Rapid Detection Of Dermatophytosis By PCR

Objective:Conventional diagnosis of dermatophytosis like potassium hydroxide (KOH)microscopy and fungal culture are frequently used, the people who takes the conventionalmethods must be capable of high professional skills to insure the accuracy of the test andthe KOH lacks the ability to make a specific diagnosis, the culture lacks the ability to makean early diagnosis. This study collectd clinical dermatophyte strains and skin specimenscoming from patients suspected of dermatophytosis, and compare the traditional methodsto the PCR. This study aims to develop an alternative method to replace the traditionaldiagnosis of dermatophytosis.Methods:In the first part, disposed all strains by the one-step method which is fast andconvenient to get DNA of strains. evaluate the specificity of universal primersCHS1(panDerm1:5’G A A G A A G A T T G T C G T T T G C A T C G T C T C3’,panDerm2:5’C T C G A G G T C AAAA G C A C G C CA G A G3’） of dermatophytesand the sensitity of the PCR used in the study through strains.The determination ofspecificity of the universal primers:80dermatophytes and50non-dermatophytes, as wellas2human were amplified by universal primers, then observed whether the specificpositive band appeared. The determination of sensitivity of PCR: in order to determine thethreshold of detection of the Derm PCR, serial dilutions of genomic DNA of dermatophytewere tested,,all the diluated DNAwere amplified,，repeated3times in PCR procedure.In the second part, the collected119skin specimens were divided into three portionsequally, and then detect by microscopy、culture and PCR, the two steps DNA extractionmethod from clinical specimens constructed by Brillowska-Dabrowsk firstly was taken inthe research. To prevent the PCR inhibitor, we designed an internal control which wasspecific plasmid obtained from Escherichia coli, the specific plasmid could be amplified byCHS1and showed510bp band, the510bp gene segment also ontained from Escherichiacoli DNA which was amplified by primes we designed, the primers were: For:5’ G AA GAA G AT T G T C G T T T G C AT C G T C T C AG G G T T T G AT T C T G G C T CAG-3，Rev:5’C T C GAG G T C AAAAG C AC G C C AGA G G TAT TAC C G C G G C G G C T G G-3. The result of electrophoresis can tell whether the specimen wasinfected or not. If it showed the specific366bp，it was diagnose as dermatophte infection,if not and the internal control band510bp appear, then it is not infected by dermatophyte,if neither of the366bp nor510bp show, then the result is false negative. Comparing theaggrement between the nested PCR and traditional diagnosisResults:In the first part, all the dermatophyte strains yielded the same band(366bp),non-dermatophyte strains and human DNA yielded no band. Diluated dermatophyte DNAsamples ranging from600ng to6pg yielded the366bp band except the600fg.In the second part, in the statistical analysis of119clinical specimens, the Kappavalue was0.832, P<0.01when comparing the PCR and traditional diagnosis which provedthe high aggrement between the two diagnosises. What’s more, the PCR method can befinished in five hours from colleting specimen to the result outcome.Conclusion:The specificity of dermatophyte primer is high, the sensitivity of PCR in the study is6pg. The PCR which was faster and more convenient can be an alternative method fordermatophytosis diagnosis.