Invasive fungal infection (IFI) is the leading cause of death in neonatal patients, yet the diagnosis of IFI remains a major challenge. At present, most IFI laboratory diagnostic methods are based on classical, but limited, methods such as fungal isolation and culture and histopathological
examination. Recently, quantitative polymerase chain reaction (qPCR) and droplet digital polymerase chain reaction (ddPCR) technology have been adopted to quantify nucleic-acid identification. In this study, we established qPCR and ddPCR assays for IFI diagnosis and quantification. qPCR and
ddPCR were carried out using identical primers and probe for the amplification of 18S rRNA. Assay results for three fungal strains were positive, whereas ten non-fungal strains had negative results, indicating 100% specificity for both ddPCR and qPCR methods. Genomic DNA of Candida albicans
was tested after a serial dilution to compare the sensitivity of the two PCR methods. The limit of detection of ddPCR was 3.2 copies/L, which was a ten-fold increase compared with that of the qPCR method (32 copies/L). Blood samples from 127 patients with high-risk factors and clinical symptoms
for IFI were collected from a NICU in Shenzhen, China, and analyzed using qPCR and ddPCR. Thirty-four blood samples from neonates had a proven or probable diagnosis of IFI, and 25 of these were positive by qPCR, whereas 30 were positive by ddPCR. Among the 93 blood samples from neonates who
had a possible IFI or no IFI, 24 were positive using qPCR, and 7 were positive using ddPCR. In conclusion, ddPCR is a rapid and accurate pan-fungal detection method and provides a promising prospect for IFI clinical screening.
Identification and Quantification of Heterogeneously-methylated DNA Fragments Using Epiallele-sensitive Droplet Digital Polymerase Chain Reaction (EAST-ddPCR)