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Fulltext Technical aspects in understanding effects of gamma irradiation on flower colour changes in Dendrobium Sonia

Sakinah Ariffin, Azhar Mohamad, Ratnam, Wickneswari

Jurnal Sains Nuklear Malaysia, 2012;24(1):91-101.
MyJurnal

Colour is one of the most important traits in orchids and has created great interest in breeding programmes. Gamma irradiation is an alternative way for generation of somaclonal variation for new flower colours. Phenotypic changes are usually observed during screening and selection of mutants. Understanding of targeted gene expression level and evaluation of the changes facilitate in the development of functional markers for selection of desired flower colour mutants. Four Dendrobium orchid sequences (NCBI accessions: AM490639, AY41319, FM209429 and DQ462460) were selected to design gene specific primers based on information for chalcone synthase (CHS) from NCBI database. Quantitative real-time PCR (qPCR) was used to understand flower colour expression quantitatively derived from the CHS gene activities in different flower tissues (petal and sepal) from control Dendrobium Sonia (red purple), mutant DS 35-1/M (purple pink) and mutant DS 35-WhiteA. It was found that expression of CHS gene was highest in sepals of white flowers and lowest in both sepals and petals of purple pink flowers. Genomic DNA was amplified and PCR products were sequenced, aligned and compared. Sequence variations of CHS partial gene in Dendrobium Sonia mutants with different flower colour showed that two protein positions have been changed as compared to the control. These non-synonymous mutations may have contributed to the colour alterations in the white and purple pink mutants. This paper describes important procedures to quantify gene expression such as RNA isolation (quantity and quality), cDNA synthesis and primer design steps for CHS genes.

Matched MeSH terms: DNA Primers
Molecular and ecological investigations on the wild populations of Glycyrrhiza L. taxa distributed in the East Mediterranean Area of Turkey

Altay V, Karahan F, Öztürk M, Hakeem KR, Ilhan E, Erayman M

J Plant Res, 2016 Nov;129(6):1021-1032.
PMID: 27655558

This paper covers studies on the molecular and ecological aspects of G. glabra var. glandulifera, G. flavescens ssp. flavescens and G. echinata collected from Hatay (Turkey); with the aim to better understand their genetic variation and ecological requirements for possible conservation programs. The material including total genomic DNA was extracted by the CTAB, and for PCR reaction, a total of 14 SSR primers developed for Medicago truncatula were used. PCR amplifications were performed in a Multigen(®) Thermal Cycler. Soil samples were analysed for their texture, pH, total soluble salts, calcium carbonate, total N content, total phosphorus and organic matter content. In order to see the association between genetic, ecological and geographical data, a similarity matrix was generated. Genetic similarity distances between genotypes were correlated with those of Eucledian distances obtained from ecological and geographical data. Analysis of molecular variance (AMOVA) was performed using GenAlEx 6.5 software to determine variation among and within genetic variations. The genetic analysis showed that the highest expected heterozygosity values were obtained from G. glabra while the lowest were obtained from G. echinata. In general heterozygosity values were low, especially for G. echinata. Therefore, variation appears to be lower within each species than among three species. The physical and chemical analysis of soil and plant samples indicates that mineral accumulation in plants is substantially affected by the soil characteristics. There is a need for identification of better strategies for the improvement of varieties, especially for small farmers managing marginal soils. More studies should be conducted in order to safeguard these taxa, especially G. glabra var. glandulifera which is collected intensively due to its economic value, the same is true for endemic taxon G. flavescens ssp. flavescens.

Matched MeSH terms: DNA Primers
Fulltext Elucidating the diet of the island flying fox (Pteropus hypomelanus) in Peninsular Malaysia through Illumina Next-Generation Sequencing

Aziz SA, Clements GR, Peng LY, Campos-Arceiz A, McConkey KR, Forget PM, et al.

PeerJ, 2017;5:e3176.
PMID: 28413729 DOI: 10.7717/peerj.3176

There is an urgent need to identify and understand the ecosystem services of pollination and seed dispersal provided by threatened mammals such as flying foxes. The first step towards this is to obtain comprehensive data on their diet. However, the volant and nocturnal nature of bats presents a particularly challenging situation, and conventional microhistological approaches to studying their diet can be laborious and time-consuming, and provide incomplete information. We used Illumina Next-Generation Sequencing (NGS) as a novel, non-invasive method for analysing the diet of the island flying fox (Pteropus hypomelanus) on Tioman Island, Peninsular Malaysia. Through DNA metabarcoding of plants in flying fox droppings, using primers targeting the rbcL gene, we identified at least 29 Operationally Taxonomic Units (OTUs) comprising the diet of this giant pteropodid. OTU sequences matched at least four genera and 14 plant families from online reference databases based on a conservative Least Common Ancestor approach, and eight species from our site-specific plant reference collection. NGS was just as successful as conventional microhistological analysis in detecting plant taxa from droppings, but also uncovered six additional plant taxa. The island flying fox's diet appeared to be dominated by figs (Ficus sp.), which was the most abundant plant taxon detected in the droppings every single month. Our study has shown that NGS can add value to the conventional microhistological approach in identifying food plant species from flying fox droppings. At this point in time, more accurate genus- and species-level identification of OTUs not only requires support from databases with more representative sequences of relevant plant DNA, but probably necessitates in situ collection of plant specimens to create a reference collection. Although this method cannot be used to quantify true abundance or proportion of plant species, nor plant parts consumed, it ultimately provides a very important first step towards identifying plant taxa and spatio-temporal patterns in flying fox diets.

Matched MeSH terms: DNA Primers
Fulltext Absence of nucleotide alteration in region of exon 34 of NOTCH1 and NOTCH2 receptor genes analysed in oral cancer samples: a preliminary observation

Nor Nasyitah Ismail, Khairani Idah Mokhtar

Archives of Orofacial Sciences, 2010;5(1):17-23.
MyJurnal

Oral cancer is one of the common cancer cases identified in the developing countries. Genetic mutation and overexpression of certain genes and proteins have been associated in the development of this cancer. Notch signalling pathway is normally involved in controlling the development process of vertebrates and invertebrates; however, deregulation of this pathway was found to be responsible in the formation of certain cancers including oral cancers. Activation of this pathway requires binding of the ligands to its receptors. Four NOTCH receptors (NOTCH 1, 2, 3 and 4) have been identified in mammals. Disruptions within these molecules might interfere with the normal functions of Notch signalling pathway. Hence, this study was conducted to detect mutations of NOTCH1 and NOTCH2 receptor genes which might be occurring in the oral cancer cases obtained from the local population. DNA extracted from fresh-frozen tissue biopsy of the tongue and buccal mucosa from 10 confirmed cases of oral cancer were subjected for polymerase chain reaction (PCR) amplification using the specific sets of primers. The PCR products were sent for sequencing before final results were analysed.
Due to time and cost limitation, only two out of four NOTCH receptor genes; NOTCH1 and NOTCH2, were used in this analysis. The results revealed absence of nucleotide changes for both NOTCH receptor genes amplified from these oral cancer samples. More samples and further analysis looking into other regions in these genes are required to conclude the involvement of NOTCH receptor genes mutation in causing oral cancer.

Matched MeSH terms: DNA Primers
Fulltext Molecular genetic approaches for the conservation of endangered orchids

Rodrigues, K. F., Yeoh, K. A., Kumar, S. V.

Buletin Persatuan Genetik Malaysia, 2006;12(1):13-14.
MyJurnal

Geographically isolated populations of endemic orchids have evolved and adapted to an existence within specifi c ecological niches. These populations are highly susceptible to anthropogenic
infl uences on their microhabitats. The primary objective of conservation programs is the restoration of endangered populations to their ecologically sustainable levels, and the fi rst stage in the process of conservation involves estimation of molecular diversity at the level of the population. The approach described in this article involves the application of RAPD, Microsatellites and Chloroplast DNA markers for the characterization of the genetic structure of Paphiopedilum rothschildianum and Phalaenopsis gigantea, two endangered and endemic orchids of Sabah. This study has isolated a total of 96 microsatellite loci in P. rothschildianum and P. gigantea, 42 specifi c primer pairs have been designed for amplifi cation of microsatellite loci and are currently being applied to screen the breeding pools. The Chloroplast DNA regions amplifi ed by the primer pairs trnH-psbA and trnL-trnF exhibit distinct polymorphisms and can be used to establish phylogenetic
relationships. The ability of microsatellite loci to cross-amplify selected varieties of orchids has been determined. The molecular markers developed will be applied to estimate population diversity
levels and to formulate long-term management strategies for the conservation of endangered species of orchids of Sabah.

Matched MeSH terms: DNA Primers
Fulltext Development of genetic markers for termitomyces identification

Tan, Chon Seng, Wee, Chien Yeong, Lau, Han Yi Kelly

Buletin Persatuan Genetik Malaysia, 2009;14(1):18-19.
MyJurnal

Termitomyces are delicious edible mushrooms found in Africa and South-East Asia including Malaysia. These mushrooms were found to grow symbiotically with termites around termite nests. Numerous efforts have been made worldwide to develop a cultivation method for these mushrooms. Unfortunately, none of those attempts were successful. The main obstacles encountered were the difficulty to identify and isolate pure termitomyces culture. The problem became prevalent as the culture gets contaminated by other fungi. Termitomyces can easily be identified by its mushroom fruiting body eventually but certainly not at the mycelium and hyphea stages. In this study a simple PCR-based genetic marker detection method for confirmation of termitomyces at any culture stage was developed. Using this method, four distinctive PCR assays
were developed using specific PCR primers designed based on the DNA sequence of the termitomyces mushroom. The PCR results showed that the PCR assays using intact termitomyces
DNA as template was not suitable for this purpose. However, PCR using BamHI and EcoRI predigested termitomyces DNA as template showed identical polymorphism pattern for both
termitomyces mushroom DNA and termitomyces culture DNA. Thus, the method reported here can be used for the identific.

Matched MeSH terms: DNA Primers
Fulltext Characterization of Escherichia coli isolated from cultured catfish by antibiotic resistance and RAPD analysis

Samuel, L., Marian, M.M.,, Apun, K., Lesley, M.B., Son, R.

International Food Research Journal, 2011;18(3):-.
MyJurnal

Antibiotic susceptibility and genetic diversity of E. coli isolated from cultured catfish and their surrounding environment were determined. The levels of resistance of the E. coli isolates towards six different antibiotics tested differed considerably. Though the isolates displayed resistance towards some of the antibiotics tested, none of the isolates showed resistant towards norfloxacin, sulphametoxazole/trimethoprim and chloramphenicol. RAPD-PCR analysis using single primer and primers combination clustered the E. coli isolates into 3 and 5 groups, respectively. The results of this study suggest that the E. coli isolates from the catfish and their surrounding environment derived from a mixture of sensitive and resistant strains with diverse genetic contents. The use of the RAPD analysis is sufficiently discriminatory for the typing of the E. coli isolates.

Matched MeSH terms: DNA Primers
Fulltext Cloning of gene encoding yeast alcohol dehydrogenase 1 (YADH 1) in Escherichia coli TOP10 for biocatalysis

Mohd Rezuan M Aspar, Rashidah Abdul Rahim, Mohamad Hekarl Uzir

Pertanika Journal of Science & Technology, 2011;19(2):423-430.
MyJurnal

Yeast producing alcohol dehydrogenase 1 (YADH 1) enzyme has been used as a biocatalyst for the synthesis of an optically active flavouring compound known as citronellol. However, the slow growth of yeast (Saccharomyces cerevisiae) has deterred the progress of biotransformation. The main purpose of this work is to clone the genes producing YADH1 enzyme from yeast into a faster growing bacteria, Escherichia coli. Initially, the sequence of the gene encoding this protein has been identified in the S. cerevisiae Genome Databases (SGD). The so-called Yadh1 gene sequence is located from coordinate 159548 to 160594 on chromosome XV of yeast. Based on this information, two primer sequences (Forward and Reverse) were constructed. Each of these primers will bind to either end of the Yadh1 gene. The Yadh1 gene was then amplified using Polymerase Chain Reaction (PCR) technique. The amplified Yadh 1 gene was successfully cloned into a cloning vector, TOPO TA plasmid. This plasmid also contains a gene which confers resistance to ampicillin. This recombinant
plasmid was then inserted into Escherichia coli TOP 10 using heat shock protocol at 42oC. Finally, the cloned bacteria containing the recombinant TOPO TA plasmid harbouring Yadh1 gene was able to grow on Luria Bertani (LB) media supplied with antibiotic.

Matched MeSH terms: DNA Primers
Fulltext Optimization of Polymerase Chain Reaction (PCR) of mitochondrial cytochrome c oxidase I (COI) gene in two Bornean fanged frogs

Ramlah Zainudin, Augustine Gawin, Dency Flenny

Pertanika Journal of Science & Technology, 2011;19(1):57-66.
MyJurnal

Limnonectes kuhlii and Limnonectes leporinus are two of the Bornean fanged frogs (without advertisement call) which are widely distributed, thus thought to exhibit different evolutionary lineages and the existence of genetically cryptic species. Yet, the two species are still under study especially at the molecular level. Hence, cytochrome c oxidase I (COI) of mitochondrial gene was used to investigate suitable parameters for DNA amplification using the Polymerase Chain Reaction (PCR) method. Three PCR programmes (varied in the temperatures and period of each PCR step) were employed to identify the most efficient parameters in amplifying PCR products for both species. From the three programmes, Programme B (Initial denaturation: 96°C for 5 min; denaturation: 95°C for 45 sec; annealing: 48-53°C for 1 min 30 sec; extension: 72°C for 1 min 30 sec; final extension: 72°C for 10 min, 30 cycles) showed the highest percentage (53%) of optimal PCR products. The other two programmes showed non-specific products or “primer-dimers”. The results also suggest that the annealing temperature of 52°C, 0.025-0.05 units/µl of 1.5mM Taq polymerase, 0.04 mM of
dNTPs mix and optimal concentrations of magnesium in 50 µl of reaction mixture were sufficient enough to amplify high quality PCR products for both species. However, using Programme B, the re-amplification of the PCR products yielded “primer-dimer”. In addition, a ‘Hot-Start’ PCR method was also applied and mostly yielded in an optimal PCR amplification. Nevertheless, further research on the second amplification of the two species should be conducted to determine the causes of the primer-dimer production.

Matched MeSH terms: DNA Primers
Fulltext Developing new primers for molecular identification of plasmodium spp. recorded in Sabah

Chua, T.H., Stanis, C.S., Song, B.K., Lau, Y.L., Jelip, P., Lau, T.Y.

Borneo Journal of Medical Sciences, 2015;9(1):3-10.
MyJurnal

Malaria is a major public health problem in tropical and subtropical areas, caused by five
species of Plasmodium (P. falciparum, P. vivax, P. malariae, P. ovale andP. knowlesi) and is the leading cause of morbidity and mortality worldwide. We have developed molecular markers for three genes viz, Cytb, dhfr and Msp-1 gene and designed a protocol for rapid molecular diagnostics of the four malaria parasites prevalent in Southeast Asia. The new primers were used on the blood
samples containing Plasmodium parasites by conventional PCR. The result was compared with
the nested PCR of Singh et al. (2004) and the microscopy method. The result shows that the new
set of primers had successfully amplified all four human malaria parasite species. These primers
were 100% sensitive and more specific than microscopy and PCR identification using these
primers was faster than the nested PCR. These alternative primers should provide powerful and
rapid molecular diagnostic method for detecting Plasmodium species as well as providing reliable
data for epidemiology study. These primers have the potential to be combined and used in
multiplex PCR.

Matched MeSH terms: DNA Primers
Fulltext Comparison of DNA extraction efficiencies using various methods for the detection of genetically modified organisms (GMOs)

Tung Nguyen, C.T., Son, R., Raha, A.R., Lai, O.M., Clemente Michael, W.V.L.

International Food Research Journal, 2009;16(1):21-30.
MyJurnal

The ability to detect the presence of transgenes in crop-derived foods depends on the quantity and quality of DNA obtained from a product to be analyzed. The efficiency of DNA extraction protocols differs due to the nature of each food product. In this paper, we described two main DNA extraction protocols and their modifications that have been applied and evaluated for DNA extraction from raw and processed food as well as animal feed. The yield and quality for five categories of food and feed samples namely, raw soybean, raw maize, animal feed, smooth tofu and soymilk are discussed. The statistical interaction analyses showed that the cetyltrimethyl ammonium bromide (CTAB) method was proven to be the best method to extract DNA from raw soybean, maize and animal feed samples which not only obtained high DNA yield of 32.7, 28.4 and 33.4 ng DNA/mg sample respectively, but also produced high quality DNA with the absorbance A260/A280 ratio of 1.9, 1.9 and 2.0, respectively. These DNA were suitable for PCR amplification which produced a 164 bp DNA fragment of the lectin gene from soybean, and a 277 bp DNA fragment of the zein gene from maize. In the processed food category, the Wizard isolation method was found to be the best for the extraction of DNA from smooth tofu and soymilk with the yield of 13.2 and 3.4 ng DNA/mg sample, and the quality of the DNA at the absorbance A260/A280 ratio ranged from 1.9 to 1.7. These DNA were successfully amplified using primers specific to the lectin gene of soybean.

Matched MeSH terms: DNA Primers
Fulltext DNA extraction from ghee and beef species identification using polymerase chain reaction (PCR) assay

Nooratiny, I., Sahilah, A.M., Alif Alfie, A.R., Mohd. Farouk, M.Y.

International Food Research Journal, 2013;20(5):2959-2961.
MyJurnal

A technique to isolate DNA from ghee was developed for the authentication of beef fat product. The method was based on pre-mixed ghee with phosphate buffer solution (PBS) prior to DNA extraction using Epicentre extraction method. The recovery of beef DNA was then analysed by polymerase chain reaction (PCR) using beef species-specific oligonucleotide primers which targeted the mitochondria DNA (mtDNA) of cytochrome b (cyt b) gene. The amplicon was 274 bp in size. The developed ghee extraction method offers a high yield of DNA providing 100 ng per μl and useful for validating beef fat product.

Matched MeSH terms: DNA Primers
Fulltext Detection of malaria parasites in Sabah by nested polymerase chain reaction: a focus of naturally acquired Plasmodium knowlesi infections

Anderios F, Zulaikah Mohamed, Ratnam S, Mohd Yusof Ibrahim, Tajul Ariffin Mohd Awang

Sains Malaysiana, 2008;37(2).

The emergence of primate malaria known as Plasmodium knowlesi in humans, which is always misdiagnosed by microscopy as P. malariae, has contribute to the needs of nucleic acid based technology to be applied in detection and differentiation of malaria parasites. The target DNA sequence of the 18SrRNA gene was amplified by a nested PCR assay for detection and identification of Plasmodium species in 31 Giemsa-stained blood smears examined as P. malariae. The assay demonstrated three samples identified as positive to genus-specific primers but negative to all species-specific primers. Three cases of misdiagnosed species were detected. The samples were diagnosed as P. malariae microscopically, but detected as P. falciparum by PCR assay. Twenty five out of 31 samples were detected as P. knowlesi. None of the samples diagnosed microscopically as P. malariae were identified as P. malariae with the nested PCR assay. Over 80.6% of all malaria cases in this study showed naturally acquired P. knowlesi infections.

Matched MeSH terms: DNA Primers
First Report of Gray Leaf Spot on Pepper Caused by Stemphylium solani in Malaysia

Nasehi A, Kadir JB, Abidin MAZ, Wong MY, Ashtiani FA

Plant Dis, 2012 Aug;96(8):1227.
PMID: 30727084 DOI: 10.1094/PDIS-03-12-0262-PDN

Symptoms of gray leaf spot were first observed in June 2011 on pepper (Capsicum annuum) plants cultivated in the Cameron Highlands and Johor State, the two main regions of pepper production in Malaysia (about 1,000 ha). Disease incidence exceeded 70% in severely infected fields and greenhouses. Symptoms initially appeared as tiny (average 1.3 mm in diameter), round, orange-brown spots on the leaves, with the center of each spot turning gray to white as the disease developed, and the margin of each spot remaining dark brown. A fungus was isolated consistently from the lesions using sections of symptomatic leaf tissue surface-sterilized in 1% NaOCl for 2 min, rinsed in sterile water, dried, and plated onto PDA and V8 agar media (3). After 7 days, the fungal colonies were gray, dematiaceous conidia had formed at the end of long conidiophores (19.2 to 33.6 × 12.0 to 21.6 μm), and the conidia typically had two to six transverse and one to four longitudinal septa. Fifteen isolates were identified as Stemphylium solani on the basis of morphological criteria described by Kim et al. (3). The universal primers ITS5 and ITS4 were used to amplify the internal transcribed spacer region (ITS1, 5.8, and ITS2) of ribosomal DNA (rDNA) of a representative isolate (2). A 570 bp fragment was amplified, purified, sequenced, and identified as S. solani using a BLAST search with 100% identity to the published ITS sequence of an S. solani isolate in GenBank (1). The sequence was deposited in GenBank (Accession No. JQ736024). Pathogenicity of the fungal isolate was tested by inoculating healthy pepper leaves of cv. 152177-A. A 20-μl drop of conidial suspension (105 spores/ml) was used to inoculate each of four detached, 45-day-old pepper leaves placed on moist filter papers in petri dishes (4). Four control leaves were inoculated similarly with sterilized, distilled water. The leaves were incubated at 25°C at 95% relative humidity for 7 days. Gray leaf spot symptoms similar to those observed on the original pepper plants began to develop on leaves inoculated with the fungus after 3 days, and S. solani was consistently reisolated from the leaves. Control leaves did not develop symptoms and the fungus was not reisolated from these leaves. Pathogenicity testing was repeated with the same results. To our knowledge, this is the first report of S. solani causing gray leaf spot on pepper in Malaysia. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) M. P. S. Camara et al. Mycologia 94:660, 2002. (3) B. S. Kim et al. Plant Pathol. J. 15:348, 1999. (4) B. M. Pryor and T. J. Michailides. Phytopathology 92:406, 2002.

Matched MeSH terms: DNA Primers
First Report of Pseudoperonospora cubensis Causing Downy Mildew of Trichosanthes cucumerina in Malaysia

Salati M, Wong MY, Sariah M, Nik Masdek H

Plant Dis, 2010 May;94(5):642.
PMID: 30754434 DOI: 10.1094/PDIS-94-5-0642A

In December 2008, infected leaves of Trichosanthes cucumerina were observed on commercial cucurbit farms located in Pontian, Johor (south of West Malaysia). Bright yellow and small necrotic lesions were observed on the adaxial surface of the leaves, whereas sporangiophores were observed on pale yellowish brown-to-brown lesions on the abaxial surface. The length and width of the sporangia ranged from 19 to 36 μm (28.6) and 11 to 23 μm (17.6), respectively. The length of the sporangiophores ranged from 310 to 450 μm, with an average length of 380 μm. The pathogen was identified as Pseudoperonospora cubensis on the basis of the morphological criteria described by Palti and Cohen (2). To confirm the morphological findings, DNA was extracted from symptomatic tissue and the internal transcribed spacer (ITS) region was PCR amplified using primers ITS5-P2 and ITS4 (3). The appropriate-sized amplicon was gel excised and column purified and then submitted for direct sequencing. The resulting 802 bp amplified ITS region was 100% identical to published P. cubensis sequences (GenBank Accession Nos. EU876603, EU876584, and AY198306). This sequence was deposited with NCBI GenBank under the Accession No. GU233293. In this study, pathogenicity tests were conducted using detached leaf disc assays (1) and a P. cubensis isolate obtained from T. cucumerina. For this purpose, leaf discs were excised from 6- to 8-week-old leaves of T. cucumerina using a 20-mm cork borer. Five leaf discs were placed with their abaxial surface facing upward on moist filter paper in petri dishes. Each of four leaf discs was inoculated with four 10-μl droplets of a 1 × 105 per ml sporangial suspension, whereas the fifth disc was inoculated with water droplets and served as a control. Three replications were completed. The leaf discs were placed in darkness at 14 ± 2°C for 24 h and subsequently incubated with a 12-h photoperiod. After 10 days, sporulation was observed on the sporangia-inoculated leaf discs with similar morphological features to the initial field samples. To our knowledge, this is the first report of P. cubensis causing downy mildew of T. cucumerina in Malaysia. References: (1) A. Lebeda and M. P. Widrlechner. J. Plant Dis. Prot. 110:337, 2003. (2) J. Palti and Y. Cohen. Phytoparasitica 8:109, 1980. (3) H. Voglmayr and O. Constantinescu. Mycol. Res. 112:487, 2008.

Matched MeSH terms: DNA Primers
Dual panel multiplex PCR assay for rapid detection of medically important fungi and resistant species of candida and aspergillus

Jacinta Santhanam, Mohd Hanif Jainlabdin, Ang LC, Tzar Mohd Nizam

Sains Malaysiana, 2018;47:489-498.

Invasive fungal infections (IFIs) have risen dramatically in recent years among high risk immunocompromised patients.
Rapid detection of fungal pathogens is crucial to timely and accurate antifungal therapy. Two multiplex polymerase
chain reaction (PCR) assays were developed to detect major fungal species that cause invasive infections and identify
resistant species. Genus specific primers for Candida, Aspergillus, Fusarium and species specific primers for Candida
glabrata, Candida krusei and Aspergillus terreus which are known to be clinically resistant species, were designed from
the internal transcribed spacer (ITS) regions of ribosomal ribonucleic acid (rRNA) gene complex. Both assays were
performed simultaneously to promote rapid detection of fungal isolates based on distinct amplicon sizes. Inclusion of the
universal fungal primers ITS 1 and ITS 4 in the genus specific assay produced a second amplicon for each isolate which
served to confirm the detection of a fungal target. The limit of detection for the genus specific assay was 1 nanogram
(ng) deoxyribonucleic acid (DNA) for Aspergillus fumigatus and Candida albicans, 0.1 ng DNA for Fusarium solani, while
the species-specific assay detected 0.1 ng DNA of A. terreus and 10 picogram (pg) DNA of C. krusei and C. glabrata. The
multiplex PCR assays, apart from universal detection of any fungal target, are able to detect clinically important fungi
and differentiate resistant species rapidly and accurately, which can contribute to timely implementation of effective
antifungal regime.

Matched MeSH terms: DNA Primers
Fulltext Discovery of novel genic-SSR markers from transcriptome dataset of an important non-human primate, Macaca fascicularis

Chang W, Ee-Uli J, Ng WL, Rovie-Ryan JJ, Tan SG, Yong CSY

Sci Rep, 2019 06 11;9(1):8504.
PMID: 31186469 DOI: 10.1038/s41598-019-44870-4

Macaca fascicularis, also known as the cynomolgus macaque, is an important non-human primate animal model used in biomedical research. It is an Old-World primate widely distributed in Southeast Asia and is one of the most abundant macaque species in Malaysia. However, the genetic structure of wild cynomolgus macaque populations in Malaysia has not been thoroughly elucidated. In this study, we developed genic-simple sequence repeat (genic-SSR) markers from an in-house transcriptome dataset generated from the Malaysian cynomolgus macaque via RNA sequencing, and applied these markers on 26 cynomolgus macaque individuals. A collection of 14,751 genic-SSRs were identified, where 13,709 were perfect SSRs. Dinucleotide repeats were the most common repeat motifs with a frequency of 65.05%, followed by trinucleotide repeats (20.55%). Subsequently, we designed 300 pairs of primers based on perfect di- and trinucleotide SSRs, in which 105 SSRs were associated with functional genes. A subset of 30 SSR markers were randomly selected and validated, yielding 19 polymorphic markers with an average polymorphism information content value of 0.431. The development of genic-SSR markers in this study is indeed timely to provide useful markers for functional and population genetic studies of the cynomolgus macaque and other related non-human primate species.

Matched MeSH terms: DNA Primers
Fulltext Rapid identification of melioidosis agent by an insulated isothermal PCR on a field-deployable device

Chua KH, Tan EW, Chai HC, Puthucheary SD, Lee PC, Puah SM

PeerJ, 2020;8:e9238.
PMID: 32518734 DOI: 10.7717/peerj.9238

Background: Burkholderia pseudomallei causes melioidosis, a serious illness that can be fatal if untreated or misdiagnosed. Culture from clinical specimens remains the gold standard but has low diagnostic sensitivity.
Method: In this study, we developed a rapid, sensitive and specific insulated isothermal Polymerase Chain Reaction (iiPCR) targeting bimA gene (Burkholderia Intracellular Motility A; BPSS1492) for the identification of B. pseudomallei. A pair of novel primers: BimA(F) and BimA(R) together with a probe were designed and 121 clinical B. pseudomallei strains obtained from numerous clinical sources and 10 ATCC non-targeted strains were tested with iiPCR and qPCR in parallel.
Results: All 121 B. pseudomallei isolates were positive for qPCR while 118 isolates were positive for iiPCR, demonstrating satisfactory agreement (97.71%; 95% CI [93.45-99.53%]; k = 0.87). Sensitivity of the bimA iiPCR/POCKIT assay was 97.52% with the lower detection limit of 14 ng/µL of B. pseudomallei DNA. The developed iiPCR assay did not cross-react with 10 types of non-targeted strains, indicating good specificity.
Conclusion: This bimA iiPCR/POCKIT assay will undoubtedly complement other methodologies used in the clinical laboratory for the rapid identification of this pathogen.

Matched MeSH terms: DNA Primers
Evaluation of new multiplex PCR primers for the identification ofPlasmodium species found in Sabah, Malaysia

Stanis CS, Song BK, Chua TH, Lau YL, Jelip J

Turk J Med Sci, 2016 Jan 05;46(1):207-18.
PMID: 27511356 DOI: 10.3906/sag-1411-114

BACKGROUND/AIM: Malaria is a major public health problem, especially in the Southeast Asia region, caused by 5 species of Plasmodium (P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi). The aim of this study was to compare parasite species identification methods using the new multiplex polymerase chain reaction (PCR) against nested PCR and microscopy.
MATERIALS AND METHODS: Blood samples on filter papers were subject to conventional PCR methods using primers designed by us in multiplex PCR and previously designed primers of nested PCR. Both sets of results were compared with microscopic identification.
RESULTS: Of the 129 samples identified as malaria-positive by microscopy, 15 samples were positive for P. falciparum, 14 for P. vivax, 6 for P. knowlesi, 72 for P. malariae, and 2 for mixed infection of P. falciparum/P. malariae. Both multiplex and nested PCR identified 12 P. falciparum single infections. For P. vivax, 9 were identified by multiplex and 12 by nested PCR. For 72 P. malariae cases, multiplex PCR identified 58 as P. knowlesi and 10 as P. malariae compared to nested PCR, which identified 59 as P. knowlesi and 7 as P. malariae.
CONCLUSION: Multiplex PCR could be used as alternative molecular diagnosis for the identification of all Plasmodium species as it requires a shorter time to screen a large number of samples.

Matched MeSH terms: DNA Primers
Fulltext Data on partial polyhydroxyalkanoate synthase genes (phaC) mined from Aaptos aaptos marine sponge-associated bacteria metagenome

Amelia TSM, Amirul AA, Bhubalan K

Data Brief, 2018 Feb;16:75-80.
PMID: 29188224 DOI: 10.1016/j.dib.2017.11.011

We report data associated with the identification of three polyhydroxyalkanoate synthase genes (phaC) isolated from the marine bacteria metagenome of Aaptos aaptos marine sponge in the waters of Bidong Island, Terengganu, Malaysia. Our data describe the extraction of bacterial metagenome from sponge tissue, measurement of purity and concentration of extracted metagenome, polymerase chain reaction (PCR)-mediated amplification using degenerate primers targeting Class I and II phaC genes, sequencing at First BASE Laboratories Sdn Bhd, and phylogenetic analysis of identified and known phaC genes. The partial nucleotide sequences were aligned, refined, compared with the Basic Local Alignment Search Tool (BLAST) databases, and released online in GenBank. The data include the identified partial putative phaC and their GenBank accession numbers, which are Rhodocista sp. phaC (MF457754), Pseudomonas sp. phaC (MF437016), and an uncultured bacterium AR5-9d_16 phaC (MF457753).

Matched MeSH terms: DNA Primers

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