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Chapter 29 : PCR

Authors: P. Lewis White1, Rosemary A. Barnes2
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Affiliations: 1: NPHS Microbiology Cardiff, University Hospital of Wales, Cardiff CF14 4XN, United Kingdom; 2: Dept. of Medical Microbiology, Cardiff University, University Hospital of Wales, Cardiff CF14 4XN, United Kingdom
Content Type: Monograph
Publication Year: 2009

Category: Clinical Microbiology; Fungi and Fungal Pathogenesis

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Abstract:

Combining molecular techniques with variations in specimen type, DNA extraction technique, PCR amplification, and minimal evidence for real-time result interpretation highlights the amount of research that still needs to be performed. The successful application of diagnostic PCR has been reported since the early 1990s. By the end of 2005 over 150 papers describing the use of clinical PCR methods had been published, but despite this no consensus PCR method had been agreed upon. An efficient DNA extraction procedure is critical for any molecular diagnostic test. In a recent study comparing the performance of PCR, galactomannan (GM), enzyme-linked immunosorbent assay (ELISA), and β-D-glucan in 27 pediatric or neonate patients with probable or possible invasive aspergillosis (IA), agreement between PCR and both GM ELISA and β-D-glucan assays was excellent and PCR generated a high negative predictive value (97%). PCR out-performed GM ELISA when testing serum from eight mice with histologically proven IA, generating sensitivities of 71 and 43%, respectively. Discovering the optimal fraction is important for standardization of the PCR, and the use of an animal model would be an ideal way to investigate this issue. As species are ubiquitous, controlling the contamination factor becomes a major issue in developing a suitable area to perform PCR. As with all diagnostic PCR systems, universal precautions to prevent contamination should be in place. Immunocompromised patients with PCR-positive BAL specimens should be considered at risk for IA, and the result may be an indication for preemptive antifungal therapy.

Citation: White P, Barnes R. 2009. PCR, p 373-388. In Latgé J, Steinbach W (ed), and Aspergillosis. ASM Press, Washington, DC. doi: 10.1128/9781555815523.ch29

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Real-Time PCR
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Nested PCR
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Enzyme-Linked Immunosorbent Assay
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Magnetic Resonance Imaging
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Aspergillus PCR
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Citation: White P, Barnes R. 2009. PCR, p 373-388. In Latgé J, Steinbach W (ed), and Aspergillosis. ASM Press, Washington, DC. doi: 10.1128/9781555815523.ch29
/content/10.1128/9781555815523.ch29.ch29fig01
Figure 1.
The number of articles describing PCR per annum (1993 to 2006).
10.1128/9781555815523/f0374-01_thmb.gif
10.1128/9781555815523/f0374-01.gif
Image of Figure 1.
Figure 1.

The number of articles describing PCR per annum (1993 to 2006).

Citation: White P, Barnes R. 2009. PCR, p 373-388. In Latgé J, Steinbach W (ed), and Aspergillosis. ASM Press, Washington, DC. doi: 10.1128/9781555815523.ch29
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Figure 2.
Performance of PCR using whole blood versus serum.
10.1128/9781555815523/f0379-01_thmb.gif
10.1128/9781555815523/f0379-01.gif
Image of Figure 2.
Figure 2.

Performance of PCR using whole blood versus serum.

Citation: White P, Barnes R. 2009. PCR, p 373-388. In Latgé J, Steinbach W (ed), and Aspergillosis. ASM Press, Washington, DC. doi: 10.1128/9781555815523.ch29
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Tables

Aspergillus PCR
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Citation: White P, Barnes R. 2009. PCR, p 373-388. In Latgé J, Steinbach W (ed), and Aspergillosis. ASM Press, Washington, DC. doi: 10.1128/9781555815523.ch29
/content/10.1128/9781555815523.ch29.ch29tab01
Table 1.
Summary of publications comparing nucleic acid extraction methods
Generic image for table
Table 1.

Summary of publications comparing nucleic acid extraction methods

Citation: White P, Barnes R. 2009. PCR, p 373-388. In Latgé J, Steinbach W (ed), and Aspergillosis. ASM Press, Washington, DC. doi: 10.1128/9781555815523.ch29
/content/10.1128/9781555815523.ch29.ch29tab02
Table 2.
Published PCR methods principally testing BAL specimens
Generic image for table
Table 2.

Published PCR methods principally testing BAL specimens

Citation: White P, Barnes R. 2009. PCR, p 373-388. In Latgé J, Steinbach W (ed), and Aspergillosis. ASM Press, Washington, DC. doi: 10.1128/9781555815523.ch29
/content/10.1128/9781555815523.ch29.ch29tab03
Table 3.
PCR performance when testing serum specimens
Generic image for table
Table 3.

PCR performance when testing serum specimens

Citation: White P, Barnes R. 2009. PCR, p 373-388. In Latgé J, Steinbach W (ed), and Aspergillosis. ASM Press, Washington, DC. doi: 10.1128/9781555815523.ch29
/content/10.1128/9781555815523.ch29.ch29tab04
Table 4.
PCR performance when testing whole blood specimens
Generic image for table
Table 4.

PCR performance when testing whole blood specimens

Citation: White P, Barnes R. 2009. PCR, p 373-388. In Latgé J, Steinbach W (ed), and Aspergillosis. ASM Press, Washington, DC. doi: 10.1128/9781555815523.ch29
/content/10.1128/9781555815523.ch29.ch29tab05
Table 5.
Molecular detection of cerebral aspergillosis, endophthalmic aspergillosis, sinusitis, and -infected tissue specimens
Generic image for table
Table 5.

Molecular detection of cerebral aspergillosis, endophthalmic aspergillosis, sinusitis, and -infected tissue specimens

Citation: White P, Barnes R. 2009. PCR, p 373-388. In Latgé J, Steinbach W (ed), and Aspergillosis. ASM Press, Washington, DC. doi: 10.1128/9781555815523.ch29

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