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Visual System Homeobox 1 (VSX1) Gene Analysis in Keratoconus: Design of Specific Primers and DNA Amplification Protocols for Accurate Molecular Characterization

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*
Fulltext Rapid Detection of Plasmodium knowlesi by Isothermal Recombinase Polymerase Amplification Assay

Lai MY, Ooi CH, Lau YL

Am J Trop Med Hyg, 2017 Nov;97(5):1597-1599.
PMID: 28820700 DOI: 10.4269/ajtmh.17-0427

In this study, we developed a recombinase polymerase amplification (RPA) assay for specific diagnosis of Plasmodium knowlesi. Genomic DNA was extracted from whole blood samples using a commercial kit. With incubation at 37°C, the samples were successfully amplified within 20 minutes. The end product of RPA was further examined by loading onto agarose gel and a specific band was observed with a size of 128 bp. The RPA assay exhibited high sensitivity with limits of detection down to one copy of the plasmid. From the specificity experiments, it was demonstrated that all P. knowlesi samples (N = 45) were positive while other Plasmodium spp. (N = 42) and negative samples (N = 6) were negative. Therefore, the RPA assay is a highly promising approach with the potential to be used in resource-limited settings. This assay can be further optimized for bedside and on field application.

Matched MeSH terms: Nucleic Acid Amplification Techniques*
Fulltext Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

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*
An ambient temperature stable and ready-to-use loop-mediated isothermal amplification assay for detection of toxigenic Vibrio cholerae in outbreak settings

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*
Fulltext Diagnostic accuracy of genetic markers and nucleic acid techniques for the detection of Leptospira in clinical samples: A meta-analysis

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*
Probe-specific loop-mediated isothermal amplification magnetogenosensor assay for rapid and specific detection of pathogenic Leptospira

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*
Evaluation of WarmStart Colorimetric Loop-Mediated Isothermal Amplification Assay for Diagnosis of Malaria

Lai MY, Ooi CH, Jaimin JJ, Lau YL

Am J Trop Med Hyg, 2020 06;102(6):1370-1372.
PMID: 32228783 DOI: 10.4269/ajtmh.20-0001

The incidence of zoonotic malaria, Plasmodium knowlesi, infection is increasing and now is the major cause of malaria in Malaysia. Here, we describe a WarmStart colorimetric loop-mediated isothermal amplification (LAMP) assay for the detection of Plasmodium spp. The detection limit for this assay was 10 copies/µL for P knowlesi and Plasmodium ovale and 1 copy/µL for Plasmodium falciparum, Plasmodium vivax, and Plasmodium malariae. To test clinical sensitivity and specificity, 100 microscopy-positive and 20 malaria-negative samples were used. The WarmStart colorimetric LAMP was 98% sensitive and 100% specific. Amplification products were visible for direct observation, thereby eliminating the need for post-amplification processing steps. Therefore, WarmStart colorimetric LAMP is suitable for use in resource-limited settings.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
Fulltext Development of a reverse transcription recombinase polymerase amplification assay for rapid and direct visual detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

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
Detection of Coconut cadang-cadang viroid (CCCVd) in oil palm by reverse transcription loop-mediated isothermal amplification (RT-LAMP)

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*
STR data for the AmpFlSTR Profiler loci from the three main ethnic population groups (Malay, Chinese and Indian) in Malaysia

Lim KB, Jeevan NH, Jaya P, Othman MI, Lee YH

Forensic Sci Int, 2001 Jun 01;119(1):109-12.
PMID: 11348801

Allele frequencies for the nine STRs genetic loci included in the AmpFlSTR Profiler kit were obtained from samples of unrelated individuals comprising 139-156 Malays, 149-153 Chinese and 132-135 Indians, residing in Malaysia.

Matched MeSH terms: Nucleic Acid Amplification Techniques/instrumentation; Nucleic Acid Amplification Techniques/methods
A microdevice for rapid, monoplex and colorimetric detection of foodborne pathogens using a centrifugal microfluidic platform

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*
Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira

Nurul Najian AB, Engku Nur Syafirah EA, Ismail N, Mohamed M, Yean CY

Anal Chim Acta, 2016 Jan 15;903:142-8.
PMID: 26709307 DOI: 10.1016/j.aca.2015.11.015

In recent years extensive numbers of molecular diagnostic methods have been developed to meet the need of point-of-care devices. Efforts have been made towards producing rapid, simple and inexpensive DNA tests, especially in the diagnostics field. We report on the development of a label-based lateral flow dipstick for the rapid and simple detection of multiplex loop-mediated isothermal amplification (m-LAMP) amplicons. A label-based m-LAMP lateral flow dipstick assay was developed for the simultaneous detection of target DNA template and a LAMP internal control. This biosensor operates through a label based system, in which probe-hybridization and the additional incubation step are eliminated. We demonstrated this m-LAMP assay by detecting pathogenic Leptospira, which causes the re-emerging disease Leptospirosis. The lateral flow dipstick was developed to detect of three targets, the LAMP target amplicon, the LAMP internal control amplicon and a chromatography control. Three lines appeared on the dipstick, indicating positive results for all representative pathogenic Leptospira species, whereas two lines appeared, indicating negative results, for other bacterial species. The specificity of this biosensor assay was 100% when it was tested with 13 representative pathogenic Leptospira species, 2 intermediate Leptospira species, 1 non-pathogenic Leptospira species and 28 other bacteria species. This study found that this DNA biosensor was able to detect DNA at concentrations as low as 3.95 × 10(-1) genomic equivalent ml(-1). An integrated m-LAMP and label-based lateral flow dipstick was successfully developed, promising simple and rapid visual detection in clinical diagnostics and serving as a point-of-care device.

Matched MeSH terms: Nucleic Acid Amplification Techniques
Fulltext Optimization of Polymerase Chain Reaction (PCR) of mitochondrial cytochrome c oxidase I (COI) gene in two Bornean fanged frogs

Ramlah Zainudin, Augustine Gawin, Dency Flenny

Pertanika Journal of Science & Technology, 2011;19(1):57-66.
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
Fulltext Advanced DNA-Based Point-of-Care Diagnostic Methods for Plant Diseases Detection

Lau HY, Botella JR

Frontiers in plant science, 2017;8:2016.
PMID: 29375588 DOI: 10.3389/fpls.2017.02016

Diagnostic technologies for the detection of plant pathogens with point-of-care capability and high multiplexing ability are an essential tool in the fight to reduce the large agricultural production losses caused by plant diseases. The main desirable characteristics for such diagnostic assays are high specificity, sensitivity, reproducibility, quickness, cost efficiency and high-throughput multiplex detection capability. This article describes and discusses various DNA-based point-of care diagnostic methods for applications in plant disease detection. Polymerase chain reaction (PCR) is the most common DNA amplification technology used for detecting various plant and animal pathogens. However, subsequent to PCR based assays, several types of nucleic acid amplification technologies have been developed to achieve higher sensitivity, rapid detection as well as suitable for field applications such as loop-mediated isothermal amplification, helicase-dependent amplification, rolling circle amplification, recombinase polymerase amplification, and molecular inversion probe. The principle behind these technologies has been thoroughly discussed in several review papers; herein we emphasize the application of these technologies to detect plant pathogens by outlining the advantages and disadvantages of each technology in detail.

Matched MeSH terms: Nucleic Acid Amplification Techniques
Fulltext Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of SARS-CoV-2

Lau YL, Ismail I, Mustapa NI, Lai MY, Tuan Soh TS, Hassan A, et al.

PeerJ, 2020;8:e9278.
PMID: 32547882 DOI: 10.7717/peerj.9278

Background: Highly sensitive real-time reverse transcription polymerase chain reaction (RT-qPCR) methods have been developed for the detection of SARS-CoV-2. However, they are costly. Loop-mediated isothermal amplification (LAMP) assay has emerged as a novel alternative isothermal amplification method for the detection of nucleic acid.
Methods: A rapid, sensitive and specific real-time reverse transcription LAMP (RT-LAMP) assay was developed for SARS-CoV-2 detection.
Results: This assay detected one copy/reaction of SARS-CoV-2 RNA in 30 min. Both the clinical sensitivity and specificity of this assay were 100%. The RT-LAMP showed comparable performance with RT-qPCR. Combining simplicity and cost-effectiveness, this assay is therefore recommended for use in resource resource-limited settings.

Matched MeSH terms: Nucleic Acid Amplification Techniques
Fulltext Identification of Mycobacterium tuberculosis-specific DNA marker using MAMA-PCR

Chin Kai Ling, Jaeyres Jani, Zainal Arifin Mustapha

Malaysian Journal of Medicine and Health Sciences, 2019;15(106):36-36.
MyJurnal

Introduction: Tuberculosis (TB), commonly caused by Mycobacterium tuberculosis (Mtb), is one of the ten leading causes of death worldwide. The gold standard, microbiological culture for detection and differentiation of mycobac-teria are time-consuming and laborious. The use of fast, easy and sensitive nucleic acid amplification tests (NAATs) for diagnosis of TB remains challenging because there is a high degree of homology within Mtb complex (MTBC) members and absence of target genes in the genome of some strains. This study aimed to identify new candidate genetic marker and to design specific primers to detect Mtb using in silico methods. Methods: Using Basic Local Alignment Search Tool (BLAST) program, Mtb H37Rv chromosome reference genome sequence was mapped with other MTBC members and a single nucleotide polymorphism (SNP) at Rv1970 was found to be specific only for Mtb strains. Mismatch amplification mutation assay (MAMA) combine with polymerase chain reaction (PCR) was used as an alternative method to detect the point mutation. MAMA primers targeting the SNP were designed using Primer-BLAST and the PCR assay was optimized via Taguchi method. Results: The assay amplified a 112 bp gene fragment and was able to detect all Mtb strains, but not the other MTBC members and non-tuberculous Mycobacte-ria. The detection limit of the assay was 60 pg/μl. Conclusion: Bioinformatics has provided predictive identification of many new target markers. The designed primers were found to be highly specific at single-gene target resolution for detection of Mtb.

Matched MeSH terms: Nucleic Acid Amplification Techniques
Fulltext Development of loop-mediated isothermal amplification (LAMP) for the detection of speB gene in invasive Streptococcus pyogenes clinical isolates

Azi Simon Onyema, Leslie Than Thian Lung, Suresh Kumar, Rukman Awang Hamat

Malaysian Journal of Medicine and Health Sciences, 2019;15(106):1-1.
MyJurnal

Introduction: Group A streptococcus (GAS) is responsible for high morbidity and mortality globally. Hence, the need to develop sensitive, reliable and cost- effective method of detection is crucial. In this study, we developed a visual detection method for the common virulence gene, streptococcal pyrogenic exotoxin B (speB) involved in invasive GAS diseases using loop-mediated isothermal amplification (LAMP) with fluorescent detection dye (calcein). Meth-ods: The LAMP reaction was optimized at 63°C for 35 minutes using five sets of primer designed with LAMP primer V5 software. When the dye was added prior to amplification, samples with speB DNA developed a characteristic green color after the reaction, but no color reactions were observed in samples with DNAs of non-GAS isolates. De-tection of speB by LAMP assay was done among 43 clinical isolates of blood, pus, wound, tissue and throat samples and ATCCs for controls. Our findings were further reconfirmed by subjecting the LAMP products to 0.5% gel electro-phoresis. Results: The detection limit of this LAMP assay for speB was 10-7 ng/μl of genomic DNA per reaction, which was 10,000-fold more sensitive than conventional PCR 10-3 ng/μl. All 100 % samples were positive for speB gene by LAMP, and 93% by conventional PCR method. Conclusion: LAMP assay could offer remarkably high sensitivity, specificity, repeatability, reliability, affordability, and visibility; it is appropriate for rapid detection of speB in Group A streptococci (GAS) as a point of care testing.

Matched MeSH terms: Nucleic Acid Amplification Techniques
Rapid in-situ detection kit (RisK): Development of loop-mediated isothermal amplification (LAMP) assay for the rapid identification of selected invasive alien fish in Malaysian freshwaters

Vythalingam LM, Hossain MAM, Bhassu S

Mol Cell Probes, 2020 Nov 28;55:101683.
PMID: 33259896 DOI: 10.1016/j.mcp.2020.101683

Invasive alien fish species have become a silent treat towards the ecosystem especially the native fish population in Malaysia. There has been a need to develop rapid identification methods that can aid management teams in identifying fish species that are not native to our ecosystem. Current visual identification methods are highly tedious and require time, delaying action towards curbing the invasion. The LAMP assay successfully identified six popular invasive fish species in Malaysia. None of the LAMP assays showed false positives and the Limit of Detection of the LAMP primers were highly sensitive and could detect DNA samples up to 1 × 10-15 ng/μl. The LAMP primers designed were highly specific to the target species and did not amplify non target species. DNA sequencing was done to ensure the accuracy of LAMP assay results. This study demonstrates that LAMP is a suitable tool in species identification efforts of invasive fish species in Malaysia.

Matched MeSH terms: Nucleic Acid Amplification Techniques
Fulltext A Sensitive, Colorimetric, High-Throughput Loop-Mediated Isothermal Amplification Assay for the Detection of Plasmodium knowlesi

Britton S, Cheng Q, Grigg MJ, William T, Anstey NM, McCarthy JS

Am J Trop Med Hyg, 2016 07 06;95(1):120-2.
PMID: 27162264 DOI: 10.4269/ajtmh.15-0670

The simian parasite Plasmodium knowlesi is now the commonest cause of malaria in Malaysia and can rapidly cause severe and fatal malaria. However, microscopic misdiagnosis of Plasmodium species is common, rapid antigen detection tests remain insufficiently sensitive and confirmation of P. knowlesi requires polymerase chain reaction (PCR). Thus available point-of-care diagnostic tests are inadequate. This study reports the development of a simple, sensitive, colorimetric, high-throughput loop-mediated isothermal amplification assay (HtLAMP) diagnostic test using novel primers for the detection of P. knowlesi. This assay is able to detect 0.2 parasites/μL, and compared with PCR has a sensitivity of 96% for the detection of P. knowlesi, making it a potentially field-applicable point-of-care diagnostic tool.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
Fulltext Loop-mediated isothermal amplification assay for detection of generic and verocytotoxin-producing Escherichia coli among indigenous individuals in Malaysia

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*

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