Rapid Identification Of Common Pathogenic Fungi Of Fungal Keratitis By PCR-RFLP

Background:With the rapid development of our society and the improvement of medical needs,fungal infections are getting more and more attention by clinicians and researchers.fungal keratitis is a common ophthalmic infection in our large agricultural country,because of lacking rapid and effective identification to pathogenic fungi and the drug spicies used in fungal keratitis is relatively less and side effects usually more,often lead to bad results;coupled with the particularity of the sick population(the majority of agricultural population)and the concealent of onset often resulted patient's own neglecence.All above reveal that we need a timely and effective treatment to idendify the common pathogenic fungi of fungal keratitis.The aim of this study was to explore a rapid and effective method for the identification of common fungal keratitis,so as to provide a basis for clinical treatment.Objective:DNA was extracted from clinical corneal scrapings which had been identified as fungal infection by microscopic morphology.We aim to establish a method concerning the technology of polymerase chain reaction-restriction fragment Length polymorphism(PCR-RFLP)for experimental research and extensive clinical applications.Methods:The universal primers were synthesized according to the mitochondrial cytochrome Coxidase subunit I(COI)gene fragment of these clinically common corneal susceptibility fungi of Fusarium and Aspergillus.The genomic DNA was extracted by using the Gen Tel TM DNA kit.After ITS1 and ITS4 were used to amplify the gene fragments,the sequences were input into Gen Bank for homologous sequence alignment.The results were compared with the results of microscopic identification.The genomic DNA was amplified by synthetic COI universal primers.Restriction endonuclease Ssp I,Vsp I and Dra I digested COI primers to amplify the target fragment,line 3% TAE agarose gel electrophoresis,ultraviolet photographic instrument to take electrophoresis pictures and analyze the comparative map,the initial detection of its specificity.Results:The results of microscopic examination of clinical corneal scrap specimens showed that Fusarium solani,Fusarium moniliforme and Fusarium oxysporum were the majority,and Aspergillus fumigatus was the second.The length of the fragment obtained by ITS1 and ITS4 was about500 bp,and the purity of the experimental strain was ensured after the sequencing.The genomic DNA of each strain was amplified by COI1(3'-ATCG-5 ')and COI2(5'-CGAT-3')primers to obtain the target fragment length of about 440 bp.The restriction endonuclease Ssp I,Dra I and Vsp I alone or in combination can be used to cut out different bands,so that differences in electrophoretic images reflect differences in the sequence of the fragments of the target fragment of COI amplification.The uniqueness of the Fusarium and Aspergillus can be identified into species.Conclusion:In the present study,16 strains of 7 strains were amplified by COI1(3'-ATCG-5 ')and COI2(5'-CGAT-3')primers to obtain about 440 bp fragment.The PCR products amplified by Ssp I,Vsp I and Dra I restriction endonuclease bands were polymorphic,suggesting that they could be used for interspecific identification of Aspergillus and Fusarium.RestrictionFragment Length Polymorphism(RFLP)method can be used to quickly and easily identify the common clinical corneal susceptible fungi to species,and the method own it's prospect.