Beta (β)- thalassaemia is a public health problem in Malaysia. The carrier rate is estimated to be 4.5% by micro-mapping studies particularly among Malays who comprise 53.5% of the population in Malaysia. The common diagnostic method in Malaysia for mutation detection is by amplification refractory mutation system (ARMS). It allows single mutation detection in each reaction but is labour intensive and time consuming when many mutations need to be identified. The purpose of this study was to develop a diagnostic tool for effective mutation detection of beta thalassaemia in Malay patients and compare its efficacy with ARMS-PCR, the current method in use. Methods: Reverse dot blot hybridization (RDBH) technique was incorporated in the development of two strip assays [RDBH-Strip M(6) and RDBH-Strip C(6)] to identify common beta thalassaemia mutations in the Malays. The panels of selected mutations were based on the mutation frequencies in Malaysia reported in previous studies. RDBH-Strip M(6) was applied as step 1 and RDBH-Strip C(6) was applied as step 2 for unidentified mutations. The strips were validated with the gold standard method, ARMS- PCR. Results: One hundred and thirty seven Malay patients with 274 alleles were studied. In Step 1 mutation detection, 238 alleles (86.9%) were identified in 119 of patients by RDBH-Strip M(6). Step 2 resulted in a further detection of 20 alleles in another 10 patients by RDBH-Strip C(6). The combination of both strips resulted in the identification of 258 alleles in 129 (94.6%) of 137 Malay patients. The strip assays were 100% sensitive and specific when compared with ARMS-PCR method. Conclusion: Two strip assays utilising the RDBH technique were developed to identify common β-thalassaemia mutations in Malays. The RDBH Strip M(6) identified 86.9% of the mutations and the RDBH-Strip C (6) detected further 7.3% alleles. This two step strategy was found to be rapid and cost effective for the direct diagnosis of β-thalassaemia mutations in the Malays. The remaining unidentified mutations would require DNA sequencing. It can serve as a specific molecular diagnostic tool for effective diagnosis of
β-thalassaemia mutations in this ethnic group.
Human herpesvirus-6 (HHV-6) levels have been considered as markers for various diseases. The aim of this study was to evaluate the prevalence of HHV-6 infection in healthy adults in Malaysia. Methods: The level of HHV-6 in saliva was investigated in 36 healthy adults, age 19 to 23 years, at Kuala Lumpur, Malaysia using variant-specific TaqmanTM quantitative real-time PCR (qPCR). Results: The amount of HHV-6 DNA in the saliva of healthy adults ranged from negative to 10,000 HHV-6 genomes/ml of saliva (median, 360 genomes/ml of saliva). Of the 36 samples tested, 30 (83%) contained HHV-6 DNA. HHV-6B was the only variant detected in the saliva of all the positive cases. Conclusions: The detection of HHV-6 DNA in saliva by real-time PCR assay provides a sensitive and specific quantitation of HHV-6. Our pilot study suggests the wide prevalence of HHV-6 in saliva from
healthy adults.
We aimed to establish a method for quantitative analysis of mixed haematopoietic chimerism based on microchip electrophoresis of selected molecular markers following PCR amplification for accurate monitoring of graft status post-transplantation. A 12-year-old girl with relapsed acute lymphoblastic leukaemia who underwent allogeneic bone marrow transplantation had qualitative chimerism analysis using short tandem repeat markers at three time points following the procedure. Her archived DNA samples were then used to test the ability to correlate her clinical course with changes in the quantity of donor chimerism at the different time points. Quantitative chimerism analysis was performed on the Agilent 2100 bioanalyser and donor-recipient ratios were calculated from generated electropherograms. Complete donor chimerism (98%) was demonstrated three weeks post- transplantation. Decreasing amount of donor chimerism to 24% was shown after three months and this concurred with clinical relapse. Following a second transplant, full donor chimerism was reestablished where donor chimerism rose to 100%. High resolution microchip electrophoresis could be useful in predicting the occurrence of increasing recipient chimerism which may herald impending relapse in patients while the disease burden is still low. This investigational approach may provide useful information for clinicians to select appropriate intervention strategies to ensure successful transplantation.
Assisted Reproductive Technology (ART) is a suite of laboratory techniques designed to rescue infertile phenotypes. While ART has led to the birth of 5 million ART babies worldwide, success rates rarely exceed 40%. One potential factor for this could be iatrogenic (‘clinician-induced’) damage to critical sperm proteins, such as phospholipase C zeta (PLCζ) and protamine, which are fundamental for oocyte activation and sperm DNA integrity, respectively. This report describes how we have begun to investigate the adverse effects of ART techniques upon these key sperm proteins. We also describe the pathway taken by Miss Suseela Yelumalai to acquire a scholarship from the Malaysian Government and her postgraduate experience at the University of Oxford. We introduce the facilities and learning opportunities available at the Institute of Reproductive Sciences (IRS) which houses Dr Kevin Coward’s research laboratory, and finally, highlight the potential for collaborative development between the Universities of Oxford and Malaya.
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.
Presence of Norovirus in food can cause viral gasteroenteritis. Recently, lots of reports relating to Norovirus in food have been published. Special attention must be paid to the raw foods as they are not subjected to further heat treatment. In this study, pegaga, kesum, tauge and ulam raja (popular salad vegetables in Malaysia) were investigated for Norovirus. A total of 32 samples from each type of salad vegetables were purchased from local market and analyzed using One-step RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction) for both genogroups namely Norovirus Genogroup I and Genogroup II. Results showed that tauge had the highest contamination with Norovirus Genogroup I (15.6%) comparing to pegaga (9.4%), kesum (12.5%)
and ulam raja (0%). Samples were free from Norovirus Genogroup II. The study showed that raw vegetables are high-risk foods and can be contaminated with Norovirus.
A total of 32 clinical strains of Vibrio cholerae, including members of the 01 and 0139 serogroup
were collected from Klang, Selangor; Penang Island; Samarahan, Sarawak and Miri, Sarawak in Malaysia. In general, all the isolates except the 0139 serotype expressed low resistance to all the antibiotics tested with their Multiple Antibiotic Resistance (MAR) indices ranged from 0.10 to 0.48. The presence of ctx gene that encoded the cholera toxin was confirmed in all these clinical isolates by polymerase chain reaction. The results from the RAPD-PCR were analyzed using the RAPDistance software (Version 1.04). From the dendrogram generated, two main groups were observed which were subdivided into two clusters each. The Selangor’s isolates and the 0139 Penang’s isolates formed one group whereas the Samarahan, Sarawak isolates and the Miri, Sarawak isolates made up the other group, thus delineating their different sources of origin based on their geographical location.
Matched MeSH terms: Random Amplified Polymorphic DNA Technique
Vibrio cholerae still represents a significant threat to human health worldwide despite the advances in hygiene, consumer knowledge, food treatment and food processing. In Malaysia, statistics in year 2009 have shown that among the food and water borne diseases, food poisoning has the highest incidence rate of 36.17 per 100,000 populations and with a mortality rate of 0.01 per 100,000 populations. In this study, 22 seafood samples comprising of fish, squid, crustacean and mollusks purchased from wet market and supermarket were analyzed. The Most Probable Number (MPN) and real time PCR was used to enumerate the Vibrio cholerae in seafood sample. The results showed that MPN-real time PCR of the samples from wet market had a maximum of >1100 MPN/g compare to 93 MPN/g enumerated from the MPN plate. The MPN-real time PCR in the samples from supermarket indicated 290 MPN/g as compared to 240 MPN/g enumerated from the MPN plate. The standard curves showed that there was a good linear correlation between the Ct values. The minimum level of detection of Vibrio cholerae standard DNA at targeted gene was 3 x 10-5 ng/μl.
Objective: to review the field of epigenetics, and present basic and recent material that may be of interest to clinical psychiatrists. We include basic molecular mechanism, a consideration of findings related to mental disorders, evidence of sustained effects, and the evidence for and implications of transgenerational epigenetic modifications. Method: we examined all the available papers for the last five years identified by PubMed using the words ‘epigenetics’ and ‘epigenetics psychiatry’, and the available leading specialized textbooks. Results: we report on molecular mechanisms including DNA and histone modifications, and non-coding RNAs. While some modifications are short-lived, others are life-long. Depression, suicide, schizophrenia, PTSD, borderline personality disorder and drug addiction are among the conditions for which epigenetic involvement has been proposed. Transgenerational epigenetics enables the environmental experience of one generation to be non-genetically inherited by subsequent generations. This has been molecularly demonstrated in laboratory animals and epidemically suggested in humans. Conclusions: epigenetics provides a new way of understanding human behavior and points to potential therapies for mental disorders. Should it transpire that transgenerational epigenetic modifications apply with force in humans as they do to laboratory animals, this will emphasize the need for cultural shift, safe societies with ample opportunities.
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked red blood cell enzymopathy common in malaria endemic areas. Individuals affected by this disease show a wide variety of clinical signs including neonatal jaundice. In this preliminary report we describe the heterogeneity of G6PD deficient gene in neonatal jaundice in the Malay population in Kelantan. Thirteen G6PD deficient Malay neonates with hyperbilirubinemia were subjected to mutation analysis of the G6PD gene for known candidate mutations. Molecular defects were identified in the 13 patients studied. Though all of these were mis-sense mutations, identified nucleotide changes were heterogeneous. Six patients were found to have a C to T nucleotide change at nucleotide 563 of the G6PD gene (C563T), corresponding to G6PD Mediterranean; three cases had a single nucleotide change at T383C (G6PD Vanua Lava), two cases had G487A (G6PD Mahidol) and two cases had G1376T (G6PD Canton). These findings suggest that there are heterogeneous mutations of the G6PD gene associated with neonatal jaundice in the Malay population in Kelantan.
(GTG)5 PCR is a type of repetitive extragenic palindromic (rep)-PCR which amplifies the (GTG)5 repetitive element that lays throughout the bacterial genome. In this study, fifty, thirty-nine and forty-nine unknown bacteria were isolated from aquaculture farms in Miri, Limbang and Lundu, respectively. (GTG)5 PCR was used to screen for clonal diversity among the isolates according to sampling sites. Banding profiles obtained from electrophoresed (GTG)5 PCR products were analyzed by RAPDistance Software to generate a dendrogram of neighbor joining tree (NJT) format. Based on the constructed dendrogram, representative isolates were selected for further identification. Conserved 16S rRNA region of the selected bacteria isolates were amplified and purified DNA products were sequenced. (GTG)5 PCR is useful in differentiation of unknown bacterial isolates and 16S rRNA analysis species identity of the bacteria in Sarawak aquaculture environment. The high diversity of bacteria in aquaculture environment may be caused by contamination from various sources.
Pathogenic Vibrio parahaemolyticus is one of the leading causes of bacterial gastroenteritis in many countries. Among the strains examined, 36 RAPD-types were found when amplified with primers OPA8 and OPA10. The analysis shows the majority of V. parahaemolyticus isolates originated from seafood were branched into four major clusters at 18.2%, 20.7% 34% and 3.4% similarity levels. This suggests that there is potential for a single strain to be distributed widely within a population and there also potential for multiple contaminating strains of different clonal lineages to be present within the same population. Optimum temperature (37ºC) was the highest and stable formation of biofilm. The total percentage of biofilm formation at 37ºC was 33.33% for each of weak, moderate and strong biofilm producers. Room temperature produces 61.1% of weak biofilm producer, while 13. 89% for moderate biofilm producers and produce 25% of strong biofilm. While a total of 91.67% weak biofilm producers at 4ºC and 8:33% for room temperature and no growth of strong biofilm. Upon analysis, strong biofilm was tracked from the largest group at 37°C and room temperature which produce 27.27% of strong biofilm producer respectively. Interestingly, they are derived from cockles.
Matched MeSH terms: Random Amplified Polymorphic DNA Technique
In Malaysia, halal certification status for some surimi-based product is still suspicious due to the incorporation of non-halal plasma protein additives as part of the food ingredient. This study was conducted to determine the presence of plasma protein additives that have been incorporated into surimi-based product using Polymerase Chain Reaction (PCR)-Southern Hybridization method which able to differentiate 7 type (beef, chicken, duck, goat, buffalo, lamb and pork) of species on a single chip. A random of 17 (n = 17*3) different brands of surimi-based product was purchased from Selangor local market in January 2013. Of 17 brands, 3 (n = 3*3) brands were positive for chicken DNA and 1 (n = 1*3) brand was positive for goat DNA, while remainder 13 brands (n = 13*3) has no DNA species detected. In presence study, it is evidence that PCR-Southern Hybridization analysis offered a reliable result due to its highly specific and sensitive properties in detecting plasma protein incorporation in surimi-based product.
The tetraspanin gene, CD151 is involved in various tumour cell progression and metastasis. Its expression is increased in high grade, estrogen receptor negative and c-erbB-2 positive breast cancer. However, the biological function and expression phenotype among different tumour status, estrogen receptor (ER) status, progesterone receptor (PR) status and c-erbB-2 expression in multi-ethnic Malaysian breast cancer patients has not been well investigated. We used quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) to measure the CD151 gene expression in 45 breast cancers. Our preliminary results revealed that CD151 expression is significantly higher in ER positive and PR positive breast cancers at 95% and 99% confidence intervals, respectively. In contrast, there is no significant correlation between CD151 expression and tumour grades or c-erbB-2 status at 95% confidence interval level. Our preliminary findings suggested that CD151 may be involved in the estrogen responsive pathways. CD151 could be a potential prognostic marker and therapeutic target in the treatment of estrogen dependent breast cancer patients.
Thalassaemia screening programme has been conducted in Malaysia since 2004. The aim of the programme was to reduce the burden of the disease by identifying thalassaemia carriers. However, the response towards the screening activities was unsatisfactory as there was lack of public awareness against the importance of thalassaemia screening. An alternative approach is to screen blood donors. The purpose of this study was to observe the prevalence of thalassaemia carriers among healthy blood donors. Seven hundred and thirty eight healthy blood donors were screened in Hospital Tengku Ampuan Rahimah, Klang from July to September 2010 using cation-exchange high performance liquid chromatography (HPLC). Cases with haemoglobin variants were further analyzed by gel electrophoresis at alkaline pH. Result shows that the blood donors consisted of 413 Malays (56%), 162 Indians (22%), 148 Chinese (20%) and 15 others (2%). There were 19 (2.6%) individuals with haemoglobin E trait, six (0.8%) with co-inheritance of haemoglobin E and αα- thalassaemia and five (0.7%) with β-thalassaemia trait. Haemoglobin Constant Spring and haemoglobin A2 prime were observed in two (0.3%); and Haemoglobin Lepore and alpha chain variant in one (0.2%). αα-thalassaemia and normal haemoglobin A2 β-thalassaemia could not be excluded in 190 cases (26%), as they required deoxyribonucleic acid (DNA) studies for identification. Thalassaemia screening in blood donors is more feasible and effective. Therefore, a wider scale population screening including blood donors could benefit the existing thalassaemia screening programme in Malaysia.
The application of PCR technique in genetic screening was demonstrated using the genetic materials from buccal cells of the students in the class. Two factors were taken into consideration when designing the experiments. The DNA region to be amplified should not be associated with any disease state. This is to eliminate any emotional and ethical problems associated with the experiments. In this practical, the presence and absence of a 38 bp sequence in the intron of COLIA2 gene were studied. The students were also shown on how to analyse the presence of homozygous and heterozygous alleles and the genetic variations that might be observed in the different ethnic groups of students. Another factor was the time taken to complete the experiment. Our experience showed that this experiment would take at least six hours to obtain and analyse the results. It is therefore suitable to be used in class teaching.
Introduction: We aim to investigate the effect of vasectomy on the histology of the testis as well as to evaluate DNA fragmentation in testicular tissue of male mice. Methods: Bilateral vasectomy was performed on 20 mature male mice; 10 control mice underwent sham-operation. After 6 weeks, the testes were evaluated for histological changes and DNA fragmentation by single cell gel electrophoresis (comet assay). Results: Marked alterations were observed in the testes of vasectomized mice, including degeneration of spermatids, thickened basement membrane, dilatation of the seminiferous tubules, exfoliation of germ cells, reduction in the seminiferous cell population, vacuolated appearance of the epithelium in the tubules and marked interstitial fibrosis. Single cell gel electrophoresis showed a highly significant (P
Many studies have shown that probiotic strains added to a number of probiotic products are not compatible to that of
claimed. It is thus of note to validate probiotic strains added to probiotic products. In this study, three probiotic drinks,
A, B and C, were cultured on MRS agar and the number of bacterial colonies was enumerated. The bacterial counts
recovered from A (9.3 ± 6.9 log CFU/ml) and C (9.0 ± 6.9 log CFU/ml) were signifi cantly higher than B (5.2 ± 3.5 log
CFU/ml) and achieved the minimal amount recommended for probiotic bacteria. All of the isolates appeared as gram
positive rods microscopically and were proven to be catalase negative. However, there were only A1, A2, B4 and C1 that
were highly tolerant to the gastrointestinal pH 3 to 6. The four isolates produced and secreted antimicrobial substances
which inhibited the growth of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). C1 showed the greatest
growth inhibition by forming 17.50-mm and 17.85-mm inhibition zones against E. coli and S. aureus, respectively. The
16s rDNA sequencing and phylogenetic analysis were performed to further identify the twelve isolates. The twelve isolates
were found to be Lactobacillus (L.), particularly L. casei and L. paracasei. However, the bacteria isolated from drink B
were incompatible to the labelled ones. In conclusion, probiotic drinks are possible to contain different bacterial counts
and probiotic strains from the labelled ones. These differences might affect health benefi ts rendered by probiotic strains
to consumers.
The human genome contains many submicroscopic copy number variations which includes deletions, duplications and insertions. Although conventional karyotyping remains an important diagnostic tool in evaluating a dysmorphic patient with mental retardation, molecular diagnostic technology such as array comparative genomic hybridization (aCGH) has proven to be sensitive and reliable in detecting these submicroscopic anomalies. A 3 month-old infant with dysmorphic facies, microcephaly and global developmental delay was referred for genetic evaluation. Preliminary karyotyping which was confounded by the quality of metaphase spread was normal; however, aCGH detected a 30.6Mb deletion from 5p15.33-p13.3. This case illustrates the usefulness of aCGH as an adjunctive investigative tool for detecting chromosomal imbalances.
Background: The tissue specimens used for extraction of DNA in this study were from rodents trapped in four states in Peninsular Malaysia, namely Kedah, Kelantan, Selangor and Johor. Methods: Histological sections of these rodent muscle tissues stained with hematoxylin and eos in showed infection with Sarcocystis spp. Based on these results, the current study was carried out to determine the phylogenetic relationship among the identified Sarcocystis spp. in these rodents.The formalin fixed paraffin embedded (FFPE) rodent muscle blocks were subjected to DNA extraction and followed with semi nested PCR targeting 5’ and 3’ regions of 18S rRNA of Sarcocystis spp. Results: Phylogenetic analysis showed two distinct groups of Sarcocystis spp. among the rodents in Peninsular Malaysia. Most of the identified Sarcocystis spp. were genetically closely related to Sarcocystis rodentifelis and Sarcocystis muris and were also observed to be genetically closely related to Sarcocystis sp. ex Columba livia and Sarcocystis sp. cyst type I ex Anser albifrons. Conclusion: Further classification to confirm these Sarcocystis spp. was not possible as only partial sequences of 18S rRNA was available and this was insufficient for
optimal differentiation.