Evaluation of a droplet digital polymerase chain reaction format for Aspergillus DNA copy number quantification in patient samples

Ref ID: 19211

Author:

J. Menotti, A. Alanio*, O. Cabaret, N. Guigue, J.-M. Costa, S. Bretagne

Author address:

Paris, Créteil, Cergy-Pontoise, FR

Full conference title:

23rd European Congress of Clinical Microbiology and
Infectious Diseases

Date: 27 April 2014

Abstract:

The use of quantitative PCR (qPCR) for diagnosing invasive aspergillosis has been hampered by the low copy numbers observed in clinical blood samples. This renders comparisons between results difficult since some samples can be negative only by chance according to Poisson’s law.
To overcome this shortcoming, we investigated the potential of droplet digital PCR (ddPCR). This is a new approach to nucleic acid detection that offers a different method for absolute quantification compared to qPCR. The goal is to isolate single molecules in droplet and to amplify them. Some droplets will contain the target molecule while others will not. Following PCR analysis and Poisson correction since there is a probability that one droplet contains more than one molecule, one generates an absolute count of the number of target molecules in the sample, without reference to standards or endogenous controls.
We first evaluated a range of different hybridization temperatures using the recent MIQE-compliant qPCR assay for Aspergillus detection (1) targeting the 18S rDNA (18SqPCR) and a serial dilution of Aspergillus fumigatus DNA (Af293 strain). Primers and probe were unchanged and the hybridization temperature (59°C) was slightly modified compared to the initial publication (1). We then tested 23 serial samples from 4 leukemic patients (median 6; range 2-8) with probable invasive aspergillosis using the Bio-Rad QX100™ Droplet Digital PCR System. DNA had been extracted from blood samples [volumes: starting (1.5 ml), elution (300 µl)] using the LightCycler SeptiFast assay for a previous study. Five µl of the remaining elution volume (stored at -80°C) were used in the 18S qPCR (tested in duplicate) and the 18S ddPCR.
As expected, the fungal load was low since the 18SqPCR positive samples had high quantification cycle (Cq>=37.6; Table 1). The ddPCR showed that the median copy number was 0.08 copies/µl (range 0-0.31).
This first ddPCR study on Aspergillus DNA detection confirmed that due to low copy number, some qPCR results (n= 6/23) can be wrongly given as negative. The reverse is also true suggesting that more droplets should be tested. This method seems of utmost interest to solve some discrepant results between different qPCR assays and to define the best blood fraction to be tested.
1- A MIQE-Compliant Real-Time PCR Assay for Aspergillus Detection. Johnson GL, et al. Plos ONE, July 2012, 7 (7) e40022.

Abstract Number: P1070

Conference Year: 2013

Link to conference website: http://registration.akm.ch/einsicht.php?XNABSTRACT_ID=162542&XNSPRACHE_ID=2&XNKONGRESS_ID=180&XNMASK

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