Bulk PCR Research Articles

Koala ocular disease grades are defined by chlamydial load changes and increases in Th2 immune responses.

This study employs bulk RNA sequencing, PCR, and ELISA assays to analyze the pathological factors affecting the outcomes of C. pecorum ocular infections in koalas. It investigates the immune responses and gene expression profiles associated with various stages of koala ocular chlamydiosis. A cohort of 114 koalas from Queensland, Australia were assessed, with 47% displaying clinical signs of ocular disease. Animals were classified into three cohorts: acute active disease (G1), chronic active disease (G2), and chronic inactive disease (G3), along with subclinical Chlamydia pecorum positive (H2) and healthy (H1) cohorts. Analysis of clinical, microbiological, humoral immune and cellular immune biomarkers revealed varying chlamydial loads and anti-chlamydial IgG levels across disease grades, with a negative correlation observed between ocular chlamydial load and anti-chlamydial IgG. Koala ocular mucosa gene expression analysis from 27 koalas identified shared expression pathways across disease cohorts, with a significant upregulation of IFNγ expression and tryptophan metabolism in all disease stages. These findings help elucidate immune response dynamics and molecular pathways underlying koala ocular chlamydiosis, providing insights crucial for disease management strategies.

Characteristics (sensitivity and specificity) of herd-level diagnostic tests for Mycobacterium avium subspecies paratuberculosis in cattle — A systematic review

Diagnostic tests for Johne’s disease in cattle are characterised by poor sensitivity and often imperfect specificity at the animal level. Because farmers and veterinarians have limited assurance or confidence from results of testing individual animals for Mycobacterium avium subsp. paratuberculosis (MAP), control programmes based on whole herd-level testing provide the best framework for classifying herds. At the herd level, there is a diverse range of testing options for MAP based on both direct and indirect testing of individual and pooled samples. The most common measures of herd test performance, herd sensitivity (HSe) and herd specificity (HSp), are important for decision-making in herd test selection, for estimating prevalence and as inputs for simulation studies. This systematic review investigated the results of herd test evaluations for MAP in cattle, through a comprehensive search of the literature and a systematic four-stage screening process to identify relevant publications.Forty-six publications with relevant results were eligible for inclusion in the final review, containing evaluations of whole-herd ELISA serological testing, bulk milk tank ELISA, culture, PCR and phage testing, pooled faecal testing and environmental sample testing. Data extracted from each publication included sample populations, methods of analysis, reference tests, cut-off points, HSe and HSp. Direct comparisons between the reported HSe and HSp estimates of different studies is challenging due to the variations in herd prevalence and test protocols used. The data in this systematic review will benefit decision-makers and researchers and highlights knowledge gaps requiring further research.

Evaluation of microfluidics-based droplet PCR combined with multiplex STR system in forensic science.

Because of its excellent monodispersity, high throughput, and low volume, microfluidics-based droplet PCR has become the core technology of digital PCR, next-generation sequencing, and other technology platforms. This study constructed a microfluidic water-in-oil droplet PCR system and amplified a commercially available forensic 22-plex short tandem repeat detection system. We analyzed the sensitivity, concordance, amplification efficiency of the droplet PCR, and influence factors of the above aspects. The droplet PCR showed high concordance with conventional bulk PCR and had high sensitivity as 0.125ng. Furthermore, we observed the performance of droplet PCR in high-order mixed DNA. As the mixture ratios from 10:1 to 30:1, droplet PCR presented more mixture proportion (Mx) increased loci from 11 (57.89%) to 17 (89.47%). In the mixture ratios 20:1, 25:1, and 30:1, significant Mx differences between droplet PCR and bulk PCR were observed (p <0.05). The results showed that the droplet PCR could improve the identification of the minor contributor's DNA in a two-person mixture and alleviate the imbalanced amplification problem. This study provides a reference and basis for the wide application of droplet PCR in forensic science.

Liquid drop of DNA libraries reveals total genome information

Conventional "bulk" PCR often yields inefficient and nonuniform amplification of complex templates in DNA libraries, introducing unwanted biases. Amplification of single DNA molecules encapsulated in a myriad of emulsion droplets (emulsion PCR, ePCR) allows the mitigation of this problem. Different ePCR regimes were experimentally analyzed to identify the most robust techniques for enhanced amplification of DNA libraries. A phenomenological mathematical model that forms an essential basis for optimal use of ePCR for library amplification was developed. A detailed description by high-throughput sequencing of amplified DNA-encoded libraries highlights the principal advantages of ePCR over bulk PCR. ePCR outperforms PCR, reduces gross DNA errors, and provides a more uniform distribution of the amplified sequences. The quasi single-molecule amplification achieved via ePCR represents the fundamental requirement in case of complex DNA templates being prone to diversity degeneration and provides a way to preserve the quality of DNA libraries.

Temporal trends in bulk tank milk antibody ELISA and PCR test results for bovine viral diarrhoea in New Zealand pastoral dairy herds

ABSTRACT Aims: To describe temporal trends in bulk milk antibody ELISA and PCR testing for bovine viral diarrhoea (BVD) in New Zealand pastoral dairy herds and to assess the use of historical accession data to predict herd-level BVD incursions. Methods: Data on all diagnostic testing of bulk milk for BVD performed by the Livestock Improvement Corporation (Hamilton, NZ) over eight lactation seasons from 1 June 2010 to 31 May 2018 were analysed. This included anonymised herd identification, geographic location, herd size, sample collection date, sample to positive (S/P) ratio for antibody ELISA results, and cycle threshold values for PCR detecting viral RNA. Multivariable logistic regression was used to explore the relationship between historical accession data and the risk of herds having at least one positive bulk milk PCR test result in the 2017 season. Results: There were 156,034 bulk milk BVD diagnostic testing accessions for 10,495 uniquely identified dairy herds over the 8-season period. The prevalence of tested herds with at least one positive bulk milk PCR test result decreased from 14.6% (407/2,786) in the 2010 season to 5.6% (355/6,309) in the 2017 season with similarly marked declines in S/P ratios. In the 2017 season, 2,961/6,309 (46.9%) herds had S/P ratios greater than the 0.75 cut-off value indicating recent or active BVD virus transmission within the herd while 1,422/6,309 (22.5%) herds were classified as having negative or low S/P ratios. Herds that cleared BVD from the milking herd experienced a mean decline in S/P ratio of 0.11 units per year (min 0.05; max 0.18). In the multivariable analysis, the overall incidence risk of herds experiencing a BVD incursion in the 2017 season was 3.8% (146/3,848) and there were three significant predictors in the final model: herd size, PCR status in the 2014 season, and change in S/P ratio between the 2014 and 2015 seasons. The area under the receiver operating curve for the final model was 0.695 indicating poor discrimination. Conclusions and clinical relevance: The prevalence of dairy herds in New Zealand with positive bulk milk PCR test results and high S/P ratios has decreased over time, suggesting fewer herds are actively infected with BVD and that herd immunity may also be declining. Although monitoring trends in bulk milk test results provides useful information on changes in individual herd status, it is difficult to accurately predict when new incursions will occur and farmers should continue to maintain good biosecurity.

Detection of osteopontin gene transcripts in bull spermatozoa

The present experiment was aimed to detect osteopontin gene transcript from bull spermatozoa. Fresh semen samples from 12 Jersey cross breed bulls were collected using artificial vagina. The volume and concentration of the individual samples were recorded. The spermatozoa were separated from bull semen samples by swim up protocol using sperm TALP. The initial concentration of semen sample was checked immediately before proceeding for RNA isolation and normalization of initial concentration was done. So, initial amount of every sample was made equal. Total RNA from the bull spermatozoa were extracted by the RNeasy® Mini Kit, Qiagen as per manufacturer’s protocol. The purity and concentration of the samples were measured at A280/260 by using Nano Drop TM 1000 spectrophotometer. The first strand cDNA was synthesized from 1μg total RNA using M-Mu LV Reverse Transcriptase Revert Aid TM H minus first strand cDNA synthesis kit (Thermo Scientific) as per manufacturer’s protocol. PCR product of about 267 bp fragment length of OPN gene was confirmed by conventional PCR. The bulk PCR product of about 100μl was purified by Gen Elute™ PCR Clean-Up Kit Sigma-Aldrich as per manufacturer’s protocol. The concentration and purity of PCR product were 64ng/μl and 1.6 respectively. The cloning and expression of OPN gene was carried out by using TA cloning kit. The ligated product was then transformed into E. coli DH5α cells. The plate was incubated for overnight at 37 ˚C for selection of the colonies and stock cultures were maintained in LB media at -80˚ C. The clones which showed the amplification of 400 bp DNA fragment were considered as positive clone carrying desired insert. Plasmid isolation was done as per the protocol followed in Hi Yield TM Plasmid Mini Kit (RBC Cat. NO. YPD100) and the eluted DNA were stored at -20 ˚C. The concentration and purity of PCR purified product were 67ng/μl and 1.6 respectively. The eluted plasmid DNA was sequenced commercially (Amnion, Bangalore) using M13 primers. The cloned partial sequence of OPN gene isolated from bovine spermatozoa was submitted at DDBJ (Accession No. AB983656). On blast analysis using NCBI nucleotide blast (Online tool), 99 per cent identity was found with reported consensus sequences except single nucleotide variation in DNA sequence at 234th nucleotide. There was substitution of adenine instead of cytosine.

Abstract 2278: A high throughput method for the optimization of digital PCR assays for personalized circulating tumor DNA detection

Abstract Single color digital PCR (sc-dPCR) is a robust approach for the quantitation of low allelic fraction mutations in clinical oncology samples. More recently this technology has been employed to identify mutations from circulating tumor DNA (ctDNA) that has been extracted from the blood samples of cancer patients. The use of digital PCR has great potential for non-invasive longitudinal monitoring via liquid biopsies. However, this application requires low input DNA volumes and relies on a single nucleotide variant (SNV) to distinguish between normal and ctDNA, necessitating that sc-dPCR primer binding is both highly efficient and specific. These stringent requirements make assay optimization a tedious process that greatly limits the rate at which personalized detection panels can be generated. We have developed a high throughput method to optimize sc-dPCR assays utilizing Next Generation Sequencing (NGS) technology to assess amplification more quickly and with more flexibility than traditional gel based analysis. Using our assay optimization approach, a segment of each gene containing a tumor specific SNV was incorporated into the genome of Saccharomyces cerevisiae. These renewable positive control colonies were cultured in a 96 well plate format and pooled to mimic the low allelic frequency conditions of ctDNA. The presence of each tumor specific SNV was confirmed by preparing and sequencing a library containing the unique barcode region of each colony. Using bulk PCR, up to 96 primer sets were tested at one annealing temperature in a singleplex format. Alternatively, we multiplexed up to 11 primers in each well, greatly increasing the number of assays that can be developed per plate. Using this multiplexed format, we introduced a thermal gradient across the plate to identify the optimal annealing temperature of each primer set in a single run. A parallel experiment with identical PCR conditions was run using NA18507 human DNA to act as a negative control for primer specificity. All amplicons in each PCR condition were uniquely indexed and sequenced using an NGS platform. Using a ratio of the number of reads associated with on target and non-mutation specific amplicon sequences for each primer set, the success of each assay was determined. This method was also used to identify specific mismatches incorporated in the primer sequence that increased binding specificity. Using a sequencing based analysis method, we have observed that sc-dPCR assays can be optimized rapidly across multiple mutations, making them more accessible for personalized monitoring. Citation Format: Maya M. Arce, Christina Wood-Bouwens, Derrick Haslem, Billy T. Lau, John Bell, Alison Almeda, Matt Kubit, Bryce Moulton, Robin Romero, Robert P. St. Onge, Lincoln Nadauld, Hanlee P. Ji. A high throughput method for the optimization of digital PCR assays for personalized circulating tumor DNA detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2278.

Abstract 1604: Highly sensitive digital detection of circulating DNA cancer mutations using synthetic genome standards

Abstract With the emergence of precision cancer medicine, there is an increasing need for new longitudinal diagnostic tests to evaluate patients at the initial diagnosis, during treatment, and for routine monitoring. As a general solution, we developed a single-color digital PCR assay technology that detects and quantifies circulating DNA cancer mutations collected from the plasma of cancer patients. The assay has a sensitivity of 0.1% mutation allelic fraction and can be designed for nearly any cancer mutation. In addition, this assay demonstrates design flexibility, low cost, and robust performance, making it a powerful approach for longitudinal monitoring of cancer patients during treatment. Positive controls are a critical component for highly sensitive detection of circulating DNA cancer mutations. However, there are minimal resources for providing standard curves. Commercially available cancer cell lines cover a very limited number of cancer mutations. Amplicons or oligonucleotides pose a high risk of contamination due to the sheer number of molecules generated, an important consideration when working with rare molecular species such as cell-free DNA. To address this challenge, we developed a novel technology that enables a single copy of synthetic human genome sequence to be introduced into a non-mammalian eukaryotic genome. The system highly-scalable and capable of producing thousands of unique clones which can be archived for future use, accessed individually, or combined to create customizable target pools on-demand. These custom designed synthetic positive controls are therefore readily renewable, require no amplification to obtain, and most importantly allow precise control over the number of molar equivalents of each target in a reaction. Using these synthetic controls, we simulate low mutation-allele fractions to validate the specificity and sensitivity of our assay. As a proof of concept, we have designed approximately 400 cancer mutation controls in driver genes such as KRAS and EGFR, and developed a design process to generate multiplex digital PCR assays targeting these driver genes. We will validate the digital PCR assays with standard bulk PCR and will validate the template by comparing to results generated using cancer cell line derived DNA. In the era of circulating DNA diagnostics, these genome engineered synthetic controls meet a critical need for reliable and customizable positive controls. Citation Format: Christina M. Wood-Bouwens, Robert P. St.Onge, Hanlee P. Ji. Highly sensitive digital detection of circulating DNA cancer mutations using synthetic genome standards [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1604.

Individuals with HIV-1 Subtype C Infection and Cryptococcal Meningitis Exhibit Viral Genetic Intermixing of HIV-1 Between Plasma and Cerebrospinal Fluid and a High Prevalence of CXCR4-Using Variants.

The genotypic properties of human immunodeficiency virus type 1 (HIV-1) subtype C in individuals presenting with cryptococcal meningitis (CM) are not well established. Employing single-genome amplification as well as bulk PCR, cloning and sequencing strategies, we evaluated the genetic properties of HIV-1 subtype C env in 16 antiretroviral therapy-naive study participants with CM. Eleven of the 16 participants had matched blood plasma and cerebrospinal fluid (CSF) evaluated, with the rest having either a plasma or CSF sample evaluated. Before antiretroviral therapy initiation, matched plasma and CSF-derived env sequences of all 11 participants displayed genetic intermixing between the two compartments. Overall, 7 of the 16 (∼43.8%) participants harbored CXCR4-using variants in plasma and/or CSF, according to coreceptor usage prediction algorithms. This study suggests that HIV-1 subtype C genetic intermixing between peripheral blood and the central nervous system is common in individuals presenting with CM, and that CXCR4 usage is present in one or both compartments in approximately 44% of individuals.

Single genome amplification and standard bulk PCR yield HIV-1 envelope products with similar genotypic and phenotypic characteristics

Recent studies suggest that single genome amplification (SGA) as compared to standard bulk PCR and virus stocks from 293T transfection versus short term passage in peripheral blood mononuclear cells (PBMC) yield a less biased representation of HIV-1 envelope characteristics. In 9 different subjects, genetic diversity, divergence, and population structure were not significantly different among SGA or bulk PCR amplified envelope V1–V3 segments. In these subjects, 293T transfection derived virus stocks with SGA or bulk PCR amplified envelopes have similar infectivity, replication kinetics, co-receptor usage, and neutralization susceptibility. While PBMC passage as compared to the 293T derived virus stocks had similar co-receptor usage, PBMC viruses were less neutralization susceptible to some specific antibodies. Our results suggest that the method of envelope sequence amplification, either SGA or bulk PCR, does not have a significant impact on the genotypic and phenotypic properties of the virus envelope quasispecies.

Minimizing inhibition of PCR-STR typing using digital agarose droplet microfluidics.

The presence of PCR inhibitors in forensic and other biological samples reduces the amplification efficiency, sometimes resulting in complete PCR failure. Here we demonstrate a high-performance digital agarose droplet microfluidics technique for single-cell and single-molecule forensic short tandem repeat (STR) typing of samples contaminated with high concentrations of PCR inhibitors. In our multifaceted strategy, the mitigation of inhibitory effects is achieved by the efficient removal of inhibitors from the porous agarose microgel droplets carrying the DNA template through washing and by the significant dilution of targets and remaining inhibitors to the stochastic limit within the ultralow nL volume droplet reactors. Compared to conventional tube-based bulk PCR, our technique shows enhanced (20 ×, 10 ×, and 16 ×) tolerance of urea, tannic acid, and humic acid, respectively, in STR typing of GM09948 human lymphoid cells. STR profiling of single cells is not affected by small soluble molecules like urea and tannic acid because of their effective elimination from the agarose droplets; however, higher molecular weight humic acid still partially inhibits single-cell PCR when the concentration is higher than 200 ng/μL. Nevertheless, the full STR profile of 9948 male genomic DNA contaminated with 500 ng/μL humic acid was generated by pooling and amplifying beads carrying single-molecule 9948 DNA PCR products in a single secondary reaction. This superior performance suggests that our digital agarose droplet microfluidics technology is a promising approach for analyzing low-abundance DNA targets in the presence of inhibitors.

Plasmatic Tumor Dna Assessments Predict Clinical Outcome in Egfr-Mutated Non-Small Cell Lung Cancer Patients Treated By Egfr Inhibitors

ABSTRACT Aim: We aimed to study the dynamics of plasmatic EGFR mutations by qualitative (Q-PCR) and quantitative (droplet PCR) methods in EGFR-mutated lung cancer patients treated by EGFR tyrosine kinase inhibitors (EGFR-TKI). Methods: We investigated a single-institution cohort of patients treated by EGFR-TKI. Plasma blood samples and RECIST assessments were repeated every 2 months. Plasmatic cell-free DNA extraction was performed with the QIAsymphony DSP Virus/Pathogen kit (Qiagen). EGFR mutations were assessed in plasmatic DNA using Q-PCR and droplets digital PCR with CAST probes (L858R and Del19). The rate of EGFR mutant/wild-type droplets was used for normalization. The prediction of plasmatic DNA changes between two consecutive samples on treatment outcome was evaluated by Receiver Operator Characteristic (ROC). Survival was estimated with the Kaplan-Meier method. Results: 22 EGFR-mutated patients treated by EGFR-TKI underwent plasmatic DNA follow-up. Patients were stage IV (n = 22), TKI naive (n = 17), or TKI pre-treated (n = 5). Median follow-up, progression-free survival (PFS) and overall survival were 19.0, 4.7 and 15.8 months, respectively. At baseline, tumor plasmatic DNA was found by Q-PCR, droplets or one of the two technics in 14, 15 and 16/20 (80%) patients, respectively. Droplet changes under treatment were more accurate than bulk PCR changes to predict RECIST outcome (progressive disease vs. clinical benefit, ie stable disease or partial response): ROC area under the curve (AUC) 0.92 and 0.35, respectively. Droplet changes also predict 2-month RECIST outcome, ROC AUC 0.94. According to ROC Youden indexes, a droplets increase of +19% best predicts immediate (sensitivity 0.90, specificity 0.84) and 2-months RECIST outcome (sensitivity 0.88, specificity 0.96). Median PFS assessed by droplets was 3.4 months (cut off +19%). Mean time from droplets increase (cut off +19%) to RECIST progression was 42 ± 23 days. Conclusions: In this feasibility cohort, droplets increase under EGFR-TKI predicts immediate and 2-month treatment outcomes. Plasmatic tumor DNA monitoring could anticipates the radiological tumor progression, allowing clinicians to change treatment before a symptomatic or lethal tumor progression occurs. The threshold of +19% droplets increase will be prospectively tested in an on-going biomarker trial. Disclosure: N. Pecuchet: I received honorarium from Roche; P. Laurent-Puig: I received honorarium from RainDance; E. Fabre: I received honorarium from Roche and AstraZeneca; V. Taly: I received honorarium from RainDance. All other authors have declared no conflicts of interest.

Rapid 16S rRNA Next-Generation Sequencing of Polymicrobial Clinical Samples for Diagnosis of Complex Bacterial Infections

Classifying individual bacterial species comprising complex, polymicrobial patient specimens remains a challenge for culture-based and molecular microbiology techniques in common clinical use. We therefore adapted practices from metagenomics research to rapidly catalog the bacterial composition of clinical specimens directly from patients, without need for prior culture. We have combined a semiconductor deep sequencing protocol that produces reads spanning 16S ribosomal RNA gene variable regions 1 and 2 (∼360 bp) with a de-noising pipeline that significantly improves the fraction of error-free sequences. The resulting sequences can be used to perform accurate genus- or species-level taxonomic assignment. We explore the microbial composition of challenging, heterogeneous clinical specimens by deep sequencing, culture-based strain typing, and Sanger sequencing of bulk PCR product. We report that deep sequencing can catalog bacterial species in mixed specimens from which usable data cannot be obtained by conventional clinical methods. Deep sequencing a collection of sputum samples from cystic fibrosis (CF) patients reveals well-described CF pathogens in specimens where they were not detected by standard clinical culture methods, especially for low-prevalence or fastidious bacteria. We also found that sputa submitted for CF diagnostic workup can be divided into a limited number of groups based on the phylogenetic composition of the airway microbiota, suggesting that metagenomic profiling may prove useful as a clinical diagnostic strategy in the future. The described method is sufficiently rapid (theoretically compatible with same-day turnaround times) and inexpensive for routine clinical use.

Abstract 3399: Detection of rare mutations in plasma by droplet digital PCR

Abstract Detection and quantitation of specific mutations in circulating plasma holds promise for earlier and less invasive diagnosis of disease. This presents significant analytical challenges, particularly as the biomarker may differ from its highly abundant wildtype by only a single nucleotide. Conventional methods have poor selectivity and fail to detect mutant sequence below 1 in 100 wildtype sequences. Compounding this, the amount of circulating nucleic acid in plasma is low. Here we present a simple strategy using droplet digital™ PCR (ddPCR™) for the detection of somatic mutations with high selectivity and sensitivity. Based on the simple principle of sample partitioning into water-in-oil microdroplets, this ddPCR method increases the abundance of a mutant DNA sequence up to 20,000 times compared to an equivalent bulk PCR reaction. Using conventional TaqMan chemistries and workflow, selectivities of up to 1/100,000 can readily be achieved in any laboratory. We evaluated ddPCR for the detection and quantitation of several clinically important mutations in the EGFR and KRAS loci from clinical samples derived from normal and tumor plasma samples. We also demonstrate the feasibility of multiplexing of Kras and EGFR assays to improve sample processing efficiency. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3399. doi:1538-7445.AM2012-3399

Abstract 4859: Ultra-sensitive detection of rare mutants by droplet digital PCR with conventional TaqMan assays

Abstract Molecular tests for genetic mutations play an important role in the diagnosis of cancer. Somatic mutations that drive the pathological features of most tumors have increasing promise as biomarkers for cancer prognosis and therapeutic efficacy. The detection of somatic mutations poses an analytical challenge due to the heterogeneous nature of most samples, where a gene carrying a mutation may differ from the highly abundant wild type sequence by only a single nucleotide. Although a variety of methods exist for mutation analysis, many have poor selectivity and fail to detect mutant sequence below 1 in 100 wildtype sequences. Methods that provide better discrimination and quantitation of somatic mutations are desirable. Here we present a simple strategy using droplet digital™ PCR (ddPCR™) for the detection of somatic mutations with high selectivity and sensitivity. Based on the simple principle of sample partitioning into water-in-oil microdroplets, this ddPCR method increases the abundance of a mutant DNA sequence up to 20,000 times compared to an equivalent bulk PCR reaction. Using conventional TaqMan chemistries and workflow, selectivities of up to 1/100,000 can readily be achieved in any laboratory. Here we present results on the use of ddPCR for the detection and quantitation of several clinically important mutations, including KRAS, c-KIT D816V and JAK2 from clinical samples such as bone marrow aspirates and FFPE. Results from ddPCR are compared to those of conventional approaches including allele specific real-time PCR and sequencing. This ddPCR method may play an important role in the earlier detection of cancer, monitoring the progress of disease and response to therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4859. doi:1538-7445.AM2012-4859

Investigating retroviral super-infection in wild chimpanzees (Pan troglodytes verus)

Background While the molecular epidemiology of retroviruses in wild primate populations has received much attention, the related question of the frequency and nature of super-infection events has remained largely neglected. Here, we explicitly investigated it, focusing on simian foamy viruses (SFV) infecting wild chimpanzees (Pan troglodytes verus), as an example. Methods We first compared the costs and benefits of end-point dilution PCR (EPD-PCR) (currently considered a gold standard method) and multiple bulk PCR cloning, when applied to non-invasive samples. For the latter method, we had to develop a specific analytical framework aimed at deciphering relevant biological information from method-induced biases. We then applied multiple bulk PCR cloning to samples collected from chimpanzees belonging to different age classes.

Genetic Identity and Biological Phenotype of a Transmitted/Founder Virus Representative of Nonpathogenic Simian Immunodeficiency Virus Infection in African Green Monkeys

Understanding the lack of disease progression in nonpathogenic simian immunodeficiency virus (SIV) infections is essential for deciphering the immunopathogenesis of human AIDS. Yet, in vivo studies have been hampered by a paucity of infectious molecular clones (IMCs) of SIV suitable to dissect the viral and host factors responsible for the nonpathogenic phenotype. Here, we describe the identification, cloning, and biological analysis of the first transmitted/founder (T/F) virus representing a nonpathogenic SIV infection. Blood was collected at peak viremia from an acutely infected sabaeus monkey (Chlorocebus sabaeus) inoculated intravenously with an African green monkey SIV (SIVagm) strain (Sab92018) that had never been propagated in vitro. To generate IMCs, we first used conventional (bulk) PCR to amplify full-length viral genomes from peripheral blood mononuclear cell (PBMC) DNA. Although this yielded two intact SIVagmSab genomes, biological characterization revealed that both were replication defective. We then performed single-genome amplification (SGA) to generate partially overlapping 5' (n = 10) and 3' (n = 13) half genomes from plasma viral RNA. Analysis of these amplicons revealed clusters of nearly identical viral sequences representing the progeny of T/F viruses. Synthesis of the consensus sequence of one of these generated an IMC (Sab92018ivTF) that produced infectious CCR5-tropic virions and replicated to high titers in Molt-4 clone 8 cells and African green monkey PBMCs. Sab92018ivTF also initiated productive infection in sabaeus monkeys and faithfully recapitulated the replication kinetics and nonpathogenic phenotype of the parental Sab92018 strain. These results thus extend the T/F virus concept to nonpathogenic SIV infections and provide an important new tool to define viral determinants of disease nonprogression.

Lentiviral Vector-Mediated Transfer of the Gamma-Globin Gene Into Normal and Beta-Thalassemic Human CD34+ Cells Results in Potentially Therapeutic Levels of Fetal Hemoglobin in Erythroid Progeny.

Abstract 3579Poster Board III-516Beta-thalassemia results from severely reduced or absent expression of the beta-globin chain of hemoglobin, leading to severe anemia which is dependent upon transfusion for survival. The pathophysiology centers on the alpha- to beta-globin chain imbalance, which results in precipitation of excess alpha chains. These precipitates caused oxidative stress, membrane damage, and apoptosis of erythroid precursors in the bone marrow (BM). Allogeneic stem cell transplant can be curative but is only available to individuals with a matched donor. It is well known that increased levels of fetal hemoglobin (α2γ2;HbF), such as in the case of hereditary persistence of fetal hemoglobin, ameliorate the clinical severity of beta-thalassemia. We therefore developed a self-inactivating (SIN), erythroid-specific, gamma-globin lentiviral vector with the goal of reducing the chain imbalance through the production of HbF in red blood cells following transduction and transplantation of autologous HSCs. The vector, termed V5m3, contains 3.1-kb of transcriptional regulatory sequences from the β-globin locus control region, a 130-bp beta-globin promoter regulating transcription of the gamma-globin gene, and 3′ untranslated sequences from the native beta-globin gene. We previously showed that this vector was effective in correcting mouse models of beta-thalassemia and sickle cell disease (SCD). We further modified V5m3 to include a 400-bp chicken HS4 insulator element. Mobilized peripheral blood (PB)- or steady state bone marrow (BM)-derived CD34+ cells from normal or thalassemic human donors were used to evaluate vector performance. A two-phase in vitro model of human erythropoiesis was used in which a seed population of CD34+ cells is first expanded and then differentiated into late stage erythroblasts over a two week period. Gene transfer is performed 48 hours after initiation of culture. In this model, we routinely observe a 1000-fold expansion in total cell numbers where the vast majority of maturing erythroid cells are late stage erythroblasts reflected by nearly complete enrichment for expression of transferrin receptor (CD71; ≥98%) and glycophorin A (CD235; ≥80%) and loss or reduced expression of CD34 and CD45. Erythroid cells generated from normal adult PB and BM CD34+ cells demonstrate an adult pattern of hemoglobin production (HbA>96%;HbF<2%), as measured by acetate gel electrophoresis and HPLC, making this model ideal to evaluate enhancement of HbF levels by gene transfer. Normal PB CD34+ cells from four independent donors were transduced with the gamma-globin vector (MOI=20) or a GFP control vector (MOI=5). Vector transduction had no effect on cell growth or differentiation as monitored by consistent increases in total cell numbers and the appearance of CD71 (transferrin receptor) and glycophorin A on most cells (≥98% and '80%, respectively). The GFP vector achieved an average transduction rate of 87+/−6% and erythroblasts expressed low levels of HbF (1.7+/−0.6). Gamma globin gene transfer with the V5m3 (N=2) and V5m3-400 (N=4) vectors resulted in HbF levels ranging from 6 to 25%, with an average vector copy number of 0.8 to 1.1. We next tested the V5m3-400 vector using BM CD34+ cells from two patients with beta-thalassemia major. High levels of gene transfer were obtained with both the GFP and the globin vector, as evidenced by bulk marking (74+/−6% GFP+) or PCR analysis of CFU for presence of the V5m3-400 vector (12/12 positive Exp 1); (18/20 Exp 2); (18/24 Exp 3). Again, gene transfer did not perturb erythroid differentiation as monitored by the appearance glycophorin A (88+/−9%; GFP control and 86+/−6%; V5m3-400) as well as cell morphology. Erythroblasts derived from GFP transduced cells had a mean HbF level of 26+/−5% whereas those derived from cells transduced with V5m3-400 demonstrated a 100% increase of HbF to 58+/−2% with an average vector copy number of 0.6-0.8. Importantly, cultures of cells transduced with the globin vector demonstrated an average of 30% reduction in apoptotic cells (10% tunnel+) cells compared to GFP transduced control cells (15% tunnel+), suggesting rescue of erythroblasts through correction of the globin chain imbalance. Our data show that potentially therapeutic levels of HbF in thalassemic erythroblasts can be obtained following gene transfer using a gamma-globin lentiviral vector. Disclosures:No relevant conflicts of interest to declare.

Functional and genetic analysis of coreceptor usage by dualtropic HIV-1 subtype C isolates

It is widely documented that a complete switch from the predominant CCR5 (R5) to CXCR4 (X4) phenotype is less common for HIV-1 subtype C (HIV-1C) compared to other major subtypes. We investigated whether dualtropic HIV-1C isolates represented dualtropic, mixed R5 and X4 clones or both. Thirty of 35 functional HIV-1 env clones generated by bulk PCR amplification from peripheral blood mononuclear cells (PBMCs) infected with seven dualtropic HIV-1C isolates utilized CXCR4 exclusively. Five of 35 clones displayed dualtropism. Endpoint dilution of one isolate did not yield a substantial proportion of R5-monotropic env clones. Sequence-based predictive algorithms showed that env sequences from PBMCs, CXCR4 or CCR5-expressing cell lines were indistinguishable and all possessed X4/dualtropic characteristics. We describe HIV-1C CXCR4-tropic env sequence features. Our results suggest a dramatic loss of CCR5 monotropism as dualtropism emerges in HIV-1C which has important implications for the use of coreceptor antagonists in therapeutic strategies for this subtype.

High-Throughput Single Copy DNA Amplification and Cell Analysis in Engineered Nanoliter Droplets

A high-throughput single copy genetic amplification (SCGA) process is developed that utilizes a microfabricated droplet generator (microDG) to rapidly encapsulate individual DNA molecules or cells together with primer functionalized microbeads in uniform PCR mix droplets. The nanoliter volume droplets uniquely enable quantitative high-yield amplification of DNA targets suitable for long-range sequencing and genetic analysis. A hybrid glass-polydimethylsiloxane (PDMS) microdevice assembly is used to integrate a micropump into the microDG that provides uniform droplet size, controlled generation frequency, and effective bead incorporation. After bulk PCR amplification, the droplets are lysed and the beads are recovered and rapidly analyzed via flow cytometry. DNA targets ranging in size from 380 to 1139 bp at single molecule concentrations are quantitatively amplified using SCGA. Long-range sequencing results from beads each carrying approximately 100 amol of a 624 bp product demonstrate that these amplicons are competent for achieving attomole-scale Sanger sequencing from a single bead and for advancing pyrosequencing read-lengths. Successful single cell analysis of the glyceraldehyde 3 phosphate dehydrogenase (GAPDH) gene in human lymphocyte cells and of the gyr B gene in bacterial Escherichia coli K12 cells establishes that SCGA will also be valuable for performing high-throughput genetic analysis on single cells.