Rapid Exonuclease Digestion of PCR-Amplified Targets for Improved Microarray Hybridization

Boissinot, Karel; Huletsky, Ann; Peytavi, Régis; Turcotte, Sophie; Veillette, Véronique; Boissinot, Maurice; Picard, François J.; Martel, Eric A.; Bergeron, Michel G.

doi:10.1373/clinchem.2007.091157

Cited by 16 publications

(12 citation statements)

References 18 publications

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“…To achieve rapid microarray hybridization, amplicons were subjected to a digestion with 15 U of lambda exonuclease (Thermo Fisher Scientific, Waltham, MA, USA) to generate single stranded DNA by digesting the non-complementary strand with a 5′ phosphorylated modification ( Figure 3 ), as described in previous studies [ 61 , 62 , 65 ]. The exonuclease digestion was performed in a final volume of 25 μL for 30 min at 37 °C, then incubated for 10 min at 95 °C, followed by an immediate addition of 25 μL of 2× Arrayit Hybridization Buffer (Arrayit).…”

Section: Methodsmentioning

confidence: 99%

Sensitive Genotyping of Foodborne-Associated Human Noroviruses and Hepatitis A Virus Using an Array-Based Platform

Quiñones

Lee

Martinsky³

et al. 2017

Sensors

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Human noroviruses (NoV) are the leading cause of human gastroenteritis in populations of all ages and are linked to most of the foodborne outbreaks worldwide. Hepatitis A virus (HAV) is another important foodborne enteric virus and is considered the most common agent causing acute liver disease worldwide. In the present study, a focused, low-density DNA microarray was developed and validated for the simultaneous identification of foodborne-associated genotypes of NoV and HAV. By employing a novel algorithm, capture probes were designed to target variable genomic regions commonly used for typing these foodborne viruses. Validation results showed that probe signals, specific for the tested NoV or HAV genotypes, were on average 200-times or 38-times higher than those detected for non-targeted genotypes, respectively. To improve the analytical sensitivity of this method, a 12-mer oligonucleotide spacer sequence was added to the capture probes and resulted in a detection threshold of less than 10 cRNA transcripts. These findings have indicated that this array-based typing sensor has the accuracy and sensitivity for identifying NoV and HAV genotypic profiles predominantly linked to food poisoning. The implementation of this typing sensor would thus provide highly relevant and valuable information for use in surveillance and outbreak attribution.

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Section: Methodsmentioning

confidence: 99%

Sensitive Genotyping of Foodborne-Associated Human Noroviruses and Hepatitis A Virus Using an Array-Based Platform

Quiñones

Lee

Martinsky³

et al. 2017

Sensors

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“…To achieve a rapid microarray hybridization (Boissinot et al, 2007), single stranded DNA targets were produced after a lambda exonuclease digestion of the PCR-amplified targets, consisting of 10 μl of the eluate, 10 U of lambda exonuclease and 1× lambda exonuclease reaction buffer (Epicenter Biotechnologies, Madison, WI, USA) in a final volume of 23 μl for 15 min at 37°C, followed by addition of 23 μl of 2× Hybridization Buffer (InDevR, Inc.). The hybridization mixture was applied to each microarray, and the slides were incubated in a humidified chamber (InDevR, Inc.) for 1 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

O-antigen and Virulence Profiling of Shiga Toxin-Producing Escherichia coli by a Rapid and Cost–Effective DNA Microarray Colorimetric Method

Quiñones¹,

Swimley²,

Narm³

et al. 2012

Front. Cell. Inf. Microbio.

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Shiga toxin-producing Escherichia coli (STEC) is a leading cause of foodborne illness worldwide. The present study developed the use of DNA microarrays with the ampliPHOX colorimetric method to rapidly detect and genotype STEC strains. A low-density 30-mer oligonucleotide DNA microarray was designed to target O-antigen gene clusters of 11 E. coli serogroups (O26, O45, O91, O103, O104, O111, O113, O121, O128, O145, and O157) that have been associated with the majority of STEC infections. In addition, the DNA microarray targeted 11 virulence genes, encoding adhesins, cytotoxins, proteases, and receptor proteins, which have been implicated in conferring increased ability to cause disease for STEC. Results from the validation experiments demonstrated that this microarray-based colorimetric method allowed for a rapid and accurate genotyping of STEC reference strains from environmental and clinical sources and from distinct geographical locations. Positive hybridization signals were detected only for probes targeting serotype and virulence genes known to be present in the STEC reference strains. Quantification analysis indicated that the mean pixel intensities of the signal for probes targeting O-antigen or virulence genes were at least three times higher when compared to the background. Furthermore, this microarray-based colorimetric method was then employed to genotype a group of E. coli isolates from watershed sediment and animal fecal samples that were collected from an important region for leafy-vegetable production in the central coast of California. The results indicated an accurate identification of O-type and virulence genes in the tested isolates and confirmed that the ampliPHOX colorimetric method with low-density DNA microarrays enabled a fast assessment of the virulence potential of STEC using low-cost reagents and instrumentation.

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“…To determine that a gene target was present in the sample, a multiplex PCR amplification was performed to generate biotinylated fragments, ranging from 163 to 223 base pairs ( Table 1). The PCR-amplified fragments were subjected to lambda exonuclease digestion to produce single-stranded DNA targets for achieving a rapid microarray hybridization (Boissinot et al, 2007). For photopolymerization to occur, a streptavidinconjugated photoinitiator is used to specifically label the microarrays that have been hybridized with the biotin-labeled, single-stranded DNA targets (Kuck and Taylor, 2008).…”

Section: A Novel Colorimetric Methods For Detection Of Pathogenic E Cmentioning

confidence: 99%

Identification ofEscherichia coliO157 by Using a Novel Colorimetric Detection Method with DNA Microarrays

Quiñones

Swimley

Taylor

et al. 2011

Foodborne Pathogens and Disease

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Shiga toxin-producing Escherichia coli O157 is a leading cause of foodborne illness worldwide. To evaluate better methods to rapidly detect and genotype E. coli O157 strains, the present study evaluated the use of ampliPHOX, a novel colorimetric detection method based on photopolymerization, for pathogen identification with DNA microarrays. A low-density DNA oligonucleotide microarray was designed to target stx1 and stx2 genes encoding Shiga toxin production, the eae gene coding for adherence membrane protein, and the per gene encoding the O157-antigen perosamine synthetase. Results from the validation experiments demonstrated that the use of ampliPHOX allowed the accurate genotyping of the tested E. coli strains, and positive hybridization signals were observed for only probes targeting virulence genes present in the reference strains. Quantification showed that the average signal-to-noise ratio values ranged from 47.73 AE 7.12 to 76.71 AE 8.33, whereas average signal-to-noise ratio values below 2.5 were determined for probes where no polymer was formed due to lack of specific hybridization. Sensitivity tests demonstrated that the sensitivity threshold for E. coli O157 detection was 100-1000 CFU=mL. Thus, the use of DNA microarrays in combination with photopolymerization allowed the rapid and accurate genotyping of E. coli O157 strains.

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Rapid Exonuclease Digestion of PCR-Amplified Targets for Improved Microarray Hybridization

Cited by 16 publications

References 18 publications

Sensitive Genotyping of Foodborne-Associated Human Noroviruses and Hepatitis A Virus Using an Array-Based Platform

Sensitive Genotyping of Foodborne-Associated Human Noroviruses and Hepatitis A Virus Using an Array-Based Platform

O-antigen and Virulence Profiling of Shiga Toxin-Producing Escherichia coli by a Rapid and Cost–Effective DNA Microarray Colorimetric Method

Identification ofEscherichia coliO157 by Using a Novel Colorimetric Detection Method with DNA Microarrays

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