The use of DNA analysis by employing DNA probes or microarrays is a novel and revolutionary approach for specifically identifying the disease causing pathogenic organisms. This is in contrast to the traditional methods of disease diagnosis by detection of enzymes, antibodies etc, besides the microscopic examination of the pathogens. Although at present it is not in wide spread use DNA analysis may soon take over the traditional diagnostic tests in the future years to come. Diagnosis of selected diseases by genetically engineered techniques or DNA probes or direct DNA analysis is briefly described below.
Detection of Tuberculosis
Tuberculosis is caused by the bacterium Mycobacterium tuberculosis. The commonly used diagnostic tests for this disease are very slow and sometimes may take several weeks to give accurate results. This is because M.tuberculosis multiplies very slowly taking about 24 hours to double the population.
A novel diagnostic test for tuberculosis was developed by genetic engineering. A gene from firefly, encoding luciferase enzyme is introduced into the bacteriophage specific for M.tuberculosis. The bacteriophage is a bacterial virus frequently referred to as luciferase reporter phage or mycophage. The genetic engineered phage is added to the culture of M.tuberculosis. The phage attaches to the bacterial cell wall, penetrates the cell wall and inserts genes along with luciferase gene into the M.tuberculosis chromosome. The enzyme luciferase is produced by the bacterium. When luciferin and ATP are added to culture medium, luciferase cleaves luciferin. This reaction is accompanied by a flash of light which can be detected by a lucimonometer. This diagnostic test is quite sensitive for the confirmation of tuberculosis.
The flash of light is specific for the identification of M.tuberculosis in the culture. For other bacteria, the genetically engineered phage cannot attach and enter inside the bacteria. Hence no flash of light would be detected.
Detection of Malaria
Malaria is mainly caused by Plasmodium falciparum and Plasmodium vivax. Malaria affects about 1/3rd of the world population. The commonly used laboratory tests for the diagnosis of malaria include microscopic examination of blood smears and detection of antibodies in the circulation. While the former is time consuming and frequently gives false negative results. The latter procedure cannot distinguish between the present and past infections.
A specific DNA diagnostic test for identification of current infection of Plasmodium falciparum has been developed. This is carried out by using a DNA probe that can bind and hybridize with a DNA fragment of P.falciparum genome and not with other species of Plasmodium. It is reported that this DNA probe can detect as little as 1 ng of P.falciparum in blood or 10 pg of its purified DNA.
Detection of Chagas' Disease
The protozoan parasite Trypanosoma cruzi causes Chagas' disease. This disease is characterised by destruction of several tissues such as liver, brain, spleen, lymph nodes by the invading parasite. Chagas' disease is diagnosed by the microscopic examination of the fresh blood samples. Immunological tests although available are not commonly used since they frequently give false positive results.
A DNA fragment with 188 bp length is found to be present in T.cruzi genome. This was however not found in any other parasite. A PCR technique is employed to amplifying of the 188 bp DNA fragment. This can be detected by using polyacrylamide gel electrophoresis. Thus PCR based amplification can be effectively used for the diagnosis of Chagas' disease.
Detection of Lyme Disease
Lyme disease is caused by the bacterium Borrelia burgodorferi. This disease is characterised by fever, skin irritation or rashes, arthritis and neurological disorders.
The diagnosis of Lyme disease is rather difficult since it is not possible to see B.burgodorferi under microscope and the antibody detection tests are not very reliable.
Some workers have used PCR to amplify the DNA of the bacterium. By using appropriate probes the bacterium causing Lyme disease can specifically be detected.
Detection of AIDS
Acute immune deficiency syndrome or AIDS is caused by the virus human immune deficiency virus or HIV. The commonly used laboratory tests for detection AIDS uses processes to detect the HIV antibodies. However it might take several weeks for the body to response and produce sufficient HIV antibodies. Consequently, the antibody test may be negative meaning false negative while HIV is present in the body. During this period being a carrier the patient may transfer the HIV to other people.
DNA probes, with radioisotope labels for HIV DNA are now available. By using PCR and DNA probes AIDS can be specifically diagnosed in the laboratory. During the short course of infection cycle HIV exists as a segment of DNA integrated into the T lymphocytes of the host. The T lymphocytes of a suspected AIDS patient are isolated and disrupted to release DNA. The so obtained DNA is amplified by PCR technique and to this DNA probes is added. If the HIV DNA is present it hybridizes with the complementary DNA sequence of the radio labelled DNA probe. This can be detected by its radioactivity. The advantage of DNA is that it can detect the virus when there are no detectable antibodies in circulating blood.
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