Identification of pathogenic yeast species by polymerase chain reaction amplification of theRPS0gene intron fragment

Abstract

Aims: This work focuses on the development of a method for the identification of pathogenic yeast. With this aim, we target the nucleotide sequence of the RPS0 gene of pathogenic yeast species with specific PCR primers. PCR analysis was performed with both the genomic DNA, whole cells of clinical isolates of Candida species and clinical samples. Methods and Results: A single pairs of primers, deduced from the nucleotide sequence of the RPS0 gene from pathogenic yeast, were used in PCR analysis performed with both the genomic DNA and whole cells of clinical isolates of Candida species and clinical samples. The primers designed are highly specific for their respective species and produce amplicons of the expected sizes and fail to amplify any DNA fragment from the other species tested. The set of primers was tested successfully for the identification of yeast from colonies, blood cultures and clinical samples. These results indicate that genes containing intron sequences may be useful for designing species‐specific primers for the identification of fungal strains by PCR. The sensitivity of the method with genomic DNA was evaluated with decreasing DNA concentrations (200 ng to 1 pg) and different cell amounts (107–105 cells). Conclusion: The results obtained show that the amplification of RPS0 sequences may be suitable for the identification of pathogenic and other yeast species. Significance and Impact of the Study: Identification of Candida species using molecular approaches with high discriminatory power is important in determining adequate measures for the interruption of transmission of this yeast. The approach described in this work is based on standard technology, and it is specific, sensitive and does not involve complex and expensive equipment. Furthermore, the method developed in this work not only can be used in eight yeast species, but also provides the basis to design primers for other fungi species of clinical, industrial or environmental interest.

Phenotypic characterization and adhesive properties of vaginal Candida spp. strains provided by the CHU Farhat Hached (Sousse, Tunisia)

“…Indeed, the RPS0 gene codes for a protein which has been extremely conserved among species and which is a component of the translational machinery. 5,16 According to previous results, PCR primers based on RPS0 gene are an important tool for the identification of yeasts and fungi of clinical and environmental interest. 38 The enzymatic activities have an important role in fungal pathogenesis.…”

Molecular evidence on the occurrence of co-infection with Pichia guilliermondii and Wuchereria bancrofti in two filarial endemic districts of India

“…The inoculums from the medium were cultured and maintained in said medium until pure culture was achieved in the yeast specific medium. From the primary culture, 100 μl of inoculums were added to the pure culture medium comprising modified YPD agar containing 2% peptone, 1% yeast extract, 2% dextrose, and 6% FBS [24], and cultured at 32°C for 48 hours. After 11 consecutive sub-cultures, pure yeast colonies were obtained and maintained for further studies.…”

“…Pathogenicity of the fungal isolate was determined by the PCR-based detection of RPS0 gene previously described by Martinez et al [24]. Selective amplification of the RPS0 gene was carried out using the primers (see Table 1) designed for the RPS0 exon region for P. guilliermondii by Martinez et al [24].…”

“…Selective amplification of the RPS0 gene was carried out using the primers (see Table 1) designed for the RPS0 exon region for P. guilliermondii by Martinez et al [24]. …”

Systematic Internal Transcribed Spacer Sequence Analysis for Identification of Clinical Mold Isolates in Diagnostic Mycology: a 5-Year Study

“…Analysis of the ITS regions is the most commonly used molecular method for identification of molds (3,4,29). Studies reporting on the use of molecularly based identification procedures in diagnostic mycology have mainly focused on method development (12,25,30,40) and include anecdotal case reports (15,17,20). There are few studies addressing the use of systematic ITS sequencing implemented as a routine tool according to a defined work flow and its impact on diagnostic performance in a medical mycology laboratory (21).…”