Table 1.
List of cholera RLDT primers used in this study.
Fig 1.
Workflow of cholera RLDT using RLDT kit.
Step 1, using the fixed volume pipette provided in the kit, add a stool sample to the Sample Processing Tube (SPT) with lysis buffer. Step 2, Cap the SPT tube and mix by inverting the tube up and down. Squeeze the SPT tube to add the lysis buffer + stool sample to the lysis tube. Step 3, lysis tube is heated for 5 minutes in a heat block or water bath. Step 4, add 25 μL of lysate to each lyophilized reaction tube (LRT) strip tube using a disposable fixed volume pipette. Step 5, place the LRTs in the reader. Scan the barcode for the cholera RLDT program. Step 6, read the results displayed on the screen real time or after 40 minutes. All the reagents and sample processing accessories required for RLDT are included in the kit. The illustration was created with BioRender.com https:doi.org/10.1371/journal.pntd.0010180.g001.
Fig 2.
Workflow of cholera RLDT from water samples.
Step 1, Filter 400ml water using a syringe filter. Step 2, cut membrane in half and insert one half in the lysis tube (A). Place the other half in APW media (B) for overnight enrichment at room temperature followed by culture on TCBS. Step 3, Heat tube A for 5 minutes in a heat block or water bath and spin down. Step 4, Transfer the supernatant from tube A into the SPT. Step 5, Squeeze the SPT tube to add the lysate to the lysis tube. Step 6, Add lysate from the lysis tube to LRTs. Step 7, Place the LRTs in the reader. Scan the barcode for the cholera RLDT program. Step 8, Read results displayed on the screen real time or after 40 minutes. The illustration was created with BioRender.com.
Fig 3.
Lowest detection limit (LOD) of cholera RLDT tested directly from the stool.
Stool samples were spiked with ten-fold serial dilutions of V. cholerae N16961. The spiked stool samples were processed and tested with the RLDT kit. LOD was determined as the lowest concentration at which the target gene was detected in all ten runs. A: O1rfb gene; B: ctxA gene.
Table 2.
List of strains used for the evaluation of the specificity of cholera RLDT.
Table 3.
Variations in cholera RLDT Time to Result (TTR) in repeatability and reproducibility analysis.
Fig 4.
Stability of the dry cholera RLDT.
The cholera lyophilized reaction tubes (LRT) strips were stored at room temperature (RT), at 37°C, and 42°C. LRT strips were tested using the RLDT kit at three months intervals and the Time to Results (TTR) were recorded.
Fig 5.
Lowest Detection Limit (LOD) of cholera RLDT from dried stool on filter paper.
Stool samples were spiked with ten-fold serial dilutions of V. cholerae N16961and 50 uL of each dilution was spotted on filter paper. The stool spots were processed and tested with an RLDT kit. LOD was determined. A: O1rfb gene; B: ctxA gene.
Fig 6.
Lowest detection limit (LOD) of cholera RLDT for environmental water samples.
Environmental water samples were spiked with ten-fold serial dilutions of V. cholerae N16961 and processed and tested with an RLDT kit. LOD was determined. A: O1rfb gene; B: ctxA gene.