The model system we used for the development of PCR was amplification from human genomic DNA of a β-globin fragment containing the codon 6 mutated in sickle cell anemia (SCA). So, the first demonstration of the diagnostic potential of this new technology was the prenatal diagnosis of SCA and, slightly later, for hemoglobinopathies in general via the detection of a large panel of β-globin mutations.
The same probe-based genotyping technology was used to develop an HLA typing test employed in the first forensic DNA test in 1986, and in 1991, in the first commercial PCR assay, the HLA-DQ-alpha Forensics test. Widely used during the ’90s, this forensics test of the highly polymorphic HLA-DQA1 locus has now been replaced with a panel of STR (short tandem repeat) markers.
Since our first case (Pennsylvania v. Pestinikis), PCR genetic typing has transformed the criminal justice system, helping convict the guilty and exonerate the wrongly convicted. These forensics genetic markers have also been used to identify missing persons and the victims of mass disasters, and for clinical diagnostic analyses of mixtures such as post stem cell transplant engraftment monitoring.
In the HLA field, serologic HLA typing, previously used for matching transplant donor and recipients as well as disease association studies, has been replaced by PCR-based typing.
The first clinical molecular diagnostics in-vitro diagnostic (IVD) test was a PCR-and-probe based assay for Chlamydia trachomatis. Arguably, the PCR diagnostic test that had the most clinical impact was the HIV viral load test. Introduced during the height of the AIDS epidemic, this assay not only allowed monitoring HIV patients’ disease but also provided a surrogate marker for testing new antiviral drugs, measuring their effect on viral load rather than by following clinical progression (slower and more subjective).
Today, most quantitative tests are based on the real-time PCR quantitative analysis with TaqMan probes. PCR-based multiplex tests for HIV, HCV, and HBV are also widely used in most blood screening programs.
Another PCR-based viral assay with a significant impact on clinical practice involves detection of human papilloma virus (HPV) in cervical swabs. PCR was instrumental in demonstrating that HPV is the cause of cervical cancer, and PCR assays—particularly those capable of distinguishing the very high-risk HPV16 from the other high-risk HPV types—have been shown to be more sensitive than the Pap test for detection of cervical cancer. Currently, a variety of PCR assays detect other pathogens as well as pathogen resistance to specific drugs.
Although most of the initial PCR-based IVD tests were for infectious disease pathogens, many PCR assays now involve genetic targets, such as cystic fibrosis carrier screening, and many focus on detecting inherited (e.g., BRACA1 and BRACA2) mutations and somatic mutations in tumors (e.g., KRAS).