The process of culture-based testing for microbes has been in common use for more than a century. In recent decades, molecular diagnostic tests have emerged as a more specific, sensitive, and faster alternative. In many cases, however, these tests require multimillion dollar molecular labs and highly skilled technicians to run them. Thus, while molecular diagnostic techniques can theoretically generate more sensitive and specific reactions in hours instead of the days needed for a culture-based test, they have remained out of reach for many organizations.
The three key steps to performing a real-time polymerase chain reaction (PCR)—sample preparation and extraction of nucleic acids, amplification of extracted nucleic acids, and detection of a target gene sequence—were first developed in the 1980s. These steps are time-consuming and are not amenable to on-demand testing. Institutions fortunate enough to have a molecular lab typically process samples in batches.
First-generation PCR assays for molecular diagnostics required manual sample extraction using reagents prepared by the laboratory itself and often involved enzymatic digestion, extraction with organic solvents, and alcohol precipitation. After drying down the nucleic acid pellet, it was then resuspended and added to a custom, laboratory-prepared buffered cocktail of enzymes, nucleotides, and oligonucleotide primers for carrying out the PCR process. After amplification, the real work of detecting the amplification products began. For best sensitivity, labeled probes were used to detect PCR amplification products on Southern blots or in microtiter plates. The two-to-three day process of carrying out the complete diagnostic procedure was labor intensive and error prone, primarily from laboratory contamination, which gave rise to false positive results.
The next milestone in the development of diagnostic applications of PCR was the development of real-time PCR assays. These assays use labeled probes that are part of the initial PCR reaction mixture; detection of fluorescence accumulation during thermal cycling is used in place of Southern blots for detection of PCR products within the closed environment reaction tube. Real-time PCR is now the most commonly used method for user-developed assays, although only a few commercial kits incorporate this technology.
Still to be refined, however, has been the sample-preparation component. No universal method for sample preparation exists. Although several kit-based methods are adaptable to a variety of specimens, these methods are still not well adapted to on-demand testing.
This paradigm has now shifted, ushering in a new era in how tests are processed, data is analyzed, and patient care delivered. Today, advances in molecular diagnostics and the ability to automate molecular reactions have the power to move the lab to the front lines of medicine more efficiently and cost-effectively. The lab is democratized as new molecular diagnostic technologies now bring the same benefits to a broad spectrum of facilities: large reference labs, regional hospitals, field research, and first response teams.
The key is automation, which now makes it possible for virtually anyone to collect a sample and carry out a molecular reaction. This is a major step in applying the technology to a growing list of public health priorities.