Research Of Metallproteinase 1 In The Identification Of Dermatophyte

Objective:To explore the sensitivity of universal primers ITS and metalloproteinase 1 in the amplification of dermatophytes,saccharomycodes,and molds;and the sensitivity and specificity of metalloproteinase 1 combining with restriction enzyme digestion in the identification of dermatophytes species.The study provides a theoretical basis for clinical diagnosis and treatment.Methods:1.To collect 125 samples from suspected tinea manus,tinea pedis,tinea corporis and onychomycosis diagnosed by The Second Hospital of Shanxi Medical University from November 2016 to May 2017.Record the information of the samples,steriliz with 75%alcohol,scrape off the dander between the affected area and the normal skin with a blunt knife,divide the sample into two parts.One is observed under a light microscope with10% KOH,and the other is inoculated.The culture of Sabouraud'sagar containing chloramphenicol was incubated at 25°C.After two,and four weeks,the culture were observed and recorded.2.Count the number of positive cases of microscopic examination,culture positive cases and record the results.3.DNA of culture-positive strains was extracted with conventional benzyl chloride methods.Then ITS4,ITS86,and metalloproteinase 1 specific primers were used to amplify and analyze the results.4.The product of metalloproteinase 1 PCR was digested with Hinf I restriction enzyme,and the strains were identified according to the fragment size after enzyme digestion.Results:1.125 cases were collected,of which 62 cases were diagnosed with fungal infections.The positive rate of microscopic examination in clinical specimens was 31 cases(35.2%),57 cases were culture positive strains(64.7%).Among them,dermatophytes is 45 cases,12 cases were belonged to saccharomycodes and 5 cases were molds.Dermatophytes were identified by restriction endonuclease digestion,and Trichophyton rubrum was detected in 40 cases and 5 cases were Microsporum canis.2.The colonies of clinical strains on the culture medium are various in morphology and can be roughly divided into three types: Saccharomyces are soft,smooth,and cheese-like;the dermatophytes are white villous,wooly,and powdery,and some colonies have base different pigmentation;Molds colony morphology was loose,dry,opaque,cotton wool,mesh and other colored colonies;3.After amplification of ITS4 and ITS86 universal primers,approximately 300-400 bp fragments appeared in the positive samples,but the fragment sizes did not differ greatly and the species could not be specifically identified.Universal primers have universal applicability for amplification of different genera.4.The colony morphology of the metalloproteinase 1 gene-specific amplification,of which fragments approximately 700 bp,appeared to similar to the dermatophytes genus.The morphology of the colonies was similar to that of saccharomycodes and molds did not show obvious target bands;After the use of Hinf I enzyme,Trichophyton rubrum can be digest into 133 bp,255bp,358bp;Microsporum canis were divided into approximately277 bp,447bp.Conclusion:1.We used microscopy and culture for clinical diagnosis.The microscopic examination method was convenient and rapid,but the positive rate was lower.The positive rate of ulture methods was higher than that of microscopic examination.Compared with the literature,the positive rate was lower.This method can identify genus,but it cannot to species,still have a certain clinical diagnostic value;2.Trichophyton rubrum is still the most common and the most common pathogenic bacteria in clinical superficial fungal diseases in our department;3.Universal primers have universal applicability,and are sensitive to common dermatophytes,yeasts,and molds.However,specific identification of strains requires combination with other methods.4.The study is the first using of metalloproteinase 1 in clinical common skin dermatophytes identification,it has a higher sensitivity,combining with specific restriction enzyme digestion,The diagnosis of dermatophytes infection is sensitivity and this method is higher value and can be used as a supplementary diagnostic method for traditional methods.