Displaying publications 1 - 20 of 80 in total

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  1. Long-Range Polymerase Chain Reaction
    Kee PS, Karunanathie H, Maggo SDS, Kennedy MA, Chua EW
    Methods Mol Biol, 2023;2967:181-192.
    PMID: 37608112 DOI: 10.1007/978-1-0716-3358-8_15
    Polymerase chain reaction (PCR) is a laboratory technique used to amplify a targeted region of DNA, demarcated by a set of oligonucleotide primers. Long-range PCR is a form of PCR optimized to facilitate the amplification of large fragments. Using the adapted long-range PCR protocol described in this chapter, we were able to generate PCR products of 6.6, 7.2, 13, and 20 kb from human genomic DNA samples. For some of the long PCRs, successful amplification was not possible without the use of PCR enhancers. Thus, we also evaluated the impact of some enhancers on long-range PCR and included the findings as part of this updated chapter.
    Matched MeSH terms: DNA Primers/genetics
  2. Fulltext Isolation and characterization of novel microsatellite markers in commercial selected golden Malaysian arowana fish, Scleropages formosus (Osteoglossidae)
    Manoharan B, Sulaimen Z, Omar F, Othman RY, Mohamed SZ, Bhassu S
    Genet. Mol. Res., 2011;10(2):712-6.
    PMID: 21523650 DOI: 10.4238/vol10-2gmr944
    Malaysian arowana (dragonfish; Scleropages formosus) is an ancient osteoglossid fish from southeast Asia. Due to the high demand of the ornamental fish trade and because of habitat loss, the species is close to extinction. We isolated and characterized 10 polymorphic microsatellites of this species, using 5'-anchored PCR. The number of alleles at the 10 microsatellite loci ranged from 2 to 28, with a mean of 7.8/locus. The observed heterozygosity ranged from 0.03 to 0.93 (mean: 0.39), whereas the expected heterozygosity ranged from 0.03 to 0.94 (mean: 0.46). Seven microsatellites deviated from Hardy-Weinberg equilibrium, and three conformed to Hardy-Weinberg equilibrium and were in linkage equilibrium. These 10 novel microsatellites should facilitate studies of genetic diversity and population structure of arowana to help plan actions for the conservation of the indigenous Malaysian arowana.
    Matched MeSH terms: DNA Primers/genetics*
  3. Fulltext Rapid Detection of Porcine DNA in Meatball Using Recombinase Polymerase Amplification Couple with Lateral Flow Immunoassay for Halal Authentication
    Yusop MHM, Bakar MFA, Kamarudin KR, Mokhtar NFK, Hossain MAM, Johan MR, et al.
    Molecules, 2022 Nov 22;27(23).
    PMID: 36500215 DOI: 10.3390/molecules27238122
    Point-of-care diagnostic methods for animal species determination are critical for rapid, simple, and accurate enforcement of food labelling. PCR is the most common method for species identification. However, the requirement of using a thermal cycler created drawbacks for the PCR application, particularly in low-resource settings. Hence, in this study, a method for porcine DNA detection using recombinase polymerase amplification (RPA), coupled with nucleic acid lateral flow immunoassay (NALFIA), was developed. Porcine-specific primers targeting pig (Sus scrofa) cytochrome b gene fragments specifically amplify a 197 bp fragment of the mitochondrial gene as being visualized by 2% agarose gel and PCRD NALFIA. The reaction temperature and time were 39 °C and 20 min, respectively. Herein, the specificity of the primers to porcine was confirmed after being assayed against six animal species, namely cow, goat, chicken, duck, dog, and rabbit. The porcine-specific RPA assay shows a high limit of detection of 0.01 ng/µL pork DNA. Based on the preliminary performance data obtained from this study, the potential of this method as a rapid and sensitive tool for porcine DNA detection in meat-based products is foreseen.
    Matched MeSH terms: DNA Primers/genetics
  4. Potential halophilic cellulases for in situ enzymatic saccharification of ionic liquids pretreated lignocelluloses
    Gunny AA, Arbain D, Edwin Gumba R, Jong BC, Jamal P
    Bioresour Technol, 2014 Mar;155:177-81.
    PMID: 24457303 DOI: 10.1016/j.biortech.2013.12.101
    Ionic liquids (ILs) have been used as an alternative green solvent for lignocelluloses pretreatment. However, being a salt, ILs exhibit an inhibitory effect on cellulases activity, thus making the subsequent saccharification inefficient. The aim of the present study is to produce salt-tolerant cellulases, with the rationale that the enzyme also tolerant to the presence of ILs. The enzyme was produced from a locally isolated halophilic strain and was characterized and assessed for its tolerance to different types of ionic liquids. The results showed that halophilic cellulases produced from Aspergillus terreus UniMAP AA-6 exhibited higher tolerance to ILs and enhanced thermo stability in the presence of high saline conditions.
    Matched MeSH terms: DNA Primers/genetics
  5. Fulltext Detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification
    Teoh BT, Sam SS, Tan KK, Johari J, Danlami MB, Hooi PS, et al.
    BMC Infect Dis, 2013;13:387.
    PMID: 23964963 DOI: 10.1186/1471-2334-13-387
    BACKGROUND: Early and rapid detection of dengue virus (DENV) infection during the febrile period is crucial for proper patient management and prevention of disease spread. An easy to perform and highly sensitive method is needed for routine implementation especially in the resource-limited rural healthcare settings where dengue is endemic.
    METHODS: A single-tube reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay with a set of nine primers was developed for the detection of all four DENV serotypes and their different genotypes. The sensitivity and specificity of the RT-LAMP were evaluated. The clinical applicability of RT-LAMP assay for detection of DENV RNA was assessed in a total of 305 sera of clinically-suspected dengue patients. The test results of RT-LAMP were statistically compared to those of quantitative reverse transcription-polymerase chain reaction (qRT-PCR), IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA).
    RESULTS: Acute DENV infection was confirmed in 171 samples (n = 305); 43.3% (74/171) and 46.8% (80/171) of the samples were positive for DENV using RT-LAMP and qRT-PCR, respectively. The combination of RT-LAMP with the dengue IgM and IgG ELISA increased detection of acute DENV infection to 97.7% (167/171), in comparison to only 70.8% (121/171) when dengue IgM and IgG ELISA alone were used. The RT-LAMP assays showed high concordance (κ = 0.939) with the qRT-PCR. The RT-LAMP assay detected up to 10 copies of virus RNA within an hour but 100% reproducibility (12/12) was achieved with 100 copies. There was no cross reactivity of RT-LAMP with other closely related arboviruses.
    CONCLUSION: The RT-LAMP assay developed in this study is sensitive, specific and simple to perform. The assay improved the detection of dengue when used in combination with serological methods.
    Matched MeSH terms: DNA Primers/genetics
  6. Fulltext Isolation and characterization of microsatellite markers for an important tropical tree, Aquilaria malaccensis (Thymelaeaceae)
    Tnah LH, Lee CT, Lee SL, Ng KK, Ng CH, Nurul-Farhanah Z, et al.
    Am J Bot, 2012 Nov;99(11):e431-3.
    PMID: 23108468 DOI: 10.3732/ajb.1200165
    Aggressive collections and trade activities in recent decades have resulted in heavy pressure on the natural stands of Aquilaria malaccensis and concerns over its long-term survival potential. To aid DNA profiling and assessment of its genetic diversity, microsatellite markers were developed for the species.
    Matched MeSH terms: DNA Primers/genetics
  7. Fulltext Elaeis oleifera genomic-SSR markers: exploitation in oil palm germplasm diversity and cross-amplification in arecaceae
    Zaki NM, Singh R, Rosli R, Ismail I
    Int J Mol Sci, 2012;13(4):4069-88.
    PMID: 22605966 DOI: 10.3390/ijms13044069
    Species-specific simple sequence repeat (SSR) markers are favored for genetic studies and marker-assisted selection (MAS) breeding for oil palm genetic improvement. This report characterizes 20 SSR markers from an Elaeis oleifera genomic library (gSSR). Characterization of the repeat type in 2000 sequences revealed a high percentage of di-nucleotides (63.6%), followed by tri-nucleotides (24.2%). Primer pairs were successfully designed for 394 of the E. oleifera gSSRs. Subsequent analysis showed the ability of the 20 selected E. oleifera gSSR markers to reveal genetic diversity in the genus Elaeis. The average Polymorphism Information Content (PIC) value for the SSRs was 0.402, with the tri-repeats showing the highest average PIC (0.626). Low values of observed heterozygosity (H(o)) (0.164) and highly positive fixation indices (F(is)) in the E. oleifera germplasm collection, compared to the E. guineensis, indicated an excess of homozygosity in E. oleifera. The transferability of the markers to closely related palms, Elaeis guineensis, Cocos nucifera and ornamental palms is also reported. Sequencing the amplicons of three selected E. oleifera gSSRs across both species and palm taxa revealed variations in the repeat-units. The study showed the potential of E. oleifera gSSR markers to reveal genetic diversity in the genus Elaeis. The markers are also a valuable genetic resource for studying E. oleifera and other genus in the Arecaceae family.
    Matched MeSH terms: DNA Primers/genetics
  8. Recent discoveries of armyworms in Japan and their species identification using DNA barcoding
    Sutou M, Kato T, Ito M
    Mol Ecol Resour, 2011 Nov;11(6):992-1001.
    PMID: 21693000 DOI: 10.1111/j.1755-0998.2011.03040.x
    Long columns of migrating larval sciarid armyworms were discovered in central and northern Japan, specifically Kanagawa, Gunma, Miyagi and Akita prefectures, as well as Hokkaido. This is the first examination of armyworms in East Asia. In Europe, armyworms have been identified as Sciara militaris, belonging to the family Sciaridae (sciarid flies or black fungus gnats), by rearing them to adulthood. In Japan, we were unable to obtain live samples for rearing; therefore, DNA barcodes were obtained from the samples of armyworms collected in the Gunma and Miyagi prefectures. The DNA barcodes were compared with those obtained from the following samples: pupae of S. militaris from UK, adults of Sciara kitakamiensis, Sciara humeralis, Sciara hemerobioides, Sciara thoracica, Sciara helvola and Sciara melanostyla from Japan, and adults of one undescribed Sciara species from Malaysia. Neighbour-joining, maximum parsimony, and maximum likelihood analyses revealed that the armyworms discovered in Japan are S. kitakamiensis. Although adults of this species have been recorded in several locations in Japan, this is the first report of migrating larval armyworms. DNA barcodes were effectively used to link different life stages of this species. The average intraspecific and interspecific pairwise genetic distances of the genus Sciara were 0.3% and 12.6%, respectively. The present study illustrates that DNA barcodes are an effective means of identifying sciarid flies in Japan.
    Matched MeSH terms: DNA Primers/genetics
  9. Characterization of Malaysian Trichoderma isolates using random amplified microsatellites (RAMS)
    Siddiquee S, Tan SG, Yusuf UK, Fatihah NH, Hasan MM
    Mol Biol Rep, 2012 Jan;39(1):715-22.
    PMID: 21553047 DOI: 10.1007/s11033-011-0790-6
    Trichoderma species are commercially applied as biocontrol agents against numerous plant pathogenic fungi due to their production of antifungal metabolites, competition for nutrients and space, and mycoparasitism. However, currently the identification of Trichoderma species from throughout the world based on micro-morphological descriptions is tedious and prone to error. The correct identification of Trichoderma species is important as several traits are species-specific. The Random Amplified Microsatellites (RAMS) analysis done using five primers in this study showed different degrees of the genetic similarity among 42 isolates of this genus. The genetic similarity values were found to be in the range of 12.50-85.11% based on a total of 76 bands scored in the Trichoderma isolates. Of these 76 bands, 96.05% were polymorphic, 3.95% were monomorphic and 16% were exclusive bands. Two bands (250 bp and 200 bp) produced by primer LR-5 and one band (250 bp) by primer P1A were present in all the Trichoderma isolates collected from healthy and infected oil palm plantation soils. Cluster analysis based on UPGMA of the RAMS marker data showed that T. harzianum, T. virens and T. longibrachiatum isolates were grouped into different clades and lineages. In this study we found that although T. aureoviride isolates were morphologically different when compared to T. harzianum isolates, the UPGMA cluster analysis showed that the majority isolates of T. aureoviride (seven from nine) were closely related to the isolates of T. harzianum.
    Matched MeSH terms: DNA Primers/genetics
  10. Isolation and cloning of microRNAs from recalcitrant plant tissues with small amounts of total RNA: a step-by step approach
    Yew CW, Kumar SV
    Mol Biol Rep, 2012 Feb;39(2):1783-90.
    PMID: 21625851 DOI: 10.1007/s11033-011-0919-7
    MicroRNAs (miRNAs) are small RNAs (sRNAs) with approximately 21-24 nucleotides in length. They regulate the expression of target genes through the mechanism of RNA silencing. Conventional isolation and cloning of miRNAs methods are usually technical demanding and inefficient. These limitations include the requirement for high amounts of starting total RNA, inefficient ligation of linkers, high amount of PCR artifacts and bias in the formation of short miRNA-concatamers. Here we describe in detail a method that uses 80 μg of total RNA as the starting material. Enhancement of the ligation of sRNAs and linkers with the use of polyethylene glycol (PEG8000) was described. PCR artifacts from the amplification of reverse-transcribed sRNAs were greatly decreased by using lower concentrations of primers and reducing the number of amplification cycles. Large concatamers with up to 1 kb in size with around 20 sRNAs/concatamer were obtained by using an optimized reaction condition. This protocol provide researchers with a rapid, efficient and cost-effective method for the construction of miRNA profiles from plant tissues containing low amounts of total RNA, such as fruit flesh and senescent leaves.
    Matched MeSH terms: DNA Primers/genetics
  11. Fulltext Isolation and characterization of microsatellite loci in an endangered palm, Johannesteijsmannia lanceolata (Arecaceae)
    Ang CC, Lee SL, Lee CT, Tnah LH, Zakaria RM, Ng CC
    Am J Bot, 2011 May;98(5):e117-9.
    PMID: 21613176 DOI: 10.3732/ajb.1000494
    Microsatellite markers were developed for Johannesteijsmannia lanceolata to assess the genetic diversity and mating system of this alarmingly endangered species.
    Matched MeSH terms: DNA Primers/genetics*
  12. Fulltext Microsatellite markers of an important medicinal plant, Eurycoma longifolia (Simaroubaceae), for DNA profiling
    Tnah LH, Lee CT, Lee SL, Ng KK, Ng CH, Hwang SS
    Am J Bot, 2011 May;98(5):e130-2.
    PMID: 21613180 DOI: 10.3732/ajb.1000469
    Microsatellite markers of an important medicinal plant, Eurycoma longifolia (Simaroubaceae), were developed for DNA profiling and genetic diversity studies.
    Matched MeSH terms: DNA Primers/genetics*
  13. Fulltext Rapidly shifting sex ratio across a species range
    Hornett EA, Charlat S, Wedell N, Jiggins CD, Hurst GD
    Curr Biol, 2009 Oct 13;19(19):1628-31.
    PMID: 19747825 DOI: 10.1016/j.cub.2009.07.071
    Sex ratios are subject to distortion by a range of inherited parasites. Although it has been predicted that the presence of these elements will result in spatial and temporal variation in host sex ratio, testing of this hypothesis has been constrained by availability of historical data. We here determine spatial and temporal variation in sex ratio in a interaction between a butterfly and male-killing Wolbachia bacteria by assaying infection presence in museum specimens, and from this inferring infection prevalence and phenotype in historical populations. Comparison of contemporary and museum samples revealed profound change in four of five populations examined. Two populations become extremely female biased, associated with spread of the male-killer bacterium. One evolved from extremely female biased to a sex ratio near parity, resulting from the infection losing male-killing activity. The final population fluctuated widely in sex ratio, associated with varying frequency of the male killer. We conclude that asynchronous invasion and decline of sex-ratio distorters combines with the evolution of host suppressors to produce a rapidly changing mosaic of sex ratio. As a consequence, the reproductive ecology of the host species is likely to be fundamentally altered over short time scales. Further, the study demonstrates the utility of museum specimens as "silent witnesses" of evolutionary change.
    Matched MeSH terms: DNA Primers/genetics
  14. A comparison of the VP1, VP2, and VP4 regions for molecular typing of human enteroviruses
    Perera D, Shimizu H, Yoshida H, Tu PV, Ishiko H, McMinn PC, et al.
    J Med Virol, 2010 Apr;82(4):649-57.
    PMID: 20166171 DOI: 10.1002/jmv.21652
    The VP4, VP2, and VP1 gene regions were evaluated for their usefulness in typing human enteroviruses. Three published RT-PCR primers sets targeting separately these three gene regions were used. Initially, from a total of 86 field isolates (36 HEV-A, 40 HEV-B, and 10 HEV-C) tested, 100% concordance in HEV-A was identified from all three gene regions (VP4, VP2, and VP1). However, for HEV-B and HEV-C viruses, only the VP2 and VP1 regions, and not VP4, showed 100% concordance in typing these viruses. To evaluate further the usefulness of VP4 in typing HEV-A enteroviruses, 55 Japanese and 203 published paired VP4 and VP1 nucleotide sequences were also examined. In each case, typing by VP4 was 100% in concordance with typing using VP1. Given these results, it is proposed that for HEV-A enteroviruses, all three gene regions (VP4, VP2, and VP1), would be useful for typing these viruses. These options would enhance the capability of laboratories in identifying these viruses and would greatly help in outbreaks of hand, foot, and mouth disease.
    Matched MeSH terms: DNA Primers/genetics
  15. The status of the Japanese and East Asian bats of the genus Myotis (Vespertilionidae) based on mitochondrial sequences
    Kawai K, Nikaido M, Harada M, Matsumura S, Lin LK, Wu Y, et al.
    Mol Phylogenet Evol, 2003 Aug;28(2):297-307.
    PMID: 12878466
    The genus Myotis includes the largest number of species in the family Vespertilionidae (Chiroptera), and its members are distributed throughout most of the world. To re-evaluate the phylogenetic position of East Asian Myotis species with respect to Myotis species worldwide, we analyzed mitochondrial gene sequences of NADH dehydrogenase subunit 1 and cytochrome b from 24 East Asian individuals as well as 42 vespertilionid bats determined previously. The results suggest that: (1) some individuals having the same species name in Europe and Japan do not form a monophyletic clade, indicating that some bat species exhibit morphological convergence, (2) Japanese Myotis mystacinus forms a sister relationship with Myotis brandtii (Palaearctic), and both species are included in the American clade implying that an ancestor of these species originated in North America, and (3) the Black whiskered bat, Myotis pruinosus, is endemic to Japan and forms sister relationships with Myotis yanbarensis and Myotis montivagus collected from Okinawa (Japan) and Selangor (Malaysia), respectively, implying that M. pruinosus originated from the south. The systematics of Japanese and East Asian Myotis bats were revisited by considering their phylogenetic relationships. Our study provides the first extensive phylogenetic hypothesis of the genus Myotis that includes East Asian and Japanese species.
    Matched MeSH terms: DNA Primers/genetics
  16. Molecular phylogeny of Crematogaster subgenus Decacrema ants (Hymenoptera: Formicidae) and the colonization of Macaranga (Euphorbiaceae) trees
    Feldhaar H, Fiala B, Gadau J, Mohamed M, Maschwitz U
    Mol Phylogenet Evol, 2003 Jun;27(3):441-52.
    PMID: 12742749
    To elucidate the evolution of one of the most species-rich ant-plant symbiotic systems, the association between Crematogaster (Myrmicinae) and Macaranga (Euphorbiaceae) in South-East Asia, we conducted a phylogenetic analysis of the ant partners. For the phylogenetic analysis partial mitochondrial cytochrome oxidase I and II were sequenced and Maximum Parsimony analysis was performed. The analyzed Crematogaster of the subgenus Decacrema fell into three distinct clades which are also characterized by specific morphological and ecological traits (queen morphology, host-plants, and colony structure). Our results supported the validity of our currently used morphospecies concept for Peninsula Malaysia. However, on a wider geographic range (including North and North-East Borneo) some morphospecies turned out to be species complexes with genetically quite distinct taxa. Our phylogenetic analysis and host association studies do not indicate strict cocladogenesis between the subgenus Decacrema and their Macaranga host-plants because multiple ant taxa occur on quite distinct host-plants belonging to different clades within in the genus Macaranga. These results support the view that host-shifting or host-expansion is common in the ants colonizing Macaranga. Additionally, the considerable geographic substructuring found in the phylogenetic trees of the ants suggests that allopatric speciation has also played a role in the diversification and the current distribution of the Decacrema ants.
    Matched MeSH terms: DNA Primers/genetics
  17. Complete genome sequence analysis of dengue virus type 2 isolated in Brunei
    Osman O, Fong MY, Devi S
    Virus Res, 2008 Jul;135(1):48-52.
    PMID: 18406488 DOI: 10.1016/j.virusres.2008.02.006
    In a previous study, we have reported the detection and isolation of dengue virus in Brunei (Osman, O., Fong, M.Y., Devi, S., 2007. A preliminary study of dengue infection in Brunei. JJID 60 (4), 205-208). DEN-2 was the predominant serotype followed by DEN-1. The full genomic sequences of 3 DEN-2 viruses isolated during the 2005-2006 dengue incident in Brunei were determined. Twenty-five primer sets were designed to amplify contiguous overlapping fragments of approximately 500-600 base pairs spanning the entire sequence of the viral genome. The amplified PCR products were sent for sequencing and their nucleotides and the deduced amino acids were determined. All three DEN-2 virus isolated were clustered in the Cosmopolitan genotype of the DEN-2 classification by Twiddy et al. This work constitutes the first complete genetic characterization of three Brunei DEN-2 virus strains.
    Matched MeSH terms: DNA Primers/genetics
  18. Two microsatellite loci from Brugia malayi show polymorphisms among isolates from Indonesia and Malaysia
    Underwood AP, Supali T, Wu Y, Bianco AE
    Mol Biochem Parasitol, 2000 Mar 05;106(2):299-302.
    PMID: 10699259
    Matched MeSH terms: DNA Primers/genetics
  19. Molecular Prevalence and Epidemiology of Trypanosoma evansi Among Cattle in Peninsular Malaysia
    Ola-Fadunsin SD, Gimba FI, Abdullah DA, Abdullah FJF, Sani RA
    Acta Parasitol, 2020 Mar;65(1):165-173.
    PMID: 31797192 DOI: 10.2478/s11686-019-00150-9
    BACKGROUND: Animal trypanosomiasis (Surra) caused by Trypanosoma evansi (T. evansi) is known to be one of the important haemoprotozoan parasites that causes great economical loss on animal production due to mortality and loss of condition.

    METHODS: A cross-sectional study was designed to evaluate the prevalence and risk factors associated with T. evansi infection among cattle in Peninsular Malaysia. Polymerase chain reaction (PCR) was employed on 1045 blood samples collected from 43 farms. A well-structured questionnaire was used to collect data on risk factors associated with T. evansi prevalence. The RoTat 1.2 set of primers was used to amplify products of 205 base pair.

    RESULTS: The overall prevalence was found to be 17.9% (187/1045; 95% CI = 15.66-20.31). Trypanosoma evansi was detected among cattle in all the States of Peninsular Malaysia. Breeds of cattle and closeness to waste area, where the risk factors significantly (p 

    Matched MeSH terms: DNA Primers/genetics
  20. Human T-cell receptor V gene segment of alpha and beta families: A revised primer design strategy
    Ch'ng ACW, Chan SK, Ignatius J, Lim TS
    Eur J Immunol, 2019 08;49(8):1186-1199.
    PMID: 30919413 DOI: 10.1002/eji.201747328
    The application of human TCR in cancer immunotherapy has gained momentum with developments in tumor killing strategies using endogenous adaptive immune responses. The successful coverage of a diverse TCR repertoire is mainly attributed to the primer design of the human TCR V genes. Here, we present a refined primer design strategy of the human TCR V gene by clustering V gene sequence homolog for degenerate primer design based on the data from IMGT. The primers designed were analyzed and the PCR efficiency of each primer set was optimized. A total of 112 alpha and 160 beta sequences were aligned and clustered using a phylogram yielding 32 and 27 V gene primers for the alpha and beta family. The new primer set was able to provide 93.75% and 95.63% coverage for the alpha and beta family, respectively. A semi-qualitative approach using the designed primer set was able to provide a relative view of the TCR V gene diversity in different populations. Taken together, the new primers provide a more comprehensive coverage of the TCR gene diversity for improved TCR library generation and TCR V gene analysis studies.
    Matched MeSH terms: DNA Primers/genetics*
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