Use Of Nucleic Acid Probes Research Articles

A carbon nanoparticle and DNase I-Assisted amplified fluorescent biosensor for miRNA analysis

Nucleic acid-based biosensors have become powerful tools in biomedical applications. But the stability issue seriously limits their wide applications. Fortunately, the emergence of carbon nanoparticles (CNPs), which can effectively protect DNA probes from enzymatic digestion and unspecific protein binding, provides a good solution. In this work, a DNase I-aided cyclic enzymatic amplification method (CEAM) for microRNA analysis has been developed based on the coupling use of nucleic acid probes with specific molecular recognition ability as well as CNPs with excellent biostability. The method is simple and sensitive, with a detection limit down to 3.2 pM. Furthermore, satisfactory results are achieved for miRNA analysis in breast cancer cell lysate, demonstrating the applicability in disease diagnosis. The ingenious combination of CNPs and nucleic acid probes can open a new chapter in the development of versatile analytical strategies that holds great potentials for clinical diagnosis, food safety, and environmental monitoring.

The new frontier of diagnostics: Molecular assays and their role in infection prevention and control.

Recent advances in technology over the last decade have propelled the microbiology laboratory into a pivotal role in infection prevention and control. The rapid adaptation of molecular technologies to the field of clinical microbiology now greatly influences infectious disease management and significantly impacts infection control practices. This review discusses recent developments in molecular techniques in the diagnosis of infectious diseases. It describes the basic concepts of molecular assays, discusses their advantages and limitations, and characterizes currently available commercial assays with respect to cost, interpretive requirements, and clinical utility.

Human papillomavirus testing methods.

Testing for human papillomavirus (HPV) relies exclusively on techniques of molecular biology using nucleic acid probes. Tests for HPV using nucleic acid probes have been commercially available since the late 1980s, but early tests were cumbersome, involving the use of nucleic acid probes labeled with radioactive phosphorus (32P). These early HPV tests did not achieve widespread use because they did not detect all oncogenic HPV genotypes. The current commercial HPV detection kit, Digene's Hybrid Capture 2 kit, detects virtually all high-risk oncogenic HPV types, as well as most low-risk nononcogenic HPV genotypes. The Hybrid Capture 2 test format is a proprietary nucleic acid hybridization signal amplification system owned by Digene Corporation. Virtually all test formats for DNA sequence analysis are amenable to applications intended to detect and perhaps quantify the various HPV genotypes. These methods can involve direct hybridization with complementary DNA probes, such as Southern blotting or in situ hybridization, signal amplification, such as the Hybrid Capture 2 method or target nucleic acid amplification, most notably the polymerase chain reaction (PCR). Polymerase chain reaction has been used for HPV detection, genotyping, and viral load determination. General or consensus primer-mediated PCR assays have enabled screening for a broad spectrum of HPV types in clinical specimens using a single PCR reaction. Following amplification using consensus primers, individual HPV genotypes are identified using a variety of methods. Using consensus primers in a test format known as real-time quantitative PCR (RQ-PCR), it is possible to generate viral load (concentration) data from reaction curves generated by monitoring PCR reaction kinetics in real time.

Use of nucleic acid probes for identification of Mycobacterium tuberculosis directly from MB/BacT bottles.

The feasibility of using nucleic acid probes directly from positive MB/BacT broth to identify mycobacteria was determined in this study. A total number of 2,727 specimens were cultured into the MB/BacT (Organon Teknika) automated system and on conventional Loweinstein-Jensen (LJ) slants. The Gen-Probe AccuProbe culture identification tests (DNA probes) were used on samples from bottles which were identified as positive for mycobacteria by MB/BacT. Samples of positive MB/BacT broth (0.1 ml) were used directly in the broth culture method for the DNA probes as published by Gen-Probe. Centrifugation of the contents of the bottle was not done prior to probe testing. The number of mycobacteria detected by MB/BacT and LJ was 253 (221 isolates of M. tuberculosis and 32 isolates of mycobacteria other than M. tuberculosis [MOTT]). A total of 96.4% (213 of 221) of the bottles growing M. tuberculosis produced a positive direct DNA probe result for M. tuberculosis complex. One hundred percent (16 of 16) of the bottles growing M. gordonae produced a positive direct DNA probe result for M. gordonae. A total of 3.6% (8 of 221) of the bottles growing M. tuberculosis did not yield a positive direct DNA probe result for M. tuberculosis complex. The testing of subcultures made onto solid media from the positive bottles by AccuProbe identified six of these eight M. tuberculosis isolates. Two (0.9%) M. tuberculosis isolates gave a negative result for the M. tuberculosis probe test applied on the MB/BacT broth and its subculture. The rest of the positive MB/BacT bottles growing MOTT (16 of 32) were negative for M. gordonae, M. avium, M. intracellulare, and M. kansasii probes. The sensitivity and specificity of AccuProbe for the identification of M. tuberculosis and M. gordonae directly from MB/BacT broth were 96.4 and 100% for M. tuberculosis and 100 and 100% for M. gordonae, respectively. The direct testing of positive MB/BacT broth by AccuProbe, without prior centrifugation, allows for the accurate and rapid identification of M. tuberculosis and M. gordonae.

Overview of flow cytometry and fluorescent probes for cytometry.

This chapter provides an introduction to the use of fluorescent probes in flow cytometry. Sample preparation for the use of surface labeling with antibodies as well as for the use of nucleic acid probes is discussed. The utility of cell sorting is also discussed.

In situ hybridization to human chromosomes of an alkaline phosphatase-labeled centromeric probe.

The main principles that underly the use of nucleic acid probes for in situ hybridization are summar- ized. These include probe design, target preparation, hybridization formats and conditions, and signal:gener- ating systems. These principles underly the specific protocol that is described, namely the use of an akaline phosphatase-labeled cloned sequence of the alphoid repeated DNA family as a centomere probe for human chromosomes. Index Entries: In situ hybridization; centromere; chromosomes; nucleic acid probes.

Comparison of algorithms for selective use of nucleic-acid probes for identification of Mycobacterium tuberculosis from BACTEC 12B bottles

We retrospectively compared the sensitivity of two approaches, a time-to-detection algorithm and the presence of serpentine cords of acid-fast bacilli, for discriminating between BACTEC 12B cultures containing either Mycobacterium tuberculosis complex (MTB) or Mycobacterium avium complex (MAC). From January 1996 through March 1997 a total of 217 of 2089 respiratory specimens received in our laboratory were positive in the BACTEC 12B radiometric culture system for either MTB (120 specimens) or MAC (97 specimens). Use of a previously published time-to-positivity algorithm would have resulted in the correct use of the MTB probe on 109 of 120 cultures (91% sensitivity), and the MAC probe on 52 of 97 cultures (54% sensitivity). The presence of serpentine cords was detected in 58 of 120 cultures containing MTB (48%), and in 3 of 97 (3%) cultures containing MAC. Using a combination of time to positivity and cord formation to determine initial probe selection would have resulted in first use of the MTB probe in 116 of 120 (97%) instances in which MTB was present in the culture. In only 49 of 97 (51%) cultures, however, from which MAC was recovered would the correct probe have been selected. These results indicate that limiting the initial use of the MTB probe to those cultures that are either identified by the time-to-detection algorithm or demonstrate serpentine cords on acid-fast smear would eliminate a considerable amount of unnecessary probe use without compromising the efficiency of identification of isolates of MTB.

Use of nucleic acid probes to identify mycobacteria directly from Difco ESP-Myco bottles.

Mycobacterial isolates were identified directly from positive ESP-Myco bottles by use of nucleic acid probes. Retrospective analysis of 360 cultures which grew either Mycobacterium tuberculosis, M. avium complex, or M. gordonae showed that 87% were identified by direct testing of an aliquot obtained at the time a positive culture was detected. Another 12% of these cultures gave results in the equivocal range, with only 1% of the isolates yielding negative results on initial testing.

Nucleic acid in situ probing. Hybridization to human chromosomes of an alkaline phosphatase labeled centromeric probe.

The main principles that underly the use of nucleic acid probes for in situ hybridization are summarized. These include probe design, target preparation, hybridization formats and conditions, and signal generating systems. These principles underly the specific protocol that is described, namely the use of an akaline phosphatase-labeled cloned sequence of the alphoid repeated DNA family as a centomere probe for human chromosomes.

Molecular epidemiology of Escherichia coli O157:H7 by restriction fragment length polymorphism using Shiga-like toxin genes.

The purpose of this study was to assess a simplified method for interstrain differentiation of Escherichia coli O157:H7 and other Shiga-like toxin-producing E. coli (SLTEC) strains. A method based on the use of nucleic acid probes from Shiga-like toxin (SLT) I and II structural genes was used to generate restriction fragment length polymorphism (RFLP) patterns of SLTEC strains, (SLT-RFLP patterns) resulting from digestion of isolated genomic DNA with four different restriction enzymes (BamHI, EcoRI, HindIII, and PvuII) used separately. A total of 165 SLTEC strains from clinical, food, and environmental sources, including O157:H7 isolates from four food-borne outbreaks in Canada and the United States, were analyzed in the study. SLT-RFLP demonstrated that E. coli O157:H7 strains from each food-borne outbreak had the same unique SLT-RFLP pattern. Fifty-two SLT-RFLP types were found among 96 E. coli O157:H7 isolates from sporadic cases of hemorrhagic colitis and hemolytic uremic syndrome in Washington state. The use of the SLT probes proved to be a very powerful method for interstrain differentiation of SLTEC strains. Although the use of each of the enzymes alone did not give enough differentiative power to be used in epidemiological studies, the combination of patterns generated by two restriction enzymes (EcoRI and PvuII, used separately) provided the desired sensitivity for such studies. The results clearly demonstrate the usefulness of the method for studying the molecular epidemiology of E. coli O157:H7. The method is also suitable for establishing an epidemiological database, in terms of both sensitivity and ease of compilation and interpretation of results.

Diagnostic Mycobacteriology Laboratory Practices

The resurgence of tuberculosis has forced clinical laboratories to improve the methods used for detection of M. tuberculosis. Current recommendations for diagnostic laboratory performance [7] include (1) daily processing of specimens (i.e., handling these specimens in the same way that all other specimens sent to the laboratory are handled); (2) inoculation of liquid media (e.g., BACTEC) for the primary culture; (3) use of nucleic acid probes or the NAP test for identifying isolates as M. tuberculosis as soon as possible; (4) determining drug susceptibility with use of liquid media; and (5) reporting results of each step to physicians in a timely manner. The immediate goals are to report identification of M. tuberculosis within 10-14 days of receipt of the specimen and to report drug susceptibilities within 15-30 days. This can be done if current technologies are fully utilized. The amplification-based systems for the identification of M. tuberculosis and the luciferase-based systems for rapid determination of drug susceptibilities should help further shorten turn-around times. The results to date demonstrate that these systems are feasible, although they must be reduced to formats that can be used routinely in clinical laboratories. The gene-amplification systems may be the most promising, and they are nearing commercial availability. If the assays function as well during routine use as they have during clinical trials, a clinical laboratory may soon be able to report confirmed M. tuberculosis infection to the physician within hours of receiving a specimen, instead of within the typical period of 2-4 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Algorithm for use of nucleic acid probes for identifying Mycobacterium tuberculosis from BACTEC 12B bottles.

Nucleic acid probes (Gen-Probe, San Diego, Calif.) can be used to identify mycobacteria in BACTEC 12B broth cultures prior to detection of growth on solid media. We developed an algorithm that can be used to make an initial choice of a probe (either Mycobacterium tuberculosis complex [MTB] or M. avium complex [MAC]) for use in testing respiratory specimens. The algorithm was based on both the fluorochrome smear result of the concentrated specimen and the time from inoculation until the BACTEC 12B broth culture is flagged (growth index 10) as presumptively positive. The MTB probe is used first for all 4+ smear specimens, 3+ smear specimens positive in 5 days, 2+ and 1+ smear specimens positive in 7 days, and smear-negative specimens positive in 11 days. The MAC probe is used for all other specimens. The algorithm is used when other information about the culture (e.g., previous positive cultures and colonial morphology of growth on solid media) is unknown. Use of the algorithm to probe 102 respiratory BACTEC 12B broth cultures (35 with MTB; 1 with MTB, MAC, and M. gordonae; 47 with MAC; and 19 with other mycobacterial species) from 1 September through 30 November 1992 resulted in the initial use of the MTB probe for 35 (97%) of the cultures positive for MTB and the use of the MAC probe for 35 (73%) of the cultures positive for MAC. Use of the algorithm aided in the efficient use of laboratory resources without delaying the time to identification of MTB isolates.

Utility of newer techniques for classification and identification of pathogenic anaerobic bacteria.

Results of genetic and biochemical analyses have broadened our understanding of taxonomic relationships among groups of anaerobic bacteria and have led to a better understanding of the pathogenesis of infection. Conventional bacteriologic methods are still of prime importance for the detection and identification of anaerobic pathogens. The use of nucleic acid probes has so far been restricted to research laboratories. A polymerase chain reaction-generated probe would be most useful for the rapid detection of toxigenic Clostridium difficile in feces. Probes are needed for detection of periodontopathogenic bacteria in dental plaque. Use of nucleic acid probes may become a useful adjunct to classic methodology in reference and teaching laboratories.

Application of new technology to the detection, identification, and antimicrobial susceptibility testing of mycobacteria.

Mycobacteria are reemerging as important causes of human disease. The increase in mycobacterial infections has prompted the development of more rapid and efficient ways of detecting and characterizing mycobacteria in the clinical microbiology laboratory. Methods currently in use or under development include more sensitive methods of direct detection, improved techniques for culture, identification, and susceptibility testing, and the use of nucleic acid probes for identification and epidemiologic typing. Broad application of rapid and sensitive methods for detection and characterization of mycobacteria are essential if we are to limit the spread of Mycobacterium tuberculosis (TB) and provide optimal care for patients infected with TB or other Mycobacterium species.

In situ analysis of the cardiac muscle gene program during embryogenesis

During the last decade, gene expression in cardiac muscle of the developing embryo has been examined in situ with the use of nucleic acid probes and antibodies. With growth and maturation from a simple tube to a four-chambered organ, the myocardium undergoes a complex series of developmental changes in the temporal and spatial expression patterns of a number of structural and transcription factor genes. Studies of embryonic hearts suggest that different populations of cardiac myocytes might appear during development. These in vivo results are summarized and discussed in the context of the morphogenetic events that shape the myocardium.

Nucleic acid probes in aquatic bacteriology

The use of nucleic acid probe should facilitate the work of fish pathologists, since results are obtained more rapidly and genome analysis is more accurate. In this short review, the principles of molecular hybridization are mentioned (probe marking; reaction patterns on solid and liquid media; hybrid detection methods). The critical analysis of the first results which have appeared in this field comprised the identification of the bacteria which cause the principle diseases observed in ichthyopathology and bacterial ecology. Analysis also concerns bacteria concentrated by aquatic animals and which are of importance to human health as regards the quality of marine food products.

Expression and characterization of ovine major histocompatibility complex class II (OLA-DR) genes.

Previous work made use of nucleic acid probes corresponding to different subtypes of the class II regions of the human and murine major histocompatibility complex (MHC) to isolate seven different alpha and 24 different beta genes of the ovine MHC from two cosmid libraries. In an attempt to identify pairs of alpha and beta genes capable of cell surface expression, all permutations of alpha and beta genes were in turn transfected into mouse L-cells. Two pairs of alpha and beta genes co-expressed and stable ovine MHC class II L-cell lines were developed. The expressed alpha genes had previously been defined as DR-alpha homologues (DRA) by differential Southern hybridization to human subtype specific class II probes. The expressed ovine beta genes were also assigned as ovine DR-beta homologues (DRB) on the basis of their sequence having a higher degree of similarity with human DRB than any other subtype. A total of eight out of 23 anti-sheep class II specific monoclonal antibodies were typed OLA-DR specific by FACScan analysis using the L-cell lines.

Genetic diversity and BVD virus

The various measures of genetic variation of BVD virus was reviewed with emphasis on the implications for future control of virus-induced disease and diagnosis. While experimental data does not support unique serotypes for BVDV, there is substantial antigenic variation among the isolates examined. This variation may permit fetal infections even in animals assumed to be well vaccinated. The genetic differences between cytopathic and noncytopathic strains of BVDV are expressed in infected cells by the production of a p80 protein by cytopathic strains. In addition, cellular gene inserts have been detected in cytopathic strains. Monoclonal antibodies have demonstrated a high degree of diversity with the pestivirus population. Grouping of BVDV isolates by monoclonal antibody analysis is suggestive at best. The use of nucleic acid probes as diagnostic reagents has been compromised by the nucleic acid sequence variation found in the BVDV isolates tested.

Nucleic Acid Probes in Microbiology

Single-stranded nucleic acid molecules can interact under certain conditions with other nucleic acids in the regions of base complementarily to form double-stranded hybrid molecules. From measurement of kinetics of the hybridisation and the extent of hybrid formation, information on nucleotide sequence heterogeneity of nucleic acid fractions, sequence properties of purified classes of molecules or the degree of homology between different samples of nucleic acids can be obtained. A specific deoxyribonucleic acid or ribonucleic acid sequence with an identifiable marker can be used as a nucleic acid probe. This paper reviews the use of nucleic acid probes for classifying the microorganisms and their detection in clinical or environmental samples.

Use of nucleic acid probes in genetic tests

Advances in molecular genetics have led to the development of clinical assays for several genetic diseases. Two general testing approaches are available: direct detection of the genetic mutation or indirect detection using DNA markers close to, or within, the defective gene. Direct testing at the nucleic acid level is available for diseases in which the basic defect is well characterized, such as sickle cell anemia. Several methods are available for detection of the point mutation that causes sickle cell anemia including: routine Southern blot analysis, allele specific oligonucleotides, and polymerase chain reaction gene amplification. For diseases such as cystic fibrosis, in which the basic genetic defect has not yet been characterized, an indirect approach is used. This approach relies on linkage analysis using DNA markers close to the genetic defect. Inheritance of the DNA marker is followed through the family. Unlike direct testing, the DNA marker does not detect the actual genetic defect. Therefore, a prediction of inheritance of the linked disease is given based on the risk of recombination between the disease locus and the DNA marker. An appreciation of the differences between the direct and indirect approaches is necessary to understand their attributes and their limitations.