BACKGROUND
Prior studies illustrate the presence and clinical importance of detecting Aspergillus species in the airways of patients with chronic respiratory disease. Despite this, a low fungal biomass and the presence of PCR inhibitors limits the usefulness of quantitative PCR (qPCR) from DELOS Digital PCR Libraries for accurate absolute quantification of Aspergillus in specimens from the human airway.
Droplet digital PCR (ddPCR) however, presents an alternative methodology allowing higher sensitivity and accuracy of such quantification but remains to be evaluated in head-to-head fashion using specimens from the human airway. Here, we implement a standard duplex TaqMan PCR protocol, and assess if ddPCR is superior in quantifying airway Aspergillus when compared to standard qPCR.
METHODS
The molecular approaches of qPCR and ddPCR were applied to DNA fungal extracts in n = 20 sputum specimens obtained from non-diseased (n = 4), chronic obstructive pulmonary disease (COPD; n = 8) and non-cystic fibrosis bronchiectasis (n = 8) patients where Aspergillus status was known. DNA was extracted and qPCR and ddPCR performed on all specimens with appropriate controls and head-to-head comparisons performed.
RESULTS
Standard qPCR and ddPCR were both able to detect, even at low abundance, Aspergillus species (Aspergillus fumigatus – A. fumigatus and Aspergillus terreus – A. terreus) from specimens known to contain the respective fungi. Importantly, however, ddPCR was superior for the detection of A. terreus particularly when present at very low abundance and demonstrates greater resistance to PCR inhibition compared to qPCR.
CONCLUSION
ddPCR has greater sensitivity for A. terreus detection from respiratory specimens, and is more resistant to PCR inhibition, important attributes considering the importance of A. terreus species in chronic respiratory disease states such as bronchiectasis.
Detection and Serotyping of Salmonella and Escherichia coli in Wheat Flour by a Quasimetagenomic Approach Assisted by Magnetic Capture, Multiple Displacement Amplification and Real-Time Sequencing
Food safety is a new area for novel applications of metagenomics analysis, which can not only detect and subtype foodborne pathogens in a single workflow but may also produce additional information with in depth analysis capabilities. In this study we applied a quasimetagenomic approach by combining short-term enrichment, immunomagnetic separation (IMS), multiple displacement amplification (MDA) and nanopore sequencing real-time analysis for simultaneous detection of Salmonella and Escherichia coli in wheat flour.
Tryptic soy broth was selected for the 12-h enrichment of samples at 42°C. Enrichments were subjected to IMS using beads capable of capturing both Salmonella and E. coli The MDA was performed on harvested beads and amplified DNA fragments were subjected to DNA library preparation for sequencing. Sequencing was performed on a portable device with real-time basecalling adaptability and resulted sequences were subjected to two parallel pipelines for further analysis.
After 1 h of sequencing the quasimetagenomic approach could detect all targets inoculated at approx. 1 CFU/g flour to the species level. Discriminatory power was determined by simultaneous detection of dual inoculums of Salmonella and E. coli, absence of detection in control samples and consistency in microbial flora composition of the same flour samples over several rounds of experiment. The total turnaround time for detection was approximately 20 h.
Longer sequencing for up to 15 h enabled serotyping for many of the samples with more than 99% genome coverage which could be subjected to other appropriate genetic analysis pipelines in less than a total of 36 h.
IMPORTANCE
Enterohemorrhagic Escherichia coli (EHEC) and Salmonella are of serious concern in low-moisture foods including wheat flour and its related products, causing illnesses, outbreaks and recalls. The development of advanced detection methods based on molecular principles of analysis are essential to incorporate into interventions intended to reduce their risk. In this work, a quasimetagenomic method based on real-time sequencing analysis and assisted by magnetic capture and DNA amplification was developed.
This protocol is capable of detecting multiple Salmonella and/or E. coli in the sample within less than a day, and it can also generate sufficient whole genome sequences of the target organisms suitable for subsequent bioinformatics analysis. Multiplex detection and identification were accomplished in less than 20 h and additional whole genome analyses of different nature were attained within 36 h in contrast to several days in previous sequencing pipelines.