Optimization Of Sporotrichosis Rapid Detection Method

Sporothrix schenckii is the causative agent of sporotrichosis, which affects primarilythe skin, subcutaneous tissue, lymph vessels, lymphnodes and rarely, visceral organs.Sporotrichosis spread widely in the world, Primary disease in our country northeastfairly pilosity,on prevail region batch to happen, harm persons heath. Now,this diseasediagnose difficulty. Raising the fungus,waste lots of time(about one week), as wellmasccline rate low; tissue pathology is nonspecific granulomatous inflammation, intelaetiology diagnosis still to remain restrict at HE and PAS dyeing, but because fungusare few, acquiring routine histology method is difficult. For the past few years, scholarshave conducted a multifaceted exploration in the diagnosis of this disease, includingserology detection, IMFA, immunopathogenesis and electron microscopy. Becauseserology detection specific problem fail to continue, although IMFA andimmunopathogenesis raise dignosis’ speed, antibody standard is difficult. Electronmicroscopy manipulate multiplicity, eyesight is little, the fungus are difficult to befinded, so to spread is impossible. This study intends to extract genomic DNA ofSporothrix using improved CTAB method, and a pair of synthetic oligonucleotideprimers were designed according to the conserved sequence of Sporothrix schenkiicalmodulin gene, combining with PCR technology, fast detection and identification ofSporothrix schenkii are explored, and then expecting to establish a rapid, sensitive,molecular biology methods to test and identify Sporothrix Schenckii in infected tissueso as to provide a reliable basis for clinical diagnosis and treatment.Objective:1.A modified method for extracting DNA from Sporothrix.2. To study arapid method to detect and identify Sporothrix schenckii by using Species-specificoligonucleotide primer PCR techniques，and lay the fundation of molecular diagnosisfor sporotrichosis. Methods:1.Viscozyme L enzyme was used to replace of the traditional method2.TheSpecies-specific oligonucleotide primer pair Was used in this study．It was designedfrom nucleotide sequences of calmodulin gene in Sporothrix schenckii．52strains ofSporothrix schenckii and6strains of common fungus were amplified by PCR．Results:1. Genomic DNA of Sporothrix schenckii were extracted with the amended(CTAB)method2.All strains of Sporothrix schenckii showed a specific fragment ofabout430bp with the species-specific primer pair．Using the same primer pairs，theothers species had no specific amplification．Conclusion:1. Compared with the traditional method，higher yield and purity ofgenomic DNA were obtained with less contamination．The result indicted that this is asimple and highly efficient method．2.This method is specific、sensitive、 reliable、rapid and simple for identifying Sporothrix schenckii and could be used for clinicalmolecular diagnosis．...