Direct antigen detection of the organism by enzyme immunoassay or direct immunofluorescence is technically easier than culture but may lack sensitivity and specificity.12,39 PCR assays have been found to be significantly more accurate, with sensitivities of 90%-100% and specificities greater than 97% for the detection of C. trachomatis from cervical or urethral specimens.9,10,11 The positive predictive values reported in these studies ranged from 89% to 100%. Because PCRallows an investigator to zero in on a potentially defective DNAsequence, it can be used effectively in the diagnosis of diseases in which a specific mutational site is in question. A PCR assay for cytomegalovirus is available for detection of the virus in plasma or cerebrospinal fluid specimens and has been useful in monitoring HIV and bone marrow transplant patients with cytomegalovirus infection. A 19-year-old student is admitted to hospital with meningitis. This may be exacerbated if an inadequate sample or very small specimen volume (i.e., < 20 μL) is available for testing. DNA fragment analysis using capillary electrophoresis has many applications: detecting deletions and duplications (30% or more of all genetic diseases); prenatal testing to detect aneuploidy; rapid identification of known mutations associated with specific diseases … PCR for the diagnosis of enteroviral meningitis using cerebrospinal fluid samples has been found to be significantly more sensitive than conventional viral isolation (14% of specimens positive v. 10% positive respectively).26,27 Moreover, the PCR assay can be completed within 1 day, whereas cultures for enteroviruses typically require up to 5 days for isolation of the virus. The performance of these tests may be erratic because factors such as inoculum size or variability in culture conditions may affect phenotypic expression of resistance. His studies have markedly increased awareness of the potential use of recombinant DNA technology in diagnosis and have provided an extraordinary example of the power of molecular biology to bring about practical benefits. These techniques are also used to locate genes on human chromosomes. A major advantage of these tests is the ability to detect Chlamydia in urine specimens. Before her admission she had received 3 courses of oral cefaclor therapy. In the laboratory, to begin with, it is very essential to identify a specific DNA sequence. In this paper we describe the principles behind PCR-based diagnosis and its applications for the diagnosis of infectious diseases. Should he receive high-dose intravenous acyclovir therapy for presumed infection with herpes simplex virus? Two tests that have undergone such evaluations, and are currently among the most widely used PCR assays in diagnostic microbiology laboratories, are nucleic acid amplification assays for the detection of C. trachomatis and M. tuberculosis from clinical specimens. Identification using culture and susceptibility tests is even more problematic than that of methicillin-resistant S. aureus, primarily because of difficulties in detecting low levels of resistance75 and because accurate identification using conventional laboratory procedures may take as long as 4-6 days. From *the Department of Microbiology, SD Laboratory Services and the Division of Infectious Diseases, Sunnybrook & Women's College Health Sciences Centre, and †the Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ont. Substantial advances in the simplification of procedures for diagnostic … Assays that are currently available commercially for use in diagnostic laboratories include tests for the detection of Chlamydia trachomatis,9,10,11,12 C. pneumoniae,8 Mycobacterium tuberculosis,14,15 Mycoplasma pneumoniae,17 Neisseria gonorrhoeae,19 herpes simplex virus30 and cytomegalovirus.24 In addition there are PCR assays available for monitoring the viral load of HIV,31,32,33 hepatitis C virus29 and hepatitis B virus.28 Unfortunately only a few of these commercially available assays have been extensively evaluated to determine their sensitivity, specificity or clinical utility. Specificity of the test may be affected by contamination of the specimen during laboratory processing, if nonspecific primers are selected for the assay or if PCR conditions are not optimal, allowing nonspecific products to amplify. This cDNA can then be amplified by standard PCR methods as described earlier. Each of these clinical scenarios presents the medical practitioner with a problem that involves establishing a diagnosis of infection in a setting where routine laboratory investigations are likely to be nondiagnostic or will not provide results in a timely manner. Reprint requests to: Dr. Andrew E. Simor, Department of Microbiology, Sunnybrook & Women's College Health Sciences Centre, Rm. Several strategies for the amplification of nucleic acids have been described, including amplification of the nucleic acid target (e.g., polymerase chain reaction [PCR], strand-displacement amplification, self-sustaining sequence replication), amplification of a nucleic acid probe (e.g., ligase chain reaction, Qβ replicase) and signal amplification (e.g., branched-probe DNA assay). As yet recombinant DNA technology does not appear to have widespread diagnostic application in pathology. As recombinant DNA technology was being developed, Dr. Kan continued to devise applications for the diagnosis of human disease. Matsumura SO, Louie L, Louie M, Simor AE. In order to accomplish this, investigators took advantage of the observation that portions of bacterial 16S ribosomal RNA sequences are highly conserved, whereas other regions are less well conserved and are species-specific. NUCLEIC ACID AMPLIFICATION AND DETECTION METHODS developed in the past decade are useful for the diagnosis and management of a variety of infectious diseases. PCR-based methods for the detection of antimicrobial resistance have been applied to bacteria including methicillin-resistant Staphylococcus aureus,58,59 vancomycin-resistant enterococci60,61,62 and multidrug-resistant M. tuberculosis.65,66 Detection of resistance to antiviral agents by molecular methods has also been described for acyclovir-resistant herpesviruses67 and HIV resistant to reverse transcriptase inhibitors69 and to protease inhibitors.70 Currently none of these assays are available commercially, but they have been used in a number of reference and research laboratories. DNA Typing (DNA Fingerprinting): 1. Moreover, cultures may yield no bacterial growth if there has been a delay in transporting the specimen to the laboratory, if the number of viable infecting organisms is low, or if the patient was taking antibiotics by the time the culture specimen was obtained. In pioneering studies using DNA to diagnose human disease, Dr. Kan has helped lay the groundwork for the rapid progress now being made in the identification of many genetic diseases and in prenatal genetic screening. Despite the obvious advantages to these newer procedures, there may be potential limitations to DNA amplification technology in the diagnostic microbiology laboratory (Table 4). Following this, the enzyme Taq polymerase and nucleotides are added to create new DNA fragments complementary to the target DNA (extension). NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail.
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