Development Of A DNA-Based Molecular Method For The Rapid Detection Of Enterococcus Species And Antimicrobial Resistance Genotypes [QR82.S78 C454 2008 f rb].

Enterococci muncul sebagai penyebab jangkitan nosokomial yang penting di kebanyakan negara di dunia semenjak kurun yang lepas. Enterococci have emerged as a significant cause of nosocomial infections in many parts of the world over the last decade

Development of integrated multisensor isothermal amplification with nurcleic acid lateral flow system for point of care biosensor assay

1 developed prototype provides a method of definitive detection multiplex for loop-mediated isothermal amplification AMP). Method of amplicons detection relates to a label-based lateral flow dipstick device. The device comprises an orbent pad and a reaction pad. The reaction pad immobilized with more than two sorts of antibodies to detect tiple dual-labeled LAMP amplicons. A first line on the reaction pad, a chromatography control line, is an Tunological component that capable of binding to an enzyme-labeled antibody. The second and third lines, known as ■ lines, are also an immunological component that capable of specifically binding to labeled site of LAMP amplicons ch are another labeled site are bound to enzyme-labeled antibody to form the sandwich complexes. The enzymeeled antibody produces a visual red color by which it is pre-coated with gold nanoparticles. In specific, the present totype simplifies the multi-steps of mLAMP reagent preparation, reduces the risk of carry-over contamination, ease of A.MP amplicons detection and improved the credibility of the study. The ready-to-use thermostabilized mLAMP gents can be potentially applied as a diagnostic kit for diagnosis of diseases on field or in clinical settings

A nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistance genes in Enterococcus species

<p>Abstract</p> <p>Background</p> <p>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 <it>E. faecalis </it>and <it>E. faecium </it>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 <it>Enterococcus </it>genus, <it>ddl </it>of <it>E. faecalis </it>and <it>E. faecium</it>, <it>aac</it>A-<it>aph</it>D that encodes high level gentamicin resistance (HLGR), multilevel vancomycin resistant genotypes such as <it>van</it>A, <it>van</it>B, <it>van</it>C and <it>van</it>D and one internal control gene.</p> <p>Results</p> <p>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 <it>E. faecalis</it>, 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.</p> <p>Conclusion</p> <p>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 <it>Enterococcus </it>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.</p

A Pentaplex PCR Assay for the Detection and Differentiation of Shigella Species

The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis of Shigella spp. is laborious and time consuming and has low sensitivity. Hence, in the present study, a molecular-based diagnostic assay which amplifies simultaneously four specific genes to identify invC for Shigella genus, rfc for S. flexneri, wbgZ for S. sonnei, andrfpB for S. dysenteriae, aswell as one internal control (ompA) gene, was developed in a single reaction to detect and differentiate Shigella spp. Validation with 120 Shigella strains and 37 non-Shigella strains yielded 100% specificity. The sensitivity of the PCR was 100 pg of genomic DNA, 5.4 × 104 CFU/ml, or approximately 120 CFU per reaction mixture of bacteria. The sensitivity of the pentaplex PCR assay was further improved following preincubation of the stool samples in Gram-negative broth. A preliminary study with 30 diarrhoeal specimens resulted in no cross-reaction with other non-Shigella strains tested. We conclude that the developed pentaplex PCR assay is robust and can provide information about the four target genes that are essential for the identification of the Shigella genus and the three Shigella species responsible for the majority of shigellosis cases

Harnessing CRISPR-Cas to combat COVID-19: from diagnostics to therapeutics

The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global threat with an ever-increasing death toll even after a year on. Hence, the rapid identification of infected individuals with diagnostic tests continues to be crucial in the on-going effort to combat the spread of COVID-19. Viral nucleic acid detection via real-time reverse transcription polymerase chain reaction (rRT-PCR) or sequencing is regarded as the gold standard for COVID-19 diagnosis, but these technically intricate molecular tests are limited to centralized laboratories due to the highly specialized instrument and skilled personnel requirements. Based on the current development in the field of diagnostics, the programmable clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system appears to be a promising technology that can be further explored to create rapid, cost-effective, sensitive, and specific diagnostic tools for both laboratory and point-of-care (POC) testing. Other than diagnostics, the potential application of the CRISPR–Cas system as an antiviral agent has also been gaining attention. In this review, we highlight the recent advances in CRISPR–Cas-based nucleic acid detection strategies and the application of CRISPR–Cas as a potential antiviral agent in the context of COVID-19

Antibacterial activity and toxicity of Duckweed, Lemna minor L. (Arales: Lemnaceae) from Malaysia

Aims: New therapeutics are needed to ease the prevailing waterborne disease, and one of the alternatives is by exploring the natural compounds with antimicrobial properties. Duckweed, Lemna sp. is recorded as a medicinal herb that known to have antifungal and antibacterial activities towards several fungi and bacteria. Suitability of duckweed (Lemna minor) as an antibacterial resource against selected waterborne bacteria were evaluated in terms of its antibacterial activity and toxicity. Methodology and results: Antibacterial activity of the duckweed methanolic extract was tested against 11 selected waterborne bacteria using disc diffusion, minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) assay. Brine shrimp lethality assay was used to determine the toxicity of this extract. The lethal concentrations of plant extract resulting in 50% mortality of the brine shrimp (LC50) were then determined. Conclusion, significance and impact of study: Results showed that duckweed extract exhibited bacteriostatic and bactericidal against the selected bacteria activity at the concentration of MIC = 1.8-2.0 mg/mL and MBC ≥ 2.0 mg/mL. This study shows that methanolic extract of L. minor may contain bioactive compounds against bacteria and potential therapeutic effect. The crude extract is slightly toxic and may not safe to be used in high concentration but is valuable in further study as a potential antitumor agent