Multiplex 16S rRNA-derived geno-biochip for detection of 16 bacterial pathogens from contaminated foods

Abstract

Foodborne diseases caused by various pathogenic bacteria occur worldwide. To prevent foodborne diseases and minimize their impacts, it is important to inspect contaminated foods and specifically detect many types of pathogenic bacteria. Several DNA oligonucleotide biochips based on 16S rRNA have been investigated to detect bacteria; however, a mode of detection that can be used to detect diverse pathogenic strains and to examine the safety of food matrixes is still needed. In the present work, a 16S rRNA gene-derived geno-biochip detection system was developed after screening DNA oligonucleotide specific capture probes, and it was validated for multiple detection of 16 pathogenic strains that frequently occur with a signature pattern. rRNAs were also used as detection targets directly obtained from cell lysates without any purification and amplification steps in the bacterial cells separated from 8 food matrixes by simple pretreatments. Thus, the developed 16S rRNA-derived geno-biochip can be successfully used for the rapid and multiple detection of the 16 pathogenic bacteria frequently isolated from contaminated foods that are important for food safety.

Keywords: Direct RNA-detection; Food matrix; Foodborne pathogen; Multiple detection; Oligonucleotide microarray.

Figure 1

Schematic diagrams of (A) the array format for the 16S rRNA‐derived geno‐biochip containing 27 specific capture probes and (B) the schematic steps of the 16S rRNA detection of bacterial cells directly separated from food matrixes.

Figure 2

The heat map plot of S/N of fluorescence intensities acquired from hybridization with each amplified target. Inner numbers indicate the S/N of each spots. The signals were marked as different colors: black for true‐positive spots; gray for false‐positive spots; and white for negative spots. Positive signals were determined when S/N was equal to or higher than 2, and negative signals were determined when S/N was lower than 2.

Figure 3

Sensitivity of the 16S rRNA‐derived geno‐biochip. The diluted PCR amplicons of S. sonnei were used for sensitivity determination. (A) The plot of dynamic detection ranges based on the fluorescence intensities according to target DNA concentration changes for each capture probe SHBO‐1 (closed circle) and SHBO‐2 (open circle). (B) Raw hybridization images for (i) 24.8 nM, (ii) 12.4 nM, and (iii) 2.48 nM 16S rDNA targets. Each value of plot was the mean of four repeated spots, and the error bars represent standard deviation.

Figure 4

The raw images of hybridization with complete RNA targets directly obtained from isolated S. Enteritidis cells in food matrixes using the rRNA‐derived geno‐biochip in Fig. 1. (A) control, (B) pork, (C) egg, (D) milk, (E) rice, (F) cheese, (G) canned corn, (H) ham, and (I) fish cake. White box indicates specific spots. Asterisks on images represent each p‐value range of POCO and SAEN calculated by paired t‐tests. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.0005.