybrid nanobioprobe for the determination of pork adulteration in 2.5-h autoclaved pork-beef binary mixtures. This hybrid probe detected as low as 1% pork in deliberately contaminated autoclaved pork-beef binary mixtures and no cross-species detection was recorded, demonstrating the feasibility of this type of probe for biosensor-based detection of pork adulteration of halal and kosher foods.
  10. Nanoparticle sensor for label free detection of swine DNA in mixed biological samples
    Ali ME, Hashim U, Mustafa S, Man YB, Yusop MH, Bari MF, et al.
    Nanotechnology, 2011 May 13;22(19):195503.
    PMID: 21430321 DOI: 10.1088/0957-4484/22/19/195503
    We used 40 ± 5 nm gold nanoparticles (GNPs) as colorimetric sensor to visually detect swine-specific conserved sequence and nucleotide mismatch in PCR-amplified and non-amplified mitochondrial DNA mixtures to authenticate species. Colloidal GNPs changed color from pinkish-red to gray-purple in 2 mM PBS. Visually observed results were clearly reflected by the dramatic reduction of surface plasmon resonance peak at 530 nm and the appearance of new features in the 620-800 nm regions in their absorption spectra. The particles were stabilized against salt-induced aggregation upon the adsorption of single-stranded DNA. The PCR products, without any additional processing, were hybridized with a 17-base probe prior to exposure to GNPs. At a critical annealing temperature (55 °C) that differentiated matched and mismatched base pairing, the probe was hybridized to pig PCR product and dehybridized from the deer product. The dehybridized probe stuck to GNPs to prevent them from salt-induced aggregation and retained their characteristic red color. Hybridization of a 27-nucleotide probe to swine mitochondrial DNA identified them in pork-venison, pork-shad and venison-shad binary admixtures, eliminating the need of PCR amplification. Thus the assay was applied to authenticate species both in PCR-amplified and non-amplified heterogeneous biological samples. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The LOD (for genomic DNA) of the assay was 6 µg ml(-1) swine DNA in mixed meat samples. We believe the assay can be applied for species assignment in food analysis, mismatch detection in genetic screening and homology studies between closely related species.
  11. Fulltext Food assimilated by two sympatric populations of the brown planthopper Nilaparvata lugens (Delphacidae) feeding on different host plants contaminates insect DNA detected by RAPD-PCR analysis
    Latif MA, Omar MY, Tan SG, Siraj SS, Ali ME, Rafii MY
    Genet. Mol. Res., 2012;11(1):30-41.
    PMID: 22290463 DOI: 10.4238/2012.January.9.4
    Contamination of insect DNA for RAPD-PCR analysis can be a problem because many primers are non-specific and DNA from parasites or gut contents may be simultaneously extracted along with that of the insect. We measured the quantity of food ingested and assimilated by two sympatric populations of brown planthopper (BPH), Nilaparvata lugens, one from rice and the other from Leersia hexandra (Poaceae), a wetland forage grass, and we also investigated whether host plant DNA contaminates that of herbivore insects in extractions of whole insects. Ingestion and assimilation of food were reduced significantly when individuals derived from one host plant were caged on the other species. The bands, OPA3 (1.25), OPD3 (1.10), OPD3 (0.80), OPD3 (0.60), pUC/M13F (0.35), pUC/M13F (0.20), BOXAIR (0.50), peh#3 (0.50), and peh#3 (0.17) were found in both rice-infesting populations of brown planthopper and its host plant (rice). Similarly, the bands, OPA4 (1.00), OPB10 (0.70), OPD3 (0.90), OPD3 (0.80), OPD3 (0.60), pUC/ M13F (0.35), pUC/M13F (0.20), and BOXAIR (0.50) were found in both Leersia-infesting populations of brown planthopper and the host plant. So, it is clear that the DNA bands amplified in the host plants were also found in the extracts from the insects feeding on them.
  12. Fulltext Impact of hydrogen concentrations on the impedance spectroscopic behavior of Pd-sensitized ZnO nanorods
    Kashif M, Ali ME, Ali SM, Hashim U, Hamid SB
    Nanoscale Res Lett, 2013;8(1):68.
    PMID: 23399029 DOI: 10.1186/1556-276X-8-68
    ZnO nanorods were synthesized using a low-cost sol-gel spin coating technique. The synthesized nanorods were consisted of hexagonal phase having c-axis orientation. SEM images reflected perpendicular ZnO nanorods forming bridging network in some areas. The impact of different hydrogen concentrations on the Pd-sensitized ZnO nanorods was investigated using an impedance spectroscopy (IS). The grain boundary resistance (Rgb) significantly contributed to the sensing properties of hydrogen gas. The boundary resistance was decreased from 11.95 to 3.765 kΩ when the hydrogen concentration was increased from 40 to 360 ppm. IS gain curve showed a gain of 6.5 for 360 ppm of hydrogen at room temperature. Nyquist plot showed reduction in real part of impedance at low frequencies on exposure to different concentrations of hydrogen. Circuit equivalency was investigated by placing capacitors and resistors to identify the conduction mechanism according to complex impedance Nyquist plot. Variations in nanorod resistance and capacitance in response to the introduction of various concentrations of hydrogen gas were obtained from the alternating current impedance spectra.
  13. Modification of gelatin-DNA interaction for optimised DNA extraction from gelatin and gelatin capsule
    Mohamad NA, Mustafa S, El Sheikha AF, Khairil Mokhtar NF, Ismail A, Ali ME
    J Sci Food Agric, 2016 May;96(7):2344-51.
    PMID: 26441285 DOI: 10.1002/jsfa.7482
    Poor quality and quantity of DNA extracted from gelatin and gelatin capsules often causes failure in the determination of animal species using PCR. Gelatin, which is mainly derived from porcine and bovine, has been a matter of concern among customers in order to fulfill religious obligation and safety precaution against several transmissible infectious diseases associated with bovine species. Thus, optimised DNA extraction from gelatin is very important for successful real-time PCR detection of gelatin species. In this work, the DNA extraction method was optimised in terms of lysis incubation period and inclusion of pre-treatment pH modification of samples.
  14. 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.
  15. 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.
  16. A suitable method for the detection of a potential fraud of bringing macaque monkey meat into the food chain
    Rashid NR, Ali ME, Hamid SB, Rahman MM, Razzak MA, Asing, et al.
    Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2015;32(7):1013-22.
    PMID: 25906074 DOI: 10.1080/19440049.2015.1039073
    Being the third-largest primate population has not made macaque (Macaca fascicularis sp.) monkeys less exposed to threats and dangers. Despite wildlife protection, they have been widely hunted and consumed in several countries because of their purported nutritional values. In addition to trading as pure bush meats in several places, monkey meat has been sold in meatball and soup products in Indonesia. Thus the possibility of macaque meat trafficking under the label of common meats is quite high. This paper reports the development of a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay with the shortest amplicon length for the confirmed detection of monkey meat under compromised states which are known to degrade DNA. We amplified a 120-bp region of d-loop gene using a pair of macaque-specific primers and confirmed their specificity for the target species through cross-challenging against 17 different species using a 141-bp site of an 18 S rRNA gene as an endogenous control for eukaryotes. This eliminated the possibilities of any false-negative detection with complex matrices or degraded specimens. The detection limit was 0.00001 ng DNA in a pure state and 0.1% of meat in mixed matrices and commercial meatball products. RFLP analysis further authenticated the originality of the PCR product and distinctive restriction patterns were found upon AluI and CViKI-1 digestion. A micro-fluidic lab-on-a-chip automated electrophoretic system separated the fragments with high resolution. The assay was validated for screening commercial meatball products with sufficient internal control.
  17. Fulltext The employment of q-PCR using specific primer targeting on mitochondrial cytochrome-b gene for identification of wild boar meat in meatball samples
    Aina GQ, Erwanto Y, Hossain M, Johan MR, Ali ME, Rohman A
    J Adv Vet Anim Res, 2019 Sep;6(3):300-307.
    PMID: 31583226 DOI: 10.5455/javar.2019.f348
    Objective: The objective of this study was to employ real-time or quantitative polymerase chain reaction (q-PCR) using novel species specific primer (SSP) targeting on mitochondrial cytochrome-b of wild boar species (CYTBWB2-wb) gene for the identification of non-halal meat of wild boar meat (WBM) in meatball products.

    Materials and Methods: The novel SSP of CYTBWB2-wb was designed by our group using PRIMERQUEST and NCBI software. DNA was extracted using propanol-chloroform-isoamyl alcohol method. The designed SSP was further subjected for validation protocols using DNA isolated from fresh meat and from meatball, which include specificity test, determination of efficiency, limit of detection and repeatability, and application of developed method for analysis of commercially meatball samples.

    Results: The results showed that CYTBWB2-wb was specific to wild boar species against other animal species with optimized annealing temperature of 59°C. The efficiency of q-PCR obtained was 91.9% which is acceptable according to the Codex Allimentarius Commission (2010). DNA, with as low as 5 pg/μl, could be detected using q-PCR with primer of CYTBWB2-wb. The developed method was also used for DNA analysis extracted from meatball samples commercially available.

    Conclusion: q-PCR using CYTBWB2-wb primers targeting on mitochondrial cytochrome-b gene (forward: CGG TTC CCT CTT AGG CAT TT; Reverse: GGA TGA ACA GGC AGA TGA AGA) can be fruitfully used for the analysis of WBM in commercial meatball samples.

  18. Analysis of pork adulteration in commercial meatballs targeting porcine-specific mitochondrial cytochrome b gene by TaqMan probe real-time polymerase chain reaction
    Ali ME, Hashim U, Mustafa S, Che Man YB, Dhahi TS, Kashif M, et al.
    Meat Sci, 2012 Aug;91(4):454-9.
    PMID: 22444666 DOI: 10.1016/j.meatsci.2012.02.031
    A test for assessing pork adulteration in meatballs, using TaqMan probe real-time polymerase chain reaction, was developed. The assay combined porcine-specific primers and TaqMan probe for the detection of a 109 bp fragment of porcine cytochrome b gene. Specificity test with 10 ng DNA of eleven different species yielded a threshold cycle (Ct) of 15.5 ± 0.20 for the pork and negative results for the others. Analysis of beef meatballs with spiked pork showed the assay can determine 100-0.01% contaminated pork with 102% PCR efficiency, high linear regression (r(2) = 0.994) and ≤ 6% relative errors. Residuals analysis revealed a high precision in all determinations. Random analysis of commercial meatballs from pork, beef, chicken, mutton and goat, yielded a Ct between 15.89 ± 0.16 and 16.37 ± 0.22 from pork meatballs and negative results from the others, showing the suitability of the assay to determine pork in commercial meatballs with a high accuracy and precision.
  19. Polymerase chain reaction assay targeting cytochrome b gene for the detection of dog meat adulteration in meatball formulation
    Rahman MM, Ali ME, Hamid SB, Mustafa S, Hashim U, Hanapi UK
    Meat Sci, 2014 Aug;97(4):404-9.
    PMID: 24769096 DOI: 10.1016/j.meatsci.2014.03.011
    A polymerase chain reaction (PCR) assay for the assessment of dog meat adulteration in meatballs was developed. The assay selectively amplified a 100-bp region of canine mitochondrial cytochrome b gene from pure, raw, processed and mixed backgrounds. The specificity of the assay was tested against 11 animals and 3 plants species, commonly available for meatball formulation. The stability of the assay was proven under extensively autoclaving conditions that breakdown target DNA. A blind test from ready to eat chicken and beef meatballs showed that the assay can repeatedly detect 0.2% canine meat tissues under complex matrices using 0.04 ng of dog DNA extracted from differentially treated meatballs. The simplicity, stability and sensitivity of the assay suggested that it could be used in halal food industry for the authentication of canine derivatives in processed foods.
  20. Quantitative Tetraplex Real-Time Polymerase Chain Reaction Assay with TaqMan Probes Discriminates Cattle, Buffalo, and Porcine Materials in Food Chain
    Hossain MAM, Ali ME, Sultana S, Asing, Bonny SQ, Kader MA, et al.
    J Agric Food Chem, 2017 May 17;65(19):3975-3985.
    PMID: 28481513 DOI: 10.1021/acs.jafc.7b00730
    Cattle, buffalo, and porcine materials are widely adulterated, and their quantification might safeguard health, religious, economic, and social sanctity. Recently, conventional polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) assays have been documented but they are just suitable for identification, cannot quantify adulterations. We described here a quantitative tetraplex real-time PCR assay with TaqMan Probes to quantify contributions from cattle, buffalo, and porcine materials simultaneously. Amplicon-sizes were very short (106-, 90-, and 146-bp for cattle, buffalo, and porcine) because longer targets could be broken down, bringing serious ambiguity in molecular diagnostics. False negative detection was eliminated through an endogenous control (141-bp site of eukaryotic 18S rRNA). Analysis of 27 frankfurters and 27 meatballs reflected 84-115% target recovery at 0.1-10% adulterations. Finally, a test of 36 commercial products revealed 71% beef frankfurters, 100% meatballs, and 85% burgers contained buffalo adulteration, but no porcine was found in beef products.
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