Displaying publications 1 - 20 of 49 in total

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  1. Kawasaki disease following coronavirus disease 2019 with prolonged fecal viral shedding
    Uda K, Okita K, Soneda K, Taniguchi K, Horikoshi Y
    Pediatr Int, 2021 05;63(5):597-599.
    PMID: 33278321 DOI: 10.1111/ped.14452
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods
  2. 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
  3. Fulltext A reverse transcription loop-mediated isothermal amplification for broad coverage detection of Asian and African Zika virus lineages
    Teoh BT, Chin KL, Samsudin NI, Loong SK, Sam SS, Tan KK, et al.
    BMC Infect Dis, 2020 Dec 11;20(1):947.
    PMID: 33308203 DOI: 10.1186/s12879-020-05585-4
    BACKGROUND: Early detection of Zika virus (ZIKV) infection during the viremia and viruria facilitates proper patient management and mosquito control measurement to prevent disease spread. Therefore, a cost-effective nucleic acid detection method for the diagnosis of ZIKV infection, especially in resource-deficient settings, is highly required.

    METHODS: In the present study, a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of both the Asian and African-lineage ZIKV. The detection limit, strain coverage and cross-reactivity of the ZIKV RT-LAMP assay was evaluated. The sensitivity and specificity of the RT-LAMP were also evaluated using a total of 24 simulated clinical samples. The ZIKV quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used as the reference assay.

    RESULTS: The detection limit of the RT-LAMP assay was 3.73 ZIKV RNA copies (probit analysis, P ≤ 0.05). The RT-LAMP assay detected the ZIKV genomes of both the Asian and African lineages without cross-reacting with other arthropod-borne viruses. The sensitivity and specificity of the RT-LAMP assay were 90% (95% CI = 59.6-98.2) and 100% (95% CI = 78.5-100.0), respectively. The RT-LAMP assay detected ZIKV genome in 9 of 24 (37.5%) of the simulated clinical samples compared to 10 of 24 (41.7%) by qRT-PCR assay with a high level of concordance (κ = 0.913, P 

    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods
  4. Clinical implications of conventional cytogenetics, fluorescence in situ hybridization (FISH) and molecular testing in chronic myeloid leukaemia patients in the tyrosine kinase inhibitor era - A review
    Ankathil R, Ismail SM, Mohd Yunus N, Sulong S, Husin A, Abdullah AD, et al.
    Malays J Pathol, 2020 Dec;42(3):307-321.
    PMID: 33361712
    Chronic myeloid leukaemia (CML) provides an illustrative disease model for both molecular pathogenesis of cancer and rational drug therapy. Imatinib mesylate (IM), a BCR-ABL1 targeted tyrosine kinase inhibitor (TKI) drug, is the first line gold standard drug for CML treatment. Conventional cytogenetic analysis (CCA) can identify the standard and variant Philadelphia (Ph) chromosome, and any additional complex chromosome abnormalities at diagnosis as well as during treatment course. Fluorescence in situ hybridization (FISH) is especially important for cells of CML patients with inadequate or inferior quality metaphases or those with variant Ph translocations. CCA in conjunction with FISH can serve as powerful tools in all phases of CML including the diagnosis, prognosis, risk stratification and monitoring of cytogenetic responses to treatment. Molecular techniques such as reverse transcriptase-polymerase chain reaction (RT-PCR) is used for the detection of BCR-ABL1 transcripts at diagnosis whereas quantitative reverse transcriptase-polymerase chain reaction (qRTPCR) is used at the time of diagnosis as well as during TKI therapy for the quantitation of BCR-ABL1 transcripts to evaluate the molecular response and minimal residual disease (MRD). Despite the excellent treatment results obtained after the introduction of TKI drugs, especially Imatinib mesylate (IM), resistance to TKIs develops in approximately 35% - 40% of CML patients on TKI therapy. Since point mutations in BCR-ABL1 are a common cause of IM resistance, mutation analysis is important in IM resistant patients. Mutations are reliably detected by nested PCR amplification of the translocated ABL1 kinase domain followed by direct sequencing of the entire amplified kinase domain. The objective of this review is to highlight the importance of regular and timely CCA, FISH analysis and molecular testing in the diagnosis, prognosis, assessment of therapeutic efficacy, evaluation of MRD and in the detection of BCR-ABL1 kinase mutations which cause therapeutic resistance in adult CML patients.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
  5. 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*
  6. Fulltext A quantitative reverse transcription-polymerase chain reaction for detection of Getah virus
    Sam SS, Teoh BT, Chee CM, Mohamed-Romai-Noor NA, Abd-Jamil J, Loong SK, et al.
    Sci Rep, 2018 12 05;8(1):17632.
    PMID: 30518924 DOI: 10.1038/s41598-018-36043-6
    Getah virus (GETV), a mosquito-borne alphavirus, is an emerging animal pathogen causing outbreaks among racehorses and pigs. Early detection of the GETV infection is essential for timely implementation of disease prevention and control interventions. Thus, a rapid and accurate nucleic acid detection method for GETV is highly needed. Here, two TaqMan minor groove binding (MGB) probe-based quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays were developed. The qRT-PCR primers and TaqMan MGB probe were designed based on the conserved region of nsP1 and nsP2 genes of 23 GETV genome sequences retrieved from GenBank. Only the qRT-PCR assay using nsP2-specific primers and probe detected all two Malaysia GETV strains (MM2021 and B254) without cross-reacting with other closely related arboviruses. The qRT-PCR assay detected as few as 10 copies of GETV RNA, but its detection limit at the 95% probability level was 63.25 GETV genome copies (probit analysis, P ≤ 0.05). Further validation of the qRT-PCR assay using 16 spiked simulated clinical specimens showed 100% for both sensitivity and specificity. In conclusion, the qRT-PCR assay developed in this study is useful for rapid, sensitive and specific detection and quantification of GETV.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
  7. Functional Analysis of Circular RNAs
    Shanmugapriya, Huda HA, Vijayarathna S, Oon CE, Chen Y, Kanwar JR, et al.
    Adv Exp Med Biol, 2018 9 28;1087:95-105.
    PMID: 30259360 DOI: 10.1007/978-981-13-1426-1_8
    Circular RNAs characterize a class of widespread and diverse endogenous RNAs which are non-coding RNAs that are made by back-splicing events and have covalently closed loops with no polyadenylated tails. Various indications specify that circular RNAs (circRNAs) are plentiful in the human transcriptome. However, their participation in biological processes remains mostly undescribed. To date thousands of circRNAs have been revealed in organisms ranging from Drosophila melanogaster to Homo sapiens. Functional studies specify that these transcripts control expression of protein-coding linear transcripts and thus encompass a key component of gene expression regulation. This chapter provide a comprehensive overview on functional validation of circRNAs. Furthermore, we discuss the recent modern methodologies for the functional validation of circRNAs such as RNA interference (RNAi) gene silencing assay, luciferase reporter assays, circRNA gain-of-function investigation via overexpression of circular transcript assay, RT-q-PCR quantification, and other latest applicable assays. The methods described in this chapter are demonstrated on the cellular model.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods
  8. Fulltext Performance of a Taqman Assay for Improved Detection and Quantification of Human Rhinovirus Viral Load
    Ng KT, Chook JB, Oong XY, Chan YF, Chan KG, Hanafi NS, et al.
    Sci Rep, 2016 10 10;6:34855.
    PMID: 27721388 DOI: 10.1038/srep34855
    Human rhinovirus (HRV) is the major aetiology of respiratory tract infections. HRV viral load assays are available but limitations that affect accurate quantification exist. We developed a one-step Taqman assay using oligonucleotides designed based on a comprehensive list of global HRV sequences. The new oligonucleotides targeting the 5'-UTR region showed high PCR efficiency (E = 99.6%, R2 = 0.996), with quantifiable viral load as low as 2 viral copies/μl. Assay evaluation using an External Quality Assessment (EQA) panel yielded a detection rate of 90%. When tested on 315 human enterovirus-positive specimens comprising at least 84 genetically distinct HRV types/serotypes (determined by the VP4/VP2 gene phylogenetic analysis), the assay detected all HRV species and types, as well as other non-polio enteroviruses. A commercial quantification kit, which failed to detect any of the EQA specimens, produced a detection rate of 13.3% (42/315) among the clinical specimens. Using the improved assay, we showed that HRV sheds in the upper respiratory tract for more than a week following acute infection. We also showed that HRV-C had a significantly higher viral load at 2-7 days after the onset of symptoms (p = 0.001). The availability of such assay is important to facilitate disease management, antiviral development, and infection control.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods
  9. Detection and Quantification of Chikungunya Virus by Real-Time RT-PCR Assay
    Wang SM, Ali UH, Sekaran SD, Thayan R
    Methods Mol Biol, 2016;1426:105-17.
    PMID: 27233265 DOI: 10.1007/978-1-4939-3618-2_10
    Real-time PCR assay has many advantages over conventional PCR methods, including rapidity, quantitative measurement, low risk of contamination, high sensitivity, high specificity, and ease of standardization (Mackay et al., Nucleic Acids Res 30:1292-1305, 2002). The real-time PCR system relies upon the measurement of a fluorescent reporter during PCR, in which the amount of emitted fluorescence is directly proportional to the amount of the PCR product in a reaction (Gibsons et al., Genome Res 6:995-1001, 1996). Here, we describe the use of SYBR Green I-based and TaqMan(®) real-time reverse transcription polymerase chain reaction (RT-PCR) for the detection and quantification of Chikungunya virus (CHIKV).
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
  10. Fulltext Validation and utilization of an internally controlled multiplex Real-time RT-PCR assay for simultaneous detection of enteroviruses and enterovirus A71 associated with hand foot and mouth disease
    Thanh TT, Anh NT, Tham NT, Van HM, Sabanathan S, Qui PT, et al.
    Virol J, 2015 Jun 09;12:85.
    PMID: 26050791 DOI: 10.1186/s12985-015-0316-2
    BACKGROUND: Hand foot and mouth disease (HFMD) is a disease of public health importance across the Asia-Pacific region. The disease is caused by enteroviruses (EVs), in particular enterovirus A71 (EV-A71). In EV-A71-associated HFMD, the infection is sometimes associated with severe manifestations including neurological involvement and fatal outcome. The availability of a robust diagnostic assay to distinguish EV-A71 from other EVs is important for patient management and outbreak response.

    METHODS: We developed and validated an internally controlled one-step single-tube real-time RT-PCR in terms of sensitivity, linearity, precision, and specificity for simultaneous detection of EVs and EV-A71. Subsequently, the assay was then applied on throat and rectal swabs sampled from 434 HFMD patients.

    RESULTS: The assay was evaluated using both plasmid DNA and viral RNA and has shown to be reproducible with a maximum assay variation of 4.41 % and sensitive with a limit of detection less than 10 copies of target template per reaction, while cross-reactivity with other EV serotypes was not observed. When compared against a published VP1 nested RT-PCR using 112 diagnostic throat and rectal swabs from 112 children with a clinical diagnosis of HFMD during 2014, the multiplex assay had a higher sensitivity and 100 % concordance with sequencing results which showed EVs in 77/112 (68.8 %) and EV-A71 in 7/112 (6.3 %). When applied to clinical diagnostics for 322 children, the assay detected EVs in throat swabs of 257/322 (79.8 %) of which EV-A71 was detected in 36/322 (11.2 %) children. The detection rate increased to 93.5 % (301/322) and 13.4 % (43/322) for EVs and EV-A71, respectively, when rectal swabs from 65 throat-negative children were further analyzed.

    CONCLUSION: We have successfully developed and validated a sensitive internally controlled multiplex assay for rapid detection of EVs and EV-A71, which is useful for clinical management and outbreak control of HFMD.

    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
  11. 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: Reverse Transcriptase Polymerase Chain Reaction/methods*
  12. Fulltext Early detection of dengue virus by use of reverse transcription-recombinase polymerase amplification
    Teoh BT, Sam SS, Tan KK, Danlami MB, Shu MH, Johari J, et al.
    J Clin Microbiol, 2015 Mar;53(3):830-7.
    PMID: 25568438 DOI: 10.1128/JCM.02648-14
    A method for the rapid diagnosis of early dengue virus (DENV) infection is highly needed. Here, a prototype reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed. The assay detected DENV RNA in <20 min without the need for thermocycling amplification. The assay enabled the detection of as few as 10 copies of DENV RNA. The designed RT-RPA primers and exo probe detected the DENV genome of at least 12 genotypes of DENV circulating globally without cross-reacting with other arboviruses. We assessed the diagnostic performance of the RT-RPA assay for the detection of DENV RNA in 203 serum samples of patients with clinically suspected dengue. The sera were simultaneously tested for DENV using a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay, quantitative RT-PCR (qRT-PCR), and IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA). Acute DENV infection was confirmed in 130 samples and 61 of the samples (46.9%) were classified as viremic with qRT-PCR. The RT-RPA assay showed good concordance (κ of ≥0.723) with the RT-LAMP and qRT-PCR assays in detecting the dengue viremic samples. When used in combination with ELISA, both the RT-RPA and RT-LAMP assays increased the detection of acute DENV infection to ≥95.7% (≥45/47) in samples obtained within 5 days of illness. The results from the study suggest that the RT-RPA assay is the most rapid molecular diagnostic tool available for the detection of DENV. Hence, it is possible to use the RT-RPA assay in a laboratory to complement routine serology testing for dengue.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
  13. Fulltext Development of conventional and real-time reverse transcription polymerase chain reaction assays to detect Tembusu virus in Culex tarsalis mosquitoes
    Petz LN, Turell MJ, Padilla S, Long LS, Reinbold-Wasson DD, Smith DR, et al.
    Am J Trop Med Hyg, 2014 Oct;91(4):666-71.
    PMID: 25114013 DOI: 10.4269/ajtmh.13-0218
    Tembusu virus (TMUV) is an important emerging arthropod-borne virus that may cause encephalitis in humans and has been isolated in regions of southeast Asia, including Malaysia, Thailand, and China. Currently, detection and identification of TMUV are limited to research laboratories, because quantitative rapid diagnostic assays for the virus do not exist. We describe the development of sensitive and specific conventional and real-time quantitative reverse transcription polymerase chain reaction assays for detecting TMUV RNA in infected cell culture supernatant and Culex tarsalis mosquitoes. We used this assay to document the replication of TMUV in Cx. tarsalis, where titers increased 1,000-fold 5 days after inoculation. These assays resulted in the detection of virus-specific RNA in the presence of copurified mosquito nucleic acids. The use of these rapid diagnostic assays may have future applications for field pathogen surveillance and may assist in early detection, diagnosis, and control of the associated arthropod-borne pathogens.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
  14. Polymerase chain reaction-based discrimination of viable from non-viable Mycoplasma gallisepticum
    Tan CG, Ideris A, Omar AR, Yii CP, Kleven SH
    Onderstepoort J Vet Res, 2014 09 02;81(1):e1-e7.
    PMID: 25686255 DOI: 10.4102/ojvr.v81i1.708
    The present study was based on the reverse transcription polymerase chain reaction (RT-PCR) of the 16S ribosomal nucleic acid (rRNA) of Mycoplasma for detection of viable Mycoplasma gallisepticum. To determine the stability of M. gallisepticum 16S rRNA in vitro, three inactivation methods were used and the suspensions were stored at different temperatures. The 16S rRNA of M. gallisepticum was detected up to approximately 20-25 h at 37 °C, 22-25 h at 16 °C, and 23-27 h at 4 °C. The test, therefore, could detect viable or recently dead M. gallisepticum (< 20 h). The RT-PCR method was applied during an in vivo study of drug efficacy under experimental conditions, where commercial broiler-breeder eggs were inoculated with M. gallisepticum into the yolk. Hatched chicks that had been inoculated in ovo were treated with Macrolide 1. The method was then applied in a flock of day 0 chicks with naturally acquired vertical transmission of M. gallisepticum, treated with Macrolide 2. Swabs of the respiratory tract were obtained for PCR and RT-PCR evaluations to determine the viability of M. gallisepticum. This study proved that the combination of both PCR and RT-PCR enables detection and differentiation of viable from non-viable M. gallisepticum.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
  15. Fulltext Transcriptional profiling of feline infectious peritonitis virus infection in CRFK cells and in PBMCs from FIP diagnosed cats
    Harun MS, Kuan CO, Selvarajah GT, Wei TS, Arshad SS, Hair Bejo M, et al.
    Virol J, 2013;10:329.
    PMID: 24209771 DOI: 10.1186/1743-422X-10-329
    BACKGROUND:
    Feline Infectious Peritonitis (FIP) is a lethal systemic disease, caused by the FIP Virus (FIPV); a virulent mutant of Feline Enteric Coronavirus (FECV). Currently, the viruses virulence determinants and host gene expressions during FIPV infection are not fully understood.

    METHODS:
    RNA sequencing of Crandell Rees Feline Kidney (CRFK) cells, infected with FIPV strain 79-1146 at 3 hours post infection (h.p.i), were sequenced using the Illumina next generation sequencing approach. Bioinformatic's analysis, based on Felis catus 2X annotated shotgun reference genome, using CLC bio Genome Workbench mapped both control and infected cell reads to 18899 genes out of 19046 annotated genes. Kal's Z test statistical analysis was used to analyse the differentially expressed genes from the infected CRFK cells. Real time RT-qPCR was developed for further transcriptional profiling of three genes (PD-1, PD-L1 and A3H) in infected CRFK cells and Peripheral Blood Mononuclear Cells (PBMCs) from healthy and FIP-diseased cats.

    RESULTS:
    Based on Kal's Z-test, with False Discovery Rate (FDR) <0.05 and >1.99 fold change on gene expressions, a total of 61 genes were differentially expressed by both samples, where 44 genes were up-regulated and the remainder were down-regulated. Most genes were closely clustered together, suggesting a homogeneous expression. The majority of the genes that were significantly regulated, were those associated with monocytes-macrophage and Th1 cell functions, and the regulation of apoptosis. Real time RT-qPCR developed focusing on 2 up-regulated genes (PD-L1 and A3H) together with an apoptosis associated gene PD-1 expressions in FIPV infected CRFK cells and in PBMCs from healthy and FIP diagnosed cats produced concordant results with transcriptome data.

    CONCLUSION:
    The possible roles of these genes, and their importance in feline coronaviruses infection, are discussed.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods
  16. Current status of diagnostic methods for henipavirus
    Tamin A, Rota PA
    Dev Biol (Basel), 2013;135:139-45.
    PMID: 23689891 DOI: 10.1159/000189236
    Hendra virus (HeV) and Nipah virus (NiV) are the causative agents of emerging transboundary animal disease in pigs and horses. They also cause fatal disease in humans. NiV has a case fatality rate of 40 - 100%. In the initial NiV outbreak in Malaysia in 1999, about 1.1 million pigs had to be culled. The economic impact was estimated to be approximately US$450 million. Worldwide, HeV has caused more than 60 deaths in horses with 7 human cases and 4 deaths. Since the initial outbreak, HeV spillovers from Pteropus bats to horses and humans continue. This article presents a brief review on the currently available diagnostic methods for henipavirus infections, including advances achieved since the initial outbreak, and a gap analysis of areas needing improvement.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods
  17. Proteomic analysis reveals that treatment with tocotrienols reverses the effect of H₂O₂ exposure on peroxiredoxin expression in human lymphocytes from young and old individuals
    Dahlan HM, Karsani SA, Rahman MA, Hamid NA, Top AG, Ngah WZ
    J Nutr Biochem, 2012 Jul;23(7):741-51.
    PMID: 21840697 DOI: 10.1016/j.jnutbio.2011.03.018
    Vitamin E has been suggested to modulate age-associated changes by altering the redox balance resulting in altered gene and/or protein expression. Here we have utilized proteomics to determine whether such regulation in protein expression occurs in human lymphocytes from two different age groups stressed with H₂O₂ and then treated with vitamin E in the form of tocotrienol-rich fraction (TRF). In this study, lymphocytes obtained from young (30-49 years old) and old (>50 years old) volunteers were first challenged with 1 mM H₂O₂. They were then treated by exposure to 50, 100 and 200 μg/ml TRF. Two-dimensional gel electrophoresis followed by MALDI-TOF/TOF (matrix-assisted laser desorption/ionization time-of-flight/time-of-flight) tandem mass spectrometry was then performed on whole-cell protein extracts to identify proteins that have changed in expression. A total of 24 proteins were found to be affected by H₂O₂ and/or TRF treatment. These included proteins that were related to metabolism, antioxidants, structural proteins, protein degradation and signal transduction. Of particular interest was the regulation of a number of proteins involved in stress response--peroxiredoxin-2, peroxiredoxin-3 and peroxiredoxin-6-all of which were shown to be down-regulated with H₂O₂ exposure. The effect was reversed following TRF treatment. The expression of peroxiredoxin-2 and peroxiredoxin-6 was confirmed by quantitative reverse transcriptase polymerase chain reaction. These results suggested that TRF directly influenced the expression dynamics of the peroxiredoxin-2, thus improving the cells ability to resist damage caused by oxidative stress.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods
  18. Real-time quantification for BCR-ABL transcripts in chronic myeloid leukaemia patients in UKMMC, Malaysia
    Wong FL, Hamidah NH, Hawa AA, Nurul AN, Leong CF, Saw F, et al.
    Malays J Pathol, 2011 Dec;33(2):107-12.
    PMID: 22299211
    Molecular pathogenesis of chronic myeloid leukemia (CML) is well established and molecular monitoring for patients with CML has become an important practice in the management of patients on imatinib therapy. In the present study, we report the use of RQ-PCR method for detection of BCR-ABL fusion gene for our CML cases. We performed a two-step RQ-PCR on bone marrow aspirates or peripheral blood of 37 CML patients. Quantitative expression of BCR-ABL fusion gene was carried out relative to the expression of a housekeeping gene as endogenous control to compensate for uneven cell numbers, RNA quality, or variations in reverse transcription efficiencies. Twenty-four of these patients were pre-treated with hydroxyurea or alpha interferon prior to the imatinib therapy. Their BCR-ABL fusion gene levels were monitored for 18 months. All samples processed were evaluable. The PCR amplification efficiency of the ABL gene is 90.5% (0.2158) and the BCR-ABL gene, 93.4% (0.1573).
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
  19. Fulltext A comparative evaluation of dengue diagnostic tests based on single-acute serum samples for laboratory confirmation of acute dengue
    Chua KB, Mustafa B, Abdul Wahab AH, Chem YK, Khairul AH, Kumarasamy V, et al.
    Malays J Pathol, 2011 Jun;33(1):13-20.
    PMID: 21874746
    A prospective study was carried out to evaluate the sensitivity of dengue NS1 antigen-capture ELISA in comparison with dengue virus isolation, conventional RT-PCR and real-time RT-PCR for laboratory confirmation of acute dengue based on single-acute serum samples. Four primary healthcare centres were involved to recruit patients with clinical diagnosis of dengue illness. Patient's demographic, epidemiological and clinical information were collected on a standardized data entry form and 5 ml of venous blood was collected upon consent. In the laboratory, six types of laboratory tests were performed on each of the collected acute serum sample. Of the 558 acute serum samples collected from 558 patients with clinical diagnosis of dengue from mid-August 2006 to March 2009, 174 serum samples were tested positive by the dengue NS1 antigen-capture ELISA, 77 by virus isolation, 92 by RT-PCR and 112 by real-time RT-PCR. A total of 190 serum samples were tested positive by either one or a combination of the four methods whereas, only 59 serum samples were tested positive by all four methods. Thus, based on single-acute serum samples, 190 of the 558 patients (34.1%) were laboratory-confirmed acute dengue. The overall test sensitivity was 91.6%, 40.5%, 48.4% and 58.9% for dengue NS1 antigen-capture ELISA, virus isolation, conventional RT-PCR and real-time RT-PCR respectively. Statistically, dengue NS1 antigen-capture ELISA was the most sensitive and virus isolation was the least sensitive test for the laboratory confirmation of acute dengue based on single-acute serum specimens. Real-time RT-PCR was significantly more sensitive than the conventional RT-PCR.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
  20. Fulltext A pentaplex real-time polymerase chain reaction assay for detection of four species of soil-transmitted helminths
    Basuni M, Muhi J, Othman N, Verweij JJ, Ahmad M, Miswan N, et al.
    Am J Trop Med Hyg, 2011 Feb;84(2):338-43.
    PMID: 21292911 DOI: 10.4269/ajtmh.2011.10-0499
    Soil-transmitted helminth infections remain a major public health burden in low- and middle-income countries. The traditional diagnosis by microscopic examination of fecal samples is insensitive and time-consuming. In this study, a pentaplex real-time polymerase chain reaction (PCR) was evaluated for the simultaneous detection of Ancylostoma, Necator americanus, Ascaris lumbricoides, and Strongyloides stercoralis. The results were compared with those obtained by conventional parasitological diagnostic methods. Real-time PCR was positive in 48 of 77 samples (62.3%) and microscopic examination was positive in six samples (7.8%) only (P < 0.05). In conclusion, the real-time PCR assay described in this study provides a specific and sensitive diagnostic tool for the detection of these four helminth species in epidemiological studies and monitoring of treatment programs.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
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