Applications in the Clinical Diagnosis of Infection and Disease

PCR is now used routinely for clinical diagnosis of disease and infection. In the diagnosis of disease, PCR is most valuable as a tool in the prenatal diagnosis of genetic disorders. In order to be useful, the mutation in the human genome that causes a particular disease must be known. An example of such a disease is sickle cell anemia, which is caused by a mutation within the β-globin gene.

In order for the disease to be evident, both allelic copies of the β-globin gene must carry this mutation. If only one copy of the gene is mutated, the patient is a carrier but will not express the severe sickle cell phenotype. The mutation in the DNA of the β-globin that causes sickle cell anemia also results in the alteration of a restriction enzyme site.

Therefore, DNA isolated from a normal β-globin gene can be cleaved by the restriction enzyme whereas DNA isolated from a mutated gene that can give rise to sickle cell anemia cannot be cleaved by the restriction enzyme. For prenatal diagnosis, fetal cells are isolated by amniocentesis and DNA is prepared from this. The DNA is then subjected to PCR using primers that flank the region that is mutated in sickle cell anemia patients. The amplified DNA is cut with the restriction enzyme, and DNA fragments are separated by electrophoresis.

The presence or absence of the restriction site in the amplified β-globin gene will indicate whether the fetus is a carrier of the disease or will develop sickle cell anemia. In addition to prenatal diagnosis, this technique can be utilized in the examination of parental DNA for genetic counseling and to determine the likelihood of any offspring developing the disease.

Although it is helpful for the specific mutation that causes a disease to be known, PCR can also be useful in the diagnosis of diseases that are caused by a variety of mutations. An example of such a disease is Duchenne muscular dystrophy (DMD). DMD is one of the most common human genetic disorders, affecting approximately 1 in 3500 male births.

The dystrophin gene, which has been shown to be mutated in affected individuals with DMD, is enormous, spanning more than 2 million base pairs of DNA. One-third of all the alterations in this gene which result in DMD have been shown to have arisen by new, previously unreported mutations. Interestingly, 60% of all cases of the disease are caused by large intragenic deletions within the dystrophin gene.

Because of the heterogeneity of the mutations that cause DMD, a single PCR reaction would not be useful in diagnosis of the disease. However, multiple primers can be used in a single PCR reaction to amplify nine separate regions of the dystrophin gene. Once these regions are amplified, the samples can be separated by electrophoresis and compared to PCR samples prepared from a normal dystrophin gene. In this way, a large region can be examined rapidly for gross deletions that may cause DMD.

Because of the heterogeneity of mutations, this PCR test is not useful for all DMD diagnoses, but the coamplification method with multiple primers has permitted the rapid detection of 80-90% of all dystrophin gene deletions.

In addition to genetic disorders, PCR can be used to diagnose viral infections. This method is most useful for the detection of viruses that are able to remain latent in individuals and may cause no overt symptoms immediately. Early identification of these types of viral infections may be important for effective treatment. An example of this is the use of PCR to detect human immunodeficiency virus (HIV). Upon infection of an individual, HIV is able to integrate its DNA into that of the patient’s genome and may remain latent for many years.

The current method used for detecting the virus is identifying antibodies against HIV in the host. There are several drawbacks in using the indirect detection of host antibodies rather than the direct detection of virus. One is that it takes some-time for antibodies to be produced following viral exposure. Therefore, if the HIV antibody test is performed prior to seroconversion, a false-negative diagnosis may occur.

Second, in infants, maternal antibodies may persist in the newborn for up to 15 months. Detection of anti-HIV antibodies in an infant may therefore result in a false-positive result. Finally, antibody detection in individuals may sometime render inconclusive results. Therefore, a second detection method is required. The PCR test for HIV detection is performed on DNA prepared from an individual’s blood samples. Although the HIV DNA sequence is highly diverse, the primers used are from regions shown to be highly conserved among all the different HIV isolates.

Because of its level of sensitivity, PCR is now routinely used to diagnose HIV as well as a large variety of other viral infections. In addition to diagnosis, PCR is also used to differentiate between active and latent viral infections. In the case of a HIV active infection, RNA transcripts are generated from the integrated viral DNA and are used to synthesize more virus. In a latent infection, no such HIV-specific transcripts are present. RT-PCR can be used to detect the presence of HIV transcripts, indicating whether the infection is active or latent and allowing appropriate treatment.