Multiplexed identification, quantification and genotyping of infectious agents using a semiconductor biochip.

The emergence of pathogens resistant to existing antimicrobial drugs is a growing worldwide health crisis that threatens a return to the pre-antibiotic era. To decrease the overuse of antibiotics, molecular diagnostics systems are needed that can rapidly identify pathogens in a clinical sample and determine the presence of mutations that confer drug resistance at the point of care. We developed a fully integrated, miniaturized semiconductor biochip and closed-tube detection chemistry that performs multiplex nucleic acid amplification and sequence analysis. The approach had a high dynamic range of quantification of microbial load and was able to perform comprehensive mutation analysis on up to 1,000 sequences or strands simultaneously in <2 h. We detected and quantified multiple DNA and RNA respiratory viruses in clinical samples with complete concordance to a commercially available test. We also identified 54 drug-resistance-associated mutations that were present in six genes of Mycobacterium tuberculosis, all of which were confirmed by next-generation sequencing.

An array-based melt curve analysis method for the identification and classification of closely related pathogen strains.

PCR-based techniques are widely used to identify disease causing bacterial and viral pathogens, especially in point-of-care or near-patient clinical settings that require rapid results and sample-to-answer workflows. However, such techniques often fail to differentiate between closely related species that have highly variable genomes. Here, a homogenous (closed-tube) pathogen identification and classification method is described that combines PCR amplification, array-based amplicon sequence verification, and real-time detection using an inverse fluorescence fluorescence-resonance energy transfer technique. The amplification is designed to satisfy the inclusivity criteria and create ssDNA amplicons, bearing a nonradiating quencher moiety at the 5'-terminus, for all the related species. The array includes fluorescent-labeled probes which preferentially capture the variants of the amplicons and classify them through solid-phase thermal denaturing (melt curve) analysis. Systematic primer and probe design algorithms and empirical validation methods are presented and successfully applied to the challenging example of identification of, and differentiation between, closely related human rhinovirus and human enterovirus strains.

A mutation in teg-4, which encodes a protein homologous to the SAP130 pre-mRNA splicing factor, disrupts the balance between proliferation and differentiation in the C. elegans germ line.

Dividing stem cells can give rise to two types of daughter cells; self-renewing cells that have virtually the same properties as the parent cell, and differentiating cells that will eventually form part of a tissue. The Caenorhabditis elegans germ line serves as a model to study how the balance between these two types of daughter cells is maintained. A mutation in teg-4 causes over-proliferation of the stem cells, thereby disrupting the balance between proliferation and differentiation. We have cloned teg-4 and found it to encode a protein homologous to the highly conserved splicing factor subunit 3 of SF3b. Our allele of teg-4 partially reduces TEG-4 function. In an effort to determine how teg-4 functions in controlling stem cell proliferation, we have performed genetic epistasis analysis with known factors controlling stem cell proliferation. We found that teg-4 is synthetic tumorous with genes in both major redundant genetic pathways that function downstream of GLP-1/Notch signaling to control the balance between proliferation and differentiation. Therefore, teg-4 is unlikely to function specifically in either of these two genetic pathways. Further, the synthetic tumorous phenotype seen with one of the genes from these pathways is epistatic to glp-1, indicating that teg-4 functions downstream of glp-1, likely as a positive regulator of meiotic entry. We propose that a reduction in teg-4 activity reduces the splicing efficiency of targets involved in controlling the balance between proliferation and differentiation. This results in a shift in the balance towards proliferation, eventually forming a germline tumor.