Optimisation of an Automated DNA Extraction Method for Bone and Teeth Samples and Applicability to Two Forensic Cases

Bones and teeth are highly challenging sources of DNA in forensic science and human remains identification, requiring multiple laborious processing steps. In this study, we compared an organic phenol–chloroform method to the QIAamp® DNA Investigator and PrepFiler Express BTA™ methods in order to identify the most efficient automated DNA extraction method for bones and teeth. Results from individual tooth powder replicates showed that the PrepFiler Express BTA™ method extracted the highest yields of DNA per mg of tooth powder, returning a minimum of 20/21 PowerPlex® 21 loci. Samples extracted using the organic extraction or QIAamp® DNA Investigator methods produced PowerPlex® 21 profiles displaying a ski-slope morphology. The improved DNA quality and yield from the PrepFiler Express BTA™ method was verified using aged samples, where higher DNA yields per mg of powder and more informative profiles were obtained. Furthermore, the PrepFiler Express BTA™ method subsequently provided useful DNA profiles for two forensic cases involving degraded bone samples. Overall, this study showed that the PrepFiler Express BTA™ chemistry is a reliable and robust method for DNA extraction from bone and teeth samples, and will allow larger numbers of samples to be efficiently extracted in the event of a Disaster Victim Identification event.

Illumina DNA Prep (M) Tagmentation Library Preparation for use on an Illumina MiSeq Sequencer v2

This procedure outlines the protocol for whole genome sequencing of bacterial organisms using the Illumina DNA Prep library preparation kit for sequencing on an Illumina MiSeq sequencer. This document applies to all laboratory personnel in the Division of Microbiology (DM) as well as laboratories in the GenomeTrakr Network. Complete in order: 1. DNA Extraction (Manual DNA Extraction or Automated DNA Extraction using the Qiacube) Step-by-step procedures to obtain high quality DNA from isolates in TSB for whole genome sequencing 2. DNA Quantitation Quantitation of extracted DNA using the Qubit Flourometer 3. Library Preparation for WGS (Included SOP or Library Preparation using Illumina Nextera XT ) Library preparation using NexteraXT or Illumina DNA Prep (previously Nextera DNA Flex) 4. Sequencing using Illumina MiSeq 5. Data Quality Checks and NCBI Submission

Evaluation of 11 DNA Automated Extraction Protocols for the Detection of the 5 Mains Candida Species from Artificially Spiked Blood

The molecular detection of Candida plays an important role in the diagnosis of candidaemia, a major cause of morbidity and mortality. The sensitivity of this diagnosis is partly related to the efficiency of yeast DNA extraction. In this monocentric study, we investigated the suitability of 11 recent automated procedures for the extraction of low and high amounts of Candida DNA from spiked blood. The efficacy of the DNA extraction procedures to detect Candida spp. in blood samples ranged from 31.4% to 80.6%. The NucliSENSTM easyMAGTM procedure was the most efficient, for each species and each inoculum. It significantly outperformed the other procedures at the lower Candida inocula mimicking the clinical setting. This study highlighted a heterogeneity in DNA extraction efficacy between the five main Candida species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei). Up to five automated procedures were appropriate for C. krusei DNA extraction, whereas only one method yielded an appropriate detection of low amount of C. tropicalis. In the era of the syndromic approach to bloodstream infection diagnosis, this evaluation of 11 automated DNA extraction methods for the PCR diagnosis of candidaemia, puts the choice of an appropriate method in routine diagnosis within the reach of laboratories.

Automated DNA Extraction Monitoring System Based on MTConnect Technology

MTConnect standard technology provides simplicity, flexibility, and scalability in integrating various equipment and operating systems and enabling accurate and consistent data collection from any MTConnect-compatible system. Using MTConnect technology, it is possible to immediately identify the cause of a problem and respond quickly when a problem occurs. Molecular genetic diagnostic point-of-care testing (POCT) devices have received attention in recent years because they enable rapid disease diagnosis. A molecular genetic diagnostic POCT device is under development by the authors. The system consists of a gene extraction process and a real-time PCR-based gene amplification process. In this study, we propose and demonstrate a system based on MTConnect technology to monitor an automated DNA extraction process. The proposed system consists of an automated DNA extraction system, an MTConnect adapter, an MTConnect agent, and a client application. The adapter and agent were developed on a Raspberry Pi single-board computer. The agent publishes the collected data in Extensible Markup Language (XML) format over a network. The performance and reliability of the system were evaluated by verifying the request response time between the implemented system’s agent and the client application. The results demonstrate the feasibility of monitoring the DNA extraction process over a network.

Comparison of DNA extraction methods and real-time PCR assays for the molecular diagnostics of chronic Chagas disease

Aim: This work aimed to compare the sensitivity of four protocols for the detection of Trypanosoma cruzi DNA in 98 blood samples from chronic Chagas disease patients. Materials & methods: Two DNA extraction (automated and manual) methods and two T. cruzi satellite DNA qPCRs (with a recent design and the usually used set of primers) were analyzed. Results: Both DNA extraction methods and qPCR assays tested in this work gave comparable qualitative results, although the lowest Ct values were obtained when samples were analyzed using the new set of primers for T. cruzi satellite DNA. Conclusion: Our results encourage the implementation of automated DNA extraction systems and the new T. cruzi qPCR for the molecular diagnostics and treatment response monitoring of chronic Chagas disease patients.

HPV Genotype Prevalence and Success of Vaccination to Prevent Cervical Cancer

Background: Nearly 500,000 new cases of cervical cancer are estimated annually worldwide. Three vaccines are currently licensed to prevent cervical cancer. The success of vaccination depends mainly on the prevalence of HPV genotypes, and many cases of HPV infection have been diagnosed after vaccination. Our aim was to search for HPV genotyping in cervical samples to verify the proportion of women that remain susceptible to infection even after vaccination. Methods: 21,017 liquid-based cervical (LBC) specimens were received for cytology and HPV detection from 2015 to 2018. Before slide preparations for cytology, a 1,000-μL aliquot was taken from the LBC fixative and subjected to automated DNA extraction and multiplex PCR followed by capillary electrophoresis to detect and classify HPV. Results: HPV was detected in 895 (4.3%) specimens. The most prevalent genotype was HPV-16, followed by HPV-58 and HPV-66. A total of 258 (28.8%) cases were positive for high-risk (HR)-HPV types (66, 59, 39, 56, 30, 35, 53, 51, 68, 82, and 70) that are not covered by the HPV vaccines. Conclusion: A significant proportion of HPV types detected in cytological specimens are representative of HR-HPV not covered by the available vaccines. The health system should be aware of the considerable percentage of women who are not being immunized and will continue to need cervical cancer screening.

Development of a Multiplex real-time PCR Assay for the Newborn Screening of SCID, SMA, and XLA

Numerous studies have shown evidence supporting the benefits of universal newborn screening for primary immunodeficiencies (PID) and for Spinal Muscular Atrophy (SMA). We have developed a four-plex, real-time PCR assay to screen for Severe Combined Immune Deficiencies (SCID), X-linked agammaglobulinemia (XLA), and SMA in DNA extracted from a single 3.2 mm punch of a dried blood spot (DBS). A simple, high-throughput, semi-automated DNA extraction method was developed for a Janus liquid handler that can process 384 DBS punches in four 96-well plates in just over one hour with sample tracking capability. The PCR assay identifies the absence of exon 7 in the SMN1 gene, while simultaneously evaluating the copy number of T-cell receptor excision circles (TREC) and Kappa-deleting recombination excision circles (KREC) molecules. Additionally, the amplification of a reference gene, RPP30, was included in the assay as a quality/quantity indicator of DNA isolated from the DBS. The assay performance was demonstrated on over 3000 DNA samples isolated from punches of putative normal newborn DBS. The reliability and analytical accuracy were further evaluated using DBS controls, and contrived and confirmed positive samples. The results from this study demonstrate the potential of future molecular DBS assays, and highlight how a multiplex assay could benefit newborn screening programs.