Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing

Espy, Mark J.; Uhl, James R.; Sloan, Lynne M.; Buckwalter, Seanne P.; Jones, Mary F.; Vetter, Emily A.; Yao, Joseph D.; Wengenack, Nancy L.; Rosenblatt, Jon E.; Cockerill, F. R.; Smith, T. F.

doi:10.1128/cmr.00022-06

Cited by 272 publications

(357 citation statements)

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“…Therefore, many different assays, including quantitative PCR (qPCR) (Cheng et al, 2006) and ligation assays (Sando et al, 2004), have been developed to meet these requirements. Although qPCR has shown extensive utility, as it combines extreme sensitivity with real-time detection and multiplexing capacity (Espy et al, 2006), several characteristics of this assay, among which a high cost, complexity of the assay and fluorescence used to monitor the amplification process in realtime, have limited its application towards POC diagnostics. In the last decade, numerous efforts have been made to use nanoparticles (NPs) in this purpose (Storhoff et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

Real-time ligation chain reaction for DNA quantification and identification on the FO-SPR

Knez

Spasić

Delport

et al. 2015

Biosensors and Bioelectronics

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Different assays have been developed in the past years to meet point-of-care diagnostic tests requirements for fast and sensitive quantification and identification of targets. In this paper, we developed the ligation chain reaction (LCR) assay on the Fiber Optic Surface Plasmon Resonance (FO-SPR) platform, which enabled simultaneous quantification and cycle-to-cycle identification of DNA during amplification. The newly developed assay incorporated FO-SPR DNA melting assay, previously developed by our group. This required establishment of several assay parameters, including buffer ionic strength and thermal ramping speed as these parameters both influence the ligation enzyme performance and the hybridization yield of the gold nanoparticles (Au NPs) on the FO-SPR sensor. Quantification and identification of DNA targets was achieved over a wide concentration range with a calibration curve spanning 7 orders of magnitude and LOD of 13.75 fM. Moreover, the FO-SPR LCR assay could discriminate single nucleotide polymorphism (SNPs) without any post reaction analysis, featuring thus all the essential requirements of POC tests.

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Section: Introductionmentioning

confidence: 99%

Real-time ligation chain reaction for DNA quantification and identification on the FO-SPR

Knez

Spasić

Delport

et al. 2015

Biosensors and Bioelectronics

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“…Conversely, inhibitors in clinical specimens or nucleic acid degradation can lead to false-negative results. False-negative results may also occur where the nucleic acid extraction step has failed (14,16).…”

Section: Limitations Of Molecular Amplification Methods In Diagnosticmentioning

confidence: 96%

“…The main disadvantage of SYBR green is that it will bind to any dsDNA, including nonspecific PCR products such as primer dimer, and so may rely on additional analyses, such as melting curve analysis, for assay specificity. For these reasons, sequence-specific probe chemistries are favored over intercalating dyes in diagnostic virology as they are specific to the target DNA sequence of interest, and so offer superior result resolution (15,16). The most commonly used sequence-specific oligonucleotide probe format used in diagnostic real-time PCR has been the dual-labeled TaqMan R probe.…”

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confidence: 99%

Molecular Amplification Methods in Diagnostic Virology

Whiley¹,

Sloots²

2010

Infectious Disease and Therapy

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Conventional PCR Detection MethodsPCR product detection was traditionally performed by direct visualization of the product using agarose gel electrophoresis with DNA-binding fluorescent dyes such as ethidium bromide. Gel-based methods, although laborious and dependent on subjective result interpretation, are still widely used in diagnostic virology, particularly for PCR-based sequencing and typing (10). In addition, gel-based visualization of PCR product remains an effective way for troubleshooting problems encountered using alternative detection methods. Thus, most diagnostic laboratories maintain gel-based methods to some extent.Enzyme immunoassays, including the enzyme-linked immunosorbent assay (ELISA), provided the first key advancement in PCR detection methodology. Briefly, the PCR-ELISA system used a colorimetric microtiter plate probe-based capture system whereby a 5 biotinylated oligonucleotide probe targeting a DNA sequence internal to the primers was used to capture Lennette's Laboratory Diagnosis of Viral Infections Downloaded from informahealthcare.com by Kainan University on 05/16/15For personal use only. MOLECULAR AMPLIFICATION METHODS IN DIAGNOSTIC VIROLOGY 21PCR product to streptavidin-coated wells (11). A major advantage of the PCR-ELISA over gel-based techniques is that it is objective, minimizing the potential for interpretation errors. In addition, the technology is particularly suitable for multiplex PCR reactions (discussed below) (12). PCR-ELISA was extensively used in "home-brew" assays as well as in several commercial assays targeting a variety of important viral pathogens, including human immunodeficiency virus (7) and hepatitis C virus (13). However, the PCR-ELISA technology has now been largely superseded by real-time PCR. Real-Time PCR DetectionThe advent of real-time PCR probably represents the greatest leap in DNA amplification technology since the development of PCR itself, and real-time PCR has been the key advancement in revolutionizing diagnostic virology. Briefly, real-time PCR is achieved through the use of fluorescent detection technology. Fluorescent molecules are added to a PCR reaction mix and interact with the PCR product to produce an increase in fluorescent signal when PCR amplification occurs. Monitoring of the fluorescent output is achieved through real-time PCR instrumentation, which measures the fluorescent signal during or following each thermal cycle. Thus, PCR amplification is monitored in "real-time," providing numerous advantages over conventional detection methods. From a practical perspective, real-time PCR removes the need for a separate detection step, which significantly reduces PCR result turnaround times and decreases staff hands on time. Also, the system is closed (i.e., reactions do not need to be opened for detection), reducing the potential for carryover contamination. The technology also provides an additional key performance characteristic, in that it has an extremely broad dynamic range for virus detection making it highly suitable for viral quan...

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“…Table 5 describes the application of molecular methods to specific CNS pathogens. PCR and other PCR-derived techniques, including reverse transcriptase (RT)-PCR, multiplex PCR, nested PCR, broad-range PCR, and real-time PCR have collectively revolutionized the diagnosis and monitoring of CNS infections (41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51). In addition to PCR, transcription-mediated amplification (TMA) or nucleic acid sequence-based amplification (NASBA) begins with the synthesis of a DNA molecule complementary to the target nucleic acid (usually RNA).…”

Section: Molecular Assays (Csf)mentioning

confidence: 99%

Infections of the Central Nervous System

Reznicek¹,

Bloch²,

Tang³

2010

Infectious Disease and Therapy

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INTRODUCTIONViral infections of the central nervous system (CNS) often present a diagnostic dilemma. The number of causative agents is vast, much greater than a century ago, when the established etiologies of CNS infections were mainly limited to rabies virus and polio virus. The physical findings of CNS infection are often nonspecific, and initial laboratory results may provide few additional clues as to the etiology. Molecular diagnostic testing has dramatically improved the ability to detect viral CNS infections, but requires expertise in the attributes and limitations of these techniques. Even discriminating infectious from noninfectious causes may be challenging, as metabolic, autoimmune, neoplastic, toxic, and endocrinologic entities may mimic meningitis or encephalitis. CNS infections represent syndromes where close collaboration between clinicians and laboratorians is crucial in providing a rapid diagnostic evaluation and an appropriate interpretation of results.This chapter concentrates on viral infections of the CNS in immunocompetent adults, with a focus on assessment and laboratory evaluation. The close anatomical proximity of meninges and brain parenchyma often blurs the distinction between meningitis and encephalitis, causing an overlap syndrome termed meningoencephalitis. For the purposes of this chapter, meningoencephalitis will be considered a subset of encephalitis, while meningitis will refer to isolated inflammation of the meninges. There is significant geographic variability with respect to the microbiology of CNS infections, and the following discussion will highlight the most significant viral pathogens in the United States. A brief overview of laboratory diagnostic techniques will initially be provided followed by a more detailed discussion specifically relating to the various viral pathogens.

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Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing

Cited by 272 publications

References 0 publications

Real-time ligation chain reaction for DNA quantification and identification on the FO-SPR

Real-time ligation chain reaction for DNA quantification and identification on the FO-SPR

Molecular Amplification Methods in Diagnostic Virology

Infections of the Central Nervous System

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