Molecular And Early Diagnostic Methods Based On Nucleic Acid Isothermal Amplification Techniques Of Candida Krusei

As we all know,Candida is the most common opportunistic pathogens in clinical practices,Such as Candida albicans,Candida tropcalis,Candida parapsilosis,Candida glabrata,Candida krusei and so on.It has been reported that non-albicans infections has on the rise in recent years,whereas infections caused by C.albicans was the most common in the past.Candida krusei can cause infections of mucosal,blood and solid organs of those patients especially with depressed immunity.The incidence of C.krusei infection has increased in recent years and cause high mortality rate with application of fluconazole to prevent fungal infection,development of organ transplantation and prescription of broad-spectrum antibiotics in clinical trails.At present,diagnostic methods of pathogenic fungi in clinical are as followings: morphological observation,biochemical metabolic identification methods,detection of serum antigens and so on.And culture result is the “Gold standard” of confirmation of fungal infections.But the consumption of long time and high rate of false positive and false negative has restrict the use of culture method in clinical.Thus we need an early,fast and specific method to identify pathogenic fungus to save patients’ lives.Nowadays molecular diagnosis techniques based on nucleic acid and protein has been put into use in clinical with the development of theory and technology of molecular biology.These methods overcome the shortcomings of traditional methods with advantages of needing less time,high specificity and sensitivity rate,and thus these methods can save lives and improve prognosis of patients in certain degree.Jacobsen MD and his colleagues found 6 housekeeping genes of Candida krusei by using multilocus sequence typing(MLST)method,namely ADE2,HIS3,LEU2,LYS2,NMT1,TRP1.MLST method has high specificity to differentiate different pathogens.In our study,we choose ADE2 as the target gene fragment by analyzing the size and the length of amplicons of these 6 gene fragments.A total of 47 clinical and reference strains(13 C.krusei,2 Issatchenkia orientalis,24 other clinical commonly yeast strains and 8 clinical commonly filamentous trains)are including in this study.Loop-mediated isothermal amplification(LAMP)and Real-time quantitative PCR(q PCR)are used to amplify and detect related DNA of different strains.And we compare the specificity and sensitivity of this two methods in diagnose infection of C.krusei.In this study,specific primers targeting ADE2 gene sequence are designed for LAMP and q PCR to amplify DNA of C.krusei.We find that LAMP and q PCR can both distinguish C.krusei correctly with 100% specificity rate,the sensitivity rate of LAMP and q PCR are 5×103 and 5×10~4 separately.In conclusion,this study provide a clue in early diagnosis of C.krusei using molecular techniques.