Utilization of Small RNA Genes to Distinguish Vibrio cholerae Biotypes via Multiplex Polymerase Chain Reaction

Abstract

The diarrheal disease "cholera" is caused by Vibrio cholerae, and is primarily confined to endemic regions, mostly in Africa and Asia. It is punctuated by outbreaks and creates severe challenges to public health. The disease-causing strains are most-often members of serogroups O1 and O139. PCR-based methods allow rapid diagnosis of these pathogens, including the identification of their biotypes. However, this necessitates the selection of specific target sequences to differentiate even the closely related biotypes of V. cholerae. Oligonucleotides for selective amplification of small RNA (sRNA) genes that are specific to these V. cholerae subtypes were designed. The resulting multiplex PCR assay was validated using V. cholerae cultures (i.e., 19 V. cholerae and 22 non-V. cholerae isolates) and spiked stool samples. The validation using V. cholerae cultures and spiked stool suspensions revealed detection limits of 10-100 pg DNA per reaction and 1.5 cells/mL suspension, respectively. The multiplex PCR assay that targets sRNA genes for amplification enables the sensitive and specific detection, as well as the differentiation of V. cholerae-O1 classical, O1 El Tor, and O139 biotypes. Most importantly, the assay enables fast and cheaper diagnosis compared with classic culture-based methods.

Figure 1.

Determination of PCR detection thresholds: Thresholds of PCR detection with genomic DNA from (A) O1 classical isolate; (B) O1 El Tor; and (C) O139: Lane L: 25-bp molecular ladder; Lane N: no-template control; PCR products were generated with serially diluted genomic DNA templates: Lane 1 (10 ng), Lane 2 (1 ng), Lane 3 (100 pg), Lane 4 (10 pg), Lane 5 (1 pg), Lane 6 (100 fg), Lane 7 (10 fg), and Lane 8 (1 fg). The lowest DNA concentration that (still) produced visible amplicon bands was 10 pg per reaction for Vibrio cholerae O1 classical (A, Lane 4), and 100 pg for O1 El Tor (B, Lane 3) and O139 (C, Lane 3). This figure appears in color at www.ajtmh.org.

Figure 2.

Determination of PCR specificity: Evaluation with genomic DNA templates from selected Vibrio cholerae O1 classical isolates, O1 El Tor, and O139: Lane L: 25-bp molecular ladder; Lane N: no-template control; Lane 1–3: O1 classical identified by the 85-bp PCR signal; Lane 4–7: O1 El Tor identified by the combination of PCR signals at 85 and 96 bp; Lane 8–11: O139 identified by the combination of PCR signals at 85, 96, and 120 bp; and Lane 12–13: Vibrio vulnificus and Vibrio parahaemolyticus, respectively. This figure appears in color at www.ajtmh.org.

Figure 3.

Evaluation of the multiplex PCR assay with spiked human stool suspensions: Test for the applicability of the assay on DNA templates prepared from enrichment cultures inoculated with human stool suspension spiked with: (A) O1 classical; (B) O1 El Tor; and (C) O139 strains. Lane L: 25-bp molecular ladder; Lane N: no-template control; Lane C: enrichment culture of human stool suspension without spiking (control); enrichment culture of human stool suspensions spiked with Vibrio cholerae cell suspensions representing cell densities of: 1.5 × 10⁷ (Lane 1), 1.5 × 10⁶ (Lane 2), 1.5 × 10⁵ (Lane 3), 1.5 × 10⁴ (Lane 4), 1.5 × 10³ (Lane 5), 150 (Lane 6), 15 (Lane 7), 1.5 (Lane 8), 0.15 (Lane 9) and 0.015 (Lane 10) cells/mL stool suspension. Detection limits for the multiplex PCR with stool samples were 1.5 × 10⁵ cells/mL stool suspension for V. cholerae O1 classical (A, Lane 3) and O1 El Tor (B, Lane 3), and 1.5 cells/mL stool suspension for O139 (C, Lane 8). This figure appears in color at www.ajtmh.org.