Application Of PCR For The Identification Of Sporothrix Scheckii

Sporotrichosis is a chronic granulomatous fungal infection caused by Sporothrix schenckii with worldwide distribution. Sporothrix schenckii is a saprophyte in nature,is commonly found on vegetation or in soil.It is mainly encroached on the cutaneous and subcutaneous tissues,and presented a nonspecific granulomatous nodules. It usually spreads along the lymphatic vessels.The pulmonary and hematogenous disseminated forms of sporotrichosis,which are especially prevalent in immunocompromised persins such as HIV,can be crippling or fatal. Recently, the incidence of sporotrichosis is increasing and it needs a more effective method of early diagnosis of the disease. The conventional method for definitive diagnosis of sporotrichosis is based on time-consuming tissue culture,However,It frequently yield negative results.Although the fluorescent antibody or immunohistochemical techniques also provide a rapid diagnosis of sporotrichosis ,they are not available in most clinical laboratories.so the development of a easy, reliable ,specific and rapid detection system of Sporothrix schenckii would be very useful.In recent years, molecular techniques are starting to play an increasingly important role in fungal disease diagnosis. Molecular analyses such as PCR techniques have greatly improved the diagnosis of Candida albicans,However,a specific detection system of Sporothrix schenckii has not been applied in clinical laboratories.The aim of the present study was to establish a rapid method to detect and identify Sporothrix schenckii by using Species-specific oligonucleotide primer PCR techniques, and lay the fundation of molecular diagnosis for sporotrichosis. The Species-specific oligonucleotide primer pair S2-R2 was used in this study. It were designed from nucleotide sequences of chitin synthase 1 gene in Sporothrix schenckii. The departed essay proved that the primer pair S2-R2 did not amplify DNA from human cells and bacteria.Their location in Sporothrix schenckii CHS1 gene were 174-199bp and 468-492bp.Polymerase chain reaction analysis with the primer pair S2-R2 was able to amplified a 318bp fragment. For the examination of specificity, 26 strains of Sporothrix schenckii ,6 strains of clinical common fungi was amplified by PCR. 26 strains of Sporothrix schenckii include 1 strain of standard,7 strains of laboratory reserved and 18 strains of clinical isolated . 6 strains of clinical common fungi include Candida albicans , Candida glabrata , Trichophyton rubum , Microsporum gypseum , Trichophyton mentagrophyts and Microsporum canis . Clinical isolates of Sporothrix schenckii obtained from sporotrichosis,they were confirmed by tissue cultures.All strains of clinical isolated were grown on a Sabouraud medium at 25℃ for one or two weeks,then identified by double phase transformation assay.To guarantee the smoothly developing of fungal genomic DNA prepared in fungal study for PCR techniques,We used two method for extraction of fungal genomic DNA involving CTAB and alkaline guanidine thiocyanate boiling.At the same time,two methods were compared with each other.Result of test indicate: (1).All strains of standard and clinical isolated Sporothrix schenckii strains showed a specific fragment of 318bp with the species-specific primer pair.Using the same primer pairs, Candida albicans,Candida glabrata , Trichophyton rubum , Microsporum gypseum , Trichophyton mentagrophytes, Microsporum canis had no specific amplification.(2).To compare CTAB method with alkaline guanidine thiocyanate boiling method, genomic DNA consistence of the later was significantly higher than the former.Our conclusions: Species-specific primer PCR is a rapid,simple and feasible method for identifying Sporothrix schenckii,and alkaline guanidine thiocyanate boiling method for extraction of fungal genomic DNA is easy ,low cost and may be applicated in clinical laboratories.