Practical Considerations for the Design of Quantitative PCR Assays

Diaco, Robert

doi:10.1016/b978-012372182-2/50009-5

Cited by 34 publications

(24 citation statements)

References 14 publications

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“…Similar results have been achieved with B. abortus assays with detections of roughly two genome copies of DNA (25). At extremely low concentrations of DNA, a failure of at least 37% of reactions is expected based on Poisson statistics and the likelihood of sampling error (14). Low-level detectability is essential for forensic applications, as well as for environmental sampling and clinical detection.…”

Section: Discussionsupporting

confidence: 69%

Real-Time PCR Assays of Single-Nucleotide Polymorphisms Defining the Major Brucella Clades

et al. 2008

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Members of the genus Brucella are known worldwide as pathogens of wildlife and livestock and are the most common organisms of zoonotic infection in humans. In general, brucellae exhibit a range of host specificity in animals that has led to the identification of at least seven Brucella species. The genomes of the various Brucella species are highly conserved, which makes the differentiation of species highly challenging. However, we found single-nucleotide polymorphisms (SNPs) in housekeeping and other genes that differentiated the seven main Brucella species or clades and thus enabled us to develop real-time PCR assays based around these SNPs. Screening of a diverse panel of 338 diverse isolates with these assays correctly identified each isolate with its previously determined Brucella clade. Six of the seven clade-specific assays detected DNA concentrations of less than 10 fg, indicating a high level of sensitivity. This SNP-based approach places samples into a phylogenetic framework, allowing reliable comparisons to be made among the lineages of clonal bacteria and providing a solid basis for genotyping. These PCR assays provide a rapid and highly sensitive method of differentiating the major Brucella groups that will be valuable for clinical and forensic applications.Brucella spp. are pathogenic bacteria that infect a wide variety of mammalian hosts worldwide, often causing reproductive failure. The genus Brucella has classically been divided into six species based on host specificity, including B. abortus (cattle and bison), B. melitensis (goats and sheep), B. suis (pigs), B. canis (dogs), B. neotomae (desert woodrat), and B. ovis (sheep) (12). Two new species have been discovered recently in marine mammals (B. cetaceae in dolphins and whales and B. pinnipediae in seals) (10). Taxonomic limits of the marine clade, however, are not fully defined, and this group may represent one to three species (8, 18). B. abortus, B. melitensis, B. suis, and B. canis are well-characterized zoonotic pathogens, annually infecting Ͼ500,000 people worldwide (26). In the United States, the first three of these species are defined as select agents due to their pathogenicity and potential use as biological weapons (11).Despite host-based segregation, Brucella spp. have proven challenging to differentiate using molecular techniques. Brucella genomes are highly conserved, with Ͼ90% homology among species based on DNA-DNA hybridization (35), identical 16S rRNA sequences among all species (15), and Ͼ90% of genes sharing Ͼ98% sequence identity (16,27). Serological methods and biochemical testing of isolates allow differentiation of species and biovars. However, PCR-based methods have been used increasingly due to their accuracy, sensitivity, and speed of identification and the ability to work with DNA as opposed to highly infectious live cultures. A wide array of genetic polymorphisms can be assayed for the differentiation of Brucella spp., including the insertion element IS711 (2, 3, 29, 31) and genes of outer membrane proteins (7,...

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Section: Discussionsupporting

confidence: 69%

Real-Time PCR Assays of Single-Nucleotide Polymorphisms Defining the Major Brucella Clades

et al. 2008

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“…We found a detection limit of 6 copies of IC DNA in 60% of the samples by PCR. Poisson statistics predict that 63% of the reactions will be positive by PCR with a single copy of DNA (12). Thus, if RNA is extracted and reverse transcribed with efficiencies of 100%, the 60% detection rate by PCR should represent 1 copy of IC DNA.…”

Section: Discussionmentioning

confidence: 99%

Highly Sensitive Assay for Detection of Enterovirus in Clinical Specimens by Reverse Transcription-PCR with an Armored RNA Internal Control

et al. 2004

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The objective of the present study was the development of a diagnostic reverse transcription (RT)-PCR for the specific detection of enterovirus (EV) RNA in clinical specimens controlled by an internal control (IC) RNA. The IC RNA contains the same primer binding sites as EV RNA but has a different probe region. The IC RNA was packaged into an MS2 phage core particle (armored) and was added to the clinical sample to allow monitoring of both extraction efficiency and RT-PCR efficiency. Serial dilutions of the IC RNA were made, and the detection limit of the RT-PCR was tested in a background of EV RNA-negative cerebrospinal fluid. The sensitivity and specificity of the RT-PCR assay were tested by using all 64 known EV serotypes, several non-EV serotypes, and two Quality Control for Molecular Diagnostics ( The human enteroviruses (EVs) are members of the family Picornaviridae, are ubiquitous, and are mainly enterically transmitted. EVs have traditionally been identified by serotype-specific antisera in a virus-neutralizing test, and 66 EV types are known to infect humans (19). The 66 EV serotypes were initially recognized and divided into five major groups: polioviruses (PV; types 1 to 3), coxsackieviruses A (CVAs; types 1 to 22 and 24), coxsackieviruses B (CVBs; types 1 to 6), echoviruses (types 1 to 7, 9, 11 to 27, and 29 to 33), and EV types 68 to 71 (17). Recent molecular analyses have proved that echovirus types 22 and 23 are genetically distinct from the members of the genus Enterovirus and have been reclassified in a separate genus, Parechovirus, in the family Picornaviridae (13,20,23).Infections with EVs cause a wide range of clinical outcomes, such as asymptomatic infections, aseptic meningitis (meningeal inflammation in the absence of a bacterial pathogen), encephalitis, paralytic poliomyelitis, and myocarditis. Although the majority of EV infections do not cause significant disease, infection can cause serious illness, especially in infants and immune-compromised patients. EV infections are the most common cause of aseptic meningitis and account for 80 to 90% of all cases of central nervous system infections for which a possible causative agent is identified (24). In the neonate, aseptic meningitis-induced complications and poor outcomes of EV infections generally occur within the first 2 days of life (1, 2). Aseptic meningitis in immune-competent adults is characterized by sudden onset of fever, but neurological abnormalities are rare, and both short-term and long-term outcomes are generally good. Encephalitis caused by EV infections is a less common but a more severe disease than aseptic meningitis (18,29,30). Immune-compromised children and adults who are infected with EV may develop chronic meningitis and encephalitis, which may last for years before becoming fatal (16).The early clinical symptoms of meningitis caused by viruses, bacteria, and fungi are quite similar and are difficult to distinguish, but the diagnosis, therapy, and outcome of disease caused by these pathogens vary considerably. A reli...

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“…Pilot studies were used to determine the conditions for linear incorporation of 32 P with the amount of input RNA (27 cycles with 0.7 g of total RNA for Sno and 0.05 g for GAPDH [glyceraldehyde-3-phosphate dehydrogenase] [ Table 1]) (17,49). One-fifth of the [ 32 P]dCTP-labeled reaction mixture was loaded on a 5% acrylamide gel, electrophoresed, exposed to a phosphor screen, and scanned for image analysis.…”

Section: Methodsmentioning

confidence: 99%

Defective T-Cell Activation Is Associated with Augmented Transforming Growth Factor β Sensitivity in Mice with Mutations in the Sno Gene

Pearson‐White

McDuffie²

2003

Molecular and Cellular Biology

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The proto-oncogene Sno has been shown to be a negative regulator of transforming growth factor beta (TGF-␤) signaling in vitro, using overexpression and artificial reporter systems. To examine Sno function in vivo, we made two targeted deletions at the Sno locus: a 5 deletion, with reduced Sno protein (hypomorph), and an exon 1 deletion removing half the protein coding sequence, in which Sno protein is undetectable in homozygotes (null). Homozygous Sno hypomorph and null mutant mice are viable without gross developmental defects. We found that Sno mRNA is constitutively expressed in normal thymocytes and splenic T cells, with increased expression 1 h following T-cell receptor ligation. Although thymocyte and splenic T-cell populations appeared normal in mutant mice, T-cell proliferation in response to activating stimuli was defective in both mutant strains. This defect could be reversed by incubation with either anti-TGF-␤ antibodies or exogenous interleukin-2 (IL-2). Together, these findings suggest that Sno-dependent suppression of TGF-␤ signaling is required for upregulation of growth factor production and normal T-cell proliferation following receptor ligation. Indeed, both IL-2 and IL-4 levels are reduced in response to anti-CD3 stimulation of mutant T cells, and transfected Sno activated an IL-2 reporter system in non-T cells. Mutant mouse embryo fibroblasts also exhibited a reduced cell proliferation rate that could be reversed by administration of anti-TGF-␤. Our data provide strong evidence that Sno is a significant negative regulator of antiproliferative TGF-␤ signaling in both T cells and other cell types in vivo.Sno and Ski are the two most closely related members of the Ski/Sno proto-oncogene family. A third family member, Dach, is very distantly related and plays a role in central nervous system, eye, and skeletal-muscle formation (9,24,26). The Sno and Ski proteins localize to the nucleus (59) and appear to function as transcriptional activators or repressors (18,43,62

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Practical Considerations for the Design of Quantitative PCR Assays

Cited by 34 publications

References 14 publications

Real-Time PCR Assays of Single-Nucleotide Polymorphisms Defining the Major Brucella Clades

Real-Time PCR Assays of Single-Nucleotide Polymorphisms Defining the Major Brucella Clades

Highly Sensitive Assay for Detection of Enterovirus in Clinical Specimens by Reverse Transcription-PCR with an Armored RNA Internal Control

Defective T-Cell Activation Is Associated with Augmented Transforming Growth Factor β Sensitivity in Mice with Mutations in the Sno Gene

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