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).
From its discovery in Malaysia in the late 1990s, the spillover of the Nipah virus from its pteropid reservoir into the human population has resulted in sporadic outbreaks of fatal encephalitis and respiratory disease. In this chapter, we revise a previously described quantitative reverse transcription polymerase chain reaction method, which now utilizes degenerate nucleotides at certain positions in the probe and the reverse primer to accommodate the sequence heterogeneity observed within the Nipah henipavirus species.
Kapsaisinoid merupakan alkaloid yang memberikan ciri kepedasan pada cili serta khusus pada genus Capsicum. Sebatian kapsaisinoid terdiri daripada dua komponen utama iaitu kapsaisin dan dihidrokapsaisin. Dalam kajian ini, pengklonan cDNA Kapsaisin sintase (Cs) telah berjaya dilakukan menerusi kaedah transkripsi berbalik PCR (RT-PCR) dan klon cDNA tersebut dinamakan CUKMCS yang bersaiz 981 pb. Pencarian homologi menggunakan program blastx dan blastp yang terdapat pada pangkalan data NCBI mendapati CUKMCS mempunyai persamaan yang sangat tinggi terhadap Cs pada Capsicum frutescens, Capsicum annuum dan Capsicum chacoense. Saiz ramalan protein CUKMCS diangggarkan sekitar 36 kDa. Penentuan pengekspresan transkrip Cs pada 5 tisu yang berbeza mendapati transkrip dikesan pada tisu plasenta, mesokarp dan biji manakala tiada transkrip Cs dikesan pada daun dan akar
Emerging SARS-CoV-2 variants of concern (VOC) have been associated with enhanced transmissibility and immune escape. Next-generation sequencing (NGS) of the whole genome is the gold standard for variant identification for surveillance but is time-consuming and costly. Rapid and cost-effective assays that detect SARS-CoV-2 variants are needed. We evaluated Allplex SARS-CoV-2 Master Assay and Variants I Assay to detect HV69/70 deletion, Y144 deletion, E484K, N501Y, and P681H spike mutations in 248 positive samples collected in Kuala Lumpur, Malaysia, between January and May 2021. Spike variants were detected in 78/248 (31.5 %), comprising 60 VOC B.1.351 (beta) and 18 B.1.1.7 (alpha). With NGS as reference for 115 samples, the sensitivity for detecting the spike mutations was 98.7 % with the Master Assay and 100 % with the Variants I Assay. The emergence of beta variants correlated with increasing COVID-19 infections in Malaysia. The prevalence of alpha VOC and lineage B.1.466.2 was low. These assays detect mutations present in alpha, beta and gamma VOCs. Of the VOCs which have subsequently emerged, the assays should detect omicron (B.1.1.529) but not B.1.617.2 (delta). In conclusion, spike variant PCR assays can be used to rapidly monitor selected SARS-CoV-2 VOCs in resource-limited settings, but require updates as new variants emerge.
The aim of the study is to determine the neuronal and glial gene expression of cultured human amniotic epithelial cells (HAECs) in serial passages. HAECs obtained from human term placentae were cultured in F12:DMEM (1:1) + 10% FBS +10ng/ml EGF in serial passages. Quantitative RT-PCR was used to assess the gene expression analysis. The results showed that the cultured HAECs expressed the neural stem cell genes (Nestin, NSE and Vimentin), mature neuronal genes (TH, MAP-2, beta-III-tubulin and NFM) and glial genes (CNPase, MBP and Olig). These neural stem cell genes increased with serial passages while the genes expression for mature neuronal and glial cells were downregulated. These results suggested that HAECs may promote or involve in neurogenesis and gliagenesis.
Increasing evidence of the association between ribosomal protein (RP) genes with nasopharyngeal carcinoma (NPC) have been derived from findings of their differential expression patterns in NPC cell lines. Nevertheless, expression data from a comprehensive list of RP gene family members is still lacking. This paper reports the assessment of two RP genes, eL13 and eL14, with regards to their expression patterns in several NPC cell lines (TW04, TW01, HK1, HONE1 and SUNE-1) relative to a non-malignant control (NP69). A conventional Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assay was employed. Analysis of eL13 has never been explored before this, whereas investigation of eL14 represents an extended study. We found a general over-expression trend of eL14 in 40% (2 of 5; TW01 and HONE-1) of the NPC cell lines studied, with higher upregulated level in only one (TW01) of them. However, this pattern of expression level is not statistically significant. Expression of eL13 was not detected in any of the cell lines used. The inconsistency of these expression patterns demonstrates an elusive nature of RP activities in the malignancy of the nasopharynx.
Tilapia lake virus (TiLV) is a highly contagious viral pathogen that affects tilapia, a globally significant and affordable source of fish protein. To prevent the introduction and spread of TiLV and its impact, there is an urgent need for increased surveillance, improved biosecurity measures, and continuous development of effective diagnostic and rapid sequencing methods. In this study, we have developed a multiplexed RT-PCR assay that can amplify all ten complete genomic segments of TiLV from various sources of isolation. The amplicons generated using this approach were immediately subjected to real-time sequencing on the Nanopore system. By using this approach, we have recovered and assembled 10 TiLV genomes from total RNA extracted from naturally TiLV-infected tilapia fish, concentrated tilapia rearing water, and cell culture. Our phylogenetic analysis, consisting of more than 36 TiLV genomes from both newly sequenced and publicly available TiLV genomes, provides new insights into the high genetic diversity of TiLV. This work is an essential steppingstone towards integrating rapid and real-time Nanopore-based amplicon sequencing into routine genomic surveillance of TiLV, as well as future vaccine development.
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.
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).
Enteroviruses are positive stranded RNA viruses belonging to the genus Enterovirus of the Picornaviridae family. Human enteroviruses are transmitted through the fecal-oral route and have been shown to cause mild to life-threatening diseases. Various diagnostic methods have been developed to detect enteroviruses from clinical specimens but many were impeded by requirements for special reagents, lengthy procedures, low sensitivity or cross-reactivity. This chapter describes rapid and highly sensitive methods of enteroviral detection directly from clinical specimens based on a conventional one-step Reverse Transcription polymerase chain reaction (RT-PCR) and a one-step real-time RT-PCR.
The VP4, VP2, and VP1 gene regions were evaluated for their usefulness in typing human enteroviruses. Three published RT-PCR primers sets targeting separately these three gene regions were used. Initially, from a total of 86 field isolates (36 HEV-A, 40 HEV-B, and 10 HEV-C) tested, 100% concordance in HEV-A was identified from all three gene regions (VP4, VP2, and VP1). However, for HEV-B and HEV-C viruses, only the VP2 and VP1 regions, and not VP4, showed 100% concordance in typing these viruses. To evaluate further the usefulness of VP4 in typing HEV-A enteroviruses, 55 Japanese and 203 published paired VP4 and VP1 nucleotide sequences were also examined. In each case, typing by VP4 was 100% in concordance with typing using VP1. Given these results, it is proposed that for HEV-A enteroviruses, all three gene regions (VP4, VP2, and VP1), would be useful for typing these viruses. These options would enhance the capability of laboratories in identifying these viruses and would greatly help in outbreaks of hand, foot, and mouth disease.
Male-specific coliphages are often used as indicators of contamination by enteric viruses. These phages can be detected in water samples by plaque assays and by polymerase chain reaction. In this study, the M13 coliphage was used to develop a real-time PCR assay for the detection of male-specific DNA coliphages. The real-time PCR was found to have a reaction efficiency of 1.45 and detection limit of 10(-3) plaque forming units per reaction mix. Repeated amplification and melting curve analyses demonstrated high specificity and reproducibility of the real-time assay. Quantitative detection with the real-time PCR should allow rapid assessment of the level of viral contamination in water.
Continuous outbreaks of the highly pathogenic H5N1 avian influenza A in Asia has resulted in an urgent effort to improve current diagnostics to aid containment of the virus and lower the threat of a influenza pandemic. We report here the development of a PCR-based assay that is highly specific for the H5N1 avian influenza A virus.
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).
Human enterovirus 71 has emerged as an important pathogen in the Asia Pacific region and it is important to be able to make a rapid and specific diagnosis for outbreak control. Recent Asian strains of Coxsackievirus A16 have changes in the VP1 gene which causes mispriming of widely used primers for human enterovirus 71 specific identification.
A study of chikungunya virus was carried out to establish Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR) as a rapid detection technique of the virus. The susceptibility of lab-colonized Aedes aegypti to chikungunya virus was also determined. Artificial membrane feeding technique was used to orally feed the mosquitoes with a human isolate of chikungunya virus. A total of 100 fully engorged female Ae. aegypti were obtained and maintained for 7 days. Seventy of them survived and then pooled at 10 individuals per pool. Total RNA was extracted from the samples and RT-PCR amplifications were carried out. Five out of 7 pools showed positive PCR band at 350-bp, indicating Ae. aegypti is a potential vector of chikungunya virus. The minimum infection rate (MIR) was 71% within these laboratory colonies. RT-PCR is a sensitive technique that is useful in detecting infected mosquitoes in epidemic areas. This technique can de used as a rapid detection method and provide an early virologic surveillance systems of chikungunya virus infected mosquitoes.
In childhood acute lymphoblastic leukaemia (ALL), cytogenetics play an important role in diagnosis, allocation of treatment and prognosis. Conventional cytogenetic analysis, involving mainly karyotyping in our experience, has not been successful in a large proportion of cases due to inadequate metaphase spreads and poor chromosome morphology. Our aim is to develop a highly sensitive and specific method to screen simultaneously for the four most frequent fusion transcripts resulting from specific chromosomal translocations, namely, both the CML- and ALLtype BCR-ABL transcripts of t(9;22), E2A-PBX1 transcript of t(1;19), the MLL-AF4 transcript of t(4;11) and TEL-AML1 (also termed ETV6-CBFA2) of the cryptic t(12;21). A multiplex reverse transcription polymerase chain reaction protocol (RT-PCR) was developed and tested out on archival bone marrow samples and leukaemia cell lines. In all samples with a known translocation detected by cytogenetic techniques, the same translocation was identified by the multiplex-PCR assay. Multiplex RT-PCR assay is an effective, sensitive, accurate and cost-effective diagnostic tool which can improve our ability to accurately and rapidly risk-stratify patients with childhood ALL.
Dengue fever and dengue haemorrhagic fever currently rank highly among the newly-emerging infectious diseases, and are considered to be the most important arboviral disease worldwide. The definitive diagnosis is culture analysis, but practical considerations limit its use. Also, the period for viral detection is limited. Within a day or two after fever subsides, rising levels of antibodies interfere with viral cultures. An alternative to this quandary is the use of viral RNA detection assays. In our laboratory, a reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed using a set of degenerate primers.
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.