"This volume should be of utmost interest to all investigators interested and involved in using RT-PCR ... the RT-PCR protocols covered in this book will be of interest to most, if not all, investigators engaged in research that uses this important technique ... a well balanced book covering the many potential uses of real-time PCR ... valuable for all those interested in RT-PCR." ... read more

from Doodys reviews 2009

Further reading: Real-Time PCR: Current Technology and Applications

Labels: book review, real-time PCR, RT-PCR

NASBA is an isothermal nucleic acid amplification method which is particularly suited to detection and quantification of genomic, ribosomal or messenger RNA. The product of NASBA is single-stranded RNA of opposite sense to the original target. The initial NASBA methods relied on liquid or gel-based probe-hybridisation for post-amplification detection of products. More recently, real-time procedures incorporating amplification and detection in a single step have been applied to a wide range of RNA and some DNA targets. Real-time NASBA has become a sensitive and specific method for detection, quantification and differentiation of RNA and DNA targets. Molecular beacons have been used in real-time NASBA in commercially-available kits in published assays. The increase in availability of fluorimeters suitable for real-time NASBA ensures that this methodology will become a realistic alternative to real-time reverse transcriptase PCR.

NASBA technology is an alternative method to standard proceduresfor the amplification and detection of a range of nucleic acid targets. The majority of applications have been developed for detection and analysis of RNA targets including viral genomes, viroids, ribosomal RNA (rRNA) and messenger RNA (mRNA). Advantages of NASBA over methods such as reverse transcriptase PCR include fast amplification kinetics and selective amplification of RNA in a background of DNA. The amplification is isothermal and thus there is no requirement for thermocycling during the procedure. Single-stranded RNA amplicons are produced by NASBA which can be used directly in subsequent rounds of amplification or probed for detection without the need for denaturation or strand separation.

Real-time NASBA assays are rapid, specific and sensitive with RNA amplification and a target-specific fluorescent signal achieved simultaneously in one tube with measurements obtained through a fluorimeter. Qualitative, quantitative, monoplex and multiplex formats of real-time NASBA have now been described. The methodology seems to be a suitable alternative to other amplification procedures without the need for expensive thermocyclers. Since the original reports of real-time NASBA in 1998 the number of applications, available kits and expansion to include DNA targets is apparent and likely to continue over the next few years.

from Fox et al in "Chapter 12: Real-Time NASBA" from Real-Time PCR: Current Technology and Applications

Further reading: Real-Time PCR: Current Technology and Applications

Labels: DNA, NASBA, real-time PCR, RNA, RT-PCR

Real-time PCR has removed many of the limitations of standard end-point PCR and since its introduction in the mid-1990s there has been an explosion both in the number of publications and available instrumentation describing real-time PCR applications across many disciplines. Real-time PCR (RT-PCR) technology is highly flexible and many alternative instruments and fluorescent probe systems have been developed recently. The decreased hands-on time, increased reliability and improved quantitative accuracy of RT-PCR methods have contributed to the adoption of RT-PCR for a wide range of new applications.

The development of instruments that allowed real-time monitoring of fluorescence within PCR reaction vessels was a significant advance. The technology is flexible and many alternative instruments and fluorescent probe systems are available. RT-PCR assays can be completed rapidly since no manipulations are required after the amplification. Identification of the amplification products by probe detection in real-time is highly accurate compared with the traditional PCR method of size analysis on gels. Analysis of the progress of the reaction allows accurate quantification of the target sequence over a very wide dynamic range, provided suitable standards are available. Further investigation of the RT-PCR products within the original reaction mixture using probes and melting analysis can detect sequence variants including single base mutations. RT-PCR has found applications in many branches of biological science. Applications include gene expression analysis, the diagnosis of infectious disease and human genetic testing. Due to their fluorimetry capabilities, these real-time machines are also compatible with alternative amplification methods such as NASBA, provided a fluorescence end-point is available.

The introduction of RT-PCR assays to the clinical microbiology laboratory has led to significant improvements in the diagnosis of infectious disease. The technology has applications in clinical bacteriology, parasitology and virology. There are few areas of clinical microbiology which remain unaffected by this new method. It has been particularly useful to detect slow growing or difficult to grow infectious agents.Its greatest impact is probably its use for the quantitation of target organisms in samples. The ability to monitor the PCR reaction in real-time allows accurate quantitation of target sequence over at least six orders of magnitude. The closed-tube format which removes the need for post-amplification manipulation of the PCR products also reduces the likelihood of amplicon carryover to subsequent reactions reducing the risk of false-positives. Standardisation of assay protocols for use in diagnostic clinical microbiology and external quality control schemes is required to ensure quality of testing.

Further reading:
Real-Time PCR: Current Technology and Applications
Real-Time PCR in Microbiology: From Diagnosis to Characterization

Labels: diagnostics, PCR, qPCR, real-time PCR, RT-PCR