A novel molecular assay using hybridisation probes and melt curve analysis for CALR exon 9 mutation detection in myeloproliferative neoplasms.

AIMS: Somatic insertions/deletions in exon 9 of the calreticulin gene have been identified in patients with essential thrombocythemia and primary myelofibrosis. Over 55 mutations have been discovered, 80% of which consist of either type 1 52-bp deletion or type 2 5-bp insertion. Other mutations (types 3-5) in conjunction with types 1 and 2 account for >87% of identified mutations. The aim of this study was development of a rapid PCR-based assay using LightCycler Hybridisation Probes for the detection of type 1-5 CALR mutations. METHOD: A real-time PCR assay using a novel HybProbe set was developed for use on the LightCycler 480 Instrument II. The acceptor probe was labelled with LC640 and Faststart DNA Master HybProbe kit was used for PCR reactions. RESULTS: Assay limit of detection was determined to be seven target copies with a probability of 95%. The specificity of the assay was determined by using synthetic constructs of CALR wild-type and CALR mutation types 1-5 with no non-specific detection observed. Samples from 21 patients with essential thrombocythemia (ET) and 12 patients with primary myelofibrosis (PMF), together with 29 control samples from patients diagnosed with various conditions, were screened using the assay. Of these, 24 were found to have mutations in CALR exon 9, with the assay detecting 8 type 1 mutations, 12 type 2 mutations, 2 type 24 mutations, 1 type 20 mutation and 1 31-bp deletion. CONCLUSIONS: The novel assay described has potential for application as a rapid, sensitive, high-throughput screening method in the clinical diagnostics setting.

Nucleic acid detection technologies and marker molecules in bacterial diagnostics.

There is a growing need for quick and reliable methods for microorganism detection and identification worldwide. Although traditional culture-based technologies are trustworthy and accurate at a relatively low cost, they are also time- and labor-consuming and are limited to culturable bacteria. Those weaknesses have created a necessity for alternative technologies that are capable for faster and more precise bacterial identification from medical, food or environmental samples. The most common current approach is to analyze the nucleic acid component of analyte solution and determine the bacterial composition according to the specific nucleic acid profiles that are present. This review aims to give an up-to-date overview of different nucleic acid target sequences and respective analytical technologies.

Label-free, multiplexed detection of bacterial tmRNA using silicon photonic microring resonators.

A label-free biosensing method for the sensitive detection and identification of bacterial transfer-messenger RNA (tmRNA) is presented employing arrays of silicon photonic microring resonators. Species specific tmRNA molecules are targeted by complementary DNA capture probes that are covalently attached to the sensor surface. Specific hybridization is monitored in near real-time by observing the resonance wavelength shift of each individual microring. The sensitivity of the biosensing platform allowed for detection down to 53 fmol of Streptococcus pneumoniae tmRNA, equivalent to approximately 3.16×10(7) CFU of bacteria. The simplicity and scalability of this biosensing approach makes it a promising tool for the rapid identification of different bacteria via tmRNA profiling.

Culture confirmation of Listeria monocytogenes using tmRNA as a diagnostics target.

16s ribosomal RNA (rRNA) is routinely used to identify bacteria in direct detection culture confirmation assays. In some instances rRNA cannot be used as a target to distinguish between phylogenetically closely related bacteria. Here we evaluate an alternative target, transfer messenger RNA (tmRNA), for the culture confirmation of Listeria monocytogenes.

Detection of NASBA amplified bacterial tmRNA molecules on SLICSel designed microarray probes.

BACKGROUND: We present a comprehensive technological solution for bacterial diagnostics using tmRNA as a marker molecule. A robust probe design algorithm for microbial detection microarray is implemented. The probes were evaluated for specificity and, combined with NASBA (Nucleic Acid Sequence Based Amplification) amplification, for sensitivity. RESULTS: We developed a new web-based program SLICSel for the design of hybridization probes, based on nearest-neighbor thermodynamic modeling. A SLICSel minimum binding energy difference criterion of 4 kcal/mol was sufficient to design of Streptococcus pneumoniae tmRNA specific microarray probes. With lower binding energy difference criteria, additional hybridization specificity tests on the microarray were needed to eliminate non-specific probes. Using SLICSel designed microarray probes and NASBA we were able to detect S. pneumoniae tmRNA from a series of total RNA dilutions equivalent to the RNA content of 0.1-10 CFU. CONCLUSIONS: The described technological solution and both its separate components SLICSel and NASBA-microarray technology independently are applicative for many different areas of microbial diagnostics.

Recent advances in the development of nucleic acid diagnostics.

Since the early 1970s, the use of nucleic acid sequences for specific diagnostic applications has followed a somewhat linear pattern of development. Early methods for restriction enzyme digestion, as well as reverse transcription, were followed in the late 1970s by Southern, northern and dot blotting, as well as DNA sequencing. In 1985, the description of PCR and the routine laboratory manipulation of sufficient quantities of DNA for diagnostics, resulted in the exponential growth of molecular biology. Subsequently, alternative DNA and RNA amplification protocols followed. The last 10 years have seen the second explosion in molecular biology with the development of real-time quantitative PCR and oligonucleotide microarrays. This advancement continues with the development of methods for 'direct' nucleic acid target detection from samples without in vitro amplification, and enhanced transduction elements for improved sensitivity of nucleic acid detection. In this article, we will describe the current state of the art in nucleic acid diagnostics, the use of nucleic acid-based diagnostics in clinical practice and the emerging technologies in the field. Finally, we will describe future trends and expected advances in the field.

tmRNA--a novel high-copy-number RNA diagnostic target--its application for Staphylococcus aureus detection using real-time NASBA.

A real-time nucleic acid sequence-based amplification assay, targeting tmRNA, was designed for the rapid identification of Staphylococcus aureus. The selectivity of the assay was confirmed against a panel of 76 Staphylococcus strains and species and 22 other bacterial species. A detection limit of 1 cell equivalent was determined for the assay. A chimeric in vitro transcribed internal amplification control was developed and included in the assay. Application of the assay in natural and artificially contaminated unpasteurized (raw) milk enabled detection of 1-10 CFUS. aureus mL(-1) in 3-4 h, without the need for culture enrichment. Staphylococcus aureus was detected in all artificially contaminated milk samples (n=20) and none of the natural milk samples (n=20). Microbiological analysis of the natural milk samples was performed in parallel according to ISO 6888-3 and confirmed the absence of S. aureus. The method developed in this study has the potential to enable the specific detection of S. aureus in raw milk in a significantly shorter time frame than current standard methods. The assay further demonstrates the usefulness of tmRNA/ssrA as a nucleic acid diagnostic target.

Fluorescent labeling of NASBA amplified tmRNA molecules for microarray applications.

BACKGROUND: Here we present a novel promising microbial diagnostic method that combines the sensitivity of Nucleic Acid Sequence Based Amplification (NASBA) with the high information content of microarray technology for the detection of bacterial tmRNA molecules. The NASBA protocol was modified to include aminoallyl-UTP (aaUTP) molecules that were incorporated into nascent RNA during the NASBA reaction. Post-amplification labeling with fluorescent dye was carried out subsequently and tmRNA hybridization signal intensities were measured using microarray technology. Significant optimization of the labeled NASBA protocol was required to maintain the required sensitivity of the reactions. RESULTS: Two different aaUTP salts were evaluated and optimum final concentrations were identified for both. The final 2 mM concentration of aaUTP Li-salt in NASBA reaction resulted in highest microarray signals overall, being twice as high as the strongest signals with 1 mM aaUTP Na-salt. CONCLUSION: We have successfully demonstrated efficient combination of NASBA amplification technology with microarray based hybridization detection. The method is applicative for many different areas of microbial diagnostics including environmental monitoring, bio threat detection, industrial process monitoring and clinical microbiology.

Application of two-step quantitative reverse-transcription PCR to bacterial diagnostics.

Use of high copy number bacterial RNA offers several advantages in a diagnostics context compared with current deoxyribonucleic acid-based assays. The opportunity to only detect viable cells by targeting labile RNA transcripts may create an opportunity for "real-time" monitoring of pathogen load in response to a treatment regimen, while the natural amplification provided by the relative abundance of the RNA target compared with its corresponding gene opens a door to potential nonamplified direct detection technologies. In this chapter, a method is described to accurately quantify specific RNA transcripts and thus determine their potential utility as "high-copy" targets. The quantification method described also has application in gene-expression analysis.