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Non-protein coding RNA-based genosensor with quantum dots as electrochemical labels for attomolar detection of multiple pathogens

Vijian D, Chinni SV, Yin LS, Lertanantawong B, Surareungchai W

Biosens Bioelectron, 2016 Mar 15;77:805-11.
PMID: 26513287 DOI: 10.1016/j.bios.2015.10.057

The ability of a diagnostic test to detect multiple pathogens simultaneously is useful to obtain meaningful information for clinical treatment and preventive measures. We report a highly sensitive and specific electrochemical biosensor assay for simultaneous detection of three gene targets using quantum dots (QDs). The targets are novel non-protein coding RNA (npcRNA) sequences of Vibrio cholerae, Salmonella sp. and Shigella sp., which cause diarrheal diseases. QDs (PbS, CdS, ZnS) were synthesized and functionalized with DNA probes that were specific to each pathogen. Electrochemical detection of QDs was performed using square wave anodic stripping voltammetry (SWASV). The QDs gave distinct peaks at 0.5 V (PbS), 0.75 V (CdS) and 1.1 V (ZnS). There was no interference in signal response when all three QDs were mixed and detected simultaneously. The detection limits of single and multiplex assays with linear targets and PCR products were in the attomolar ranges. The high assay sensitivity, in combination with specific npcRNA sequences as novel diagnostic targets, makes it a viable tool for detecting pathogens from food, environment and clinical samples.
Electrochemical DNA detection of hepatitis E virus genotype 3 using PbS quantum dot labelling

Ngo DB, Chaibun T, Yin LS, Lertanantawong B, Surareungchai W

Anal Bioanal Chem, 2021 Feb;413(4):1027-1037.
PMID: 33236225 DOI: 10.1007/s00216-020-03061-1

The aim of this study was to develop a highly specific electrochemical DNA sensor using functionalized lead sulphide (PbS) quantum dots for hepatitis E virus genotype 3 (HEV3) DNA target detection. Functionalized-PbS quantum dots (QDs) were used as an electrochemical label for the detection of HEV3-DNA target by the technique of square wave anodic stripping voltammetry (SWASV). The functionalized-PbS quantum dots were characterized by UV-vis, FTIR, XRD, TEM and zeta potential techniques. As-prepared, functionalized-PbS quantum dots have an average size of 4.15 ± 1.35 nm. The detection platform exhibited LOD and LOQ values of 1.23 fM and 2.11 fM, respectively. HEV3-DNA target spiked serum is also reported.Graphical abstract.
A nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistance genes in Enterococcus species

Yean CY, Yin LS, Lalitha P, Ravichandran M

BMC Microbiol, 2007 Dec 11;7:112.
PMID: 18070365

BACKGROUND: Enterococci have emerged as a significant cause of nosocomial infections in many parts of the world over the last decade. The most common enterococci strains present in clinical isolates are E. faecalis and E. faecium which have acquired resistant to either gentamicin or vancomycin. The conventional culture test takes 2-5 days to yield complete information of the organism and its antibiotic sensitivity pattern. Hence our present study was focused on developing a nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistant enterococci (V-BiA-RE). This assay simultaneously detects 8 genes namely 16S rRNA of Enterococcus genus, ddl of E. faecalis and E. faecium, aacA-aphD that encodes high level gentamicin resistance (HLGR), multilevel vancomycin resistant genotypes such as vanA, vanB, vanC and vanD and one internal control gene.
RESULTS: Unique and specific primer pairs were designed to amplify the 8 genes. The specificity of the primers was confirmed by DNA sequencing of the nanoplex PCR products and BLAST analysis. The sensitivity and specificity of V-BiA-RE nanoplex PCR assay was evaluated against the conventional culture method. The analytical sensitivity of the assay was found to be 1 ng at the DNA level while the analytical specificity was evaluated with 43 reference enterococci and non-enterococcal strains and was found to be 100%. The diagnostic accuracy was determined using 159 clinical specimens, which showed that 97% of the clinical isolates belonged to E. faecalis, of which 26% showed the HLGR genotype, but none were vancomycin resistant. The presence of an internal control in the V-BiA-RE nanoplex PCR assay helped us to rule out false negative cases.
CONCLUSION: The nanoplex PCR assay is robust and can give results within 4 hours about the 8 genes that are essential for the identification of the most common Enterococcus spp. and their antibiotic sensitivity pattern. The PCR assay developed in this study can be used as an effective surveillance tool to study the prevalence of enterococci and their antibiotic resistance pattern in hospitals and farm animals.
Fulltext Efficient extraction of small and large RNAs in bacteria for excellent total RNA sequencing and comprehensive transcriptome analysis

Heera R, Sivachandran P, Chinni SV, Mason J, Croft L, Ravichandran M, et al.

BMC Res Notes, 2015;8(1):754.
PMID: 26645211 DOI: 10.1186/s13104-015-1726-3

Next-generation transcriptome sequencing (RNA-Seq) has become the standard practice for studying gene splicing, mutations and changes in gene expression to obtain valuable, accurate biological conclusions. However, obtaining good sequencing coverage and depth to study these is impeded by the difficulties of obtaining high quality total RNA with minimal genomic DNA contamination. With this in mind, we evaluated the performance of Phenol-free total RNA purification kit (Amresco) in comparison with TRI Reagent (MRC) and RNeasy Mini (Qiagen) for the extraction of total RNA of Pseudomonas aeruginosa which was grown in glucose-supplemented (control) and polyethylene-supplemented (growth-limiting condition) minimal medium. All three extraction methods were coupled with an in-house DNase I treatment before the yield, integrity and size distribution of the purified RNA were assessed. RNA samples extracted with the best extraction kit were then sequenced using the Illumina HiSeq 2000 platform.
Gold-nanoparticle based electrochemical DNA sensor for the detection of fish pathogen Aphanomyces invadans

Kuan GC, Sheng LP, Rijiravanich P, Marimuthu K, Ravichandran M, Yin LS, et al.

Talanta, 2013 Dec 15;117:312-7.
PMID: 24209346 DOI: 10.1016/j.talanta.2013.09.016

Epizootic ulcerative syndrome (EUS) is a devastating fish disease caused by the fungus, Aphanomyces invadans. Rapid diagnosis of EUS is needed to control and treat this highly invasive disease. The current diagnostic methods for EUS are labor intensive. We have developed a highly sensitive and specific electrochemical genosensor towards the 18S rRNA and internal transcribed spacer regions of A. invadans. Multiple layers of latex were synthesized with the help of polyelectrolytes, and labeled with gold nanoparticles to enhance sensitivity. The gold-latex spheres were functionalized with specific DNA probes. We describe here the novel application of this improved platform for detection of PCR product from real sample of A. invadans using a premix sandwich hybridization assay. The premix assay was easier, more specific and gave higher sensitivity of one log unit when compared to the conventional method of step-by-step hybridization. The limit of detection was 0.5 fM (4.99 zmol) of linear target DNA and 1 fM (10 amol) of PCR product. The binding positions of the probes to the PCR amplicons were optimized for efficient hybridization. Probes that hybridized close to the 5' or 3' terminus of the PCR amplicons gave the highest signal due to minimal steric hindrance for hybridization. The genosensor is highly suitable as a surveillance and diagnostic tool for EUS in the aquaculture industry.
Enzyme-linked amperometric electrochemical genosensor assay for the detection of PCR amplicons on a streptavidin-treated screen-printed carbon electrode

Yean CY, Kamarudin B, Ozkan DA, Yin LS, Lalitha P, Ismail A, et al.

Anal Chem, 2008 Apr 15;80(8):2774-9.
PMID: 18311943 DOI: 10.1021/ac702333x

A general purpose enzyme-based amperometric electrochemical genosensor assay was developed wherein polymerase chain reaction (PCR) amplicons labeled with both biotin and fluorescein were detected with peroxidase-conjugated antifluorescein antibody on a screen-printed carbon electrode (SPCE). As a proof of principle, the response selectivity of the genosensor was evaluated using PCR amplicons derived from lolB gene of Vibrio cholerae. Factors affecting immobilization, hybridization, and nonspecific binding were optimized to maximize sensitivity and reduce assay time. On the basis of the background amperometry signals obtained from nonspecific organisms and positive signals obtained from known V. cholerae, a threshold point of 4.20 microA signal was determined as positive. Under the optimum conditions, the limit of detection (LOD) of the assay was 10 CFU/mL of V. cholerae. The overall precision of this assay was good, with the coefficient of variation (CV) being 3.7% using SPCE and intermittent pulse amperometry (IPA) as an electrochemical technique. The assay is sensitive, safe, and cost-effective when compared to conventional agarose gel electrophoresis, real-time PCR, and other enzyme-linked assays for the detection of PCR amplicons. Furthermore, the use of a hand-held portable reader makes it suitable for use in the field.
Target Induced-DNA strand displacement reaction using gold nanoparticle labeling for hepatitis E virus detection

Ngamdee T, Yin LS, Vongpunsawad S, Poovorawan Y, Surareungchai W, Lertanantawong B

Anal Chim Acta, 2020 Oct 16;1134:10-17.
PMID: 33059855 DOI: 10.1016/j.aca.2020.08.018

DNA strand displacement is an attractive, enzyme-free target hybridization strategy for nano-biosensing. The target DNA induces a strand displacement reaction by replacing the pre-hybridized strand that is labeled with gold nanoparticles (AuNPs). Thus, the amount of displaced-AuNP-labeled strand is proportional to the amount of target DNA in the sample. The use of a magnetogenosensing technique to isolate the target DNA allows for a simple, one-pot detection approach, which minimizes possible carry-over contamination and pipetting errors. We sought a proof-of-concept for this technology in its ability to detect DNA-equivalent of hepatitis E virus (HEV), which causes acute viral hepatitis for which rapid and simple diagnostic methods remain limited. Signal detection was done via visual observation, spectrophotometry, and electrochemistry. The sensor demonstrated good sensitivity with detection limits of 10 pM (visual), 10 pM (spectrophotometry) and 1 fM (electrochemical). This sensor also exhibited high specificity for real target amplicons and could discriminate between perfect and mismatched sequences. Lyophilized biosensor reagents stored at 4 °C, 25 °C, and outdoor ambient temperature, were stable for up to 90, 50, and 40 days, respectively. The integration of magnetic separation and target DNA-induced strand displacement reaction in a dry reagent form makes the sensing platform easy-to-use and suitable for field settings.
Fulltext Knowledge and Practice of ICDAS and ICCMS in Young Children: A Cross-sectional Study among Final-year Dental Students in Malaysia

Yin LS, Tamilselvam K, Abdulla AM, Heng AP, Jayaraman J

Int J Clin Pediatr Dent, 2021;14(2):278-285.
PMID: 34413607 DOI: 10.5005/jp-journals-10005-1912

BACKGROUND: To evaluate the association of sex, type of institution, and satisfaction of educational activities with dental students' knowledge of International Caries Detection and Assessment System (ICDAS) and International Caries Classification and Management System (ICCMS) in young children.
MATERIALS AND METHODS: This was a cross-sectional study involving final-year dental students from all accredited dental institutions across Malaysia. A self-constructed questionnaire was given through an identified personal contact from each school. The students' responses on the association of sex and institution type with the satisfaction of education activities and ICDAS and ICCMS knowledge were tested using the independent t-test. The relationship between satisfaction of educational activities and knowledge of ICDAS and ICCMS among dental students was evaluated using Spearman's correlation analysis. Statistical significance was set at p < 0.05.
RESULTS: A total of 440 responses were obtained. Out of this, 312 (70.9%) were female and 126 (28.6%) were male respondents, and 2 with unknown sex. There were 231 responses (52.5%) from the private institution and 209 responses (47.5%) from the government institution. Males had a higher level of satisfaction toward the education activities. No significant difference was observed between the knowledge of ICCMS and ICDAS and sex or institution type.
CONCLUSION: Male dental students felt more satisfied with pediatric lectures conducted during preclinical years and were more prepared to treat patients independently upon graduation. Sex and type of institution did not play a role in the knowledge and dental students were more knowledgeable in ICDAS compared to ICCMS.
HOW TO CITE THIS ARTICLE: Yin LS, Tamilselvam K, Abdulla AM, et al. Knowledge and Practice of ICDAS and ICCMS in Young Children: A Cross-sectional Study among Final-year Dental Students in Malaysia. Int J Clin Pediatr Dent 2021;14(2):278-285.
Fulltext Complete genome sequence of Serratia marcescens D1_6, isolated from peat soil

Isaac P, Mutusamy P, Yin LS, Jing Wei Y, Mohd Salleh F, Bin Abu Bakar MAL, et al.

Microbiol Resour Announc, 2024 Nov 12;13(11):e0029924.
PMID: 39311881 DOI: 10.1128/mra.00299-24

We present a complete genome of Serratia marcescens D1_6 isolated from peat swamp forest. The complete genome for the isolate D1_6 was constructed using data from Oxford Nanopore Technologies and Illumina. The genome of D1_6 has a total length of 4,996,151 bp, comprising a chromosome and a plasmid.