Internal PCR Control Research Articles

Antimicrobial Resistance and the Prevalence of the Panton-Valentine Leukocidin Gene among Clinical Isolates of Staphylococcus aureus in Lithuania.

This study aimed to determine resistance to antimicrobials of Staphylococcus aureus strains isolated from clinical specimens in Lithuanian hospitals and to identify the genes conferring resistance and virulence. The study was carried out from June 2019 to September 2021. S. aureus strains were isolated from skin, soft tissues, blood, lower respiratory tract, urine and other specimens. Antibiotic susceptibility testing was performed using the disc diffusion method according to EUCAST guidelines. All isolates were analyzed for detection of the ermA, ermC, mecA, mecC, tetK, tetM, and lukF-PV genes by multiplex real-time PCR. The 16S rRNA coding sequence was applied as an internal PCR control. Altogether, 745 S. aureus strains were analyzed. Antimicrobial susceptibility testing revealed that all isolates were susceptible to rifampin and vancomycin. Of the 745 strains, 94.8% were susceptible to tetracycline, 94.5% to clindamycin, and 88.3% to erythromycin. The lowest susceptibility rate was found for penicillin (25.8%). Six percent of the tested strains were methicillin-resistant S. aureus (MRSA). The majority of methicillin-resistant strains were isolated from skin and soft tissues (73.3%), with a smaller portion isolated from blood (17.8%) and respiratory tract (8.9%). The ermC gene was detected in 41.1% of erythromycin-resistant S. aureus strains, whereas ermA was detected in 32.2% of erythromycin-resistant S. aureus strains. 69.2% of tetracycline-resistant S. aureus strains had tetK gene, and 28.2% had tetM gene. 7.3% of S. aureus isolates harbored lukF-PV gene. The frequency of the pvl gene detection was significantly higher in MRSA isolates than in methicillin-susceptible S. aureus isolates (p < 0.0001).

MOLECULAR GENETIC STATUS OF PIGS OF UKRAINIAN BREEDS SUITABLE FOR USE IN XENOTRANPLANTATION

Thousands of people in Ukraine and the world need organ transplants. However, the main obstacle to the wider use of transplantation remains the shortage of donor organs and tissues. To prevent human infection with viruses during organ transplantation, pigs are carefully screened for PERV retroviruses and animals with low expression levels of PERV-A and PERV-C are selected. The article is devoted to the discussion of the results of research on the frequency of retrovirus PERV types A and C in populations of Ukrainian pig breeds. The research was carried out on samples of pigs of the following breeds: Mirgorod, Great White, Poltava meat, Ukrainian meat, Ukrainian Steppe Ryaba, Landras, Pietren, Vietnamese Vyslobryukha, Vietnamese Meishan, and wild pig. Biomaterial samples (venous blood, bristles with hair bulbs) were collected for molecular genetic analysis. Isolation of genomic DNA from the samples was carried out by the salt method and using the Chelex-100 ion exchange resin. Genotyping was performed by the method of allele-specific (PCR-SSP) multiplex polymerase chain reaction using primers complementary to regions of the PERV-C, PERV-A gene loci. A fragment of the domestic pig alpha-actin locus (α-Actin) was used as an internal PCR control. Amplification was carried out in a thermal cycler "Tertsik-2" ("DNA-technology", RF). Electrophoretic separation of amplified DNA sections in multiplex PCR format was performed in a 2% agarose gel in a tris-borate electrophoresis buffer. It was established that among the studied groups of pigs, the highest concentration of retrovirus PEVR-C was found in animals of the breeds Vietnamese Vyslobrukha (100%), Poltava meat (75%), Ukrainian steppe spotted (55%), large white (50%), landrace (50%). The largest share of PEVR-A retrovirus carrier animals is characterized by the breeds of Vietnamese Vyslobrukha (100%), Poltava meat 95%), Pietren (80%) and Ukrainian meat (73%). The largest number of individuals free from PERV-A and PERV-C retroviruses were found in experimental groups of breeds of Ukrainian steppe spotted (75 and 45%, respectively), Myrhorod (32 and 75%, respectively), landrace (75 and 50%, respectively) and wild pig. Pigs these breeds can become promising organ donors for xenotransplantation.

A comparison of five DNA extraction methods from degraded human skeletal remains

Extracting DNA from degraded human remains poses a challenge for any forensic genetics laboratory, as it requires efficient high-throughput methods. While little research has compared different techniques, silica in suspension has been identified in the literature as the best method for recovering small fragments, which are often present in these types of samples. In this study, we tested five DNA extraction protocols on 25 different degraded skeletal remains. Including the humerus, ulna, tibia, femur, and petrous bone. The five protocols were organic extraction by phenol/chloroform/isoamyl alcohol, silica in suspension, High Pure Nucleic Acid Large Volume silica columns (Roche), InnoXtract™ Bone (InnoGenomics), and PrepFiler™ BTA with AutoMate™ Express robot (ThermoFisher). We analysed five DNA quantification parameters (small human target quantity, large human target quantity, human male target quantity, degradation index, and internal PCR control threshold), and five DNA profile parameters (number of alleles with peak height higher than analytic and stochastic threshold, average relative fluorescence units (RFU), heterozygous balance, and number of reportable loci) were analysed. Our results suggest that organic extraction by phenol/chloroform/isoamyl alcohol was the best performing method in terms of both quantification and DNA profile results. However, Roche silica columns were found to be the most efficient method.

Developmental validation of the Investigator Argus Y-28 QS Kit

The analysis of Y-chromosomal STRs is an integral part of forensic practice, especially in the context of sexual offenses, where it enables the identification of a male even in a high background of female DNA. We have developed and validated the new Investigator Argus Y-28 QS Kit as an assay to co-amplify 27 Y-STR markers. The new Fast Reaction Mix 3.0 ensures not only robust and fast PCR amplification with improved inhibitor resistance and easy handling, but also provides enhanced robustness towards DNA overloading and stability. Highly sensitive, yielding rapid and reliable results from trace DNA, the kit is suitable for all forensic applications. It can also be used for direct amplification of reference samples like blood or saliva on FTA cards or crude lysates from buccal swabs. Furthermore, the assay uses a 6-dye technology to keep the amplicon length of the markers short and to avoid marker overlap. The kit contains QIAGEN’s Quality Sensor System (QS1 and QS2) as an internal PCR control.

Predicting the optimal STR profile amplification set up from Quantifiler™ Trio data

Samples collected for forensic case work may be of varying quality and quantity. The sample DNA is often quantified prior to short tandem repeats (STR) profile analysis with methods such as Quantifiler™Trio (QFT). The QFT measures the quantity of DNA as well as an internal PCR control (IPC) and a degradation index (DI).The aim of this study was to use IPC and DI measurements to identify samples, which would benefit from a modified PCR amplification set-up when generating the STR profiles. The sample quality of 6287 single source case work samples were categorized as 'Good’, ‘Partly degraded’, ‘Highly degraded’, ‘Inhibited’ and ‘Degraded and Inhibited’ based on the peak height ratios in the electropherogram data. The DI and IPC were correlated with the assigned quality categories of the samples. Samples categorized into the degraded and/or inhibited categories were found to have statistically significantly different DI and IPC compared to samples categorized as ‘Good’. This indicates that the additional information gained from the QFT can be useful to identify degraded and/or inhibited samples prior to the STR-profile analysis. Future work will re-evaluate the criteria of inclusion in the sample quality groups and implement multi source samples.

Recovery of DNA from bone without demineralization

This study assessed the performance of five different DNA extraction methods for the recovery of DNA from bone: ChargeSwitch® gDNA Plant Kit, DNA IQ™ System Kit, DNeasy® Blood & Tissue Kit, PrepFiler® BTA Forensic DNA Extraction Kit and phenol-chloroform-isoamyl alcohol. DNA was extracted from pig rib and femur bones that was fresh, had undergone surface decomposition for three months, and had undergone surface decomposition for one year. Extracted DNA was analyzed using real-time PCR and amplification of an in-house PCR multiplex that assessed the quality and quantity of DNA and for the presence of inhibitors. The phenol-chloroform-based method consistently yielded the highest amounts of DNA and DNA IQ the lowest; however, all methods produced relatively high yields of DNA from both pig rib and femur samples that could be amplified without any detected inhibition. The data demonstrate that with reasonable quality bone samples any of the tested methods can isolate DNA that can be successfully analyzed. The effective use of internal PCR controls is also demonstrated.

Comparison of DNA extraction techniques for the recovery of bovine DNA from fly larvae crops.

Forensic entomology aids investigations using insects and is primarily associated with the estimation of post-mortem interval (PMI). Studies have shown that human DNA can be recovered from the crops of fly larvae. While several factors regarding the recovery of human DNA from crops have been studied, DNA extraction methods have not been thoroughly assessed. Determining a method for optimal extraction could aid crime laboratories in implementing DNA extraction from larvae and streamlining future research. Bovine DNA was used as a substitute for human DNA to test several DNA extractions kits. Four DNA extraction kits (Chelex®, PDQeX forensicGEM, EZ1® DNA Investigator, DNeasy® Powersoil® Pro Kit) were evaluated based on the quantity and quality of bovine DNA extracted. Extractions were performed on whole fly larvae and dissected crops. Quantification was performed using real-time PCR (qPCR) on a StepOne™ Real-Time PCR System with SYBR® Green using bovine-specific cytochrome b primers. The quality of extracts was determined by checking for inhibition using commercial qPCR chemistries with an internal PCR control (IPC). When using whole fly larvae, Powersoil® Pro yielded the highest average DNA yield (n=10, 0.668±0.458ng/μl), while EZ1® DNA Investigator yielded the highest average with crops (n=10, 0.605±0.403ng/μl). Chelex and forensicGEM yielded low amounts of bovine DNA, and its extracts were inhibited, unlike EZ1® and Powersoil® Pro, which have purification steps. Therefore, it is recommended to use EZ1® DNA Investigator coupled with automation on EZ1® Advanced XL to recover DNA from fly larvae crops.

The performance of quality controls in the Investigator® Quantiplex® Pro RGQ and Investigator® 24plex STR kits with a variety of forensic samples

Forensic DNA laboratories process database reference samples on FTA® cards or buccal swabs, which commonly contain adequate amounts of quality DNA resulting in full STR profiles and high first-pass rates. However, some reference samples and many forensic casework samples are exposed to a variety of insults that may lead to low quantities of DNA, DNA degradation, DNA mixtures, and/or PCR inhibition, posing a challenge to downstream genotyping success. The inclusion of multiple amplification targets and internal PCR controls (IPCs) in DNA quantification kits, and quality sensors within STR amplification kits can aid in the accurate interpretation of sample/profile quality, and guide more efficient rework strategies when needed. In order to assess the effectiveness of these quality systems we subjected database-type samples (buccal swabs and blood or saliva on FTA® cards), mock casework samples (low-template, degraded, inhibited, DNA mixtures), and authentic post-coital samples to various challenging conditions. Concordance between the quality flags in the Investigator® Quantiplex® Pro RGQ kit (QIAGEN), the QS markers in QIAGEN’s Investigator® 24plex QS kit, and overall STR profile quality was evaluated for all casework-type samples. To assess the value of the QS markers in the Investigator® 24plex QS and GO! STR kits, samples with partial or failed STR profiles were reworked based on the quality of the electropherogram first with the QS markers redacted, and second in conjunction with the QS markers. Results from each of the rework approaches were compared to determine which strategy, if any, improved the STR profile quality and the number of reportable alleles. The QS markers in the 24plex STR kits correctly confirmed sample quality in 99.9% of databasing samples and 98% of mock casework samples. Quality flags during DNA quantification were concordant with downstream STR profiles for the majority (77%) of the mock casework samples. Additionally, when samples with partial STR profiles were reworked, more loci were obtained for 80% of the samples regardless of the rework strategy used. However, the most notable improvement in STR completeness was observed in inhibited samples that were reworked based on the information provided by the STR quality sensors, with an average increase of 56% reportable alleles.

Degradation modeling of water environmental DNA: Experiments on multiple DNA sources in pond and seawater

AbstractEnvironmental DNA (eDNA) analysis methods have been developed to detect organism distribution and abundance/biomass in various environments. eDNA degradation is critical for eDNA evaluation. However, the dynamics and mechanisms of eDNA degradation are largely unknown, especially when considering different eDNA sources, for example, cells and fragmental DNA. We experimentally evaluated the degradation rates of eDNA derived from multiple sources, including fragmental DNA (internal PCR control [IPC]), free cells (from Oncorhynchus kisutch), and resident species. We conducted the experiment with pond and seawater to evaluate the differences between freshwater and marine habitats. We quantified the eDNA copies of free cells, fragmental DNA, and resident species (Cyprinus carpio in the pond and Trachurus japonicus in the sea). We found that eDNA derived from both cells and fragmental DNA decreased exponentially in both the sea and pond samples. The degradation of eDNA from resident species showed similar behavior to the cell‐derived eDNA. We evaluated three degradation models with different assumptions and degradation steps and found that a simple exponential model was effective in most cases. Our findings on cell‐ and fragmental DNA‐derived eDNA provide fundamental information about the eDNA degradation process and can be applied to quantify eDNA behavior in natural environments.

Development of the Investigator® 26plex QS Kit: A New multiplex PCR Kit for Global STR analysis

STR assays for forensic casework must conform to national DNA standards. QIAGEN developed the Investigator 26plex QS Kit as an assay to co-amplify 25 markers, including the current Chinese, CODIS and European standard markers in one reaction. The kit is designed for purified DNA from casework and reference samples. The new Fast Reaction Mix 3.0 ensures not only robust and fast PCR amplification with improved inhibitor resistance and easy handling, but also provides enhanced robustness towards DNA overloading and stability. Highly sensitive, yielding rapid and reliable results from trace DNA, the kit is suitable for all forensic applications and paternity testing. It can also be used for direct amplification of reference samples like blood or buccal cells on FTA cards or swabs. Furthermore, with the inclusion of Penta E and Penta D in the marker set, the Investigator 26plex QS Kit offers very high discriminatory power. The assay uses a 6-dye technology to keep the amplicon length of the markers short and to avoid marker overlap. The kit contains QIAGEN’s Quality Sensor System (QS1 and QS2) as an internal PCR control.

Validation study and forensic applications of investigator 24plex QS Kit

The Investigator 24plex QS Kit (Qiagen) is used for multiplex PCR in forensic human identity and paternity testing. The PCR simultaneously amplifies 22 polymorphic STR markers: the Combined DNA Index System (CODIS) core loci, the European Standard Set (ESS) loci, SE33, DYS391 and Amelogenin. The Investigator 24plex QS Kit is specifically designed for rapid and reliable generation of DNA profiles from blood, buccal swabs, and forensic stains. The kit utilizes QIAGEN’s fast-cycling PCR technology, allowing amplification in around 60 min and provides highly robust results with inhibitor-resistant chemistry. The Investigator 24plex QS Kit Primer Mix contains two innovative internal PCR controls (Quality Sensor QS1 and QS2), which are amplified simultaneously with the polymorphic STR markers, to yield helpful information about the efficiency of the PCR and the presence of PCR inhibitors. The Investigator 24plex QS Kit User Guide describes instrument setup and sample preparation of PCR products using the Applied Biosystems 3500 Series Data Collection Software and the GeneMapper ID-X Software, but an internal validation is always needed.In this study, we report the successful analysis of amplicons obtained with Investigator 24plex QS Kit on the 3500 Genetic Analyzer. We performed internal validation studies following the European Network of Forensic Science Institutes (ENFSI) and the European DNA Profiling Group (EDNAP) guidelines, testing several critical areas of kit performance such as sensibility, sensitivity, DNA mixtures and inhibited samples.

Anaplasma phagocytophilum, Bartonella spp., haemoplasma species and Hepatozoon spp. in ticks infesting cats: a large-scale survey

BackgroundTicks derived from cats have rarely been evaluated for the presence of pathogens. The aim of this study was to determine the prevalence of Anaplasma phagocytophilum, Bartonella spp., haemoplasma species and Hepatozoon spp. in ticks collected from cats in the UK.MethodsFive hundred and forty DNA samples extracted from 540 ticks collected from cats presenting to veterinarians in UK practices were used. Samples underwent a conventional generic PCR assay for detection of Hepatozoon spp. and real-time quantitative PCR assays for detection of Anaplasma phagocytophilum and three feline haemoplasma species and a generic qPCR for detection of Bartonella spp. Feline 28S rDNA served as an endogenous internal PCR control and was assessed within the haemoplasma qPCR assays. Samples positive on the conventional and quantitative generic PCRs were submitted for DNA sequencing for species identification.ResultsFeline 28S rDNA was amplified from 475 of the 540 (88.0%) ticks. No evidence of PCR inhibition was found using an internal amplification control. Of 540 ticks, 19 (3.5%) contained DNA from one of the tick-borne pathogens evaluated. Pathogens detected were: A. phagocytophilum (n = 5; 0.9%), Bartonella spp. (n = 7; 1.3%) [including Bartonella henselae (n = 3; 0.6%) and Bartonella clarridgeiae (n = 1; 0.2%)], haemoplasma species (n = 5; 0.9%), “Candidatus Mycoplasma haemominutum” (n = 3; 0.6%), Mycoplasma haemofelis (n = 1; 0.2%), “Candidatus Mycoplasma turicensis” (n = 1; 0.2%), Hepatozoon spp. (n = 2; 0.4%), Hepatozoon felis (n = 1; 0.2%) and Hepatozoon silvestris (n = 1; 0.2%).ConclusionThese data provide important information on the prevalence of tick-borne pathogens in ticks infesting cats, with the identification of haemoplasma species, A. phagocytophilum, H. felis and Bartonella spp. (including B. henselae and B. clarridgeiae). This study also documents the first report of H. silvestris in ticks collected from domestic cats.

Abstract LB-B11: Development and validation of ColoScape™: A new colorectal cancer mutation detection assay™

Abstract Introduction: Colorectal cancer is a highly preventable disease as early detection increases rates of patient survival to near 100%. Herein we report the development and validation of ColoScape™, a highly sensitive test powered by XNA technology to detect novel multigene mutation biomarkers in CRC by real-time PCR. The assay allows the sensitive detection of the presence or absence of mutations in the targeted regions of the genes interrogated in tissue biopsy (FFPE) and plasma samples.Methodology: The high sensitivity of this multigene biomarker assay is achieved due to xenonucleic acid (XNA) probe technology. XNA probes are novel backbone modified oligomers with natural nucleoside bases (A, T, C and G) that hybridize by Watson-Crick base pairing to natural DNA and RNA with much higher binding affinity. XNA probes are designed that bind to the selected wild-type sequences at the respective genetic loci in the target genes. These XNA probes cannot be extended by DNA polymerase thus suppress amplification of WT DNA templates and only allow amplification of the target mutant DNA templates in the sample. For each of the selected mutation sites, primers and FAM-labeled TaqMan probes were designed and tested together with the selected XNA oligomers. An internal PCR control selected in the Human -Actin (ACTB) gene was employed utilizing an HEX-labeled TaqMan probe.Results: The ColoScape™ kit was demonstrated to have robust analytical performance and clinical accuracy for FFPE, stool and plasma samples. The rapid, precise and sensitive molecular assay for mutation detection in colon cancer has key benefits listed below. The assays showed a sensitivity of as low as 0.1% mutation in 5-10 ng of WT DNA/well. No cross-reactivity was observed with wild-type up to 320ng purified gDNA and up to 20ng FFPE DNA per reaction demonstrating high specificity of the ColoScape™ assay. Intra-assay, inter-assay, lot-to-lot and operator variation comparison showed CV% between 3% and 8%. Excluding pre-cancer samples, the assay clinical specificity and sensitivity were 95% and 100%, respectively. Pre-cancer detection sensitivity was 60% and 62.5% for stool samples. For tested FFPE clinical samples, the assay specificity and sensitivity were 95% and 91% respectively while the assay clinical specificity and sensitivity were both 100% for plasma samples.Conclusion: The ColoScape™ Colorectal Cancer Mutation Detection qPCR assay is shown to be a sensitive tool intended to aide in the early detection of colorectal cancer, disease monitoring and therapeutic interventions. Citation Format: Michael J. Powell, Elena Peletskaya, Qing Sun, Larry Pastor, Aiguo Zhang, Kamila Koprowska, Walter Bodmer. Development and validation of ColoScape™: A new colorectal cancer mutation detection assay™ [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B11.

STUDY OF CUCUMBER MOSAIC VIRUS GENE EXPRESSION IN CAPSICUM ANNUUM

Cucumber mosaic virus (CMV) is a plant pathogenic virus in the genus Cucumovirus, family Bromoviridae. It has the potential and reputation of having the widest host range of any known plant virus including monocotyledons and dicotyledons, herbaceous plants, shrubs and trees. CMV is one of the major diseases in Capsicum annum (chilli). Chilli plant samples exhibiting virus-like disease symptoms were collected from Taman Pertanian Indera Mahkota (location 1) and Greenhouse 12 of Horticulture Research Centre, Serdang (location 2). Viral disease was detected based on symptoms like mosaic-mottling, yellow ringspots and cholorotic that appeared on the leaves. The isolation of total RNA was done by using Vivantis GF-1 total RNA extraction kit. RT-PCR technique was used to detect the presence of virus disease symptoms gene in chillies. Identification of causal agents was based on cDNA amplified product size, using virus-specific oligonucleotides. Actin was used as the internal PCR control. The product size of the DNA fragment was 315 bp. From RT-PCR, the expression of CMV can be detected in chilli plants that exhibited the virus-like disease symptoms. This research revealed that some of the chilli plants at the Taman Pertanian Indera Mahkota and Greenhouse 12 of Horticulture Research Centre have been affected by this viral disease.

Development of Internal PCR Control (IPC) for Human Mitochondrial DNA Typing Kit

Development of Internal PCR Control (IPC) for Human Mitochondrial DNA Typing Kit

Development and validation of the Investigator® Quantiplex Pro Kit for qPCR-based examination of the quantity and quality of human DNA in forensic samples

We developed and validated the Investigator Quantiplex Pro Kit which quantifies human and male DNA in a sample using quantitative real-time PCR. An internal PCR control (IPC) is incorporated into the kit to reliably and consistently identify PCR inhibitors that may interfere with downstream processes, such as STR PCR. In addition, the effect of DNA degradation on downstream STR analysis can be accurately assessed. The kit works with any human ID workflow, regardless of upstream sample prep or downstream STR kit, and has been designed to complement the Quality Sensor found in several of QIAGEŃs Investigator STR PCR assays (Investigator ESSplex SE QS, Investigator 24plex QS and Investigator 24plex GO!), enabling an unparalleled level of workflow optimization.

Abstract 728: Development and validation of ColoScape™ - a new colorectal cancer mutation detection assay

Abstract Introduction: Colorectal cancer is a highly preventable disease as early detection increases rates of patient survival to near 100%. Herein we report the development and validation of a novel multigene mutation biomarker real-time PCR based assay for qualitative detection of colorectal cancer associated biomarkers that comprise tumor specific mutations in the following genes: APC (Exon 15), KRAS (Exon 2), BRAF (Exon 15) and CTNNB1 (Exon 3) called ColoScapeTM . The assay allows the sensitive detection of the presence or absence of mutations in the targeted regions of the genes interrogated. Methodology: The high sensitivity of this multigene biomarker assay is achieved due to xenonucleic acid (XNA) probe technology. XNA probes are novel backbone modified oligomers having natural nucleoside bases (A,T,C and G) that hybridize by Watson-Crick base pairing to natural DNA and RNA with much higher binding affinity than natural deoxyribonucleic acid oligomers of the same sequence. XNA probes are designed that bind to the selected wild-type sequences at the respective genetic loci in the target genes. These XNA probes have a much higher Tm than the primer annealing temperature and suppress amplification of WT DNA templates and only allow amplification of the target mutant DNA templates in the sample. Both single nucleotide polymorphisms (SNP’s) and insertion/deletions (indels) mutations can be detected. For each of the selected mutation sites primers and FAM-labeled TaqMan hydrolysis probes were designed and tested together with the selected XNA oligomers and analytical assay performance confirmed. An internal PCR control amplicon selected in the Human β-Actin (ACTB) gene was employed utilizing a HEX-labeled TaqMan probe. Performance parameters of the assay were established on DNA of colorectal cancer patients extracted from FFPE as well as reference DNA materials (synthetic and cell line derived DNA). The assay demonstrates high sensitivity and specificity in detection of colon cancer and adenoma samples based on the set of biomarkers involved in colorectal cancer neogenesis and disease progression. Conclusion: The ColoScapeTM Colorectal Cancer Mutation Detection assay is shown to be a sensitive tool intended to facilitate research in colon cancer development, early detection, disease monitoring and therapeutic interventions. Citation Format: Michael J. Powell, Elena Peletskaya, Anne Vallerga, Qing Sun, Larry Pastor, Aiguo Zhang. Development and validation of ColoScape™ - a new colorectal cancer mutation detection assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 728. doi:10.1158/1538-7445.AM2017-728

Abstract 492: Automated circulating cell-free DNA purification from large volume draws

Abstract Circulating, cell-free DNA (ccfDNA) has emerged as an important tool in molecular oncology research. Because it is highly fragmented, present in small quantities, and highly susceptible to degradation, purification of ccfDNA poses unique challenges to researchers. While plasma is the main focus of many researchers for ccfDNA, recent work has shown that it is present in other biological fluids. Promega has developed a unique chemistry to selectively purify ccfDNA from plasma. The Maxwell RSC ccfDNA Plasma system is a completely automated system that allows purification of ccfDNA from 1ml of plasma. To add flexibility, we adapted the chemistry to automated platforms in 24-well configurations. These platforms include the Hamilton Microlab STAR series and the KingFisher Flex Processor. Because the biomarkers in ccfDNA can be of very low frequency, many researchers prefer to process volumes of samples &amp;gt;1ml which can exceed the capacity of many purification systems. Using a sequential bind strategy, we demonstrate that ccf DNA can be purified from at least 8ml sample draw using a fully automated method. Liquid draws tested include plasma and urine. DNA quantity was assessed by qPCR using an autosomal target, and quality was assessed using an internal PCR control. This study shows the flexibility and robustness of the Maxwell RSC ccfDNA chemistry. We successfully adapted it for purification from relatively large volume liquids in a fully automated manner making this a convenient option for early biomarker research. Citation Format: Robert Ray, Mark Bratz, Doug Wieczorek, Douglas White, Douglas Horejsh, Eric Vincent, Trista Schagat. Automated circulating cell-free DNA purification from large volume draws. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 492.

Human DNA quantification and sample quality assessment: Developmental validation of the PowerQuant¨r) system

Quantification of the total amount of human DNA isolated from a forensic evidence item is crucial for DNA normalization prior to short tandem repeat (STR) DNA analysis and a federal quality assurance standard requirement. Previous commercial quantification methods determine the total human DNA and total human male DNA concentrations, but provide limited information about the condition of the DNA sample. The PowerQuant¨r) System includes targets for quantification of total human and total human male DNA as well as targets for evaluating whether the human DNA is degraded and/or PCR inhibitors are present in the sample. A developmental validation of the PowerQuant¨r) System was completed, following SWGDAM Validation Guidelines, to evaluate the assay tm)s specificity, sensitivity, precision and accuracy, as well as the ability to detect degraded DNA or PCR inhibitors. In addition to the total human DNA and total human male DNA concentrations in a sample, data from the degradation target and internal PCR control (IPC) provide a forensic DNA analyst meaningful information about the quality of the isolated human DNA and the presence of PCR inhibitors in the sample that can be used to determine the most effective workflow and assist downstream interpretation.

Real-Time PCR Assays for the Detection of Puccinia psidii.

Puccinia psidii (Myrtle rust) is an emerging pathogen that has a wide host range in the Myrtaceae family; it continues to show an increase in geographic range and is considered to be a significant threat to Myrtaceae plants worldwide. In this study, we describe the development and validation of three novel real-time polymerase reaction (qPCR) assays using ribosomal DNA and β-tubulin gene sequences to detect P. psidii. All qPCR assays were able to detect P. psidii DNA extracted from urediniospores and from infected plants, including asymptomatic leaf tissues. Depending on the gene target, qPCR was able to detect down to 0.011 pg of P. psidii DNA. The most optimum qPCR assay was shown to be highly specific, repeatable, and reproducible following testing using different qPCR reagents and real-time PCR platforms in different laboratories. In addition, a duplex qPCR assay was developed to allow coamplification of the cytochrome oxidase gene from host plants for use as an internal PCR control. The most optimum qPCR assay proved to be faster and more sensitive than the previously published nested PCR assay and will be particularly useful for high-throughput testing and to detect P. psidii at the early stages of infection, before the development of sporulating rust pustules.