 Other BGS
| Enrichment Article | Quiz #2 
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The article below discusses the rise of a molecular genetics technique, the polymerase chain reaction (PCR), to detect tiny amounts of viral RNA or DNA. PCR was first presented to the world at a scientific meeting in 1984 by Kary Mullis, its primary inventor. In describing PCR Mullis has written that molecular biologists, when first hearing about PCR, almost universally reacted by saying "Why didn't I think of that?" (Sci Am 1990;262:65). Of interest, in 1993 Kary Mullis received the Nobel Prize in chemistry for his discovery of PCR almost 10 years earlier.
Simply put, PCR is an in vitro method of amplifying RNA or DNA sequences. The sequence to be amplified is called the target. The nucleoside sequences that flank each side of the target are used to design two synthetic single-stranded oligonucleotides that will serve as primers for the in vitro synthesis of the target DNA. PCR is now widely used in medical laboratories, including to diagnose infection. However, applying the method to routine screening of blood donors is not imminent (see Hewlett IK, Epstein JS. Food and Drug Administration Conference on the feasibility of genetic technology to close the HIV window in donor screening. Transfusion 1997;37:346-51). Note that since the article below was written , beginning in 1996, blood centers have initiated routine screening of donors for HIV infection by testing for p24 antigen.
Currently, all but one of the transmissible disease tests are immunoassays, designed to identify antibodies or antigens associated with the agent of concern. Estimates of the risk of disease transmission to blood recipients suggest that donor screening and testing measures are remarkably successful. Residual risk of HIV-1 infection is thought to be less than one in 225,000 per blood component; corresponding figures for HBV, HCV and HTLV-I/II are less than one in 50,000, 3,300 and 50,000, respectively.
Despite the success of immunologically based donor tests, it is widely appreciated that these tests do have limitations. First, tests based upon immunologic methods have practical and theoretical limits to their analytic sensitivity; in general, an immunologic test is unable to detect less than about 50 picograms of analyte. Second, those tests that depend upon the detection of antibodies are limited by the fact that a finite time is required for the infected host to develop an immunologic response and synthesize detectable levels of antibody. It must be recognized that the antibody response represents a very high degree of amplification; many antibody molecules are generated for each infecting organism. Nevertheless, there is increasing interest in the concept of using tests that have the potential of directly identifying infectious agents in blood samples. Immunologic tests are not generally sensitive enough to achieve this objective, so there is considerable focus on diagnostic or detection systems based upon the identification of viral or bacterial nucleic acids.
There are four areas in which modern molecular biology contributes to diagnostic or screening procedures. The first is the identification and characterization of infectious agents, as exemplified by the recent work on the hepatitis C virus, which has been characterized almost entirely as a result of gene isolation and sequencing. Second, the development of optional reagents for immunologic tests contributes to diagnostic or screening procedures. Understanding protein and/or genetic sequences permits the preparation of recombinant or synthetic peptides that can be used as capture reagents or probes in immunologic tests. Currently, one manufacturer's combination test for antibodies to HIV-1 and HIV-2 depends upon this approach. A benefit may be enhanced sensitivity and specificity of test methods. Third, there are a variety of hybridization procedures in which a known genomic sequence can be labelled and used to probe directly for the presence of viral or bacterial genomes in tissue, in solution or in gels. Hybridization techniques do not, in themselves, seem to offer great promise for donor screening, despite their increasing role in diagnostic applications. Finally, and perhaps of most interest, is the area of gene amplification procedures, which have the capacity to specifically replicate selected fragments of a genome fragment by a factor of many millionfold. The product of this amplification is then readily detectable. Gene amplification has the potential for detecting a single virus within a complex sample. Best known in this context is the polymerase chain reaction (PCR). The remainder of this report will deal with gene amplification techniques.
Gene amplification techniques involve manipulation of the normal process by which nucleic acids replicate. In essence, a known segment of nucleic acid is identified; each end of the sequence is defined by a short primer sequence. When the primers are added to the test sample, if the target sequence (i.e., viral DNA or RNA) is present, each replication step favors the sequence between the primers. Consequently, each time a replication cycle is completed, the target sequence is essentially doubled. This means that, in theory, after 20 replication cycles, the gene sequence is amplified by a millionfold. In practice, several millionfold amplification is usually achieved after 30-35 cycles.
PCR technology essentially permits the detection of a single viral genome from a background population of more than 10,0000 human cells. Thus, for example, during the early phase of HIV infection, prior to positivity in a test for anti-HIV, PCR assays can define the actual presence of the virus in appropriate samples. A recent advance has been the development of techniques permitting the direct detection of RNA as well as DNA, offering the promise of detection of HIV and HCV, in addition to cells infected with these viruses. Until recently, PCR could only be considered to be a research laboratory technique, since meticulous care is necessary to prevent cross-contamination between samples and amplified DNA. Such contamination rapidly leads to a profound loss of specificity. In addition, the technique itself requires lengthy incubation and research level techniques for the detection of the amplified products. At this time, Roche Biomedical is in the process of developing an accessible, microplate format PCR assay that is free of the problems of cross-contamination and that can be completed and evaluated in about five hours, using routine laboratory equipment. It is anticipated that clinical trials of a PCR assay for HIV will be initiated in the near future; it is initially intended for confirmation of serologic results.
A variety of data suggest that gene amplification technology has the potential for the detection of many different infectious agents. It seems likely that there are a number of samples in which the only evidence of infection with HIV, HTLV, HCV or HBV is the ability to amplify and detect viral gene sequences. It is not yet clear, however, whether the performance characteristics of PCR are such that it could be used in place of, rather than supplementing, current immunologic techniques.
There is still considerable concern about the potential for transmission of HIV from seronegative blood donors in the window period. As pointed out above, this risk is probably less than 1 in 225,000 currently. The suggestion that PCR for HIV may reduce this risk requires careful evaluation. For example, at least in the case of retroviruses, it is clear that it is necessary to perform gene amplification procedures on nucleic acid preparations obtained from cellular blood samples, rather than from serum. At this time, the PCR technology is not accessible for routine screening applications in transfusion medicine. However, data reflecting the potential sensitivity of the method continue to be developed. In addition, the technology has been modified and improved to the point where it might be accessible for routine applications within the next year or two.
Terms to look up
| Other BGS
| Enrichment Article | Quiz #2
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