Three Decades of Quantitative PCR

17th August 2018

Category: Biosafety

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By: Ifigeneia Stavrou - Biosafety Scientist,

In April of 1983 while on a late-night Friday “moonlit drive through the mountains of California” to his weekend cabin in Mendocino, Kary Mullis, a researcher at the Cetus Corporation biotechnology company, experienced a “Eureka” moment that would change the world forever (1). The idea made him stop the car on the side of the highway so he could grab a pen and paper and start performing calculations, and kept him awake through the night “drawing diagrams on every horizontal surface that would take pen, pencil or crayon” (2) in his cabin. Ten years later, Mullis was awarded the 1993 Nobel Prize in Chemistry for his invention of the Polymerase Chain Reaction (PCR). Since then, the discovery of the PCR has revolutionized the course of molecular sciences, with its applications spanning numerous biological disciplines, including basic research, disease diagnostics and forensics. The pioneering technique can amplify a DNA segment into several orders of magnitude in a straightforward, reliable and inexpensive manner, and today is one of the most common technologies used in the lab.

At around the same time as the 1993 Nobel Prize ceremony, another ground-breaking PCR innovation took place. Russel Higuchi described the use of an optical sensing system and a fluorescent label binding to the accumulating PCR product, as a tool to measure the accumulation of DNA in real time. Today, quantitative real-time PCR (qPCR) is considered as the “gold standard” in the quatitation of neucleic acid. This powerful method has tremendous applications within a broad range of scientific fields and industries, including the growing sector of Contract Testing & Research Organizations (CTOs & CROs).

With strict product safety requirements constantly being placed upon biopharmaceutical companies by health authorities, biosafety testing is one of the most crucial types of services provided by Contract Research Companies. The qPCR technology is at the molecular forefront of biologics safety testing due to its good reproducibility, high sensitivity and value for money and, at Sartorius Stedim BioOutsource, qPCRs are routinely implemented to ensure that clinical batches, cell and virus banks, raw materials of animal origin and bulk harvest are free of potential contaminants. The range of contaminants that can be measured through qPCR is vast, thanks to the technique’s extensive potential in identifying and quantifying nucleic acids in numerous types of biological matrix.

Quantitative real-time PCR can, for example, quantify residual host cell DNA in drug substances manufactured in  bioreactors, which ultimately determines that a drug is safe for human use. Moreover, the technique is implemented to detect an array of bovine and porcine viruses, recognized by regulatory agencies such as the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) as potential contamination threat to biopharmaceuticals and vaccines. Interestingly, the use of qPCR for virus detection circumvents the drawbacks of the more traditional in vitro cultivation and immune-staining methods applied, which require a longer timeframe and are dependent on which viral strains are able to grow well in cell culture (3). In addition, as of 2012, qPCR has been accepted by the FDA as a technique for mycoplasma detection in biologics, replacing traditional and laborious mycoplasma testing assays and consequently, rapidly reporting key biosafety information back to manufacturers (4). Finally, qPCR is also sensitive enough to detect yeast and fungi in test materials.

Quantitative real-time PCR assays offered at Sartorius Stedim BioOutsource for virus detection are validated according to ICH Q2 (R1) guidelines and are performed in accordance with Good Manufacturing Practice (GMP).  Sartorius Stedim BioOutsource has invested in fully validated instrumentation which is 21 CFR compliant, meeting current regulatory requirements for data integrity and computer software validation (CSV).

Despite the important methodological advances that have been implemented over the past two decades (just imagine that prior to the invention of automated thermocyclers, PCR reactions required manual incubations of tubes in waterbaths at different temperatures for varying time periods), there is a constant need for evolution of the technique towards a more sensitive, less variable and more reliable approach. Key steps in the qPCR method, such as the mechanism of DNA priming and the reverse transcription of mRNA into cDNA are susceptible to technical variability between assays and, thus, must be optimally designed and executed to reduce the yield of non-reproducible results. In parallel to the optimization of the PCR method itself, improvements to pre- and post- qPCR steps, including sample preparation and data analysis, can also mitigate some of the shortcomings. For example, use of instruments that can achieve high-throughput, fully automated nucleic acid extraction allows for a more reproducible workflow and, consequently, more consistent measurement results.

Ultimately, the continuous combination of these advances with more prosaic biological considerations, such as the use of prime quality biological material, and the ever-increasing array of evolving qPCR platforms and protocols enables high quality biosafety testing and ultimately impacts the international biopharmaceutical industry.

Interested to discover more about our biosafety testing services led by our expert scientists? Get in touch with us today.

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1.      Mullis, K. (1990) The Unusual Origin of the Polymerase Chain Reaction. Scientific American,  262 (4), 56-65.

2.      Nobel Lecture: The Polymerase Chain Reaction 1993, The Nobel Prize in Chemistry 1993, Available from: <https://www.nobelprize.org/nobel_prizes/chemistry/laureates/1993/mullis-lecture.html>. [11 July 2018].

3.      Bovine and Porcine Assays n.d., Biosafety Testing, Available from: < https://www.biooutsource.com/biosafety/assays/bovine-porcine-assays/>. [16 July 2018].

4.       Mycoplasma Testing n.d., Biosafety Testing, Available from: < https://www.biooutsource.com/biosafety/assays/mycoplasma-testing/>. [16 July 2018].

 

 

 

 

 

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