DIARRHOEAGENICE. COLI PCR KIT(EHEC, EIEC, EPEC, ETEC, EAEC)

SSI Diagnostica

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Diarrhoeagenic E. coli PCR Kit (EHEC, EIEC, EPEC, ETEC, EAEC) for detection of diarrhoeagenic E. coli (DEC)

For in vitro diagnostic use

ApplicationThe Diarrhoeagenic E. coli PCR Kit (EHEC, EIEC, EPEC, ETEC, EAEC) is for in vitro diagnostic PCR detection of diarrhoeagenic E. coli (DEC)1. The kit detects the 5 most important diarrhoeagenic groups that are pathogenic for humans; the enterohaemorrhagic E. coli (EHEC), entero-invasive E. coli (EIEC) , enteropathogenic

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E. coli (EPEC), attaching and effacing E. coli (A/EEC), enterotoxigenic E. coli (ETEC), and ente-roaggregative E. coli (EAEC)2.

Materials providedPrimer Mix PCR positive DNA Control 1 and 2PCR ReadyMix (including loading buffer) TE-buffer (10 mM Tris-HCL, 1 mM EDTA, pH 8) 10 % Chelex-100 in TE-buffer (magnetic stir bar added)

Materials Required but not ProvidedAgar plates Inoculation loops Tubes for template preparation Tube cap locks 100 bp DNA marker1.5-2.0 % agarose gels

DescriptionThe Primer Mix amplifies a specific fragment of the following genes: intimin (eae), verocytotoxin 1 (vtx1), verocytotoxin 2 (vtx2), heat stable entero-toxin, human variant (estA-human), heat stable enterotoxin, porcine variant (estA-porcine), heat labile enterotoxin (eltA), invasive plasmid antigen (ipaH), dispersin transporter protein (aatA), transcriptional activator (aggR), AaiC secreted

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protein (aaiC) and 16S rDNA (internal positive control). All primers are synthetic single-stranded oligonucleotides with free 5’- and 3’- hydroxyl ends. The individual concentration of each primer pair is adjusted for optimal performance in the multiplex PCR. The two positive PCR controls, Control 1 and 2, consist of purified DNA from different DEC strains. Together, the two controls represent all 10 virulence genes and the internal positive control (16S rDNA). The table below shows the combination of virulence genes as well as the amplicon size of the fragments for the two controls:

Control 1 Control 2 Amplicon size (bp)

16S rDNA 16S rDNA 1062ipaH 647

aatA 550eltA 479

vtx2 420eae 377

aggR 323vtx1 260

aaiC 214estA-porcine 160

estA-human 151

Each control DNA vial contains 150 μL corre-sponding to at least 25 PCR tests. Sample preparation and PCR set-up should be performed in dedicated areas free of possible

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contamination. If running only a few PCRs at a time, dispense the Primer Mix and the PCR Rea-dyMix into several aliquots and use only one at a time. The kit contains reagents to perform 100 PCR reactions.

PrincipleThe DEC is detected using template prepared from E. coli colonies grown on an agar plate. The genes used for identification of the particular DEC and the amplicon sizes are listed below.

Gene DEC Amplicon size (bp)

16S rDNAa - 1062ipaHb EIEC 647aatA EAEC 550eltA ETEC 479vtx2 EHEC 420eae EPEC, A/EECc 377aggR EAEC 323vtx1d EHEC 260aaiC EAEC 214estA-porcinee ETEC 160estA-humane ETEC 151

a) Amplifies a fragment from most Gram-negative bacteria, allowing an evaluation of the PCR.

b) Might also be present in Shigella spp.c) EPEC and A/EEC distinction is based on serotype.d) Might also be present in Shigella dysenteriae I.e) Amplicon sizes for estA-porcine and estA-human are very similar.

LimitationsIt is not possible to use the stool sample directly in the PCR reaction.

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Procedure DNA preparation1. Plate the stool sample on a selective agar

plate overnight.

2. Place the bottle with 10 % Chelex-100 on a magnetic stirrer and pipette 200 μL/tube while the Chelex-100 is homogeneous. Pick up to 10 plate-grown E. coli colonies (both lactose positive and negative) and suspend them in the aliquoted 10 % Chelex-100 solution.

3. Boil the suspension for 5 min. (remember tube cap locks) and centrifuge briefly (5 min. at approx. 2200 x g).

4. Dilute 15.0 μL of the supernatant in 100 μL TE-buffer and use 4.0 μL directly in the PCR.

PCR Set-up1. Prepare the total master mix for the number

of samples to be run and dispense 16.0 μL of the mixture in each tube.

PCR ReadyMix 10.0 μLPrimer Mix 6.0 μL

Total 16.0 μL

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2. Add 4.0 μL template DNA (sample or positive control) to each tube and mix. Prepare a negative control by adding 4.0 μL TE-buffer to one of the tubes.

3. Run the PCR amplification in a thermocycler at the following conditions.

Step Temp. (ºC) Time

Initial denaturation 95 2 min.

35 cycles of:

Denaturation 94 50 sec.

Annealing 62 40 sec.

Extension 72 50 sec.

Final extension 72 3 min.

4. Run 18 μL of each completed PCR reaction in separate wells on an agarose gel (1.5-2.0 %) capable of separating the particular amplicon sizes.

Interpretation of ResultsFigure 1 shows the PCR results containing a variety of different templates compared to a 100 bp DNA marker. If very high template concentrati-ons are used in the PCR analysis of EIEC strains, an unspecific fragment of ~750 bp may be amplified in addition to the 647 bp long ipaH fragment. The in-tensities of the two bands depend on the template

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concentration. Dilution of the template may there-fore prevent amplification of the unspecific band. A DEC positive sample giving a strong band for one or more of the virulence genes will often result in a weak internal control band.

Figure 1. Analysis of 10 different templates.Lane 1: 100 bp DNA marker, lane 2 (control 1): ipaH, vtx2, eae, vtx1 and estA-porcine, lane 3 (control 2): aatA, eltA, aggR, aaiC and estA-human, lane 4: aatA, aggR and aaiC, lane 5: aatA and aggR, lane 6: vtx2, eae and vtx1, lane 7: vtx2 and eae, lane 8: eltA, lane 9: eltA and estA-human, lane 10: ipaH, lane 11: estA-porcine, lane 12: 100 bp DNA marker.

Recovery The Diarrhoeagenic E. coli PCR Kit (EHEC, EIEC, EPEC, ETEC, EAEC) has been tested with a panel of 161 E. coli (reference strains and clinical iso- lates) from The National Escherichia and Kleb-siella Centre, Statens Serum Institut, Denmark. The panel represented a broad variety of diffe-rent serotypes and virulence gene combinations.

The recovery (percentage of samples giving the

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expected result) was 99% when compared to results obtained by doing PCR, sequencing, and/or DNA hybridisation technique at The National Escherichia and Klebsiella Centre. 14 non-E. coli species were also tested, all of which only pro-duced the 16S rDNA control band.

Storage and Shelf Life

Reagent Temperature

Primer Mix -20˚CDNA controls -20˚CPCR ReadyMix -20˚CTE-buffer Room temp.10% Chelex-100 Room temp.

The Primer Mix and the controls in use can be stored at 2-8 ºC for up to 2 weeks. The expiry date of the kit is printed on the label.

Available Products

Art. No. Product No. of test Packing

97004 Diarrhoeagenic E. coli PCR Kit (EHEC, EIEC, EPEC, ETEC, EAEC)

100 1 box

62870 DEC Primer Mix 100 1 box94724 EAEC PCR Kit 100 1 box94831 DEC & EAEC PCR Kit 2 x 100 2 boxes73410 DEC PCR Kit 100 1 box91346 E. coli vtx1 and vtx2

Suptyping PCR Kit200 1 box

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References1. Persson, S., Olsen, K. E. P., Scheutz,

F., Krogfelt, K. A. and Gerner-Smidt, P. 2007. A method for fast and simple detection of major diarrhoeagenic Escherichia coli in the routine diagnostic laboratory. Clin MicrobiolInfect. 13:516-524.

2. Nataro, J. P. and J. B. Kaper. 1998. Diarrhoeagenic Escherichia coli. Clin. Micro-biol Rev. 11:142-201.

Quality Certificate SSI Diagnostica’s development, production and sales of in vitro diagnostics are quality assured and certified in accordance with ISO 9001 and ISO 13485.

Information and OrderingSSI Diagnostica A/SHerredsvejen 23400 HillerødDenmarkT +45 4829 9100F +45 4829 9179@ info@ssidiagnostica.com W ssidiagnostica.com

SSI Diagnostica A/S • 4th Edition • January 2017 • 97989

SSI Diagnostica A/SHerredsvejen 23400 HillerødDenmark

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