Displaying publications 1 - 20 of 85 in total

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  1. Fulltext A Nested Allele-Specific Multiplex Polymerase Chain Reaction Method for the Detection of DRD2 Polymorphisms
    Zahari Z, Salleh MR, Zahri Johari MK, Musa N, Ismail R
    Malays J Med Sci, 2011 Oct;18(4):44-57.
    PMID: 22589672 MyJurnal
    The dopamine D2 receptor gene (DRD2) plays a role in many diseases such as schizophrenia, Parkinson's disease, and addictive behaviour. Methods currently available for the detection of DRD2 polymorphisms are costly and cannot detect all 8 polymorphisms of our research interest simultaneously (Val96Ala, Leu141Leu, Val154Ile, Pro310Ser, Ser311Cys, TaqI A, A-241G, and -141C Ins/Del). Therefore, we developed a nested multiplex polymerase chain reaction (PCR) for simultaneous detection of these polymorphisms.
    Matched MeSH terms: Multiplex Polymerase Chain Reaction
  2. Fulltext A Thermostabilized, One-Step PCR Assay for Simultaneous Detection of Klebsiella pneumoniae and Haemophilus influenzae
    Khazani NA, Noor NZ, Yean Yean C, Hasan H, Suraiya S, Mohamad S
    J Trop Med, 2017;2017:7210849.
    PMID: 28386286 DOI: 10.1155/2017/7210849
    Klebsiella pneumoniae and Haemophilus influenzae are two common pathogens associated with respiratory tract infections. The identification of these pathogens using conventional molecular diagnostic tests requires trained personnel, cold-chain transportation, and storage-dependance, which does not render them user-friendly. The aim of this study was to develop a thermostabilized, cold-chain-free, one-step multiplex PCR for simultaneous detection of K. pneumoniae and H. influenzae. The multiplex PCR assay was designed to amplify the php gene of K. pneumoniae (202 bp) and p6 gene of H. influenzae (582 bp). In addition, the specific primer to amplify glm gene of Helicobacter pylori (105 bp) was included as an internal amplification control. Subsequently, the designed primers and all PCR reagents were thermostabilized by lyophilization. The stability of the thermostabilized PCR was evaluated using the Q(10) method. The sensitivity and specificity of performances for thermostabilized PCR were evaluated using 127 clinical isolates and were found to be 100% sensitive and specific. The thermostabilized PCR mix was found to be stable for 30 days and the Q10 accelerated stability was found to be 3.02 months. A cold-chain-free, PCR assay for easy, rapid, and simultaneous detection of K. pneumoniae and H. influenzae was successfully developed in this study.
    Matched MeSH terms: Multiplex Polymerase Chain Reaction
  3. Fulltext A higher sensitivity and efficiency of common primer multiplex PCR assay in identification of meat origin using NADH dehydrogenase subunit 4 gene
    Hanapi UK, Desa MN, Ismail A, Mustafa S
    J Food Sci Technol, 2015 Jul;52(7):4166-75.
    PMID: 26139881 DOI: 10.1007/s13197-014-1459-7
    A Common Primer Multiplex PCR (CP-M-PCR) was developed to detect meat origin of four groups of animal (pig, ruminant, avian and rabbit). This method demonstrated higher sensitivity and efficiency than the conventional multiplex PCR. In this approach, a common forward primer was designed in the 5' end of a homologous region of mitochondrial NADH dehyrogenase subunit 4 (Nad 4) gene sequences of all the animal groups. Specific adapter reverse primers were designed by adding an adapter sequence at the 5' end. The same adapter sequence was used as the common adapter reverse primer. The primers generated specific fragments of 267, 370, 504, and 548 bp lengths for pig, ruminant, avian and rabbit meats, respectively. The use of adapter sequence at the 5' end of the common adapter reverse primers increased the efficiency of the amplification and the application of a common forward primer solved the complexity in multiplex PCR system. Bands of specific amplification can be detected in the PCR assays containing as low as 10(-6) μM of adapter reverse primer. This result indicated that the sensitivity was tremendously increased as compared to the conventional multiplex PCR (10(-3) μM). CP-M-PCR detection limit of the DNA samples was 0.1 ng for the four groups of meats. CP-M-PCR has greatly improved the sensitivity and efficiency of the PCR system for a more reliable and accurate outcome than conventional multiplex PCR system.
    Matched MeSH terms: Multiplex Polymerase Chain Reaction
  4. Fulltext A new nested allele-specific multiplex polymerase chain reaction method for haplotyping of VKORC1 gene to predict warfarin sensitivity
    Chua YA, Abdullah WZ, Yusof Z, Gan SH
    Biomed Res Int, 2014;2014:316310.
    PMID: 24790995 DOI: 10.1155/2014/316310
    The vitamin K epoxide reductase complex 1 gene (VKORC1) is commonly assessed to predict warfarin sensitivity. In this study, a new nested allele-specific multiplex polymerase chain reaction (PCR) method that can simultaneously identify single nucleotide polymorphisms (SNPs) at VKORC1 381, 861, 5808, and 9041 for haplotype analysis was developed and validated. Extracted DNA was amplified in the first PCR DNA, which was optimized by investigating the effects of varying the primer concentrations, annealing temperature, magnesium chloride concentration, enzyme concentration, and the amount of DNA template. The amplification products produced from the first round of PCR were used as templates for a second PCR amplification in which both mutant and wild-type primers were added in separate PCR tubes, followed by optimization in a similar manner. The final PCR products were resolved by agarose gel electrophoresis and further analysed by using a VKORC1 genealogic tree to infer patient haplotypes. Fifty patients were identified to have H1H1, one had H1H2, one had H1H7, 31 had either H1H7 or H1H9, one had H1H9, eight had H7H7, and one had H8H9 haplotypes. This is the first method that is able to infer VKORC1 haplotypes using only conventional PCR methods.
    Matched MeSH terms: Multiplex Polymerase Chain Reaction/methods*
  5. Fulltext An insight into the isolation, enumeration, and molecular detection of Listeria monocytogenes in food
    Law JW, Ab Mutalib NS, Chan KG, Lee LH
    Front Microbiol, 2015;6:1227.
    PMID: 26579116 DOI: 10.3389/fmicb.2015.01227
    Listeria monocytogenes, a foodborne pathogen that can cause listeriosis through the consumption of food contaminated with this pathogen. The ability of L. monocytogenes to survive in extreme conditions and cause food contaminations have become a major concern. Hence, routine microbiological food testing is necessary to prevent food contamination and outbreaks of foodborne illness. This review provides insight into the methods for cultural detection, enumeration, and molecular identification of L. monocytogenes in various food samples. There are a number of enrichment and plating media that can be used for the isolation of L. monocytogenes from food samples. Enrichment media such as buffered Listeria enrichment broth, Fraser broth, and University of Vermont Medium (UVM) Listeria enrichment broth are recommended by regulatory agencies such as Food and Drug Administration-bacteriological and analytical method (FDA-BAM), US Department of Agriculture-Food and Safety (USDA-FSIS), and International Organization for Standardization (ISO). Many plating media are available for the isolation of L. monocytogenes, for instance, polymyxin acriflavin lithium-chloride ceftazidime aesculin mannitol, Oxford, and other chromogenic media. Besides, reference methods like FDA-BAM, ISO 11290 method, and USDA-FSIS method are usually applied for the cultural detection or enumeration of L. monocytogenes. most probable number technique is applied for the enumeration of L. monocytogenes in the case of low level contamination. Molecular methods including polymerase chain reaction, multiplex polymerase chain reaction, real-time/quantitative polymerase chain reaction, nucleic acid sequence-based amplification, loop-mediated isothermal amplification, DNA microarray, and next generation sequencing technology for the detection and identification of L. monocytogenes are discussed in this review. Overall, molecular methods are rapid, sensitive, specific, time- and labor-saving. In future, there are chances for the development of new techniques for the detection and identification of foodborne with improved features.
    Matched MeSH terms: Multiplex Polymerase Chain Reaction
  6. Analysis of four PCR/SNaPshot multiplex assays analyzing 52 SNPforID markers
    Goodwin W, Alimat S
    Electrophoresis, 2017 04;38(7):1007-1015.
    PMID: 28008628 DOI: 10.1002/elps.201600383
    The SNPforID consortium identified a panel of 52 SNPs for forensic analysis that has been used by several laboratories worldwide. The original analysis of the 52 SNPs was based on a single multiplex reaction followed by two single-base-extension (SBE) reactions each of which was analyzed using capillary electrophoresis. The SBE assays were designed for high throughput genetic analyzers and were difficult to use on the single capillary ABI PRISM 310 Genetic Analyzer and the latest generation 3500 Genetic Analyzer, as sensitivity on the 310 was low and separation of products on the 3500 with POP-7™ was poor. We have modified the original assay and split it into four multiplex reactions, each followed by an SBE assay. These multiplex assays were analyzed using polymer POP-4™ on ABI 310 PRISM® and polymers POP-4™, POP-6™ and POP-7™ on the 3500 Genetic Analyzer. The assays were sensitive and reproducible with input DNA as low as 60 pg using both the ABI 310 and 3500. In addition, we found that POP-6™ was most effective with the 3500, based on the parameters that we assessed, achieving better separation of the small SBE products; this conflicted with the recommended use of POP-7™ by the instrument manufacturer. To support the use of the SNP panel in casework in Malaysia we have created an allele frequency database from 325 individuals, representing the major population groups within Malaysia. Population and forensic parameters were estimated for all populations and its efficacy evaluated using 51 forensic samples from challenging casework.
    Matched MeSH terms: Multiplex Polymerase Chain Reaction/methods*
  7. Fulltext Association of GSTM1 and GSTT1 with ageing in auto repair shop workers
    Eshkoor SA, Marashi SJ, Ismail P, Rahman SA, Mirinargesi M, Adon MY, et al.
    Genet. Mol. Res., 2012;11(2):1486-96.
    PMID: 22653598 DOI: 10.4238/2012.May.21.5
    We evaluated the possible influence of glutathione S-transferase mu (GSTM1) and glutathione S-transferase theta (GSTT1) genes on genetic damage due to occupational exposure, which contributes to accelerate ageing. This study was conducted on 120 car auto repair workshop workers exposed to occupational hazards and 120 controls without this kind of exposure. The null and non-null genotypes of GSTM1 and GSTT1 genes were determined by multiplex PCR. Micronucleus frequency, Comet tail length and relative telomere length differences between the null and non-null genotypes of the GSTM1 gene were significantly greater in the exposed group. Lack of GSTT1 did not affect the damage biomarkers significantly (P > 0.05), while lack of GSTM1 was associated with greater susceptibility to genomic damage due to occupational exposure. It was concluded that early ageing is under the influence of these genes and the environmental and socio-demographic factors. Duration of working time was significantly associated with micronucleus frequency, Comet tail length and relative telomere length.
    Matched MeSH terms: Multiplex Polymerase Chain Reaction
  8. Fulltext Clinical diagnosis of early dengue infection by novel one-step multiplex real-time RT-PCR targeting NS1 gene
    Kim JH, Chong CK, Sinniah M, Sinnadurai J, Song HO, Park H
    J Clin Virol, 2015 Apr;65:11-9.
    PMID: 25766980 DOI: 10.1016/j.jcv.2015.01.018
    BACKGROUND: Dengue is a mosquito-borne disease that causes a public health problem in tropical and subtropical countries. Current immunological diagnostics based on IgM and/or nonstructural protein 1 (NS1) antigen are limited for acute dengue infection due to low sensitivity and accuracy.
    OBJECTIVES: This study aimed to develop a one-step multiplex real-time RT-PCR assay showing higher sensitivity and accuracy than previous approaches.
    STUDY DESIGN: Serotype-specific primers and probes were designed through the multiple alignment of NS1 gene. The linearity and limit of detection (LOD) of the assay were determined. The assay was clinically validated with an evaluation panel that was immunologically tested by WHO and Malaysian specimens.
    RESULTS: The LOD of the assay was 3.0 log10 RNA copies for DENV-1, 2.0 for DENV-3, and 1.0 for DENV-2 and DENV-4. The assay showed 95.2% sensitivity (20/21) in an evaluation panel, whereas NS1 antigen- and anti-dengue IgM-based immunological assays exhibited 0% and 23.8-47.6% sensitivities, respectively. The assay showed 100% sensitivity both in NS1 antigen- and anti-dengue IgM-positive Malaysian specimens (26/26). The assay provided the information of viral loads and serotype with discrimination of heterotypic mixed infection.
    CONCLUSIONS: The assay could be clinically applied to early dengue diagnosis, especially during the first 5 days of illness and approximately 14 days after infection showing an anti-dengue IgM-positive response.
    Matched MeSH terms: Multiplex Polymerase Chain Reaction/methods*
  9. Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis
    Hussain H, Chong NF
    Biomed Res Int, 2016;2016:8041532.
    PMID: 27995143
    The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40-45 PCR cycles. A total of eight mutagenesis experiments were successfully carried out, with each experiment mutating between two to six base pairs. Up to four adjacent codons were changed in a single experiment. This method is especially useful for codon optimization, where doublet or triplet rare codons can be changed using a single mutagenic primer set, in a single experiment.
    Matched MeSH terms: Multiplex Polymerase Chain Reaction/methods*
  10. Fulltext Combined PCR and MAT improves the early diagnosis of the biphasic illness leptospirosis
    Philip N, Affendy NB, Masri SN, Yuhana MY, Than LTL, Sekawi Z, et al.
    PLoS One, 2020;15(9):e0239069.
    PMID: 32915919 DOI: 10.1371/journal.pone.0239069
    The diagnosis of leptospirosis remains a challenge due to its non-specific symptoms and the biphasic nature of the illness. A comprehensive diagnosis that includes both molecular (polymerase chain reaction (PCR)) and serology is vital for early detection of leptospirosis and to avoid misdiagnosis. However, not all samples could be subjected to both tests (serology and molecular) due to budget limitation, infrastructure, and technical expertise at least in resource-limited countries. We evaluated the usefulness of testing the clinically suspected leptospirosis cases with both techniques on all samples collected from the patients on the day of admission. Among the 165 patient's blood/serum samples tested (from three hospitals in Central Malaysia), 43 (26%) showed positivity by microscopic agglutination test (MAT), 63 (38%) by PCR, while 14 (8%) were positive by both MAT and PCR. For PCR, we tested two molecular targets (lipL32 by qPCR and 16S rDNA or rrs by nested PCR) and detected lipL32 in 47 (29%) and rrs gene in 63 (38%) patients. The use of more than one target gene for PCR increased the detection rates. Hence, a highly sensitive multiplex PCR targeting more than one diagnostic marker is recommended for the early detection of Leptospira in suspected patients. When the frequencies for positivity detected either by MAT or PCR combined, leptospirosis was diagnosed in a total of 92 (56%) patients, a higher frequency compared to when samples were only tested by a single method (MAT or PCR). The results from this study suggest the inclusion of both serology and molecular methods for every first sample irrespective of the days post-onset of symptoms (DPO) collected from patients for early diagnosis of leptospirosis.
    Matched MeSH terms: Multiplex Polymerase Chain Reaction
  11. Fulltext Comparative Study of CDST & Multiplex PCR to Detect MBL Producing Gram-Negative Bacilli among VAP Patients Admitted in a Public Medical College Hospital of Bangladesh
    Nusrat T, Akter N, Haque M, Rahman NAA, Dewanjee AK, Ahmed S, et al.
    Pathogens, 2019 Sep 12;8(3).
    PMID: 31547453 DOI: 10.3390/pathogens8030151
    BACKGROUND: Ventilator-associated pneumonia (VAP) is the most common nosocomial infection in intensive care units (ICU), which accounts for 25% of all ICU infection. Documenting carbapenem-resistant gram-negative bacilli is very important as these strains may often cause outbreaks in the ICU setting and are responsible for the increased mortality and morbidity or limiting therapeutic options. The classical phenotypic method cannot provide an efficient means of diagnosis of the metallo-β-lactamases (MBLs) producer. Polymerase chain reaction (PCR) assays have lessened the importance of the phenotypic approach by detecting metallo-β-lactamase resistance genes such as New Delhi metallo-β-lactamase (NDM), Imipenemase (IMP), Verona integron-encoded metallo-β-lactamase (VIM), Sao Paulo metallo-β-lactamase (SPM), Germany Imipenemase (GIM).

    OBJECTIVE: To compare the results of the Combined Disc Synergy Test (CDST) with that of the multiplex PCR to detect MBL-producing gram-negative bacilli.

    MATERIALS AND METHOD: A total of 105 endotracheal aspirates (ETA) samples were collected from the ICU of a public school in Bangladesh. This cross-sectional study was carried out in the Department of Microbiology, Chittagong for quantitative culture, CDST test, and multiplex PCR for blaIMP, blaVIM, blaNDM genes of MBL producers.

    RESULTS: Among the 105 clinically suspected VAP cases, the quantitative culture was positive in 95 (90%) and among 95 g-negative bacilli isolated from VAP patients, 46 (48.42%) were imipenem resistant, 30 (65.22%) were MBL producers by CDST, 21 (45.65%) were identified as MBL producers by multiplex PCR.

    CONCLUSION: PCR was highly sensitive and specific for the detection of MBL producers.

    Matched MeSH terms: Multiplex Polymerase Chain Reaction
  12. Fulltext Comparison of three molecular methods for the detection and speciation of five human Plasmodium species
    Lau YL, Lai MY, Anthony CN, Chang PY, Palaeya V, Fong MY, et al.
    Am J Trop Med Hyg, 2015 Jan;92(1):28-33.
    PMID: 25385862 DOI: 10.4269/ajtmh.14-0309
    In this study, three molecular assays (real-time multiplex polymerase chain reaction [PCR], merozoite surface antigen gene [MSP]-multiplex PCR, and the PlasmoNex Multiplex PCR Kit) have been developed for diagnosis of Plasmodium species. In total, 52 microscopy-positive and 20 malaria-negative samples were used in this study. We found that real-time multiplex PCR was the most sensitive for detecting P. falciparum and P. knowlesi. The MSP-multiplex PCR assay and the PlasmoNex Multiplex PCR Kit were equally sensitive for diagnosing P. knowlesi infection, whereas the PlasmoNex Multiplex PCR Kit and real-time multiplex PCR showed similar sensitivity for detecting P. vivax. The three molecular assays displayed 100% specificity for detecting malaria samples. We observed no significant differences between MSP-multiplex PCR and the PlasmoNex multiplex PCR kit (McNemar's test: P = 0.1489). However, significant differences were observed comparing real-time multiplex PCR with the PlasmoNex Multiplex PCR Kit (McNemar's test: P = 0.0044) or real-time multiplex PCR with MSP-multiplex PCR (McNemar's test: P = 0.0012).
    Matched MeSH terms: Multiplex Polymerase Chain Reaction
  13. DNA barcoding relates Trichuris species from a human and a man's best friend to non-human primate sources
    Brandon-Mong GJ, Ketzis JK, Choy JS, Boonroumkaew P, Tooba M, Sawangjaroen N, et al.
    Trop Biomed, 2018 Dec 01;35(4):1131-1139.
    PMID: 33601860
    Trichuris trichiura, the whipworm of humans, is one of the most prevalent soiltransmitted helminths (STH) reported worldwide. According to a recent study, out of 289 STH studies in Southeast Asia, only three studies used molecular methods. Hence, the genetic assemblages of Trichuris in Southeast Asia are poorly understood. In this study, we used partial mitochondrial DNA (cytochrome c oxidase subunit 1 or COI) sequences for analysis. Trichuris grouped in a same clade with different hosts indicate the potential of cross infection between hosts. Based on COI, the adult Trichuris isolated from a Malaysian patient was most closely related to Trichuris isolated from Papio anubis (olive baboons) from the USA. The Trichuris isolated from the dog from Malaysia was genetically similar to a Trichuris species isolated from Macaca silenus (lion-tailed macaque) from Czech Republic. Both the human and dog isolated Trichuris grouped in clades with different hosts indicating the potential of cross infection between hosts. Specific PCR primers based on the partial COI of T. trichiura isolated from African green monkey and T. serrata were designed and successfully amplified using multiplex PCR of the pooled DNA samples. Our results suggest a complex parasite-host relationship, and support the theory of cross infection of Trichuris between humans and non-human primates as suggested in previous publications.
    Matched MeSH terms: Multiplex Polymerase Chain Reaction
  14. Fulltext Detection of Respiratory Viruses from ARTI Patients by xTAG RVP Fast v2 Assay and Conventional Methods
    Kuan CS, Yew SM, Hooi PS, Lee LM, Ng KP
    Malays J Med Sci, 2017 Oct;24(5):33-43.
    PMID: 29386970 MyJurnal DOI: 10.21315/mjms2017.24.5.4
    Introduction: Acute respiratory tract infections (ARTIs) are a major cause of morbidity and mortality in paediatric patients. Therefore, early detection of the viral aetiologies of ARTIs is essential for patient management and infection control. In this study, we evaluated the performance of a new multiplex polymerase chain reaction (PCR) assay (xTAG Respiratory Viral Panel [RVP] Fast v2) in the detection of respiratory viruses by comparing it with that of viral culture and direct immunofluorescence (IF) staining.

    Methods: Nasopharyngeal swab and aspirate samples were collected prospectively from 199 patients who presented with ARTIs at the University Malaya Medical Centre (UMMC) in Kuala Lumpur, Malaysia during a 10-month period. The PCR assay was conducted in parallel with conventional culture and direct IF staining methods.

    Results: The positive rate of the xTAG RVP Fast v2 assay (78.4%) in detecting respiratory viruses was higher than that of the viral isolation (7.5%) and direct IF (23.1%) methods. Using the xTAG RVP Fast v2 assay, human enterovirus/human rhinovirus (HEV/HRV) was the most frequently detected (46.2%). The xTAG RVP Fast v2 assay revealed mixed infection caused by two or three respiratory viruses in 40 specimens, and these were undetected by the viral isolation and direct IF methods.

    Conclusion: The xTAG RVP Fast v2 assay was superior to conventional methods in the identification of common respiratory viruses, with higher sensitivity and shorter turnaround times for laboratory results.

    Matched MeSH terms: Multiplex Polymerase Chain Reaction
  15. Fulltext Detection of Vibrio cholerae in street food (satar and otak-otak) by Loop-Mediated Isothermal Amplification (LAMP), multiplex Polymerase Chain Reaction (mPCR) and plating methods
    Tang, J-Y-H., Farhana Sakinah, M.R., Nakaguchi, Y., Nishibuchi, M., Chai, L-C., New, C.Y., et al.
    Food Research, 2018;2(5):447-452.
    MyJurnal
    This goal of this study was to investigate the presence of Vibrio cholerae in street food,
    namely satar and otak-otak, using Loop-Mediated Isothermal Amplification (LAMP),
    multiplex Polymerase Chain Reaction (mPCR) and conventional plating on Thiosulphate
    Citrate Bile-Salt Sucrose (TCBS) agar methods. A total of 78 satar and 35 otak-otak were
    purchased from different districts of Terengganu (Besut, Setiu, Kuala Terengganu and
    Kemaman). V. cholerae was found in satar with LAMP (10.3%), mPCR (10.3%) and
    plating (0%). No V. cholerae was found in otak-otak using the three methods. This might
    be due to V. cholerae able to survive in satar after grilling due to its thickness which may
    contribute to undercooking. This study concluded that low presence of V. cholerae in satar
    and otak-otak can be detected by molecular methods but not the conventional plating
    method. LAMP assay is a useful tool for rapid detection of pathogens in food due to its
    simplicity, highly sensitive and visual interpretation capability. Though the prevalence of
    V. cholerae was low in the samples, proper handling of this food will help in reducing the
    risk of acquiring infection from V. cholerae in contaminated samples.
    Matched MeSH terms: Multiplex Polymerase Chain Reaction
  16. Fulltext Detection of selected intestinal helminths and protozoa at Hospital Universiti Sains Malaysia using multiplex real-time PCR
    Basuni M, Mohamed Z, Ahmad M, Zakaria NZ, Noordin R
    Trop Biomed, 2012 Sep;29(3):434-42.
    PMID: 23018507
    Intestinal parasites are the causative agents of a number of important human infections in developing countries. The objective of this study was to determine the prevalence of selected helminths and protozoan infections among patients admitted with gastrointestinal disorders at Hospital Universiti Sains Malaysia, Kelantan, Malaysia using multiplex real-time PCR. In addition microscopic examination was also performed following direct smear, zinc sulphate concentration and Kato-Katz thick smear techniques; and the presence of protozoan parasites was confirmed using trichrome and acid-fast stains. Of the 225 faecal samples analysed, 26.2% were positive for intestinal parasites by the multiplex real-time PCR, while 5.3% were positive by microscopy. As compared to microscopy, the multiplex real-time PCR detected 5.8 and 4.5 times more positives for the selected helminth and protozoan infections respectively. Among the selected helminths detected in this study, hookworm was the most prevalent by real-time PCR, while Ascaris lumbricoides was detected the most by microscopy. Meanwhile, among the selected protozoa detected in this study, Entamoeba histolytica was the most prevalent by real-time PCR, however microscopy detected equal number of cases with E. histolytica and Giardia lamblia. This study showed that real-time PCR can be used to obtain a more accurate prevalence data on intestinal helminths and protozoa.
    Matched MeSH terms: Multiplex Polymerase Chain Reaction
  17. 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: Multiplex Polymerase Chain Reaction
  18. Fulltext Development and evaluation of a Multiplex Polymerase Chain Reaction for the detection of Salmonella species
    Thong KL, Teh CS, Chua KH
    Trop Biomed, 2014 Dec;31(4):689-97.
    PMID: 25776594 MyJurnal
    The present study aims to develop a system which consists of four pairs of primers that specifically detects Salmonella spp., Salmonella serovar Typhi and Salmonella serovar Paratyphi A with an internal amplification control. The system, when applied in Polymerase Chain Reaction (PCR) under specific conditions, reaction mixture and cycling temperatures produced four bands; 784 bp, 496 bp, 332 bp and 187 bp. The DNA band 784 bp is present in all Salmonella spp., while the bands of 496 bp and 332 bp are only present in S. Paratyphi A and S. Typhi, respectively. An internal amplification control as indicated by the 187 bp shows the system is working in optimum condition in all the tests. This multiplex PCR was evaluated on 241 bacterial cultures and 691 naturally contaminated samples. Overall, this multiplex PCR detection system provides a single step for simultaneous detection of DNAs of Salmonella spp., S. Typhi and S. Paratyphi A.
    Matched MeSH terms: Multiplex Polymerase Chain Reaction/methods*; Multiplex Polymerase Chain Reaction/standards
  19. Development of a multiplex PCR assay for rapid identification of Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex
    Koh SF, Tay ST, Sermswan R, Wongratanacheewin S, Chua KH, Puthucheary SD
    J Microbiol Methods, 2012 Sep;90(3):305-8.
    PMID: 22705921 DOI: 10.1016/j.mimet.2012.06.002
    We have developed a multiplex PCR assay for rapid identification and differentiation of cultures for Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex. The assay is valuable for use in clinical and veterinary laboratories, and in a deployable laboratory during outbreaks.
    Matched MeSH terms: Multiplex Polymerase Chain Reaction*
  20. Fulltext Development of conventional and real-time multiplex PCR assays for the detection of nosocomial pathogens
    Anbazhagan D, Mui WS, Mansor M, Yan GO, Yusof MY, Sekaran SD
    Braz J Microbiol, 2011 Apr;42(2):448-58.
    PMID: 24031653 DOI: 10.1590/S1517-83822011000200006
    Nosocomial infections are major clinical threats to hospitalised patients and represent an important source of morbidity and mortality. It is necessary to develop rapid detection assays of nosocomial pathogens for better prognosis and initiation of antimicrobial therapy in patients. In this study, we present the development of molecular methods for the detection of six common nosocomial pathogens including Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. Conventional multiplex PCR and SYBR Green based real time PCR assays were performed using genus and species specific primers. Blind testing with 300 clinical samples was also carried out. The two assays were found to be sensitive and specific. Eubacterial PCR assay exhibited positive results for 46 clinical isolates from which 43 samples were detected by real time PCR assay. The sensitivity of the assay is about 93.7% in blind test isolates. The PCR results were reconfirmed using the conventional culture method. This assay has the potential to be a rapid, accurate and highly sensitive molecular diagnostic tool for simultaneous detection of Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. This assay has the potential to detect nosocomial pathogens within 5 to 6 hours, helping to initiate infection control measures and appropriate treatment in paediatric and elderly (old aged) patients, pre-and post surgery patients and organ transplant patients and thus reduces their hospitalization duration.
    Matched MeSH terms: Multiplex Polymerase Chain Reaction
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