Target Nucleic Acid Sequences Research Articles

Development, implementation, and trend analysis of real-time PCR tests for the clinical microbiology laboratory

In the past, culture-based techniques for the detection of microorganisms have been the standard for clinical microbiology laboratories. Recently, the detection of many nucleic acid target sequences by real-time PCR has been demonstrated to have superior test performance characteristics (sensitivity and specificity) compared with conventional culture methods and standard PCR (heating block amplification of target nucleic acid and product detection by gel electrophoresis, followed by Southern blotting, ELISA, or chemiluminescence). Implementation of real-time PCR in the routine clinical microbiology laboratory has been facilitated by two outstanding features of this technology: (i) target amplification and detection in a closed system, thereby essentially eliminating carryover amplicon contamination, and (ii) using hybridization fluorescence resonance energy transfer probes with the ability to perform melting-curve analysis on amplified target nucleic acid to characterize products or to perform genotypic analysis. Herpes simplex virus (HSV) and varicella-zoster virus (VZV) real-time assays replaced traditional cell culture methods in our laboratory about 4 years ago (May 2000). Detection of group A streptococcus (GAS) as a replacement for rapid antigen and culture was implemented in August 2002. We describe a unique standardized clinical laboratory workflow process to facilitate rapid PCR testing, reporting, and treatment of GAS infections. In addition, we review the development, implementation, and performance of the HSV and VZV assays and trend analysis of these routine real-time PCR test procedures.

A universal nucleic acid sequence biosensor with nanomolar detection limits.

A quantitative universal biosensor was developed on the basis of olignucleotide sandwich hybridization for the rapid (30 min total assay time) and highly sensitive (1 nM) detection of specific nucleic acid sequences. The biosensor consists of a universal membrane and a universal dye-entrapping liposomal nanovesicle. Two oligonucleotides, a reporter and a capture probe that can hybridize specifically with the target nucleic acid sequence, can be coupled to the universal biosensor components within a 10-min incubation period, thus converting it into a specific assay. The liposomal nanovesicles bear a generic oligonucleotide sequence on their outer surface. The reporter probes consist of two parts: the 3' end is complementary to the generic liposomal oligonucleotide, and the 5' end is complementary to the target sequence. Streptavidin is immobilized in the detection zone of the universal membranes. The capture probes are biotinylated at the 5' end and are complementary to another segment in the target sequence. Thus, by incubating the liposomal nanovesicles with the reporter probes, the target sequence, and the capture probes in a hybridization buffer for 20 min, a sandwich complex is formed. The mixture is applied to the membrane, migrates along the strip, and is captured in the detection zone via streptavidin-biotin binding. The biosensor assay was optimized with respect to hybridization conditions, concentrations of all components, and length of the generic probe. It was tested using synthetic DNA sequences and authentic RNA sequences isolated and amplified using nucleic acid sequence-based amplification (NASBA) from Escherichia coli, Bacillus anthracis, and Cryptosporidium parvum. Dose-response curves were carried out using a portable reflectometer for the instantaneous quantification of liposomal nanovesicles in the detection zone. Limits of detection of 1 fmol per assay (1 nM) and dynamic ranges between 1 fmol and at least 750 fmol (1-750 nM) were obtained. The universal biosensors were compared to specific RNA biosensors developed earlier and were found to match or exceed their performance characteristics. In addition, no changes to hybridization conditions were required when switching to the detection of a new target sequence or when using actual nucleic acid sequence-based amplified RNA sequences. Therefore, the universal biosensor described is an excellent tool for use in laboratories or at test sites for rapidly investigating and quantifying any nucleic acid sequence of interest.

Relevance of commercial amplification methods for direct detection of Mycobacterium tuberculosis complex in clinical samples.

Mycobacteria are a group of acid-fast, aerobic, slow-growing organisms whose genus includes more than 90 different species. The causative agents of tuberculosis (TB), which is currently considered a global emergency, with more than 2 million people dying every year and 8 million new cases, belong to the Mycobacterium tuberculosis complex (MTB). Moreover, although of lesser public health importance, many other species referred to as nontuberculous mycobacteria (NTM) have also been associated with human disease with increasing frequency worldwide (65). As MTB is highly infectious for humans, it is of paramount importance that TB be diagnosed as early as possible to stop the spread of the disease. Active TB is currently diagnosed by conventional laboratory procedures including specimen digestion and decontamination, microscopic examination for the presence of acid-fast bacilli (AFB), isolation by culture on solid and/or liquid media, and identification and drug susceptibility testing of the recovered isolate. Because of the slow growth of mycobacteria, the above-reported laboratory procedures may require turnaround times of 3 to 4 weeks or longer. During the last decade, several molecular methods have been developed for direct detection and identification of MTB in clinical specimens. These methods, being able to potentially reduce the diagnostic time from weeks to days, have been acquiring greater and greater relevance in the field of laboratory TB diagnosis. The basic principle of any molecular diagnostic test is the detection of a specific nucleic acid sequence by hybridization to a complementary sequence, a probe, followed by detection of the hybrid. However, the sensitivity of nucleic acid probe tests that do not involve amplification is much lower than that of amplified ones. Any portion of nucleic acid can be copied by using the specific polymerase, provided that some sequence data are known for the setup of appropriate primers. In general, amplification of target nucleic acid sequences is composed of three parts: denaturation, primer annealing, and primer extension. Discovery of PCRs in 1986 made this process reiterative, leading to an exponential increase in the production of the amplified target. Soon after, alternative amplification techniques were developed and patented by companies, which used different enzymes and strategies, but they are all based on reiterative reactions. Many different amplification targets including both DNA or RNA fragments have been proposed. The target most frequently amplified in MTB is the IS6110 (31) repetitive element, of which 10 to 16 copies are present in most clinical isolates. Numerous techniques for nucleic acid extraction have been proposed, as have different types of controls for monitoring the efficacy of nucleic acid extraction and amplification procedures. Currently, the U.S. Food and Drug Administration (FDA) requires that culture (still considered the “gold standard” for TB diagnosis) must be done in conjunction with the performance of each amplification-based test. In this paper, we review and discuss the currently available commercial methods which are capable of detecting MTB directly from clinical samples.

Patent News

Peptech TNF Patent Granted in the US. Methods and Compositions for Diagnosing and Treating Chromosome-18p Related Disorders. Method for Detecting an HBV-Derived Nucleic Acid Target Sequence. Merck Wins European Patent Infringement Case Against Pharmacia/Searle.

Correlation of human papillomavirus 16 and 18 with cervical neoplasia in histological typing and clinical stage in Taiwan: an in-situ polymerase chain reaction approach.

In situ polymerase chain reaction (ISPCR) promises to considerably enhance our ability to detect a few copies of target nucleic acid sequences in fixed tissues and cells. The aim of this study was to investigate cervical carcinoma to determine the human papillomavirus (HPV) types on paraffin-embedded tissue sections by ISPCR and standard in situ hybridization. The results will correlate the morphological characteristics of lesions with viral typing results. This study examined prevalence of HPV 16 and 18 DNA in biopsies from 85 cervical cancer patients by ISPCR, employing HPV 16, 18 consensus primers. There are 45 patients with squamous cell carcinomas, 13 with adenocarcinoma, 2 with adenosquamous carcinomas, 3 with small cell carcinomas, and 22 carcinoma in situ. The relation between the types of HPV detected, tumor type, and clinical stage were analyzed. Fifty-two of 85 biopsies were HPV 16- or 18-positive, HPV 16 being the most prevalent type. Squamous cell carcinoma had a high prevalence of HPV 16 and adenocarcinoma had a high prevalence of HPV 18. HPV 18 was the predominant type among high clinical stage (III-IV) cases while HPV 16 and mixed HPV 16 with HPV18 were significantly correlated with low clinical stage (0-I-II). Our results indicate that certain malignant cervical tumor phenotypes and stages correlate with specific HPV type, and that ISPCR is a sensitive and fast method to detect HPV in these patients.

Methods for quantification of in situ hybridization signals obtained by film autoradiography and phosphorimaging applied for estimation of regional levels of calmodulin mRNA classes in the rat brain

Comparative analysis of the regional abundances of the various mRNAs in neural tissues requires the quantitation of target nucleic acid sequences while their tissue distribution is preserved. A quantitative in situ hybridization protocol is presented for the assessment of regional levels of calmodulin (CaM) I, II and III mRNAs in the rat brain. Coronal brain cryostat sections were hybridized with multiple CaM [ 35S]cRNA probes and co-exposed to an autoradiographic film or storage phosphor screen, together with a membrane-based radioactive standard scale. The membrane scale was calibrated against a brain paste standard scale. Regression analyses of the sensitometric graphs of standard scales corresponding to the autoradiographic film and to the storage phosphor screen were performed by means of exponential (ROD= p 1(1−exp[− p 2 x])) and linear (LI= ax) functions, respectively (ROD is relative optical density, LI is labeling intensity, and x is radioactivity). The ROD/LI values for the hybridized brain regions were converted into cRNA probe copy numbers (estimations of mRNA copy numbers) through use of the above standard scales. This method was applied to compare the regional abundances of multiple CaM mRNAs in the brains of control, dehydrated, chronic ethanol-treated and ethanol withdrawal-treated animals.

Single-copy Gene Detection Using Branched DNA (bDNA) In Situ Hybridization

We have developed a branched DNA in situ hybridization (bDNA ISH) method for detection of human papillomavirus (HPV) DNA in whole cells. Using human cervical cancer cell lines with known copies of HPV DNA, we show that the bDNA ISH method is highly sensitive, detecting as few as one or two copies of HPV DNA per cell. By modifying sample pretreatment, viral mRNA or DNA sequences can be detected using the same set of oligonucleotide probes. In experiments performed on mixed populations of cells, the bDNA ISH method is highly specific and can distinguish cells with HPV-16 from cells with HPV-18 DNA. Furthermore, we demonstrate that the bDNA ISH method provides precise localization, yielding positive signals retained within the subcellular compartments in which the target nucleic acid sequences are localized. As an effective and convenient means for nucleic acid detection, the bDNA ISH method is applicable to the detection of cancers and infectious agents. (J Histochem Cytochem 49:603-611, 2001)

In Situ Hybridization Detection of Calcitonin mRNA in Routinely Fixed, Paraffin-Embedded Tissue Sections: A Comparison of Different Types of Probes Combined With Tyramide Signal Amplification

In situ hybridization (ISH) is a powerful molecular tool used to visualize nucleic acids in tissues and cells. However, its use is limited by the relative lack of sensitivity in detecting low copy numbers of nucleic acids. Several strategies have been developed to improve the threshold levels of in situ detection of nucleic acid by amplification of either target nucleic acid sequences before ISH (such as in situ polymerase chain reaction) or after the hybridization procedure (such as tyramide signal amplification [TSA]). The authors compared the use of different types of probes to detect calcitonin mRNA in 10 cases of formalin-fixed, paraffin-embedded medullary thyroid carcinoma with and without TSA. In addition, dot blot hybridization was used to compare the signal amplification by TSA with oligonucleotide. cDNA, and cRNA probes. These results show that cRNA probes are the most sensitive types of probes for detecting mRNA molecules in formalin-fixed, paraffin-embedded tissue sections and that tyramide amplification can increase the sensitivity for detection of calcitonin mRNA in formalin-fixed, paraffin-embedded tissue sections at least 2- to 4-fold with cRNA probes.

In Situ Hybridization Detection of Calcitonin mRNA in Routinely Fixed, Paraffin-Embedded Tissue Sections: A Comparison of Different Types of Probes Combined With Tyramide Signal Amplification

In situ hybridization (ISH) is a powerful molecular tool used to visualize nucleic acids in tissues and cells. However, its use is limited by the relative lack of sensitivity in detecting low copy numbers of nucleic acids. Several strategies have been developed to improve the threshold levels of in situ detection of nucleic acid by amplification of either target nucleic acid sequences before ISH (such as in situ polymerase chain reaction) or after the hybridization procedure (such as tyramide signal amplification [TSA]). The authors compared the use of different types of probes to detect calcitonin mRNA in 10 cases of formalin-fixed, paraffin-embedded medullary thyroid carcinoma with and without TSA. In addition, dot blot hybridization was used to compare the signal amplification by TSA with oligonucleotide, cDNA, and cRNA probes. These results show that cRNA probes are the most sensitive types of probes for detecting mRNA molecules in formalin-fixed, paraffin-embedded tissue sections and that tyramide amplification can increase the sensitivity for detection of calcitonin mRNA in formalin-fixed, paraffin-embedded tissue sections at least 2-to 4-fold with cRNA probes.

Microgravimetric DNA sensor based on quartz crystal microbalance: comparison of oligonucleotide immobilization methods and the application in genetic diagnosis

We report on the study of immobilization DNA probes onto quartz crystal oscillators by self-assembly technique to form variety types of mono- and multi-layered sensing films towards the realization of DNA diagnostic devices. A 18-mer DNA probe complementary to the site of genetic β-thalassaemia mutations was immobilized on the electrodes of QCM by covalent bonding or electrostatic adsorption on polyelectrolyte films to form mono- or multi-layered sensing films by self-assembled process. Hybridization was induced by exposure of the QCMs immobilized with DNA probe to a test solution containing the target nucleic acid sequences. The kinetics of DNA probe immobilization and hybridization with the fabricated DNA sensors were studied via in-situ frequency changes. The characteristics of QCM sensors containing mono- or multi-layered DNA probe constructed by direct chemical bonding, avidin–biotin interaction or electrostatic adsorption on polyelectrolyte films were compared. Results indicated that the DNA sensing films fabricated by immobilization of biotinylated DNA probe to avidin provide fast sensor response and high hybridization efficiencies. The effects of ionic strength of the buffer solution and the concentration of target nucleic acid used in hybridization were also studied. The fabricated DNA biosensor was used to detect a set of real samples. We conclude that the microgravimetric DNA sensor with its direct detection of amplified products provide a rapid, low cost and convenient diagnostic method for genetic disease.

Measurement of In Situ Hybridization

Hybridization of labeled specific molecular probes to nucleic acids in tissues allows geometric and functional location of gene expression or of foreign genome sequences. Estimates of amounts and location of target nucleic acid sequence can be made with phosphor storage imaging and molecular controls.

Detection of different mRnas expressed in the thyro-parathyroid complex of the rat by in situ hybridization using digoxigenin-labelled oligonucleotide probes.

The effects have been examined of different methods and regimens for tissue fixation, preservation, permeabilization and immunostaining of different mRNAs detected by in situ hybridization in paraffin-embedded samples. The three main hormone mRNAs expressed in the thyro-parathyroid glands, namely thyroglobulin, calcitonin and parathyroid hormone mRNAs, were chosen as the target nucleic acid sequences to be detected using digoxigenin-labelled probes. Our results suggest that chemical fixation and permeabilization of tissue samples are restrictive steps. Thus, paraformaldehyde fixation provides excellent signal intensities and non-detectable background levels whereas routine formalin and Bouin's solution give unsatisfactory results. A clear linear correlation was also found between signal intensity and proteinase K permeabilization. Moreover, the optimization of immunohistochemical steps, such as anti-digoxigenin antibody concentration and colour development times, enhance the intensity and specificity of hybrid signals. Furthermore, our results show that, in contrast to some data in the literature, paraffin-embedded tissue is suitable for detection of mRNAs by in situ hybridization. It gives equivalent intensities of specific signal and superior histological and cellular resolutions when compared to cryopreserved tissue.

Homogeneous detection of a target nucleic acid sequence by combination of the intercalation activating fluorescence DNA probe and the isothermal sequence amplification.

We demonstrated the homogeneous real-time detection of RNA produced during isothermal TRC amplification by INAF probe.

CARD In Situ Hybridization: Sights and Signals.

During the last decade, several strategies have been developed to improve the detection sensitivity of in situ hybridization (ISV) by amplification of either target nucleic acid sequences prior to ISH (e.g., in situ PCRX or the detection signals after the hybridization procedures (signal amplification). Here we outline the principles of tyramide signal amplification using the catalyzed reporter deposition (CARD) technique, summarize applications as well as possible limitations of CARD 15K, and discuss some future directions of in situ nucleic acid detection using this amplification strategy.

Detection of Nucleic Acids by Cycling Probe Technology on Magnetic Particles: High Sensitivity and Ease of Separation

Cycling Probe Technology (CPT) is a signal amplification system that allows detection of nucleic acid target sequences without target amplification. CPT employs a sequence specific chimeric probe, typically DNA-RNA-DNA, which hybridizes to a complementary target DNA sequence and becomes a substrate for RNase H. Cleavage occurs at the RNA internucleotide linkages and results in dissociation of the probe from the target, thereby making it available for the next probe molecule. This communication describes the use of oligonucleotides attached to solid supports for target capture and release followed by solution and solid phase cycling. Through the attachment of chimeric probes to Sera-MagTM magnetic particles (SMP) a simple and effective method of separating the cleaved probe from non-cycled probe has been developed. By capturing the target DNA on particles and separating it from the extraneous non-specific DNA we are able to dramatically reduce background and thus discriminate between samples of Methicillin Resistant (MRSA) and Methicillin Sensitive (MSSA) Staphylococcus Aureus. We conjugated oligonucleotide probes to SMPs (∼1 um) and Nylon beads (NB) which were coated with ID Biomedical's proprietary coating materials (R, patent pending). The general structure of the constructs is shown below:

Amplification methods to increase the sensitivity of in situ hybridization: play card(s).

In situ hybridization (ISH) has proved to be an invaluable molecular tool in research and diagnosis to visualize nucleic acids in their cellular environment. However, its applicability can be limited by its restricted detection sensitivity. During the past 10 years, several strategies have been developed to improve the threshold levels of nucleic acid detection in situ by amplification of either target nucleic acid sequences before ISH (e.g., in situ PCR) or the detection signals after the hybridization procedures. Here we outline the principles of tyramide signal amplification using the catalyzed reporter deposition (CARD) technique, present practical suggestions to efficiently enhance the sensitivity of ISH with CARD, and discuss some applications and possible future directions of in situ nucleic acid detection using such an amplification strategy.

In situ RT-PCR and hybridization techniques.

AbstractIn situ polymerase chain reaction (PCR) is a very powerful tool, which enhances our ability to detect minute quantities of a rare, single copy number, target nucleic acid sequences in freshly frozen or paraffin-embedded intact cells or tissue sections (1–10). In 1986, the introduction of PCR methods opened new horizons and revolutionized research in all areas of molecular biology (11,12). Dr. Hasse and his coworkers in 1990 used multiple primers and successfully amplified the target nucleic acid sequences in intact cells by combining a traditional in situ hybridization protocol with a powerful PCR technology (13).KeywordsPolymerase Chain ReactionPolymerase Chain Reaction ReactionPolymerase Chain Reaction CycleRubber CementPolymerase Chain Reaction MachineThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Antisense‐Oligonucleotide – ein universelles Therapieprinzip

AbstractIn recent years, a nova1 class of pharmaceuticals has been developed which bind in a predictable way to certain nucleic acid target sequences aiming at selective inhibition of expression of disease‐causing genes. The chemical structure of these so‐called antisense oligonucleotide compounds must be altered relative to their natural models to render them stable under in vivo conditions and to allow their penetration to the site of action inside cells. In this article, the principle of antisense oligonucleotide function, the structure of antisense oligonucleotide analogues, the different strategies for improvement of their biological potency, and selected reports on successful in vivo studies and first clinical investigations using these antisense oligonucleotides are discussed.

Matrix-based comparative genomic hybridization: Biochips to screen for genomic imbalances

Comparative genomic hybridization (CGH) to metaphase chromosomes has been widely used for the genome-wide screening of genomic imbalances in tumor cells. Substitution of the chromosome targets by a matrix consisting of an ordered set of defined nucleic acid target sequences would greatly enhance the resolution and simplify the analysis procedure, both of which are prerequisites for a broad application of CGH as a diagnostic tool. However, hybridization of whole genomic human DNA to immobilized single-copy DNA fragments with complexities below the megabase pair level has been hampered by the low probability of specific binding because of the high probe complexity. We developed a protocol that allows CGH to chips consisting of glass slides with immobilized target DNAs arrayed in small spots. High-copy-number amplifications contained in tumor cells were rapidly scored by use of target DNAs as small as a cosmid. Low-copy-number gains and losses were identified reliably by their ratios by use of chromosome-specific DNA libraries or genomic fragments as small as 75 kb cloned in PI or PAC vectors as targets, thus greatly improving the resolution achievable by chromosomal CGH. The ratios obtained for the same chromosomal imbalance by matrix CGH and by chromosomal CGH corresponded very well. The new matrix CGH protocol provides a basis for the development of automated diagnostic procedures with biochips designed to meet clinical needs.

Target and signal amplification: approaches to increase the sensitivity of in situ hybridization.

In situ hybridization (ISH) has proven to be a very important molecular tool in research and diagnosis. However, its applicability can be limited by its restricted detection sensitivity. During the last few years, several strategies have been developed to improve the threshold levels for ISH detection by amplification of either target nucleic acid sequences prior to ISH or the detection signals after hybridization procedures. In this overview, we outline and analyze the principles, applications, and limitations of in situ polymerase chain reaction, in situ self-sustained sequence replication, primed in situ labeling (PRINS), and in situ transcription as examples of target amplification methods, and catalyzed reporter deposition using biotinylated tyramine as an approach to signal amplification in ISH. We also provide a detailed, 1-day protocol for non-radioactive oligonucleotide ISH including signal amplification, which is suitable for diagnostic purposes. Furthermore, future directions of ISH including combined strategies of target and signal amplification as well as automation are discussed.