RReal-time PCR (polymerase chain reaction) provides clinical diagnostic labs with the ability to specifically target a particular sequence of human or pathogen DNA or RNA and amplify it to a level that can be detected and measured. This type of test is sensitive enough to pick up small variations in genetic sequence and can be quantified to determine the relative amount of DNA in a sample. Before PCR, this level of specificity, sensitivity, and speed did not exist. In terms of infectious disease diagnostics, even miniscule amounts of pathogen-specific DNA or RNA can be artificially amplified, detected, and measured in just a few hours without the need to culture the specimen—a task that can be very difficult for some infectious agents.

“PCR is a fast, accurate, and highly sensitive method that can be used for a broad range of genetic tests that identify infectious organisms or influence cancer therapy,” said David Persing, MD, PhD, executive VP and chief medical and technology officer at Cepheid, Sunnyvale, Calif.

Until recently, the use of PCR has been restricted to a small number of laboratories that have the dedicated space and technical skill to perform complicated molecular diagnostics procedures. Even then, specimens are often run in periodic batches, which can add several days of waiting time to a 1-hour PCR test. As the technology advances and costs drop, however, clinical labs are increasingly incorporating PCR techniques into their repertoire of diagnostic tests.

RT-PCR: A Primer

The polymerase chain reaction—usually known simply as PCR—is so named in part for the polymerase enzyme that uses primers bound to a DNA template to assemble a new strand of nucleic acids. The chain reaction occurs as the template is heated to strip apart the double helix, cooled to allow the polymerase to synthesize a new strand of DNA using one of the parent strands as a template, and then allowed to zip back up before the process starts again. As each new strand of DNA then becomes a template for subsequent rounds of replication, the amount of target sequence is dramatically amplified. RNA can also be detected by first applying a reverse transcriptase enzyme to generate a complementary DNA (cDNA) template before proceeding to PCR.

The recipe is simple: add the DNA template, a polymerase enzyme, nucleotides, and primers in a buffer, place the sample in a thermal cycler instrument, set the program to cycle through the necessary temperatures, and work on something else while it runs. During real-time PCR (RT-PCR), the concentration of DNA in the sample is detected and quantified in real time by an instrument measuring the amount of a dye that fluoresces when bound to double-stranded DNA.1 Fluorescence increases as the concentration of DNA in the sample increases.

At the end of a RT-PCR cycle, the sample temperature is raised to determine the melting point. Since melting points should be equal for samples amplified with the same pair of primers, plotting the decrease in fluorescence as strands separate will determine homogeneity and purity. Curve variations can indicate subtle sequence variations like a single base mutation, or problems such as contamination.

COBAS TaqMan®

For many clinical labs, concerns over ease of use and standardization of assay design remain formidable barriers to the adoption of PCR-based diagnostics.2 Several companies are developing products specifically designed to address these concerns.

Roche Diagnostics Automates RT-PCR

Molecular diagnostics can be applied to four areas of clinical testing: rapid identification, screening, classification, and monitoring. Each category has different clinical, operational, and economic needs required to maximize the benefits to patient care. Roche Diagnostics, Basel, Switzerland, markets its portfolio of instruments, which includes the COBAS TaqMan® and LightCycler® analyzers, to meet these distinct needs.

The COBAS TaqMan Analyzer is a RT-PCR system that automates amplification and detection of DNA or RNA for up to 96 samples and four assays at one time. This system can be paired with the COBAS AmpliPrep Instrument for a truly “sample in, result out” workflow. AmpliPrep automatically purifies nucleic acids from closed input tubes by lysing, washing, resuspending, and transferring samples directly to output tubes. When the two systems are docked together, the target template and a master mix of PCR reagents move directly to the COBAS TaqMan Analyzer for RT-PCR analysis. Without human intervention, the workflow is streamlined and the risk of contamination is minimized.

LightCycler Analyzer

COBAS TaqMan is uniquely qualified to meet the needs for monitoring viral load. “A patient with HIV, for example, is tested regularly to confirm that the viral load is below a certain threshold,” explains Harsha Mokashi, marketing manager at Roche Diagnostics. “Since any increase in viral load could mandate a change in treatment, measurements made over time must be consistent. The COBAS system delivers this continuity of care.”

Roche’s COBAS AmpliPrep/TaqMan system has been rigorously tested for its sensitivity and specificity in detecting pathogens such as HIV-1. Now, a TaqMan test for measuring Hepatitis B virus (HBV) DNA recently became the first of its kind approved in the United States.3 Tests for Hepatitis C virus (HCV) and cytomegalovirus (CMV) are currently in development.

To meet the needs of rapid identification, Roche also offers the LightCycler Analyzer, a carousel-based thermal cycler. With this instrument, RT-PCR can be performed in as little as 30 minutes in small batch sizes, eliminating the need to wait for enough samples to complete the batch before testing.

“Molecular diagnostics has redefined what it means to be timely. The LightCycler delivers speed to those tests where trimming off hours can deliver clinical impact,” Mokashi says.

GeneXpert Infinity-48

Cepheid Offers an Entire PCR Facility in a Cartridge

The GeneXpert Infinity-48 high-throughput system (Cepheid, Sunnyvale, Calif) automates the management of the entire molecular testing workflow. After a sample is loaded into a GeneXpert cartridge, the Infinity-48 system does the rest by managing the sample data, cartridge loading and unloading, and reporting of test results. It uses a robotic cartridge handling, managed by Cepheid’s Xpertise™ software, to run up to 1,300 different tests during any 24-hour period.

“With each of the system’s 48 testing modules managed as independent testing sites, the GeneXpert system can start test runs anytime samples are collected—24 hours per day, 365 days per year in under 2 hours,” Persing says. “It’s designed to improve workflow while accelerating time-to-result, enabling health care providers to make more informed patient-management and treatment decisions.”

Industry trends in automation are moving toward high-throughput systems, but these platforms still rely on batch testing. “While batch testing may seem ideal for the select handful of national and regional reference laboratories looking for that level of throughput, it does not meet the needs of the average hospital,” Persing says. “Cepheid’s technology has democratized diagnostics, making it possible for almost anyone to perform a sophisticated molecular diagnostic test.”

Currently, the Infinity-48 system is designed specifically for the detection of methicillin-resistant Staphylococcus aureus, enteroviral meningitis, and Group B Streptococcus. The platform also supports other assays for infectious diseases, genetic markers, and, soon, oncology.

AmpliSpeed

Advalytix Simplifies Single-Cell Analysis

Genetic heterogeneity, even within the same patient, can push clinical decisions to depend on analysis at the single-cell level. According to Frank Feist, executive director of Advalytix Business at Olympus America Inc, Concord, Mass, “Oncogene expression can vary from one cell to another in a single tumor, and immune cells can differ depending on their pathogen specificity, so clinical tests are increasingly looking to exploit the information contained in a single cell to inform diagnosis, prognosis, and treatment.”

To simplify this process, Advalytix has developed the AmpliGrid and AmpliSpeed systems. AmpliGrid looks like a standard microscope slide, but is processed like a semiconductor chip to allow the surface to hold up to 48 1-µL samples safely in place. The drop can be placed by pipetting, laser capture microdissection, or flow cytometry, and visually verified under a microscope. Next, with the surface tension preventing any dripping or sliding, samples are covered with a mastermix of PCR reagents. Then, the whole slide drops right into the AmpliSpeed cycler, a miniature PCR thermal cycler customized for the AmpliGrid.

“The AmpliSpeed is so small you could take it home in your bag to run another cycle during dinner,” Feist jokes. “It saves energy by relying on moving parts to create a temperature profiles, instead of bulky fans and heaters.

In just a couple of hours from start to finish, DNA from a single cell is amplified by the AmpliSpeed instrument and ready for standard RT-PCR analysis.

Thermo Fisher Scientific Changes the Paradigm in Sample Measurement

Accurate RT-PCR results rely on consistent loading of an appropriate concentration of template. The NanoDrop™ (Thermo Fisher Scientific Inc, Wilmington, Del) further streamlines molecular workflow by measuring the concentration of DNA or RNA in 10 seconds with only 1 µL.

Measuring the concentration of a nucleic acid sample usually requires sacrificing a substantial amount of sample to make dilutions, transferring it to a cuvette, and measuring the absorbancy with a standard spectrophotometer.

“The NanoDrop simply takes away the need for a container,” says Philippe Desjardins, scientific marketing manager at Thermo Fisher Scientific Inc.

The NanoDrop brings speed and reproducibility to PCR-based diagnostics. The inherent surface tension of 1 µL of undiluted sample holds it in place as the drop is stretched between two points and a narrow optical beam is fired precisely in the middle, accurately reading a range of 2 to 3700 ng/µL of undiluted nucleic acid. The instrument is then simply wiped clean and ready for the next sample.

“The basic idea for the NanoDrop came from hearing molecular biologists complain about wasting their sample to take measurements,” Desjardins says. “Waste is especially a shame when you have a limited amount, and that factor becomes even more important in clinical settings.”

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Overcoming Limitations

No one wants to wait 2 days for a result when a health care decision is in the balance, yet the cost of speedy RT-PCR instruments and reagents can be prohibitively expensive. Risk of contamination is also a huge issue, since results can easily be skewed by the unintentional amplification of just a few stray copies of DNA. Thus, clinical technicians must be well trained in RT-PCR techniques and assays must be standardized, whether by purchasing manufacturer-designed kits or developing in-house protocols. PCR experts encourage technicians to educate themselves by attending regional trade shows or talking to vendors that can properly demonstrate how to use these instruments.

In the end, the value of applying RT-PCR in the clinical laboratory environment comes down to a cost/benefit analysis. As Persing summarizes, “The technology comes at a premium price, but most users figure out very quickly that they can save money in labor and overhead, and most importantly by delivering the most effective health care.”

Heather Buschman is a freelance science journalist based in San Diego.

References

  1. Kubista M, Andrade JM, Bengtsson M, et al. The real-time polymerase chain reaction. Mol Aspects Med. 2006;27(2-3):95-125.
  2. Read SJ, Burnett D, Fink CG. Molecular techniques for clinical diagnostic virology. J Clin Pathol. 2000;53:502-506.
  3. Chevaliez S, Bouvier-Alias M, Laperche S, Pawlotsky JM. Performance of the COBAS AmpliPrep/COBAS TaqMan real-time PCR assay for hepatitis B virus DNA quantification. J Clin Microbiol. 2008;46:1716-1723.