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Chapter 12.2 : Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens

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Abstract:

The detection of pathogenic microorganisms directly in clinical specimens by molecular methods has been investigated extensively using a variety of nucleic acid probe hybridization, target amplification, and signal-generating formats. Commercial product development has focused on direct diagnosis of blood-borne and sexually transmitted diseases and respiratory pathogens using solid-and solution-phase hybridization with nonisotopic nucleic acid probes and several different target amplification methods, including conventional PCR, strand displacement amplification, transcription-mediated amplification, and, more recently, real-time PCR. Many laboratories are developing their own “home brew” assays for pathogen detection using real-time PCR for organisms not addressed by commercial kits (Table 12.1-4).

Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
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Figure 12.2.3-1

GeneXpert cartridge. S, sample; 1, reagent 1; 2, reagent 2.

Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
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Melting peaks corresponding to and

Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
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References

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Tables

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Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
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Untitled

Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
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Table 12.2.3-1

Commercially available, FDA-cleared and tests

This table contains examples of FDA-cleared or -approved nucleic acid amplification tests that are commercially available in the United States. It is not intended to be all-inclusive. Websites of the manufacturers are useful sources of the most up-to-date information.

FCU, first-catch urine.

SDA, strand displacement amplification.

Inhibition control is optional with manual assay performance but cannot be run on Viper platform.

ThinPrep PreservCyt Liquid PAP medium for collection of cervical cells.

Roche PCR is not cleared for testing on female urine.

Clearance granted to Cytyc Corporation, Boxborough, MA, not Roche Molecular Systems, Inc.

Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
Generic image for table
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Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
Generic image for table
Untitled

Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
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Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
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Table 12.2.3-2

Commercially available, FDA-cleared HIV-1 quantitative tests

This table contains examples of FDA-cleared or -approved nucleic acid tests that are commercially available in the United States. It is not intended to be all-inclusive and does not contain tests used for screening of blood products. Websites of the manufacturer are useful sources of the most up-to-date information. Abbreviations: NASBA, nucleic acid sequence-based amplification; bDNA, branched-chain DNA; PPT, plasma preparation tube.

Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
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Untitled

Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
Generic image for table
Untitled

Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
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Table 12.2.3-3

PCR primers and probes for the detection of HSV-1 and HSV-2

6-FAM and QUASAR 670 represent the 5' reporter fluorophores for the HSV-1 and the HSV-2 probes, respectively. BHQ-1 and BHQ-2 are the quencher molecules for the HSV-1 and the HSV-2 probes, respectively. These reporter and quencher molecules are covalently linked to the oligonucleotides.

Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
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Table 12.2.3-4

Primer-probe mix formulations

IPC, internal positive control; Rx, reaction.

Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
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Table 12.2.3-5

PCR primers and probe for detection of in respiratory specimens

Label probe 5′ end with biotin.

Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
Generic image for table
Untitled

Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
Generic image for table
Untitled

Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
Generic image for table
Untitled

Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
Generic image for table
Untitled

Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
Generic image for table
Untitled

Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
Generic image for table
Untitled

Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
Generic image for table
Untitled

Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
Generic image for table
Untitled

Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
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Table 12.2.3-6

Thermal cycler parameters for real-time PCR

Cont., continuous.

Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
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Table 12.2.3-7

PCR primers and probe for quantitation of human CMV

6-FAM and TAMRA represent the 5′ reporter fluorophore and the 3′ quencher, respectively. These molecules are covalently linked to the oligonucleotide.

Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2
Generic image for table
Table 12.2.3-8

Primer-probe mix formulation

IPC, internal positive control; Rx, reaction.

Citation: Garcia L. 2010. Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens, p 275-365. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.2

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