Bacteriology Colorimetric detection of Helicobacter pylori DNA using isothermal helicase-dependent amplification and gold nanoparticle probes Pooria Gill a,b, ⁎ , Amir-Houshang Alvandi c , Hossein Abdul-Tehrani b , Majid Sadeghizadeh a,d a Department of Nanobiotechnology, Faculty of Basic Sciences, Tarbiat Modares University, Tehran 14115-175, Iran b Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran 14115-331, Iran c Department of Bacteriology, Faculty of Paramedicine, Iran University of Medical Sciences and Health Care, Tehran 14155-6183, Iran d Department of Genetics, Faculty of Basic Sciences, Tarbiat Modares University, Tehran 14115-175, Iran Received 14 March 2008; accepted 5 May 2008 Abstract This study describes a nanodiagnostic method using thermophilic helicase-dependent isothermal amplification (tHDA) and gold nanoparticle probes for colorimetric detection of Helicobacter pylori DNA. The primers targeting ureC gene were used for the amplification of bacterial DNA by the isothermal tHDA reaction, resulting in the accumulation of DNA amplicons. The amplicons were hybridized with specific gold nanoparticle probes. The hybrids were colorimetrically detected by the assembly of gold nanoparticles. Using this method, we detected as little as 10 CFU mL -1 of H. pylori within less than 1 h. Results obtained from the gastric biopsy samples showed 92.5% and 95.4% of sensitivity and specificity, respectively, in comparison with culture results, and 100% and 98.8% of sensitivity and specificity, respectively, in comparison with those of the histologic studies. Owing to its ease of operation, this assay significantly reduces the time and cost needed for the molecular diagnosis of H. pylori and has the potential to facilitate early detection of this pathogen. © 2008 Elsevier Inc. All rights reserved. Keywords: Gold nanoparticle probes; tHDA; Helicobacter pylori; Colorimetric detection; ureC 1. Introduction Helicobacter pylori is a common agent of chronic gastritis, gastric adenocarcinoma, gastric and duodenal ulcers, and mucosa-associated lymphoid tissue lymphoma (Ernst and Gold, 2000). Rapid diagnosis is essential for the effective treatment and management of these infections, and several diagnostic methods have been described for this microorganism (Brooks et al., 2004; Monteiro et al., 2001). We have also described the thermophilic helicase-dependent isothermal amplification (tHDA)–ELISA detection system for molecular identifying of this pathogen previously (Gill et al., 2007). This technique did not require thermocycler machine because of tHDA of DNA targets coupled with ELISA to allow simple analysis of several samples simul- taneously. In this study, we describe a colorimetric method using tHDA and gold nanoparticle probes (Figs. 1 and 2) for more simple, rapid, and cost-effective diagnosis of this bac- terium in gastrointestinal biopsies. 2. Materials and methods 2.1. Bacteria The H. pylori (ATCC 49503) strain was provided from the Department of Bacteriology, Faculty of Paramedicine, Iran University of Medical Sciences, Tehran, Iran, preserved in Luria-Bertani (LB) broth medium and 20% sterile glycerol. The Escherichia coli (ATCC 11775) was used to evaluate the specificity of the test (tHDA and gold nanoparticle probes) and grown in tryptic soy broth. 2.2. Specimens One hundred fourteen human gastric biopsies were obtained from patients and analyzed as they were received. Available online at www.sciencedirect.com Diagnostic Microbiology and Infectious Disease 62 (2008) 119 – 124 www.elsevier.com/locate/diagmicrobio ⁎ Corresponding author. Department of Nanobiotechnology, Faculty of Basic Sciences, Tarbiat Modares University, Tehran, P.O. Box 14115-175, Iran. Tel.: +98-9127615950, +98-2122944230; fax: +98-2122944230. E-mail address: pooriagill@yahoo.com (P. Gill). 0732-8893/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2008.05.003