Development and performance of a next generation sequencing (NGS) assay for monitoring of dd-cfDNA post solid organ transplantation.

The aim of this study was to evaluate the analytical performance of a novel NGS assay, intended for monitoring of donor-derived cell-free DNA (dd-cfDNA), and describe its validity in clinical plasma samples from kidney transplanted patients. Artificial and clinical samples with increasing amounts of patient DNA were evaluated using NGS analysis of indel markers. Monitoring of dd-cfDNA with the NGS assay presented herein demonstrated a sensitivity of ≥0.

Development and performance of a next generation sequencing (NGS) assay for monitoring of mixed chimerism.

The aim of this study was to evaluate the performance of a novel NGS-based assay to monitor mixed chimerism (MC) and compare its technical capacity to established techniques for chimerism analysis. Artificial and clinical samples with increasing amounts of patient DNA were compared using real-time PCR detection of indels and SNP, fragment analysis of short-tandem repeats (STR) and NGS analysis of indels. Real-time PCR displayed excellent sensitivity (>0,01%) but poor accuracy (>20 CV% at MC > 20%), while fragment analysis exhibited good accuracy (<5 CV% at MC > 20%) with limited sensitivity (>2,5%).

Hepatitis B virus DNA quantitation and detection of core promoter, precore and polymerase mutations in chronic hepatitis B: evaluation and clinical usefulness of three new commercial assays.

Background: HBV-DNA quantitation, the HBe antigen status and the appearance of mutations in the core promoter, precore and polymerase regions are important elements in the management of chronic HBV infection.

Methods: We performed a technical evaluation of 3 new kits, affigene HBV VL, affigene HBV mutant VL and affigene HBV DE/3TC assays (Sangtec Molecular Diagnostics) in comparison with the Amplicor HBV Monitor assay (Manual Test, Roche), direct sequencing and direct sequencing/Inno-LIPA HBV DR (Innogenetics), respectively. We evaluated the clinical application of these tests in the management of patients with chronic (HBeAg positive) hepatitis B.

Automated quantitative analysis of hepatitis B virus DNA by using the Cobas Amplicor HBV monitor test.

A highly sensitive method of quantitative analysis of hepatitis B virus (HBV) DNA in serum, the Cobas Amplicor HBV Monitor (Cobas-AM) test, was evaluated. Following a manual extraction of viral DNA, amplification, colorimetric detection, and quantitative determination are all automatically performed in the Cobas analyzer. Serially diluted samples with known HBV DNA concentrations were analyzed blindly.

An integrated system using immunomagnetic separation, polymerase chain reaction, and colorimetric detection for diagnosis of Plasmodium falciparum.

An integrated system for sample preparation and DNA detection of the malaria parasite using immunomagnetic separation in combination with the polymerase chain reaction (PCR) and colorimetric analysis is described. A cocktail of three monoclonal antibodies towards merozoite surface antigen-1 was used for magnetic capture of parasites from microliter amounts of whole blood. A sensitivity down to a parasitemia of 10(-6)% was achieved using cultured parasites as a model.