PCR expands its domains


Research Unit of Hospital Universitario La Paz, Madrid

Author analyzes platform evolution that has undergone diagnostic technique of chain reaction polymerase, born as a tool for molecular biology and has slowly been expanding its indications to be key in identifying viruses and retroviruses .

Since Kary B. Mullis invented the PCR (polymerase chain reaction), in early 1980s, on horizon of this field has been appearing a lot of applications to clinical diagnosis. This technique has been so influential in the development of biology and therefore in medicine, such as the discovery of the double helix structure of the genetic material (DNA). Ability to amplify minute amounts of DNA specifically has led to its application in the detection of fastidious organisms, recent viral infections, disease-causing polymorphisms and cancer markers, among many other applications. Today, medicine can not walk without it.

Exploiting the original function of polymerases-enzymes whose activity is to copy DNA sequences, this technique allows us to perform a molecular copying a portion of the genetic material. Therefore, presence of minute amounts of a specific sequence such as the sequence that characterizes a virus, can be amplified until it becomes visible and hence detectable. Initially PCR was only applicable to the detection of DNA, however, by using a previous step using the retrotranscriptasas , we can also detect RNA, which is present in so-called retroviruses, such as HIV or hepatitis genetic material C. This variation is known as RT-PCR (Polymerase Chain Reaction Retrotranscriptase).

Amplifying a portion of the genetic material belonging to a virus or a microorganism only informs us of presence or absence of the infection in patient examined. Latter is known as qualitative PCR. However, if the amplification process of the sequence in question is monitored in real time , we can quantify the amount of genetic material present in the sample. Q -PCR in real time enables us not only to know the presence of infection but also quantify the number of existing copies, becoming crucial for determining viral load instrument. Moreover, the possibility to amplify multiple specific sequences of the same specimen (multi- PCR) gives us a complete information of the presence of infection.

New indications

Clearly this tool, which was originally understood as another molecular biology, has a wide application in diagnosis. In the field of microbiology, PCR is called to completely replace large amount of current methods for detection of pathogens. Not only win by a large difference in terms of the ability to detect the presence of fastidious organisms, facilitates have reliable results in short-timespan-hours, while crops take days.

Currently have developed PCR assays for the detection of Listeria, Legionella, Borrelia, Leptospira, Chlamydia, Neisseria and Treponema, among others.

PCR results demonstrated erfectamente correlate with information provided by the crops, except that for the latter, had to wait about seven days , whereas PCR results were obtained in hours. Note that the sensitivity of this technique is a thousand times greater than that having a crop. Other interesting applications, and increasingly widespread, are determining antibiotic resistance isolated from patients and detecting parasites such as Toxoplasma, Cryptosporidium Trypanosoma and bacteria.

Cancer and viruses

In cancer diagnosis application of PCR has been essential for the presence of specific markers of the development of this disease and the occurrence of polymorphisms that induce cancer events. A clear example is the expression levels of factors such as HER -2 and topo IIa , important in the development of breast cancer easily detectable by real-time PCR genes. Levels indicate a poor prognosis for the patient. Moreover, the location of mutations in genes such as p53 is another application of PCR to diagnosis in the field of oncology. However, one of the areas where this technique has had a major impact is in the detection and quantitation of virus and retrovirus. Three factors contributed to this: are pathogens with a very simple genetic material , isolation of DNA or RNA is simple and, finally, the diagnosis by culture is difficult and laborious.

A long list of PCR assays has been established for viruses . This section deserve special mention retroviruses causing AIDS, since the PCR has helped eliminate the window period in detection. More traditional methods, based on the appearance of antibodies or proteins of the virus detection (ELISA or Western Blot), require high levels of contamination or long incubation period (three to six months after infection).

However, the PCR can you recognize her 48-72 hours elapsed presence of pollution, being more prudent to wait a week to eliminate possible false negatives. Moreover, it provides the possibility of quantifying existing copies helps in adjusting treatment. Accuracy of the PCR does distinguish presence of different subtypes of the same virus as “appearance” is indistinguishable by more conventional methods.

Other applications of this technique are neurodegenerative diseases and prenatal diagnosis. While in the first group using the PCR is limited to the detection of polymorphisms that are used for classification of these diseases , in the case of prenatal diagnosis is applied to identify possible genetic flaws in fetuses. Recently it has opened a wide range of possibilities for non-invasive prenatal diagnosis using the PCR , because it has identified the presence of fetal DNA in the maternal circulation role, allowing the extraction of fetal genetic material from maternal plasma.

Accuracy and reliability of this technique has eliminated the old fear raised by the occurrence of false positives/negatives . His many and varied possibilities make us recommend the study of the peculiarities of each diagnosis where PCR is applied, tending another bridge between molecular biology and clinical diagnosis.

Interview with Dr. Eduardo Lopez Collazo Medical Journal published on Monday, September 4, 2006.