This method provide a high throughput platform for the screening of Salmonella and Shigella. The main advantage of this technique is repeat, specific, sensitive, and high throughput. This technique combine NAAT fast method and culture in which real-time PCR screening was applied first, and then positive ones was sent for bacteria culture and identification.
Though this method focuses on Salmonella and Shigella screening, it can also be applied to other pathogens such as Vibrios, Staphylococcus, E.Coli, and et cetera. Visual demonstration of this method is critical as the selection of the correct colony is difficult due to background flora in samples. To begin, add three milliliters of nutrient broth to each sample.
Then, incubate the samples at 36 degrees Celsius for six hours. After this, centrifuge the pre-enrichment culture at 800 gs for two minutes. Then, transfer the supernatant to a new tube, and centrifuge it again for five minutes at 12, 000 gs.
After discarding the supernatant, add 100 microliters of DNA extraction solution to the pellet. Then, vortex the tube for one minute. Next, heat the sample at 1, 000 degrees Celsius in the dry bath.
After repeating the centrifugation, collect the supernatant into a new tube. First, create the reaction mixture and set the cycling program according to the text protocol. Then, perform real-time PCR on a fluorescent real-time PCR machine.
First, add 100 microliters of the pre-enrichment culture to five milliliters of selenite crystal medium. Then, incubate the culture at 36 degrees Celsius for 18 to 24 hours. Next, collect one loop of the culture and spread it onto a plate.
Then, incubate the plate at 36 degrees Celsius for 18 to 24 hours. After incubation, select suspicious colonies and transfer them to a plate. Incubate the plate at 36 degrees Celsius for 18 to 24 hours.
Then, identify the suspicious colony using an automated microbial identification system. To begin O-antigen characterization, add one drop of the O-antigen polyvalent sera to a clean slide. Transfer one loop of the colony to the slide and grind it in the sera.
If the bacteria look like flowing sand, repeat the previous treatment with O-antigen monovalent sera until the antigen is characterized. To begin H-antigen characterization, add one drop of H-antigen polyvalent sera to a clean slide. Then, collect one loopful of the colony and grid it in the sera.
Use H-antigen monovalent sera to repeat treatment until the specific H-antigen is characterized. To separate the culture, collect one loop of the pre-enrichment culture and spread it onto a plate. Then, incubate the place at 36 degrees Celsius for 18 to 24 hours.
After incubation, collect a suspicious looking colony onto a plate. Incubate the plate at 36 degrees Celsius for 17 to 24 hours. Then, subject the colony to biochemical identification using an automated microbial identification system.
Next, apply one drop of Shigella species polyvalent sera to a clean slide. Then, use a micro-loop to collect some of the bacteria and grind it into the sera. Finally, if the sera resemble flowing sand, use Shigella species monovalent sera to further characterize the bacteria.
Using this protocol, stool samples from patients were screened for Salmonella and Shigella species. Using real-time PCR, there was successful amplification in the HEX channel, indicating that the sample was positive for Salmonella species. Further real-time PCR indicated that sample two was positive for Salmonella and was chosen for guided Salmonella species culture.
In the guided culture of Salmonella species, the pink and purple colonies were plated separately and subjected to biochemical identification. The results indicated that the colonies from sample two were Salmonella paratyphi B.Using real-time PCR, there was successful amplification in the FAM channel, indicating that the sample was positive for Shigella species. Further real-time PCR indicated that sample 10 was positive for Shigella and was chosen for guided culture.
In the guided culture of Shigella species, the pink-red and colorless colonies were plated separately and subjected to biochemical identification. The results indicated that sample 10 contained Shigella sonnei phase II bacteria. While attempting this procedure, it's important to remember the limitation of the method due to sequence variation.
Following this procedure, other methods, like direct culture, can be performed in order to avoid false negative real-time PCR results. This technique paves the way for researchers in the field of Salmonella and Shigella detection, as the new protocol could increase the positive rate by twofolds and decrease workload and TAT significantly. Don't forget that working with Salmonella and Shigella can be extremely dangerous, and the personal protective equipment, PPE, should be always be taken while performing this procedure.
