Nucleic acids are the ultimate biomarker and real-time PCR (qPCR) is firmly established as the method of choice for nucleic acid detection. Together, they allow the accurate, sensitive and specific identification of pathogens, and the use of qPCR has become routine in diagnostic laboratories. The reliability of qPCR-based assays relies on a combination of optimal sample selection, assay design ...

Real time RT-PCR is the most sensitive method for quantitation of gene expression levels. The accuracy can be dependent on the mathematical model on which the quantitative methods are based. The generally accepted mathematical model assumes that amplification efficiencies are equal at the exponential phase of the reactions for the same amplicon. However, no methods are available to test the ass...

A quantitative TaqMn Real-Time PCR assay was developed and its diagnostic value on human serum and livestock samples were evaluated. Brucella species could be distributed through communities as a biological agent. Rapid detection of biological threat agents is critical for timely therapeutic administration. Quantitative real-time PCR provides a rapid, sensitive and specific tool for molecular i...

We describe here a quantitative real-time PCR assay for the detection of single-base-pair differences that does not require fluorescently labeled gene-specific probes or complicated primer combinations. Following PCR or RT-PCR of a gene segment that may contain allele-specific differences, 100 pg amplified product are used for a real-time PCR with allele-specific primers and SYBR Green. The use...

Extensive detail on the application of the real-time quantitative polymerase chain reaction (QPCR) for the analysis of gene expression is provided in this unit. The protocols are designed for high-throughput, 384-well-format instruments, such as the Applied Biosystems 7900HT, but may be modified to suit any real-time PCR instrument. QPCR primer and probe design and validation are discussed, and...

Basically, diagnosis inherited diseases in gestation is very important. But amplication of the progress in early stage of pregnance is the serious and great aim for rapid application of risk assesment for both and fetus. Fetal genetic tissues collected through techniques such as amniocentesis and chorionic villus sampling (CVS) for prenatal diagnosis of fetal genetic diseases. These procedures ...

This chapter describes a method for the rapid assessment of gene copy number in laser microdissected material using multiplex real-time polymerase chain reaction (PCR). Here a putative oncogene (ZNF217) was evaluated in a series of colon tumors, but the method is applicable to any locus for which a nucleic acid sequence is available. The preparation, laser microdissection, and optimum storage o...

Advances in the biologic sciences and technology are providing molecular targets for diagnosis and treatment of cancer. Lymphoma is a group of cancers with diverse clinical courses. Gene profiling opens new possibilities to classify the disease into subtypes and guide a differentiated treatment. Real-time PCR is characterized by high sensitivity, excellent precision and large dynamic range, and...

A real-time PCR assay was developed for the detection of Ehrlichia chaffeensis. The assay is species specific and provides quantitative results in the range 10 to 10(10) gene copies. The assay is not inhibited by the presence of tick, human, or mouse DNA and is compatible with high sample throughput. The assay was compared with previously described assays for E. chaffeensis.

The MIQE (minimum information for the publication of quantitative real-time PCR) guidelines were published in 2009 with the twin aims of providing a blueprint for good real-time quantitative polymerase chain reaction (qPCR) assay design and encouraging the comprehensive reporting of qPCR protocols. It had become increasingly clear that variable pre-assay conditions, poor assay design, and incor...