Polymerase chain reaction

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Molecular technologies have increased the speed of antigen detection methods. The most widely used of these methods is PCR, a technique that enables the amplification of specific sequences of nucleic acids. The technique was originally described by Saiki and coworkers and subsequently perfected by Mullis in 1987. PCR can amplify minute amounts of target DNA (10 to 100 copies in clinical samples) within a few hours. In the laboratory PCR is used for DNA sequencing, cloning, gene isolation, and analysis of gene expression, and for sequencing of mitochondrial and genomic DNA in the human genome organization project. PCR can be combined with other techniques to determine whether the amplification products contain a mutation, such as restriction enzyme digestion, allelic specific oligonucle-otide hybridization, and single-strand conformation polymorphism analysis.

Applications in microbiology and infectious diseases have included the diagnosis of infection caused by slow-growing or fastidious microorganisms; detection of infectious agents that cannot be cultured; presence of novel microorganisms (Tropheryma whippelii) [14]; and recognition of newly emerging infectious diseases (nearly 100 kinds of organisms have been detected by PCR). The procedure also improves the accuracy of subtyping pathogens in epidemiologic studies, quantifies the viral load [15], and allows rapid identification of antimicrobial resistance. PCR can also be used to detect RNA viruses [16-18] (eg, hepatitis C virus); a specific messenger RNA (mRNA) transcribed by a microorganism; DNA virus in autoimmune conditions treated with immunosuppressive drugs [19-21]; and to identify cases of HIV patients with rheumatologic conditions (Box 1). It is important to understand how PCR-based techniques are used to detect the presence of infectious agents in which there are too few organisms present for detection by other means. This is illustrated by the use of PCR for the detection of Mycobacterium infection, especially tuberculosis in RA patients treated with tumor necrosis factor-a inhibitors. Tuberculosis among RA patients before the use of tumor necrosis factor-a inhibitors was approximately 6 cases

Box 1. Applications of PCR

Genetics

Detection of genetic defects associated with inherited diseases Detection of mutations associated with genetic diseases Determination of genetic susceptibility to a disease Determination of disease risk to offspring in families with affected members Detection of cancer and determination of the extent of residual disease

Ability to detect clonality with a high sensitivity (0.001%-0.1%) Detection of gene polymorphism

Detection of nonself cells or occult neoplastic cells in tissues Detection of genetic markers (receptor cell rearrangements, major histocompatibility complex system) Forensic determination of identity

Infectious diseases

Ability to diagnose infections caused by slow-growing or fastidious microorganisms Detection of infectious agents that cannot be cultured or organisms that have not yet been identified Specie identification in the mycobacterium Useful for screening, diagnosis, and management of viral infections (hepatitis viruses, human herpes virus 8, HIV) Recognition of newly emerging infectious disease Detection of RNA viruses (eg, hepatitis C virus) or specific mRNA

transcribed by a microorganism Recognition of viral load to monitor therapy Detection of infections in autoimmune conditions treated with immunosuppressive drugs Diagnosis of viral encephalitis

Identification of bacterial DNA for the diagnosis of septic arthritis and reactive arthritis Allows rapid identification of antimicrobial resistance per 100,000 patients; it has increased to 24 cases per 100,000 patients following the institution of tumor necrosis factor-a inhibitors. Tuberculosis has been reported with the use of all tumor necrosis factor-a inhibitors including etanercept, infliximab, and adalimumab. It is frequently caused by reactivation of latent tuberculosis, or new infection with Mycobacterium avium and M avium-intracellulare. These nontuberculous mycobacteria have overlapping phenotypic properties that make their speciation difficult to determine by conventional methods; also, their clinical picture can be atypical with isolated fever, and these presentations may lead to delays in the diagnosis with subsequent dissemination. Smear can be negative 50% of the time, particularly in immunosuppressed individuals; cultures can take a long time, which makes the diagnostic approach problematic. PCR can provide both rapid results and an improved diagnostic accuracy of the involved mycobacteria (Mycobacterium tuberculosis from nontuberculous mycobacteria) in the disease process, and lead to the right therapeutic approach in a short period of time [8,11,22].

PCR is also used to detect mutations associated with genetic diseases, for tissue typing, and to diagnose neoplastic diseases. It is also a valuable tool for amplification of T-cell receptor genes to distinguish neoplastic proliferation from reactive T cells. Moreover, many diseases not traditionally thought to be genetic are now being evaluated in terms of inherited susceptibilities, or as genetically mediated maladaptive responses to external stimuli, such as autoimmune diseases.

PCR also facilitates antibody engineering, such as monoclonal drugs antibodies. The isolation of an individual antigen-binding B cell is sufficient to isolate the relevant antibody-binding variable (V) region, by using oligonu-cleotide primers to amplify antibody V regions. In addition, the V region of the monoclonal antibody can be fused to the constant region to produce the desired isotype and subclass; by this method the entire antibody repertoire in the form of recombinant antibody libraries can be obtained (see Box 1).

In the past few years a variant of this technique, called broad-range PCR, has enabled researchers to identify a number of uncultivable microbial pathogens. This approach, which uses ribosomal RNA (rRNA) gene sequences (which are found in all prokaryotes and eukaryotes and contain highly conserved regions interspersed with species-specific ones), makes it possible to perform PCR on infected tissue containing extremely small numbers of bacteria.

First, the highly conserved regions of the bacterial rRNA gene are used to "prime" the synthesis of the remainder of the gene. The resulting PCR amplified product is then sequenced to identify bacteria-specific variable regions of the gene. Such an approach has enabled researchers to identify a variety of microbes, including the etiologic agents of bacillary angiomatosis and Whipple's disease.

An enzymatic in vitro DNA method PCR can be coupled with a number of other detection methods to identify any gene (by DNA) or its expression

(by mRNA) directly from clinical samples, regardless of the amount of target molecules present [23]. This application requires reverse transcription (RT) of the RNA isolated from the sample into DNA; the product of this reaction, termed "complementary DNA'' (cDNA), then serves as the template for amplification by PCR. Caution must be exercised in these cases because RNA is a labile molecule and its degradation could lead to false-negative results. This process depends on a uniquely synthesized pair of oligonucleotide primers that flank and define the DNA segment of interest. In the initial step of the procedure, nucleic acid (eg, DNA) is extracted from the microorganism or clinical specimen of interest. A thermally stable DNA polymerase uses the target DNA strand to which the primer has bound as a template to synthesize a complementary strand of DNA in an automated instrument known as a "thermocycler." Heat (90°C-95°C) is used to separate the extracted double-stranded DNA into single strands (denaturation). Cooling to 55°C then allows primers specifically designed to flank the target nucleic acid sequence to adhere to the target DNA (annealing). The enzyme Taq polymerase and nucleotides are then added to create new DNA fragments complementary to the target DNA (extension). This completes one cycle of PCR. This process of denaturation, annealing, and extension is repeated numerous times in the thermocycler. At the end of a cycle, each newly synthesized DNA sequence acts as a new target for the next cycle, so that after 30 or more cycles, millions of copies of the original target DNA are created. The result is the accumulation of a specific PCR product with sequences located between the two flanking primers. Repeated cycles result in a 2n exponential increase of the template DNA, where n is the number of cycles. Detection of the amplified products can be done by visualization with agarose gel electrophoresis, an enzyme immunoassay format using probe-based calorimetric detection, or by fluorescence emission technology.

A major strength of PCR is that the DNA segment of interest does not need to be purified from the background DNA. Furthermore, PCR can be used to diagnose disease using almost any tissue or body fluid, including fresh and archival specimens. The exponential amplification of PCR provides for great sensitivity and the use of unique primers provides for the specificity. Unlike other assay systems in which sensitivity is often compromised for the sake of specificity, an increase in one of these parameters leads to increases in the other [24,25].

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