This article contains data of the sequence variation in the mitochondrial DNA D-loop region of the Malayan gaur (Bos gaurus hubbacki), locally known as the seladang, from two captive centers. Thirty fecal samples of Malayan gaur were collected from Jenderak Selatan Wildlife Conservation Center (Pahang) and the Sungkai Wildlife Reserve (Perak) for DNA extraction and amplification with polymerase chain reactions. DNA sequences were then analyzed using neighbor joining (NJ) and maximum parsimony (MP) methods. Based on the 652 base pairs obtained, we found seven variable characters with a value of 1%. The genetic distance between the two captive centers was 0.001. Haplotype analyses detected only four haplotypes between these two captive centers. Both NJ and MP trees demonstrate that all individuals in the Jenderak and Sungkai captive centers are in the same clade. Genetic variation of the Malayan gaur in these centers is considered low, possibly because individuals share the same common parent. This sequence variation data are of paramount importance for designing a proper breeding and management program of the Malayan gaur in the future.
Wild-cultivated medicinal mushroom Ganoderma lucidum was morphologically identified and sequenced using phylogenetic software. In submerged-liquid fermentation (SLF), biomass, exopolysaccharide (EPS) and intracellular polysaccharide (IPS) production of the identified G.lucidum was optimised based on initial pH, starting glucose concentration and agitation rate parameters using response surface methodology (RSM). Molecularly, the G. lucidum strain QRS 5120 generated 637 base pairs, which was commensurate with related Ganoderma species. In RSM, by applying central composite design (CCD), a polynomial model was fitted to the experimental data and was found to be significant in all parameters investigated. The strongest effect (p < 0.0001) was observed for initial pH for biomass, EPS and IPS production, while agitation showed a significant value (p < 0.005) for biomass. By applying the optimized conditions, the model was validated and generated 5.12 g/L of biomass (initial pH 4.01, 32.09 g/L of glucose and 102 rpm), 2.49 g/L EPS (initial pH 4, 24.25 g/L of glucose and 110 rpm) and 1.52 g/L of IPS (and initial pH 4, 40.43 g/L of glucose, 103 rpm) in 500 mL shake flask fermentation. The optimized parameters can be upscaled for efficient biomass, EPS and IPS production using G. lucidum.
Family Scolopacidae includes the sandpipers, shanks, snipes, godwits and curlews. Systematic classifications of shorebirds
at the higher level have been successfully resolved. Nevertheless, the phylogeny of shorebirds in the familial level is still
poorly understood. Thus, this phylogenetic study on Scolopacidae was conducted upon the framework provided by the first
sequence-based species-level phylogeny within the shorebirds to determine the phylogenetic relationships among family
members of Scolopacidae in West Borneo, Sarawak using combined gene markers, mtDNA Cytochrome Oxidise I (COI)
and nucDNA Recombinant Activating Gene 1 (RAG1). A total of 1,342 base pair (bp) were inferred from both COI and RAG1
gene from 45 sequences constituted of 15 species Scolopacidae sampled from Sarawak namely Xenus cinereus, Actitis
hypoleucos, Tringa totanus, Tringa glareola, Tringa stagnatilis, Heteroscelus brevipes, Calidris alba, Calidris ruficollis,
Calidris ferruginea, Calidris tenuirostris, Calidris alpina, Gallinago stenura, Gallinago megala, Numenius arquata, and
Numenius phaeopus. The phylogenetic tree was constructed with Charadrius mongulus derived as an outgroup. The
Bayesian Inference (BI) tree constructed supported grouping of species into several lineages of Numeniinae, Calidrinae,
Scolopacinae and Tringinae. The groupings of species into several lineages correlate with morphological features that
contribute to their adaptation and ability of the species to fit to their ecosystems.
Present study investigates the genetic diversity and genetic distribution of the longtail tuna Thunnus tonggol collected from east Malaysia (Borneo states of Sabah and Sarawak) based on mitochondrial DNA D-loop sequence analysis. 58 fish samples were obtained, specifically from Kota Kinabalu, KK (n = 22), Miri, MR (n=20) and Bintulu, BT (n = 17). DNA template was isolated using the salt extraction method. Final length of 404 base pair (bp) D-loop sequences revealed 52 haplotypes that comprise of 77 variable sites (38 of parsimony informative and 39 singleton). A total of 20 haplotypes were found in KK, 19 haplotypes in MR and 16 haplotypes in BT. Molecular diversity indices revealed high haplotype diversity and low nucleotide diversity in all populations; KK (h = 0.9913 ± 0.0165, π = 0.00239 ± 0.0127), MR (h = 0.9942 ± 0.0193, π = 0.0226 ± 0.0121) and BT (h = 0.9926 ± 0.0230, π = 0.0196 ± 0.0171). Population comparison pairwise FST show that KK and BT were significantly genetically differentiated. The result from this study will be beneficial for fisheries management and also to provide information on the population genetics of T. tonggol in East Malaysian waters.
A thermostabilized, multiplex polymerase chain reaction (mPCR) assay was developed in this study for the detection of six respiratory bacterial pathogens. Specific primers were designed for an internal amplification control (IAC) and six target sequences from Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Haemophilus influenzae. The resultant seven-band positive amplification control (PAC) of this heptaplex PCR assay corresponded to 105 base pairs (bp) of IAC, 202 bp of K. pneumoniae, 293 bp of S. aureus, 349 bp of S. pneumoniae, 444 bp of P. aeruginosa, 505 bp of M. tuberculosis, and 582 bp of H. influenzae. Results found that 6% (w/v) of the stabilizer was optimum to preserve the functional conformation of Taq DNA polymerase enzyme. This assay was stable at ambient temperature for at least 6 months. The sensitivity and specificity of this assay were both 100% when testing on the intended target organisms (n = 119) and non-intended species (n = 57). The mPCR assay developed in this study enabled accurate, rapid, and simple detection of six respiratory bacteria.
Three restriction enzymes were used in Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) using the mitochondrial cytochrome b region to establish a differential diagnosis which detect and discriminate between three meat species: pork, cow and chicken. DNA was extracted from samples containing meat of a single animal such as raw pork (Sus scrofa domesticus), chicken (Gallus gallus) and cow (Bos taurus) as well as mixed samples of two species of animals in different ratios. The amplified 359 base pairs (bp) portion of the mitochondrial cyt b gene from pure or mixed samples in different ratios was cut using three different restriction enzymes resulting in species specific restriction fragment length polymorphism (RFLP). This technique proved to be extremely reliable in detecting the presence of low levels of target DNA obtained from a 0.25 mg component in a particular mixed meat sample. This revealed the cyt b region as highly conserved and consequently a good molecular marker for diagnostic studies. Thus, this technique can be applied to food authentication for the identification of different species of animals in food products.
A method for species identification from pork and lard samples using polymerase chain reaction (PCR) analysis of a conserved region in the mitochondrial (mt) cytochrome b (cyt b) gene has been developed. Genomic DNA of pork and lard were extracted using Qiagen DNeasy(®) Tissue Kits and subjected to PCR amplification targeting the mt cyt b gene. The genomic DNA from lard was found to be of good quality and produced clear PCR products on the amplification of the mt cyt b gene of approximately 360 base pairs. To distinguish between species, the amplified PCR products were cut with restriction enzyme BsaJI resulting in porcine-specific restriction fragment length polymorphisms (RFLP). The cyt b PCR-RFLP species identification assay yielded excellent results for identification of pig species. It is a potentially reliable technique for detection of pig meat and fat from other animals for Halal authentication.
Chikungunya virus (CHIKV) is maintained in the sylvatic cycle in West Africa and is transmitted by Aedes mosquito species to monkeys. In 2006, four verified CHIKV isolates were obtained during a survey of arboviruses in monkeys (Macaca fascicularis) in Pahang state, Peninsular Malaysia. RNA was extracted from the CHIKV isolates and used in reverse transcription polymerase chain reactions (RT-PCR) to amplify PCR fragments for sequencing. Nucleic acid primers were designed to generate overlapping PCR fragments that covered the whole viral sequence. A total of 11,238 base pairs (bp) corresponding to open reading frames (ORFs) from our isolates and 47 other registered isolates in the National Center for Biotechnology Information (NCBI) were used to elucidate sequences, amino acids, and phylogenetic relationships and to estimate divergence times by using MEGA 7.0 and the Bayesian Markov chain Monte Carlo method. Phylogenetic analysis revealed that all CHIKV isolates could be classified into the Asian genotype and clustered with Bagan Panchor clades, which are associated with the chikungunya outbreak reported in 2006, with sequence and amino acid similarities of 99.9% and 99.7%, respectively. Minor amino acid differences were found between human and non-human primate isolates. Amino acid analysis showed a unique amino acid at position 221 in the nsP1region, at which a glycine (G) was found only in monkey isolates, whereas arginine (R) was found at the same position only in human isolates. The time to the most recent common ancestor (MRCA) estimation indicated that CHIKV probably started to diverge from human to non-human primates in approximately 2004 in Malaysia. The results suggested that CHIKV in non-human primates probably resulted from the spillover of the virus from humans. The study will be helpful in understanding the movement and evolution of CHIKV in Malaysia and globally.
The activities of lipase produced by five lipases-producing thermophilic bacteria strains (SY1, SY5, SY6, SY7 and SY9) isolated from Selayang Hot Spring in the western part of Peninsular Malaysia were analyzed and compared. SY7 and SY9 had considerably higher lipolytic activities than those of SY1, SY5 and SY6. Thermostabilities of lipase produced by all strains were determined after heating at 80°C for 30 minutes. Strain SY7 retained the highest lipolytic activity of 77%, while others had infinitesimally low thermostability (retaining less than 34% of their original activity) at the same temperature and time. SY7 was chosen for further characterization because it showed exceptionally high lipase activity and thermostability. It was identified as belonging to Bacillus species by the conventional Gram-staining technique, Biochemical tests and Biolog Microstation system. By using 16S rRNA gene sequencing, strain SY7 generated the same expected PCR product with molecular weight of 1500 base pair. It displayed 98% sequence similarity to Bacillus cereus strain J-1 16S rRNA gene partial sequence with accession number: AY305275 and has been deposited in the database of Genbank.
Common wild rice (Oryza rufipogon) plays an important role by contributing to modern rice breeding. In this paper, we report the sequence and analysis of a 172-kb genomic DNA region of wild rice around the RM5 locus, which is associated with the yield QTL yld1.1. Comparative sequence analysis between orthologous RM5 regions from Oryza sativa ssp. japonica, O. sativa ssp. indica and O. rufipogon revealed a high level of conserved synteny in the content, homology, structure, orientation, and physical distance of all 14 predicted genes. Twelve of the putative genes were supported by matches to proteins with known function, whereas two were predicted by homology to rice and other plant expressed sequence tags or complementary DNAs. The remarkably high level of conservation found in coding, intronic and intergenic regions may indicate high evolutionary selection on the RM5 region. Although our analysis has not defined which gene(s) determine the yld1.1 phenotype, allelic variation and the insertion of transposable elements, among other nucleotide changes, represent potential variation responsible for the yield QTL. However, as suggested previously, two putative receptor-like protein kinase genes remain the key suspects for yld1.1.
Vibrio parahaemolyticus is a gram negative bacterium and causes gastrointestinal illness in humans. In this study, twenty five out of fifty cockle samples from Padang, Indonesia produced purple colonies when they were grown on selective medium, CHROMagarTM Vibrio. Specific–PCR for toxR gene detection gave positive results in which a band with 368 base pairs size appeared on the gel for all the isolates that confirmed the presence of V. parahaemolyticus. In the virulence properties test, all the isolates showed negative results for tdh and trh genes detection. The results indicate that the isolates under this study do not contain virulence properties that correlate to the ability of infection and diseases, which means that they are nonpathogenic.
We describe the characteristics of complete mitogenome of C. brachyotis in this article. The complete mitogenome of C. brachyotis is 16,701 bp long with a total base composition of 32.4% A, 25.7% T, 27.7% C and 14.2% G. The mitogenome consists of 13 protein-coding genes (11,408 bp), (KM659865) two rRNA (12S rRNA and 16S rRNA) genes (2,539 bp), 22 tRNA genes (1518 bp) and one control region (1239 bp).
Malaysia is one of the countries that are loaded with mega biodiversity which
includes microbial communities. Phages constitute the major component in the microbial
communities and yet the numbers of discovered phages are just a minute fraction of
its population in the biosphere. Taking into account of a huge numbers of waiting to be
discovered phages, a new bacteriophage designated as Escherichia phage YD-2008.s
was successfully isolated using Escherichia coli ATCC 11303 as the host. Phage YD-2008.s poses icosahedral head measured at 57nm in diameter with a long non-contractile
flexible tail measured at 107nm; proving the phage as one of the members of Siphoviridae
family under the order of Caudovirales. Genomic sequence analyses revealed phage
YD-2008.s genome as linear dsDNA of 44,613 base pairs with 54.6% G+C content.
Sixty-two open reading frames (ORFs) were identified on phage YD-2008.s full genome,
using bioinformatics annotation software; Rapid Annotation using Subsystem Technology
(RAST). Among the ORFs, twenty-eight of them code for functional proteins. Thirty two are
classified as hypothetical proteins and there are two unidentified proteins. Even though
majority of the coded putative proteins have high amino acids similarities to phages from the
genus Hk578likevirus of the Siphoviridae family, yet phage YD-2008.s stands with its’ own
distinctiveness. Therefore, this is another new finding to Siphoviridae family as well as to the
growing list of viruses in International Committee on Taxonomy of Viruses (ICTV) database.
Yardlong bean (Vigna unguiculata subsp. sesquipedalis) is extensively cultivated in Indonesia for consumption as a green vegetable. During the 2008 season, a severe outbreak of a virus-like disease occurred in yardlong beans grown in farmers' fields in Bogor, Bekasi, Subang, Indramayu, and Cirebon of West Java, Tanggerang of Banten, and Pekalongan and Muntilan of Central Java. Leaves of infected plants showed severe mosaic to bright yellow mosaic and vein-clearing symptoms, and pods were deformed and also showed mosaic symptoms on the surface. In cv. 777, vein-clearing was observed, resulting in a netting pattern on symptomatic leaves followed by death of the plants as the season advanced. Disease incidence in the Bogor region was approximately 80%, resulting in 100% yield loss. Symptomatic leaf samples from five representative plants tested positive in antigen-coated plate-ELISA with potyvirus group-specific antibodies (AS-573/1; DSMZ, German Resource Center for Biological Material, Braunschweig, Germany) and antibodies to Cucumber mosaic virus (CMV; AS-0929). To confirm these results, viral nucleic acids eluted from FTA classic cards (FTA Classic Card, Whatman International Ltd., Maidstone, UK) were subjected to reverse transcription (RT)-PCR using potyvirus degenerate primers (CIFor: 5'-GGIVVIGTIGGIWSIGGIAARTCIAC-3' and CIRev: 5'-ACICCRTTYTCDATDATRTTIGTIGC-3') (3) and degenerate primers (CMV-1F: 5'-ACCGCGGGTCTTATTATGGT-3' and CMV-1R: 5' ACGGATTCAAACTGGGAGCA-3') specific for CMV subgroup I (1). A single DNA product of approximately 683 base pairs (bp) with the potyvirus-specific primers and a 382-bp fragment with the CMV-specific primers were amplified from ELISA-positive samples. These results indicated the presence of a potyvirus and CMV as mixed infections in all five samples. The amplified fragments specific to potyvirus (four samples) and CMV (three samples) were cloned separately into pCR2.1 (Invitrogen Corp., Carlsbad, CA). Two independent clones per amplicon were sequenced from both orientations. Pairwise comparison of these sequences showed 93 to 100% identity among the cloned amplicons produced using the potyvirus-specific primers (GenBank Accessions Nos. FJ653916, FJ653917, FJ653918, FJ653919, FJ653920, FJ653921, FJ653922, FJ653923, FJ653924, FJ653925, and FJ653926) and 92 to 97% with a corresponding nucleotide sequence of Bean common mosaic virus (BCMV) from Taiwan (No. AY575773) and 88 to 90% with BCMV sequences from China (No. AJ312438) and the United States (No. AY863025). The sequence analysis indicated that BCMV isolates from yardlong bean are more closely related to an isolate from Taiwan than with isolates from China and the United States. The CMV isolates (GenBank No. FJ687054) each were 100% identical and 96% identical with corresponding sequences of CMV subgroup I isolates from Thailand (No. AJ810264) and Malaysia (No. DQ195082). Both BCMV and CMV have been documented in soybean, mungbean, and peanut in East Java of Indonesia (2). Previously, BCMV, but not CMV, was documented on yardlong beans in Guam (4). To our knowledge, this study represents the first confirmed report of CMV in yardlong bean in Indonesia and is further evidence that BCMV is becoming established in Indonesia. References: (1) J. Aramburu et al. J. Phytopathol. 155:513, 2007. (2) S. K. Green et al. Plant Dis. 72:994, 1988. (3) C. Ha et al. Arch. Virol. 153:25, 2008. (4) G. C. Wall et al. Micronesica 29:101, 1996.
Nanoparticles have been investigated as flagging tests for the sensitive DNA recognition that can be utilized as a part of field applications to defeat restrictions. Gold nanoparticles (AuNPs) have been widely utilized due to its optical property and capacity to get functionalized with a mixed bag of biomolecules. This study exhibits the utilization of AuNPs functionalized with single-stranded oligonucleotide (AuNP-oligo test) for fast the identification of Human Papillomavirus (HPV). This test is displayed on interdigitated electrode sensor and supported by colorimetric assay. DNA conjugated AuNP has optical property that can be controlled for the applications in diagnostics. With its identification abilities, this methodology incorporates minimal effort, strong reagents and basic identification of HPV.
DNA barcoding of the cytochrome oxidase subunit I (COI) gene was utilized to assess the species diversity of the freshwater halfbeak genus Hemirhamphodon. A total of 201 individuals from 46 locations in Peninsular Malaysia, north Borneo (Sarawak) and Sumatra were successfully amplified for 616 base pairs of the COI gene revealing 231 variable and 213 parsimony informative sites. COI gene trees showed that most recognized species form monophyletic clades with high bootstrap support. Pairwise within species comparisons exhibited a wide range of intraspecific diversity from 0.0% to 14.8%, suggesting presence of cryptic diversity. This finding was further supported by barcode gap analysis, ABGD and the constructed COI gene trees. In particular, H. pogonognathus from Kelantan (northeast Peninsular Malaysia) diverged from the other H. pogonognathus groups with distances ranging from 7.8 to 11.8%, exceeding the nearest neighbor taxon. High intraspecific diversity was also observed in H. byssus and H. kuekanthali, but of a lower magnitude. This study also provides insights into endemism and phylogeographic structuring, and limited support for the Paleo-drainage divergence hypothesis as a driver of speciation in the genus Hemirhamphodon.
The phylogenetic relationships of long-tailed macaque (Macaca fascicularis fascicularis) populations distributed in Peninsular Malaysia in relation to other regions remain unknown. The aim of this study was to reveal the phylogeography and population genetics of Peninsular Malaysia's M. f. fascicularis based on the D-loop region of mitochondrial DNA. Sixty-five haplotypes were detected in all populations, with only Vietnam and Cambodia sharing four haplotypes. The minimum-spanning network projected a distant relationship between Peninsular Malaysian and insular populations. Genetic differentiation (F(ST), Nst) results suggested that the gene flow among Peninsular Malaysian and the other populations is very low. Phylogenetic tree reconstructions indicated a monophyletic clade of Malaysia's population with continental populations (NJ = 97%, MP = 76%, and Bayesian = 1.00 posterior probabilities). The results demonstrate that Peninsular Malaysia's M. f. fascicularis belonged to Indochinese populations as opposed to the previously claimed Sundaic populations. M. f. fascicularis groups are estimated to have colonized Peninsular Malaysia ~0.47 million years ago (MYA) directly from Indochina through seaways, by means of natural sea rafting, or through terrestrial radiation during continental shelf emersion. Here, the Isthmus of Kra played a central part as biogeographical barriers that then separated it from the remaining continental populations.
Betulinic acid (BA) is a pentacyclic triterpene found in several botanical sources that has been shown to cause apoptosis in a number of cell lines. This study was undertaken to determine the in vitro cytotoxic properties of BA towards the human mammary carcinoma cell line MDA-MB-231 and the human promyelocytic leukaemia cell line HL-60 and the mode of the induced cell death. The cytotoxicity and mode of cell death of BA were determined using the MTT assay and DNA fragmentation analysis, respectively. In our study, the compound was found to be cytotoxic to MDA-MB-231 and HL-60 cells with IC50 values of 58 μg/mL and 134 μg/mL, respectively. Cells treated with high concentrations of BA exhibited features characteristic of apoptosis such as blebbing, shrinking and a number of small cytoplasm body masses when viewed under an inverted light microscope after 24h. The incidence of apoptosis in MDA-MB-231 was further confirmed by the DNA fragmentation analysis, with the formation of DNA fragments of oligonucleosomal size (180-200 base pairs), giving a ladder-like pattern on agarose gel electrophoresis. BA was more cytotoxic towards MDA-MB-231 than HL-60 cells, and induced apoptosis in MDA-MB-231 cells.
Candida speciation is vital for epidemiology and management of candidiasis. Nonmolecular conventional methods often fail to identify closely related germ tube positive yeasts from clinical specimens. The present study was conducted to identify these yeasts and to highlight issues in conventional versus molecular methods of identification. A total of 98 germ tube positive yeasts from high vaginal swabs were studied over a 12-month period. Isolates were examined with various methods including growth at 42 °C and 45 °C on Sabouraud dextrose agar (SDA), color development on CHROMagar Candida medium, chlamydospore production on corn meal agar at 25 °C, carbohydrate assimilation using ID 32C system, and polymerase chain reaction using a single pair of primers targeting the hyphal wall protein 1 (Hwp1) gene. Of all the isolates studied, 97 were molecularly confirmed as C. albicans and one isolate was identified as C. dubliniensis. No C. africana was detected in this study. The molecular method used in our study was an accurate and useful tool for discriminating C. albicans, C. dubliniensis, and C. africana. The conventional methods, however, were less accurate and riddled with many issues that will be discussed in further details.
Alteration of the tumor suppressor gene p53 is considered to be a critical step in the development of human cancer. Changes in this gene have been detected in a wide range of human tumours, including gliomas. In glioma, the presence of p53 gene alterations has been associated with worse prognosis.