Research Of Nucleic Acid Sequence-based Amplification Assay For Detecting And Diagnosis Of Cryptococcal Infction

Objective : Cryptococcosis is a deep fungal infectious disease that seriously endangers human health.The clinical lack of high sensitivity,specificity and rapid and reliable diagnostic methods enables patients to treat early and improve prognosis.This study intends to establish a Nucleic acid sequence-based amplification(NASBA)technology to detect Cryptococcus,combined with Polymerase chain reaction(PCR)technology and capsular polysaccharide antigen detection method for clinical specimen detection to evaluate the clinical application of NASBA method value.Methods : A pair of primers with Cryptococcus specificity were designed to construct NASBA amplification of Cryptococcus RNA and the sensitivity and specificity of the method were evaluated.According to European Organization forResearch and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group(EORTC/MSG)criteria,positive cases of cryptococcal infection and negative cases of cryptococcalinfection were collected,and cryptococcal capsular polysaccharide antigen detection(colloidal gold method),common PCR and NASBA method established in this study were used for detection.Various performance indicators of the diagnostic method were used to evaluate the diagnostic performance of the three methods.Results : The method of NASBA detection of cryptococcus was successfully established in the laboratory with good specificity and sensitivity of 10 cfu/ml.The sensitivity of NASBA,capsular polysaccharide antigen colloidal gold and PCR were 87.5%(95% CI,66.53-96.71%),100%(95% CI,82.83-100%),and 66.67%,respectively(95% CI,44.69-The specificity was 84.59%(95% CI,80.47-99.06%),81.08%(95% CI,64.29-91.44%),and 78.37%(95% CI,61.34-81.58%).Conclusions:The use of NASBA to detect cryptococcal RNA is fast,accurate,and easy to operate.And the whole experimental process does not require special instruments,it is suitable for development in ordinary clinical laboratories,and it is expected to become a new diagnostic method for new cryptococcal infection.