Multiplexed, lateral flow, polymerase chain reaction (PCR) techniques for biological identification
W.M. Nelson, G.W. Long and L.M. Cockrell, Tetracore, Inc., USA
Abstract:
This chapter describes the history and development of the real-time polymerase chain reaction (PCR). It describes the types of real-time PCR chemistries and analyses the advantages of real-time PCR compared with other techniques. The chapter goes on to look at relevant considerations when designing a real-time PCR assay – such as contamination, controls and reverse transcription real-time PCR. Instrument platforms are covered, including portable real-time PCR detection, with examples of available systems.
Key words
real-time polymerase chain reaction; instrumentation platform; contamination; reverse transcription
2.1 Introduction
The polymerase chain reaction (PCR) has markedly changed the life sciences field, with its influence touching many areas of science, including molecular biology, medicine, anthropology and forensics, among many others. With the publication of numerous genomes, PCR assays can now be designed to specifically target a unique nucleic acid sequence within a particular organism, allowing the detection and identification of a multitude of biological organisms. Kary B. Mullis, PhD, first invented PCR in 1983, at which time he was a DNA chemist in the Department of Chemistry at Cetus Corporation. Two years later, the PCR technique was published in a Science journal article, which showed successful exponential amplification of a specific sequence within the human beta-globin gene.1 For this, Mullis was awarded the Nobel Prize in chemistry in 1993. PCR is distinguished not only by its strength as a molecular biology technique, but also by its ease of use.
The reason why PCR has made such an impact in the life sciences field is that it filled two gaps that were present in molecular biology. First, scientists needed a method to specifically address a particular region in the nucleic acid sequence of an organism – for example, they needed to focus on a gene of 2500 base pairs among a total genomic sequence of approximately 3 billion base pairs (in humans). Second, scientists needed to be able to amplify this gene ortarget region in order that it could be more easily manipulated with molecular techniques. Before PCR, these techniques were laborious and time-consuming. With the introduction of PCR, they could now be completed in a matter of one to a few days.
2.1.1 Overview of the polymerase chain reaction (PCR)
At its core, the PCR is comparable to the way in which cells replicate DNA each time they undergo mitosis.2 To overcome the need for multiple enzymes, the reaction is exposed instead to repetitive temperature changes. Traditional PCR includes three temperature steps that together make up a reaction ‘cycle’. During the PCR, these cycles are repeated many times; this ‘thermocycling’ allows DNA to accumulate at the completion of each cycle. Because the DNA products produced in each cycle act as templates for amplification in the subsequent cycle, DNA accumulation becomes exponential. These temperature changes are performed automatically by instruments called thermal cyclers.
The initial step in the PCR process is a high-temperature denaturation step. The temperatures used in this step (typically 92–98 °C) cause breakage of the hydrogen bonds that hold the DNA double helix together. This results in separation of the double-stranded DNA into single-stranded DNA. The neighboring base pairs within a strand remain intact, because the strength of the covalent bonds that hold the base pairs together is not impacted even at this high temperature.
In the next step of PCR, the temperature is lowered to allow the primers to anneal to a homologous target sequence within the single-strand DNA template. Primers are synthetic single-stranded nucleic acid sequences that are designed by the user. Typically short (40–70 bp in length, although more current assays require only 18–20 bp in length), these sequences are complementary to the outer edges of the target DNA sequence. Each binds, or ‘anneals’, to the 5′ region of the target sequence within the single-stranded DNA template. The two primers are referred to as forward and reverse primers; the former is complementary to the (+) DNA strand, and the latter is complementary to the (–) strand. At the lower temperatures typically used in this annealing step (usually between 45 °C and 65 °C), hydrogen bonds are again allowed to form between the nucleic acids that make up the primer and their target complementary sequence within the template. The choice of temperature is dependent upon the primer’s melting temperature, or Tm – that is, the temperature at which one-half of the nucleic acid sequence will dissociate and become single-stranded DNA.
The elongation step makes up the third and final step of each temperature cycle. During the elongation step, the DNA polymerase enzyme binds to the DNA strand at the 3′ end of the primer/template DNA complex. The polymerase then progresses along the single-stranded DNA strand, moving in a 5′→3′ direction. As it travels, it incorporates free nucleotides in solution (dNTPs) that are complementary to thetarget DNA sequence. Elongation typically occurs at approximately 72 °C, although this is dependent upon the polymerase enzyme used and can also be affected by DNA sequence.
This entire three-step cycle is repeated for a number of cycles – up to 35–45. At the end of the PCR reaction, the number of DNA molecules that can be amplified approaches 2x, where 2 is the number of copies of target single-stranded DNA molecules present after the initial denaturation, and x is the number of PCR cycles. Because of the high efficiency of PCR, the rate of exponential amplification nears 100%. Thus, a PCR reaction comprised of 35 cycles and starting with just one copy of the target DNA template can generate over 34 billion DNA molecules. In most cases, PCR reactions have a markedly higher number of starting DNA copies to serve as the template; thus, the total DNA accumulation is increased accordingly.
Although the polymerase enzyme is able to attach to the 3′ end of the primer when bound to the target DNA sequence, with this site serving as the initiation of DNA replication, it is unable to identify where the target nucleic acid sequence ends. This becomes unnecessary, though, because as the PCR reaction continues each newly generated strand serves as the template for amplification in the subsequent cycle. As the reaction progresses, it becomes increasingly less likely that the initial DNA sequence will be used as the DNA template strand. Instead, the shorter amplified pieces will have a greater likelihood of being used as the template. Thus, after the first few cycles of PCR, only the region defined by the primers is used as the template. Amplified products are known as ‘amplicons’.
Although the high temperatures during the DNA denaturation step are necessary to fully separate the double-stranded DNA into single-stranded DNA templates, they are detrimental for most polymerases. To overcome this, initial PCR reactions required the investigator to spike the reaction with fresh enzyme after each denaturation step. The discovery of a thermostable polymerase enzyme – Taq polymerase – was a major step towards the widespread implementation of PCR. The Taq DNA polymerase was originally purified from Thermus aquaticus, a thermophilic bacterium identified from a hot spring located in Yellowstone National Park.3 This bacterial species grows naturally at 92.5 °C, and therefore its complement of cellular machinery is able to function at this temperature. After its important impact on PCR was realized, recombinant forms of Taq polymerase began to be produced by a number of biotechnology companies, allowing more widespread distribution.
2.1.2 PCR modifications
Since its discovery nearly three decades ago, numerous modifications of the original PCR method have been reported – two-step PCR, nested PCR, immuno-PCR, lateral flow PCR, and far more than can be mentioned in this chapter. One of these PCR modifications – real-time PCR – will be described in greater detail here. This modification was chosen as a reflection of its amenability and utility for use in the rapid and simple detection and identification of biological samples.
2.2 Real-time PCR: development and description
Traditional PCR is not without its limitations. One of the most important among these is its inherent semi-quantitative nature (at best). Initial development of more quantitative PCR amplification was described in 1991 by Holland and colleagues from the Cetus Corporation.4 These researchers exploited the use of the 5′→3′ exonuclease activity of the Taq enzyme, whereby a small strand of DNA – termed a ‘probe’ and labeled with a radioactive isotope – annealed to the target sequence, yielding a substrate that was specifically cleaved by the enzyme. When separated on a gel, the cleaved probe could be differentiated from the uncleaved version. Because the probe would only be cleaved when bound to the amplified DNA target, this allowed an indirect measurement of DNA amplification. However, this method still relied upon measuring the product at the completion of thermocycling – a concept termed endpoint detection.
Soon after, in 1992–1993, a research group from Roche Molecular Systems, Inc. reported on the use of a video camera to capture the accumulation of doublestranded DNA.5,6 In these papers, ethidium bromide was used as a read-out, as it selectively fluoresces when bound to double-stranded (as opposed to singlestranded) nucleic acids. The authors noted that one of the key concepts afforded by monitoring DNA amplification in real time is the ability to quantitate the results. The number of cycles it took to produce detectable fluorescence was inversely proportional to the amount of starting DNA template – that is, fewer cycles were indicative of a greater amount of target DNA sequence. These papers provided the first example of real-time PCR, showing that it differed from traditional PCR in that the amount of DNA amplified is measured after each cycle, instead of waiting until the completion of the entire PCR reaction.
Since these initial experiments, real-time PCR technology has advanced to incorporate novel detection strategies that overall improve the sensitivity and specificity of detection compared with what had been achieved with radioactive isotopes and ethidium bromide. In addition, specific instrumentation now allows real-time PCR to be performed routinely on the laboratory bench. This has become the foundation for more portable versions, some of which are also substantially ruggedized. These features allow the instruments to travel even to distant places to bring detection and identification techniques to the source, instead of requiring sample transport back to a reference laboratory.
2.2.1 Real-time PCR kinetics
Because the amount of DNA present in each cycle is doubled, PCR reactions follow exponential kinetics. The PCR reaction can be depicted using a sigmoidal-shapedamplification plot, showing the amount of DNA generated throughout the entire amplification process. This sigmoidal amplification plot is only theoretical for traditional PCR reactions, because the amplified DNA is not visualized until the end of the reaction. The sigmoidal curve culminates in a ‘plateau phase’, at which time PCR efficiency is decreased as enzymes, cofactors and other reaction components are used up, and amplification no longer follows exponential kinetics. Because it is no longer possible to calculate DNA amplification predictably during this phase, the visualized product (run on an agarose gel) cannot be used to determine the amount of starting DNA template. Additionally, it is not possible to accurately compare between samples. Instead, for accurate calculations the DNA generated during the PCR reaction must be quantitated as it is being amplified during the exponential phase. The cycles that comprise the exponential phase of each PCR reaction will differ, however, based on a number of variables, such as the concentration of each PCR component as well as the starting amount of DNA template. Thus, it is difficult to predict at what point during the PCR reaction the exponential phase will occur for a given reaction.
Several points can be seen on an amplification plot of a real-time PCR reaction. For example, the normalized reporter (Rn) signal is graphed on the y-axis. Rn is specific for a given real-time PCR instrument, and is a ratio of the fluorescence emitted from the reporter dye divided by the fluorescence emission from a passive reference dye. Passive reference dyes are used to supply an internal fluorescence standard that permits the reporter dye signal to be normalized and corrected for, accounting for fluctuations not due to the PCR itself. The ROX reference dye is often used as the passive reference dye, although other dyes may be used dependent upon the fluorescence channel of the reporter dye.
Another aspect of the real-time PCR amplification plot is the baseline period, which comprises the baseline fluorescence signal present during the initial cycles of the PCR reaction. The baseline period is typically made up of the first 12 to 15 cycles of the PCR reaction, although this differs by instrument. During the baseline period, the fluorescence signal changes very little. Therefore, any fluorescence measured during this period is considered as the background or signal noise of the PCR reaction. Often the baseline period is defined by the instrument’s software, but some instruments allow the user to empirically manipulate which cycles are considered to be part of the baseline period for a given PCR reaction. When doing so, the baseline period should not include the portion of the PCR reaction during which the signal fluorescence begins to rise above the background signal. Additionally, the baseline period should be the same across PCR reactions that are being compared.
A third important point on a real-time PCR reaction plot is the threshold, defined as the Rn determined to be a statistically significant increase over the baseline signal. Like the baseline signal and the Rn itself, the threshold is also specific to the real-time instrument used. While it is typically determined by the instrument’s software, the threshold can be changed by the user. Typically, thethreshold is set to be just above the baseline signal, where the exponential growth portion of the amplification curve begins. This threshold is used to determine the threshold cycle (Ct), or the PCR cycle number at which the Rn of the reaction crosses the threshold. PCR reactions with large amounts of starting DNA templates take less time to cross the threshold compared with PCR reactions that have smaller starting DNA amounts; thus, the Ct value is considered to be inversely proportional to the starting DNA template amount. A 1-log change in starting DNA template concentration correlates with an approximate three-fold change in Ct value. Based on the exponential algorithm of PCR (2x), a single cycle difference in Ct value correlates with an approximate two-fold difference in the amount of starting DNA material.
2.2.2 Real-time PCR chemistries
A multitude of systems have been published as means to detect and measure realtime PCR DNA amplification. As previously discussed, initial real-time PCR assays used ethidium bromide for DNA detection; ethidium bromide was selected for its ability to preferentially bind the double-stranded DNA amplified during the reaction. Subsequent real-time methodologies relied upon isotopic-labeled reagents for detection. However, both ethidium bromide and isotopic reagents have several associated limitations; chief among these are their hazardous characteristics, which require special precautions to be undertaken when they are used.
Most real-time PCR detection systems in current use are based upon a fluorescence chemistry. Fluorescent molecules have the advantage of being highly sensitive and non-hazardous. Although fluorescence detection is employed in a variety of ways, they all share detection of an increase in fluorescence signal as a marker of DNA amplification. Each method of fluorescence detection relies upon specifically designed components that must be carefully designed to ensure optimal efficiency.
DNA-binding dyes
Fluorescent DNA-binding dyes represent the simplest application of fluorescent chemistry to real-time PCR. Fluorescent DNA-binding dyes intercalate within the DNA sequence, inserting among the base pairs that make up the DNA sequence. These dyes have little or no fluorescence when bound to single-stranded DNA, but their fluorescence greatly increases when bound to double-stranded DNA. For this reason, an increase in fluorescence signal is considered to be proportional to an increase in the amount of DNA amplified in each cycle. Fluorescent DNA-binding dyes detect amplicons independent of the DNA sequence; thus, they are a lower-cost alternative that requires little optimization, and are flexible enough to be used across a broad range of PCR assays.
A number of DNA-binding dyes are now commercially available for real-time PCR detection. One of the most widely used of these is SYBR® Green I. Compared with its fluorescence in solution, SYBR®Green I exhibits an approximately 1000-fold increase in fluorescence emission when bound to double-stranded DNA.7 The next-generation DNA-binding dye EvaGreen® may have a higher fluorescent signal, due to an unique dye construction and an ability to use an increased dye concentration in the PCR reaction. Several other DNA-binding dyes have also been reported, including BEBO, YOYO-1 and TOTO-1, among others.8–10
Although simple and low-cost, DNA-binding dyes have the disadvantage of binding all double-stranded DNA present in a reaction. As a result, even nonspecific DNA products will be detected, including primer dimers and off-target amplicons. Due to their non-specific binding, it is not possible to differentiate these non-specific products from the desired DNA target amplicon.
Hydrolysis probes
Fluorescence probes have quickly become the most relied-upon chemistry for realtime PCR detection. Because each probe is designed to recognize and bind only the amplified target DNA sequence, probe-based detection is associated with a higher degree of specificity as compared with DNA-binding dyes. Another advantage of the use of probe-based detection chemistry is the availability of a number of fluorescent dye labels. As a result, different PCR assays can be multiplexed into a single reaction, using uniquely labeled probes that detect different amplified targets. Their disadvantage revolves around their increased design complexity and often a need for a greater degree of optimization, leading to increased cost.
The most widely used fluorescent probe for real-time PCR detection is the TaqMan® probe. The name of this probe refers to its use with the Taq polymerase, which has the characteristics needed for this probe to function. TaqMan® probes are designed to have a reporter fluorescent dye molecule on one end (typically the 5′ end), and a fluorescent quencher molecule labeled on the other end. The fluorescence emitted by the reporter fluorophore is transferred intact to the quencher fluorophore, a reaction that occurs over the very small distance of the length of the probe. This fluorescently labeled probe binds to a complementary sequence located between the forward and reverse primer pair. The Taq polymerase used in conjunction with TaqMan® chemistry has a 5′ to 3′ exonuclease activity; therefore, as the polymerase travels along the template DNA strand, it encounters the probe and excises the probe nucleotides base-by-base. The reporter fluorophore is released from the probe, placing a greater distance between it and the quencher fluorophore. Once separate, the fluorophore produces a fluorescence that is detected when excited at an appropriate wavelength.
Molecular beacons are an alternative probe that can be used in real-time PCR applications. While also dual-labeled probes, molecular beacons differ fromTaqMan probes in that they have short (5–7 bp) complementary sequences added to both the 5′ and the 3′ end of the probe. The hairpin or stem-loop conformation that results from these sequences binding brings the fluorophore and the quencher into close proximity. During the denaturation step of PCR, the conformation is relaxed and the probe is able to bind to its complementary DNA sequence during the annealing step.
With a growing list of fluorophores now commercially available, the use of probes as the detection system allows the real-time PCR reaction to be multiplexed. In multiplexed real-time PCR reactions, two (or more) separate DNA sequences are each targeted by unique primer/probe combinations. Each different probe is labeled with a different fluorophore that is read by the instrument in a different channel. Thus, the number of reactions that may be multiplexed is theoretically only limited by the number of different channels on the real-time PCR instrument. In practice, though, multiplexing reactions often requires optimization of the concentrations of each primer/probe combination.
2.2.3 Real-time PCR reaction components
In addition to the DNA-binding dyes or probes that are specific for real-time PCR reactions, a mixture of reagents are used in the synthesis of new DNA.
Primers
One of the most important design elements in PCR is that of the primers. Primer design constraints for real-time PCR are generally similar to those for traditional PCR, with some exceptions. First, the primers should be designed so that the final amplicon produced is less than 200 bp in size. Although longer amplicons could be used, this requires more time and often decreases the efficiency of the reaction. Typically, the final concentration of each primer ranges between 50 and 500 nM – often 300 nM is a good concentration to begin optimization, from which the primer concentration can be titrated up and down as necessary. Another major difference in the primers used for real-time PCR as compared with traditional PCR is the length. While primers for traditional PCR may be designed to be up to 40 bp in length, the primers used in real-time PCR are generally shorter (between 18 and 24 bp).
When designing real-time PCR primers, a 50% GC content should be the goal. When possible, the 3′ end of the primer should be designed to end in a GC-rich sequence, to boost binding to the DNA template. Additionally, unusual sequences including long chains of base repeats should be avoided. While primers can be designed directly by the user, it is often more efficient to input the desired DNA sequence into one of several primer design programs (either free or for sale) that are now available. Finally, before settling on a particular set of primers, each primer sequence should be used in a BLAST search to avoid non-specific amplification.
DNA Polymerase
Due to its speed and robust activity, Taq polymerase enzyme has become the most widely used polymerase for real-time PCR. It is now widely available from a multitude of commercial suppliers, although differences may exist in purity, robustness and cost.
Even at low temperatures (such as the ambient temperature during reaction set-up), Taq polymerase shows residual activity. This can result in non-specific amplification, as the primers in the reaction mixture are more prone to anneal to incorrect target sequences at these lower temperatures. Though small, these initial non-specific amplifications can ultimately bias the entire reaction.
To overcome this, ‘hot-start’ versions of Taq polymerase were developed. Although several strategies fall under the term ‘hot-start’, they all share in common an ability to inhibit the polymerase’s enzymatic activity at low temperatures. This inhibition is relieved when the reaction undergoes the initial high temperatures of PCR. Depending on the hot-start strategy employed, the real-time PCR reaction protocol may be modified to hold at 95–98 °C from a few seconds to several minutes. Hot-start techniques currently available include chemical modifications of amino acids in the Taq polymerase active site, wax barrier methods, or binding of an antibody to the enzyme itself.
Other additives
Completing the real-time PCR reaction mixture includes a compilation of other reagents and additives (used as necessary). PCR reaction buffer is often supplied with the Taq polymerase. Most buffers are comprised of Tris-HCl and potassium chloride; magnesium chloride (or magnesium sulfate) may or may not be included in the buffer. It is sometimes beneficial to use PCR reaction buffers that do not contain magnesium, instead titrating the magnesium into the reaction until the optimal concentration is achieved. Another important component of the PCR reaction mixture is dNTPs, which are a mixture of dATP, dTTP, dCTP and dGTP Like the primers, the dNTPs should be included in excess concentration in order to let the PCR reaction proceed in the exponential phase.
Some additives often used in traditional PCR may also be necessary for particular real-time PCR reactions. Bovine serum albumin, for example, may help stabilize the Taq polymerase and extend its half-life in the reaction. Formamide, dimethyl sulfoxide (DMSO) and glycerol may be used to enhance amplification of difficult target sequences, such as those high in GC content. Many companies advertise PCR additive solutions to enhance problematic targets. While the make-up of these solutions is most often proprietary, it is likely they contain some combination of these reagents.
2.3 Considerations when developing a real-time PCR assay
2.3.1 Contamination
Although an important consideration for all PCR reactions, contamination is an especially important consideration in real-time PCR assays, given their particular sensitivity. Contamination may occur from an amplicon produced in a previous PCR reaction. For this reason, it is imperative never to open a PCR reaction tube after the PCR has completed, as the amplicon is present in very high concentrations and can be easily spread through aerosol or spray. To help prevent amplicon contamination, also called carryover contamination, some laboratories include a system comprised of dUTP and uracil N-glycosylase (UNG) in their PCR reaction. This strategy first relies on the substitution of dUTP in the place of dTTP during the PCR reaction. All subsequent reactions then include UNG, an enzyme which excises uracil from DNA sequences. A pretreatment of the PCR reaction with UNG will ensure that any carryover contamination from amplicons containing the uracil bases is eliminated, and therefore the only products formed should be specific to that reaction.
Contamination may also occur from the samples used in PCR. For example, if a genomic DNA extraction from a bacterial culture is used as the template in PCR, contamination may occur when that sample tube is opened, and when any subsequent tips or tubes that came into contact with that sample are manipulated. Contaminating DNA can be insidious, and often users are unaware when contamination occurs. For this reason, even practiced technicians should observe careful laboratory techniques, including frequent glove changes, use of filtered pipette tips, and placing used tips and tubes immediately in a bleach solution (such as 100% bleach in a plastic pitcher with a flip-top lid). Laboratory flow should be considered, meaning that samples should be extracted and prepped in an area that is separate from where the PCR reaction mixture is made.
2.3.2 Controls for real-time PCR
In addition to controls appropriate for a particular sample, two reaction controls are often included in real-time PCR. First is the no template control (NTC). This control is a blank, in which either water or Tris-EDTA (or whatever vehicle the sample was diluted in) is added to a PCR reaction tube in place of the sample. NTCs are imperative to check for contamination, and are particularly important when the more non-specific DNA-binding dyes are used instead of probes.
Second is an internal control (IC). The IC is a PCR reaction in itself, comprised of a template (often a short synthetic sequence) and primers and probe to detect that template. These components are all added into the PCR reaction mixture, and undergo PCR amplification simultaneously with the target sequence. Because this is essentially a multiplexed reaction, the IC probe must be labeled with a different fluorophore than the one used for the target probe. While not always necessary, the IC can provide confidence in a negative result. For example, if an experimental sample is shown to be negative in a particular assay, but the IC is positive, than the user can have more confidence that the negative was a true negative, and not due to sample inhibition or the PCR reaction mixture not working correctly.
2.3.3 Reverse transcription real-time PCR
Although PCR requires DNA as a template, expansion of the protocol to include a reverse transcription step allows the user to start with an RNA template that is subsequently converted to a DNA sequence, which then becomes the template for the PCR reaction. In this variation of PCR, the RNA template is converted into a complementary DNA (cDNA) sequence by a reverse transcriptase enzyme. This step typically requires the addition of a long (30–60 min) incubation step at a relatively low (37–55 °C) temperature before the PCR thermal cycling begins. However, newer and more robust enzymatic formulations now offer increased speed, allowing this time to be shortened.
2.4 Real-time PCR instrument platforms
Since the inception of real-time PCR, the number and type of applicable platforms compatible with this type of reaction have markedly grown. Each instrument has its own characteristics which can make it more or less useful for particular assays, and in specific laboratory situations. These characteristics, in addition to cost, should be weighed heavily when considering which platform to invest in, as the extent of the instrument’s capabilities will largely dictate the type and sensitivity of detection assay available to the investigator. A further consideration in the field of pathogen detection is the potential need for portable or field-based systems that can provide on-site or point-of-care real-time molecular identification.
2.4.1 Portable real-time PCR detection
The scientific literature is replete with examples of portable, field-deployed and point-of-care real-time PCR detection of biological organisms. For example, one study developed a real-time reverse transcription-PCR assay designed to detect all subtypes of the influenza A virus.11 This assay, in combination with a portable real-time PCR platform, successfully detected virus-positive samples among 104 clinical specimens. The authors further demonstrated that this diagnostic could successfully be performed in an on-site near-patient environment. Separately, a real-time reverse transcription-PCR assay was used under field conditions tosuccessfully detect foot and mouth disease virus using oral swab specimens from cattle in an outbreak zone.12
Characteristics important to consider when comparing portable real-time platforms include the ability to run autonomously (without a laptop or computer connection), battery type and life, and form factor (including size, weight and space envelope). Another important consideration is the availability of stable reagents that can be transported and stored at ambient temperatures. While the development of portable real-time thermocyclers is relatively recent, great strides have been made in the ruggedization of these sensitive pieces of equipment. Portable real-time instruments from two companies, representative of the industry standards, are discussed below.
Portable thermocyclers available from Idaho Technology, Inc.
The Ruggedized Advanced Pathogen Identification Device (R.A.P.I.D®) Biodetection System (Idaho Technology, Inc.) was the first real-time PCR instrument that was specifically designed for ruggedized field use. This early version of a portable thermocycler adapted an existing platform – the LightCycler® real-time instrument – by integrating it into a portable impact-resistant case. The R.A.P.I.D.® system has a sample capacity of 32, must use freeze-dried reagents supplied by Idaho Technology, Inc. and requires a laptop computer for operation. The R.A.P.I.D.® system served as the basis for the US Joint Biological Agent Identification and Diagnostic System (JBAIDS) program, which granted a contract for the development of rapid positive identification and diagnostic confirmation of biological warfare agents and other pathogens of operational concern for the four branches of the US military.
The RAZOR™ EX is the next-generation field-deployable real-time PCR platform designed by Idaho Technology, Inc. This instrument, which has an even smaller footprint than the R.A.P.I.D.®, is unique in that it uses a patented pouch system to easily combine the sample of interest with freeze-dried reagents. After the prepared pouch is inserted into the instrument, an automated process moves the sample between heat zones to achieve temperature cycling. During the run, a fluorimeter takes fluorescence readings in real time. The RAZOR™ EX only has the capability to detect one dye channel, so assays cannot be multiplexed. A barcode scanner is included to simplify use, allowing the operator to scan the pouch and be directed on pouch preparation; once the reagent-specific pouch is scanned, the instrument selects the appropriate program for PCR.
One of the newest portable real-time technologies introduced by Idaho Technology, Inc. is the FilmArray® BioSurveillance System, a fully automated detection system that integrates sample preparation and purification steps as well as reverse transcription and a two-stage nested multiplex real-time PCR process in a process that takes approximately 1 h. Similarly to the RAZOR™ EX, the FilmArray® uses pouches pre-filled with room temperature freeze-dried reagents, available for the detection of either biothreat agents or respiratory pathogens (the latter of which is cleared by the US Food and Drug Administration). Also, like the RAZOR™ EX, the FilmArray® uses vacuum-controlled syringe loading to deliver predetermined volumes of rehydration solution and the sample of interest into the pouch. Once both liquids are loaded, the pouch is inserted into the FilmArray® and a barcode reader is used to enter the sample into the associated computer. Once the run begins, all remaining steps are completely automated. The cells and viral particles present in the sample are first lysed in a bead-beating process, which agitates ceramic beads at high speeds. Nucleic acids released in this step are bound to magnetic beads and shunted to an adjoining area of the pouch, where they are washed to remove impurities and debris, as well as inhibitors which may interfere with subsequent PCR. The nucleic acids are then eluted off the beads and transferred to the first PCR reaction chamber. A nonspecific reverse transcription step is performed to convert any viral RNA into DNA; this is followed by a highly multiplexed nested PCR process to amplify target DNA in the sample. The resulting PCR products are diluted into a second PCR chamber, where they are mixed with fresh reaction components and aliquoted into individual wells of an array. Each of the wells of this array is pre-spotted with one pair of primers designed to detect sequences that were amplified in the first PCR step. The dual-stage PCR reactions combine to reduce non-specific amplification and detection. Each array well is pre-spotted with primers against one specific target gene. Thus, detection of the amplified product, using a doublestranded DNA-binding dye, can be traced to any given individual well, thus allowing target identification. Positive and negative calls are made by the FilmArray® software.
T-COR™ portable thermocyclers from Tetracore®, Inc.
The T-COR™ (Tetracore®, Inc.) line of real-time portable thermocyclers is unique in that these instruments were designed as open platforms – that is, they are compatible with most commercially available off-the-shelf reagents of the user’s choice. The first generation of these, the T-COR™ 4, is considered field-deployable and is supplied in a ruggedized plastic carrying case with all necessary components. The success of the T-COR™ 4 led to the design and development of a next-generation instrument, the T-COR™ 8. In addition to field use, this newer version is more amenable to point-of-care clinical environments such as clinics, emergency rooms and individual hospital units.
The independent wells of the T-COR™ thermocyclers permit separate protocols to be run concurrently. Further, these independent wells also mean that several samples can be analyzed simultaneously – a capability that could prove especially important for use in cases where there is the potential for high numbers of samples that require testing, or if the pathogen of interest is not known and must be identified from a number of potential culprits. The capability of the T-COR™ thermocyclers is extended by the use of several dye channels, which allows multiplex detection (either multiple targets plus an IC or multiple targets only). Each T-COR™ thermocycler can be powered using either a standard electrical outlet or a rechargeable lithium ion battery. In an example of the true fieldability of the instrument, the T-COR™ 4 was powered via a car charger adapter. The user has a choice of operating the T-COR™ thermocyclers either in stand-alone mode or connected to a personal or laptop computer. Stand-alone operation provides a rapid and simple interface to minimize the need for user input, with runs called by the instrument’s software and results displayed on the instrument’s screen. Computer control of the T-COR™ thermocycler confers the ability to create and edit protocols, visualize and adjust data, and view past runs; in this mode, multiple T-COR™ instruments may be simultaneously run from one system.
Another characteristic that sets the T-COR™ thermocyclers apart from other real-time instruments is the use of a Smart Ct™ value to call a run as positive or negative. As described above, the Ct of a run is typically defined as the PCR cycle at which the fluorescence signal crosses a predetermined threshold specific for a particular platform. In contrast, the T-COR™ thermocyclers use a Smart Ct™, which is defined as the PCR cycle at which an exponential rise in fluorescence signal is first detected. Thus, the Smart Ct™ value is a surrogate for the beginning of the exponential phase of DNA amplification. Compared with the typical Ct value, the Smart Ct™ value is associated with a much lower variance, making it more consistent and reliable for run comparisons. When a database of over 3600 real-time PCR standard curves was used to compare the typical Ct value with the Smart Ct™ value, the latter was associated with a four-fold decrease in the number of false positives and a seven-fold decrease in the incidence of false negatives.