Displaying publications 1 - 20 of 50 in total

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  1. Yaseen SG, Ahmed SA, Johan MF, Kiron R, Daher AM
    Transfus. Apher. Sci., 2013 Dec;49(3):647-51.
    PMID: 23890575 DOI: 10.1016/j.transci.2013.07.003
    Transmission of infectious diseases is a recognized complication of blood transfusion and blood products. Nucleic acid testing (NAT) may contribute to improved efficiency of blood screening and thereby increase the safety margin for transfused blood.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/instrumentation; Nucleic Acid Amplification Techniques/methods*
  2. 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*
  3. Wee YC, Tan KL, Tan PC, Yap SF, Tan JAMA
    Med. J. Malaysia, 2005 Oct;60(4):447-53.
    PMID: 16570706
    Haemoglobin Bart's hydrops foetalis syndrome (--SEA/--SEA) is not compatible with life and contributes to a majority of the hydropic foetuses in the Malaysian Chinese alpha-thalassaemia carriers who possess the 2-alpha-gene deletion in cis (--SEA/alphaalpha). A duplex-PCR which simultaneously amplifies a normal 136 bp sequence between the psialpha-alpha2-globin genes and a 730 bp Southeast Asian deletion-specific sequence (--SEA) between the psialpha2-theta1-globin genes was established. The duplex-PCR which detects the --SEA deletion in both chromosomes serves as a rapid and cost-effective confirmatory test in the antenatal diagnosis of Haemoglobin Bart's hydrops foetalis syndrome in Malaysia. In addition, the duplex-PCR is simple to perform as both the normal and deletion-specific alpha-globin gene sequences are amplified in the same PCR reaction.
    Matched MeSH terms: Nucleic Acid Amplification Techniques
  4. Wang LY, Wang YS, Cheng H, Zhang JP, Yeok FS
    Ecotoxicology, 2015 Oct;24(7-8):1705-13.
    PMID: 26044931 DOI: 10.1007/s10646-015-1502-0
    Chitinases in terrestrial plants have been reported these are involved in heavy metal tolerance/detoxification. This is the first attempt to reveal chitinase gene (AcCHI I) and its function on metal detoxification in mangroves Aegiceras corniculatum. RT-PCR and RACE techniques were used to clone AcCHI I, while real-time quantitative PCR was employed to assess AcCHI I mRNA expressions in response to Cadmium (Cd). The deduced AcCHI I protein consists of 316 amino acids, including a signal peptide region, a chitin-binding domain (CBD) and a catalytic domain. Protein homology modeling was performed to identify potential features in AcCHI I. The CBD structure of AcCHI I might be critical for metal tolerance/homeostasis of the plant. Clear tissue-specific differences in AcCHI I expression were detected, with higher transcript levels detected in leaves. Results demonstrated that a short duration of Cd exposure (e.g., 3 days) promoted AcCHI I expression in roots. Upregulated expression was also detected in leaves under 10 mg/kg Cd concentration stress. The present study demonstrates that AcCHI I may play an important role in Cd tolerance/homeostasis in the plant. Further studies of the AcCHI I protein, gene overexpression, the promoter and upstream regulation will be necessary for clarifying the functions of AcCHI I.
    Matched MeSH terms: Nucleic Acid Amplification Techniques
  5. Thanarajoo SS, Kong LL, Kadir J, Lau WH, Vadamalai G
    J. Virol. Methods, 2014 Jun;202:19-23.
    PMID: 24631346 DOI: 10.1016/j.jviromet.2014.02.024
    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) detected Coconut cadang-cadang viroid (CCCVd) within 60 min at 60 °C in total nucleic acid extracted from oil palm leaves infected with CCCVd. Positive reactions showed colour change from orange to green in the reaction mix after the addition of fluorescent reagent, and a laddering pattern band on 2% agarose gel electrophoresis. Conventional RT-PCR with LAMP primers produced amplicons with a sequence identical to the 297-nt CCCVd oil palm variant with the primers being specific for CCCVd and not for other viroids such as PSTVd and CEVd. RT-LAMP was found to be rapid and specific for detecting oil palm CCCVd.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  6. 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*
  7. Teh CS, Chua KH, Lim YA, Lee SC, Thong KL
    ScientificWorldJournal, 2014;2014:457839.
    PMID: 24967435 DOI: 10.1155/2014/457839
    We have successfully developed a Loop-mediated isothermal amplification (LAMP) assay that could specifically detect generic Escherichia coli (E. coli). This assay was tested on 85 bacterial strains and successfully identified 54 E. coli strains (average threshold time, Tt = 21.26). The sensitivity of this assay was evaluated on serial dilutions of bacterial cultures and spiked faeces. The assay could detect 10(2) CFU/mL for bacterial culture with Tt = 33.30 while the detection limit for spiked faeces was 10(3) CFU/mL (Tt = 31.12). We have also detected 46 generic E. coli from 50 faecal samples obtained from indigenous individuals with 16% of the positive samples being verocytotoxin-producing E. coli (VTEC) positive. VT1/VT2 allele was present in one faecal sample while the ratio of VT1 to VT2 was 6 : 1. Overall, our study had demonstrated high risk of VTEC infection among the indigenous community and most of the asymptomatic infection occurred among those aged below 15 years. The role of asymptomatic human carriers as a source of dissemination should not be underestimated. Large scale screening of the VTEC infection among indigenous populations and the potential contamination sources will be possible and easy with the aid of this newly developed rapid and simple LAMP assay.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  8. Tay BY, Ahmad N, Hashim R, Mohamed Zahidi J, Thong KL, Koh XP, et al.
    BMC Infect. Dis., 2015;15:220.
    PMID: 26033227 DOI: 10.1186/s12879-015-0958-0
    Brucellosis is one of the most common zoonotic diseases worldwide. It can cause acute febrile illness in human and is a major health problem. Studies in human brucellosis in Malaysia is limited and so far no genotyping studies has been done on Brucella isolates. The aim of the study was to determine the genetic diversity among Brucella species isolated from human brucellosis, obtained over a 6-year period (2009-2014).
    Matched MeSH terms: Nucleic Acid Amplification Techniques
  9. Tan le V, Tuyen NT, Thanh TT, Ngan TT, Van HM, Sabanathan S, et al.
    J. Virol. Methods, 2015 Apr;215-216:30-6.
    PMID: 25704598 DOI: 10.1016/j.jviromet.2015.02.011
    Enterovirus A71 (EV-A71) has emerged as the most important cause of large outbreaks of severe and sometimes fatal hand, foot and mouth disease (HFMD) across the Asia-Pacific region. EV-A71 outbreaks have been associated with (sub)genogroup switches, sometimes accompanied by recombination events. Understanding EV-A71 population dynamics is therefore essential for understanding this emerging infection, and may provide pivotal information for vaccine development. Despite the public health burden of EV-A71, relatively few EV-A71 complete-genome sequences are available for analysis and from limited geographical localities. The availability of an efficient procedure for whole-genome sequencing would stimulate effort to generate more viral sequence data. Herein, we report for the first time the development of a next-generation sequencing based protocol for whole-genome sequencing of EV-A71 directly from clinical specimens. We were able to sequence viruses of subgenogroup C4 and B5, while RNA from culture materials of diverse EV-A71 subgenogroups belonging to both genogroup B and C was successfully amplified. The nature of intra-host genetic diversity was explored in 22 clinical samples, revealing 107 positions carrying minor variants (ranging from 0 to 15 variants per sample). Our analysis of EV-A71 strains sampled in 2013 showed that they all belonged to subgenogroup B5, representing the first report of this subgenogroup in Vietnam. In conclusion, we have successfully developed a high-throughput next-generation sequencing-based assay for whole-genome sequencing of EV-A71 from clinical samples.
    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. 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
  12. Sayad A, Ibrahim F, Mukim Uddin S, Cho J, Madou M, Thong KL
    Biosens Bioelectron, 2018 Feb 15;100:96-104.
    PMID: 28869845 DOI: 10.1016/j.bios.2017.08.060
    Outbreaks of foodborne diseases have become a global health concern; hence, many improvements and developments have been made to reduce the risk of food contamination. We developed a centrifugal microfluidic automatic wireless endpoint detection system integrated with loop mediated isothermal amplification (LAMP) for monoplex pathogen detection. Six identical sets were designed on the microfluidic compact disc (CD) to perform 30 genetic analyses of three different species of foodborne pathogens. The consecutive loading, mixing, and aliquoting of the LAMP primers/reagents and DNA sample solutions were accomplished using an optimized square-wave microchannel, metering chambers and revulsion per minute (RPM) control. We tested 24 strains of pathogenic bacteria (Escherichia coli, Salmonella spp and Vibrio cholerae), with 8 strains of each bacterium, and performed DNA amplification on the microfluidic CD for 60min. Then, the amplicons of the LAMP reaction were detected using the calcein colorimetric method and further analysed via the developed electronic system interfaced with Bluetooth wireless technology to transmit the results to a smartphone. The system showed a limit of detection (LOD) of 3 × 10-5ngμL-1 DNA by analysing the colour change when tested with chicken meat spiked with the three pathogenic bacteria. Since the entire process was performed in a fully automated way and was easy to use, our microdevice is suitable for point-of-care (POC) testing with high simplicity, providing affordability and accessibility even to poor, resource-limited settings.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/economics; Nucleic Acid Amplification Techniques/instrumentation*
  13. Ramlah Zainudin, Augustine Gawin, Dency Flenny
    MyJurnal
    Limnonectes kuhlii and Limnonectes leporinus are two of the Bornean fanged frogs (without advertisement call) which are widely distributed, thus thought to exhibit different evolutionary lineages and the existence of genetically cryptic species. Yet, the two species are still under study especially at the molecular level. Hence, cytochrome c oxidase I (COI) of mitochondrial gene was used to investigate suitable parameters for DNA amplification using the Polymerase Chain Reaction (PCR) method. Three PCR programmes (varied in the temperatures and period of each PCR step) were employed to identify the most efficient parameters in amplifying PCR products for both species. From the three programmes, Programme B (Initial denaturation: 96°C for 5 min; denaturation: 95°C for 45 sec; annealing: 48-53°C for 1 min 30 sec; extension: 72°C for 1 min 30 sec; final extension: 72°C for 10 min, 30 cycles) showed the highest percentage (53%) of optimal PCR products. The other two programmes showed non-specific products or “primer-dimers”. The results also suggest that the annealing temperature of 52°C, 0.025-0.05 units/µl of 1.5mM Taq polymerase, 0.04 mM of
    dNTPs mix and optimal concentrations of magnesium in 50 µl of reaction mixture were sufficient enough to amplify high quality PCR products for both species. However, using Programme B, the re-amplification of the PCR products yielded “primer-dimer”. In addition, a ‘Hot-Start’ PCR method was also applied and mostly yielded in an optimal PCR amplification. Nevertheless, further research on the second amplification of the two species should be conducted to determine the causes of the primer-dimer production.
    Matched MeSH terms: Nucleic Acid Amplification Techniques
  14. Piera KA, Aziz A, William T, Bell D, González IJ, Barber BE, et al.
    Malar. J., 2017 01 13;16(1):29.
    PMID: 28086789 DOI: 10.1186/s12936-016-1676-9
    BACKGROUND: Plasmodium knowlesi is the most common cause of malaria in Malaysia. However, microscopic diagnosis is inaccurate and rapid diagnostic tests (RDTs) are insufficiently sensitive. PCR is sensitive and specific but not feasible at a district level. Loop-mediated isothermal amplification (LAMP) shows potential with only basic requirements. A commercially available LAMP assay, the Eiken Loopamp™ MALARIA Pan Detection kit, is sensitive for Plasmodium falciparum and Plasmodium vivax, but has not previously been evaluated for P. knowlesi. This study aims to determine the sensitivity of this LAMP assay for detecting P. knowlesi infection.

    METHODS: Study participants included 73 uncomplicated malaria patients with PCR species confirmation: 50 P. knowlesi, 20 P. falciparum and 3 P. vivax. Nineteen malaria-negative, non-endemic area controls were also included. The sensitivity of the Eiken Loopamp™ MALARIA Pan Detection kit (Pan LAMP) for detecting each Plasmodium species was evaluated. Sensitivity and specificity of the Eiken Loopamp™ MALARIA Pf Detection kit (Pf LAMP) for P. falciparum were also determined. The limit of detection for each LAMP assay was evaluated, with results compared to PCR. All P. knowlesi patients were also tested by CareStart™ (Pf/VOM) and OptiMAL-IT™ (Pan/Pf) RDTs.

    RESULTS: The sensitivity of the Pan LAMP assay was 100% for P. knowlesi (95% CI 92.9-100), P. falciparum (95% CI 83.2-100), and P. vivax (95% CI 29.2-100). The Pf LAMP was 100% sensitive and specific for P. falciparum detection, with all P. knowlesi samples having a negative reaction. LAMP sensitivity was superior to both RDTs, with only 10 and 28% of P. knowlesi samples testing positive to CareStart™ and OptiMAL-IT™, respectively. Limit of detection using the Pan LAMP for both P. knowlesi and P. vivax was 2 parasites/μL, comparable to PCR. For P. falciparum both the Pan LAMP and Pf LAMP demonstrated a limit of detection of 20 parasites/μL.

    CONCLUSIONS: The Eiken Loopamp™ MALARIA Pan Detection kit is sensitive for detection of P. knowlesi in low parasitaemia clinical infections, as well as P. falciparum and P. vivax. However, a P. knowlesi-specific field assay in a simpler format would assist correct species identification and initiation of optimal treatment for all malaria patients.

    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  15. 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*
  16. 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*
  17. Michelle Wong Tzeling J, Yean Yean C
    Analyst, 2016 Feb 21;141(4):1246-9.
    PMID: 26783560 DOI: 10.1039/c5an01741f
    A shelf-stable loop-mediated isothermal amplification (LAMP) reagent for Burkholderia pseudomallei detection is described. The coupling of LAMP reagents with the indirect colorimetric indicator and consequently its lyophilization enable the simple evaluation of results without the need for any advance laboratory instruments. The reagents were found to have a stable shelf life of at least 30 days with well-maintained sensitivity and specificity.
    Matched MeSH terms: Nucleic Acid Amplification Techniques
  18. 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*
  19. Loh Q, Omar N, Glökler J, Lim TS
    Anal. Biochem., 2014 Oct 15;463:67-9.
    PMID: 24972268 DOI: 10.1016/j.ab.2014.06.012
    Immunoassays are often coupled to peroxidase activity for antigen detection. Sensitivity and speed of detection has been increased by the advent of hybrid methods such as immuno-PCR (polymerase chain reaction). However, a more simplified immunoassay that retains both colorimetric peroxidase detection and effective DNA amplification in a setting closer to field application conditions has been nonexistent. Here we describe a method that successfully combines a competitive immunoassay with the new isothermal quadruplex-primed amplification (QPA) to generate excess quadruplex reporter molecules with intrinsic peroxidase DNAzyme activity.
    Matched MeSH terms: Nucleic Acid Amplification Techniques*
  20. Lim KT, Teh CS, Thong KL
    Biomed Res Int, 2013;2013:895816.
    PMID: 23509796 DOI: 10.1155/2013/895816
    Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is an important human pathogen that produces a variety of toxins and causes a wide range of infections, including soft-tissue infections, bacteremia, and staphylococcal food poisoning. A loop-mediated isothermal amplification (LAMP) assay targeting the arcC gene of S. aureus was developed and evaluated with 119 S. aureus and 25 non-S. aureus strains. The usefulness of the assay was compared with the PCR method that targets spa and arcC genes. The optimal temperature for the LAMP assay was 58.5°C with a detection limit of 2.5 ng/μL and 10(2) CFU/mL when compared to 12.5 ng/μL and 10(3) CFU/mL for PCR (spa and arcC). Both LAMP and PCR assays were 100% specific, 100% sensitive, 100% positive predictive value (PPV), and 100% negative predictive value (NPV). When tested on 30 spiked blood specimens (21 MRSA, eight non-S. aureus and one negative control), the performance of LAMP and PCR was comparable: 100% specific, 100% sensitive, 100% PPV, and 100% NPV. In conclusion, the LAMP assay was equally specific with a shorter detection time when compared to PCR in the identification of S. aureus. The LAMP assay is a promising alternative method for the rapid identification of S. aureus and could be used in resource-limited laboratories and fields.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
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