Identification of Fungal Pathogens by Visible Microarray System in Combination with Isothermal Gene Amplification

Abstract

The increasing incidence of infectious diseases caused by fungi in immunocompromised patients has encouraged researchers to develop rapid and accurate diagnosis methods. Identification of the causative fungal species is critical in deciding the appropriate treatment, but it is not easy to get satisfactory results due to the difficulty of fungal cultivation and morphological identification from clinical samples. In this study, we established a microarray system that can identify 42 species from 24 genera of clinically important fungal pathogens by using a chemical color reaction in the detection process. The array uses the internal transcribed spacer region of the rRNA gene for identification of fungal DNA at the species level. The specificity of this array was tested against a total of 355 target and nontarget fungal species. The fungal detection was succeeded directly from 103 CFU/ml for whole blood samples, and 50 fg DNA per 1 ml of serum samples indicating that the array system we established is sensitive to identify infecting fungi from clinical sample. Furthermore, we conducted isothermal amplification in place of PCR amplification and labeling. The successful identification with PCR-amplified as well as isothermally amplified target genes demonstrated that our microarray system is an efficient and robust method for identifying a variety of fungal species in a sample.Electronic supplementary materialThe online version of this article (doi:10.1007/s11046-014-9756-2) contains supplementary material, which is available to authorized users.

Recombinase Polymerase Amplification for Diagnostic Applications

“…RPA was successfully used to detect major human pathogens including bacteria (6,(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23), viruses (9, 24 -32 ), fungi (33 ), and parasites (34,35 ), as well as genetically modified organisms (36,37 ) and genetic alterations observed in cancer cells (38,39 ). RPA was also used for HIV diagnosis in low-resource settings (40,41 ) (Tables 2 and 3).…”

Development of real-time recombinase polymerase amplification assay for rapid and sensitive detection of canine parvovirus 2

“…Similar to conventional PCR, the use of two opposing primers allows exponential amplification of the target sequence in RPA, but the latter is tolerant to 5-9 mismatches in the primer and probe without influencing the performance of the assay. RPA possesses superiority in speed, portability and accessibility compared to PCR, and it has been used to replace the PCR method for the molecular detection of diverse pathogens, such as fungi, parasites, bacteria and viruses [20][21][22]. Based on our previous study, we developed here a real-time RPA assay for simple, rapid, convenient and POC detection of CPV-2, which utilizes an exo probe and a portable, userfriendly POC tube scanner.…”

Rapid and sensitive detection of canine parvovirus type 2 by recombinase polymerase amplification

“…A number of geneamplification-based assays have been developed for CPV-2 diagnosis, such as polymerase chain reaction (PCR), nested PCR, real-time PCR, loop-mediated isothermal amplification, and insulated isothermal PCR (iiPCR) [6][7][8][9][10]. Recombinase polymerase amplification (RPA), a novel isothermal gene amplification technique, employs three core enzymes: a recombinase, a single-stranded DNAbinding protein (SSB), and a strand-displacing polymerase [11][12][13]. The reaction initiates with the recombinase binding to the primer to form filaments (protein-DNA complex) that are capable of pairing the primer with a homologous sequence in the target DNA.…”

“…When opposing primers are used, exponential amplification typically runs to completion in about 30 minutes. RPA has been explored for the molecular detection of diverse pathogens, e.g., bacteria, fungi, parasites and viruses [12][13][14][15][16][17], as it possesses superiority in speed, portability and accessibility [17]. In this study, a novel RPA-based method was developed for the detection of CPV-2.…”