Carbon nanoparticles in lateral flow methods to detect genes encoding virulence factors of Shiga toxin-producing Escherichia coli

Abstract

The use of carbon nanoparticles is shown for the detection and identification of different Shiga toxin-producing Escherichia coli virulence factors (vt1, vt2, eae and ehxA) and a 16S control (specific for E. coli) based on the use of lateral flow strips (nucleic acid lateral flow immunoassay, NALFIA). Prior to the detection with NALFIA, a rapid amplification method with tagged primers was applied. In the evaluation of the optimised NALFIA strips, no cross-reactivity was found for any of the antibodies used. The limit of detection was higher than for quantitative PCR (q-PCR), in most cases between 104 and 105 colony forming units/mL or 0.1–0.9 ng/μL DNA. NALFIA strips were applied to 48 isolates from cattle faeces, and results were compared to those achieved by q-PCR. E. coli virulence factors identified by NALFIA were in very good agreement with those observed in q-PCR, showing in most cases sensitivity and specificity values of 1.0 and an almost perfect agreement between both methods (kappa coefficient larger than 0.9). The results demonstrate that the screening method developed is reliable, cost-effective and user-friendly, and that the procedure is fast as the total time required is <1 h, which includes amplification.FigureResults achieved with multi-analyte NALFIA for E. coli virulence factors. First strip: blank; second to sixth strip: each of the STEC factors; seventh strip: all factorsElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-010-4334-z) contains supplementary material, which is available to authorized users.

Enhancing the Signal of Lateral Flow Immunoassays by Using Different Developing Methods

“…The anti-Texas Red assay consistently generated higher signal intensities than the anti-Cy5 assay, presumably due to differences in the affinity between antigen and antibody pairs. (10) …”

“…In addition, borate buffer (containing both H 3 BO 3 and Na 2 B 4 O 7 ) was previously reported to have superior performance for lateral flow immunoassays that adopt a hapten-antibody detection system. (9)(10)(11) We thus compared a boric buffer (H 3 BO 3 ) with a borate buffer (Na 2 B 4 O 7 ), in the presence of 1% BSA as the blocking molecule. We also compared these buffers with the standard immunoassay buffers TBS-T and TENTC.…”

A Novel, Universal and Sensitive Lateral-Flow Based Method for the Detection of Multiple Bacterial Contamination in Platelet Concentrations

“…7,8 Recently, several studies have been conducted to apply this method to detect specific nucleic acid fragments. 9,10 During the assay, genes were first amplified by PCR, and then hybridized by a set of probes that were specific to the conserved region of the target.…”

Carbon Nanoparticles as Detection Label for Diagnostic Antibody Microarrays

“…The image was processed using image analysis software to produce the pixel grey volume of the spots generated by the label. As an alternative label to gold and latex, carbon nanoparticles have been used to develop lateral flow immunoassays (LFIA) for over 15 years (Aldus et al 2003;Capps et al 2004;Kalogianni et al 2011;Koets et al 2006;Koets et al 2011;Lonnberg et al 2008;Noguera et al 2011;Posthuma-Trumpie et al 2008;van Amerongen et al 1993;van Amerongen & Koets 2005;van Dam et al 2004). The possibility to use the pixel grey volume of the carbon particles in data processing following digitization by a CCD camera and image analysis was already shown in 1994 in a comparison between a simple one-step lateral flow immunoassay and a radioimmunoassay specific for the human choriogonadotropin hormone (van Amerongen et al 1994).…”

“…In this one-step format, the labelled amplicons were mixed with the conjugate of neutravidin and carbon nanoparticles in incubation buffer, immediately applied and detected after one to several hours. Such mixed immuno-DNA formats have been used in lateral flow and microfluidic detection assays (Baeumner 2004;Blazkova et al 2009;Blazkova et al 2011;Corstjens et al 2001;Koets et al 2009;Kozwich et al 2000;Mens et al 2008;Noguera et al 2011;van Amerongen & Koets 2005;Wang et al 2006). To get proof of concept for the use of carbon nanoparticles as signal labels in antibody microarrays we studied two applications in which the antigens consisted of double-tagged DNA amplicons: the detection of L. monocytogenes and the detection of three antibiotic resistance genes from Salmonella spp.…”