A PCR-RFLP Method For Rapid Identification Of Fusarium Keratitis

Purpose Isolate and extract DNA from the corneal scrapings of thepresumed infectious keratisis patients. Establish a diagnostic method forcommon inflectious fungal with polymerase chain reaction-restrictionfragment length polymorphism (PCR-RFLP) method for experimentalstudies and clinical applications.Methods Synthesize the universal primers of the gene fragment from themitochondrial cytochrome C oxidase subunit I(COI) in the commonclinical pathogenic fungi, including Fusarium, Penicillium, Paecilomyces,Beauveria bassiana, red yeast. Collect clinical specimens from cornealulcers, isolate the fungal strains, then extract the DNA by the improvedGenTLETMmethod. Multiplex polymerase chain reaction(PCR) with theuniversal primers of the ITS and COI. Search and analyze thehomologous sequences of nucleic acid sequenses in the GenBankdatabase. Digest the COI gene fregment with the restriction endonucleaseSSPI and VSPI, use the2.0%TAE agarose gel for electrophoresis, takephotographs, then compare and analyze the restriction maps topreliminarily test its specificity.Results The genomic DNA were amplified by polymerased chain reaction with primer ITS1and ITS4.The fragment length of the products wereabout550bp.The PCR products were sequenced after recover andpurification. Compare the sequerence with the corresponding sequencesin GenBank to identify.The fragment length of the product amplified withprimer COI-all1and COI－all2ware about450bp. Restrictionendonuclease analysis were did after the DNA sequencing.Digest thetarget DNA fragment with the restriction enzyme SspI and VspI. Thenidentify the fungi to species with the restrition fragment map.Conclusion In this study, the15fungi of6species cound be amplified byPCR with primer COI-all1and COI－all2, the longth of the fragmentwere about450bp.The PCR products of Fusarium solani, Fusariumoxysporum and Fusarium moniliforme were digested by the restrictionendonuclease SSPI and VSPI, use the2.0%TAE agarose gel forelectrophoresis, take photographs, then compare and analyze therestriction maps to preliminarily test its specificity to thespecies.Restriction fragment length polymorphism analysis method canbe used to quickly and easily identification for the Fusarium species tospecies, there are good prospects.