Molecular Biological Identification On The Pathogenic Geotrichum

Objective: The species of pathogentic Geotrichum have Geotrichum Silvicola and Geotrichum candidum and so on. a new strain in Geotrichum, was firstly isolated from Brazil fly and Indian oak silkworm. In 2002, we isolated a strain from a child's kerion-like lesions, which firstly confirmed that Geotrichum silvicola can infect human. Then it was isolated from blood of the patient suffered from toxic epidermal necrolysis in our department after three years.In 2006, the Geotrichum Silvicola was isolated from a case of foot ulcer by HUANG Wen Ming ,which demonstrated that this strain can cause systemic infection. Geotrichum candidum is a typical strain of Geotrichum, which can cause systemic infections such as respiratory system, alimentary system, recirculating system, ear, article and skin. But the bronchus to be infected at mostly. In 2007, a case of invasive cutaneous infected with Geotrichum candidum was reported by Sfakianakis A. And a case of congenital immunodeficiency disease accompaniting the infection of candida albicoms and Geotrichum candidum was reported by ZHONG Hua in the same year. In 2008, a case of vaginitis infected by Geotrichum candidum was reported by LI Xiu Li at the first time in china. Geotrichum silvicola was intimate studied about morphology, and physiology, Pathogenicity,Animal experiment. but nowadays, Geotrichum candidum was studied about exploitation and utilization of its tropina ,there are fewer researches on its pathogenicity ,and it was relied on phenotypic identification. We will study about molecular biological identification of Geotrichum Silvicola and Geotrichum candidum.In recent years ,with the development of technology , the molecular biology technique was widespreadly applied within laboratory diagnosis of fungus ,and it has been demonstrated that the technique was a effective method for the identification of fungus. In 1900,the arbitrarily primed polymerase chain reaction (AP-PCR) was established in different laboratory at the same time. It displayed enormous potential about genotype and appreciation of fungus. To study the significance of AP-PCR in identification and subtyping, two species were amplified by AP-PCR. We compared the differences between Geotrichum silvicola and Geotrichum candidum of distinct DNA band patterns in different species with nine different primers and we amplified the templates isolated from different strains of same species, and obtained the uniform DNA band patterns. AP-PCR is a rapid, simple and dependable method for species identification of Geotrichums. The results of AP-PCR could provide the guidance to approach infection sources,route of transmission of pathogenic microorganism and epidemiologic survey ,thereby it profited initial clinical diagnosis and medication.Methods:. Geotrichum candidum was identified by china Institute of Microbiology in Acdemt Science.we afforded it from here. Geotrichum Silvicola was identified by china Institute of Microbiology in Acdemt Science, it was conserved in a laboratory of Fungus Department of Derma tology. the Fourth Hospital Hebei Medical University. All the strains of Geotrichums were identified by cultivation, micromorphology,morphology and biochemical test .We isolated genomic DNAs from fungi using E.Z.N.A. Yeast DNA Kit. And the DNAs of Geotrichum Silvicola and Geotrichum candidum were amplified using the random primers(AP3,ATG,RP2,OPA-10,S034,S040,S101,S167,S368) by AP-PCR. We analyzed variation among GeotrichumsResults: we extracted the DNA of Geotrichums very succeedly. The purity and concentration of the DNAs could satisfy requisition for PCR reaction. The result of identifying Geotrichum species: Distinct DNA band patterns were observed in different species with nine different primers. The more distinct patterns were observed between species, especially using primers S040,S167,S368,AP3,OPA-10 and RP2, We amplified the same template many times, and the uniform DNA band patterns were obtained. We amplified the templates isolated from different strains of same species, and obtained the uniform DNA band patterns using primers S167,ATG,AP3,RP2 ,and obtained the distinct DNA band patterns using primers S034,S040 and S368.Conclusion: (1) the DNAs extracted by using E.Z.N.A Yeast DNA Kit could be used for PCR reaction (2) AP-PCR may be applied in the identification and subtyping of Geotrichums using AP3,ATG,RP2,OPA-10,S034,S040,S101,S167,S368 as primers. AP-PCR is a rapid, simple and dependable method for species identification of Geotrichums (3) The more distinct patterns were observed between species, especially using primers S040,S167,S368,AP3,OPA-10 and RP2.(4) Uniform DNA band patterns were observed with primer S167,ATG,AP3 and RP2 between Geotrichum silvicola isolated from the skin lesion and Geotrichum silvicola isolated from blood .Distinct DNA band patterns were observed with primer S034,S040 and S368 between Geotrichum silvicola isolated from the skin lesion and Geotrichum silvicola isolated from blood .