Discriminating Bacterial and Viral Infection Using a Rapid Host Gene Expression Test.

Objectives: Host gene expression signatures discriminate bacterial and viral infection but have not been translated to a clinical test platform. This study enrolled an independent cohort of patients to describe and validate a first-in-class host response bacterial/viral test.

Design: Subjects were recruited from 2006 to 2016.

Immune Profiling Panel: A Proof-of-Concept Study of a New Multiplex Molecular Tool to Assess the Immune Status of Critically Ill Patients.

Background: Critical illness such as sepsis is a life-threatening syndrome defined as a dysregulated host response to infection and is characterized by patients exhibiting impaired immune response. In the field of diagnosis, a gap still remains in identifying the immune profile of critically ill patients in the intensive care unit (ICU).

Methods: A new multiplex immune profiling panel (IPP) prototype was assessed for its ability to semiquantify messenger RNA immune-related markers directly from blood, using the FilmArray System, in less than an hour.

The influence of nucleotide sequence and temperature on the activity of thermostable DNA polymerases.

Extension rates of a thermostable, deletion-mutant polymerase were measured from 50°C to 90°C using a fluorescence activity assay adapted for real-time PCR instruments. Substrates with a common hairpin (6-base loop and a 14-bp stem) were synthesized with different 10-base homopolymer tails. Rates for A, C, G, T, and 7-deaza-G incorporation at 75°C were 81, 150, 214, 46, and 120 seconds(-1).

Influence of PCR reagents on DNA polymerase extension rates measured on real-time PCR instruments.

Background: Radioactive DNA polymerase activity methods are cumbersome and do not provide initial extension rates. A simple extension rate assay would enable study of basic assumptions about PCR and define the limits of rapid PCR.

Methods: A continuous assay that monitors DNA polymerase extension using noncovalent DNA dyes on common real-time PCR instruments was developed.

Stopped-flow DNA polymerase assay by continuous monitoring of dNTP incorporation by fluorescence.

DNA polymerase activity was measured by a stopped-flow assay that monitors polymerase extension using an intercalating dye. Double-stranded DNA formation during extension of a hairpin substrate was monitored at 75°C for 2 min. Rates were determined in nucleotides per second per molecule of polymerase (nt/s) and were linear with time and polymerase concentration from 1 to 50 nM.

Symmetric snapback primers for scanning and genotyping of the cystic fibrosis transmembrane conductance regulator gene.

Background: High-resolution melting of PCR products is an efficient and analytically sensitive method to scan for sequence variation, but detected variants must still be identified. Snapback primer genotyping uses a 5' primer tail complementary to its own extension product to genotype the resulting hairpin via melting. If the 2 methods were combined to analyze the same PCR product, the residual sequencing burden could be reduced or even eliminated.

High-resolution DNA melting analysis in clinical research and diagnostics.

Among nucleic acid analytical methods, high-resolution melting analysis is gaining more and more attention. High-resolution melting provides simple, homogeneous solutions for variant scanning and genotyping, addressing the needs of today's overburdened laboratories with rapid turnaround times and minimal cost. The flexibility of the technique has allowed it to be adopted by a wide range of disciplines for a variety of applications.

Spatial DNA melting analysis for genotyping and variant scanning.

A continuous-flow, temperature gradient microfluidic device was used to demonstrate spatial DNA melting analysis with the resolution and reproducibility necessary for clinical SNP scanning and genotyping of human genomic DNA. With a steady-state temperature gradient of 20-30 degrees C across a sample, melting curves were constructed from a single fluorescence data acquisition. This technique was used to scan for heterozygotes and to fully genotype single base changes using unlabeled probes.

Scanning the cystic fibrosis transmembrane conductance regulator gene using high-resolution DNA melting analysis.

Background: Complete gene analysis of the cystic fibrosis transmembrane conductance regulator gene (CFTR) by scanning and/or sequencing is seldom performed because of the cost, time, and labor involved. High-resolution DNA melting analysis is a rapid, closed-tube alternative for gene scanning and genotyping.

Methods: The 27 exons of CFTR were amplified in 37 PCR products under identical conditions.

Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis.

This protocol permits the simultaneous mutation scanning and genotyping of PCR products by high-resolution DNA melting analysis. This is achieved using asymmetric PCR performed in the presence of a saturating fluorescent DNA dye and unlabeled oligonucleotide probes. Fluorescent melting curves of both PCR amplicons and amplicon-probe duplexes are analyzed.