Name: a high-throughput platform for the screening of salmonella spp./shigella spp.
Uploaded: 2018-11-07T14:00:00
Description: Watch this Scientific Journal Video about A High-throughput Platform for the Screening of Salmonella spp./Shigella spp. at JoVE.com

This work was supported by the Science and Technology Program of Zhuhai, China (grant number 20171009E030064), the Science and Technology Program of Guangdong, China (grant number 2015A020211004) and the Science and Technology Program of General Administration of Quality Supervision, Inspection and Quarantine of the People&#39;s Republic of China (grant number 2016IK302, 2017IK224).

The protocol follows the guidelines set by the human research ethics committee of Zhuhai International Travel Healthcare Center. Please use standard sterile operation during the experiment.
1. Culture Media Composition and Preparation
<ol>
	<li>Prepare Nutrient Broth: Dissolve 1% peptone, 0.3% beef extract, 0.5% sodium chloride, 0.1% glucose in H2O, adjust pH to 7.5 and autoclave it in 121 &#176;C for 15 min.</li>
	<li>Prepare Selenite Cystine medium: Dissolve 0.5% peptone, 0.4% l...

<ol>
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(24), 3497-3500 (2014).</li><li>Blanco, G., Diaz de Tuesta, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture-+and+molecular-based+detection+of+swine-adapted+Salmonella+shed+by+avian+scavengers.">Culture- and molecular-based detection of swine-adapted Salmonella shed by avian scavengers.</a> Science of the Total Environment. 634, 1513-1518 (2018).</li><li>Tang, X. J., Yang, Z., Chen, X. B., Tian, W. F., Tu, C. N., Wang, H. 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Since Salmonella spp./Shigella spp. are often associated with food poisoning and fecal-oral transmission of acute gastroenteritis28,29 and the routine method is either labor-intensive or time-consuming7, we describe a high-throughput platform for the Salmonella spp./Shigella spp. screening, using real-time PCR combined with guided culture.
There are several steps that need con...

Diarrhea is still a common health issue with a high incidence rate globally1,2. Though the mortality is relatively low, some patients show various symptoms for weeks (such as loose and watery stools, an urgency to go to the bathroom), which make the socioeconomic impact very high3,4. More seriously, some patients may even develop irritable bowel syndrome if left untreated5. There are various kinds of bacteria, viruses and parasites that can cause diarrhea6. Salmonella spp./Shigella spp. are among the most common bacteria for the transmission of acute gastroenteritis7,8,9,10,11. Therefore, many counties have issued laws or regulations for regular Salmonella spp./Shigella spp. screening among people who would handle food and water. For example, the Chinese government have issued laws for obligatory Salmonella spp./Shigella spp. screening once a year.
The gold standard method for Salmonella spp./Shigella spp. detection is bacteria culture. Through bacteria culture and successive biochemical identification and serological characterization, we can identify the species of bacteria, which could facilitate disease outbreak management and antimicrobial profiling to aid the treatment of patients12. It could also help trace the source of infection during the Salmonella spp./Shigella spp. outbreak13. However, this method is labor-intensive (requiring manual operation) and time-consuming (taking several days), especially for the testing of large numbers of samples7. Moreover, viable but non-culturable (VBNC) Salmonella spp./Shigella spp.may exist in some stool samples14. In view of these drawbacks, many laboratories have tried to develop new techniques for the detection of Salmonella spp./Shigella spp.15,16,17,18,19,20,21,22,23,24,25. All these methods use the nuclear acid amplification test (NAAT), among which the polymerase chain reaction (PCR) is the most common. One major limitation of these NAAT based methods is that dead bacteria, even bacterial debris containing incomplete genomic DNA, could show positive results26, which could largely influence the accurate diagnosis of disease. Blanco et al. showed that molecular assay is highly sensitive, not only to viable Salmonella in cultures, but also to partial genomes and dead or unviable bacteria from past infections or contamination26. Therefore, new technology should be developed.
Here, we described a novel method that combines the NAAT based method and culturing. As shown in Figure 1, this new method applies real-time PCR screening first and then positive samples are sent for bacteria culture and identification.

<table border="0" cellpadding="0" cellspacing="0" style="width:732px;" width="732">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Tris</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:119px;">10708976001</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">EDTA</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:119px;">798681</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">NP40</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:119px;">11332473001</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:249px;">ddH2O</td>
			<td style="width:112px;">Takara</td>
			<td style="width:119px;">9012</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">PrimeSTAR HS (Premix)</td>
			<td style="width:112px;">Takara</td>
			<td style="width:119px;">R040Q</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Nutrient Broth</td>
			<td style="width:112px;">LandBridge</td>
			<td style="width:119px;">CM106</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Nutrient agar</td>
			<td style="width:112px;">LandBridge</td>
			<td style="width:119px;">CM107</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Selenite Cystine medium</td>
			<td style="width:112px;">LandBridge</td>
			<td style="width:119px;">CM225</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">XLD</td>
			<td style="width:112px;">LandBridge</td>
			<td style="width:119px;">CM219</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">MAC&#160;</td>
			<td style="width:112px;">LandBridge</td>
			<td style="width:119px;">CM908</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Salmonella chromogenic agar</td>
			<td style="width:112px;">CHROMagar</td>
			<td style="width:119px;">SA130</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Salmonella diagnostic serum</td>
			<td style="width:112px;">Tianrun</td>
			<td style="width:119px;">SAL60</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Shigella diagnostic serum</td>
			<td style="width:112px;">Tianrun</td>
			<td style="width:119px;">SHI54</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">anal swab (collecting tube plus)</td>
			<td style="width:112px;">Huachenyang</td>
			<td style="width:119px;"></td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">slide</td>
			<td style="width:112px;">Mingsheng</td>
			<td style="width:119px;">7102</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">micro-loop</td>
			<td style="width:112px;">Weierkang</td>
			<td style="width:119px;">W511</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">incubator</td>
			<td style="width:112px;">Jinghong</td>
			<td style="width:119px;">DNP-9082</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">autoclave</td>
			<td style="width:112px;">AUL</td>
			<td style="width:119px;">SS-325</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">dry bath</td>
			<td style="width:112px;">Jinghong</td>
			<td style="width:119px;">KB-20</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">automated microbial identification system</td>
			<td style="width:112px;">bioM&#233;rieux</td>
			<td style="width:119px;">VITEK2</td>
			<td style="width:252px;">other equivalent system could be used</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">fluorescent real-time PCR machine</td>
			<td style="width:112px;">ThermoFisher</td>
			<td style="width:119px;">ABI7500</td>
			<td style="width:252px;">other equivalent machine could be used</td>
		</tr>
	</tbody>
</table>

a high-throughput platform for the screening of salmonella spp./shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

The protocol was applied for the screening of Salmonella spp./Shigella spp. in anal stool samples from people who would handle food and water.
In the real-time PCR step, as shown in Figure 5A, there was a successful amplification in HEX channel, which meant that the mixed sample was positive for Salmonella spp. Then a further real-time PCR was conducted on...

Watch this Scientific Journal Video about A High-throughput Platform for the Screening of Salmonella spp./Shigella spp. at JoVE.com

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

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.

Research

JoVE Journal

Immunology and Infection

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Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many ...

High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence

Bacterial persisters are defined as a small subpopulation of phenotypic variants with the capability of tolerating high concentrations of antibiotics. ...