A Pilot Research On The Detection Of Deep-fungi By Liquid Phase Hybridizations For PCR-ELISA

Objective To establish a rapid .sensitive and specific method to detect and identitydeep-fungi by a liquid phase hybridizations for PCR-ELISA.Methods According to the high-conserved sequence of fungal 28S rDNA gene ,we used anuniversal primers for fungal kingdom labeled with biotion at 5 ' end .After the PCR, theproduct mixed with universal probe or species-specific probe labeled with digoxin at 5'-end .hybridization was carried out in a thermocycler for 2min at 90℃and Imin at 55℃. Themixture was captured on microplate wells coated with streptavidin and was detected with aperoxiase- labeled antibody and substrate.Results 1. All 9 strains fungi can be amplified by the universal primer pair ,the productswere about 260bp,which consist with other past reports. 2. The universal probe can detect all9 fungi PCR product in our PCR-ELISA. 3. Our PCR -ELISA can detect as little as 1 fgfungal DNA, which is 32 times more sensitive than agrose gel electrophoresis. 4. Candidaalbican -specific probe, Aspergillusfomigatus-specific probe, cryptocoeous neoformans-specific probe showed high specificity in our research. 5. Our liquid phase hybridizationfor PCR-ELISA can detect multi-fungi at the same time. , 6. The repeatablity in ourPCR-ELISA is satisfactory.Conclusion Liquid hybridization phase for PCR-ELISA is a simple, sensitive ,rapid andspecific method to detect deep-fungi ,which may be very useful in clinic.