A Study On The Vitality Of Crypotococcus Neoformans Through Detecting RRNA With The Method Of RT-PCR

Cryptococcus neoformans is an important fungal pathogen that causes life-threateningmeningitis which needs long time treatment and is of high rates of relapse. In the treatmentof cryptococcal meningitis and other systemic infections, an issue that perplexed theclinicians is that no accurate and efficatious judgements in regard to the vitality ofcryptococcus neoformans can be made so that an objective benchmark of time can beprovided for treatment. rRNA and rRNA genes test can help to judge the vitality ofmicrobe, with noted specificity and sensitiveness to microbes that are alive but notculturable.The aim of this study is to discover the specific rRNA that can judge the vitality ofcryptococcus neoformans through detecting rRNA and rRNA genes clips of alive and deadcryptococcus neoformans using RT-PCR method, which may contribute to follow-upstudies.To begin with, culture some standarded strains of Cryptococcus neoformans B3501taken from the fungal laboratory (professional central laboratory of Cryptococcusneoformans) of the dermatology deparment of Chang Zheng hospital affiliated to theSecond Military Medical University with Sabourand’s Agar solid medium for three days.After that， an integral-loop-thallus from the Sabourand’s Agar solid medium wassubcultivated into YEPD liquid medium and then stirred to be a mixture. Keep the mixturein the200r-per-minute vibration incubator for one night so that fungus theoreticalconcentration should be106-107cfu/ml. Take100ml of concentration into flask A, which isdefined as contral group（the alive fungus group）. Take the rest into flask B, which isdefined as group B (dead fungus group), and heat to121℃in an autoclave for15min tillthe fungus reaches the state of inactivation. Extract Cryptococcus neoformans from thecontrol and experimental groups using centrifuge(5000rpm), then add Trizol into thecentrifuge tube, keep the mixture in room temperature and homogenate them for at least3minutes with electronic grinders to ensure the total fracture of thallus capsule, which canimprove the extraction efficiency. Use kit to do the reverse transcription in order to synthetize cDNA template and then use cDNA as the template to conduct real-time PCRexperiment. In the end, analyse the data by SPSS18.0, and the result is performed inmean±standarded deviation（x±s）. Using independent-samples test between two groups,if P is below0.05, then the contrast is significant differences, if P is below0.01, then thecontrast is more significant.After testing four small subunits-5s，5.8s，18s，28s, we found the expression level isdifferent both in the dead thallus group and the alive thallus group. The CT value indescending order is18s，5.8s，28s，5s respectively, all of statistical significance（P＜0.001）. Take the5s subunits, the least significant in CT difference value, as internalparameters and transform the Ct value of all four subunits into△Ct value. The resultshow that the difference gap in expression of dead and alive thallus of18s and5.8ssubunits are45times and14times respectively.In conclusion, we can make a pre-judgement of the vitality of Cryptococcusneoformans by testing four subunits in RT-PCR, especially the18s and5.8s subunits.