Name: Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR) (Video) | JoVE Journal
Uploaded: 2016-11-04T13:00:00.000+00:00
Duration: 8 min 51 s
Description: Learn ribosomal spacer PCR for genotyping Staphylococcus aureus in bovine mastitis research. This high-throughput method uses miniaturized electrophoresis for rapid, cost-effective strain analysis.

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5 minutes

The author thanks R. Boss, A. Cosandey, C. Fournier, I. Ivanovic, and J. Naskova for their excellent work.

1. Staphylococcus aureus Isolates
<ol>
	<li>Spread 10 &#181;l of S. aureus stock culture or one colony grown on a selective medium on Columbia agar plates containing 5% sheep blood (BA).</li>
	<li>Incubate aerobically at 37 &#176;C for 18 hr to 24 hr.</li>
</ol>
2. DNA Extraction
<ol>
	<li>Prepare TEL buffer containing 10 mM Tris-HCl and 10 mM Na2EDTA, pH 8.5. Prepare aliquots and autoclave at 121 &#176;C for 15 min.</li>
	<li>Collect 1 colony from BA using a sterile plastic loop or a sterile wooden tooth pick and resuspend in 100 &#181;l TEL. Use 1.5 ml tubes with screw caps.</li>
	<li>Incubate at 95 &#176;C for 10 min. Afterwards, place the samples immediately on wet ice.</li>
	<li>Dilute the samples 1:100 in H2O suitable for PCR (= template DNA for PCR). The non-diluted and diluted samples may be kept at -20 &#176;C for 1 month.</li>
</ol>
3. RS-PCR
<ol>
	<li>For each sample pipette into a PCR tube 1.5 &#181;l H2O, 12.5 &#181;l Taq Master Mix, 2 &#181;l G1 primer 10 &#181;M, 2 &#181;l L1 primer 10 &#181;M, and 7 &#181;l of template DNA that corresponds to about 30 ng of pure DNA 2.</li>
	<li>Run the following cycling program in a standard PCR cycler: 15 min 95 &#176;C, followed by 27 cycles comprising 94 &#176;C for 1 min, followed by a 2 min ramp and annealing at 55 &#176;C for 7 min. After a further 2 min ramp, extend at 72 &#176;C for 2 min. Terminate PCR by a final extension at 72 &#176;C for 10 min followed by cooling down to 4 &#176;C. 
	NOTE: The PCR products can be stored at -20 &#176;C for 5 days.</li>
	<li>For each run of RS-PCR include a positive control, i.e.,&#160;a sample with a known genotype (normally GTB), and a negative control (H2O).</li>
</ol>
4. Electrophoresis
<ol>
	<li>Use a commercial miniaturized electrophoresis kit for DNA (MED kit) together with a bioanalyzer connected to a PC and the installed software. Study the corresponding manuals of the manufacturer carefully.</li>
	<li>Set up the chip priming station and adjust the syringe clip according to the manual of the manufacturer.</li>
	<li>Prepare the gel-dye mix according to the manual of the manufacturer.</li>
	<li>Loading the gel-dye mix (according to the manual of the manufacturer).
	<ol>
		<li>Equilibrate the gel-dye mix to RT for 30 min before use.</li>
		<li>Put new DNA chip on the chip priming station. Pipette 9 &#181;l of gel-dye in the well-marked G.</li>
		<li>Make sure that the plunger is positioned at 1 ml and then close the chip priming station. Press plunger until it is held by the syringe clip. Wait exactly for 30 sec then release the syringe clip.</li>
		<li>Wait for 5 sec. Slowly pull back plunger to 1 ml position.</li>
		<li>Open chip priming station and pipette 9 &#181;l of gel-dye in both wells marked &#39;G&#39;.</li>
	</ol>
	</li>
	<li>Loading the Marker
	<ol>
		<li>Pipette 5 &#181;l of marker in all 12 sample wells and in the ladder well. Do not leave any of these 13 wells empty.</li>
	</ol>
	</li>
	<li>Loading the Ladder and the Samples.
	<ol>
		<li>Pipette 1 &#181;l of DNA ladder to the well-marked with the ladder sign.</li>
		<li>Pipette 1 &#181;l of sample (used wells) or 1 &#181;l of deionized water (unused wells).</li>
		<li>Put the chip horizontally in the adapter and vortex for 1 min at 2,400 rpm.</li>
		<li>Run the chip in the bioanalyzer within 5 min.</li>
	</ol>
	</li>
	<li>Running the Analysis
	<ol>
		<li>Start the bioanalyzer and the connected computer. Open the software.</li>
		<li>Open the lid of the bioanalyzer and place the chip into the chip holder (the chip fits only one way). Close the lid carefully.</li>
		<li>In the Instrument context of the software select &#34;Electrophoresis&#34; &#62; &#34;dsDNA&#34; &#62; &#34;DNA 7500&#34;. Accept the current File Prefix of the software or modify it.</li>
		<li>Click the start button present in the software. Enter the sample names in the software. When the chip run is finished (after approximately 30 min) the End of Run message appears in the bioanalyzer software.</li>
		<li>Remove the chip and clean the electrodes of the bioanalyzer according to the instructions of the manufacturer.</li>
	</ol>
	</li>
</ol>
5. Inferring the Genotype from the Electrophoresis Profile
<ol>
	<li>Load the Mahal software (software is freely available from the authors).</li>
	<li>Import reference data into the Mahal software: &#34;File&#34; &#62; &#34;Import Reference Data&#34;</li>
	<li>Open the electrophoresis run in the Data context of the bioanalyzer software. Select an electrophoresis profile of one sample.</li>
	<li>Check for the relevant peaks.
	<ol>
		<li>Use the Mahal software to check for the relevant peaks: &#34;Tools&#34; &#62; &#34;Mean Fluorescence&#34;. Insert into the &#34;Fluorescence&#34; box the fluorescence values indicated as fluorescence units (FU) as they can be read out from the bioanalyzer software. Insert the 4 (3) peaks with highest fluorescence in ascending order and separated by comma. Example: 126,130,132,137. 
		NOTE: If not all of these peaks FU are indicated by the bioanalyzer software, they need to be estimated by reading them out from the plot.</li>
	</ol>
	</li>
	<li>Press the &#34;Calculate&#34; button. A peak is relevant if the observed fluorescence exceeds the value given in the box &#34;Minimal peak height&#34;.</li>
	<li>Infer the genotype.
	<ol>
		<li>Insert into the Mahal textbox &#34;Input Peaks (bp)&#34; the sizes in bp of all relevant peaks in ascending order and separated by comma. Example: 440,462,519,579,637. 
		NOTE: If not all of these peak sizes are indicated by the bioanalyzer software, they need to be estimated by reading them out from the plot.</li>
	</ol>
	</li>
	<li>Press the &#34;Calculate&#34; button.</li>
	<li>In the list box, look for the minimal squared Mahalanobis distance &#34;d2m&#34;. If the corresponding P value right to it is &#8805;0.05 then the probability is &#8805;5% that the null hypothesis H0 is rejected (H0: the observed and the reference profile are identical) meaning that the observed profile is accepted to be identical to the reference profile of the indicated genotype.</li>
	<li>If d2m is minimal but the field of the corresponding P value is empty, reject H0 with a probability &#60;5%. 
	NOTE: This means that the profiles are unequal although d2m is minimal. Such an outcome may result from non-average electrophoresis conditions.
	<ol>
		<li>To correct for these deviations, insert into the text box &#34;Observed Peak (bp)&#34; the value 395 and press the &#34;Calculate&#34; button. Check again for minimal d2m and a corresponding P value &#8805;0.05. If not successful, try the values 405, 415, and 425. 
		NOTE: Sometimes smaller steps are even indicated. If still not successful, the observed profile is not present in the Reference Data, it may be a new genotype or variant.</li>
	</ol>
	</li>
	<li>Various Profiles
	<ol>
		<li>Repeat steps 5.6 to 5.9 for each electrophoresis profile separately.</li>
	</ol>
	</li>
</ol>

<ol><li>Sears, P. M., McCarthy, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Management+and+treatment+of+staphylococcal+mastitis%5BTitle%5D)+AND+%22Vet.Clin.North+Am.Food+Anim.Pract%22%5BJournal%5D)">Management and treatment of staphylococcal mastitis.</a> Vet.Clin.North Am.Food Anim.Pract. 19 (1), 171-185 (2003).</li>
<li>Fournier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bovine+Staphylococcus+aureus%3A+association+of+virulence+genes%2C+genotypes+and+clinical+outcome%5BTitle%5D)+AND+%22Res.Vet.Sci%22%5BJournal%5D)">Bovine Staphylococcus aureus: association of virulence genes, genotypes and clinical outcome.</a> Res.Vet.Sci. 85 (3), 439-448 (2008).</li>
<li>Cosandey, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Staphylococcus+aureus+genotype+B+and+other+genotypes+isolated+from+cow+milk+in+European+countries%5BTitle%5D)+AND+%22J.Dairy+Sci%22%5BJournal%5D)">Staphylococcus aureus genotype B and other genotypes isolated from cow milk in European countries.</a> J.Dairy Sci. 99 (1), 529-540 (2016).</li>
<li>Boss, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bovine+Staphylococcus+aureus%3A+Subtyping%2C+evolution%2C+and+zoonotic+transfer%5BTitle%5D)+AND+%22J.Dairy+Sci%22%5BJournal%5D)">Bovine Staphylococcus aureus: Subtyping, evolution, and zoonotic transfer.</a> J.Dairy Sci. 99 (1), 515-528 (2016).</li>
<li>Graber, H. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mastitis-related+subtypes+of+bovine+Staphylococcus+aureus+are+characterized+by+different+clinical+properties%5BTitle%5D)+AND+%22J.Dairy+Sci%22%5BJournal%5D)">Mastitis-related subtypes of bovine Staphylococcus aureus are characterized by different clinical properties.</a> J.Dairy Sci. 92 (4), 1442-1451 (2009).</li>
<li>Heiniger, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kosten-Nutzen-Analyse+einer+Intervention+zur+Verbesserung+der+Eutergesundheit+in+Schweizer+Milchviehbetrieben%5BTitle%5D)+AND+%22Schweiz.Arch.Tierheilkd%22%5BJournal%5D)">Kosten-Nutzen-Analyse einer Intervention zur Verbesserung der Eutergesundheit in Schweizer Milchviehbetrieben.</a> Schweiz.Arch.Tierheilkd. 156 (10), 473-481 (2014).</li>
<li>Hamann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Definition+of+the+physiological+cell+count+threshold+based+on+changes+in+milk+composition%5BTitle%5D)+AND+%22IDF+Mastitis+Newsletter%22%5BJournal%5D)">Definition of the physiological cell count threshold based on changes in milk composition.</a> IDF Mastitis Newsletter. 25, 9-12 (2003).</li>
<li>Hwang, S. Y., Park, Y. K., Koo, H. C., Park, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((spa+typing+and+enterotoxin+gene+profile+of+Staphylococcus+aureus+isolated+from+bovine+raw+milk+in+Korea%5BTitle%5D)+AND+%22J.Vet.Sci%22%5BJournal%5D)">spa typing and enterotoxin gene profile of Staphylococcus aureus isolated from bovine raw milk in Korea.</a> J.Vet.Sci. 11 (2), 125-131 (2010).</li>
<li>Sakwinska, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Link+between+genotype+and+antimicrobial+resistance+in+bovine+mastitis-related+Staphylococcus+aureus+strains%2C+determined+by+comparing+Swiss+and+French+isolates+from+the+Rhone+Valley%5BTitle%5D)+AND+%22Appl.Environ.Microbiol%22%5BJournal%5D)">Link between genotype and antimicrobial resistance in bovine mastitis-related Staphylococcus aureus strains, determined by comparing Swiss and French isolates from the Rhone Valley.</a> Appl.Environ.Microbiol. 77 (10), 3428-3432 (2011).</li>
<li>Rabello, R. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multilocus+sequence+typing+of+Staphylococcus+aureus+isolates+recovered+from+cows+with+mastitis+in+Brazilian+dairy+herds%5BTitle%5D)+AND+%22J.Med.Microbiol%22%5BJournal%5D)">Multilocus sequence typing of Staphylococcus aureus isolates recovered from cows with mastitis in Brazilian dairy herds.</a> J.Med.Microbiol. 56, 1505-1511 (2007).</li>
<li>Tavakol, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bovine-associated+MRSA+ST398+in+the+Netherlands%5BTitle%5D)+AND+%22Acta+Vet.Scand%22%5BJournal%5D)">Bovine-associated MRSA ST398 in the Netherlands.</a> Acta Vet.Scand. 54, 28(2012).</li>
<li>Harmsen, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Typing+of+methicillin-resistant+Staphylococcus+aureus+in+a+university+hospital+setting+by+using+novel+software+for+spa+repeat+determination+and+database+management%5BTitle%5D)+AND+%22J.Clin.Microbiol%22%5BJournal%5D)">Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management.</a> J.Clin.Microbiol. 41 (12), 5442-5448 (2003).</li>
<li>Zadoks, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+pulsed-field+gel+electrophoresis+and+binary+typing+as+tools+in+veterinary+clinical+microbiology+and+molecular+epidemiologic+analysis+of+bovine+and+human+Staphylococcus+aureus+isolates%5BTitle%5D)+AND+%22J.Clin.Microbiol%22%5BJournal%5D)">Application of pulsed-field gel electrophoresis and binary typing as tools in veterinary clinical microbiology and molecular epidemiologic analysis of bovine and human Staphylococcus aureus isolates.</a> J.Clin.Microbiol. 38 (5), 1931-1939 (2000).</li>
<li>Cremonesi, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genomic+characteristics+of+Staphylococcus+aureus+strains+associated+with+high+within-herd+prevalence+of+intramammary+infections+in+dairy+cows%5BTitle%5D)+AND+%22J.Dairy+Sci%22%5BJournal%5D)">Genomic characteristics of Staphylococcus aureus strains associated with high within-herd prevalence of intramammary infections in dairy cows.</a> J.Dairy Sci. 98 (10), 6828-6838 (2015).</li>
<li>Enright, M. C., Day, N. P., Davies, C. E., Peacock, S. J., Spratt, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multilocus+sequence+typing+for+characterization+of+methicillin-resistant+and+methicillin-susceptible+clones+of+Staphylococcus+aureus%5BTitle%5D)+AND+%22J.Clin.Microbiol%22%5BJournal%5D)">Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus.</a> J.Clin.Microbiol. 38 (3), 1008-1015 (2000).</li>
</ol>

The authors have nothing to disclose.

The most critical step of the whole procedure is when there is an excess of DNA in RS-PCR (&#62;30 ng pure DNA/assay) 2. In general, resolution of a profile is optimal if all of its peaks start and end at the baseline. If two peaks are too close together, resolution is incomplete. Under these conditions, the bioanalyzer software may lead to inappropriate peak identification so that manual identification is required.
All the visibly separated and relevant peaks expressed as bp or FU ...

Staphylococcus (S. aureus) is known as the most important pathogen worldwide responsible for intramammary infection (IMI) in cattle 1. The study by Fournier et al. 2 demonstrated in Swiss cows and the studies by Cosandey et al. 3 and Boss et al. 4 in cows of 12 European countries that S. aureus isolated from bovine IMI is a genetically heterogeneous group. By PCR amplification of the 16S-23S rRNA intergenic spacer region (RS-PCR), a total of 17 genotypes were initially detected in 101 epidemiologically independent isolates 2. Genotype B and genotype C (GTC) were most common (80%), whereas the other 15 genotypes (GTs) occurred rarely, each making 1 to 4% of all the isolates. At the European level 3, GTB, GTC, GTF, GTI, and GTR were the most important genotypes comprising 76% of all analyzed strains (n = 456). GTB was situated in central Europe whereas the other genotypes were widely disseminated. The remaining 24% of the strains included 41 genotypes, many of them, however, were observed only once.
The studies by Fournier et al. 2, Cosandey et al. 3, and Graber et al. 5 further demonstrated that the genotypes were highly associated with their virulence gene pattern, as well as at the Swiss as at the European level. The studies further revealed massive differences in IMI prevalence among S. aureus GTB, GTC and the other genotypes 2,5: considering GTB, up to 87% of cows in a herd were infected by this genotype. In the case of GTC and of the other genotypes, however, IMI was only found in 1 to 2 cows of a herd.
IMI caused by S. aureus, normally results in inflammation of the corresponding mammary glands and an increase of inflammatory cells in milk. As a consequence, the somatic cell counts in milk (SCC), the key indicator of milk quality in most countries, is increased leading to a decreased milk price or even delivery stop.
Besides the RS-PCR of Fournier et al. 2, other typing methods have been described for subtyping bovine strains of S. aureus 8-11. Two common ones include spa typing 12 and Multi Locus Sequence Typing (MLST) 13. The former one is based on DNA sequencing the variable spacer region of the staphylococcal spa gene coding for protein A, whereas the latter one requires the sequencing of 7 housekeeping genes. This means that one 12 and seven PCRs 13, respectively, need to be performed, followed by amplicon purification, sequencing reaction and analysis using specific equipment. Compared to RS-PCR, these methods require a considerable additional effort in the laboratory resulting in a low sample throughput and high costs. The throughput is particularly low for PFGE. Considering the resolution of these methods for bovine strains of S. aureus, RS-PCR is at least as good as spa typing 4 and better than MLST 4 or PFGE 15.
All these methods including binary typing 13 demonstrated their effectiveness in obtaining further insight into the pathogenesis of bovine IMI caused by S. aureus, but because of the mentioned restrictions they are less appropriate for large clinical investigations. In addition, genotyping by RS-PCR as described by Fournier et al. 2 allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine IMI, two key values in clinical veterinary medicine.

<table><tbody><tr><td>Tris(hydroxymethyl)-aminomethan</td><td>Merck</td><td>1.08382.0500</td><td>Mw = 121.14 g/mol</td></tr><tr><td>Titriplex III</td><td>Merck</td><td>1.08418.0250</td><td>Mw = 372.24 g/mol; Substance is equivalent to Na&lt;sub&gt;2&lt;/sub&gt;EDTA&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Hydrochloric acid 5 mol/L</td><td>Merck</td><td>1.09911.0001</td><td></td></tr><tr><td>Columbia agar+5% sheep blood</td><td>bioM&amp;eacute;rieux</td><td>43049</td><td></td></tr><tr><td>Agilent DNA7500 Kit</td><td>Agilent Technologies</td><td>5067-1504</td><td></td></tr><tr><td>Agilent 2100 Bioanalyzer</td><td>Agilent Technologies</td><td>G2940CA</td><td></td></tr><tr><td>Agilent 2100 expert sofware</td><td>Agilent Technologies</td><td></td><td></td></tr><tr><td>HotStarTaq master mix&amp;nbsp;</td><td>Qiagen</td><td>203445</td><td></td></tr><tr><td>Primer L1</td><td>Mycrosinth</td><td></td><td>5&#039;CAA GGC ATC CAC CGT3&#039;</td></tr><tr><td>Primer G1</td><td>Mycrosinth</td><td></td><td>5&#039;GAA GTC GTA ACA AGG3&#039;</td></tr></tbody></table>

genotyping of staphylococcus aureus by ribosomal spacer pcr (rs-pcr)

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Definition of a genotype and its variants
An electrophoretic profile differing in more than one band from all identified genotypes is considered as a new genotype. New genotypes are named and extended according to Fournier et al. 5 leading to the genotypes GTA to GTZ, followed by the genotypes GTAA to GTAZ, and GTBA to GTBZ, and GTCA to GTCC at present. Variation in just one band is regarded as a genotypic va...

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

The overall goal of this procedure is to obtain genotypes of Staphylococcus aureus, and to link them with disease in hosts. This method can help answer key questions in the field of bovine mastitis, to protect contagiousness and passaginicity of Staphylococcus aureus, isolate it from cows with intramammary infection. Demonstrating the procedure will be Ivana Ivanovic, a technician from my laboratory.To begin, spread 10 microliters of Staphylococcus aureus stock culture, or one colony grown on selective medium, on Columbia Agar plates containing 5%sheep blood, or BA.Incubate the culture aerobically at 37 degrees Celsius for 18 to 24 hours. Use a sterile plastic loop or wooden toothpick to collect one bacterial colony from a BA agar plate, and re-suspend it in 100 microliters of TEL in a 1.5 milliliter screw cap tube. Incubate the samples at 95 degrees Celsius for 10 minutes and immediately place them on wet ice.Then, dilute the samples one to 100 in water suitable for PCR. Prepare a master mix by adding to a tube the reagents listed here. Then, distribute it to PCR tubes and add the sample DNA.Include a positive control, DNA generating a known genotype, and water as a negative control. Use a standard PCR cycler to run the program discussed in the text protocol. After approximately six and three quarters hours of cycling, stop the machine and take out the tubes.To carry out electrophoresis, use a commercial miniaturized electrophoresis kit for DNA, together with a Bioanalyzer, connected to a PC and the installed software. Set up the chip priming station and adjust the syringe clip according to the manufacturer's instructions. Next, put a new DNA chip in the chip priming station.Then, after equilibrating the gel dye mix to room temperature, pipette nine microliters of the gel dye into the well marked G.Make sure that the plunger is positioned at one milliliter, and close the chip priming station. Press the plunger until it is held by the syringe clip, then wait for exactly 30 seconds and release the syringe clip. After waiting for five seconds, slowly pull back the plunger to the one milliliter position.Open the chip priming station, and pipette nine microliters of gel dye into both wells marked G.To load the marker, pipette five microliters of marker into all 12 sample wells, and into the well marked with the ladder sign, ensuring that all 13 wells are loaded. Pipette one microliter of DNA ladder into the ladder well. Then, pipette one microliter of sample into the sample wells and one microliter of deionized water into the unused wells.Place the chip horizontally in the adaptor and vortex for one minute at 2, 400 RPM. Within five minutes of vortexing the chip, start the Bioanalyzer and computer, and open the software. Open the lid of the Bioanalyzer and place the chip into the chip holder.Then, carefully close the lid. In the instrument context of the software, select electrophoresis, double stranded DNA, DNA 7500. Accept the current file prefix of the software, or modify it.Click the start button present in the software, then enter the sample names. When the chip run is complete, the end of run message will appear in the Bioanalyzer software. Remove the chip and clean the Bioanalyzer electrodes according to the manufacturer's instructions.Finally, infer the genotype from the electrophoresis profile according to the text protocol. As seen in this Bioanalyzer pseudo-gel of 12 RS-PCR products and their inferred genotypes, variation in just one band is regarded as a genotypic variant. Each lane is characterized by a pattern of three or more PCR bands, and a marker band in front, and a marker band at the end.This electrophoretic profile for sample 161 measures the intensity of the bands as individual peaks of fluorescent units. Another electrophoretic profile, for sample 211, reports a peak for each band as the number of base pairs. Peak reading in fluorescent units is required for evaluating the relevant peaks of a profile in the Mahal software.And reading in base pairs is for inferring the genotype. The relevant peaks identified for sample 211 are shown here, inserted in the Mahal software. The calculated squared Mahalanobis distance, or D2M, between the queried and indicated genotypic profile, then identifies a genotype that is minimal and not significantly different than the one queried.In this case, GTB. Once mastered, the complete analysis of 96 samples requires one night for PCR and one day for electrophoresis and evaluation. Pipetting for sample dilution and setting up the PCR can be massively simplified if a robot is applied.While attempting this procedure, it's important to remember to include positive and negative PCR controls, such as DNA for a particular genotype, and water respectively. Follow this procedure. Other methods like spa typing, or analysis of virulence genes, can be performed in order to further characterize the strains of Staphylococcus aureus.After its development, this technique paved the way for researchers in the field of bovine mastitis to explore highly sensitive and specific qPCR assays for clinically relevant genotypes of Staphylococcus aureus. Notably, genotype B.After watching this video, you should have a good understanding of how to establish and drum RS-PCR, particularly to infer the Staphylococcus genotypes. Don't forget that working with Staphylococcus aureus can be hazardous, and good laboratory practices should always be applied while performing this procedure.

genotyping-of-staphylococcus-aureus-by-ribosomal-spacer-pcr-rs-pcr

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We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

Here, we present a protocol for tracing genomic DNA (gDNA) contamination in RNA samples. The presented method utilizes primers specific for the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) genes. The method is suited for reliable and sensitive detection of DNA contamination in most eukaryotes and prokaryotes.

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

The feasibility of whole-genome sequencing (WGS) strategies using benchtop instruments has simplified the genome interrogation of every microbe of public health relevance in a lab setting. A methodological adaptation of the workflow for bacterial WGS is described and a bioinformatics pipeline for analysis is also presented.

Characterization of a Pathogenic Escherichia coli Strain Derived from Oreochromis spp. Farms Using Whole-Genome Sequencing

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Murine skin and soft tissue infection model is utilized for assessing the virulence function of methicillin resistant Staphylococcus aureus (MRSA) and the host immunological responses. Here, we presented a subcutaneous infection model for skin and soft tissue infection.

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

Learn ribosomal spacer PCR for genotyping Staphylococcus aureus in bovine mastitis research. This high-throughput method uses miniaturized electrophoresis for rapid, cost-effective strain analysis.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

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