Primer and probe for LF-RPA assay were designed based on the consensus sequence according to the guidelines of TwistAmp® DNA amplification kit (TwistDx Ltd., UK). DNAMAN software (Lynnon LLC., California, USA) was used to obtain the consensus sequence by multiple sequence alignment. gattii were downloaded from the GenBank® database ( ). A total of 139 available ITS sequences of C. The internal transcribed spacer (ITS) sequences of ribosomal RNA gene are highly variable and useful for species differentiation. To establish a nucleic acid-based detection method, the starting point is to identify the research target. gattii in clinical CSF samples from patients. In this study, we have assessed the performance of LF-RPA assay for detecting genomic DNA of C. At present, RPA combined with lateral flow strips (LF-RPA assay) has been successfully used for the rapid and visual detection of several pathogens including parasites, viruses, and bacteria. Recently, recombinase polymerase amplification (RPA), an isothermal in vitro nucleic acid amplification technique, appeared as a novel molecular technology for simple, robust (less sensitive to inhibitors), rapid, reliable, and low-resource diagnostics. Although detection of CrAg has demonstrated good sensitivity and specificity, extremely high concentrations of CrAg can yield negative test results in extreme instances, known as high dose hook effect. Serological diagnosis of CM, such as latex agglutination, enzyme-linked immunosorbent assays and lateral flow assay, relies usually on specific monoclonal antibodies to detect cryptococcal antigen polysaccharide (CrAg). However, culture may be negative if exposure to antifungal therapy or in non-HIV CM and might need longer incubation periods up to several weeks. The definitive diagnosis of CM relies on culture on standard Sabouraud dextrose agar (SDA) or using routine and automated culture systems inoculated with CSF incubated at 30 ☌. The sensitivity of India ink staining of the CSF is up to 70–90%, which tends to be lower in HIV-negative patients, but this value is dependent on both the fungal burden and operator. India ink staining and culture are the traditional important methods for rapid detection of Cryptococcus spp. must be identified within CSF from the patients. For a definitive diagnosis of CM, Cryptococcus spp. Lumbar puncture, also known as spinal tap, and cerebral spinal fluid (CSF) analysis should be performed in patients with suspected CM. Įarly diagnosis and treatment of cryptococcosis reduces mortality. gattii are found particularly concentrated in soil and eucalyptus trees and responsible for the most cases of human cryptococcosis. The wide dispersal environmental fungi Cryptococcus neoformans and C. Cryptococcal meningitis (CM) is a subacute meningoencephalitis and the most common cause of adult meningitis with very high mortality rates as well as vision and hearing loss in survivors. The most common presentations of cryptococcosis are meningitis and meningoencephalitis. in cerebral spinal fluid and might be useful for clinical preliminary screening of cryptococcal meningitis. The LF-RPA system described here is shown to be a sensitive and specific method for the visible, rapid, and accurate detection of Cryptococcus spp. As amplification template for LF-RPA assay, both cell lysates and genomic DNA produce similar experimental results. The overall sensitivity and specificity were 95.2 and 95.8% respectively. The system could work well at a wide range of temperature from 25 to 45 ☌. neoformans per reaction within 10 min and was highly specific for Cryptococcus spp. The LF-RPA assay could detect 0.64 pg of genomic DNA of C. A brief analysis and comparison of different DNA extraction methods was discussed, too. The diagnostic parameters were first calculated using 114 clinical specimens and then compared with that of other diagnostic method. The optimal detection time and amplification temperature were also analyzed. The specificity was assessed by excessive amount of other pathogens genomic DNA. The detection limit was evaluated using serial dilutions of C. The lateral flow strips combined with recombinase polymerase amplification (LF-RPA) assay was constructed to detect the specific DNA sequences of C. Therefore, the aim of this study was to introduce a new rapid and simple detection method of Cryptococcus neoformans and C. Although sensitive and specific enough, detection of cryptococcal antigen polysaccharide has a high dose hook effect. such as India ink staining and culture are not ideal. The traditional methods for detecting Cryptococcus spp. gattii must be identified within cerebral spinal fluid from the patients. For definitive diagnosis of cryptococcal meningitis, Cryptococcus neoformans and/or C.
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