Experimental Study On Establishment And Evaluation Of AllGlo Fluorescence PCR Diagnostic Techniques For Invasive Aspergillosis In Hematologic Malignancies Patients

Objective: The aim of this study was to develop and evaluate a AllGlo fluorescence PCR diagnostic techniques for Invasive Aspergillosis (IA) in hematologic malignancies patients with designing primers and probes. We expect to provide a rapid and accurate AllGlo fluorescent PCR assay for the clinical early diagnosis of IA.Methods:⑴According to the GenBank database, we used the Blast and DNAMan tools to search conservative sequence of Aspergillus. One pair of AllGlo fluorescent PCR primers and fluorescent probes were designed with Primer Express2.0 software. The standards were established and used to establish standard curves which were evaluated later.⑵we respectively used Lyticase enzyme digestion method,glass beads salting-out method and QIAamp–S method to extract of fungal DNA in simulated blood samples of fungal bacteremia, and analyzed the extraction efficiency of the three methods.⑶we extracted the fungal DNA in the blood samples collected, then detected them with AllGlo fluorescence PCR assay in a single-blind manner so as to statistically evaluate the value of their diagnosis.Results:⑴The AllGlo fluorescence PCR standard curve was completed Y∧=- 3.003Χ+36.825,R2=0.981(A.flavus); Y∧= - 3.052Χ+38.016,R2=0.994 (A.fumigatus),which were able to detect at least 10 CFU/ml,and had high specificityand reproducibility.⑵The comparison of A260/A280 had no statistical significance(P>0.05).The extraction efficiency of concentrations had some statistical significance(P<0.001).⑶Compared to the AllGlo fluorescence PCR,the statistic data for GM assay indicated a less positive predictive value and a negative predictive value,There is no statistical significance between them on sensitivity(P>0.05),AllGlo fluorescence PCR assay was superior to GM assay on specificity(P<0.05).Compared to the conventional blood culture,the statistic data for the AllGlo fluorescence PCR assay indicated higher sensitivity.Conclusion:The AllGlo fluorescence PCR diagnostic techniques for invasive aspergillosis in hematologic malignancies patients was successfully established,which had high sensitivity,specificity and reproducibility,which show many advantages than GM assay and blood culture.