In this study polymerase chain reaction (PCR) was used to identify yeast in domestic ragi obtained
from two local markets in Sarawak and Pahang. These ragi are normally used as a dry starter in food fermentation (tapai) for Pahang (ST2) and Sarawak (ST3) and tuak (ST1) which is an alcoholic drink in Sarawak. Universal primer, NL1 and NL4 were used as a primer in this study to amplify D1/D2 fragment. Based on the result from the sequencing and after the BLAST search of the nucleotide sequences, the strain was confirmed as Candida glabrata (FN424108.) partial 26S rRNA gene, strain IMUFRJ 51955 for ST1, Saccharomyces cerevisiae(EU285514.1) isolate 35 26S ribosomal RNA gene, partial sequence for ST2 sample and Candida glabrata (FN393990.1) partial 26S rRNA gene, strain MUCL 51244 for ST3. All these strains were found in domestic ragi used for food fermentation.
Mushroom can be used as a biological indicator in assessing radiological impact on the
environment. Radiological effect would be reflected through morphological changes as well as
those changes at molecular level. For this purpose, a preliminary work was conducted, which
included DNA isolation, optimization of PCR parameters for Inter-Simple Sequence Repeat (ISSR)
and primers screening on Pleurotus sajor caju mushroom strains from Nuclear Malaysia’s
Sterifeed Mushrooms Collection Centre. In this work, DNA isolation technique from cap and stalk
of fruit body were optimized and quantified. It was found that stalk produced highest amount of
genomic DNA at 304.01ng/µl and cap at 149.00ng/µl. A total of 100 ISSR primers were tested and
51 primers were successfully amplified. These primers will be used further for dose response
evaluation and molecular profiling in mushroom species.
The main source of E. coli 0157:H7 is cattle, but recent studies showed high percentage of outbreaks
contributed by contaminated water. The occurrence of E. coli O157:H7 in environmental water samples poses a potential threat to human health. The aim of this study was to establish a protocol for the detection of the pathogen E. coli O157:H7 and E. coli virulence genes (eaeA, rfbE, hly, stx1, and stx2) in a multiplex PCR protocol using six specific primer pairs. The target genes produced species-specific amplicons at 625 bp, 397 bp, 296 bp, 166 bp, 210 bp and 484 bp for E. coli O157:H7 (fliCh7 gene) and virulence genes (eaeA, rfbE, hly, stx1, and stx2) respectively. The results obtained show that the established PCR protocol is suitable for a rapid and specific analysis of the pathogenic E. coli O157:H7 in environmental water samples for the assessment of microbiological risks.
In this study, RAPD-PCR and ERIC-PCR were used to study the epidemiology of V. parahaemolyticus isolated from cockles in Padang, Indonesia. The Gold Oligo OPAR3 primer produced bands ranged from 1-8 with sizes from 0.2 – 5.0 kb and the Gold Oligo OPAR8 primer produced 1-7 bands with sizes 0.7 – 1.5 kb. Both primers produced twenty five RAPD patterns with a few isolates failed to produce any products. Based on phylogenetic dendrogram, all the isolates can be divided into 6 major clusters with similarity between 0 to 52%. For the ERIC primer, it produced bands ranged from 3-15 with sizes from 0.1 – 5.0 kb and twenty seven different ERIC patterns. Construction of the phylogenetic dendogram showed the isolates can be divided into 4 major clusters with similarity between 56 to 86%. The high diversity of both processes may be due to the multiple contamination sources of V. parahaemolyticus.
A total of 78 samples comprising different types of street foods, sold in different locations in Malaysia, were examined for the presence of Enterobacter cloacae. E. cloacae contamination was recorded in 9% of the samples examined. Tests for susceptibility to 12 different antibiotics showed that all were resistant to six or more antibiotics, but susceptible to chloramphenicol and gentamicin. Plasmids of four different sizes were detected from the three plasmid positive isolates. RAPD analysis using four primers yielded completely different banding patterns for all E. cloacae studied. In Malaysia, no published information on street foods in the epidemiological investigation of E.cloacae related disease is available. However, their occurrences have provided compelling evidence that the risk of disease transmission caused by E. cloacae through street foods is moderate.
In Malaysia, harmful algal blooms often occur along the coastal waters of west Sabah, where one of the causative organisms is the toxin-producing dinoflagellate, Pyrodinium bahamense var. compressum. A total of five P. bahamense var. compressum isolates were obtained from four locations and were cultured in f/2 medium. A Polymerase Chain Reaction (PCR) based technique was developed and used to screen for the presence of the dinoflagellate, P. bahamense var. compressum. A dinoflagellate-specific primer pair was designed based on sequences of P. bahamense var. compressum to amplify the 18S small subunit ribosomal DNA (rDNA) sequences. The rDNA of the P. bahamense var. compressum isolates were obtained. A species-specific primer pair was designed to target a 600 bp rDNA sequence of the target dinoflagellate. The primer pair targeting P. bahamense var. compressum did not yield any product with the fifteen algae cultures used as negative controls, but only amplified the rDNA of P. bahamense var. compressum cultures. The PCR method for identification of P. bahamense var. compressum was also applied on twenty field samples collected with plankton net. P. bahamense var. compressum cells were detected by PCR in five field samples and were confirmed by direct sequencing. From this study, a species-specific primer pair was obtained to identify the target species, P. bahamense var. compressum, among the natural complex communities of seawater.
The genus Streptomonospora is a group of extremely halophilic filamentous actinomycetes that form a distinct branch in the 16S rRNA gene phylogenetic tree adjacent to the genera Nocardiopsis and Thermobifida, family Norcadiopsaceae. To date, genus Streptomonospora only contain two validly described species which are Streptomonospora salina and Streptomonospora alba. During a biodiversity study on halophilic filamentous actinomycetes from 18 co-ordinates in Barrientos Island, Antarctic, numerous actinomycetes strains were isolated. To identify whether these isolates were members of the genus Streptomonospora, a genus specific primer that allow the rapid detection of the genus Streptomonospora by means of PCR amplification was used. Furthermore molecular cloning was performed to make identical and multiple copies of the target gene. In addition, morphological characteristic identification was performed to validate isolates with positive amplification during PCR.
Basal stem rot (BSR) caused by species Ganoderma, is one of the most serious disease of oil palm in Malaysia. As far as the disease problem to oil palm in Malaysia is concerned, BSR is the only disease requiring urgent solution. The BSR is not new to Malaysia, it has been known to attack oil palm since the early years when the crop was introduced into this country. There is an indication that there are differences in susceptibility to basal stem rot between germplasm materials from different genetic origin [2]. This provides hope in generating oil palm varieties with reduced level of susceptibility using existing genetic materials. There is also interest in developing diagnostic tools such as using PCR primers for detection of the pathogen in oil palms [1]. Altered expression of several classes of genes was observed in plants in response to fungal infection. These include genes associated with cell maintenance and development, genes involved in biosynthesis of lignin and phenolics and genes implicated in oxidative burst, programmed cell death or hypersensitive response [5].
This study looked at mutagenic effectiveness of gamma rays d on two varieties of Zingiber officinale Roscoe: Bentong and Tanjung Sepat. The rhizomes were exposed to different doses (0, 5, 7, 9, 11, 13 and 15 Gy) using Caesium-137 as source of the gamma rays. The effect of different gamma doses on the crude fibre composition of irradiated ginger was studied and genetic variability was assessed using molecular marker technique, RAPD. Findings showed different doses of gamma rays could induce variability in these two ginger varieties and the effect was found to be variety-dependent. Bentong variety irradiated with 9 Gy recorded 8.53% of crude fibre composition while Tanjung Sepat irradiated ginger with 5 Gy recorded 8.70% of crude fibre which gave the lowest composition compared with other irradiated ginger. A total of nine different arbitrary decamers were used as primers to amplify DNA from mutant plant material to assess their polymorphism level of ginger mutant lines. Polymorphism of all mutant lines was 97.62% indicating that there were significant changes in genetic sequences in irradiated ginger genotypes.
The present study was conducted to assess the rapid molecular identification and characterization of 45 Vibrio parahaemolyticus isolates from 15 samples of 3 different types of fish (Kembung, Bawal and Sangeh) in the Kuching-Samarahan district. Polymerase chain reaction (PCR) based confirmation was done targeting the 450 bp fragment of the thermolabile (tl) gene, while DNA fingerprinting was performed using Randomly Amplified Polymorphic DNA (RAPD) PCR with the primer GEN15008. All the 45 V. parahaemolyticus isolates were positive for the tl gene, however, only 34 were typable via RAPD-PCR with bands sizes ranging from slightly over 250 bp to 2.5 kbp. The degree of diversity was then determined via the Simpson Index which showed a value of 0.891, indicating high diversity among the isolates. Data from the RAPD-PCR fingerprints were later used to construct a dendrogram for clustal analysis. From the dendrogram, the 34 isolates were grouped into 2 major clusters containing 26 and 8 isolates, respectively. Further analyses of the dendrogram also indicated that the 34 isolated were clustered according to the period of sampling. This is an interesting observation as it shows the high discriminatory capability of RAPD-PCR to be used as molecular epidemiological tool to study the temporal distribution of V. parahaemolyticus.
Three polymerase chain reaction (PCR) techniques were compared to analyse the genetic diversity of Clinacanthus nutans eight populations in the northern region of Peninsular Malaysia. The PCR techniques were random amplified polymorphic deoxyribonucleic acids (RAPD), inter-simple sequence repeats (ISSR) and random amplified microsatellite polymorphisms (RAMP). Leaf genomic DNA was PCR amplified using 17 RAPD, 8 ISSR and 136 RAMP primers . However, only 10 RAPD primers, 5 ISSR primers and 37 RAMP primers produced reproducible bands. The results were evaluated for polymorphic information content (PIC), marker index (MI) and resolving power (RP). The RAMP marker was the most useful marker compared to RAPD and ISSR markers because it showed the highest average value of PIC (0.25), MI (11.36) and RP (2.86). The genetic diversity showed a high percentage of polymorphism at the species level compared to the population level. Furthermore, analysis of molecular variance revealed that the genetic diversity was higher within populations, as compared to among populations of C. nutans. From the results, the RAMP technique was recommended for the analysis of genetic diversity of C. nutans.
This study reports the results of the partial DNA sequence analysis of the 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) gene in glyphosate-resistant (R) and glyphosate-susceptible (S) biotypes of Eleusine indica (L.) Gaertn from Peninsular Malaysia. Sequencing results revealed point mutation at nucleotide position 875 in the R biotypes of Bidor, Chaah and Temerloh. In the Chaah R population, substitution of cytosine (C) to adenine (A) resulted in the change of threonine (Thr106) to proline (Pro106) and from C to thymidine (T) in the Bidor R population, leading to serine (Ser106) from Pro106. As for the Temerloh R, C was substituted by T resulting in the change of Pro106 to Ser106. A new mutation previously undetected in the Temerloh R was revealed with C being substituted with A, resulting in the change of Pro106 to Thr106 indicating multiple founding events rather than to the spread of a single resistant allele. There was no point mutation recorded at nucleotide position 875 previously demonstrated to play a pivotal role in conferring glyphosate resistance to E. indica for the Lenggeng, Kuala Selangor, Melaka R populations. Thus, there may be another resistance mechanism yet undiscovered in the resistant Lenggeng, Kuala Selangor and Melaka populations.
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.
The knowledge of genetic diversity of tree crop is very important for breeding and improvement program for the purpose of improving the yield and quality of its produce. Genetic diversity study and analysis of genetic relationship among 20 Moringa oleifera were carried out with the aid of twelve primers from, random amplified polymorphic DNA marker. The seeds of twenty M. oleifera genotypes from various origins were collected and germinated and raised in nursery before transplanting to the field at University Agricultural Park (TPU). Genetic diversity parameter, such as Shannon's information index and expected heterozygosity, revealed the presence of high genetic divergence with value of 1.80 and 0.13 for Malaysian population and 0.30 and 0.19 for the international population, respectively. Mean of Nei's gene diversity index for the two populations was estimated to be 0.20. In addition, a dendrogram constructed, using UPGMA cluster analysis based on Nei's genetic distance, grouped the twenty M. oleifera into five distinct clusters. The study revealed a great extent of variation which is essential for successful breeding and improvement program. From this study, M. oleifera genotypes of wide genetic origin, such as T-01, T-06, M-01, and M-02, are recommended to be used as parent in future breeding program.
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.
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.
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.
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.
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.
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.