Nucleic Acid-based Detection Research Articles

Comparison of nucleic acid-based detection of avian influenza H5N1 with virus isolation

Nucleic acid sequence-based amplification with electrochemiluminescent detection (NASBA/ECL) of avian influenza virus was compared with viral culture in embryonated chicken eggs. Virus was isolated from blood or anal swabs of chickens artificially infected with highly pathogenic avian influenza A/Chicken/Hong Kong/1000/97 (H5N1). Viral nucleic acid was detected in blood samples by NASBA/ECL immediately prior to death, whilst nucleic acid extracted from anal swabs was detected from the day following artificial infection until death. Thus, blood and/or anal swabs are a suitable source of material for the detection of avian influenza in dead birds, but anal swabs are more suitable for detection of viral genetic material in live birds. Dilution of a known viral standard was used to determine the limit of sensitivity for both NASBA/ECL and egg culture detection methods. The NASBA/ECL method was equivalent in sensitivity to egg culture. The NASBA/ECL results agreed with egg culture data in 71/94 (75.5%) tissue samples obtained from artificially infected birds.

Direct detection methods for Lyme Borrelia, including the use of quantitative assays.

Direct detection of Borrelia burgdorferi sensu lato, the etiologic agent of Lyme borreliosis, is the most reliable laboratory diagnostic tool. Several methods have been developed for direct detection of B. burgdorferi in infected vectors, host tissues, and clinical specimens from patients with Lyme borreliosis. These include microscope-based assays, antigen detection assays, in vitro cultivation, and nucleic acid-based detection of B. burgdorferi. The sensitivity and specificity of these methods depend on various factors and are also variable among laboratories. To date, only in vitro cultivation of B. burgdorferi has been widely accepted to confirm clinical diagnosis of Lyme borreliosis. Nevertheless, various polymerase chain reaction-based molecular assays have shown increasing significance in the laboratory diagnosis of Lyme borreliosis because of their high sensitivity, specificity, and capability for quantification and typing of spirochetes in clinical specimens. In this review, the currently available methods for direct detection of B. burgdorferi in clinical samples and quantitative analysis of spirochete load in different biological sources are discussed.

A review of molecular recognition technologies for detection of biological threat agents

The present review summarizes the state of the art in molecular recognition of biowarfare agents and other pathogens and emphasizes the advantages of using particular types of reagents for a given target, (e.g. detection of bacteria using antibodies versus nucleic acid probes). It is difficult to draw firm conclusions as to type of biorecognition molecule to use for a given analyte. However, the detection method and reagents are generally target-driven and the user must decide on what level (genetic versus phenotypic) the detection should be performed. In general, nucleic acid-based detection is more specific and sensitive than immunological-based detection, while the latter is faster and more robust. This review also points out the challenges faced by military and civilian defense components in the rapid and accurate detection and identification of harmful agents in the field. Although new and improved sensors will continue to be developed, the more crucial need in any biosensor may be the molecular recognition component (e.g. antibody, aptamer, enzyme, nucleic acid, receptor, etc.). Improvement in the affinity, specificity and mass production of the molecular recognition components may ultimately dictate the success or failure of detection technologies in both a technical and commercial sense. Achieving the ultimate goal of giving the individual soldier on the battlefield or civilian responders to an urban biological attack or epidemic, a miniature, sensitive and accurate biosensor may depend as much on molecular biology and molecular engineering as on hardware engineering. Fortunately, as this review illustrates, a great deal of scientific attention has and is currently being given to the area of molecular recognition components. Highly sensitive and specific detection of pathogenic bacteria and viruses has increased with the proliferation of nucleic acid and immuno-based detection technologies. If recent scientific progress is a fair indicator, the future promises remarkable new developments in molecular recognition elements for use in biosensors with a vast array of applications.

RRNA stability in heat-killed and UV-irradiated enterotoxigenic Staphylococcus aureus and Escherichia coli O157:H7.

Differentiation of viable cells from nonviable cells is of considerable importance in the development of methods to detect foodborne pathogens. To study the suitability of 16S rRNA as an indicator of cell viability in nucleic acid-based detection assays, we examined rRNA stability in two representative foodborne pathogens, Escherichia coli O157:H7 and enterotoxigenic Staphylococcus aureus, which were inactivated by extreme heat, moderate heat, and UV irradiation. Cell death under all conditions was confirmed by a failure to grow in brain heart infusion broth after incubation for 48 h at 37 degrees C. rRNA stability was monitored by a Northern blot analysis, and detection was evaluated by using reverse transcription (RT)-PCR performed with two primer sets (which produced 325- and 1, 400-bp amplicons). rRNA of neither pathogen was detected by Northern blot analysis and RT-PCR after cells were killed by autoclaving at 121 degrees C for 15 min. In contrast, intact rRNA of both pathogens were detected by Northern blotting and could be amplified by RT-PCR up to 48 h after cells were killed by heat treatment at 80 degrees C and UV irradiation at 254 nm. rRNA was a suitable target molecule for monitoring bacterial viability under extreme heat conditions, but the presence of rRNA was not correlated with viability following moderate heat inactivation or UV irradiation of cells.

Detection of Blueberry Scorch Virus Strain NJ2by Reverse Transcriptase-Polymerase Chain Reaction Amplification

A reverse transcription-polymerase chain reaction (RT-PCR) assay has been developed for the detection of strain NJ2 of blueberry scorch carlavirus (BBScV), which causes an economically important disease of blueberry. Four extraction methods were compared for their ability to yield nucleic acid from blueberry tissue that could be amplified by specific RT-PCR primers. Two extraction methods were found suitable when they were followed by an Elutip-r column purification step. These procedures were successful with blueberry blossoms and leaves. RT-PCR was shown to be considerably more sensitive than spot hybridization using a 32 P-labeled DNA probe. These purification systems enable use of nucleic acid-based RNA virus detection from blueberry.

Molecular detection of hepatitis C virus: impact of detection methodology on clinical and laboratory correlations.

The clinical manifestations of hepatitis C virus (HCV) infection are generally indistinguishable from other causes of viral hepatitis. HCV infections are usually anicteric, asymptomatic, and rarely cause acute fulminant liver failure. Serological testing for HCV in conjunction with epidemiological studies have verified that HCV was the major cause of parenterally transmitted non-A, non-B hepatitis (NANBH). With the widespread introduction of serological screening of blood products for HCV antibody, the risk of transfusion-associated HCV infection has been dramatically reduced (to < 3 cases per 10,000 units transfused). Despite the virtual elimination of transfusion-associated infections, the diagnosis of HCV remains important because > 50% of infections are sporadic in origin, 50 to 70% of infected individuals develop chronic hepatitis, and these individuals are at risk of developing cirrhosis (> 20%) as well as hepatocellular carcinoma. Although currently available anti-HCV immunoassays function well as blood-donor screening assays, they are poor at detecting acute infection because of the prolonged lag time between infection and detection of seroconversion (approximately 10 to 26 weeks for second-generation immunoassays). In contrast, polymerase chain reaction (PCR)-based detection of HCV RNA in serum can detect infection in as little as 1 to 2 weeks after exposure. This review focuses on the impact of modern serologic and nucleic acid-based HCV detection methodology on the clinical understanding of HCV infection, its associated illnesses, and its transmissability. Quantitative and reproducible nucleic acid-based detection assays will be required to provide additional insights into the clinical spectrum of HCV infections as well as to assess the efficacy of antiviral agents.

A novel gene tag for identifying microorganisms released into the environment

A novel method using a moc (mannityl opine catabolism) region from the Agrobacterium tumefaciens Ti plasmid pTi15955 was developed as a tag to identify genetically modified microorganisms released into the environment. Pseudomonas fluorescens 1855.344, a plant-growth-promoting rhizosphere bacterium, was chosen as the organism in which to develop and test the system. moc genes carried by pYDH208, a cosmid clone containing a 20-kb segment of the octopine-mannityl opine-type Ti plasmid, conferred on P. fluorescens strains the capacity to utilize mannopine and agropine (AGR) as a sole source of carbon and energy. Modified P. fluorescens strains containing moc or moc::nptII inserted into a chromosomal site were constructed by marker exchange. One such modified strain, PF5MT12, utilized AGR as a sole carbon source and contained detectable levels of mannopine cyclase, an easily assayable enzyme encoded by the moc region. Catabolism of AGR could be used to recover selectively the marked strain from mixed populations containing a large excess of closely related bacteria. Nucleic acid-based detection strategies were developed on the basis of the unique fusion region between Agrobacterium DNA and Pseudomonas DNA in strain PF5MT12. The specificity and sensitivity of detection of PF5MT12 were enhanced by amplifying the fused DNA region by using PCR. The target fragment could be detected at levels of sensitivity comparable to those of other described PCR-based gene tags, even in the presence of high levels of Agrobacterium, Pseudomonas, or Escherichia coli DNA. This gene tag strategy gives a method for direct selection and enumeration of the marked strain from mixtures containing a large excess of closely related bacteria and a sensitive and highly specific system for detection by PCR amplification of the target fragment even in the presence of large amounts of DNA from related or unrelated organisms.

Advances in nucleic acid-based detection methods

Laboratory techniques based on nucleic acid methods have increased in popularity over the last decade with clinical microbiologists and other laboratory scientists who are concerned with the diagnosis of infectious agents. This increase in popularity is a result primarily of advances made in nucleic acid amplification and detection techniques. Polymerase chain reaction, the original nucleic acid amplification technique, changed the way many people viewed and used nucleic acid techniques in clinical settings. After the potential of polymerase chain reaction became apparent, other methods of nucleic acid amplification and detection were developed. These alternative nucleic acid amplification methods may become serious contenders for application to routine laboratory analyses. This review presents some background information on nucleic acid analyses that might be used in clinical and anatomical laboratories and describes some recent advances in the amplification and detection of nucleic acids.

Advances in nucleic acid-based detection methods.

Advances in nucleic acid-based detection methods.

Diagnosis with oligonucleotides

Protein-based immunohistochemical techniques like immunoperoxidase methods are now routinely used in many diagnostic dermatopathology laboratories. This is possible because of the availability of antibodies that react with specific, diagnostically useful proteins and because there are simple but sensitive detection systems. Despite their promise of equal or greater specificity, techniques based on the detection of specific nucleic acids have not been as useful as immunoperoxidase techniques because of a relative lack of such reagents and detection systems. This chapter describes how oligonucleotides, short single-stranded pieces of DNA or RNA, may be used to circumvent some of the problems associated with current in situ hybridization techniques and how they may eventually allow the use of nucleic acid-based detection systems for diagnosis in dermatopathology.

Nucleic acid detection systems for enteroviruses.

The enteroviruses comprise nearly 70 human pathogens responsible for a wide array of diseases including poliomyelitis, meningitis, myocarditis, and neonatal sepsis. Current diagnostic tests for the enteroviruses are limited in their use by the slow growth, or failure to grow, of certain serotypes in culture, the antigenic diversity among the serotypes, and the low titer of virus in certain clinical specimens. Within the past 6 years, applications of molecular cloning techniques, in vitro transcription vectors, automated nucleic acid synthesis, and the polymerase chain reaction have resulted in significant progress toward nucleic acid-based detection systems for the enteroviruses that take advantage of conserved genomic sequences across many, if not all, serotypes. Similar approaches to the study of enteroviral pathogenesis have already produced dramatic advances in our understanding of how these important viruses cause their diverse clinical spectra.

Nucleic acid detection systems for enteroviruses.

The enteroviruses comprise nearly 70 human pathogens responsible for a wide array of diseases including poliomyelitis, meningitis, myocarditis, and neonatal sepsis. Current diagnostic tests for the enteroviruses are limited in their use by the slow growth, or failure to grow, of certain serotypes in culture, the antigenic diversity among the serotypes, and the low titer of virus in certain clinical specimens. Within the past 6 years, applications of molecular cloning techniques, in vitro transcription vectors, automated nucleic acid synthesis, and the polymerase chain reaction have resulted in significant progress toward nucleic acid-based detection systems for the enteroviruses that take advantage of conserved genomic sequences across many, if not all, serotypes. Similar approaches to the study of enteroviral pathogenesis have already produced dramatic advances in our understanding of how these important viruses cause their diverse clinical spectra.

A density gradient method for fractionating microsporidan spores

Nosema bombycis is an obligate intracellular parasite infecting Bombyx mori to cause pebrine disease. The disease is transmitted transovarially and spreads faster in silkworm seed multiplication farms. Pebrine is the only quarantine requirement in silkworm egg production and is usually monitored by mother moth examination and light microscopy for characteristic spores. Practically spore detection is little technical, laborious and the life cycle stages of N. bombycis could also be visualized by microscopy. However, routine monitoring of pebrine disease through light microscopy is limited by specificity and sensitivity. The more practical and feasible diagnostic methods for pebrine monitoring have been met with the development of immunodiagnostic and nucleic acid based detection techniques. Immunodiagnostic assays such as latex agglutination and ELISA employing polyclonal and monoclonal antibodies were developed for the diagnosis and characterization of microsporidian infections. However, false positives and lower level infections made immunoassays to be less-reliable. Nucleic acid-based detection methods utilizing DNA sequences specific to N. bombycis could detect lower levels of infection specifically. Major DNA diagnostics to detect N. bombycis include PCR, Loop-Mediated Isothermal Amplification (LAMP) and lateral flow assays which are high-throughput and quantitative, also enabling simultaneous detection for multiple diseases. Molecular diagnostic techniques are more advantageous than microscopy due to increased sensitivity, specificity, faster and easy result interpretations. The molecular diagnostic tests described for the identification of microsporidia infecting mulberry silkworm are provided in this chapter.