Displaying publications 1 - 20 of 314 in total

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  1. Fulltext Predominance of G to A codon 12 mutation K-ras gene in Dukes' B colorectal cancer
    Zulhabri O, Rahman J, Ismail S, Isa MR, Wan Zurinah WN
    Singapore Med J, 2012 Jan;53(1):26-31.
    PMID: 22252179
    K-ras gene mutations in codons 12 and 13 are one of the earliest events in colon carcinogenesis.
    Matched MeSH terms: Polymerase Chain Reaction/methods
  2. An improved rapid genotyping method for the study of beta-2 adrenergic receptor gene polymorphisms using single tube allele specific multiplex PCR
    Zilfalil BA, Hoh BP, Nizam MZ, Liza-Sharmini AT, Teh LK, Ismail R
    J Clin Pharm Ther, 2006 Dec;31(6):637-40.
    PMID: 17176369
    Seventeen single nucleotide polymorphisms (SNPs) have been identified so far, within the beta-2 receptor (beta(2) AR) gene. The presence of so many SNPs within the beta(2) AR gene causes a problem, for those studying beta(2) AR pharmacogenetics, in relation to which SNPs to choose. Most of the work has focused on the three common SNPs within the coding block (alleles 16, 27 and 164) and the techniques developed have been for these three functionally important alleles.
    Matched MeSH terms: Polymerase Chain Reaction/methods*
  3. Fulltext An alternate method for DNA and RNA extraction from clotted blood
    Zakaria Z, Umi SH, Mokhtar SS, Mokhtar U, Zaiharina MZ, Aziz AT, et al.
    Genet. Mol. Res., 2013;12(1):302-11.
    PMID: 23408417 DOI: 10.4238/2013.February.4.4
    We developed an alternative method to extract DNA and RNA from clotted blood for genomic and molecular investigations. A combination of the TRIzol method and the QIAamp spin column were used to extract RNA from frozen clotted blood. Clotted blood was sonicated and then the QIAamp DNA Blood Mini Kit was used for DNA extraction. Extracted DNA and RNA were adequate for gene expression analysis and copy number variation (CNV) genotyping, respectively. The purity of the extracted RNA and DNA was in the range of 1.8-2.0, determined by absorbance ratios of A(260):A(280). Good DNA and RNA integrity were confirmed using gel electrophoresis and automated electrophoresis. The extracted DNA was suitable for qPCR and microarrays for CNV genotyping, while the extracted RNA was adequate for gene analysis using RT-qPCR.
    Matched MeSH terms: Polymerase Chain Reaction/methods
  4. A simple method for the detection of CYP2C9 polymorphisms: nested allele-specific multiplex polymerase chain reaction
    Zainuddin Z, Teh LK, Suhaimi AW, Salleh MZ, Ismail R
    Clin Chim Acta, 2003 Oct;336(1-2):97-102.
    PMID: 14500040 DOI: 10.1016/s0009-8981(03)00319-x
    BACKGROUND: Cytochrome P4502C9 (CYP2C9), a principle drug-metabolizing enzyme is polymorphic in humans and is responsible for important pharmacokinetic and pharmacodynamic variations of CYP2C9 substrates. We developed an allele-specific multiplex polymerase chain reaction (PCR) method for the detection of common CYP2C9 alleles.
    METHOD: Genomic DNA was extracted from blood obtained from 40 unrelated healthy Malaysian Indian volunteers. The DNA was subjected to a first PCR that was used to amplify both exons 3 and 7 simultaneously in one reaction tube and a second PCR that was used to detect the polymorphic sites of CYP2C9 alleles using allele-specific primers. Sequencing was performed to validate the test results.
    RESULTS: We were successful in amplifying the fragments of interest from the DNA samples. The method was also reproducible and specific. The amplified sequences showed 100% homology to CYP2C9 sequence.
    CONCLUSION: This is the first nested allele-specific multiplex PCR method reported to allow for the simultaneously detection of five CYP2C9 alleles.
    Matched MeSH terms: Polymerase Chain Reaction/methods*
  5. Detection of Cryptosporidium parvum in HIV-infected patients in Malaysia using a molecular approach
    Zaidah AR, Chan YY, Asma HS, Abdullah S, Nurhaslindawati AR, Salleh M, et al.
    Southeast Asian J. Trop. Med. Public Health, 2008 May;39(3):511-6.
    PMID: 18564692
    This cross-sectional study determined the prevalence of cryptosporidiosis in HIV-infected patients using polymerase chain reaction (PCR). Stool specimens were collected from HIV infected patients who were admitted to Hospital Raja Perempuan Zainab II, Kota Bharu, Malaysia, for various indications from December 2004 to December 2005. A modified acid-fast stain was performed on the direct stool smears, then the stool specimens were further tested using nested PCR targeting the 18S rRNA gene of Cryptosporidium parvum, with a built-in internal control (IC). Out of 59 samples, 11 were positives. Nested PCR identified a total of nine samples (16%) compared to microscopy, which identified only three samples. All PCR negative results showed IC amplicons, suggesting that these samples were true negatives and were not due to inhibition of PCR. This study highlights the importance of molecular diagnosis in determining the true prevalence and epidemiology of C. parvum.
    Matched MeSH terms: Polymerase Chain Reaction/methods
  6. Fulltext Distribution of Candida in the oral cavity and its differentiation based on the internally transcribed spacer (ITS) regions of rDNA
    Zahir RA, Himratul-Aznita WH
    Yeast, 2013 Jan;30(1):13-23.
    PMID: 23208647 DOI: 10.1002/yea.2937
    This study aimed to determine the distribution of Candida species in the oral cavity and differentiate the species based on PCR amplification, including HinfI and MspI digestion, in order to assess the effectiveness of using the rDNA region for species identification. Samples from saliva as well as palate, tongue and cheek mucosa surfaces were collected from 45 individuals, consisting of three groups: periodontal disease patients; denture-wearers; and the control group. The samples were serially diluted, spread on BHI and YPD agar plates and scored for colony-forming units (CFUs). Fifteen random candidal colonies were isolated and subjected to genomic DNA extraction, based on glass beads disruption. Four primers were used to amplify regions in the rDNA, and the ITSI-5.8S-ITSII PCR product was digested by HinfI and MspI restriction enzymes. The microbial loads on all sites of the denture-wearers were found to be significantly higher than control, while in the periodontal disease group only the microbial loads on the tongue were significantly higher than control. Meanwhile, there was no significant difference at other sites. The restriction fragment lengths of the clinical samples were compared to those of seven control species, allowing the differentiation of all seven species and the identification of 14 species from the clinical samples. The MspI restriction digest was not able to distinguish between C. albicans and C. dubliniensis, whereas the HinfI digest could not distinguish between C. tropicalis and C. parapsilosis. It was concluded that PCR-RFLP of the candidal rDNA region has potential for species identification. This study demonstrates the potential use of candidal rDNA as a means for identifying Candida species, based on genotype. The results also indicate the possibility of constructing genetic probes that target specific restriction fragments in the ITSI-5.8S-ITSII region, enabling swift and precise identification of Candida species.
    Matched MeSH terms: Polymerase Chain Reaction/methods*
  7. Molecular heterogeneity of glucose-6-phosphate dehydrogenase deficiency in Malays in Malaysia
    Yusoff NM, Shirakawa T, Nishiyama K, Ghazali S, Ee CK, Orita A, et al.
    Int J Hematol, 2002 Aug;76(2):149-52.
    PMID: 12215013 DOI: 10.1007/BF02982577
    Multiplex polymerase chain reaction (PCR) using multiple tandem forward primers and a common reverse primer (MPTP) was recently established as a comprehensive screening method for mutations in X-linked recessive diseases. In the work reported here, MPTP was used to scan for mutations of the glucose-6-phosphate dehydrogenase (G6PD) gene. Mutations in exons 3,4,5,6,7,9, 11, and 12 of the G6PD gene were screened by MPTP in 93 unrelated Malaysian patients with G6PD deficiency. Of the 93 patients, 80 (86%) had identified mutations. Although all of these were missense mutations, identified nucleotide changes were heterogeneous, with 9 mutations involving various parts of the exons. These 9 mutations were G-to-A nucleotide changes at nucleotide 871 of the G6PD gene (G871A), corresponding to G6PD Viangchan, G6PD Mediterranean (C563T), G6PD Vanua Lava (T383C), G6PD Coimbra (C592T), G6PD Kaiping (G1388A), G6PD Orissa (C131G), G6PD Mahidol (G487A), G6PD Canton (G1376T), and G6PD Chatham (G1003A). Our results document heterogeneous mutations of the G6PD gene in the Malaysian population.
    Matched MeSH terms: Polymerase Chain Reaction/methods
  8. Enzymatic electrochemical detection of epidemic-causing Vibrio cholerae with a disposable oligonucleotide-modified screen-printed bisensor coupled to a dry-reagent-based nucleic acid amplification assay
    Yu CY, Ang GY, Chan KG, Banga Singh KK, Chan YY
    Biosens Bioelectron, 2015 Aug 15;70:282-8.
    PMID: 25835520 DOI: 10.1016/j.bios.2015.03.048
    In this study, we developed a nucleic acid-sensing platform in which a simple, dry-reagent-based nucleic acid amplification assay is combined with a portable multiplex electrochemical genosensor. Preparation of an amplification reaction mix targeting multiple DNA regions of interest is greatly simplified because the lyophilized reagents need only be reconstituted with ultrapure water before the DNA sample is added. The presence of single or multiple target DNAs causes the corresponding single-stranded DNA (ssDNA) amplicons to be generated and tagged with a fluorescein label. The fluorescein-labeled ssDNA amplicons are then analyzed using capture probe-modified screen-printed gold electrode bisensors. Enzymatic amplification of the hybridization event is achieved through the catalytic production of electroactive α-naphthol by anti-fluorescein-conjugated alkaline phosphatase. The applicability of this platform as a diagnostic tool is demonstrated with the detection of toxigenic Vibrio cholerae serogroups O1 and O139, which are associated with cholera epidemics and pandemics. The platform showed excellent diagnostic sensitivity and specificity (100%) when challenged with 168 spiked stool samples. The limit of detection was low (10 colony-forming units/ml) for both toxigenic V. cholerae serogroups. A heat stability assay revealed that the dry-reagent amplification reaction mix was stable at temperatures of 4-56 °C, with an estimated shelf life of seven months. The findings of this study highlight the potential of combining a dry-reagent-based nucleic acid amplification assay with an electrochemical genosensor in a more convenient, sensitive, and sequence-specific detection strategy for multiple target nucleic acids.
    Matched MeSH terms: Polymerase Chain Reaction/methods*
  9. Fulltext Rapid detection and serotyping of dengue virus by multiplex RT-PCR and real-time SYBR green RT-PCR
    Yong YK, Thayan R, Chong HT, Tan CT, Sekaran SD
    Singapore Med J, 2007 Jul;48(7):662-8.
    PMID: 17609830
    Dengue fever and dengue haemorrhagic fever currently rank highly among the newly-emerging infectious diseases, and are considered to be the most important arboviral disease worldwide. The definitive diagnosis is culture analysis, but practical considerations limit its use. Also, the period for viral detection is limited. Within a day or two after fever subsides, rising levels of antibodies interfere with viral cultures. An alternative to this quandary is the use of viral RNA detection assays. In our laboratory, a reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed using a set of degenerate primers.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
  10. A modified in situ RT-PCR method for localizing fungal-specific gene expression in Candida-infected mice renal cells
    Yong VC, Ong KW, Sidik SM, Rosli R, Chong PP
    J Microbiol Methods, 2009 Nov;79(2):242-5.
    PMID: 19737582 DOI: 10.1016/j.mimet.2009.08.019
    In situ Reverse Transcriptase PCR (in situ RT-PCR) can amplify mRNA and localize gene expression in cells. However, this method is not feasible in fungi as the thick fungal cell wall constitutes a barrier to this procedure. We developed a two step in situ RT-PCR procedure which enabled the detection and localization of Candida tropicalis mRNA expression in formalin-fixed, paraffin-embedded (FFPE) mouse kidney sections. This in situ hybridization study revealed the first direct evidence for deposition of Candida tropicalis secreted aspartic proteinase 2 (CtSAP2) in the tip of pseudohyphae and its involvement in acute systemic candidiasis. We conclude that in situ RT-PCR can be successfully applied to FFPE tissues and will offer new perspectives in studying gene expression in Candida species.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
  11. Male-specific RNA coliphages detected by plaque assay and RT-PCR in tropical river waters and animal fecal matter
    Yee SY, Fong NY, Fong GT, Tak OJ, Hui GT, Su Ming Y
    Int J Environ Health Res, 2006 Feb;16(1):59-68.
    PMID: 16507481
    Male-specific RNA coliphages (FRNA) have been recommended as indicators of fecal contamination and of the virological quality of water. In this study, 16 river water and 183 animal fecal samples were examined for the presence of FRNA coliphages by a plaque assay using Salmonella typhimurium WG49 and WG25 to differentiate between male-specific and somatic phages, a RNase spot test to differentiate between DNA and RNA phages and a reverse transcriptase-polymerase chain reaction (RT-PCR) for the specific identification of FRNA phages. The overall recovery rate for F-specific coliphages was 8.0%. (4.4% from animal fecal matter and 50% from river water samples). Plaque counts were generally low (< 6 x 10(2) pfu per g feces or ml water), with FRNA (6.5%) and Male-specific DNA coliphages (FDNA) (7.0%) phages occurring at almost equal frequencies. The RT-PCR was positive in all FRNA plaques and was able to identify FRNA phages in mixed populations of FRNA, FDNA and somatic phages.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods
  12. Enzyme-linked amperometric electrochemical genosensor assay for the detection of PCR amplicons on a streptavidin-treated screen-printed carbon electrode
    Yean CY, Kamarudin B, Ozkan DA, Yin LS, Lalitha P, Ismail A, et al.
    Anal Chem, 2008 Apr 15;80(8):2774-9.
    PMID: 18311943 DOI: 10.1021/ac702333x
    A general purpose enzyme-based amperometric electrochemical genosensor assay was developed wherein polymerase chain reaction (PCR) amplicons labeled with both biotin and fluorescein were detected with peroxidase-conjugated antifluorescein antibody on a screen-printed carbon electrode (SPCE). As a proof of principle, the response selectivity of the genosensor was evaluated using PCR amplicons derived from lolB gene of Vibrio cholerae. Factors affecting immobilization, hybridization, and nonspecific binding were optimized to maximize sensitivity and reduce assay time. On the basis of the background amperometry signals obtained from nonspecific organisms and positive signals obtained from known V. cholerae, a threshold point of 4.20 microA signal was determined as positive. Under the optimum conditions, the limit of detection (LOD) of the assay was 10 CFU/mL of V. cholerae. The overall precision of this assay was good, with the coefficient of variation (CV) being 3.7% using SPCE and intermittent pulse amperometry (IPA) as an electrochemical technique. The assay is sensitive, safe, and cost-effective when compared to conventional agarose gel electrophoresis, real-time PCR, and other enzyme-linked assays for the detection of PCR amplicons. Furthermore, the use of a hand-held portable reader makes it suitable for use in the field.
    Matched MeSH terms: Polymerase Chain Reaction/methods*
  13. A nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistance genes in Enterococcus species
    Yean CY, Yin LS, Lalitha P, Ravichandran M
    BMC Microbiol, 2007 Dec 11;7:112.
    PMID: 18070365
    BACKGROUND: Enterococci have emerged as a significant cause of nosocomial infections in many parts of the world over the last decade. The most common enterococci strains present in clinical isolates are E. faecalis and E. faecium which have acquired resistant to either gentamicin or vancomycin. The conventional culture test takes 2-5 days to yield complete information of the organism and its antibiotic sensitivity pattern. Hence our present study was focused on developing a nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistant enterococci (V-BiA-RE). This assay simultaneously detects 8 genes namely 16S rRNA of Enterococcus genus, ddl of E. faecalis and E. faecium, aacA-aphD that encodes high level gentamicin resistance (HLGR), multilevel vancomycin resistant genotypes such as vanA, vanB, vanC and vanD and one internal control gene.

    RESULTS: Unique and specific primer pairs were designed to amplify the 8 genes. The specificity of the primers was confirmed by DNA sequencing of the nanoplex PCR products and BLAST analysis. The sensitivity and specificity of V-BiA-RE nanoplex PCR assay was evaluated against the conventional culture method. The analytical sensitivity of the assay was found to be 1 ng at the DNA level while the analytical specificity was evaluated with 43 reference enterococci and non-enterococcal strains and was found to be 100%. The diagnostic accuracy was determined using 159 clinical specimens, which showed that 97% of the clinical isolates belonged to E. faecalis, of which 26% showed the HLGR genotype, but none were vancomycin resistant. The presence of an internal control in the V-BiA-RE nanoplex PCR assay helped us to rule out false negative cases.

    CONCLUSION: The nanoplex PCR assay is robust and can give results within 4 hours about the 8 genes that are essential for the identification of the most common Enterococcus spp. and their antibiotic sensitivity pattern. The PCR assay developed in this study can be used as an effective surveillance tool to study the prevalence of enterococci and their antibiotic resistance pattern in hospitals and farm animals.

    Matched MeSH terms: Polymerase Chain Reaction/methods*
  14. Human Fc gamma receptor IIA (FcgammaRIIA) genotyping and association with systemic lupus erythematosus (SLE) in Chinese and Malays in Malaysia
    Yap SN, Phipps ME, Manivasagar M, Tan SY, Bosco JJ
    Lupus, 1999;8(4):305-10.
    PMID: 10413210 DOI: 10.1191/096120399678847876
    SLE is an autoimmune and polygenic disorder characterized by an accumulation and deposition of immune complexes. Several studies have indicated differential impact of FcgammaR polymorphism genotypes in different ethnic groups studied. The Fc receptor for IgG class IIA gene (FcgammaRIIA) occurs in two allelic forms. The allele FcgammaRIIA-H131 encodes a receptor with a histidine at the 131 amino acid position; the other allele FcgammaRIIA-R131 encodes an arginine. This polymorphism is believed to determine the affinity of the receptor for hIgG2 in immune complexes. FcgammaRIIA-H131 has a higher capacity for hIgG2 compared to FcgammaRIIA-R131 as measured by in vitro studies of insoluble immune complex clearance. We have investigated the polymorphism for FcgammaRIIA using a novel polymerase chain reaction-allele specific primer (PCR-ASP) method designed specifically to distinguish the two allelic forms. Our studies were based on 175 Chinese and 50 Malays SLE patients as well as 108 and 50 ethnically matched healthy controls for the respective groups. Analysis of the data (chi2 test with Yates correction factors and odds ratios) revealed that there were no significant differences between SLE patients and controls. We have not found evidence of a protective effect conferred by FcgammaRIIA-H131 in the ethnic groups studied.
    Matched MeSH terms: Polymerase Chain Reaction/methods
  15. Quantification of human PPARgamma1 gene expression by competitive PCR using an homologous internal standard
    Yaacob NS, Bakar RA, Norazmi MN
    Ann Clin Lab Sci, 2004;34(1):47-56.
    PMID: 15038667
    The polymerase chain reaction (PCR) is useful for amplifying specific mRNAs, particularly those present in low copy numbers. However, due to the exponential nature of the amplification process, PCR cannot readily be used to quantify gene expression. A competitive PCR technique was developed to address this shortcoming. An internal standard that is 100% homologous to, but shorter than, the target gene was constructed. The practicality of the method was demonstrated by determining the expression levels of a human transcription factor, peroxisome proliferator-activated receptor gamma 1 (hPPARgamma1) which is normally present in low copy numbers in selected cells. A mock system was used to test the accuracy and sensitivity of the method, which was subsequently used to determine the expression of this receptor in lipopolysaccharide (LPS)-activated monocytes, which are known to express hPPARgamma1 differentially during cellular activation. Densitometric analysis showed that the competitive PCR method reliably estimated the expression levels of hPPARgamma1 at the attomole (10(-18)) level in monocytes.
    Matched MeSH terms: Polymerase Chain Reaction/methods*
  16. Fulltext A RT-PCR assay for the detection of coronaviruses from four genera
    Xiu L, Binder RA, Alarja NA, Kochek K, Coleman KK, Than ST, et al.
    J Clin Virol, 2020 07;128:104391.
    PMID: 32403008 DOI: 10.1016/j.jcv.2020.104391
    BACKGROUND: During the past two decades, three novel coronaviruses (CoVs) have emerged to cause international human epidemics with severe morbidity. CoVs have also emerged to cause severe epidemics in animals. A better understanding of the natural hosts and genetic diversity of CoVs are needed to help mitigate these threats.

    OBJECTIVE: To design and evaluate a molecular diagnostic tool for detection and identification of all currently recognized and potentially future emergent CoVs from the Orthocoronavirinae subfamily.

    STUDY DESIGN AND RESULTS: We designed a semi-nested, reverse transcription RT-PCR assay based upon 38 published genome sequences of human and animal CoVs. We evaluated this assay with 14 human and animal CoVs and 11 other non-CoV respiratory viruses. Through sequencing the assay's target amplicon, the assay correctly identified each of the CoVs; no cross-reactivity with 11 common respiratory viruses was observed. The limits of detection ranged from 4 to 4 × 102 copies/reaction, depending on the CoV species tested. To assess the assay's clinical performance, we tested a large panel of previously studied specimens: 192 human respiratory specimens from pneumonia patients, 5 clinical specimens from COVID-19 patients, 81 poultry oral secretion specimens, 109 pig slurry specimens, and 31 aerosol samples from a live bird market. The amplicons of all RT-PCR-positive samples were confirmed by Sanger sequencing. Our assay performed well with all tested specimens across all sample types.

    CONCLUSIONS: This assay can be used for detection and identification of all previously recognized CoVs, including SARS-CoV-2, and potentially any emergent CoVs in the Orthocoronavirinae subfamily.

    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
  17. Alcanivorax hongdengensis sp. nov., an alkane-degrading bacterium isolated from surface seawater of the straits of Malacca and Singapore, producing a lipopeptide as its biosurfactant
    Wu Y, Lai Q, Zhou Z, Qiao N, Liu C, Shao Z
    Int J Syst Evol Microbiol, 2009 Jun;59(Pt 6):1474-9.
    PMID: 19502338 DOI: 10.1099/ijs.0.001552-0
    A taxonomic study was carried out on strain A-11-3(T), which was isolated from an oil-enriched consortia from the surface seawater of Hong-Deng dock in the Straits of Malacca and Singapore. Cells were aerobic, Gram-negative, non-spore-forming irregular rods. The strain was catalase- and oxidase-negative. It grew on a restricted spectrum of organic compounds, including some organic acids and alkanes. 16S rRNA gene sequence comparisons showed that strain A-11-3(T) was most closely related to the type strains of Alcanivorax jadensis (96.8 % sequence similarity), Alcanivorax borkumensis (96.8 %), Alcanivorax dieselolei (94.8 %), Alcanivorax venustensis (94.2 %) and Alcanivorax balearicus (94.0 %). The predominant fatty acids were C(16 : 0) (31.2 %), C(18 : 1)omega7c (24.8 %), C(18 : 0) (9.6 %), C(12 : 0) (8.3 %), C(16 : 1)omega7c (8.3 %) and C(16 : 0) 3-OH (5.1 %). The G+C content of the genomic DNA was 54.7 mol%. Moreover, the strain produced lipopeptides as its surface-active compounds. According to physiological and biochemical tests, DNA-DNA hybridization results and sequence comparisons of the 16S-23S internal transcribed spacer, the gyrB gene and the alkane hydroxylase gene alkB1, strain A-11-3(T) was affiliated with the genus Alcanivorax but could be readily distinguished from recognized Alcanivorax species. Therefore strain A-11-3(T) represents a novel species of the genus Alcanivorax for which the name Alcanivorax hongdengensis sp. nov. is proposed. The type strain is A-11-3(T) (=CGMCC 1.7084(T)=LMG 24624(T)=MCCC 1A01496(T)).
    Matched MeSH terms: Polymerase Chain Reaction/methods
  18. One-step species-specific high resolution melting analysis for nosocomial bacteria detection
    Wong YP, Chua KH, Thong KL
    J Microbiol Methods, 2014 Dec;107:133-7.
    PMID: 25307691
    Nosocomial infections are a major public health concern worldwide. Early and accurate identification of nosocomial pathogens which are often multidrug resistant is crucial for prompt treatment. Hence, an alternative real-time polymerase chain reaction coupled with high resolution melting-curve analysis (HRMA) was developed for identification of five nosocomial bacteria. This assay targets species-specific regions of each nosocomial bacteria and produced five distinct melt curves with each representing a particular bacterial species. The melting curves were characterized by peaks of 78.8 ± 0.2 °C for Acinetobacter baumannii, 82.7 ± 0.2 °C for Escherichia coli, 86.3 ± 0.3 °C for Klebsiella pneumoniae, 88.8 ± 0.2 °C for Pseudomonas aeruginosa and 74.6 ± 02 °C for methicillin-resistant Staphylococcus aureus. The assay was able to specifically detect the five bacterial species with an overall detection limit of 2 × 10(-2) ng/μL. In conclusion, the HRM assay developed is a simple and rapid method for identification of the selected nosocomial pathogens.
    Matched MeSH terms: Polymerase Chain Reaction/methods*
  19. Fulltext Molecular characterisation and frequency of Ggamma Xmn I polymorphism in Chinese and Malay beta-thalassaemia patients in Malaysia
    Wong YC, George E, Tan KL, Yap SF, Chan LL, Tan JA
    Malays J Pathol, 2006 Jun;28(1):17-21.
    PMID: 17694955
    The molecular basis of variable phenotypes in P-thalassaemia patients with identical genotypes has been associated with co-inheritance of alpha-thalassaemia and persistence of HbF production in adult life. The Xmn I restriction site at -158 position of the Ggamma-gene is associated with increased expression of the Ggamma-globin gene and higher production of HbF This study aims to determine the frequency of the digammaferent genotypes of the Ggamma Xmn I polymorphism in P-thalassaemia patients in two ethnic groups in Malaysia. Molecular characterisation and frequency of the Ggamma Xmn I polymorphism were studied in fifty-eight Chinese and forty-nine beta-thalassaemia Malay patients by Xmn I digestion after DNA amplification of a 650 bp sequence. The in-house developed technique did not require further purification or concentration of amplified DNA before restriction enzyme digestion. The cheaper Seakem LE agarose was used instead of Nusieve agarose and distinct well separated bands were observed. Genotyping showed that the most frequent genotype observed in the Malaysian Chinese was homozygosity for the absence of the Xmn I site (-/-) (89.7%). In the Malays, heterozygosity of the Xmn I site (+/-) was most common (63.3%). Homozygosity for the Xmn I site (+/+) was absent in the Chinese, but was confirmed in 8.2% of the Malays. The ratio of the (+) allele (presence of the Xmn I site) to the (-) allele (absence of the Xmn I site)) was higher in the Malays (0.66) compared to the Chinese (0.05). The (+/-) and (+/+) genotypes are more commonly observed in the Malays than the Chinese in Malaysia.
    Matched MeSH terms: Polymerase Chain Reaction/methods*
  20. COVID-19 testing and diagnosis: A comparison of current approaches
    Wong RSY
    Malays J Pathol, 2021 Apr;43(1):3-8.
    PMID: 33903299
    The severe acute respiratory syndrome coronavirus 2 is a novel coronavirus that causes the coronavirus disease 2019 (COVID-19). COVID-19 has been declared a pandemic by the World Health Organisation since March 2020. To date, the number of confirmed COVID-19 cases has exceeded 47 million and more than 1.2 million people have lost their lives to the disease. The disease is spreading at an exponential rate with no signs of slowing down. COVID-19 testing and early diagnosis play a crucial role in not just patient management, but also the prevention of the further spread of the disease. Various diagnostic approaches have been applied to detect SARS-CoV-2 infection. This article will critically review these diagnostic approaches and compare each with the gold-standard, which is viral RNA detection using reverse transcriptase-polymerase chain reaction (RT-PCR).
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods
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