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Apolipoprotein E genotyping in the Malay, Chinese and Indian ethnic groups in Malaysia - a study on the distribution of the different apoE alleles and genotypes

Seet WT, Mary Anne TJ, Yen TS

Clin Chim Acta, 2004 Feb;340(1-2):201-5.
PMID: 14734213 DOI: 10.1016/j.cccn.2003.11.001

BACKGROUND: Apolipoprotein E (apoE) is encoded by a polymorphic gene located on chromosome 19. The three common apoE alleles are epsilon2, epsilon3 and epsilon4. We studied the frequencies of the apoE alleles and genotypes in the three ethnic groups-Malay, Chinese and Indian-in Malaysia using DNA amplification followed by agarose gel electrophoresis.
METHODS: EDTA blood was collected and DNA was extracted using proteinase K-SDS digestion and purified by phenol-chloroform extraction. The apoE gene sequence was amplified using the PCR and apoE genotyping was performed by restriction enzyme digestion with HhaI.
RESULTS: Genotyping of the apoE gene produces six genotypes-E2/E2, E2/E3, E3/E3, E2/E4, E3/E4 and E4/E4. The most common apoE genotype in the Malays, Chinese and Indians studied was E3/E3, thus the most common apoE allele was epsilon3. The three common apoE genotypes were E3/E3 followed by E3/E4 and E2/E3, except in the Indians where E2/E3 was not detected. The three apoE alleles were confirmed in the Malays, Chinese and Indians except for the epsilon2 allele which was absent in the Indians.
CONCLUSION: The combined frequency of the apoE alleles in the Malays, Chinese and Indians was 0.058, 0.829 and 0.114 for epsilon2, epsilon3 and epsilon4, respectively.

Matched MeSH terms: DNA/genetics
Non-Invasive DNA Sampling for Molecular Analysis of Beta-Thalassemia: Amiable Alternative Sampling Methods with Accurate Results for Pediatric Patients

Abd Rahim MR, Kho SL, Kuppusamy UR, Tan JA

Clin. Lab., 2015;61(9):1325-30.
PMID: 26554253

BACKGROUND: Beta-thalassemia is the most common genetic disorder in Malaysia. Confirmation of the β-globin gene mutations involved in thalassemia is usually carried out by molecular analysis of DNA extracted from leukocytes in whole blood. Molecular analysis is generally carried out when affected children are around 1 - 2 years as clinical symptoms are expressed during this period. Blood taking at this age can be distressing for the child. High yield and pure DNA extracted from non-invasive sampling methods can serve as alternative samples in molecular studies for genetic diseases especially in pediatric cases.
METHODS: In this study, mouthwash, saliva, and buccal cytobrush samples were collected from β-thalassemia major patients who had previously been characterized using DNA extracted from peripheral blood. DNA was extracted from mouthwash, saliva, and buccal cytobrush samples using the conventional inexpensive phenol-chloroform method and was measured by spectrophotometry for yield and purity. Molecular characterization of β-globin gene mutations was carried out using the amplification refractory mutation system (ARMS).
RESULTS: DNA extracted from mouthwash, saliva, and buccal cytobrush samples produced high concentration and pure DNA. The purified DNA was successfully amplified using ARMS. Results of the β-globin gene mutations using DNA from the three non-invasive samples were in 100% concordance with results from DNA extracted from peripheral blood.
CONCLUSIONS: The conventional in-house developed methods for non-invasive sample collection and DNA extraction from these samples are effective and negate the use of more expensive commercial kits. In conclusion, DNA extracted from mouthwash, saliva, and buccal cytobrush samples provided sufficiently high amounts of pure DNA suitable for molecular analysis of β-thalassemia.

Matched MeSH terms: DNA/genetics
Development of a DNA vaccine against chicken anemia virus by using a bicistronic vector expressing VP1 and VP2 proteins of CAV

Moeini H, Omar AR, Rahim RA, Yusoff K

Comp Immunol Microbiol Infect Dis, 2011 May;34(3):227-36.
PMID: 21146874 DOI: 10.1016/j.cimid.2010.11.006

In the present study, we describe the development of a DNA vaccine against chicken anemia virus. The VP1 and VP2 genes of CAV were amplified and cloned into pBudCE4.1 to construct two DNA vaccines, namely, pBudVP1 and pBudVP2-VP1. In vitro and in vivo studies showed that co-expression of VP1 with VP2 are required to induce significant levels of antibody against CAV. Subsequently, the vaccines were tested in 2-week-old SPF chickens. Chickens immunized with the DNA-plasmid pBudVP2-VP1 showed positive neutralizing antibody titer against CAV. Furthermore, VP1-specific proliferation induction of splenocytes and also high serum levels of Th1 cytokines, IL-2 and IFN-γ were detected in the pBudVP2-VP1-vaccinated chickens. These results suggest that the recombinant DNA plasmid co-expressing VP1 and VP2 can be used as a potential DNA vaccine against CAV.

Matched MeSH terms: Vaccines, DNA/genetics
On fuzzy semantic similarity measure for DNA coding

Ahmad M, Jung LT, Bhuiyan MA

Comput Biol Med, 2016 Feb 1;69:144-51.
PMID: 26773936 DOI: 10.1016/j.compbiomed.2015.12.017

A coding measure scheme numerically translates the DNA sequence to a time domain signal for protein coding regions identification. A number of coding measure schemes based on numerology, geometry, fixed mapping, statistical characteristics and chemical attributes of nucleotides have been proposed in recent decades. Such coding measure schemes lack the biologically meaningful aspects of nucleotide data and hence do not significantly discriminate coding regions from non-coding regions. This paper presents a novel fuzzy semantic similarity measure (FSSM) coding scheme centering on FSSM codons׳ clustering and genetic code context of nucleotides. Certain natural characteristics of nucleotides i.e. appearance as a unique combination of triplets, preserving special structure and occurrence, and ability to own and share density distributions in codons have been exploited in FSSM. The nucleotides׳ fuzzy behaviors, semantic similarities and defuzzification based on the center of gravity of nucleotides revealed a strong correlation between nucleotides in codons. The proposed FSSM coding scheme attains a significant enhancement in coding regions identification i.e. 36-133% as compared to other existing coding measure schemes tested over more than 250 benchmarked and randomly taken DNA datasets of different organisms.

Matched MeSH terms: DNA/genetics*
Fulltext Prevalence of the CYP2D6*10 (C100T), *4 (G1846A), and *14 (G1758A) alleles among Iranians of different ethnicities

Bagheri A, Kamalidehghan B, Haghshenas M, Azadfar P, Akbari L, Sangtarash MH, et al.

Drug Des Devel Ther, 2015;9:2627-34.
PMID: 25999696 DOI: 10.2147/DDDT.S79709

The presence of polymorphisms in the CYP2D6 gene may modulate enzyme level and activity, thereby affecting individual responses to pharmacological treatment. Here, we compared the prevalence of the CYP2D6*10, *4, and 14* alleles in an Iranian population of different ethnicities with those of other populations. Allele and genotype frequency distributions of CYP2D6*10 variants and predicted phenotypes including extensive metabolizers, intermediate metabolizers, and poor metabolizers were analysed in blood samples of 300 unrelated healthy individuals in an Iranian population using polymerase chain reaction (PCR)-restriction fragment length polymorphism, PCR-single-strand conformation polymorphism, and direct genomic DNA sequencing. The CYP2D6*4 (G1846A) and *14 (G1758A) allelic frequencies were not detected in different ethnicities, demonstrating the absence of a significant contribution of these alleles in Iranian populations. However, the T/T, C/T, and C/C genotype frequencies of the CYP2D6*10 allele were significantly different (P<0.01) in all Iranian ethnic groups. Additionally, the frequency of the homozygous T/T variant of the CYP2D6*10 allele was significantly high in the Lure (P<0.017) and low in the Kurd (P<0.002) ethnicities. The frequency of the T/T variant of the CYP2D6*10 allele in central Iran was the highest (P<0.001), while the south of Iran had the lowest frequency (P<0.001). The frequency of the C/T variant of the CYP2D6*10 allele was significantly a bit high (P<0.001) in females compare to males, while the frequencies of the T/T variant in females is similar to males, which are 24.4% and 24.3%, respectively. In contrast to absence of the CYP2D6*4 (G1846A) and *14 (G1758A) alleles in Iranian populations of different ethnicities, the prediction of the CYP2D6*10 allele is required in drug research and routine treatment, where the information would be helpful for clinicians to optimize therapy or identify persons at risk of adverse drug reactions before clinical trials. Approximately 39.3% of subjects (24.3% homozygous T/T CYP2D6*10 as poor metabolizers and 15% heterozygous C/T CYP2D6*10 as intermediate metabolizers) had this allele; therefore, the harmful effects of drugs are relatively common among Iranians.

Matched MeSH terms: DNA/genetics
The use of transgenic parasites in malaria vaccine research

Othman AS, Marin-Mogollon C, Salman AM, Franke-Fayard BM, Janse CJ, Khan SM

Expert Rev Vaccines, 2017 Jul;16(7):1-13.
PMID: 28525963 DOI: 10.1080/14760584.2017.1333426

INTRODUCTION: Transgenic malaria parasites expressing foreign genes, for example fluorescent and luminescent proteins, are used extensively to interrogate parasite biology and host-parasite interactions associated with malaria pathology. Increasingly transgenic parasites are also exploited to advance malaria vaccine development. Areas covered: We review how transgenic malaria parasites are used, in vitro and in vivo, to determine protective efficacy of different antigens and vaccination strategies and to determine immunological correlates of protection. We describe how chimeric rodent parasites expressing P. falciparum or P. vivax antigens are being used to directly evaluate and rank order human malaria vaccines before their advancement to clinical testing. In addition, we describe how transgenic human and rodent parasites are used to develop and evaluate live (genetically) attenuated vaccines. Expert commentary: Transgenic rodent and human malaria parasites are being used to both identify vaccine candidate antigens and to evaluate both sub-unit and whole organism vaccines before they are advanced into clinical testing. Transgenic parasites combined with in vivo pre-clinical testing models (e.g. mice) are used to evaluate vaccine safety, potency and the durability of protection as well as to uncover critical protective immune responses and to refine vaccination strategies.

Matched MeSH terms: Vaccines, DNA/genetics
The requirements for sterol regulatory element-binding protein (Srebp) and stimulatory protein 1 (Sp1)-binding elements in the transcriptional activation of two freshwater fish Channa striata and Danio rerio elovl5 elongase

Goh PT, Kuah MK, Chew YS, Teh HY, Shu-Chien AC

Fish Physiol Biochem, 2020 Aug;46(4):1349-1359.
PMID: 32239337 DOI: 10.1007/s10695-020-00793-w

Fish are a major source of beneficial n-3 LC-PUFA in human diet, and there is considerable interest to elucidate the mechanism and regulatory aspects of LC-PUFA biosynthesis in farmed species. Long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis involves the activities of two groups of enzymes, the fatty acyl desaturase (Fads) and elongase of very long-chain fatty acid (Elovl). The promoters of elovl5 elongase, which catalyses the rate-limiting reaction of elongating polyunsaturated fatty acid (PUFA), have been previously described and characterized from several marine and diadromous teleost species. We report here the cloning and characterization of elovl5 promoter from two freshwater fish species, the carnivorous snakehead fish (Channa striata) and zebrafish. Results show the presence of sterol-responsive elements (SRE) in the core regulatory region of both promoters, suggesting the importance of sterol regulatory element-binding protein (Srebp) in the regulation of elovl5 for both species. Mutagenesis luciferase and electrophoretic mobility shift assays further validate the role of SRE for basal transcriptional activation. In addition, several Sp1-binding sites located in close proximity with SRE were present in the snakehead promoter, with one having a potential synergy with SRE in the regulation of elovl5 expression. The core zebrafish elovl5 promoter fragment also directed in vivo expression in the yolk syncytial layer of developing zebrafish embryos.

Matched MeSH terms: DNA/genetics
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/genetics
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/genetics*
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.

Matched MeSH terms: DNA/genetics*
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.

Matched MeSH terms: DNA/genetics*
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

Asing, Ali E, Hamid SB, Hossain M, Ahamad MN, Hossain SM, et al.

Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2016 Nov;33(11):1643-1659.
PMID: 27643977

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.

Matched MeSH terms: DNA/genetics
Nanoplate-based digital PCR for highly sensitive pork DNA detection targeting multi-copy nuclear and mitochondrial genes

Mokhtar NFK, Shun YQ, Raja Nhari RMH, Mohamad NA, Shahidan NM, Warsanah IH, et al.

Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2024 Feb;41(2):120-133.
PMID: 38190283 DOI: 10.1080/19440049.2023.2298476

The inclusion of ingredients derived from pigs in highly processed consumer products poses a significant challenge for DNA-targeted analytical enforcement, which could be overcome by using digital PCR. However, most species detection methods use digital PCR to target single-copy nuclear genes, which limits their sensitivity. In this work, we examined the performance of a nanoplate-based digital PCR method that targets multi-copy nuclear (MPRE42) and mitochondrial (Cytb) genes. Poor separation of positive and negative partitions, as well as a 'rain effect' were obtained in the porcine-specific MPRE42 assay. Among the optimization strategies examined, the inclusion of restriction enzymes slightly improved the separation of positive and negative partitions, but a more extensive 'rain effect' was observed. The high copy number of the MPRE42 amplicon is hypothesized to contribute to the saturation of the positive signal. In contrast, the porcine-specific Cytb assay achieved perfect separation of positive and negative partitions with no 'rain effect'. This assay can detect as little as 0.4 pg of pork DNA, with a sensitivity of 0.05% (w/w) in a pork-chicken mixture, proving its applicability for detecting pork in meat and meat-based products. For the MPRE42 assay, potential applications in highly degraded products such as gelatin and lard are anticipated.

Matched MeSH terms: DNA/genetics
A review of CRISPR-Cas and PCR-based methods for the detection of animal species in the food chain-current challenges and future prospects

Sultana S, Azlan A, Mohd Desa MN, Mahyudin NA, Anburaj A

Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2024 Mar;41(3):213-227.
PMID: 38284970 DOI: 10.1080/19440049.2024.2304577

Regular testing and systematic investigation play a vital role to ensure product safety. Until now, the existing food authentication techniques have been based on proteins, lipids, and nucleic acid-based assays. Among various deoxyribonucleic acid (DNA)-based methods, the recently developed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based bio-sensing is an innovative and fast-expanding technology. The CRISPR/Cas-9 is known as Clustered Regularly Interspaced Short Palindromic Repeats due to the flexibility and simplicity of the CRISPR/Cas9 site-specific editing tool has been applied in many biological research areas such as Gene therapy, cell line development, discovering mechanisms of disease, and drug discovery. Nowadays, the CRISPR-Cas system has also been introduced into food authentication via detecting DNA barcodes of poultry and livestock both in processed and unprocessed food samples. This review documents various DNA based approaches, in an accessible format. Future CRISPR technologies are forecast while challenges are outlined.

Matched MeSH terms: DNA/genetics
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/genetics
Tetraplex PCR assay involving double gene-sites discriminates beef and buffalo in Malaysian meat curry and burger products

Hossain MA, Ali ME, Hamid SB, Hossain SM, Asing, Nizar NN, et al.

Food Chem, 2017 Jun 01;224:97-104.
PMID: 28159299 DOI: 10.1016/j.foodchem.2016.12.062

Replacement of beef by buffalo and vice versa is frequent in global markets, but their authentication is challenging in processed foods due to the fragmentation of most biomarkers including DNA. The shortening of target sequences through use of two target sites might ameliorate assay reliability because it is highly unlikely that both targets will be lost during food processing. For the first time, we report a tetraplex polymerase chain reaction (PCR) assay targeting two different DNA regions in beef (106 and 120-bp) and buffalo (90 and 138-bp) mitochondrial genes to discriminate beef and buffalo in processed foods. All targets were stable under boiling, autoclaving and microwave cooking conditions. A survey in Malaysian markets revealed 71% beef curries contained buffalo but there was no buffalo in beef burgers. The assay detected down to 0.01ng DNA and 1% meat in admixed and burger products.

Matched MeSH terms: DNA/genetics
Universal mini COI barcode for the identification of fish species in processed products

Sultana S, Ali ME, Hossain MAM, Asing, Naquiah N, Zaidul ISM

Food Res Int, 2018 03;105:19-28.
PMID: 29433207 DOI: 10.1016/j.foodres.2017.10.065

Species substitution, the use of a low value fish in place of a high value fish, is the biggest problem in international trade and the leading cause of fraud in the fisheries arena sector. Current DNA barcoding systems have partly solved this problem but also failed in many instances to amplify PCR targets from highly processed products because of the degradation of a longer barcode marker (~650bp). In the present study, a novel mini barcode marker (295bp) was developed to discriminate fish species in raw and processed states forms. The barcode primers were cross-tested against 33 fish species and 15 other animal species and found to be universal for all the tested fish varieties. When 20 commercial fish products of five different categories were screened, all commercial fish sample yielded positive bands for the novel fish barcode. PCR product was sequenced to retrieve the species IDs that reflected 55% (11/20) of Malaysian fish products were mislabeled.

Matched MeSH terms: DNA/genetics
Haplotype diversity of 17 Y-chromosomal STRs in three native Sarawak populations (Iban, Bidayuh and Melanau) in East Malaysia

Chang YM, Swaran Y, Phoon YK, Sothirasan K, Sim HT, Lim KB, et al.

Forensic Sci Int Genet, 2009 Jun;3(3):e77-80.
PMID: 19414156 DOI: 10.1016/j.fsigen.2008.07.007

17 Y-STRs (DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635 or Y-GATA C4, DYS392, Y-GATA H4, DYS437, DYS438 and DYS448) have been analyzed in 320 male individuals from Sarawak, an eastern state of Malaysia on the Borneo island using the AmpFlSTR Y-filer (Applied Biosystems, Foster City, CA). These individuals were from three indigenous ethnic groups in Sarawak comprising of 103 Ibans, 113 Bidayuhs and 104 Melanaus. The observed 17-loci haplotypes and the individual allele frequencies for each locus were estimated, whilst the locus diversity, haplotype diversity and discrimination capacity were calculated in the three groups. Analysis of molecular variance (AMOVA) indicated that 87.6% of the haplotypic variation was found within population and 12.4% between populations (fixation index F(ST)=0.124, p=0.000). This study has revealed that the indigenous populations in Sarawak are distinctly different to each other, and to the three major ethnic groups in Malaysia (Malays, Chinese and Indians), with the Melanaus having a strikingly high degree of shared haplotypes within. There are rare unusual variants and microvariants that were not present in Malaysian Malay, Chinese or Indian groups. In addition, occurrences of DYS385 duplications which were only noticeably present in Chinese group previously was also observed in the Iban group whilst null alleles were detected at several Y-loci (namely DYS19, DYS392, DYS389II and DYS448) in the Iban and Melanau groups.

Matched MeSH terms: DNA/genetics
Fulltext Finding the factors of reduced genetic diversity on X chromosomes of Macaca fascicularis: male-driven evolution, demography, and natural selection

Osada N, Nakagome S, Mano S, Kameoka Y, Takahashi I, Terao K

Genetics, 2013 Nov;195(3):1027-35.
PMID: 24026095 DOI: 10.1534/genetics.113.156703

The ratio of genetic diversity on X chromosomes relative to autosomes in organisms with XX/XY sex chromosomes could provide fundamental insight into the process of genome evolution. Here we report this ratio for 24 cynomolgus monkeys (Macaca fascicularis) originating in Indonesia, Malaysia, and the Philippines. The average X/A diversity ratios in these samples was 0.34 and 0.20 in the Indonesian-Malaysian and Philippine populations, respectively, considerably lower than the null expectation of 0.75. A Philippine population supposed to derive from an ancestral population by founding events showed a significantly lower ratio than the parental population, suggesting a demographic effect for the reduction. Taking sex-specific mutation rate bias and demographic effect into account, expected X/A diversity ratios generated by computer simulations roughly agreed with the observed data in the intergenic regions. In contrast, silent sites in genic regions on X chromosomes showed strong reduction in genetic diversity and the observed X/A diversity ratio in the genic regions cannot be explained by mutation rate bias and demography, indicating that natural selection also reduces the level of polymorphism near genes. Whole-genome analysis of a female cynomolgus monkey also supported the notion of stronger reduction of genetic diversity near genes on the X chromosome.

Matched MeSH terms: DNA/genetics
Fulltext Morphometric and molecular analysis of mackerel (Rastrelliger spp) from the west coast of Peninsular Malaysia

Darlina MN, Masazurah AR, Jayasankar P, Jamsari AF, Siti AM

Genet. Mol. Res., 2011;10(3):2078-92.
PMID: 21968625 DOI: 10.4238/vol10-3gmr1249

Mackerel (Scombridae; Rastrelliger) are small commercially important pelagic fish found in tropical regions. They serve as a cheap source of animal protein and are commonly used as live bait. By using a truss morphometrics protocol and RAPD analysis, we examined morphological and genetic variation among 77 individual mackerel that were caught using long lines and gillnets at 11 locations along the west coast of Peninsular Malaysia. Nineteen morphometric traits were evaluated and genetic information was estimated using five 10-base RAPD random primers. Total DNA was extracted from muscle tissue. Morphometric discriminant function analysis revealed that two morphologically distinct groups of Rastrelliger kanagurta and a single group of R. brachysoma can be found along the west coast of Peninsular Malaysia. We also found that the head-related characters and those from the anterior part of the body of Rastrelliger spp significantly contribute to stock assessment of this population. RAPD analysis showed a trend similar to that of the morphometric analysis, suggesting a genetic component to the observed phenotypic differentiation. These data will be useful for developing conservation strategies for these species.

Matched MeSH terms: DNA/genetics

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