Application Of Sequencing Analysis Of The Internal Transcribed Spacer Regions For Identification Of Yeast-like Fungi

Object:The aim of this study is to establish an internal transcribed spacer (ITS) sequencing method suitable for the specifical clinical lab and to evaluate this method in comparison with the traditional identification method of fungi for clinical practice. The application prospect of ITS as an direct method for clinical use has also been discussed in this study.Methods:In this study, a pair of general primer was designed and tested with several standard fungi strains. The length of the target segment was supposed to within range of 400 to 600, the variety of the sequence can be used to identify different isolations of yeast-like-fungi.135 positive strains were isolates from the clinical specimen and were tested using spectrometry, API system and Candida color culture medium. The accuracy and sensitivity of the three methods were evaluated in this study taking spectrometry as a gold standard.To evaluate this ITS method,20 specimen tested positive by traditional clinical identification method were tested directly.Results:The subjects of this study were identified through the ITS sequence alignments and the results were agreed with the clinical identification in perfect concordance. This method can also be used to identify the uncultured clinical specimen to the species level which is comparable to clinical method.All the standard fungi strains are identified successfully by the ITS method. From all the 135 positive strains, the results of ITS was in accordance with spectrometry, API system and Candida color culture medium respectively in 98.5%(133/135),78.5%(106/135) and 59.3%(80/135), the differences were statistical significant by x2 analysis(x 2=51.996, P<0.05). From all the 135 strains, one strain was identified Exophiala Dermatitides by ITS sequencing which has not been able identified by MS method. The ITS method has also identified pathogenic yeast fungi from the 20 original specimen in concordance with other methods.Conclusion:The ITS sequencing method can be used to identify the yeast-like fungi more accurately. This method can also be applied to identify the uncultured clinical specimen in a rapid way.