Identification ofSporothix schenckiiof various mtDNA types by Nested PCR Assay

Abstract

We employed a nested PCR assay to detect Sporothrix schenckii DNA of 38 strains (including all the 24 mtDNA types) collected from different areas of the world, in tissue of eight mice infected with ATCC10268 strain of the fungus, and the skin biopsies of nine patients with sporotrichosis. In addition, the same procedures were used with two strains of Ceratocystis minor and isolates of 10 species of other pathogenic fungi. The outer primers SS(1) and SS(2) and inner primers SS(3) and SS(4) of the 18S rRNA gene of S. schenckii were employed. A 152 bp fragment was detected in all 38 strains of S. schenckii, eight animal specimens and nine human skin biopsies, but not samples of C. minor and the other fungal species. The detection limit of 50 fg of S. schenckii DNA extract was determined with ethidium bromide staining. In summary, we demonstrated that nested PCR assay could identify S. schenckii of all the mtDNA types and in isolates recovered from different areas of the world. The nested PCR assay seems to be highly sensitive and specific and provides a rapid method for diagnosis of sporotrichosis under conditions of contamination avoidance.

Cerebrospinal fluid PCR: A new approach for the diagnosis of CNS sporotrichosis

“…The assay was successfully used to detect S . schenckii DNA from strains from different areas of the world [ 7 ]. However, Mendoza and colleagues [ 8 ] compared the previously described nested PCR with conventional diagnostic methods, and the molecular methodology presented lower efficacy.…”

“…Liu and colleagues [ 9 ], using the primer pair S2-R2 targeting the CHS1 gene in the PCR of biopsy tissue, verified positive results in 25 out of 30 cases (83.3%). The nested PCR targeting the partial sequence of the 18S rRNA gene was the best choice in terms of sensitivity due to a low fungal burden in CSF [ 6 – 7 ].…”

Sporotrichosis in Mexico

“…The most informative loci used for species recognition are located in regions encoding proteins such as calmodulin [74,75], beta-tubulin [74,76,77], the Translation Elongation Factor [4,64,77], and the "fungal barcoding" regions (the ribosomal internal transcribed spacers) [78,79]. Several molecular techniques, as the nested PCR [80,81]; the Random Amplification of Polymorphic DNA (RAPD) [82]; the Restriction Fragment Length Polymorphism (RFLP) [83]; the Random Amplified Polymorphic DNA (RAPD) [84]; the Amplified Fragment Length Polymorphism (AFLP) [85], and the Rolling Circle Amplification (RCA) [86], have been used successfully. However, the end-point PCR and the real-time multiplex PCR, using fluorescent probes to identify S. globosa, S. schenckii, and S. brasiliensis, predominate [87].…”

Fast diagnosis of sporotrichosis caused by Sporothrix globosa, Sporothrix schenckii, and Sporothrix brasiliensis based on multiplex real-time PCR

“…With the development of molecular biology, an increasing number of methods based on nucleic acid detection have been applied for the rapid diagnosis of infectious disease. Many molecular diagnostic tests, including DNA sequencing of “barcoding” genes[20–23], nested PCR[24–25], PCR fingerprinting[26], restriction fragment length polymorphism (RFLP) of different gene targets[27], random amplified polymorphic DNA (RAPD)[7], amplified fragment length polymorphism (AFLP) [8], rolling circle amplification (RCA) [28] and species-specific primers[29], have been developed for Sporothrix spp. detection.…”

“…However, there are still some shortcomings, such as time-consuming procedures (PCR sequencing for at least 12 h); complicated operation steps, which can increase the chance of contamination (nest PCR); insufficient sensitivity (RCA, 3 × 10 6 copies); and so on. Most of them can only identify isolates from culture, and only a few methods have been evaluated with clinical samples[24–25]. In addition, none of the above methods can detect co-infection simultaneously.…”