Digital PCR as a tool to measure HIV persistence

Although antiretroviral therapy is able to suppress HIV replication in infected patients, the virus persists and rebounds when treatment is stopped. In order to find a cure that can eradicate the latent reservoir, one must be able to quantify the persisting virus. Traditionally, HIV persistence studies have used real-time PCR (qPCR) to measure the viral reservoir represented by HIV DNA and RNA. Most recently, digital PCR is gaining popularity as a novel approach to nucleic acid quantification as it allows for absolute target quantification. Various commercial digital PCR platforms are nowadays available that implement the principle of digital PCR, of which Bio-Rad's QX200 ddPCR is currently the most used platform in HIV research. Quantification of HIV by digital PCR is proving to be a valuable improvement over qPCR as it is argued to have a higher robustness to mismatches between the primers-probe set and heterogeneous HIV, and forfeits the need for a standard curve, both of which are known to complicate reliable quantification. However, currently available digital PCR platforms occasionally struggle with unexplained false-positive partitions, and reliable segregation between positive and negative droplets remains disputed. Future developments and advancements of the digital PCR technology are promising to aid in the accurate quantification and characterization of the persistent HIV reservoir

The Digital MIQE Guidelines Update: Minimum Information for Publication of Quantitative Digital PCR Experiments for 2020

Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimental protocols, maximize efficient utilization of resources, and further enhance the impact of this powerful technology

Precision cancer monitoring using a novel, fully integrated, microfluidic array partitioning digital PCR platform.

A novel digital PCR (dPCR) platform combining off-the-shelf reagents, a micro-molded plastic microfluidic consumable with a fully integrated single dPCR instrument was developed to address the needs for routine clinical diagnostics. This new platform offers a simplified workflow that enables: rapid time-to-answer; low potential for cross contamination; minimal sample waste; all within a single integrated instrument. Here we showcase the capability of this fully integrated platform to detect and quantify non-small cell lung carcinoma (NSCLC) rare genetic mutants (EGFR T790M) with precision cell-free DNA (cfDNA) standards. Next, we validated the platform with an established chronic myeloid leukemia (CML) fusion gene (BCR-ABL1) assay down to 0.01% mutant allele frequency to highlight the platform's utility for precision cancer monitoring. Thirdly, using a juvenile myelomonocytic leukemia (JMML) patient-specific assay we demonstrate the ability to precisely track an individual cancer patient's response to therapy and show the patient's achievement of complete molecular remission. These three applications highlight the flexibility and utility of this novel fully integrated dPCR platform that has the potential to transform personalized medicine for cancer recurrence monitoring

Digital Droplet PCR for Influenza Vaccine Development

AbstractDevelopment of influenza vaccine processes requires virus quantification to optimize conditions in cell culture or in the associated downstream purification steps. Modern methods include qPCR, which utilizes TaqMan chemistry to detect and quantify viral RNA by comparison of a RNA standard of known concentration. Digital droplet PCR (ddPCR) is similar to qPCR in that it shares the same chemistry for nucleic acid detection. However, in ddPCR, the sample is diluted into partitions (‘droplets’) in order to separate and isolate single molecules. Upon PCR amplification, the droplet's fluorescent intensity depends on the presence or absence of the target; as such, positive and negative droplets are identified, which allows for absolute quantification of the viral genomes. The digital approach has enabled several key advantages. First, a standard is no longer required. Second, efficiency of the reverse transcription and the kinetics of the amplification, principles in qPCR, have no impact on the final digital PCR quantification. For this reason, the extracted RNA does not need to be purified from the reagents needed to lyse the virus. Also, viral associated RNA released by infected cells can be measured directly, further improving the quality of the data generated. Additional improvements to the approach include duplexing with a second assay that measures host cell DNA concentration. The method has been successfully implemented with automation in support of multiple upstream and downstream process development efforts for influenza vaccine manufacturing

Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA Copy Number Quantification

ABSTRACT: Droplet digital polymerase chain reaction (ddPCR) is a new technology that was recently commercialized to enable the precise quantification of target nucleic acids in a sample. ddPCR measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions. This novel ddPCR format offers a simple workflow capable of generating highly stable partitioning of DNA molecules. In this study, we assessed key performance parameters of the ddPCR system. A linear ddPCR response to DNA concentration was obtained from 0.16 % through to 99.6 % saturation in a 20,000 droplet assay corresponding to more than 4 orders of magnitude of target DNA copy number per ddPCR. Analysis of simplex and duplex assays targeting two distinct loci in the Lambda DNA genome using the ddPCR platform agreed, within their expanded uncertainties, with values obtained using a lower density microfluidic chamber based digital PCR (cdPCR). A relative expanded uncertainty under 5 % was achieved for copy number concentration using ddPCR. This level of uncertainty is much lower than values typically observed for quantification of specific DNA target sequences using currently commercially available real-time and digital cdPCR technologies

Metrology and Molecular Diagnosis of Infection

Metrology, the study of measurement, is an emerging concept within molecular diagnosis of infection. Metrology promotes high-quality, reproducible data to be used in clinical management of infection, through characterisation of technical error and measurement harmonisation. This influences measurement accuracy, which has implications for setting thresholds between healthy and disease states, monitoring disease progression, and establishing cures. This thesis examines the placing of metrology in molecular diagnosis of infectious diseases. Sources of experimental error in advanced methodologies – dPCR and MALDI-TOF MS – that can influence measurement accuracy for RNA, DNA and protein biomarkers were investigated for HIV-1, methicillin-resistant Staphylococcus spp and organisms associated with hospital transmission. Measurement error introduced at different stages of a method can directly impact upon clinical results. A 30% bias was introduced between dPCR and qPCR quantification of HIV-1 DNA in clinical samples, owing to instability in the qPCR calibration material. In addition, experimental variability was found to influence classification of protein profiles which can limit the resolution of MALDI-TOF MS for strain typing bacteria. This thesis also addresses the prospective role of these advanced methods in supporting accurate clinical measurements. dPCR offers precise measurements of RNA and DNA targets and could be used to support qPCR, or for value assignment of reference materials to harmonise inter-laboratory results. MALDI-TOF MS demonstrated potential for strain typing Acinetobacter baumannii; results correlated with epidemiological data and WGS, although were not consistent with reference typing. Further work should examine the extent to which MALDI-TOF MS can support or replace contemporary strain typing methods for identifying nosocomial outbreaks. Molecular approaches possess a crucial role in the detection, quantification and characterisation of pathogens, and are invaluable tools for managing emerging diseases. Supporting accuracy and reproducibility in molecular measurements could help to strengthen diagnostic efforts, streamline clinical pathways and provide overall benefit to patient care

Miniaturizing High Throughput Droplet Assays For Ultrasensitive Molecular Detection On A Portable Platform

Digital droplet assays – in which biological samples are compartmentalized into millions of femtoliter-volume droplets and interrogated individually – have generated enormous enthusiasm for their ability to detect biomarkers with single-molecule sensitivity. These assays have untapped potential for point-of-care diagnostics but are mainly confined to laboratory settings due to the instrumentation necessary to serially generate, control, and measure millions of compartments. To address this challenge, we developed an optofluidic platform that miniaturizes digital assays into a mobile format by parallelizing their operation. This technology has three key innovations: 1. the integration and parallel operation of hundred droplet generators onto a single chip that operates \u3e100x faster than a single droplet generator. 2. the fluorescence detection of droplets at \u3e100x faster than conventional in-flow detection using time-domain encoded mobile-phone imaging, and 3. the integration of on-chip delay lines and sample processing to allow serum-to-answer device operation. By using this time-domain modulation with cloud computing, we overcome the low framerate of digital imaging, and achieve throughputs of one million droplets per second. To demonstrate the power of this approach, we performed a duplex digital enzyme-linked immunosorbent assay (ELISA) in serum to show a 1000x improvement over standard ELISA and matching that of the existing laboratory-based gold standard digital ELISA system. This work has broad potential for ultrasensitive, highly multiplexed detection, in a mobile format. Building on our initial demonstration, we explored the following: (i) we demonstrated that the platform can be extended to \u3e100x multiplexing by using time-domain encoded light sources to detect color-coded beads that each correspond to a unique assay, (ii) we demonstrated that the platform can be extended to the detection of nucleic acid by implementing polymerase chain reaction, and (iii) we demonstrated that sensitivity can be improved with a nanoparticle-enhanced ELISA. Clinical applications can be expanded to measure numerous biomarkers simultaneously such as surface markers, proteins, and nucleic acids. Ultimately, by building a robust device, suitable for low-cost implementation with ultrasensitive capabilities, this platform can be used as a tool to quantify numerous medical conditions and help physicians choose optimal treatment strategies to enable personalized medicine in a cost-effective manner

Overview and recommendations for the application of digital PCR

The digital Polymerase Chain Reaction (dPCR), for the detection and absolute quantification of DNA, is a relatively new technique but its application in analytical laboratories is steadily increasing. In contrast to quantitative real-time PCR, DNA (fragments) can be quantified without the need for standard curves. Using dPCR, the PCR mix containing the (target) DNA is partitioned – depending on the device used – currently into a maximum of 10,000,000 small compartments with a volume as low as a few picoliters. These can be either physically distinct compartments on a chip (referred to as chamber-based digital PCR [cdPCR]), or these compartments correspond to water-in-oil droplets (referred to as droplet digital [ddPCR]). Common to both approaches, once PCR has been carried out simultaneously in all compartments/droplets, the number of positive and negative signals for each partition is counted by fluorescence measurement. With this technique, an absolute quantification of DNA copy numbers can be performed with high precision and trueness, even for very low DNA copy numbers. Furthermore, dPCR is considered less susceptible than qPCR to PCR inhibitory substances that can be co-extracted during DNA extraction from different sources. Digital PCR has already been applied in various fields, for example for the detection and quantification of GMOs, species (animals, plants), human diseases, food viruses and bacteria including pathogens. When establishing dPCR in a laboratory, different aspects have to be considered. These include, but are not limited to, the adjustment of the type of the PCR master mix used, optimised primer and probe concentrations and signal separation of positive and negative compartments. This document addresses these and other aspects and provides recommendations for the transfer of existing real-time PCR methods into a dPCR format.JRC.F.5-Food and Feed Complianc

Uuden molekulaarisen menetelmän soveltaminen syöpädiagnostiikkaan : AR-V7 mRNA:n detektio

Huolimatta viimeaikaisista edistysaskelista syöpädiagnostiikan ja syöpähoitojen saralla, on tämä kompleksi ja monitahoinen tauti yhä yksi maailman yleisimmistä kuolinsyistä. Uusia ja nopeita diagnostisia menetelmiä tarvitaan syöpätautien tunnistamiseen niiden aikaisessa kehitysvaiheessa, jotta tautien aiempi prognoosi, parempi riskienhallinta ja tehokkaampi hoito olisivat mahdollisia. Kiinnostus spesifisiin molekulaarisiin biomerkkiaineisiin, jotka toimivat syövän tunnusmerkkeinä, on vähitellen kasvamassa. Näiden merkkiaineiden tunnistaminen nestemäisistä näytemateriaaleista kehittyneiden molekulaaristen diagnostiikkamenetelmien avulla tarjoaa huomattavia etuja perinteisiin onkologiassa käytettäviin kuvantamismenetelmiin verrattuna. Tämän tutkielman tavoite oli tutkia uuden molekulaarisen menetelmän, SIBA®:n (Strand Invasion Based Amplification), soveltuvuutta syöpämerkkiaineiden tunnistamiseen, sekä kehittää testi androgeenireseptorin silmukointivariantti 7 (AR-V7) mRNA:n tunnistamiseen. AR-V7:ä on esitetty hoitovaste-biomerkkiaineeksi potilaissa, joilla on metastaattinen kastraatioresistentti eturauhassyöpä (mCRPC). Tämän variantin ekspressio voi ilmaista kehittynyttä resistenssiä edistyneen eturauhassyövän hoitoon käytetyille hormonaalisille hoidoille. Eturauhassyöpä on maailmanlaajuisesti toiseksi yleisin miehillä esiintyvä syöpä keuhkosyövän jälkeen, ja se voi vähitellen kehittyä pitkälle edenneeksi kuolettavaksi metastaattiseksi kastraatioresistentiksi eturauhassyöväksi, johon androgeeni-deprivaatiohoito ei enää toimi. Positiivisen AR-V7-statuksen on esitetty edustavan tämän pitkälle edenneen eturauhassyövän fenotyyppiä, ja sen tunnistaminen voi auttaa sopivan hoitomuodon valinnassa mCRPC-potilaille. SIBA on uusi isotermaalinen menetelmä nukleiinihappojen monistamiseen ja tunnistamiseen. Teknologia tarjoaa merkittäviä etuja perinteiseen molekulaariseen tunnistusmenetelmään, polymeraasiketjureaktioon (PCR) verrattuna, sillä SIBA-monistus tapahtuu vakiolämpötilassa eikä vaadi lämpösykliseen monistamiseen tarvittavaa hienostunutta laboratoriovälineistöä. Käänteistranskriptio-SIBA (RT-SIBA) mahdollistaa RNA:n käänteistranskription cDNA:ksi samanaikaisesti cDNA:n monistuksen ja tunnistuksen kanssa yksivaiheisessa reaktiossa ja isotermaalisissa olosuhteissa. Menetelmä on osoittanut korkeaa analyyttistä herkkyyttä sekä spesifisyyttä kohdenukleiinihapoille. RT-SIBA-teknologiaa ei ole aiemmin sovellettu ihmisperäisen DNA:n tai RNA:n tunnistamiseen. Tämän tutkielman tärkein havainto oli, että RT-SIBA-teknologiaa voidaan soveltaa molekulaaristen syöpämerkkiaineiden, kuten AR-V7 mRNA:n, nopeaan ja spesifiseen tunnistamiseen. Tässä tutkimuksessa kehitettiin ja optimoitiin kaksi RT-SIBA-testiä, jotka kohdistuivat täyspitkän androgeenireseptori (AR-FL) mRNA:n sekä androgeenireseptorin silmukointivariantti 7 (AR-V7) mRNA:n tunnistamiseen. Näiden testien suorituskykyä arvioitiin testaamalla RNA:ta, joka oli eristetty AR-V7 positiivisista sekä negatiivisista eturauhassyöpäsoluista. Reaktiossa oli samanaikaisesti läsnä myös nestemäistä näytemateriaalia; kokoverta tai plasmaa. Kehitetyt RT-SIBA-testit olivat analyyttisesti erittäin spesifisiä ja herkkiä: ne monistivat alhaisia kopiomääriä kohde-mRNA:ta alle 20 minuutissa ilman epäspesifisten amplikonien muodostumista. Tulokset osoittavat, että RT-SIBA-teknologiaa voidaan hyödyntää AR-V7 ja AR-FL mRNA:n helppoon ja nopeaan tunnistukseen suoraan nestemäisestä näytemateriaalista ilman aikaa vievää näytteenkäsittelyä. Jatkokokeet todellisilla AR-V7-positiivisilla mCRPC-potilaiden kliinisillä näytteillä ovat tarpeellisia, jotta kehitetyt testit voidaan validoida luotettavasti.Despite recent advances in understanding, diagnosis and treatment of cancer, this complex and versatile disease remains one of the leading causes of death worldwide. New and rapid diagnostic methods are needed to detect cancers at their early stages of development, thus enabling earlier prognosis, better risk assessment and more efficient treatment of the disease. There has been an increasing interest in specific molecular biomarkers as the hallmark for cancer research, and the detection of these markers from liquid biopsies using advanced molecular diagnostics methods provides major advantages over the conventional imaging methods currently used in oncology. The aims of this thesis were to examine the applicability of a novel molecular method, SIBA® (Strand Invasion Based Amplification), for the detection of cancer biomarkers, and to develop an assay targeting androgen receptor splice variant 7 (AR-V7) mRNA. The AR-V7 is proposed as a treatment-response biomarker in patients with castration-resistant metastatic prostate cancer (mCRPC). The expression of this variant can indicate resistance to hormonal therapies used for the treatment of advanced prostate cancer. Prostate cancer is the most common cancer after lung cancer in men worldwide and can gradually develop into a highly advanced lethal form, mCRPC, that is not responsive to androgen deprivation therapies. Positive AR-V7 status is suggested to represent the phenotype of this advanced stage of prostate cancer, and its detection can assist in treatment selection for the mCRPC patients. SIBA is a novel isothermal method for the amplification and detection of nucleic acids. The technology offers significant advantages over the more conventional molecular detection method, polymerase chain reaction (PCR), since the amplification reaction occurs at constant temperature and does not require sophisticated laboratory equipment for the thermal cycling. Reverse transcription SIBA (RT-SIBA) enables reverse transcription of RNA to cDNA as well as the simultaneous amplification and detection of the cDNA in one-step reaction under isothermal conditions. The method displays both high analytical sensitivity and specificity to the target nucleic acids. The RT-SIBA technology has not formerly been applied for the detection of human DNA or RNA. The main finding of this thesis was, that the RT-SIBA technology can be applied for rapid detection of specific molecular cancer biomarkers such as the AR-V7 mRNA. In this study, two RT-SIBA assays targeting the full-length androgen receptor (AR-FL) mRNA and the AR splice variant 7 mRNA were developed and optimized. Performance of the assays were evaluated by testing RNA isolates from AR-V7 positive and negative prostate cancer cell lines in the presence of human whole blood and plasma in the reaction. The developed RT-SIBA assays provided high analytical sensitivity and specificity: low copies of the target mRNA were amplified within 20 minutes without the production of non-intended amplicons. The results suggest that the RT-SIBA technology can be utilized for easy and rapid detection of AR-V7 and AR-FL mRNA directly from liquid sample material without a need for time-consuming sample treatment. Further performance evaluation using real AR-V7 positive clinical samples from mCRPC patients is necessary for the reliable validation of the developed assays

The Application of Microbial Source Tracking to aid in Site Prioritization for Remediation in Lower Michigan

Non-point source fecal pollution is a threat to both the environment and public health. Climate change, aging infrastructure, and intensified agricultural practices are predicted to accentuate this issue. In Michigan, due to the high instance of aging infrastructure and intensified agriculture, non-point source fecal pollution has caused many waterbodies to exceed the state standards posing a risk to recreational activities and source water. Due to this threat, there is an increased effort to identify and remediate these sources. My study focused on improving the identification of non-point source fecal pollution through a combination of culture-based and molecular fecal indicator bacteria (FIB) identification, combined with geospatial and statistical modeling approaches. In Chapter 2, I assessed associations between measured FIB and key watershed characteristics in two watersheds located in Ottawa County, Michigan: Bass River and Deer Creek. Results indicated several associations between watershed characteristics and monitored FIB, which should be considered in future non-point source monitoring efforts. In Chapter 3, I created a new tool to aid stakeholders in interpreting FIB monitoring results. This tool was applied to FIB data from the previous chapter as well as FIB data from five public beaches in Macomb County, Michigan. Results indicated that the framework could improve the interpretation of monitored data. Using this tool, stakeholders can better identify and remediate the most impaired areas first, maximizing their impact and minimizing costs. In Chapter 4, I assessed potential improvements to components of a commonly used geospatial model, the Agricultural Conservation Planning Framework (ACPF), and looked at the model’s ability to assess non-point source fecal pollution from runoff derived events. To determine this, I first updated the sediment delivery ratio (SDR) in runoff risk and compared the updated outputs to measured FIB to identify ACPF’s ability to assess FIB concentrations. Results indicated a significant difference between model outputs, but limitations in experimental design precluded an adequate assessment of my objective for this chapter. Recommendations on future studies to properly assess these objectives were offered