GMO detection using a bioluminescent real time reporter (BART) of loop mediated isothermal amplification (LAMP) suitable for field use

<p>Abstract</p> <p>Background</p> <p>There is an increasing need for quantitative technologies suitable for molecular detection in a variety of settings for applications including food traceability and monitoring of genetically modified (GM) crops and their products through the food processing chain. Conventional molecular diagnostics utilising real-time polymerase chain reaction (RT-PCR) and fluorescence-based determination of amplification require temperature cycling and relatively complex optics. In contrast, isothermal amplification coupled to a bioluminescent output produced in real-time (BART) occurs at a constant temperature and only requires a simple light detection and integration device.</p> <p>Results</p> <p>Loop mediated isothermal amplification (LAMP) shows robustness to sample-derived inhibitors. Here we show the applicability of coupled LAMP and BART reactions (LAMP-BART) for determination of genetically modified (GM) maize target DNA at low levels of contamination (0.1-5.0% GM) using certified reference material, and compare this to RT-PCR. Results show that conventional DNA extraction methods developed for PCR may not be optimal for LAMP-BART quantification. Additionally, we demonstrate that LAMP is more tolerant to plant sample-derived inhibitors, and show this can be exploited to develop rapid extraction techniques suitable for simple field-based qualitative tests for GM status determination. We also assess the effect of total DNA assay load on LAMP-BART quantitation.</p> <p>Conclusions</p> <p>LAMP-BART is an effective and sensitive technique for GM detection with significant potential for quantification even at low levels of contamination and in samples derived from crops such as maize with a large genome size. The resilience of LAMP-BART to acidic polysaccharides makes it well suited to rapid sample preparation techniques and hence to both high throughput laboratory settings and to portable GM detection applications. The impact of the plant sample matrix and genome loading within a reaction must be controlled to ensure quantification at low target concentrations.</p

Treatment with an anti-CD14 monoclonal antibody delays and inhibits lipopolysaccharide-induced gene expression in humans in vivo

CD14 is a receptor important for activation of cells by lipopolysaccharide (LPS). Treatment with the CD14 antibody IC14 was previously found to attenuate the release of proinflammatory cytokines and some chemokines into the circulation of healthy humans intravenously injected with LPS. To determine the role of circulating leukocytes in CD14-dependent gene expression, 16 healthy volunteers received LPS preceded by either IC14 or placebo. At different time points, mRNA was isolated from whole blood and gene expression was determined by multiplex ligation-dependent probe amplification (MLPA). LPS induced MIP-1alpha, MIP-1beta, IL-8, IL-1beta, and IL-1Ra mRNA production, which was delayed by 1 hr and reduced twofold by IC14 treatment. TNFR1 was unresponsive, whereas other investigated cytokines remained undetectable. Further, LPS showed differential effects on NFkappaB gene expression. LPS induced IkappaBalpha production, whereas p50 was unresponsive and p65 and p49/p100 remained undetectable. LPS induced IkappaBalpha expression was delayed (1 hr) and reduced by IC14. Gene expression profiles in blood cells corresponded poorly with observed changes in plasma levels. These data suggest that peripheral blood cells are of negligible importance in LPS-induced production of inflammatory mediators in vivo and that LPS may activate genes via a CD14-independent pathway that is slower and less efficien

Large genomic deletions and duplications in the BRCA1 gene identified by a novel quantitative method

We applied a novel method to detect single or multiple exon deletions and amplifications in the BRCA1 gene. The test, called multiplex ligation-dependent probe amplification (MLPA), uses probes designed to hybridize adjacently to the target sequence. After ligation, the joined probes are amplified and quantified. Our two diagnostic laboratories have tested in the recent years 805 families by conventional PCR-based techniques, and found 116 BRCA1 and 28 BRCA2 mutation-positive families. Using MLPA, we have tested the remaining 661 noninformative breast cancer families and identified five distinct BRCA1 germ-line mutations in five families: a deletion of exon 8, a deletion of exons 20-22, a duplication of exon 13 and exons 21-23, respectively, and a triplication, encompassing exons 17-19. Genomic deletions of BRCA1 constitute a substantial fraction of mutations in Dutch breast cancer families. If MLPA had been included in our initial BRCA1 testing, 33 families with a deletion or duplication would have been identified, representing 27% of the total 121 BRCA1 mutation-positive families. The MLPA test for BRCA1 ensures a sensitive and comprehensive high-throughput screening test for genomic rearrangement and can easily be implemented in the molecular analysis of BRCA1

Concentrations of substances that do not interfere with the detection of <i>C. difficile</i> by HE-LAMP-BART.

<p>Concentrations of substances that do not interfere with the detection of <i>C. difficile</i> by HE-LAMP-BART.</p

Model describing low copy number non-detection for dilutive methods.

<p>The right hand Y-axis (% amplification) shows the increase in amplification associated with dilution of inhibitors from a fecal sample. A range of starting target concentrations (CFU/gram) is plotted with the left hand Y-axis showing the copy number present in a 50 µl reaction volume (Illumigene). The reduced inhibition and increasing % amplification with increasing dilution is concomitant with a reduction in copy number. At lower CFU/gram samples, the dilution places the copy number per reaction beneath the sensitivity of the assay. HE-LAMP-BART used a 50 fold dilution together with the inhibitor removal such that a higher copy number can be present in the reaction.</p

Example <i>C. difficile</i> HE-LAMP-BART results.

<p>One HE-LAMP-BART run of 14 samples from the Illumigene comparison. Panel A shows the <i>C. difficile</i> LAMP-BART peaks obtained and includes a positive control of 10⁴ copies of <i>C. difficile</i> genomic DNA (PTC) and a no template control (NTC). Panel B shows the Inhibitor Control LAMP-BART results obtained simultaneously from the same set of HE eluates and a buffer uninhibited control (UIC).</p

Example <i>C. difficile</i> HE-LAMP-BART results.

<p>Peak time and results called for the example LAMP-BART data set presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083808#pone-0083808-g001" target="_blank">Figure 1</a>. For the <i>C. difficile</i> assay Positive control of 10⁴ copies of <i>C. difficile</i> genomic DNA (PTC) and a no template control (NTC) were used. For the inhibitor control LAMP-BART results a buffer uninhibited control (UIC) was used.</p

Comparison between the reference method and PHE-Cambridge multiplex RT-PCR and HE-LAMP-BART.

<p>Comparison between the reference method and PHE-Cambridge multiplex RT-PCR and HE-LAMP-BART.</p

Primers and probes used for real-time PCR.

<p>Primers and probes used for real-time PCR.</p

Comparison of a subset of samples from the Illumigene-HE-LAMP-BART study with GeneXpert and RIDAQuick against the reference method.

<p>Comparison of a subset of samples from the Illumigene-HE-LAMP-BART study with GeneXpert and RIDAQuick against the reference method.</p