E.coli generic detection of betaD-glucuronidase (uidA) gene
&
shiga toxin (STX2A) gene
shiga toxin (STX2A)
producing Escherichia
coli
PCRmax Ltd qPCR test
TM
150 tests
For general laboratory and research use only
1
Escherichia coli are one of many species of bacteria living in the lower intestines of
mammals, known as gut flora. When located in the large intestine, it assists with waste
processing, vitamin K production, and food absorption. Discovered in 1885 by Theodor
Escherich, a German pediatrician and bacteriologist, E. coli are abundant: the number of
individual E. coli bacteria in the faeces that a human defecates in one day averages
between 100 billion and 10 trillion. However, the bacteria are not confined to the
environment, and specimens have also been located, for example, on the edge of hot
springs. The bacteria are Gram-negative, rod-shaped, flagellated and non-spore forming.
Most strains are non-pathogenic but some cause food poisoning in humans with
transmission largely being through the faecal-oral route. E.coli have a circular, DNA
genome of approximately 4.6 Mb but also carry plasmids.
Shiga toxin-producing E. coli (STEC) are a form of enterohaemorrhagic E.coli that cause
illness ranging from mild intestinal disease to severe kidney disease. The shiga toxin can
cause haemorrhagic colitis, the source of the bloody diarrhoea associated with E. coli
O157:H7 infections, as well as being responsible for haemolytic uremic syndrome (HUS).
Shiga toxins derive their name from the organism where they were first classified, Shigella
dysenteriae. When the shiga toxin is released, it can translocate to organs other than the
digestive tract such as the kidneys and central nervous system. The ability of the shiga
toxins to pass through cell barriers is possibly due to the increased permeability of the
intestinal epithelial cells resulting from effects of the body’s own immune system. The body
increases permeability of cell barriers so that important cells of the immune system
(neutrophils/PMN’s) can reach the E. coli infection. Shiga toxin may use this opportunity to
break through the walls of the digestive tract, enter the blood stream, and bind white blood
cells for transport to locations such as the kidney or brain.
Enterohaemorrhagic E. coli are found in humans, cattle, and goats. There are a number of
E. coli serogroups that produce shiga toxin such as O157:H7, O26, O111, and O103.
Typical symptoms include severe abdominal cramping, sudden onset of watery diarrhoea,
frequently bloody, and sometimes vomiting and a low-grade fever. Most often the illness is
mild and self-limited generally lasting 1-3 days. However, serious complications such as
haemorrhagic colitis, haemolytic uremic syndrome (HUS), or post diarrhoeal thrombotic
thrombocytopenic purpura (TTP) can occur in up to 10% of cases. The incubation period
ranges from 1 to 8 days, and transmission is predominantly through consumption of
contaminated foods.
Introduction to shiga toxin (STX2A) producing
Escherichia coli
2
MIN
MAX
This kit detects stx2A variations a,b,c,d,e, and g.
This kit does not detect stx2A variation f. This f subtype is not believed to be relevant to
human disease.
The generic E.coli portion of this kit also detects:
Shigella sonnei, Shigella flexneri, Shigella dysenteriae, Shigella boydii, Rhizobium, Carica
papaya, Arabidopsis thaliana, Phytophthora capsici.
The PCR Max qPCR Kit for shiga toxin (STX2A) producing Escherichia coli (E.
coli_STX2A ) genomes is designed for the in vitro quantification of E.coli_STX2A
genomes. The kit is designed to have the broadest detection profile possible whilst
remaining specific to the E.coli_STX2A genome.
The primers and probe sequences in this kit have 100% homology with a broad range of
E.coli_STX2A sequences based on a comprehensive bioinformatics analysis.
If you require further information, or have a specific question about the detection profile of
this kit then please send an e.mail to help@pcrmax.com and our bioinformatics team will
answer your question.
Specificity
3
Kit Contents
• E.coli_STX2A specific primer/probe mix (150 reactions BROWN)
FAM labelled
• E.coli_STX2A positive control template (for Standard curve RED)
• Internal extraction control primer/probe mix (150 reactions BROWN)
VIC labelled as standard
• Internal extraction control DNA (150 reactions BLUE)
• Endogenous control primer/probe mix (150 reactions BROWN)
FAM labelled
• RNAse/DNAse free water (WHITE)
for resuspension of primer/probe mixes and internal extraction control DNA
• Template preparation buffer (YELLOW)
for resuspension of positive control template and standard curve preparation
Reagents and equipment to be supplied by the user
Real-Time PCR Instrument
DNA extraction kit
This kit is designed to work well with all processes that yield high quality DNA with
minimal PCR inhibitors.
Lyophilised 2x qPCR Mastermix
This kit is designed to be compatible with all commercially available Mastermixes that run
with standard cycling conditions.
Pipettors and Tips
Vortex and centrifuge
Thin walled 1.5 ml PCR reaction tubes
4
Kit storage and stability
This kit is stable at room temperature but should be stored at -20ºC on arrival. Once the
lyophilised components have been resuspended they should not be exposed to
temperatures above -20ºC for longer than 30 minutes and unnecessary repeated
freeze/thawing should be avoided. The kit is stable for six months from the date of
resuspension under these circumstances.
If a standard curve dilution series is prepared this can be stored frozen for an extended
period. If you see any degradation in this serial dilution a fresh standard curve can be
prepared from the positive control.
PCRmax does not recommend using the kit after the expiry date stated on the pack.
Suitable sample material
All kinds of sample material suited for PCR amplification can be used. Please ensure the
samples are suitable in terms of purity, concentration, and DNA integrity (An internal PCR
control is supplied to test for non specific PCR inhibitors). Always run at least one negative
control with the samples. To prepare a negative-control, replace the template DNA sample
with RNAse/DNAse free water.
Dynamic range of test
Under optimal PCR conditions PCRmax E.coli_STX2A detection kits have very high
priming efficiencies of >95% and can detect less than 100 copies of target template.
Notices and disclaimers
This product is developed, designed and sold for research purposes only. It is not intended for human diagnostic or drug
purposes or to be administered to humans unless clearly expressed for that purpose by the Food and Drug Administration in the
USA or the appropriate regulatory authorities in the country of use. During the warranty period PCRmax detection kits allow
precise and reproducible data recovery combined with excellent sensitivity. For data obtained by violation to the general GLP
guidelines and the manufacturer’s recommendations the right to claim under guarantee is expired. PCR is a proprietary
technology covered by several US and foreign patents. These patents are owned by Roche Molecular Systems Inc. and have
been sub-licensed by PE Corporation in certain fields. Depending on your specific application you may need a license from
Roche or PE to practice PCR. Additional information on purchasing licenses to practice the PCR process may be obtained by
contacting the Director of Licensing at Roche Molecular Systems, 1145 Atlantic Avenue, Alameda, CA 94501 or Applied
Biosystems business group of the Applera Corporation, 850 Lincoln Centre Drive, Foster City, CA 94404. In addition, the 5'
nuclease assay and other homogeneous amplification methods used in connection with the PCR process may be covered by U.
S. Patents 5,210,015 and 5,487,972, owned by Roche Molecular Systems, Inc, and by U.S. Patent 5,538,848, owned by The
Perkin-Elmer Corporation.
Trademarks
PCRmax™ is a trademark of PCRmax Ltd.
The PCR process is covered by US Patents 4,683,195, and 4,683,202 and foreign equivalents owned by Hoffmann-La Roche
AG. BI, ABI PRISM® GeneAmp® and MicroAmp® are registered trademarks of the Applera Genomics (Applied Biosystems
Corporation). BIOMEK® is a registered trademark of Beckman Instruments, Inc.; iCycler™ is a registered trademark of Bio-Rad
Laboratories, Rotor-Gene is a trademark of Corbett Research. LightCycler™ is a registered trademark of the Idaho Technology
Inc. GeneAmp®, TaqMan® and AmpliTaqGold® are registered trademarks of Roche Molecular Systems, Inc., The purchase of
the PCRmax reagents cannot be construed as an authorization or implicit license to practice PCR under any patents held by
Hoffmann-LaRoche Inc.
5
Principles of the test
Real-time PCR
A E.coli_STX2A specific primer and probe mix is provided and this can be detected
through the FAM channel.
The primer and probe mix provided exploits the so-called TaqMan® principle. During PCR
amplification, forward and reverse primers hybridize to the E.coli_STX2A DNA. A
fluorogenic probe is included in the same reaction mixture which consists of a DNA probe
labeled with a 5`-dye and a 3`-quencher. During PCR amplification, the probe is cleaved
and the reporter dye and quencher are separated. The resulting increase in fluorescence
can be detected on a range of real-time PCR platforms.
Positive control
For copy number determination and as a positive control for the PCR set up, the kit
contains a positive control template. This can be used to generate a standard curve of E.
coli_STX2A copy number / Cq value. Alternatively the positive control can be used at a
single dilution where full quantitative analysis of the samples is not required. Each time the
kit is used, at least one positive control reaction must be included in the run. A positive
result indicates that the primers and probes for detecting the target E.coli_STX2A gene
worked properly in that particular experimental scenario. If a negative result is obtained
the test results are invalid and must be repeated. Care should be taken to ensure that the
positive control does not contaminate any other kit component which would lead to falsepositive results. This can be achieved by handling this component in a Post PCR
environment. Care should also be taken to avoid cross-contamination of other samples
when adding the positive control to the run. This can be avoided by sealing all other
samples and negative controls before pipetting the positive control into the positive control
well.
Negative control
To validate any positive findings a negative control reaction should be included every time
the kit is used. For this reaction the RNAse/DNAse free water should be used instead of
template. A negative result indicates that the reagents have not become contaminated
while setting up the run.
E.coli_STX2A DNA is known to be highly prevalent within the air and environment
generally and the negative control may therefore give a late positive signal due to
environmental contamination. The interpretation of results section of this handbook gives
guidance on how to interpret results where environmental contamination is evident.
6
Internal DNA extraction control
When performing DNA extraction, it is often advantageous to have an exogenous source
of DNA template that is spiked into the lysis buffer. This control DNA is then co-purified
with the sample DNA and can be detected as a positive control for the extraction process.
Successful co-purification and real-time PCR for the control DNA also indicates that PCR
inhibitors are not present at a high concentration.
A separate primer and probe mix are supplied with this kit to detect the exogenous DNA
using real-time PCR. The primers are present at PCR limiting concentrations which allows
multiplexing with the target sequence primers. Amplification of the control DNA does not
interfere with detection of the E.coli_STX2A target DNA even when present at low copy
number. The Internal control is detected through the VIC channel and gives a Cq value of
28+/-3.
Endogenous control
To confirm extraction of a valid biological template, a primer and probe mix is included to
detect an endogenous gene. Detection of the endogenous control is through the FAM
channel and it is NOT therefore possible to perform a multiplex with the E.coli_STX2A
primers. A poor endogenous control signal may indicate that the sample did not contain
sufficient biological material.
Carry-over prevention using UNG (optional)
Carry over contamination between PCR reactions can be prevented by including uracil-Nglycosylase (UNG) in the reaction mix. Some commercial Mastermix preparations contain
UNG or alternatively it can be added as a separate component. UNG can only prevent
carry over from PCR reactions that include deoxyuridine triphosphate (dUTP) in the
original PCR reaction. PCRmax recommend the application of 0.2U UNG per assay with a
15 minute incubation step at 37°C prior to amplification. The heat-labile UNG is then
inactivated during the Taq polymerase activation step.
7
Component - resuspend in water
Volume
E.coli_STX2A primer/probe mix (BROWN)
165 µl
Internal extraction control DNA (BLUE)
600 µl
Internal extraction control primer/probe mix (BROWN)
Endogenous control primer/probe mix (BROWN)
Pre-PCR pack
Pre-PCR heat-sealed foil
165 µl
165 µl
Reconstitution Protocol
To minimize the risk of contamination with foreign DNA, we recommend that all pipetting
be performed in a PCR clean environment. Ideally this would be a designated PCR lab or
PCR cabinet. Filter tips are recommended for all pipetting steps.
1. Pulse-spin each tube in a centrifuge before opening.
This will ensure lyophilised primer and probe mix is in the base of the tube and is not
spilt upon opening the tube.
2. Reconstitute the kit components in the RNAse/DNAse free water supplied,
according to the table below:
To ensure complete resuspension, vortex each tube thoroughly.
* This component contains high copy number template and is a VERY significant contamination risk. It
must be opened and handled in a separate laboratory environment, away from the other components.
DNA extraction
The internal extraction control DNA can be added either to the DNA lysis/extraction buffer
or to the DNA sample once it has been resuspended in lysis buffer.
DO NOT add the internal extraction control DNA directly to the unprocessed biological
sample as this will lead to degradation and a loss in signal.
1. Add 4µl of the Internal extraction control DNA (BLUE) to each sample in DNA
lysis/extraction buffer per sample.
2. Complete DNA extraction according to the manufacturers protocols.
3. Reconstitute the positive control template in the template preparation buffer
supplied, according to the table below:
To ensure complete resuspension, vortex the tube thoroughly.
500 µl
Post-PCR heat-sealed foil
Component - resuspend in template preparation buffer
Volume
E.coli_STX2A Positive Control Template (RED) *
8
Component
Volume
Lyophilised 2x qPCR Mastermix
1 µl
E.coli_STX2A primer/probe mix (BROWN)
Final Volume
1 µl
15 µl
10 µl
Internal extraction control primer/probe mix (BROWN)
RNAse/DNAse free water (WHITE)
3 µl
Component
Volume
Lyophilised 2x qPCR Mastermix
1 µl
Endogenous control primer/probe mix (BROWN)
Final Volume
15 µl
10 µl
RNAse/DNAse free water (WHITE)
4 µl
Real-time PCR detection protocol
1. For each DNA sample prepare a reaction mix according to the table below:
Include sufficient reactions for positive and negative controls.
2. For each DNA sample prepare an endogenous control reaction according to the
table below (Optional):
This control reaction will provide crucial information regarding the quality of the
biological sample.
3. Pipette 15µl of each mix into individual wells according to your real-time PCR
experimental plate set up.
4. Prepare sample DNA templates for each of your samples.
5. Pipette 5µl of DNA template into each well, according to your experimental plate
set up.
For negative control wells use 5µl of RNAse/DNAse free water. The final volume in
each well is 20µl.
6. If a standard curve is included for quantitative analysis prepare a reaction mix
according to the table below:
Component
Volume
Lyophilised 2x qPCR Mastermix
1 µl
E.coli_STX2A primer/probe mix (BROWN)
Final Volume
15 µl
10 µl
RNAse/DNAse free water (WHITE)
4 µl9Standard Curve
Copy Number
Tube 1 Positive control (RED)
2 x 105 per µl
Tube 2
Tube 3
Tube 4
Tube 5
Tube 6
2 x 104 per µl
2 x 103 per µl
2 x 102 per µl
20 per µl
2 per µl
Step
UNG treatment (if required) **
Enzyme activation
Denaturation
DATA COLLECTION *
Time
Temp
15 mins
2 mins
10 secs
60 secs
37 oC
95 oC
95 oC
60 oC
Cycling x50
7. Preparation of standard curve dilution series.
1) Pipette 90µl of template preparation buffer into 5 tubes and label 2-6
2) Pipette 10µl of Positive Control Template (RED) into tube 2
3) Vortex thoroughly
4) Change pipette tip and pipette 10µl from tube 2 into tube 3
5) Vortex thoroughly
Repeat steps 4 and 5 to complete the dilution series
8. Pipette 5µl of standard template into each well for the standard curve according
to your experimental plate set up.
The final volume in each well is 20µl.
Amplification Protocol
Amplification conditions using Lyophilsed 2x qPCR Mastermix.
* Fluorogenic data should be collected during this step through the FAM and VIC channels
** Required if your Mastermix includes UNG to prevent PCR carryover contamination
10
Interpretation of Results
NO
commensal
enviro
+ / -
+
> 35*-+-
SAMPLE PREPARATION FAILED
+ / -
-
+ / -
EXPERIMENT FAILED
EXPERIMENT FAILED
due to test contamination
+ / -
+
≤ 35
++-
NEGATIVE RESULT
--+
POSITIVE QUALITATIVE RESULT
do not report copy number as this
may be due to poor sample extraction
POSITIVE QUANTITATIVE RESULT
calculate copy number
-++
+ / -
-
POSITIVE QUANTITATIVE RESULT
calculate copy number
+
Interpretation
Positive
control
Negative
control
Internal
control
(VIC)
+ / -
≤ 30
Target
(FAM)
> 30
+ / -
> 30
--+ / -
*Where the test sample is positive and the negative control is positive with a Cq > 35, the
sample must be reinterpreted based on the relative signal strength of the two results:
Positive control template (RED) is expected to amplify between Cq 16 and 23. Failure to
satisfy this quality control criterion is a strong indication that the experiment has been
compromised.
Sample
Negative
control
∆Cq$>$3
SAMPLE$POSITIVE
Sample
Negative
control
∆Cq$<$3
SAMPLE$NEGATIVE
If the sample amplifies < 3 Cq earlier than
the negative control then the positive
sample result is invalidated and a negative
call is the correct result.
If the sample amplifies > 3 Cq earlier than
the negative control then the sample
should be reinterpreted (via the table
above) with the negative control verified as
negative.
11
Internal PCR control
The Cq value obtained with the internal control will vary significantly depending on the
extraction efficiency, the quantity of DNA added to the PCR reaction and the individual
machine settings. Cq values of 28±3 are within the normal range. When amplifying a E.
coli_STX2A sample with a high genome copy number, the internal extraction control may
not produce an amplification plot. This does not invalidate the test and should be
interpreted as a positive experimental result.
Endogenous control
The signal obtained from the endogenous control primer and probe set will vary according
to the amount of biological material present in a given sample. An early signal indicates
the presence of a good yield of biological material. A late signal suggests that little
biological material is present in the sample.
12
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