Efficient Identification of Clinically Relevant Candida Yeast Species by Use of an Assay Combining Panfungal Loop-Mediated Isothermal DNA Amplification with Hybridization to Species-Specific Oligonucleotide Probes

Abstract

The occurrence of invasive mycoses has progressively increased in recent years. Yeasts of the genus Candida remain the leading etiologic agents of those infections. Early identification of opportunistic yeasts may contribute significantly to improved disease management and the selection of appropriate antifungal therapy. We developed a rapid and reliable molecular identification system for clinically relevant yeasts that makes use of nonspecific primers to amplify a region of the 26S rRNA gene, followed by reverse hybridization of the digoxigenin-labeled products to a panel of species-specific oligonucleotide probes arranged on a nylon membrane macroarray format. DNA amplification was achieved by the recently developed loop-mediated isothermal DNA amplification technology, a promising option for the development of improved laboratory diagnostic kits. The newly developed method was successful in distinguishing among the major clinically relevant yeasts associated with bloodstream infections by using simple, rapid, and cost-effective procedures and equipment.

Rapid Identification of Chikungunya and Dengue Virus by a Real-Time Reverse Transcription-Loop-Mediated Isothermal Amplification Method

“…The most attractive characteristic of the LAMP method is the visual determination of nucleic acid amplification, 18 which cannot only be used in well-equipped hospitals or laboratories in developed countries, but also in small-scale hospitals or even in field detection. 37 The LAMP method has been used in the differential diagnosis of the pathogens with close genetic relationships 16,17,38,39 and even in the differentiation of DEN virus serotypes. 40 The appearance and use of a turbidimeter allowed the LAMP method to achieve real-time detection.…”

A Universal Method for the Identification of Bacteria Based on General PCR Primers

“…Lanes represent Level reactions in the same order (1)(2)(3)(4)(5)(6)(7)(8)(9). Interpreted results are shown in the table Good sequencing results but poor BLAST alignment result, 2 indicates an independent repeat of sample processing on a different day e Different colony morphologies f H. pylori specific primers were used to confirm isolate, according to reference [8], while PCR product was generated with QUGP-F6.R3, no sequencing was attempted for this bacterium PA-SS-F and PA-SS-R [18] were utilized with this isolate.…”

“…Currently, two fundamental molecular applications are being extensively utilized in bacterial detection and identification; these are based on hybridization and nucleotide sequencing. Hybridization based applications such as Southern [7,8], PCR [9], real-time PCR [10,11], microarray [12,13], universal tagging method [14], and loop-mediated isothermal amplification (LAMP) [7] are sensitive and specific techniques for the detection and identification of microbes. Specific PCR primers have been employed to confirm the presence or absence of target microorganisms or specific features associated with them such as antibiotic resistance and virulence factors [1,[15][16][17][18].…”

Instrument-free nucleic acid amplification assays for global health settings

“…Second, the specificity of common isothermal amplification and detection schemes has been variable. 16,20,21,22,23 Finally, most isothermal amplification schemes require high heat treatment of the raw specimen and are reliant upon solid phase purification with either a centrifuge or vacuum manifold. 24 To date, these factors have all limited the penetration of isothermal amplification technologies into LRS.…”