Use of the human albumin gene as an internal control for multiplex real-time PCR in bone marrow transplant patient routine screening
Abstract number: 1734_96
McCulloch E., Harvey-Wood K., Jones B., Ramage G., Williams C.
Objectives: Real-time PCR methods to monitor bone marrow transplant patients for viral pathogens are established as part of recognized screening protocols. Real-time PCR for fungal pathogens are being developed. Real-time PCR assays are used because of their high negative predictive value. However to provide confidence in the negative results, the PCR assay must be closely controlled. The use of an albumin gene, found in blood of patients, even those who are profoundly neutropenic, has the advantage over spiking samples with foreign DNA, as it provides a marker that will confirm both success of the extraction process and the PCR reaction. This is particularly important in fungal PCR due to the increased processing of whole blood samples, which require pre-treatment with buffers that remove various components of the blood and disrupt cells prior to automated extraction of DNA.
When using any internal control it is important that this is part of a multiplex reaction rather than separate single amplifications, which provide opportunity for error.
Methods: In our Aspergillus real-time PCR assay, DNA is extracted using the EZ1 BioRobot (Qiagen) and a tissue extraction kit, then amplified and detected using an ABI Prism 7000 sequence detector and Taqman probes (Applied Biosystems). Each sample is performed in duplicate using a duplex reaction with which simultaneously amplifies the Aspergillus target and albumin gene allowing detection of the internal control and the DNA of interest in a single well.
Results: Our results show that the real-time PCR reaction for albumin and Aspergillus can be successfully multiplexed without decreasing the sensitivity of the amplification. This allows Aspergillus PCR results to be reported with confidence as true negatives or true positives. Samples where negativity is present with a decreased detection of albumin suggests PCR inhibition or ineffective extraction of the sample. Conversely positive Aspergillus with a negative albumin indicates fungal contamination in the assay, in both these cases the samples could be repeated.
Conclusion: The use of human albumin gene in a multiplex real-time PCR limits the margin for error and provides greater confidence in the negative results obtained. This gene could be used in many other real-time PCR assays.
|Session name:||European Society of Clinical Microbiology and Infectious Diseases|
|Location:||ICC, Munich, Germany|
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