FlashPCR: Revolutionising qPCR by Accelerating Amplification through Low ∆T Protocols.

Versatility, sensitivity, and accuracy have made the real-time polymerase chain reaction (qPCR) a crucial tool for research, as well as diagnostic applications. However, for point-of-care (PoC) use, traditional qPCR faces two main challenges: long run times mean results are not available for half an hour or more, and the requisite high-temperature denaturation requires more robust and power-demanding instrumentation. This study addresses both issues and revises primer and probe designs, modified buffers, and low ∆T protocols which, together, speed up qPCR on conventional qPCR instruments and will allow for the development of robust, point-of-care devices. Our approach, called "FlashPCR", uses a protocol involving a 15-second denaturation at 79 °C, followed by repeated cycling for 1 s at 79 °C and 71 °C, together with high Tm primers and specific but simple buffers. It also allows for efficient reverse transcription as part of a one-step RT-qPCR protocol, making it universally applicable for both rapid research and diagnostic applications.

Maximising the Use of Scarce qPCR Master Mixes.

The COVID-19 pandemic resulted in a universal, immediate, and vast demand for comprehensive molecular diagnostic testing, especially real-time quantitative (qPCR)-based methods. This rapidly triggered a global shortage of testing capacity, equipment, and reagents. Even today, supply times for chemicals from date of order to delivery are often much longer than pre-pandemic. Furthermore, many companies have ratcheted up the price for minimum volumes of reaction master mixes essential for qPCR assays, causing additional problems for academic laboratories often operating on a shoestring. We have validated two strategies that stretch reagent supplies and, whilst particularly applicable in case of scarcity, can readily be incorporated into standard qPCR protocols, with appropriate validation. The first strategy demonstrates equivalent performance of a selection of "past expiry date" and newly purchased master mixes. This approach is valid for both standard and fast qPCR protocols. The second validates the use of these master mixes at less than 1x final concentration without loss of qPCR efficiency or sensitivity.

RT-qPCR Detection of SARS-CoV-2: No Need for a Dedicated Reverse Transcription Step.

Reverse transcription of RNA coupled to amplification of the resulting cDNA by the polymerase chain reaction (RT-PCR) is one of the principal molecular technologies in use today, with applications across all areas of science and medicine. In its real-time, fluorescence-based usage (RT-qPCR), it has long been a core technology driving the accurate, rapid and sensitive laboratory diagnosis of infectious diseases. However, RT-qPCR protocols have changed little over the past 30 years, with the RT step constituting a significant percentage of the time taken to complete a typical RT-qPCR assay. When applied to research investigations, reverse transcription has been evaluated by criteria such as maximum yield, length of transcription, fidelity, and faithful representation of an RNA pool. Crucially, however, these are of less relevance in a diagnostic RT-PCR test, where speed and sensitivity are the prime RT imperatives, with specificity contributed by the PCR component. We propose a paradigm shift that omits the requirement for a separate high-temperature RT step at the beginning of an RT-qPCR assay. This is achieved by means of an innovative protocol that incorporates suitable reagents with a revised primer and amplicon design and we demonstrate a proof of principle that incorporates the RT step as part of the PCR assay setup at room temperature. Use of this modification as part of a diagnostic assay will of course require additional characterisation, validation and optimisation of the PCR step. Combining this revision with our previous development of fast qPCR protocols allows completion of a 40 cycle RT-qPCR run on a suitable commercial instrument in approximately 15 min. Even faster times, in combination with extreme PCR procedures, can be achieved.

Digital PCR can augment the interpretation of RT-qPCR Cq values for SARS-CoV-2 diagnostics.

Coronavirus disease 2019 (COVID-19) is an infectious, acute respiratory disease caused mainly by person-to-person transmission of the coronavirus SARS-CoV-2. Its emergence has caused a world-wide acute health crisis, intensified by the challenge of reliably identifying individuals likely to transmit the disease. Diagnosis is hampered by the many unknowns surrounding this disease, including those relating to infectious viral burden. This uncertainty is exacerbated by disagreement surrounding the clinical relevance of molecular testing using reverse transcription quantitative PCR (RT-qPCR) for the presence of viral RNA, most often based on the reporting of quantification cycles (Cq), which is also termed the cycle threshold (Ct) or crossing point (Cp). Despite it being common knowledge that Cqs are relative values varying according to a wide range of different parameters, there have been efforts to use them as though they were absolute units, with Cqs below an arbitrarily determined value, deemed to signify a positive result and those above, a negative one. Our results investigated the effects of a range of common variables on Cq values. These data include a detailed analysis of the effect of different carrier molecules on RNA extraction. The impact of sample matrix of buccal swabs and saliva on RNA extraction efficiency was demonstrated in RT-qPCR and the impact of potentially inhibiting compounds in urine along with bile salts were investigated in RT-digital PCR (RT-dPCR). The latter studies were performed such that the impact on the RT step could be separated from the PCR step. In this way, the RT was shown to be more susceptible to inhibitors than the PCR. Together, these studies demonstrate that the consequent variability of test results makes subjective Cq cut-off values unsuitable for the identification of infectious individuals. We also discuss the importance of using reliable control materials for accurate quantification and highlight the substantial role played by dPCR as a method for their development.

RT-qPCR Diagnostics: The "Drosten" SARS-CoV-2 Assay Paradigm.

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an established tool for the diagnosis of RNA pathogens. Its potential for automation has caused it to be used as a presence/absence diagnostic tool even when RNA quantification is not required. This technology has been pushed to the forefront of public awareness by the COVID-19 pandemic, as its global application has enabled rapid and analytically sensitive mass testing, with the first assays targeting three viral genes published within days of the publication of the SARS-CoV-2 genomic sequence. One of those, targeting the RNA-dependent RNA polymerase gene, has been heavily criticised for supposed scientific flaws at the molecular and methodological level, and this criticism has been extrapolated to doubts about the validity of RT-qPCR for COVID-19 testing in general. We have analysed this assay in detail, and our findings reveal some limitations but also highlight the robustness of the RT-qPCR methodology for SARS-CoV-2 detection. Nevertheless, whilst our data show that some errors can be tolerated, it is always prudent to confirm that the primer and probe sequences complement their intended target, since, when errors do occur, they may result in a reduction in the analytical sensitivity. However, in this case, it is unlikely that a mismatch will result in poor specificity or a significant number of false-positive SARS-CoV-2 diagnoses, especially as this is routinely checked by diagnostic laboratories as part of their quality assurance.

COVID-19 and Diagnostic Testing for SARS-CoV-2 by RT-qPCR-Facts and Fallacies.

Although molecular testing, and RT-qPCR in particular, has been an indispensable component in the scientific armoury targeting SARS-CoV-2, there are numerous falsehoods, misconceptions, assumptions and exaggerated expectations with regards to capability, performance and usefulness of the technology. It is essential that the true strengths and limitations, although publicised for at least twenty years, are restated in the context of the current COVID-19 epidemic. The main objective of this commentary is to address and help stop the unfounded and debilitating speculation surrounding its use.

CoV2-ID, a MIQE-compliant sub-20-min 5-plex RT-PCR assay targeting SARS-CoV-2 for the diagnosis of COVID-19.

Accurate, reliable and rapid detection of SARS-CoV-2 is essential not only for correct diagnosis of individual COVID-19 disease but also for the development of a rational strategy aimed at lifting confinement restrictions and preparing for possible recurrent waves of viral infections. We have used the MIQE guidelines to develop two versions of a unique five plex RT-qPCR test, termed CoV2-ID, that allows the detection of three viral target genes, a human internal control for confirming the presence of human cells in a sample and a control artificial RNA for quality assessment and potential quantification. Viral targets can be detected either individually with separate fluorophores or jointly using the same fluorophore, thus increasing the test's reliability and sensitivity. It is robust, can consistently detect two copies of viral RNA, with a limit of detection of a single copy and can be completed in around 15 min. It was 100% sensitive and 100% specific when tested on 23 RNA samples extracted from COVID-19 positive patients and five COVID-19 negative patients. We also propose using multiple cycle fluorescence detection, rather than real-time PCR to reduce significantly the time taken to complete the assay as well as assuage the misunderstandings underlying the use of quantification cycles (Cq). Finally, we have designed an assay for the detection of the D614G mutation and show that all of the samples isolated in the Chelmsford, Essex area between mid-April and June 2020, have the mutant genotype whereas a sample originating in Australia was infected with the wild type genotype.

RT-qPCR Testing of SARS-CoV-2: A Primer.

Testing for the presence of coronavirus is an essential diagnostic tool for monitoring and managing the current COVID-19 pandemic. The only reliable test in current use for testing acute infection targets the genome of SARS-CoV-2, and the most widely used method is quantitative fluorescence-based reverse transcription polymerase chain reaction (RT-qPCR). Despite its ubiquity, there is a significant amount of uncertainty about how this test works, potential throughput and reliability. This has resulted in widespread misrepresentation of the problems faced using this test during the current COVID-19 epidemic. This primer provides simple, straightforward and impartial information about RT-qPCR.

Parameters for Successful PCR Primer Design.

Primers are critical components of any PCR assay, as they are the main determinants of its specificity, sensitivity, and robustness. Despite the publication of numerous guidelines, the actual design of many published assays is often unsound: primers lack the claimed specificity, they may have to compete with secondary structures at their binding sites, primer dimer formation may affect the assay's sensitivity or they may bind only within a narrow temperature range. This chapter provides simple guidance to avoid these most common issues.

Talking the talk, but not walking the walk: RT-qPCR as a paradigm for the lack of reproducibility in molecular research.

Poorly executed and inadequately reported molecular measurement methods are amongst the causes underlying the lack of reproducibility of much biomedical research. Although several high impact factor journals have acknowledged their past failure to scrutinise adequately the technical soundness of manuscripts, there is a perplexing reluctance to implement basic corrective measures. The reverse transcription real-time quantitative PCR (RT-qPCR) is probably the most straightforward measurement technique available for RNA quantification and is widely used in research, diagnostic, forensic and biotechnology applications. Despite the impact of the minimum information for the publication of quantitative PCR experiments (MIQE) guidelines, which aim to improve the robustness and the transparency of reporting of RT-qPCR data, we demonstrate that elementary protocol errors, inappropriate data analysis and inadequate reporting continue to be rife and conclude that the majority of published RT-qPCR data are likely to represent technical noise.

Improving the reliability of peer-reviewed publications: We are all in it together.

The current, and welcome, focus on standardization of techniques and transparency of reporting in the biomedical, peer-reviewed literature is commendable. However, that focus has been intermittent as well as lacklustre and so failed to tackle the alarming lack of reliability and reproducibly of biomedical research. Authors have access to numerous recommendations, ranging from simple standards dealing with technical issues to those regulating clinical trials, suggesting that improved reporting guidelines are not the solution. The elemental solution is for editors to require meticulous implementation of their journals' instructions for authors and reviewers and stipulate that no paper is published without a transparent, complete and accurate materials and methods section.

Variability of the reverse transcription step: practical implications.

BACKGROUND: The reverse transcription (RT) of RNA to cDNA is a necessary first step for numerous research and molecular diagnostic applications. Although RT efficiency is known to be variable, little attention has been paid to the practical implications of that variability. METHODS: We investigated the reproducibility of the RT step with commercial reverse transcriptases and RNA samples of variable quality and concentration. We quantified several mRNA targets with either singleplex SYBR Green I or dualplex probe-based reverse transcription real-time quantitative PCR (RT-qPCR), with the latter used to calculate the correlation between quantification cycles (Cqs) of mRNA targets amplified in the same real-time quantitative PCR (qPCR) assay. RESULTS: RT efficiency is enzyme, sample, RNA concentration, and assay dependent and can lead to variable correlation between mRNAs from the same sample. This translates into relative mRNA expression levels that generally vary between 2- and 3-fold, although higher levels are also observed. CONCLUSIONS: Our study demonstrates that the variability of the RT step is sufficiently large to call into question the validity of many published data that rely on quantification of cDNA. Variability can be minimized by choosing an appropriate RTase and high concentrations of RNA and characterizing the variability of individual assays by use of multiple RT replicates.

Biomarkers for invasive aspergillosis: the challenges continue.

The incidence of invasive aspergillosis (IA), an opportunistic infection in immunocompromised individuals, is rising, but its early diagnosis remains challenging and treatment options are limited. Hence there is an urgent need to improve existing diagnostic procedures as well as develop novel approaches. The clinical usefulness of galactomannan and ß-d-glucan, widely used assays detecting cell-wall antigens of Aspergillus, is unclear and depends on clinicians' awareness of their practical limitations. This leaves room for new methods that utilise genomic, proteomic and metabolomics approaches as well as novel detection procedures, for example point-of-care lateral-flow devices. Each of these strategies has its own limitations and it is likely that a combination of methods will be required to achieve optimal performance for the diagnosis of IA and subsequent appropriate patient management.

Minimum information necessary for quantitative real-time PCR experiments.

The MIQE (minimum information for the publication of quantitative real-time PCR) guidelines were published in 2009 with the twin aims of providing a blueprint for good real-time quantitative polymerase chain reaction (qPCR) assay design and encouraging the comprehensive reporting of qPCR protocols. It had become increasingly clear that variable pre-assay conditions, poor assay design, and incorrect data analysis were leading to the routine publication of data that were often inconsistent, inaccurate, and wrong. The problem was exacerbated by a lack of transparency of reporting, with the details of technical information inadequate for the purpose of assessing the validity of published qPCR data. This had, and continues to have serious implications for basic research, reducing the potential for translating findings into valuable applications and potentially devastating consequences for clinical practice. Today, the rationale underlying the MIQE guidelines has become widely accepted, with more than 2,200 citations by March 2014 and editorials in Nature and related publications acknowledging the enormity of the problem. However, the problem we now face is rather serious: thousands of publications that report suspect data are populating and corrupting the peer-reviewed scientific literature. It will be some time before the many contradictions apparent in every area of the life sciences are corrected.

Gene expression analysis by quantitative real-time PCR for floral tissues.

Real-time, or quantitative, reverse transcription polymerase chain reaction (qRT-PCR), is a powerful method for rapid and reliable quantification of mRNA abundance. Although it has not featured prominently in flower development research in the past, the availability of novel techniques for the synchronized induction of flower development, or for the isolation of cell-specific mRNA populations, suggests that detailed quantitative analyses of gene expression over time and in specific tissues and cell types by qRT-PCR will become more widely used. In this chapter, we discuss specific considerations for studying gene expression by using qRT-PCR, such as the identification of suitable reference genes for the experimental setup used. In addition, we provide protocols for performing qRT-PCR experiments in a multiwell plate format (with the LightCycler(®) 480 system, Roche) and with nanofluidic arrays (BioMark™ system, Fluidigm), which allow the automatic combination of sets of samples with sets of assays, and significantly reduce reaction volume and the number of liquid-handling steps performed during the experiment.

The need for transparency and good practices in the qPCR literature.

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.

The digital MIQE guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments.

There is growing interest in digital PCR (dPCR) because technological progress makes it a practical and increasingly affordable technology. dPCR allows the precise quantification of nucleic acids, facilitating the measurement of small percentage differences and quantification of rare variants. dPCR may also be more reproducible and less susceptible to inhibition than quantitative real-time PCR (qPCR). Consequently, dPCR has the potential to have a substantial impact on research as well as diagnostic applications. However, as with qPCR, the ability to perform robust meaningful experiments requires careful design and adequate controls. To assist independent evaluation of experimental data, comprehensive disclosure of all relevant experimental details is required. To facilitate this process we present the Minimum Information for Publication of Quantitative Digital PCR Experiments guidelines. This report addresses known requirements for dPCR that have already been identified during this early stage of its development and commercial implementation. Adoption of these guidelines by the scientific community will help to standardize experimental protocols, maximize efficient utilization of resources, and enhance the impact of this promising new technology.

Real-time quantitative PCR, pathogen detection and MIQE.

Nucleic acids are the ultimate biomarker and real-time PCR (qPCR) is firmly established as the method of choice for nucleic acid detection. Together, they allow the accurate, sensitive and specific identification of pathogens, and the use of qPCR has become routine in diagnostic laboratories. The reliability of qPCR-based assays relies on a combination of optimal sample selection, assay design and validation as well as appropriate data analysis and the "Minimal Information for the Publication of real-time PCR" (MIQE) guidelines aim to improve both the reliability of assay design as well as the transparency of reporting, essential conditions if qPCR is to remain the benchmark technology for molecular diagnosis.

In silico tools for qPCR assay design and data analysis.

qPCR instruments are supplied with basic software packages that enable the measurement of fluorescent changes, calculations of quantification cycle (Cq) values, the generation of standard curves and subsequent relative target nucleic acid quantity determination. However, detailed assessments of the technical parameters underlying Cq values and their translation into biological meaningful results require validation of these basic calculations through further analyses such as qPCR efficiency correction, normalization to multiple reference genes, averaging and statistical tests. Some instruments incorporate some of these features, while others offer additional tools to complement the basic running software, in many cases providing those that are described below. In this chapter, there is a detailed description of some of these programs and recommended strategies for the design of robust qPCR assays. Some of the packages available for validation of the resulting Cq data and detailed statistical analysis are described.