Sensitive detection of multiple pathogens using a single DNA probe

Abstract

A simple but promising electrochemical DNA nanosensor was designed, constructed and applied to differentiate a few food-borne pathogens. The DNA probe was initially designed to have a complementary region in Vibrio parahaemolyticus (VP) genome and to make different hybridization patterns with other selected pathogens. The sensor was based on a screen printed carbon electrode (SPCE) modified with polylactide-stabilized gold nanoparticles (PLA-AuNPs) and methylene blue (MB) was employed as the redox indicator binding better to single-stranded DNA. The immobilization and hybridization events were assessed using differential pulse voltammetry (DPV). The fabricated biosensor was able to specifically distinguish complementary, non-complementary and mismatched oligonucleotides. DNA was measured in the range of 2.0×10(-9)-2.0×10(-13)M with a detection limit of 5.3×10(-12)M. The relative standard deviation for 6 replications of DPV measurement of 0.2µM complementary DNA was 4.88%. The fabricated DNA biosensor was considered stable and portable as indicated by a recovery of more than 80% after a storage period of 6 months at 4-45°C. Cross-reactivity studies against various food-borne pathogens showed a reliably sensitive detection of VP.

Electrochemical sensory detection ofSus scrofamtDNA for food adulteration using hybrid ferrocenylnaphthalene diimide intercalator as a hybridization indicator

“…1a) as a redox label. 15 In the past, methylene blue (MB) has oen been employed in electrochemical detection methods due to its stronger affinity for exposed guanine in single-stranded DNA; 16,17 however in this work, we have used a new indicator (FND) that specically binds to double-stranded DNA (dsDNA). The FND molecule binds to dsDNA through a threading intercalation mode, 18,19 in which the ferrocene molecule is inserted between the duplex and its two peripheral substituents (Fig.…”

A simple, portable, electrochemical biosensor to screen shellfish for Vibrio parahaemolyticus

“…In the work presented here, the above-mentioned disadvantages were anticipated and overcome. A previously constructed electrochemical mechanism (Nordin et al 2016 ) was modified and a simplified approach for fabrication of the DNA biosensor was based on a screen-printed carbon electrode reader with polylactide—stabilised gold nanoparticles and methylene blue as the redox indicator. Results for the analytical parameters of this sensor (selectivity, reproducibility, sensitivity and cross-reactivity) compare favourably with results from a range of other DNA sensors as recently reviewed by Wang et al (2016) .…”

“…Gold (III) chloride trihydrate (Sigma-Aldrich) and poly (lactic acid) resin, commercial grade 4042D (NatureWorks) were of analytical grade and were used without further purification. Gold nanoparticles (AuNPs) and polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) were synthesized and characterized, as described in detail in our previous report which formed the foundation for this improvement (Nordin et al 2016 ). Electrochemical measurements were performed using a portable single-technique customized potentiostat (DropSens, Spain) for use with electrochemical sensor consisting of a screen printed electrode.…”

“…A number of attempts to develop portable biosensors for monitoring foodborne pathogens have been reported (Ferguson et al 2011 ; Lee et al 2016 ; Qin et al 2016 ), but there would appear to be none which take this approach to detection of V. parahaemolyticus . Polylactide (PLA)—stabilized gold nanoparticles (AuNPs) have been widely used in a variety of analytical sensing applications (Han et al 2014 ; Nordin et al 2016 ; Song et al 2006 ; Wu et al 2011 ) and here gold nanoparticles (AuNPs) stabilized by the nanofiber were used to modify the electrode surface to increase the active surface area of the working electrode (WE) (Ding et al 2012 ; Wu et al 2011 ).…”

Rapid identification and detection of Vibrio parahaemolyticus via different types of modus operandi with LAMP method in vivo

“…With the advent of nucleic acid-based methods, such as multiplex PCR, real-time PCR, RAPD-PCR, REP-PCR, ERIC-PCR, GS-PCR, nucleic acid sequence-based amplification (NASBA), fluorescence in situ hybridization (FISH), and micro arrays (Di Pinto et al 2012;Letchumanan et al 2014;Nordin et al 2016;Cho et al 2017;Xu et al 2018), the detection of V. parahaemolyticus is becoming more convenient. In addition, biosensor and immunological-based methods have also become available (Kumar et al 2011).…”