Molecular diagnostics in the clinical lab

Nov. 20, 2014

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Upon completion of the three CE articles, the reader will be able to:

  1. Describe methods for automating PCR technology.
  2. Identify clinical applications for DNA sequencing.
  3. Describe current trends in multiplex molecular diagnostic testing.

Sample-to-answer PCR improves patient care

PCR (polymerase chain reaction) is improving patient care in the molecular laboratory through accuracy and timely results. The goal of molecular diagnostics is to create a sample-in, answer-out system that is easy to use, fast, and cost-effective in order to drive faster time to result. 

To make PCR more relevant and enable it to be more widely used, the emphasis has been on automating the workflow from sample to answer. Every step—sample introduction, lysis, nucleic acid purification, PCR amplification, read-out of results—is being integrated into a solution that provides actionable results in less time in order to have a positive impact on patient care.

The sample type depends on the disease being searched for based on clinical presentation and, perhaps, symptom panels. Complex laboratory-developed tests have been available for many years to detect a wide array of pathogens. Common sample types include sputum, blood, fecal samples, aspirates, urine, swabs, and cerebrospinal fluid. The goal is to move a broad menu of tests out of the high-complexity lab and eventually to the bedside.

Currently, the approach is toward simple, walk-away sample loading. This has been accomplished with swabs for respiratory and sexually transmitted infections by expressing the sample into an elution, vortexing (if required), and introduction into a machine. Blood culture tests are also available for gram-negative and positive bacteria and yeast. Fecal samples can be introduced into a system to test for bacteria, parasites, and viruses.


Lysis is currently accomplished through application of pressure, heat, chemicals1 (including chaotropic salts to be used later in purification), sonication,2 and/or mechanical lysis3 (for instance using zirconium3 or silica2 or ceramic4,5 beads). The sample is usually mixed with lysate buffer and held at a specified temperature. With some types of samples and pathogens, chemicals and temperature incubation for several minutes are sufficient to fully lyse the pathogens. With others, such as fungi, spores, yeast, and protozoa, agitating beads and/or sonication may be required for complete lysis.

Nucleic acid purification

The seminal work in nucleic acid purification was done in 1990 by Boom et al,6 at the Academisch Medisch Centrum in the Netherlands. Since then, many improvements have been made in the basic silica-based nucleic acid binding and elution chemistry using chaotropic salts. Silica membranes, magnetic beads, and paramagnetic beads have also been developed. Cellulosic membranes7,8 that use a different chemical binding and elution chemistry, and do not require ethanol, are also available. This is an important consideration as ethanol is difficult to store and inhibits PCR.

In most systems, the lysate, which may already contain a chaotropic salt, is allowed to mix with the binding material, immobilizing the nucleic acid. After a series of washes, the nucleic acid is eluted into water or buffer.

PCR amplification

PCR, invented by Mullis et al, at Cetus Corporation in 1983, has been developed into a powerful diagnostic technique that is capable of single copy pathogen detection sensitivity with sequence specificity.9 PCR continues to be one of the most sensitive and specific assays available to the clinician.

The eluent typically flows to a PCR amplification zone after purification. A short (~10 cycle) preamplification may occur. This pre-amplification has the tradeoff of lower absolute sensitivities for easier multiplexing. Additionally, reverse-transcription may be performed to allow the amplification of RNA pathogens.

At this stage, it is important to note that a relatively large sample (milliliters) has been concentrated to a very small sample (microliters). The sample must start as a large volume in order to meet the sensitivity requirements of most assays.

In order to perform PCR, a sample must be continually heated between the DNA denature temperature and the primer anneal temperature. Several thermal cycling strategies are currently employed to achieve this. Heating and cooling via a plastic membrane by Peltier junctions (a semiconductor used for cooling and heating) is one approach; shuttling between two or more constant temperatures zones is another.

Each PCR reaction must have an internal control that always amplifies and is detected to ensure that conditions were suitable for amplification to take place. This can be done with a “process control” that is added to the sample3 and controls for every step from sample introduction to detection. In the case of reverse transcription, RNA may be added as the process control. The RNA is typically stored in its native organism, although stabilized RNA standards can also be used.  An easier-to-store DNA template may also be present in the PCR reaction chamber to be amplified as a PCR-only control.

It is desirable that all reagents be stored in a single-use vessel for ease of use, sterility, and storage. Some companies ship buffers and ethanol in separate plastic ampules to be poured or injected into the vessel at time of use. On-vessel storage is a particular advantage of cellulosic nucleic acid purification because, in contrast to silica extraction, ethanol (which is difficult to store) is not needed. In every case, the moisture vapor transmission rate (MVTR) of suitably engineered containment vessels has been optimized for many months of storage at ambient temperature.

A major effort has been made over the years in thermally stabilizing the PCR reaction chemistries. While PCR relies on thermally stable polymerases, storage of these enzymes over time is difficult to achieve. Of particular difficulty in storage are the reverse-transcriptases that may become active at temperatures as low as 25°C. Nucleotides, co-factors, and primers must be stabilized as well, although these agents are generally better able to withstand variability in time and temperature.

The general strategy for preserving the activity of a PCR reaction is to bind the PCR reaction chemicals in a sugar crystal that maintains the components at bond angles similar to those maintained in water. When reconstituted, typically with eluent from the purification stage, the sugars must not inhibit the PCR reaction. Typical processes used are lyophilization and anhydrobiosis.10 There are also proprietary formats that encapsulate the PCR components in a soluble bead.

Read-out of results

The ultimate objective of performing sample-to-answer PCR testing is achieving efficient read-out of results. The clinician needs fast, accurate, easy- to-interpret information. Results are typically read out through optical or electrical means. Using optical means, a fluorescent probe or a fluorescent intercalating dye is used to measure amplification. As the reaction proceeds, the fluorescent signal increases. Many systems report only the end-point value as clinically relevant, qualitative data. In this manner, the presence or absence of the pathogen can be clearly identified, but not its concentration in the sample. In the case of optical methods, multiplexing results are either limited to the fluorescence channels available (typically six or fewer), or sensitivity is reduced by using smaller volume, separate chambers, each with its own optical measurement. Additionally, proprietary electronic methods can be used to determine endpoint signal. In the case of electronic detection, highly sensitive, large volume PCR is performed and the product is read out after amplification. Electronic detection systems can be capable of large multiplexing capacity.

Molecular diagnostic test methods that utilize PCR technology positively impact patient care by delivering high sensitivity and specificity and fast turnaround time. Simple operation has reduced user training needs, moving the technology out of the CLIA high-complexity lab. Technology is driving toward long-term temperature stable reagent storage, lower cost-per-test, higher vessel throughput, smaller instrument size, and faster time to result. The future looks promising for developing innovative sample-to-answer molecular diagnostics to deliver highly accurate, actionable information to physicians in significantly shorter timeframes to improve patient care.

George Maltezos, PhD, serves as Technology Advisor for San Diego-based GenMark Diagnostics.


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The rise of multiplexing in the molecular laboratory

The completion of the mapping of the human genome more than a decade ago has catalyzed a dramatic shift in approaches to diagnostics. Today we stand on the threshold of the “Genomic Era,” in a time when genetic testing and pharmacogenomics have become mainstays in the diagnosis and treatment of disease. From genotyping patients, to predicting drug response, to stratifying patients according to the risk of a disease, molecular testing has become mainstream, and the promise of personalized medicine has become the norm.1 

There are now more than 15,000 tests for more than 2,800 genes. The molecular diagnostics market has become the fastest growing segment of the IVD industry, with assays serving multiple disease areas and breaking new boundaries in personalized healthcare.2-3 

With the emphasis on personalization of treatment, recent years have witnessed pharmaceutical companies shifting their pipeline strategy from low-value, “one-size-fits-all” therapies to high-value, targeted therapies applicable to genetically defined populations. Within the genetic laboratory setting, the emergence of highly targeted multiplex molecular diagnostic assays has enabled healthcare systems to provide personalized medicine in a cost-effective and clinically relevant way.

The case for multiplex assays

The benefits of adopting multiplex assays can be seen particularly in the area of infectious disease, where detection of co-infections is vital in understanding the mechanism of infection. In a recent paper by Memish et al, published in June 2014, a study into the etiology of severe community-acquired pneumonia (CAP) during the 2013 Hajj highlighted the presence of co-infections in pilgrims diagnosed with severe CAP and admitted to a healthcare facility. The study found that co-infections were present in 68.4% of patients, with 80.7% of patients harboring more than one respiratory pathogen.4

Both bacterial and viral infections were present in 65.3% of patients, demonstrating the complexity of respiratory infections and emphasising the need for further investigation to accurately detect all pathogens present in order to better measure and manage infection.4 This is particularly necessary in respiratory and sexually transmitted infections (STIs), where the inappropriate use of antibiotics can prolong infection exposure and add to the burden of antibiotic resistance currently threatening public health worldwide.5-7

However, despite the availability of powerful molecular multiplex diagnostic assays across a range of conditions, the use of such tests in the area of inherited disease is far from routine. Many “traditional” diagnostic tests continue to under-diagnose, leading to missed opportunities for early and appropriate therapy intervention for potentially life-threatening diseases. One prime example in which a multiplex molecular diagnostic approach can improve health is testing for familial hypercholesterolemia (FH).

Familial hypercholesterolemia

FH is an inherited genetic disorder, affecting the body’s ability to clear LDL-cholesterol (LDL-C), which, if untreated, can lead typically to atherosclerosis and premature onset of cardiovascular disease in men in their 40s and women in their 50s.8-10 The main gene responsible for the genetic defects associated with FH is the low-density lipoprotein receptor (LDLR) gene. At least one in 500 people in the general population harbors mutations in this gene.8,10

While FH can be diagnosed clinically or genetically, it is recommended by UK, U.S., and international guidelines that probable or possible FH patients undergo a DNA test to confirm the diagnosis of FH. Recommendations also advocate that once an activating mutation has been found in one family member (the index case), cascade screening of that mutation in first-degree relatives of the index case should be conducted to further assist in case identification and improve patient outcomes.11

Diagnosis of FH through lipid profiling (LDL, HDL, and total cholesterol, for example) is not sufficient to distinguish FH from other hyperlipidemias and will not guide appropriate treatment. FH requires a much more aggressive treatment regime, with higher levels of statins or combined therapies to effectively reduce the risk of CHD and stroke, so definitive mutation analysis is a more accurate option. 

Further, FH management can be dictated by the type of mutation harbored by the patient, such as the poorer response to lipid-lowering therapy observed with specific LDLR mutations. Consequently, accurately identifying the gene mutation involved can potentially help the clinician decide how aggressive the treatment strategy should be.12 Genetic diagnosis of FH through adoption of routine screening will allow patients to receive appropriate therapeutic and interventional treatment to reduce morbidity and mortality associated with cardiovascular disease.

Pioneering multiplex diagnostic assays, tailored to incorporate the relevant FH-causing mutations, provide a promising future for both the genetic laboratories, where a rapid, cost-effective approach to determine mutational status in cases of suspected FH is enabled, and the patient, whose treatment and care pathway is managed effectively.

Determining mutational status

To date, more than 1,200 different LDLR mutations have been reported, with prevalence varying among different populations.13 The abundance of different FH mutations can make genetic testing labor-intensive and costly, with many laboratories defaulting to performing expensive and lengthy next generation sequencing (NGS) tests in an effort to ensure a comprehensive mutational screen. 

However, as our understanding of the genetic drivers of FH, as well as common population-specific mutations, increases, new novel multiplex assays are being developed to meet the clinical need and to screen for the most relevant mutations in FH. These assays delivering accurate results within hours rather than weeks at a significantly lower cost.8

Martin Crockard, PhD, Molecular Diagnostics Manager for Randox Laboratories, leads a team of 20 molecular biologists in the company’s Molecular Diagnostics group. He has more than 20 years’ experience leading molecular biology projects, 13 at Randox. Dr. Crockard works closely with the company’s engineering, regulatory affairs, marketing, and manufacturing departments to develop both multiplex arrays and complementary analyzers.


  1. Philips KA, Sakowski JA, Trosman J, Douglas MP, Liang S-Y, Neumann P. The economic value of personalized medicine tests: what we know and what we need to know. Genet Med. 2014;16(3):251–257.
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  3. Markets and Markets. Molecular Diagnostics Market by Application (Infectious Disease, Oncology, Genetics, Microbiology), Technology (PCR, INAAT, DNA Sequencing), End User (Hospital, Laboratories), Product (Instruments, Reagent, Service, Software) – Global Forecasts to 2018. Report code MD 2521, June 2014. Available online: Accessed September 19, 2014.
  4. Memish ZA, Almasri M, Turkestani A, Al-Shangiti AM, Yezli S. Etiology of severe community-acquired pneumonia during the 2013 Hajj—part of the MERS-CoV surveillance program. Int J Infect Dis. 2014;25(8):186-190.
  5. Zumla A, Memish ZA, Maeurer M, et al. Emerging novel and antimicrobial-resistant respiratory tract infections: new drug development and therapeutic options. Lancet Infect Dis 2014. Accessed  September 23, 2014.
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  7. World Health Organization. Antimicrobial Resistance Global Report on Surveillance. 2014. Available online: Accessed September 23, 2014.
  8. Futema M, Whittall RA, Kiley A, et al. Analysis of the frequency and spectrum of mutations recognised to cause FH in routine clinical practice in a UK specialist hospital lipid clinic. Atherosclerosis. 2013;229(1):161-168.
  9. Williams RR, Hunt SC, Schumacher CM et al. Diagnosing heterozygous familial hypercholesterolemia ysing new practical criteria validated by molecular genetics. Amer J Cardiol. 1993;72(2):171-176.
  10. Rader, DJ., Cohen J, Hobbs HH. Monogenic hypercholesterolemia: new insights in pathogenesis and treatment. J Clin Invest. 2003;111(12):1795-1803.
  11. Braenne I, Reiz B, Medack A, et al. Whole-exome sequencing in an extended family with myocardial infarction unmasks familial hypercholesterolemia. BMC Cardiovasc Dis. 2014; 14:108.
  12. Marks D, Thorogood M, Farrer JM, Humphries SE. Census of clinics providing specialist lipid services in the United Kingdom. J Pub Health. 2004;26(4):353-354.
  13. Nordestgaard, BG, Chapman MJ, Humphries SE, et al. Familial hypercholesterolemia is underdiagnosed and undertreated in the general population: guidance for clinicians to prevent coronary heart disease. Consensus Statement of the European Atherosclerosis Society. Eur Heart J. 2013. Accessed September 23, 2014.

The benefits of next-generation sequencing for diagnostic purposes

In the 35 years since its invention, the Sanger method for sequencing, based on biochemical synthesis in the presence of chain inhibitors, has been the dominant approach used for DNA sequencing. The commercial introduction in 2005 of the first system allowing parallel sequencing of massive amounts of DNA by pyrosequencing opened the era of next generation sequencing (NGS). This is a rapidly evolving set of technologies that is revolutionizing genomic research and is being embraced by clinical practice, especially in the area of molecular diagnostics. The emergence of positional sequencing NGS technology is providing unique advantages for the generation of highly accurate sequence information quickly at a much reduced cost, holding great potential for the development of novel diagnostics.

The introduction in 1998 of automated capillary sequencers based on the Sanger method brought the level of throughput necessary to complete the sequencing of the first human genome. This was a massive effort that required the collaboration of more than 20 laboratories for more than a decade at a cost of $3 billion. Since then, advancements in microfabrication and bioinformatics, combined with higher sequencing throughput, have driven the rapid development of NGS technologies. An additional enabler has been the precipitous drop in sequencing costs that make this technology reasonably priced for academic and clinical institutions.1

From generation to generation

The initial NGS sequencers, now termed second generation (2G), relied on the clonal amplification of DNA templates and generated data with very high throughput but short read lengths, amplification-based bias, and increased error rates. To overcome these limitations, 3G systems based on single-molecule sequencing and cycle-free chemistry were introduced in 2008. Currently, 4G sequencers that rely on nanopore technologies are being developed. They can generate much longer reads and employ electronic detection, a much cheaper alternative to light-based detection. 4G is also run without the need for expensive reagents. Positional sequencing technology, one of the most recent innovations in 4G NGS, delivers accurate position measurements between predictable DNA landmarks. It has the capability of providing simultaneously sequenced and positional information. This has good fit for a wide range of applications, from genome sequence assembly and finishing to sequence-based diagnostics. 

In the short time since the first NGS commercial platforms have been available, the technology has significantly impacted genomic research. This has happened through the ability of NGS to facilitate studies that were not previously feasible or affordable, in areas including genomic analysis, metagenomics, transcriptome sequencing, and chromatin analysis.

Clinical applications

This impact is now translating into clinical molecular diagnostics, especially in the context of diseases requiring the interpretation of large amounts of sequence data, with quantitative or high-sensitivity detection. The field that first embraced NGS-based diagnostics was oncology, where there is a need to characterize a complex range of mutations from biopsies, biofluid samples, and genetic diseases, and sequencing of large genomic regions is necessary. Pediatric and pre-natal genomics can greatly benefit from NGS-based diagnostics. For example, NGS is successfully used for the detection of fetal chromosomal aneuploidy from cell-free fetal nucleic acids present in maternal blood during pregnancy. In the context of mitochondrial disorders, NGS can be used to sequence the entire mitochondrial genome in a single run, to measure the degree of mutation heteroplasmy, and to analyze panels of nuclear genes involved in mitochondrial metabolism. In the field of infectious disease, NGS is uniquely positioned to support clinical epidemiologic studies by sequencing genomes of infective organisms such as mycoplasma, enabling the quantitation of viral quasi-species, or monitoring the emergence of drug resistance. 

A new positional sequencing technology that has been recently developed holds great promise for all these clinical diagnostic applications. This is due to its unique ability to quickly generate highly accurate and extensive datasets, providing information about the location, size, and orientation of large-scale structural variants. The platform uses highly scalable semiconductor chips similar to those found on everyday electronic devices. DNA molecules flow physically through these chips, and are read electronically as they pass through chip nanochannels at extremely high rates (above one million bases per second, per channel). The electronic detection system is able to identify sequence tags placed at different intervals, which are then used to generate long-range information for assembling or mapping genomes. The sequence reads are much longer than those obtained from alternative NGS systems, allowing the generation of large-scale information in a cost-effective way. Positional sequencing can provide higher resolution and accuracy, with very short time-to-results and a low cost. This is accomplished through detection that is done electronically and is much less expensive than that of standard NGS methods.

The future is now

Considerable work still needs to be done needed to bring NGS to the field of clinical diagnostics, but improvements on the technical front are occurring at a meteoric pace. NGS is already being used to catalogue mutations driving common multifactorial diseases like cancer, or to elucidate the complex molecular networks underlying disease. Long-read, positional sequencing platforms can generate genomic and transcriptomic information with unprecedented accuracy, speed, throughput, and scalability, all with very low data burden and at low cost. The technology can interpret sequences over lengths large enough to reveal genome structural variants like those present in many cancers. Because the method involves analysis of single DNA molecules, it can enable the discrimination and quantitation of the multiple genome variants present in samples containing heterogeneous cell populations, such as biofluids or tumor samples. Positional sequencing is currently being used for genome mapping and assembly, and for structural variant detection, which has direct impact for cancer diagnostics. In the future, additional applications not easily within the reach of current technologies, such as mapping of heterogeneous tumor samples, will be possible with positional sequencing.

Darren Lee serves as vice president of marketing & business development for Nabsys, Inc., a life sciences company pioneering semiconductor-based tools for genomic analysis.


  1. Wetterstrand KA. DNA sequencing costs: data from the NHGRI Genome Sequencing Program (GSP): Accessed September 19, 2014.