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Displaying publications 41 - 49 of 49 in total

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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
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*
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
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*
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*
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*
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
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
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