Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens

doi:10.1128/9781555817435.ch12.2

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

Editor: Lynne S. Garcia¹

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Affiliations: 1: LSG & Associates, Santa Monica, California

Content Type: Reference

Publication Year: 2010

Category: Clinical Microbiology

Book DOI: 10.1128/9781555817435

Chapter DOI: 10.1128/9781555817435.ch12.2

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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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Figures

Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens

/content/10.1128/9781555817435.chap12.2.fig12.2.3-1

Figure 12.2.3-1

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

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Figure 12.2.3-1

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

/content/10.1128/9781555817435.chap12.2.fig12.2.3-2

Figure 12.2.3-2

Melting peaks corresponding to B. parapertussis and B. pertussis

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Figure 12.2.3-2

Melting peaks corresponding to B. parapertussis and B. pertussis

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Tables

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Table 12.2.3-1

Commercially available, FDA-cleared Chlamydia trachomatis and Neisseria gonorrhoeae tests a

a 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.

b FCU, first-catch urine.

c SDA, strand displacement amplification.

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

e ThinPrep PreservCyt Liquid PAP medium for collection of cervical cells.

f Roche PCR is not cleared for Neisseria gonorrhoeae testing on female urine.

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

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Table 12.2.3-1

Commercially available, FDA-cleared Chlamydia trachomatis and Neisseria gonorrhoeae tests a

b FCU, first-catch urine.

c SDA, strand displacement amplification.

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

e ThinPrep PreservCyt Liquid PAP medium for collection of cervical cells.

f Roche PCR is not cleared for Neisseria gonorrhoeae testing on female urine.

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

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Table 12.2.3-2

Commercially available, FDA-cleared HIV-1 quantitative tests a

a 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.

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Table 12.2.3-2

Commercially available, FDA-cleared HIV-1 quantitative tests a

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Table 12.2.3-3

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

a 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.

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Table 12.2.3-3

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

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Table 12.2.3-4

Primer-probe mix formulations a

a IPC, internal positive control; Rx, reaction.

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Table 12.2.3-4

Primer-probe mix formulations a

a IPC, internal positive control; Rx, reaction.

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Table 12.2.3-5

PCR primers and probe for detection of Mycoplasma pneumoniae in respiratory specimens

a Label probe 5′ end with biotin.

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Table 12.2.3-5

PCR primers and probe for detection of Mycoplasma pneumoniae in respiratory specimens

a Label probe 5′ end with biotin.

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Table 12.2.3-6

Thermal cycler parameters for B. pertussis/B. parapertussis real-time PCR

a Cont., continuous.

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Table 12.2.3-6

Thermal cycler parameters for B. pertussis/B. parapertussis real-time PCR

a Cont., continuous.

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Table 12.2.3-7

PCR primers and probe for quantitation of human CMV

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

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Table 12.2.3-7

PCR primers and probe for quantitation of human CMV

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

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Table 12.2.3-8

Primer-probe mix formulation a

a IPC, internal positive control; Rx, reaction.

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Table 12.2.3-8

Primer-probe mix formulation a

a IPC, internal positive control; Rx, reaction.