Development of a serotype-specific DNA microarray for identification of some Shigella and pathogenic Escherichia coli strains

Abstract

Shigella and pathogenic Escherichia coli are major causes of human infectious diseases and are responsible for millions of cases of diarrhea worldwide every year. A convenient and rapid method to identify highly pathogenic serotypes of Shigella and E. coli is needed for large-scale epidemiologic study, timely clinical diagnosis, and reliable quarantine of the pathogens. In this study, a DNA microarray targeting O-serotype-specific genes was developed to detect 15 serotypes of Shigella and E. coli, including Shigella sonnei; Shigella flexneri type 2a; Shigella boydii types 7, 9, 13, 16, and 18; Shigella dysenteriae types 4, 8, and 10; and E. coli O55, O111, O114, O128, and O157. The microarray was tested against 186 representative strains of all Shigella and E. coli O serotypes, 38 clinical isolates, and 9 strains of other bacterial species that are commonly present in stool samples and was shown to be specific and reproducible. The detection sensitivity was 50 ng genomic DNA or 10(4) CFU per ml in mock stool specimens. This is the first report of a microarray for serotyping Shigella and pathogenic E. coli. The method has a number of advantages over traditional bacterial culture and antiserum agglutination methods and is promising for applications in basic microbiological research, clinical diagnosis, food safety, and epidemiological surveillance.

FIG. 1.

Probe positions on the slide. Numbers 1-1 to 15-2 are the specific probes for the target strains. Numbers 16-1 and 16-2 are the positive control probes based on the 16S rRNA genes of all Shigella and E. coli strains. Number 17 is the negative control probe. Number 18 is the positional reference and printing control probe.

FIG. 2.

Agrose gel electrophoresis of multiplex PCR products. Lanes: Mr, molecular weight standards (lambda DNA/EcoRI plus HindIII marker); A, S. boydii type 7; B, S. boydii type 9; C, S. boydii type 13; D, S. boydii type 16; E, S. boydii type 18; F, S. dysenteriae type 4; G, S. dysenteriae type 8; H, S. dysenteriae type 10; I, S. flexneri type 2a; J, S. sonnei; K, E. coli O55; L, E. coli O111; M, E. coli O114; N, E. coli O128; O, E. coli O157. Two bands were generated from the PCR products: one was the 16S rRNA gene (1.2 kb), and the other was the gene specific to the individual serotype.

FIG. 3.

Microarray differentiation of the pathogens. (1) S. flexneri type 2a and E. coli O13, O129, and O135. (2) S. sonnei. (3) S. boydii type 7. (4) S. boydii type 13. (5) S. boydii type 16. (6) S. boydii type 18. (7) S. dysenteriae type 4. (8) S. dysenteriae type 8. (9) S. dysenteriae type 10. (10) E. coli O55. (11) E. coli O111. (12) E. coli O114. (13) E. coli O128. (14) E. coli O157. (15) S. boydii type 9. (16) Other serotype strains of E. coli or Shigella, Salmonella, and V. cholerae. (17) B. cereus and S. aureus. (18) DNAs from healthy-adult stool specimens.