Research Of Enzyme-linked Immunosorbent Assay Of Nucleic Acid Sequence-based Amplification For Detecting And Diagnosis Of Invasive Aspergillosis

ObjectiveInvasive aspergillosis(IA)is a life-threatening infection in immunocompromised patients,rapid and sensitive detection of Aspergillus from clinical samples has been a major challenge in the early diagnosis of IA.An enzyme-linked immunosorbent assay of nucleic acid sequence-based amplification(NASBA-ELISA)was developed for facilitating the clinical diagnosis of these infections in the first place.Then GM test and reverse transcription PCR(RT-PCR)were also evaluated to find out the optimal clinical laboratory diagnostic strategy of these infections.MethodsThe primers targeting 18 S rRNA were selected for the amplification of Aspergillus RNA by the isothermal digoxigenin(DIG)-labeling NASBA process.The DIG-labeled RNA amplicons were hybridized with a specific biotinylated DNA probe immobilized on streptavidin-coated microtiter plate.The hybrids were colorimetrically detected by the addition of an anti-DIG antibodies linked to ALP and substrate(disodium 4-nitrophenyl phosphate).The specificity and sensibility of this method were evaluated.Finally,blood samples from 86 patients at a high risk for IA were tested by NASBA-ELISA,RT-PCR and GM test and their diagnostic performance were evaluated,respectively.Prospective comparison of these three methods was performed to find out the optimal clinical laboratory diagnostic strategy of these infections.ResultsThe NASBA-ELISA assay was established successfully in the laboratory.this method could detect as little as 1 CFU aspergillums spore and Its specificity was fine.The absorbance value was linearly correlated with the logarithm of the spore amount(y=0.278 x + 0.119,R2 = 0.887).The sensitivity of NASBA-ELISA,GM-ELISA and RT-PCR was 80.56%,58.33%,72.22%,respectively,and the specificity was 80.00%,82.00%,84.00%.The efficiency of the three methods in various combinations was also evaluated.Combination of NASBA-ELISA and RT-PCR testing achieved perfect specificity(100%)and perfect positive predictive value(100%).The best sensitivity(97.22%)and the highest Youden index(0.652)were obtained by testing with both NASBA and RT-PCR in parallel.ConclusionsNASBA-ELISA platform for semi-quantification Aspergillus was feasible.The NASBA-ELISA method has the advantages in sensitivity,simplicity and specificity,so this assay makes it possible for the NASBA based RNA diagnosis to become a routine work in laboratories and provides a new way for the molecular epidemiologic investigation and early diagonosis of IA.