OTHER SEROLOGICAL TESTS

OTHER SEROLOGICAL TESTS Other serological tests for laboratory diagnosis of syphilis which utilize specific T. pallidum antigens include the following. The Treponema pallidum immobilization (TPI) test has proved to be an important technique in the diagnosis of syphilis. Live, virulent T.pallidum (Nichols strain) and complement are placed in a test tube containing patient’s serum. The tube is then incubated at 34ºC for 18 hours. If the patient has syphilis, his serum will contain antibodies which immobilize the spirochetes. Serum from a nonsyphilitic patient does not contain immobilizing antibodies. The percent of motile spirochetes used in the test is determined by dark-field microscope examination. This test requires painstaking attention to detail and experience with the procedure and is useful for diagnosis of syphilis in patients who may give false positive reactions by other methods. The Treponema pallidum immune adherence (TPIA) test is based on the fact that when T.pallidum is placed in a test tube with complement, patient’s serum containing syphilitic antibodies, and red blood cells, the organisms adhere to the blood cells. In the presence of normal nonsyphilitic serum the treponema do not adhere to them. If possible, red blood cells from the patient are used, but if they cannot be obtained, group O cells may be substituted. Tubes containing the mixture are centrifuged gently but sufficiently to sediment the blood cells. The supernatant fluid is examined under the dark-field microscope. If spirochetes are found, adherence has not taken place and the test is negative. FLUORESCENT ANTIBODY TECHNIQUE The fluorescent-antibody technique for rapid identification of microorganism is an indirect immunochemical technique for visualization of a reaction which takes place when a known antibody, tagged with a flourochrome dye, combines with its homologous antigen. If a mixed culture or specimen is placed on a slide, mixed with serum containing known fluorescent antibody, and viewed by ordinary dark-field microscopy, all organisms present will appear bright on a dark background. However, if the same preparation is viewed with fluorescence microscopy using an appropriate ultraviolet-light source, only the antigen (organisms) which has been “cooted” with antibody will be visible. Thus, only a few organisms need be present to be observed, and laboratory confirmation of clinical diagnosis of many infectious diseases can be made quickly and accurately. The method is potentially useful for the identification of any microorganism to which antibodies can be produces. With certain modifications the basic technique is used for the rapid serological diagnosis of syphilis.
The Polymerase Chain Reaction, or PCR, refers to a widely used technique in molecular biology that has become quintessential in many aspects of DNA analysis with broad-based applications in medicine and forensic investigations. PCR is the amplification of specific sequences of genomic DNA, the genetic material found in virtually all living cells. This technology was conceived by the Californian geneticist Kary B. Mullis (1944), who won a Nobel Prize in chemistry in 1993 for developing PCR. It was

A scientist at the U.S. Army's biodefense laboratory at Ft. Detrick, Maryland, performs PCR analysis on anthrax samples.
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first applied to basic science research and later revolutionized modern medicine by improving the diagnosis of human diseases through enhanced genetic testing and medical research. More recently, PCR technology has significantly contributed to both domestic and international forensic sciences as well as applications aimed at improving United States homeland security.
PCR requires specialized equipment that is customized to fluctuate between specifically timed temperature variations. Before PCR is performed, DNA must be isolated from peripheral blood, hair follicles, cheek cells, or tissue samples. Isolated DNA is double stranded, meaning that there are two sequences of letters or nucleotide bases (A or adenine, G or guanine, C or cytosine, and T or thymine). The double stranded DNA is held together by complementary base pairings in that A binds to T, C binds to G and vice versa. Therefore, knowing of the sequence of one strand will reveal the sequence of the complementary strand. Amplification is necessary because there are 3.9 billion bases, and although there is a lot of total DNA, there is not enough to properly analyze specific gene or gene segments. Amplification, therefore, makes it possible to obtain ample quantities of specific sequences of DNA to perform a variety of analyses.
PCR requires "primers," or two sequences about 20–25 bases long with one binding to the beginning sequence of interest and the other binding at the end of the same sequence. In order to get the primers to bind to the targeted sequences in the genome, the PCR machines will undergo several cycles at different temperatures. In the first cycle, the DNA is heated to break apart the two strands. The temperature is then reduced so that the primers can bind or anneal to their complementary base sequence in the DNA. Finally, an enzyme called Taq polymerase adds letters from a pool of bases or letters included in the reaction to the position next to the last base of each the primer. Synthesis of one strand of DNA is in the opposite direction of the other. The result is a double stranded DNA sequence. These cycles are repeated several times and amplification of first the DNA sequence in the genome is copied and this copied DNA is re-copied in the next cycle resulting in exponential growth of the specific sequence. Thirty cycles amplified the target DNA between 100,000- to 10,000,000-fold. However, only DNA sequences of 100 to 2000 bases long are ideally suitable for PCR amplification. In this way, a gene of interest or part of the gene can be amplified to quantities that make genetic studies possible.
PCR, therefore, is rapid, inexpensive, and a relatively easy way of producing a large number of copies of a specific DNA sequence. This is particularly advantageous when there is very little or poor quality DNA. RNA, which is converted from DNA into protein, can also be amplified in the same manner as DNA, however, DNA is much more stable and is easier to isolate. Since each individual inherits sequences of DNA that are different from other individuals, the importance of DNA and PCR technology in identifying an individual is exemplified in the courtroom. DNA analysis can be a powerful tool in criminal investigations, especially those classified as homicides, theft, and sexual assault. Physical evidence left at the scene of any crime can be helpful in reconstructing the sequence of events and potentially reveal the criminal. It can also reveal non-paternity if the pattern of DNA in the offspring does not match the pattern of DNA in the assumed father.
Forensic science relies heavily on PCR technology to amplify specific sequences of DNA that will establish a connection between a specific suspect and a crime scene. Amplification of DNA is critical in cases where the source of DNA is minimal or the integrity is compromised. DNA evidence is also a powerful tool that has been used to ultimately prove the innocence of previously convicted individuals. Additionally, DNA can reveal many characteristics that can help forensic scientists and law enforcement officers identify the perpetrator. This is becoming increasingly applicable to national security as well as international intelligence. PCR has revolutionized law enforcement in this way and will continue to enhance the justice system in the future. For example, using complex algorithms and known sequences of DNA, it is possible to analyze the genetic DNA pattern from an unknown person to predict eye color, gender, and even ethnicity.
In 1985 American geneticist Alec Jeffereys, Ph.D. used PCR technology to amplify regions in the human genome that were highly variable. These DNA fragments were comprised of specific sequences that were repeated. The repeat number was found to be highly variable from individual to individual with the exception of identical twins. These DNA fragments could be amplified using PCR and then studied for variable fragment lengths of repeats. This technology was collectively referred as genetic fingerprinting and became widely used. In a highly publicized case called the Narborough Murder Enquiry, criminal investigators were able to identify the perpetrator using DNA fingerprinting.