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Chapter 31 : Broad-Range PCR for Detection and Identification of Bacteria

Author: Matthias Maiwald1
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Affiliations: 1: Department of Pathology and Laboratory Medicine, KK Women’s and Children’s Hospital, Singapore 229899, Singapore
Content Type: Reference
Publication Year: 2011

Category: Clinical Microbiology; Bacterial Pathogenesis

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Abstract:

The two main molecules that are suitable for bacterial broad-range PCR are the 16S rRNA gene, consisting of approximately 1,540 bp (in , 1,542 bp), and the 23S rRNA gene, consisting of approximately 2,900 bp (in , 2,904 bp). While the impact of this appears to be somewhat smaller for diagnostic broad-range PCR in cases of suspected monomicrobial infections, it can be dramatic for polymicrobial infections, microbial flora studies, or environmental studies, where certain bacterial taxa may become significantly over- or under-represented after PCR amplification. In addition, the inherent nonselective nature of broad-range PCR makes it susceptible to minute amounts of any bacterial DNA that might be encountered along the various steps of testing. The classical way to detect and identify bacteria by broad-range PCR is via visualization of PCR products by standard gel electrophoresis, followed by sequencing, preferably for both DNA strands of the products. Broad-range PCR offers two potential benefits: it lacks selectivity for particular groups of bacteria, and it can detect as well as identify culture-resistant, fastidious, damaged, and slow-growing microorganisms. Bacterial broad-range PCR has also attracted interest in transfusion medicine, in order to assess blood products for bacterial contamination. Conducting broad-range PCR analysis at a level of high analytical and clinical sensitivity is a complex task and remains one of the most difficult and challenging PCR applications. Sequence-based identification of positive diagnostic broad-range PCR products is generally advisable, even when real-time technology is used.

Citation: Maiwald M. 2011. Broad-Range PCR for Detection and Identification of Bacteria, p 491-505. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch31
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Broad-Range PCR for Detection and Identification of Bacteria
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Citation: Maiwald M. 2011. Broad-Range PCR for Detection and Identification of Bacteria, p 491-505. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch31
/content/10.1128/9781555816834.ch31.ch31fig01
FIGURE 1
(A) Schematic drawing of the bacterial 16S rRNA gene, with conserved (open bars) and variable (shaded bars) regions. The location of broad-range primers ( Table 1 ) is shown. Modified from reference , with permission from the American Society for Microbiology. (B) Conservation profile of 16S rRNA across the domain , constructed with the software package ARB ( ) and the SILVA ( ) data set. The graph shows the relative frequency ( axis) of the most common nucleotide for each position ( axis) in 16S rRNA. Courtesy of Frank Oliver Glöckner and Elmar Pruesse (Max Planck Institute for Marine Microbiology, Bremen, Germany).
10.1128/9781555816834/f0492-01_thmb.gif
10.1128/9781555816834/f0492-01.gif
Image of FIGURE 1
FIGURE 1

(A) Schematic drawing of the bacterial 16S rRNA gene, with conserved (open bars) and variable (shaded bars) regions. The location of broad-range primers ( Table 1 ) is shown. Modified from reference , with permission from the American Society for Microbiology. (B) Conservation profile of 16S rRNA across the domain , constructed with the software package ARB ( ) and the SILVA ( ) data set. The graph shows the relative frequency ( axis) of the most common nucleotide for each position ( axis) in 16S rRNA. Courtesy of Frank Oliver Glöckner and Elmar Pruesse (Max Planck Institute for Marine Microbiology, Bremen, Germany).

Citation: Maiwald M. 2011. Broad-Range PCR for Detection and Identification of Bacteria, p 491-505. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch31
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FIGURE 2
Logarithmic amplification plot of a broad-range bacterial 16S rRNA gene PCR in real-time format. This amplification pattern has arisen in a study in which blood from healthy subjects was compared with negative controls ( ). Samples were analyzed in triplicate and for 40 cycles. The value is derived from the number of cycles needed for the amplified DNA to reach the quantification cycle (threshold). ΔRn, relative fluorescence. Reprinted from reference , with permission from the American Society for Microbiology.
10.1128/9781555816834/f0497-01_thmb.gif
10.1128/9781555816834/f0497-01.gif
Image of FIGURE 2
FIGURE 2

Logarithmic amplification plot of a broad-range bacterial 16S rRNA gene PCR in real-time format. This amplification pattern has arisen in a study in which blood from healthy subjects was compared with negative controls ( ). Samples were analyzed in triplicate and for 40 cycles. The value is derived from the number of cycles needed for the amplified DNA to reach the quantification cycle (threshold). ΔRn, relative fluorescence. Reprinted from reference , with permission from the American Society for Microbiology.

Citation: Maiwald M. 2011. Broad-Range PCR for Detection and Identification of Bacteria, p 491-505. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch31
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Tables

Broad-Range PCR for Detection and Identification of Bacteria
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Citation: Maiwald M. 2011. Broad-Range PCR for Detection and Identification of Bacteria, p 491-505. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch31
/content/10.1128/9781555816834.ch31.ch31tab01
TABLE 1
Commonly used primers and probes for broad-range PCR with bacterial 16S and 23S rRNA genes
Generic image for table
TABLE 1

Commonly used primers and probes for broad-range PCR with bacterial 16S and 23S rRNA genes

Citation: Maiwald M. 2011. Broad-Range PCR for Detection and Identification of Bacteria, p 491-505. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch31
/content/10.1128/9781555816834.ch31.ch31tab02
TABLE 2
Methods to reduce bacterial background DNA in PCR reagents
Generic image for table
TABLE 2

Methods to reduce bacterial background DNA in PCR reagents

Citation: Maiwald M. 2011. Broad-Range PCR for Detection and Identification of Bacteria, p 491-505. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch31

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