Real-time PCR in the microbiology laboratory

Abstract

Use of PCR in the field of molecular diagnostics has increased to the point where it is now accepted as the standard method for detecting nucleic acids from a number of sample and microbial types. However, conventional PCR was already an essential tool in the research laboratory. Real-time PCR has catalysed wider acceptance of PCR because it is more rapid, sensitive and reproducible, while the risk of carryover contamination is minimised. There is an increasing number of chemistries which are used to detect PCR products as they accumulate within a closed reaction vessel during real-time PCR. These include the non-specific DNA-binding fluorophores and the specific, fluorophore-labelled oligonucleotide probes, some of which will be discussed in detail. It is not only the technology that has changed with the introduction of real-time PCR. Accompanying changes have occurred in the traditional terminology of PCR, and these changes will be highlighted as they occur. Factors that have restricted the development of multiplex real-time PCR, as well as the role of real-time PCR in the quantitation and genotyping of the microbial causes of infectious disease, will also be discussed. Because the amplification hardware and the fluorogenic detection chemistries have evolved rapidly, this review aims to update the scientist on the current state of the art. Additionally, the advantages, limitations and general background of real-time PCR technology will be reviewed in the context of the microbiology laboratory.

Evaluation of Reference Genes for the Relative Quantification of Apple stem grooving virus and Apple mosaic virus in Apple Trees

“…Relative quantification employing reference internal control genes was used for several plant virus detection and quantification studies [5,6]. A comparative study showed that the relative quantification assay may be as accurate as absolute quantification when appropriate housekeeping genes are used and normalized with target genes [6,10]. In this paper we have chosen endogenous housekeeping genes for quantification of viruses in apple trees in a relative quantification system.…”

The comparison of pyrosequencing molecular Gram stain, culture, and conventional Gram stain for diagnosing orthopaedic infections

“…[20][21][22] Conventional PCR, and more recently real-time PCR, have been recognized as efficient tools for clinically applicable assays. 11,12,[23][24][25] The use of these tools is of particular interest in orthopedics, wherein the adhesion of bacteria within a biofilm on the surface of an implant may make the organism difficult to detect by conventional techniques. [15][16][17] Although some of these tools have been used to study orthopedic infections, much remains to be learned.…”

“…9,10 Clinical microbiologists are more and more commonly using the polymerase chain reaction (PCR) and other nucleic acid amplification assays for the detection and identification of microbial pathogens and to determine the most important mechanisms of antimicrobial resistance. Realtime or homogeneous PCR is a relatively new modification of the PCR, 11,12 wherein amplification and detection occur in the same reaction vessel secondary to the use of fluorescent detector molecules. In the clinical laboratory, this translates into easier to perform assays that are more rapid, features that increase the possible clinical applications of PCR assay.…”

GENETIC METHODS FOR DETECTION OF ANTIBIOTIC RESISTANCE: FOCUS ON EXTENDED-SPECTRUM Β-Lactamases

“…This is due to the reduced cycle times, removal of separate post-PCR detection procedures and the use of sensitive fluorescence detection equipment, allowing earlier amplicon detection. Unlike conventional PCR, real-time PCR allows continual monitoring of accumulating amplicon in real time by labeling primers, oligoprobes or amplicons with molecules capable of fluorescing 53 . There are several types of detection (intercalating fluorescent dyes such as SybrGreen or LCGreen, dual-labeled probes, FRET probe systems) used in real-time amplification…”