Displaying publications 1 - 20 of 37 in total

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  1. 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: Real-Time Polymerase Chain Reaction/methods
  2. 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: Real-Time Polymerase Chain Reaction/methods*
  3. Fulltext Development of triplex real-time PCR and detection of Toxoplasma gondii DNA in infected mice tissues and spiked human samples
    Rahumatullah A, Khoo BY, Noordin R
    Trop Biomed, 2015 Jun;32(2):376-85.
    PMID: 26691266 MyJurnal
    Toxoplasma gondii is an important pathogen in veterinary and human medicine. In this study, a new multiplex TaqMan real-time PCR for detection of T. gondii DNA was developed. This assay consisted of new sets of primers and probes which targeted B1 gene and ITS-1 region of T. gondii, with Vibrio cholera gene as internal control. The B1 gene primers were designed to detect T. gondii RH strain, while the ITS-1 region primers detected most T. gondii strains. Specificity test using common protozoal and bacterial DNA revealed that the assay was very specific to T. gondii. Standard curves constructed using human body fluids spiked with T. gondii (RH and ME49 strains) showed that the sensitivity of the assay was one parasite, with R² value of 0.975 to 0.999 and efficiency of 97% to 99% for all types of samples. The assay performed on DNA extracted from tissues of mice infected with T. gondii showed that liver contained the highest parasite load for both strains of T. gondii. The multiplex real-time PCR developed in this study would be potentially useful for detection of T. gondii in human and animal samples.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
  4. Fulltext Development of multiplex real-time PCR for the rapid detection of five bacterial causes of community acquired pneumonia
    Al-Marzooq F, Imad MA, How SH, Kuan YC
    Trop Biomed, 2011 Dec;28(3):545-56.
    PMID: 22433883 MyJurnal
    Establishing a microbial diagnosis for patients with community-acquired pneumonia (CAP) is still challenging and is often achieved in only 30-50% of cases. Polymerase chain reaction (PCR) has been shown to be more sensitive than conventional microbiological methods and it could help to increase the microbial yield for CAP patients. This study was designed to develop, optimize and evaluate multiplex real-time PCR as a method for rapid differential detection of five bacterial causes of CAP namely Streptococcus pneumoniae, Burkholderia pseudomallei and atypical bacterial pathogens, Mycoplasma pneumoniae, Chlamydophila pneumoniae and Legionella pneumophila. Duplex and triplex real-time PCR assays were developed using five sets of primers and probes that were designed based on an appropriate specific gene for each of the above CAP pathogens. The performance of primers for each organism was tested using SYBR Green melt curve analysis following monoplex realtime PCR amplification. Monoplex real-time PCR assays were also used to optimize each primers-probe set before combining them in multiplex assays. Two multiplex real-time PCR assays were then optimized; duplex assay for the differential detection of S. pneumoniae and B. pseudomallei, and triplex assay for the atypical bacterial pathogens. Both duplex and triplex real-time PCR assays were tested for specificity by using DNA extracted from 26 related microorganisms and sensitivity by running serial dilutions of positive control DNAs. The developed multiplex real-time PCR assays shall be used later for directly identifying CAP causative agents in clinical samples.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
  5. Fulltext The use of multiplex real-time PCR improves the detection of the bacterial etiology of community acquired pneumonia
    Mustafa MI, Al-Marzooq F, How SH, Kuan YC, Ng TH
    Trop Biomed, 2011 Dec;28(3):531-44.
    PMID: 22433882 MyJurnal
    Community-acquired pneumonia (CAP) is still a major cause of morbidity and mortality especially to children and compromised hosts, such as the old and those with underlying chronic diseases. Knowledge of pathogens causing CAP constitutes the basis for selection of antimicrobial treatment. Previous data have shown that etiological agents can be identified in only up to 50% of patients, but this figure can be improved by using polymerase chain reaction (PCR). This study was designed to evaluate multiplex real-time PCR as a method for rapid differential detection of five bacterial causes of CAP (Streptococcus pneumoniae, Burkholderia pseudomallei and atypical bacterial pathogens namely Mycoplasma pneumoniae, Chlamydophila pneumoniae and Legionella pneumophila) in CAP patients attending Hospital Tengku Ampuan Afzan (HTAA)/ Kuantan, Pahang, Malaysia. Two previously developed multiplex real-time PCR assays, duplex for the differential detection of S. pneumoniae and B. pseudomallei and triplex for the atypical bacterial pathogens, were used to detect a bacterial cause of CAP in blood and respiratory samples. Thus, 46 blood and 45 respiratory samples collected from 46 adult CAP patients admitted to HTAA were analysed by multiplex real-time PCR assays and conventional methods. The microbial etiology of CAP could be established for 39.1% (18/46) of CAP patients by conventional methods and this was increased to 65.2% (30/46) with the additional use of real-time PCR. The most frequently detected pathogens were S. pneumoniae (21.7% - all by PCR alone), Klebsiella pneumoniae (17.3%), B. pseudomallei (13% - 83% of them positive by PCR alone and 17% by both culture and PCR), Pseudomonas aeruginosa (6.5%), M. pneumoniae (6.5% - all by serology), C. pneumoniae (4.3% - all positive by both PCR and serology), L. pneumophila (2.1% - all by PCR alone), Escherichia coli (4.3%). Haemophilus infuenzae, Acinetobacter lwoffii and Acinetobacter baumannii were detected by conventional methods (2.1% for each).
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
  6. 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: Real-Time Polymerase Chain Reaction/methods*
  7. Selection of reference genes for quantitative studies in acute myeloid leukaemia
    Lee C, Yiau KXS, Lee LJ, Chong PP, Chang KM, Abdullah M
    Malays J Pathol, 2019 Dec;41(3):313-326.
    PMID: 31901916
    INTRODUCTION: Quantitative polymerase chain reaction (qPCR) is commonly used in the investigation of acute myeloid leukaemias (AML). Stable reference genes (RG) are essential for accurate and reliable reporting but no standard method for selection has been endorsed.

    MATERIALS AND METHODS: We evaluated simple statistics and published model-based approaches. Multiplex-qPCR was conducted to determine the expression of 24 candidate RG in AMLs (N=9). Singleplex-qPCR was carried out on selected RG (SRP14, B2M and ATP5B) and genes of interest in AML (N=15) and healthy controls, HC (N=12).

    RESULTS: RG expression levels in AML samples were highly variable and coefficient of variance (CV) ranged from 0.37% to 10.17%. Analysis using GeNorm and Normfinder listed different orders of most stable genes but the top seven (ACTB, UBE2D2, B2M, NF45, RPL37A, GK, QARS) were the same. In singleplex-qPCR, SRP14 maintained the lowest CV in AML samples. B2M, one of most stable reference genes in AML, was expressed near significantly different in AML and HC. GeNorm selected ATP5B+SRP14 while Normfinder chose SRP14+B2M as the best two RG in combination. The median expressions of combined RG genes in AML compared to HC were less significantly different than individually implying smaller expression variation after combination. Genes of interest normalised with RG in combination or individually, displayed significantly different expression patterns.

    CONCLUSIONS: The selection of best reference gene in qPCR must consider all sample sets. Model-based approaches are important in large candidate gene analysis. This study showed combination of RG SRP14+B2M was the most suitable normalisation factor for qPCR analysis of AML and healthy individuals.

    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods
  8. Multiplex real-time RT-PCR assay for transfusion transmitted viruses in sero-negative allogeneic blood donors: an experience from Southern Pakistan
    Sultan S, Nasir MI, Rafiq S, Baig MA, Akbani S, Irfan SM
    Malays J Pathol, 2017 Aug;39(2):149-154.
    PMID: 28866696
    BACKGROUND: Blood transfusion safety commences with healthy donor recruitment. The threat of transfusion transmitted infections is greatly minimized by serological tools but not entirely eliminated. Recently, nucleic-acid testing for blood donor screening has virtually eliminated this jeopardy.

    METHODS: This prospective study was conducted from February 2015 to February 2016. Samples from seronegative donors were run on multiplex assay (Cobas, S-201 system platform, Roche) in a batch of six [MP-NAT]. In case of reactive pool, tests were run on every individual sample [IDNAT].

    RESULTS: Of 16957 donors, 16836 (99.2%) were replacement donors and the remaining 121 (0.7%) were voluntary donors, with a mean age of 29.09 ± 7.04 years. After serologic screening of all 16957 donors, 955 (5.6%) were found to be reactive; 291(1.71%) were reactive for hepatitis-B surface antigen, 361 (2.12%) for antibody to hepatitis C virus (anti-HCV), 14 (0.08%) for antibody to human immunodeficiency virus, 287 (1.69%) for syphilis and 2 (0.01%) for malaria. 14 (0.08%) NAT reactive donors were identified after testing the 16002 seronegative donors, with an overall NAT yield of one reactivity out of 1143 blood donations; 10 donors for HBV-DNA (HBV NAT yield-1:1600) and remaining 4 for HCV-RNA (HCV-NAT yield-1:4000). None were HIV positive.

    CONCLUSION: NAT has improved the safety attributes in blood products. Although the positivity rate for NAT testing is low but in view of the high prevalence of transfusion transmitted infections in our country, we recommend the parallel use of both serology and NAT screening of all donated blood.

    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
  9. Tricalcium phosphate/hydroxyapatite (TCP-HA) bone scaffold as potential candidate for the formation of tissue engineered bone
    Sulaiman SB, Keong TK, Cheng CH, Saim AB, Idrus RB
    Indian J Med Res, 2013 Jun;137(6):1093-101.
    PMID: 23852290
    Various materials have been used as scaffolds to suit different demands in tissue engineering. One of the most important criteria is that the scaffold must be biocompatible. This study was carried out to investigate the potential of HA or TCP/HA scaffold seeded with osteogenic induced sheep marrow cells (SMCs) for bone tissue engineering.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods
  10. Fulltext The Use of NS1 Rapid Diagnostic Test and qRT-PCR to Complement IgM ELISA for Improved Dengue Diagnosis from Single Specimen
    Teoh BT, Sam SS, Tan KK, Johari J, Abd-Jamil J, Hooi PS, et al.
    Sci Rep, 2016 06 09;6:27663.
    PMID: 27278716 DOI: 10.1038/srep27663
    Timely and accurate dengue diagnosis is important for differential diagnosis and immediate implementation of appropriate disease control measures. In this study, we compared the usefulness and applicability of NS1 RDT (NS1 Ag Strip) and qRT-PCR tests in complementing the IgM ELISA for dengue diagnosis on single serum specimen (n = 375). The NS1 Ag Strip and qRT-PCR showed a fair concordance (κ = 0.207, p = 0.001). While the NS1 Ag Strip showed higher positivity than qRT-PCR for acute (97.8% vs. 84.8%) and post-acute samples (94.8% vs. 71.8%) of primary infection, qRT-PCR showed higher positivity for acute (58.1% vs. 48.4%) and post-acute (50.0% vs.41.4%) samples in secondary infection. IgM ELISA showed higher positivity in samples from secondary dengue (74.2-94.8%) than in those from primary dengue (21.7-64.1%). More primary dengue samples showed positive with combined NS1 Ag Strip/IgM ELISA (99.0% vs. 92.8%) whereas more secondary samples showed positive with combined qRT-PCR/IgM ELISA (99.4% vs. 96.2%). Combined NS1 Ag Strip/IgM ELISA is a suitable combination tests for timely and accurate dengue diagnosis on single serum specimen. If complemented with qRT-PCR, combined NS1 Ag Strip/IgM ELISA would improve detection of secondary dengue samples.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
  11. Fulltext Single-tube, dual channel pentaplexing for the identification of Candida strains associated with human infection
    Jainlabdin MH, Batra A, Sánchez Paredes E, Hernández Hernández F, Fu G, Tovar-Torres J
    Sci Rep, 2019 10 11;9(1):14692.
    PMID: 31604994 DOI: 10.1038/s41598-019-51198-6
    Invasive candidiasis is one of the most common nosocomial fungal infections worldwide. Delayed implementation of effective antifungal treatment caused by inefficient Candida diagnosis contributes to its notoriously high mortality rates. The availability of better Candida diagnostic tools would positively impact patient outcomes. Here, we report on the development of a single-tube, dual channel pentaplex molecular diagnostic assay based on Multiplex Probe Amplification (MPA) technology. It allows simultaneous identification of C. auris, C. glabrata and C. krusei, at species-level as well as of six additional albicans and non-albicans pathogenic Candida at genus level. The assay overcomes the one-channel one-biomarker limitation of qPCR-based assays. Assay specificities are conferred by unique biomarker probe pairs with characteristic melting temperatures; post-amplification melting curve analysis allows simple identification of the infectious agent. Alerting for the presence of C. auris, the well-characterised multi-drug resistant outbreak strain, will facilitate informed therapy decisions and aid antifungal stewardship. The MPA-Candida assay can also be coupled to a pan-Fungal assay when differentiation between fungal and bacterial infections might be desirable. Its multiplexing capacity, detection range, specificity and sensitivity suggest the potential use of this novel MPA-Candida assay in clinical diagnosis and in the control and management of hospital outbreaks.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods
  12. 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: Real-Time Polymerase Chain Reaction/methods*
  13. Fulltext Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture
    Chan PL, Rose RJ, Abdul Murad AM, Zainal Z, Low ET, Ooi LC, et al.
    PLoS One, 2014;9(6):e99774.
    PMID: 24927412 DOI: 10.1371/journal.pone.0099774
    The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
  14. Fulltext Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence
    Gholami K, Loh SY, Salleh N, Lam SK, Hoe SZ
    PLoS One, 2017;12(6):e0176368.
    PMID: 28591185 DOI: 10.1371/journal.pone.0176368
    Real-time quantitative PCR (qPCR) is the most reliable and accurate technique for analyses of gene expression. Endogenous reference genes are being used to normalize qPCR data even though their expression may vary under different conditions and in different tissues. Nonetheless, verification of expression of reference genes in selected studied tissue is essential in order to accurately assess the level of expression of target genes of interest. Therefore, in this study, we attempted to examine six commonly used reference genes in order to identify the gene being expressed most constantly under the influence of testosterone in the kidneys and hypothalamus. The reference genes include glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), beta-2 microglobulin (B2m), hypoxanthine phosphoribosyltransferase 1 (HPRT), peptidylprolylisomerase A (Ppia) and hydroxymethylbilane synthase (Hmbs). The cycle threshold (Ct) value for each gene was determined and data obtained were analyzed using the software programs NormFinder, geNorm, BestKeeper, and rank aggregation. Results showed that Hmbs and Ppia genes were the most stably expressed in the hypothalamus. Meanwhile, in kidneys, Hmbs and GAPDH appeared to be the most constant genes. In conclusion, variations in expression levels of reference genes occur in kidneys and hypothalamus under similar conditions; thus, it is important to verify reference gene levels in these tissues prior to commencing any studies.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
  15. Fulltext Assessment of Aedes albopictus reference genes for quantitative PCR at different stages of development
    Dzaki N, Azzam G
    PLoS One, 2018;13(3):e0194664.
    PMID: 29554153 DOI: 10.1371/journal.pone.0194664
    Members of the Aedes genus of mosquitoes are widely recognized as vectors of viral diseases. Ae.albopictus is its most invasive species, and are known to carry viruses such as Dengue, Chikugunya and Zika. Its emerging importance puts Ae.albopictus on the forefront of genetic interaction and evolution studies. However, a panel of suitable reference genes specific for this insect is as of now undescribed. Nine reference genes, namely ACT, eEF1-γ, eIF2α, PP2A, RPL32, RPS17, PGK1, ILK and STK were evaluated. Expression patterns of the candidate reference genes were observed in a total of seventeen sample types, separated by stage of development and age. Gene stability was inferred from obtained quantification data through three widely cited evaluation algorithms i.e. BestKeeper, geNorm, and NormFinder. No single gene showed a satisfactory degree of stability throughout all developmental stages. Therefore, we propose combinations of PGK and ILK for early embryos; RPL32 and RPS17 for late embryos, all four larval instars, and pupae samples; eEF1-γ with STK for adult males; eEF1-γ with RPS17 for non-blood fed females; and eEF1-γ with eIF2α for both blood-fed females and cell culture. The results from this study should be able to provide a more informed selection of normalizing genes during qPCR in Ae.albopictus.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods
  16. Fulltext Development of a reverse transcription recombinase polymerase amplification assay for rapid and direct visual detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)
    Lau YL, Ismail IB, Mustapa NIB, Lai MY, Tuan Soh TS, Haji Hassan A, et al.
    PLoS One, 2021;16(1):e0245164.
    PMID: 33406112 DOI: 10.1371/journal.pone.0245164
    Rapid diagnosis is an important intervention in managing the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak. Real time reverse transcription polymerase chain reaction (RT-qPCR) remains the primary means for diagnosing the new virus strain but it is time consuming and costly. Recombinase polymerase amplification (RPA) is an isothermal amplification assay that does not require a PCR machine. It is an affordable, rapid, and simple assay. In this study, we developed and optimized a sensitive reverse transcription (RT)-RPA assay for the rapid detection of SARS-CoV-2 using SYBR Green I and/or lateral flow (LF) strip. The analytical sensitivity and specificity of the RT-RPA assay were tested by using 10-fold serial diluted synthetic RNA and genomic RNA of similar viruses, respectively. Clinical sensitivity and specificity of the RT-RPA assay were carried out using 78 positive and 35 negative nasopharyngeal samples. The detection limit of both RPA and RT-qPCR assays was 7.659 and 5 copies/μL RNA, respectively with no cross reactivity with other viruses. The clinical sensitivity and specificity of RT-RPA were 98% and 100%, respectively. Our study showed that RT-RPA represents a viable alternative to RT-qPCR for the detection of SARS-CoV-2, especially in areas with limited infrastructure.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
  17. Fulltext Real-time PCR assay in differentiating Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii infections in Orang Asli settlements in Malaysia
    Lau YL, Anthony C, Fakhrurrazi SA, Ibrahim J, Ithoi I, Mahmud R
    Parasit Vectors, 2013;6(1):250.
    PMID: 23985047 DOI: 10.1186/1756-3305-6-250
    Amebiasis caused by Entamoeba histolytica is the third leading cause of death worldwide. This pathogenic amoeba is morphologically indistinguishable from E. dispar and E. moshkovskii, the non-pathogenic species. Polymerase chain reaction is the current method of choice approved by World Health Organization. Real-time PCR is another attractive molecular method for diagnosis of infectious diseases as post-PCR analyses are eliminated and turnaround times are shorter. The present work aimed to compare the results of Entamoeba species identification using the real-time assay against the established nested PCR method.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
  18. Fulltext Diagnostic tools in childhood malaria
    Amir A, Cheong FW, De Silva JR, Lau YL
    Parasit Vectors, 2018 01 23;11(1):53.
    PMID: 29361963 DOI: 10.1186/s13071-018-2617-y
    Every year, millions of people are burdened with malaria. An estimated 429,000 casualties were reported in 2015, with the majority made up of children under five years old. Early and accurate diagnosis of malaria is of paramount importance to ensure appropriate administration of treatment. This minimizes the risk of parasite resistance development, reduces drug wastage and unnecessary adverse reaction to antimalarial drugs. Malaria diagnostic tools have expanded beyond the conventional microscopic examination of Giemsa-stained blood films. Contemporary and innovative techniques have emerged, mainly the rapid diagnostic tests (RDT) and other molecular diagnostic methods such as PCR, qPCR and loop-mediated isothermal amplification (LAMP). Even microscopic diagnosis has gone through a paradigm shift with the development of new techniques such as the quantitative buffy coat (QBC) method and the Partec rapid malaria test. This review explores the different diagnostic tools available for childhood malaria, each with their characteristic strengths and limitations. These tools play an important role in making an accurate malaria diagnosis to ensure that the use of anti-malaria are rationalized and that presumptive diagnosis would only be a thing of the past.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods
  19. Identification of reference genes for real-time polymerase chain reaction gene expression studies in Nile rats fed Water-Soluble Palm Fruit Extract
    Leow SS, Lee WK, Khoo JS, Teoh S, Hoh CC, Fairus S, et al.
    Mol Biol Rep, 2020 Dec;47(12):9409-9427.
    PMID: 33222119 DOI: 10.1007/s11033-020-06003-3
    The Nile rat (Arvicanthis niloticus) is a novel diurnal carbohydrate-sensitive rodent useful for studies on type 2 diabetes mellitus (T2DM) and the metabolic syndrome. Hepatic responses to T2DM and any interventions thereof can be evaluated via transcriptomic gene expression analysis. However, the study of gene expression via real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) requires identification of stably expressed reference genes for accurate normalisation. This study describes the evaluation and identification of stable reference genes in the livers from Control Nile rats as well as those supplemented with Water-Soluble Palm Fruit Extract, which has been previously shown to attenuate T2DM in this animal model. Seven genes identified as having stable expression in RNA-Sequencing transcriptome analysis were chosen for verification using real-time RT-qPCR. Six commonly used reference genes from previous literature and two genes from a previous microarray gene expression study in Nile rats were also evaluated. The expression data of these 15 candidate reference genes were analysed using the RefFinder software which incorporated analyses performed by various algorithms. The Hpd, Pnpla6 and Vpp2 genes were identified as the most stable across the 36 samples tested. Their applicability was demonstrated through the normalisation of the gene expression profiles of two target genes, Cela1 and Lepr. In conclusion, three novel reference genes which can be used for robust normalisation of real-time RT-qPCR data were identified, thereby facilitating future hepatic gene expression studies in the Nile rat.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
  20. High-Throughput RT-qPCR for the Analysis of Circulating MicroRNAs
    Tan GW, Tan LP
    Methods Mol Biol, 2017;1580:7-19.
    PMID: 28439823 DOI: 10.1007/978-1-4939-6866-4_2
    Reverse transcription followed by real-time or quantitative polymerase chain reaction (RT-qPCR) is the gold standard for validation of results from transcriptomic profiling studies such as microarray and RNA sequencing. The current need for most studies, especially biomarker studies, is to evaluate the expression levels or fold changes of many transcripts in a large number of samples. With conventional low to medium throughput qPCR platforms, many qPCR plates would have to be run and a significant amount of RNA input per sample will be required to complete the experiments. This is particularly challenging when the size of study material (small biopsy, laser capture microdissected cells, biofluid, etc.), time, and resources are limited. A sensitive and high-throughput qPCR platform is therefore optimal for the evaluation of many transcripts in a large number of samples because the time needed to complete the entire experiment is shortened and the usage of lab consumables as well as RNA input per sample are low. Here, the methods of high-throughput RT-qPCR for the analysis of circulating microRNAs are described. Two distinctive qPCR chemistries (probe-based and intercalating dye-based) can be applied using the methods described here.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
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