Displaying publications 1 - 20 of 41 in total

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  1. Glökler J, Lim TS, Ida J, Frohme M
    Crit Rev Biochem Mol Biol, 2021 12;56(6):543-586.
    PMID: 34263688 DOI: 10.1080/10409238.2021.1937927
    The introduction of nucleic acid amplification techniques has revolutionized the field of medical diagnostics in the last decade. The advent of PCR catalyzed the increasing application of DNA, not just for molecular cloning but also for molecular based diagnostics. Since the introduction of PCR, a deeper understanding of molecular mechanisms and enzymes involved in DNA/RNA replication has spurred the development of novel methods devoid of temperature cycling. Isothermal amplification methods have since been introduced utilizing different mechanisms, enzymes, and conditions. The ease with which isothermal amplification methods have allowed nucleic acid amplification to be carried out has had a profound impact on the way molecular diagnostics are being designed after the turn of the millennium. With all the advantages isothermal amplification brings, the issues or complications surrounding each method are heterogeneous making it difficult to identify the best approach for an end-user. This review pays special attention to the various isothermal amplification methods by classifying them based on the mechanistic characteristics which include reaction formats, amplification information, promoter, strand break, and refolding mechanisms. We would also compare the efficiencies and usefulness of each method while highlighting the potential applications and detection methods involved. This review will serve as an overall outlook on the journey and development of isothermal amplification methods as a whole.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  2. Lau YL, Lai MY, Teoh BT, Abd-Jamil J, Johari J, Sam SS, et al.
    PLoS One, 2015;10(9):e0138694.
    PMID: 26384248 DOI: 10.1371/journal.pone.0138694
    Dengue is usually diagnosed by isolation of the virus, serology or molecular diagnostic methods. Several commercial kits for the diagnosis of dengue are existing, but concerns have arisen regarding to the affordability and performance characteristics of these kits. Hence, the loop-mediated isothermal amplification (LAMP) is potentially ideal to be used especially in resource limited environments. Serum was collected from healthy donors and patients diagnosed with dengue infection. RNA extracted from the serum samples were tested by reverse-transcription-LAMP assay developed based on 3'-NCR gene sequences for DENV 1-4. Results were interpreted by a turbidity meter in real time or visually at the end of the assay. Sensitivity and specificity of RT-LAMP results were calculated and compared to qRT-PCR and ELISA. RT-LAMP is highly sensitive with the detection limit of 10 RNA copies for all serotypes. Dengue virus RNA was detected in all positive samples using RT-LAMP and none of the negative samples within 30-45 minutes. With continuing efforts in the optimization of this assay, RT-LAMP may provide a simple and reliable test for detecting DENV in areas where dengue is prevalent.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  3. Lai MY, Ooi CH, Lau YL
    Am J Trop Med Hyg, 2018 03;98(3):700-703.
    PMID: 29260656 DOI: 10.4269/ajtmh.17-0738
    The aim of this study was to develop a recombinase polymerase amplification (RPA) combined with a lateral flow (LF) strip method for specific diagnosis of Plasmodium knowlesi. With incubation at 37°C, the 18S rRNA gene of P. knowlesi was successfully amplified within 12 minutes. By adding a specifically designed probe to the reaction solution, the amplified RPA product can be visualized on a LF strip. The RPA assay exhibited high sensitivity with limits of detection down to 10 parasites/μL of P. knowlesi. Nonetheless, it was demonstrated that all P. knowlesi (N = 41) and other Plasmodium sp. (N = 25) were positive while negative samples (N = 8) were negative. Therefore, a combination of RPA and LF strip detection is a highly promising approach with the potential to be suitable for use in resource-limited settings.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  4. Chua EW, Maggo S, Kennedy MA
    Methods Mol Biol, 2017;1620:65-74.
    PMID: 28540699 DOI: 10.1007/978-1-4939-7060-5_3
    Polymerase chain reaction (PCR) is an oft-used preparatory technique in amplifying specific DNA regions for downstream analysis. The size of an amplicon was initially limited by errors in nucleotide polymerization and template deterioration during thermal cycling. A variant of PCR, designated long-range PCR, was devised to counter these drawbacks and enable the amplification of large fragments exceeding a few kb. In this chapter we describe a protocol for long-range PCR, which we have adopted to obtain products of 6.6, 7.2, 13, and 20 kb from human genomic DNA samples.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  5. Selvarajah D, Naing C, Htet NH, Mak JW
    Malar J, 2020 Jun 19;19(1):211.
    PMID: 32560728 DOI: 10.1186/s12936-020-03283-9
    BACKGROUND: The global malaria decline has stalled and only a few countries are pushing towards pre-elimination. The aim of the malaria elimination phase is interruption of local transmission of a specified malaria parasite in a defined geographical area. New and improved screening tools and strategies are required for detection and management of very low-density parasitaemia in the field. The objective of this study was to synthesize evidence on the diagnostic accuracy of loop-mediated isothermal amplification (LAMP) test for the detection of malaria parasites among people living in endemic areas.

    METHODS: This study adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis for Diagnostic Test Accuracy (PRISMA-DTA) guideline. Relevant studies in the health-related electronic databases were searched. According to the criteria set for this study, eligible studies were identified. The quality of included studies was evaluated with the use of a quality assessment checklist. A summary performance estimates such as pooled sensitivity and specificity were stratified by type of LAMP. Bivariate model for data analyses was applied. Summary receiver operating characteristics plots were created to display the results of individual studies in a receiver operating characteristics space. Meta-regression analysis was performed to investigate the sources of heterogeneity among individual studies.

    RESULTS: Twenty-seven studies across 17 endemic countries were identified. The vast majority of studies were with unclear risk of bias in the selection of index test. Overall, the pooled test performances were high for Pan LAMP (sensitivity: 0.95, 95% CI 0.91 to 0.97; specificity: 0.98, 95% CI 0.95 to 0.99), Plasmodium falciparum (Pf) LAMP (sensitivity: 0.96, 95% CI 0.94 to 0.98; specificity: 0.99, 95% CI 0.96 to 1.00) or for Plasmodium vivax (Pv) LAMP from 6 studies (sensitivity: 0.98, 95% CI 0.92 to 0.99; specificity: 0.99, 95% CI 0.72 to 1.00). The area under the curve for Pan LAMP (0.99, 95% CI 0.98-1.00), Pf LAMP (0.99, 95% CI 0.97-0.99) and Pv LAMP was (1.00, 95% CI 0.98-1.00) indicated that the diagnostic performance of these tests were within the excellent accuracy range. Meta-regression analysis showed that sample size had the greatest impact on test performance, among other factors.

    CONCLUSIONS: The current findings suggest that LAMP-based assays are appropriate for detecting low-level malaria parasite infections in the field and would become valuable tools for malaria control and elimination programmes. Future well-designed larger sample studies on LAMP assessment in passive and active malaria surveillances that use PCR as the reference standard and provide sufficient data to construct 2 × 2 diagnostic table are needed.

    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  6. Chaibun T, Puenpa J, Ngamdee T, Boonapatcharoen N, Athamanolap P, O'Mullane AP, et al.
    Nat Commun, 2021 02 05;12(1):802.
    PMID: 33547323 DOI: 10.1038/s41467-021-21121-7
    Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnosis of COVID-19 depends on quantitative reverse transcription PCR (qRT-PCR), which is time-consuming and requires expensive instrumentation. Here, we report an ultrasensitive electrochemical biosensor based on isothermal rolling circle amplification (RCA) for rapid detection of SARS-CoV-2. The assay involves the hybridization of the RCA amplicons with probes that were functionalized with redox active labels that are detectable by an electrochemical biosensor. The one-step sandwich hybridization assay could detect as low as 1 copy/μL of N and S genes, in less than 2 h. Sensor evaluation with 106 clinical samples, including 41 SARS-CoV-2 positive and 9 samples positive for other respiratory viruses, gave a 100% concordance result with qRT-PCR, with complete correlation between the biosensor current signals and quantitation cycle (Cq) values. In summary, this biosensor could be used as an on-site, real-time diagnostic test for COVID-19.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods
  7. Hamidah NH, Munirah AR, Hafiza A, Farisah AR, Shuhaila A, Norzilawati MN, et al.
    Malays J Pathol, 2014 Dec;36(3):163-8.
    PMID: 25500514 MyJurnal
    Prenatal diagnosis is essential in the new era of diagnosis and management of genetic diseases in obstetrics. Multiple ligation-dependent probe amplification (MLPA) is a recent technique for prenatal diagnosis for the relative quantification of 40 different nucleic acid sequences in one single reaction. We had utilized the MLPA technique in detecting aneuploidies in amniotic fluid samples from 25 pregnant women from the Obstetrics and Gynaecology Department UKMMC, versus the quantitative fluorescent polymerase chain reaction (QF-PCR) method. Conclusive results were obtained in 18 cases and all were concordant with that of the QF-PCR. All four cases of trisomies were correctly identified including one case with maternal cell contamination.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  8. Teoh BT, Sam SS, Tan KK, Johari J, Danlami MB, Hooi PS, et al.
    BMC Infect Dis, 2013;13:387.
    PMID: 23964963 DOI: 10.1186/1471-2334-13-387
    BACKGROUND: Early and rapid detection of dengue virus (DENV) infection during the febrile period is crucial for proper patient management and prevention of disease spread. An easy to perform and highly sensitive method is needed for routine implementation especially in the resource-limited rural healthcare settings where dengue is endemic.
    METHODS: A single-tube reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay with a set of nine primers was developed for the detection of all four DENV serotypes and their different genotypes. The sensitivity and specificity of the RT-LAMP were evaluated. The clinical applicability of RT-LAMP assay for detection of DENV RNA was assessed in a total of 305 sera of clinically-suspected dengue patients. The test results of RT-LAMP were statistically compared to those of quantitative reverse transcription-polymerase chain reaction (qRT-PCR), IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA).
    RESULTS: Acute DENV infection was confirmed in 171 samples (n = 305); 43.3% (74/171) and 46.8% (80/171) of the samples were positive for DENV using RT-LAMP and qRT-PCR, respectively. The combination of RT-LAMP with the dengue IgM and IgG ELISA increased detection of acute DENV infection to 97.7% (167/171), in comparison to only 70.8% (121/171) when dengue IgM and IgG ELISA alone were used. The RT-LAMP assays showed high concordance (κ = 0.939) with the qRT-PCR. The RT-LAMP assay detected up to 10 copies of virus RNA within an hour but 100% reproducibility (12/12) was achieved with 100 copies. There was no cross reactivity of RT-LAMP with other closely related arboviruses.
    CONCLUSION: The RT-LAMP assay developed in this study is sensitive, specific and simple to perform. The assay improved the detection of dengue when used in combination with serological methods.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  9. Siddiquee S, Tan SG, Yusuf UK, Fatihah NH, Hasan MM
    Mol Biol Rep, 2012 Jan;39(1):715-22.
    PMID: 21553047 DOI: 10.1007/s11033-011-0790-6
    Trichoderma species are commercially applied as biocontrol agents against numerous plant pathogenic fungi due to their production of antifungal metabolites, competition for nutrients and space, and mycoparasitism. However, currently the identification of Trichoderma species from throughout the world based on micro-morphological descriptions is tedious and prone to error. The correct identification of Trichoderma species is important as several traits are species-specific. The Random Amplified Microsatellites (RAMS) analysis done using five primers in this study showed different degrees of the genetic similarity among 42 isolates of this genus. The genetic similarity values were found to be in the range of 12.50-85.11% based on a total of 76 bands scored in the Trichoderma isolates. Of these 76 bands, 96.05% were polymorphic, 3.95% were monomorphic and 16% were exclusive bands. Two bands (250 bp and 200 bp) produced by primer LR-5 and one band (250 bp) by primer P1A were present in all the Trichoderma isolates collected from healthy and infected oil palm plantation soils. Cluster analysis based on UPGMA of the RAMS marker data showed that T. harzianum, T. virens and T. longibrachiatum isolates were grouped into different clades and lineages. In this study we found that although T. aureoviride isolates were morphologically different when compared to T. harzianum isolates, the UPGMA cluster analysis showed that the majority isolates of T. aureoviride (seven from nine) were closely related to the isolates of T. harzianum.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods
  10. Tan NH, Palmer R, Wang R
    J Obstet Gynaecol Res, 2010 Feb;36(1):19-26.
    PMID: 20178523 DOI: 10.1111/j.1447-0756.2009.01110.x
    Array-based comparative genomic hybridization (array CGH) is a new molecular technique that has the potential to revolutionize cytogenetics. However, use of high resolution array CGH in the clinical setting is plagued by the problem of widespread copy number variations (CNV) in the human genome. Constitutional microarray, containing only clones that interrogate regions of known constitutional syndromes, may circumvent the dilemma of detecting CNV of unknown clinical significance.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods
  11. Isa MN, Sulong S, Sidek MR, George PJ, Abdullah JM
    PMID: 15115103
    Telomerase, the enzyme that stabilizes telomere length is reactivated with almost all cancer types, and may be a useful diagnostic marker for malignancy. Telomerase activity has been detected in germ line cells and most cancer cells, whereas most normal somatic cells have no clearly detectable telomerase activity. In our study, we aim to detect telomerase activity in 20 human central nervous system tumors from Malaysian patients. Telomerase activity was detected based on a highly sensitive procedure consisting of a CHAPS detergent-based extraction from frozen tissues and a PCR-based telomeric repeat amplification protocol (TRAP) using a TRAPEZE Telomerase Detection Kit (Intergen, Co). Telomerase activity was considered positive when a ladder of products was observed starting at 50bp, with 6bp increments. The activity was detected in 30% of the samples analysed, included glioblastoma multiforme, meduloblastoma, paraganglioma and oligodendroglioma. The result of Fisher's exact test indicated that there was a significant association between telomerase activity status with tumor grade (p=0.003). These results suggest that telomerase activity may be an important marker for tumor malignancy.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods
  12. Mallepaddi PC, Lai MY, Podha S, Ooi CH, Liew JW, Polavarapu R, et al.
    Am J Trop Med Hyg, 2018 09;99(3):704-708.
    PMID: 29943720 DOI: 10.4269/ajtmh.18-0177
    The present study aims to develop a method for rapid diagnosis of malaria using loop-mediated isothermal amplification (LAMP) combined with a lateral flow device (LFD). By adding the biotin-labeled and fluorescein amidite-labeled loop primers to the LAMP reaction solution, the end product can be visualized on a LFD. The entire procedure takes approximately 42 minutes to complete, LAMP assay exhibited high sensitivity, as the detection limit was 0.01 pg/μL for all five Plasmodium species. It was demonstrated that all Plasmodium knowlesi (N = 90) and Plasmodium vivax (N = 56) were positively amplified by LAMP-LFD assay, whereas healthy donor samples (N = 8) were negative. However, not all mixed infections were positive, and other infected nonmalaria samples were negative. Loop-mediated isothermal amplification-LFD represents a robust approach with potential suitability for use in resource-constrained laboratories. We believe that LAMP-LFD has a potential to be developed as point-of-care diagnostic tool in future.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  13. Chan SK, Kuzuya A, Choong YS, Lim TS
    SLAS Discov, 2019 01;24(1):68-76.
    PMID: 30063871 DOI: 10.1177/2472555218791743
    The inherent ability of nucleic acids to recognize a complementary pair has gained wide popularity in DNA sensor applications. DNA molecules can be produced in bulk and easily incorporated with various nanomaterials for sensing applications. More complex designs and sophisticated DNA sensors have been reported over the years to allow DNA detection in a faster, cheaper, and more convenient manner. Here, we report a DNA sensor designed to function like a switch to turn "on" silver nanocluster (AgNC) generation in the presence of a specific DNA target. By defining the probe region sequence, we are able to tune the color of the AgNC generated in direct relation to the different targets. As a proof of concept, we used dengue RNA-dependent RNA polymerase conserved sequences from all four serotypes as targets. This method was able to distinguish each dengue serotype by generating the serotype-respective AgNCs. The DNA switch was also able to identify and amplify the correct target in a mixture of targets with good specificity. This strategy has a detection limit of between 1.5 and 2.0 µM depending on the sequence of AgNC. The DNA switch approach provides an attractive alternative for single-target or multiplex DNA detection.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  14. Ng JB, Poh RY, Lee KR, Subrayan V, Deva JP, Lau AY, et al.
    Clin. Lab., 2016 Sep 01;62(9):1731-1737.
    PMID: 28164597 DOI: 10.7754/Clin.Lab.2016.160144
    BACKGROUND: Keratoconus is an ocular degeneration characterized by the thinning of corneal stroma that may lead to varying degrees of myopia and visual impairment. Genetic factors have been reported in the pathology of keratoconus where Asians have a higher incidence, earlier onset, and undergo earlier corneal grafts compared to Caucasians. The visual system homeobox 1 (VSX1) gene forms part of a paired-like homeodomain transcription factor which is responsible for ocular development. The gene was marked as a candidate in genetic studies of keratoconus in various populations. Single nucleotide polymorphisms (SNPs) in the VSX1 gene have been reported to be associated with keratoconus. The detection of the SNPs involves DNA amplification of the VSX1 gene followed by genomic sequencing. Thus, the objective of this study aims to establish sensitive and accurate screening protocols for the molecular characterization of VSX1 polymorphisms.

    METHODS: Keratoconic (n = 74) and control subjects (n = 96) were recruited based on clinical diagnostic tests and selection criteria. DNA extracted from the blood samples was used to genotype VSX1 polymorphisms. In-house designed primers and optimization of PCR conditions were carried out to amplify exons 1 and 3 of the VSX1 gene. PCR conditions including percentage GC content, melting temperatures, and differences in melting temperatures of primers were evaluated to produce sensitive and specific DNA amplifications.

    RESULTS: Genotyping was successfully carried out in 4 exons of the VSX1 gene. Primer annealing temperatures were observed to be crucial in enhancing PCR sensitivity and specificity. Annealing temperatures were carefully evaluated to produce increased specificity, yet not allowing sensitivity to be compromised. In addition, exon 1 of the VSX1 gene was amplified using 2 different sets of primers to produce 2 smaller amplified products with absence of non-specific bands. DNA amplification of exons 1 and 3 consistently showed single band products which were successfully sequenced to yield reproducible data.

    CONCLUSIONS: The use of in-house designed primers and optimized PCR conditions allowed sensitive and specific DNA amplifications that produced distinct single bands. The in-house designed primers and DNA amplification protocols established in this study provide an addition to the current repertoire of primers for accurate molecular characterization of VSX1 gene polymorphisms in keratoconus research.

    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  15. Wong YP, Othman S, Lau YL, Radu S, Chee HY
    J Appl Microbiol, 2018 Mar;124(3):626-643.
    PMID: 29165905 DOI: 10.1111/jam.13647
    Loop-mediated isothermal amplification (LAMP) amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions by using a DNA polymerase with high displacement strand activity and a set of specifically designed primers to amplify targeted DNA strands. Following its first discovery by Notomi et al. ( Nucleic Acids Res 28: E63), LAMP was further developed over the years which involved the combination of this technique with other molecular approaches, such as reverse transcription and multiplex amplification for the detection of infectious diseases caused by micro-organisms in humans, livestock and plants. In this review, available types of LAMP techniques will be discussed together with their applications in detection of various micro-organisms. Up to date, there are varieties of LAMP detection methods available including colorimetric and fluorescent detection, real-time monitoring using turbidity metre and detection using lateral flow device which will also be highlighted in this review. Apart from that, commercialization of LAMP technique had also been reported such as lyophilized form of LAMP reagents kit and LAMP primer sets for detection of pathogenic micro-organisms. On top of that, advantages and limitations of this molecular detection method are also described together with its future potential as a diagnostic method for infectious disease.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  16. Engku Nur Syafirah EAR, Nurul Najian AB, Foo PC, Mohd Ali MR, Mohamed M, Yean CY
    Acta Trop, 2018 Jun;182:223-231.
    PMID: 29545156 DOI: 10.1016/j.actatropica.2018.03.004
    Cholera, caused by Vibrio cholerae is a foodborne disease that frequently reported in food and water related outbreak. Rapid diagnosis of cholera infection is important to avoid potential spread of disease. Among available diagnostic platforms, loop-mediated isothermal amplification (LAMP) is regarded as a potential diagnostic tool due to its rapidity, high sensitivity and specificity and independent of sophisticated thermalcycler. However, the current LAMP often requires multiple pipetting steps, hence is susceptible to cross contamination. Besides, the strict requirement of cold-chain during transportation and storage make its application in low resource settings to be inconvenient. To overcome these problems, the present study is aimed to develop an ambient-temperature-stable and ready-to-use LAMP assay for the detection of toxigenic Vibrio cholerae in low resource settings. A set of specific LAMP primers were designed and tested against 155 V. cholerae and non-V. cholerae strains. Analytical specifity showed that the developed LAMP assay detected 100% of pathogenic V. cholerae and did not amplified other tested bacterial strains. Upon testing against stool samples spiked with toxigenic V. cholerae outbreak isolates, the LAMP assay detected all of the spiked samples (n = 76/76, 100%), in contrast to the conventional PCR which amplified 77.6% (n = 59/76) of the tested specimens. In term of sensitivity, the LAMP assay was 100-fold more sensitive as compared to the conventional PCR method, with LOD of 10 fg per μL and 10 CFU per mL. Following lyophilisation with addition of lyoprotectants, the dry-reagent LAMP mix has an estimated shelf-life of 90.75 days at room temperature.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  17. Lau YL, Ismail IB, Izati Binti Mustapa N, Lai MY, Tuan Soh TS, Hassan AH, et al.
    Am J Trop Med Hyg, 2020 Dec;103(6):2350-2352.
    PMID: 33098286 DOI: 10.4269/ajtmh.20-1079
    A simple and rapid reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of SARS-CoV-2. The RT-LAMP assay was highly specific for SARS-CoV-2 and was able to detect one copy of transcribed SARS-CoV-2 RNA within 24 minutes. Assay validation performed using 50 positive and 32 negative clinical samples showed 100% sensitivity and specificity. The RT-LAMP would be valuable for clinical diagnosis and epidemiological surveillance of SARS-CoV-2 infection in resource-limited areas as it does not require the use of sophisticated and costly equipment.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods
  18. 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: Nucleic Acid Amplification Techniques/methods
  19. Lam JY, Low GK, Chee HY
    PLoS Negl Trop Dis, 2020 02;14(2):e0008074.
    PMID: 32049960 DOI: 10.1371/journal.pntd.0008074
    BACKGROUND: Leptospirosis is often difficult to diagnose because of its nonspecific symptoms. The drawbacks of direct isolation and serological tests have led to the increased development of nucleic acid-based assays, which are more rapid and accurate. A meta-analysis was performed to evaluate the diagnostic accuracy of genetic markers for the detection of Leptospira in clinical samples.

    METHODOLOGY AND PRINCIPLE FINDINGS: A literature search was performed in Scopus, PubMed, MEDLINE and non-indexed citations (via Ovid) by using suitable keyword combinations. Studies evaluating the performance of nucleic acid assays targeting leptospire genes in human or animal clinical samples against a reference test were included. Of the 1645 articles identified, 42 eligible studies involving 7414 samples were included in the analysis. The diagnostic performance of nucleic acid assays targeting the rrs, lipL32, secY and flaB genes was pooled and analyzed. Among the genetic markers analyzed, the secY gene showed the highest diagnostic accuracy measures, with a pooled sensitivity of 0.56 (95% CI: 0.50-0.63), a specificity of 0.98 (95% CI: 0.97-0.98), a diagnostic odds ratio of 46.16 (95% CI: 6.20-343.49), and an area under the curve of summary receiver operating characteristics curves of 0.94. Nevertheless, a high degree of heterogeneity was observed in this meta-analysis. Therefore, the present findings here should be interpreted with caution.

    CONCLUSION: The diagnostic accuracies of the studies examined for each genetic marker showed a significant heterogeneity. The secY gene exhibited higher diagnostic accuracy measures compared with other genetic markers, such as lipL32, flaB, and rrs, but the difference was not significant. Thus, these genetic markers had no significant difference in diagnostic accuracy for leptospirosis. Further research into these genetic markers is warranted.

    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  20. Nurul Najian AB, Foo PC, Ismail N, Kim-Fatt L, Yean CY
    Mol Cell Probes, 2019 04;44:63-68.
    PMID: 30876924 DOI: 10.1016/j.mcp.2019.03.001
    This study highlighted the performance of the developed integrated loop-mediated isothermal amplification (LAMP) coupled with a colorimetric DNA-based magnetogenosensor. The biosensor operates through a DNA hybridization system in which a specific designed probe captures the target LAMP amplicons. We demonstrated the magnetogenosensor assay by detecting pathogenic Leptospira, which causes leptospirosis. The color change of the assay from brown to blue indicated a positive result, whereas a negative result was indicated by the assay maintaining its brown color. The DNA biosensor was able to detect DNA at a concentration as low as 200 fg/μl, which is equivalent to 80 genomes/reaction. The specificity of the biosensor assay was 100% when it was evaluated with 172 bacterial strains. An integrated LAMP and probe-specific magnetogenosensor was successfully developed, promising simple and rapid visual detection in clinical diagnostics and service as a point-of-care device.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
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