Displaying publications 1 - 20 of 307 in total

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  1. Garbuglia AR, Lapa D, Pauciullo S, Raoul H, Pannetier D
    Viruses, 2023 Oct 07;15(10).
    PMID: 37896839 DOI: 10.3390/v15102062
    Nipah virus (NiV) is a paramyxovirus responsible for a high mortality rate zoonosis. As a result, it has been included in the list of Blueprint priority pathogens. Bats are the main reservoirs of the virus, and different clinical courses have been described in humans. The Bangladesh strain (NiV-B) is often associated with severe respiratory disease, whereas the Malaysian strain (NiV-M) is often associated with severe encephalitis. An early diagnosis of NiV infection is crucial to limit the outbreak and to provide appropriate care to the patient. Due to high specificity and sensitivity, qRT-PCR is currently considered to be the optimum method in acute NiV infection assessment. Nasal swabs, cerebrospinal fluid, urine, and blood are used for RT-PCR testing. N gene represents the main target used in molecular assays. Different sensitivities have been observed depending on the platform used: real-time PCR showed a sensitivity of about 103 equivalent copies/reaction, SYBRGREEN technology's sensitivity was about 20 equivalent copies/reaction, and in multiple pathogen card arrays, the lowest limit of detection (LOD) was estimated to be 54 equivalent copies/reaction. An international standard for NiV is yet to be established, making it difficult to compare the sensitivity of the different methods. Serological assays are for the most part used in seroprevalence studies owing to their lower sensitivity in acute infection. Due to the high epidemic and pandemic potential of this virus, the diagnosis of NiV should be included in a more global One Health approach to improve surveillance and preparedness for the benefit of public health. Some steps need to be conducted in the diagnostic field in order to become more efficient in epidemic management, such as development of point-of-care (PoC) assays for the rapid diagnosis of NiV.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  2. Andrew A, Citartan M, Wong KA, Tang TH, Magdline Sia Henry S, Ch'ng ES
    Microbiol Spectr, 2023 Aug 17;11(4):e0008823.
    PMID: 37272795 DOI: 10.1128/spectrum.00088-23
    Due to the general symptoms presented by the Chikungunya virus (CHIKV)-infected patients, a laboratory test is needed to differentiate CHIKV from other viral infections. The reverse transcription-quantitative real-time PCR (RT-qPCR) is a rapid and sensitive diagnostic tool, and several assays have been developed for detecting and quantifying CHIKV. Since real-time amplification efficiency varies within and between laboratories, an assay must be validated before being used on patient samples. In this study, the diagnostic performance of a TaqMan RT-qPCR assay was evaluated using synthetic RNA and archived patient samples. The cutoff quantification cycle (Cq) value for the assay was determined by experimental evidence. We found the in-house assay was highly sensitive, with a detection limit of 3.95 RNA copies/reaction. The analytical specificity of the assay was 100%. The analytical cutoff Cq value was 37, corresponding to the mean Cq value of the detection limit. Using archived samples characterized previously, the sensitivity and specificity of the assay were 76% and 100%, respectively. The in-house assay was also compared with a commercial assay, and we found that the in-house assay had higher sensitivity. Although further evaluation with prospective patient samples is needed in the future, this validated RT-qPCR was sensitive and specific, which shows its potential to detect CHIKV in clinical samples. IMPORTANCE Chikungunya virus causes chikungunya fever, a disease characterized by fever, rash, and joint pain. In the early phase of infection, chikungunya fever is always misdiagnosed as other arbovirus infections, such as dengue. Laboratory tests such as RT-qPCR are therefore necessary to confirm CHIKV infection. We evaluated the performance of an in-house RT-qPCR assay, and our study shows that the assay could detect CHIKV in clinical samples. We also show the cutoff determination of the assay, which provides important guidance to scientists or researchers when implementing a new RT-qPCR assay in a laboratory.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  3. Rashid SA, Nazakat R, Muhamad Robat R, Ismail R, Suppiah J, Rajendran K, et al.
    Front Public Health, 2023;11:1208348.
    PMID: 37965510 DOI: 10.3389/fpubh.2023.1208348
    Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) may transmit through airborne route particularly when the aerosol particles remain in enclosed spaces with inadequate ventilation. There has been no standard recommended method of determining the virus in air due to limitations in pre-analytical and technical aspects. Furthermore, the presence of low virus loads in air samples could result in false negatives. Our study aims to explore the feasibility of detecting SARS-CoV-2 ribonucleic acid (RNA) in air samples using droplet digital polymerase chain reaction (ddPCR). Active and passive air sampling was conducted between December 2021 and February 2022 with the presence of COVID-19 confirmed cases in two hospitals and a quarantine center in Klang Valley, Malaysia. SARS-CoV-2 RNA in air was detected and quantified using ddPCR and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The comparability of two different digital PCR platforms (QX200 and QIAcuity) to RT-PCR were also investigated. Additionally negative staining transmission electron microscopy was performed to visualize virus ultrastructure. Detection rates of SARS-CoV-2 in air samples using ddPCR were higher compared to RT-PCR, which were 15.2% (22/145) and 3.4% (5/145), respectively. The sensitivity and specificity of ddPCR was 100 and 87%, respectively. After excluding 17 negative samples (50%) by both QX200 and QIAcuity, 15% samples (5/34) were found to be positive both ddPCR and dPCR. There were 23.5% (8/34) samples that were detected positive by ddPCR but negative by dPCR. In contrast, there were 11.7% (4/34) samples that were detected positive by dPCR but negative by ddPCR. The SARS-CoV-2 detection method by ddPCR is precise and has a high sensitivity for viral RNA detection. It could provide advances in determining low viral titter in air samples to reduce false negative reports, which could complement detection by RT-PCR.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods
  4. Chin KL, Teoh BT, Sam SS, Loong SK, Tan KK, Azizan NS, et al.
    Trop Biomed, 2022 Dec 01;39(4):518-523.
    PMID: 36602210 DOI: 10.47665/tb.39.4.005
    Zika virus (ZIKV) infection has emerged as a global health concern following epidemic outbreaks of severe neurological disorders reported in Pacific and Americas since 2016. Therefore, a rapid, sensitive and specific diagnostic test for ZIKV infection is critical for the appropriate patient management and the control of disease spread. A TaqMan minor groove binding (MGB) probe-based quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed based on the conserved sequence regions of 463 ZIKV NS2B genes. The designed ZIKV qRT-PCR assay was evaluated for its detection limit, strain coverage and cross-reactivity. We further assessed the clinical applicability of qRT-PCR assay for ZIKV RNA detection using a total 18 simulated clinical specimens. The detection limit of the qRT-PCR assay was 11.276 ZIKV RNA copies at the 95% probability level (probit analysis, p<= 0.05). Both Asian and African ZIKV strains were detected by the qRT-PCR assay without cross-reacting with DENV-1, DENV-2, DENV-3, DENV-4, CHIKV, JEV, LGTV, GETV and SINV. The qRT-PCR assay demonstrated a perfect agreement (k = 1.000, P < 0.001) with the reference assay; the sensitivity and specificity of the qRT-PCR assay were 100% (95% CI= 79.6-100) and 100% (95% CI= 43.9-100) respectively. The qRT-PCR assay developed in this study is a useful diagnostic tool for the broad coverage detection and quantification of both the Asian and African ZIKV strains.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  5. Ramírez AM, Tang THT, Suárez ML, Fernández AÁ, García CM, Hisam S, et al.
    Am J Trop Med Hyg, 2021 Oct 12;105(6):1732-1737.
    PMID: 34662870 DOI: 10.4269/ajtmh.21-0406
    Malaria control and elimination require prompt diagnosis and accurate treatment. Conventional methods such as rapid diagnostic tests (RDTs) and microscopy lack the characteristics to detect low parasitemias, commonly found in asymptomatic parasitemias and/or submicroscopic malaria carriers. On the contrary, molecular methods have higher sensitivity and specificity. This study evaluated the performance of two commercial real-time polymerase chain reaction (PCR) assays, RealStar® Malaria PCR (RealStar-genus) and RealStar Malaria Screen&Type PCR (RealStar-species), compared with the reference Nested Multiplex Malaria PCR, for the detection of the main five Plasmodium species affecting humans. A total of 121 samples were evaluated. Values of sensitivity (98.9% and 97.8%) and specificity (100% and 96.7%) of the RealStar-genus and the RealStar-species assays, respectively, were very good. The limit of detection (LoD) for the RealStar-genus assay showed a mean value of 0.28 parasites/µL with Plasmodium falciparum samples; while, the LoD of the RealStar-species assay ranged from 0.09 parasites/µL for P. vivax to two parasites/µL for P. ovale. The time to complete a diagnosis was established in 4 hours. Our findings showed a very good concordance of both assays compared with the reference method, with a very good analytical sensitivity. RealStar-species assay was able to correctly characterize double and triple infections. Therefore, these RealStar assays have shown to be useful tools in malaria diagnosis in non-endemic countries and even endemic countries, and for malaria control in general, detecting low parasitemias with sensitivity similar to the most sensitive methods as nested PCR, but with lower time to get the results.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction*
  6. Ng DC, Chin L, Choo PPL, Paramasivam U
    BMJ Case Rep, 2021 May 31;14(5).
    PMID: 34059550 DOI: 10.1136/bcr-2021-243783
    We report a case of COVID-19 in a 29-week preterm infant. This child is the youngest reported case of SARS-CoV-2 infection in Malaysia, and to the best of our knowledge, one of the youngest documented cases of established vertical transmission of SARS-CoV-2 reported in literature. Our report highlights the clinical course, timelines of viral shedding by real-time reverse transcription-PCR and antibody seroconversion in a premature infant infected with SARS-CoV-2. In addition, we discuss the challenges faced in managing a preterm infant infected with SARS-CoV-2 and the knowledge gaps that need to be explored.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  7. Azova M, Timizheva K, Ait Aissa A, Blagonravov M, Gigani O, Aghajanyan A, et al.
    Biomolecules, 2021 05 20;11(5).
    PMID: 34065198 DOI: 10.3390/biom11050763
    This study investigated the renin-angiotensin-aldosterone system (RAAS) gene polymorphisms as possible genetic risk factors for the restenosis development in patients with drug-eluting stents. 113 participants had coronary artery disease and underwent stenting. The control group consisted of 62 individuals with intact coronary arteries. Patients were divided into two groups: with in-stent restenosis (ISR) and without it. The patients with ISR were classified into subgroups by the terms of the restenosis development and age. Real-time PCR and Restriction Fragment Length Polymorphism-PCR were used to genotype the study participants for RAAS gene polymorphisms. We found that the development of restenosis is generally associated with the minor A allele for renin (REN) rs2368564 and the major TT genotype for angiotensinogen (AGT) rs699. The heterozygous genotype for AGT rs4762 acts as a protective marker. A minor A allele for angiotensin II type 2 receptor (AGTR2) rs1403543 is associated with a risk of restenosis in people under 65 years old. Among patients with the early ISR, heterozygotes for angiotensin II type 1 receptor (AGTR1) rs5186 are more frequent, as well as A allele carriers for AGTR2 rs1403543. A minor homozygous genotype for REN rs41317140 and heterozygous genotype for aldosterone synthase (CYP11B2) rs1799998 are predisposed to the late restenosis. Thus, to choose the effective treatment tactics for patients with coronary artery disease, it is necessary to genotype patients for the RAAS polymorphisms, which, along with age and clinical characteristics, will allow a comprehensive assessment of the risk of the restenosis development after stenting.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  8. Awang H, Hamzah FH, Ahmad MH, Mahmood MF, Wahab A, Embong K, et al.
    Infect Dis (Lond), 2021 05;53(5):390-392.
    PMID: 33512265 DOI: 10.1080/23744235.2021.1876913
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  9. Sadeghi A, Tahmasebi S, Mahmood A, Kuznetsova M, Valizadeh H, Taghizadieh A, et al.
    J Cell Physiol, 2021 04;236(4):2829-2839.
    PMID: 32926425 DOI: 10.1002/jcp.30047
    In the course of the coronavirus disease 2019 (COVID-19), raising and reducing the function of Th17 and Treg cells, respectively, elicit hyperinflammation and disease progression. The current study aimed to evaluate the responses of Th17 and Treg cells in COVID-19 patients compared with the control group. Forty COVID-19 intensive care unit (ICU) patients were compared with 40 healthy controls. The frequency of cells, gene expression of related factors, as well as the secretion levels of cytokines, were measured by flow cytometry, real-time polymerase chain reaction, and enzyme-linked immunosorbent assay techniques, respectively. The findings revealed a significant increase in the number of Th17 cells, the expression levels of related factors (RAR-related orphan receptor gamma [RORγt], IL-17, and IL-23), and the secretion levels of IL-17 and IL-23 cytokines in COVID-19 patients compared with controls. In contrast, patients had a remarkable reduction in the frequency of Treg cells, the expression levels of correlated factors (Forkhead box protein P3 [FoxP3], transforming growth factor-β [TGF-β], and IL-10), and cytokine secretion levels (TGF-β and IL-10). The ratio of Th17/Treg cells, RORγt/FoxP3, and IL-17/IL-10 had a considerable enhancement in patients compared with the controls and also in dead patients compared with the improved cases. The findings showed that enhanced responses of Th17 cells and decreased responses of Treg cells in 2019-n-CoV patients compared with controls had a strong relationship with hyperinflammation, lung damage, and disease pathogenesis. Also, the high ratio of Th17/Treg cells and their associated factors in COVID-19-dead patients compared with improved cases indicates the critical role of inflammation in the mortality of patients.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  10. Teh CSJ, Lau MY, Chong CW, Ngoi ST, Chua KH, Lee WS, et al.
    J Microbiol Methods, 2021 04;183:106184.
    PMID: 33662480 DOI: 10.1016/j.mimet.2021.106184
    Diseases caused by typhoidal and non-typhoidal Salmonella remain a considerable threat to both developed and developing countries. Based on the clinical symptoms and serological tests, it is sometimes difficult to differentiate the Salmonella enterica serovar Paratyphi A (S. enterica serovar Paratyphi A) from serovar Typhi (S. enterica serovar Typhi). In this study, we developed a quadruplex real-time polymerase chain reaction (PCR) assay with an internal amplification control (IAC), to simultaneously differentiate S. enterica serovar Paratyphi A from serovar Typhi and to detect other Salmonella serovars which cause salmonellosis in humans. This assay was evaluated on 155 salmonellae and non-salmonellae strains and demonstrated 100% specificity in species differentiation. Inclusion of an IAC did not affect the efficiency of the assay. Further evaluation using a blind test on spiked stool, blood and food specimens showed that the detection limit was at 103 -104 CFU/mL (or g) and a high PCR efficiency with different targets (R2 > 0.99), except for S. enterica serovar Paratyphi A in blood. This assay has been applied to clinical specimens to detect the causative agents of gastrointestinal infections and has successfully identified 6 salmonellosis patients from the 50 diarrhoea patients. The quadruplex real-time PCR developed in this study could enhance the detection and differentiation of salmonellae. This assay could be applied to stools, blood and food based on the notable performance in the simulation tests and field evaluation.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
  11. Rao M, Rashid FA, Sabri FSAH, Jamil NN, Seradja V, Abdullah NA, et al.
    J Med Virol, 2021 Apr;93(4):2461-2466.
    PMID: 33393672 DOI: 10.1002/jmv.26773
    An optimal clinical specimen for accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by minimizing the usage of consumables and reduce hazard exposure to healthcare workers is an urgent priority. The diagnostic performance of SARS-CoV-2 detection between healthcare worker-collected nasopharyngeal and oropharyngeal (NP + OP) swabs and patient performed self-collected random saliva was assessed. Paired NP + OP swabs and random saliva were collected and processed within 48 h of specimen collection from two cohort studies which recruited 562 asymptomatic adult candidates. Real-time reverse-transcription polymerase chain reaction targeting Open reading frame 1a (ORF1a) and nucleocapsid (N) genes was performed and the results were compared. Overall, 65 of 562 (28.1%) candidates tested positive for COVID-19 based on random saliva, NP + OP swabs, or both testing techniques. The detection rate of SARS-CoV-2 was higher in random saliva compared to NP + OP testing (92.3%; 60/65 vs. 73.8%; 48/65; p 
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  12. Makhtar ST, Tan SW, Nasruddin NA, Abdul Aziz NA, Omar AR, Mustaffa-Kamal F
    BMC Vet Res, 2021 Mar 23;17(1):128.
    PMID: 33757494 DOI: 10.1186/s12917-021-02837-6
    BACKGROUND: Morbilliviruses are categorized under the family of Paramyxoviridae and have been associated with severe diseases, such as Peste des petits ruminants, canine distemper and measles with evidence of high morbidity and/or could cause major economic loss in production of livestock animals, such as goats and sheep. Feline morbillivirus (FeMV) is one of the members of Morbilliviruses that has been speculated to cause chronic kidney disease in cats even though a definite relationship is still unclear. To date, FeMV has been detected in several continents, such as Asia (Japan, China, Thailand, Malaysia), Europe (Italy, German, Turkey), Africa (South Africa), and South and North America (Brazil, Unites States). This study aims to develop a TaqMan real-time RT-PCR (qRT-PCR) assay targeting the N gene of FeMV in clinical samples to detect early phase of FeMV infection.

    RESULTS: A specific assay was developed, since no amplification was observed in viral strains from the same family of Paramyxoviridae, such as canine distemper virus (CDV), Newcastle disease virus (NDV), and measles virus (MeV), and other feline viruses, such as feline coronavirus (FCoV) and feline leukemia virus (FeLV). The lower detection limit of the assay was 1.74 × 104 copies/μL with Cq value of 34.32 ± 0.5 based on the cRNA copy number. The coefficient of variations (CV) values calculated for both intra- and inter-assay were low, ranging from 0.34-0.53% and 1.38-2.03%, respectively. In addition, the clinical sample evaluation using this assay showed a higher detection rate, with 25 (35.2%) clinical samples being FeMV-positive compared to 11 (15.5%) using conventional RT-PCR, proving a more sensitive assay compared to the conventional RT-PCR.

    CONCLUSIONS: The TaqMan-based real-time RT-PCR assay targeting the N gene described in this study is more sensitive, specific, rapid, and reproducible compared to the conventional RT-PCR assay targeting the N gene, which could be used to detect early infection in cats.

    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods; Real-Time Polymerase Chain Reaction/veterinary*
  13. Zhao MY, Li D
    Food Environ Virol, 2021 03;13(1):74-83.
    PMID: 33449335 DOI: 10.1007/s12560-020-09452-y
    Hepatitis E virus (HEV) has been frequently detected from pork liver and liver products, which can usually cause self-limiting diseases in healthy adults, yet may result in fatality in immunosuppressed groups. Nevertheless, there is so far no standardized method for HEV detection available from pork liver and/or liver products. The present study aimed to optimize the virus extraction method of HEV from raw pork liver, which is often consumed in Asia undercooked to avoid a grainy texture. By comparing different sample preparation protocols and by applying the selected protocol to 60 samples collected from Singapore retail markets, we demonstrated that homogenization of 0.25 g raw pork liver with FastPrep™ Lysing Matrix Y containing yttria-stabilized zircondium oxide beads in 2 ml tubes and with harsh mechanical force at 6 ms-1, 40 s/cycle, for 5 cycles with 300 s pause time after each cycle is promising in both releasing the potentially intracellular viruses and resulting in satisfactory virus recovery rates (> 1%). A high prevalence (52%) of HEV genome was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) from the 60 samples collected from Singapore retail markets imported from Indonesia, Australia and Malaysia. However, RNase treatment decreased the HEV prevalence to 33.3%, and all of the 20 positive samples were with high RT-qPCR Ct values above 35, suggesting that the positive RT-qPCR signals maybe largely due to the inactive viruses and/or exposed HEV RNA traces in raw pork liver products. Therefore, conscious care should be taken when interpreting molecular detection results of viruses from food samples to be correlated with public health risks.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  14. Kok SY, Namasivayam P, Ee GC, Ong-Abdullah M
    J Proteomics, 2021 02 10;232:104052.
    PMID: 33262095 DOI: 10.1016/j.jprot.2020.104052
    To gain insights on protein changes in fruit setting and growth in oil palm, a comparative proteomic approach was undertaken to study proteome changes during its early development. The variations in the proteome at five early developmental stages were investigated via a gel-based proteomic technique. A total of 129 variant proteins were determined using mass spectrometric analysis, resulting in 80 identifications. The majority of the identified protein species were classified as energy and metabolism, stress response/defence and cell structure during early oil palm development representing potential candidates for the control of final fruit size and composition. Seven prominent protein species were then characterised using real-time polymerase chain reaction to validate the mRNA expression against the protein abundant profiles. Transcript and protein profiles were parallel across the developmental stages, but divergent expression was observed in one protein spot, indicative of possible post-transcriptional events. Our results revealed protein changes in early oil palm fruit development provide valuable information in the understanding of fruit growth and metabolism during early stages that may contribute towards improving agronomic traits. BIOLOGICAL SIGNIFICANCE: Two-dimensional gel electrophoresis coupled with mass spectrometry approach was used in this study to identify differentially expressed proteins during early oil palm fruit development. A total of 80 protein spots with significant change in abundance were successfully identified and selected genes were analysed using real time PCR to validate their expression. The dynamic changes in oil palm fruit proteome during early development were mostly active in primary and energy metabolism, stress responses, cell structure and protein metabolism. This study reveals the physiological processes during early oil palm fruit development and provides a reference proteome for further improvements in fruit quality traits.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  15. Guan Q, Kong W, Zhu D, Zhu W, Dufresne C, Tian J, et al.
    J Proteomics, 2021 01 16;231:104019.
    PMID: 33075550 DOI: 10.1016/j.jprot.2020.104019
    Salinity can induce Mesembryanthemum crystallinum to shift its photosynthesis from C3 to crassulacean acid metabolism (CAM), leading to enhanced plant water use efficiency. Studying how M. crystallinum changes its carbon fixation pathways is important for potential translation into crops and enhancing crop resilience. In this study, we examined proteomic changes in guard cells and mesophyll cells in the course of the C3 to CAM transition. We collected enriched guard cells and mesophyll cells during a short period of transition. A total of 1153 proteins were identified and quantified in the two cell-types. During the transition, proteins in the guard cells and mesophyll cells exhibited differential changes. For example, we observed nocturnal carbon fixation in mesophyll cells and proteins involved in cell growth in the two cell-types. Proteins involved in osmotic adjustment, ion transport, energy metabolism and light response may play important roles in the C3 to CAM transition. Real-time PCR experiments were conducted to determine potential correlations between transcript and protein levels. These results have highlighted potential molecular mechanisms underlying the C3 to CAM transition of guard cells and mesophyll cells of the important facultative CAM plant. BIOLOGICAL SIGNIFICANCE: Fresh water resource for agricultural food production is a global challenge. Nature has evolved crassulacean acid metabolism (CAM) plants with enhanced water use efficiency. Using single cell-type proteomics, this study revealed molecular changes taking place in guard cells and mesophyll cells during the shift of ice plant photosynthesis from C3 to CAM. The results have provided important insights into the CAM transition and may facilitate effort toward enhancing crop resilience for global food security.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  16. Yu CY, Chan KG, Yean CY, Ang GY
    Diagnostics (Basel), 2021 Jan 01;11(1).
    PMID: 33401392 DOI: 10.3390/diagnostics11010053
    The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began as a cluster of pneumonia cases in Wuhan, China before spreading to over 200 countries and territories on six continents in less than six months. Despite rigorous global containment and quarantine efforts to limit the transmission of the virus, COVID-19 cases and deaths have continued to increase, leaving devastating impacts on the lives of many with far-reaching effects on the global society, economy and healthcare system. With over 43 million cases and 1.1 million deaths recorded worldwide, accurate and rapid diagnosis continues to be a cornerstone of pandemic control. In this review, we aim to present an objective overview of the latest nucleic acid-based diagnostic tests for the detection of SARS-CoV-2 that have been authorized by the Food and Drug Administration (FDA) under emergency use authorization (EUA) as of 31 October 2020. We systematically summarize and compare the principles, technologies, protocols and performance characteristics of amplification- and sequencing-based tests that have become alternatives to the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel. We highlight the notable features of the tests including authorized settings, along with the advantages and disadvantages of the tests. We conclude with a brief discussion on the current challenges and future perspectives of COVID-19 diagnostics.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  17. Balachandra D, Ahmad H, Arifin N, Noordin R
    Eur J Clin Microbiol Infect Dis, 2021 Jan;40(1):27-37.
    PMID: 32729057 DOI: 10.1007/s10096-020-03949-x
    Laboratory diagnosis of Strongyloides infections can be grouped into direct and indirect detection methods, and a combination of the two methods is often needed to reach an accurate and timely diagnosis. This review focuses on non-conventional direct detection via molecular and antigen detection assays. Conventional PCR is the most commonly used molecular diagnostic for Strongyloides. Real-time PCR is accurate and highly sensitive for quantitative and qualitative analysis. Meanwhile, PCR-RFLP can efficiently distinguish human and dog isolates of S. stercoralis, S. fuelleborni (from monkey), and S. ratti (from rodent). Loop-mediated isothermal amplification (LAMP) amplifies DNA isothermally with high specificity, efficiency, and rapidity, and has potential for point-of-care (POC) translation. As for antigen detection assay, coproantigen detection ELISAs for strongyloidiasis traditionally relied on raising rabbit polyclonal antibodies against the parasite antigens for use as capture or detection reagents. Subsequently, hybridoma technology using animals has enabled the discovery of monoclonal antibodies specific to Strongyloides antigens and was utilised to develop antigen detection assays. In recent times, phage display technology has facilitated the discovery of scFv antibody against Strongyloides protein that can accelerate the development of such assays. Improvements in both direct detection methods are being made. Strongyloides molecular diagnostics is moving from the detection of a single infection to the simultaneous detection of soil-transmitted helminths. Meanwhile, antigen detection assays can also be multiplexed and aptamers can be used as antigen binders. In the near future, these two direct detection methods may be more widely used as diagnostic tools for strongyloidiasis.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  18. Hashimoto T, Ozaki A, Bhandari D, Sawano T, Murayama A, Shrestha S, et al.
    Travel Med Infect Dis, 2021;43:102145.
    PMID: 34298174 DOI: 10.1016/j.tmaid.2021.102145
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  19. 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*
  20. 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*
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