Visual Detection of Bacterial Pathogens via PNA-Based Padlock Probe Assembly and Isothermal Amplification of DNAzymes

Abstract

We have developed a self-reporting isothermal system for visual bacterial pathogen detection with single base resolution. The new DNA diagnostic is based on combination of peptide nucleic acid (PNA) technology, rolling circle amplification (RCA) and DNAzymes. PNAs are used as exceedingly selective chemical tools that bind genomic DNA at a predetermined sequence under nondenaturing conditions. After assembly of the PNA-DNA construct a padlock probe is circularized on the free strand. The probe incorporates a G-quadruplex structure flanked by nicking enzyme recognition sites. The assembled circle serves as a template for a novel hybrid RCA strategy that allows for exponential amplification and production of short single-stranded DNA pieces. These DNA fragments fold into G-quadruplex structures and when complexed with hemin become functional DNAzymes. The catalytic activity of each DNAzyme unit leads to colorimetric detection and provides the second amplification step. The combination of PNA, RCA, and DNAzymes allows for sequence-specific and highly sensitive detection of bacteria with a colorimetric output observed with the naked eye. Herein, we apply this method for the discrimination of Escherichia coli, Salmonella typhimurium, and Clostridium difficile genomes.

Scalable DNA-Based Magnetic Nanoparticle Agglutination Assay for Bacterial Detection in Patient Samples

“…17 In addition, as each assay only requires a single padlock probe for identification with minimal interactions between probes for different sites, highly multiplexed analysis of pathogens and resistance markers can routinely be achieved. 18 Detection of RCA products is often based on colorimetric 19,20 or fluorescence 21,22 analysis and recently other methods employing MNPs for readout of RCA coils have been established. 23,24 Colorimetric detection is simple but consists of multiple reaction steps, which is sub-optimal for microfluidic integration.…”

Rolling circle amplification as isothermal gene amplification in molecular diagnostics

“…Gomez et al developed a colorimetric detection method of bacterial pathogens using HRCA combined with nicking endonuclease, which they called exponential linear RCA (ELRCA) (Figure 4) 32 . They used bis-PNA openers to expose target genomic DNA sequence for padlock probe binding.…”

“…In addition, RCA can amplify target DNA sequences with high specificity and sensitivity; 1 copy of target DNA in 100,000 copies of non-target DNAs can be amplified by RCA 10 . High sensitivity and specificity makes RCA as a feasible tool for detection of single nucleotide polymorphisms (SNPs) 8,[11][12][13][14][15][16][17] , microRNAs (miRNA) 9,18-29 , bacterial [30][31][32][33][34] , and viral nucleic acids [35][36][37][38][39] . Amplified DNA products can be detected with diverse methods including fluorescence measurement 8 , colorimetric assay 32,37 , enzymatic luminescence assay 20,21 , or elec-tric signals 33 .…”

Timing readout in paper device for quantitative point-of-use hemin/G-quadruplex DNAzyme-based bioassays

“…The sensitive, specific detection of a wide range of analytes, including metal ions (Hua et al, 2012;Li et al,2013;Liu et al, 2014;Yang et al, 2010;Wang and Irudayaraj, 2011;Wang et al, 2012;Wang et al, 2014c), small molecules (Garai-Ibabe et al, 2014;Wang et al, 2012;Wang et al, 2014g), DNAs (Wang et al, 2011;Wang et al, 2013;Wang et al, 2014a;Zhou et al, 2013), micro RNAs (Wen et al, 2012), proteins (Fu et al, 2011;He et al, 2012;Hou et al, 2014;Huang et al, 2013;Wang et al, 2014f;Tang et al, 2012;Zong et al, 2014), and bacterial pathogens (Gomez et al, 2014), implies the great potential of peroxidase-mimicking DNAzymes in broad applications ranging from medical diagnosis, environmental monitoring to food safety testing. These DNAzymes can effectively catalyze H 2 O 2 -mediated oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) (Fu et al, 2011;Gomez et al, 2014;He et al, 2012;Huang et al, 2013;Wang et al, 2012;Wang et al, 2014a;Wen et al, 2012;Tang et al,2012;Zhou et al, 2013), 3,3',5,5'-tetrazmethyl benzidinesulfate (TMB) (Yang et al, 2010), or luminol (Wang et al, 2011;Zong et al, 2014). As such, the existing quantitative hemin/G-quadruplex DNAzyme-based bioassays generally transducer recognition chemistry using several techniques, with spectrophotometry (Fu et al, 2011;…”

“…These DNAzymes can effectively catalyze H 2 O 2 -mediated oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) (Fu et al, 2011;Gomez et al, 2014;He et al, 2012;Huang et al, 2013;Wang et al, 2012;Wang et al, 2014a;Wen et al, 2012;Tang et al,2012;Zhou et al, 2013), 3,3',5,5'-tetrazmethyl benzidinesulfate (TMB) (Yang et al, 2010), or luminol (Wang et al, 2011;Zong et al, 2014). As such, the existing quantitative hemin/G-quadruplex DNAzyme-based bioassays generally transducer recognition chemistry using several techniques, with spectrophotometry (Fu et al, 2011;Gomez et al, 2014;He et al, 2012;Huang et al, 2013;Tang et al, 2012;Wang et al, 2012;Wang et al,2014a;Wen et al, 2012;Yang et al, 2010;Zhou et al, 2013) and chemiluminometry (Wang et al, 2011;Zong et al, 2014) being the two most common types. Other methods established recently include fluorescence (Hua et al, 2012;Liu et al, 2014), electrochemistry (Hou et al, 2014;Li et al, 2013;Wang et al, 2013;Wang et al, 2014c;Wang et al, 2014f;Wang et al, 2014g) and Raman scattering (Wang and Irudayaraj, 2011) techniques.…”