A Baumannii

Acinetobacter baumannii

Quantification of

For general laboratory and research use only

hypothetical protein sequence gene

PrimerDesign LtdTM

150 testsAdvanced kit

1Quantification of Acinetobacter baumannii genomes.genesig Advanced kit handbook HB10.03.05

Acinetobacter baumannii is a Gram-negative bacterium of the Moraxellaceae family. Thegenome of this bacterium is around 4Mbp in length, arranged in a single, circularchromosome. The bacterium usually resides in soil or water but increasingly is found innosocomial infections where antibiotic resistance is increasing. Porins in the outer cellmembrane of the bacterium are greatly reduced in size compared to other Gram-negativespecies and contribute to increased antibiotic resistance.

A. baumannii has been found to be the cause of up to 10% of Gram-negative bacterialinfections in Europe and the US. Individuals with weakened immune systems are particularlysusceptible to infection by this species. Due to the ability of the bacterium to proliferate onmany surfaces and environments including dry and static surfaces, nosocomial infections mayoccur by patient contact with hospital equipment, especially invasive apparatus where thebacterium can easily be introduced into the blood stream.

A. baumanni infections are known to be involved in respiratory and urinary tract infections aswell as causing pneumonia, septicaemia and meningitis.

Introduction to Acinetobacter baumannii


Specificity

The primers and probe sequences in this kit have 100% homology with a broad range ofclinically relevant reference sequences based on a comprehensive bioinformatics analysis.

The PrimerDesign™ genesig Kit for Acinetobacter baumannii (A. baumannii) Genomes isdesigned for the in vitro quantification of A. baumannii genomes. The kit is designed to havethe broadest detection profile possible whilst remaining specific to the A. baumannii genome.

The primers and probe sequences in this kit have 100% homology with a broad range ofclinically relevant reference sequences based on a comprehensive bioinformatics analysis.

If you require further information, or have a specific question about the detection profile ofthis kit then please send an e.mail to [email protected] and our bioinformaticsteam will answer your question.


Kit Contents

• A. baumannii specific primer/probe mix (150 reactions BROWN) FAM labeled, BHQ quenched

• A. baumannii positive control template (for Standard curve RED)

• Internal extraction control DNA (150 reactions BLUE)

• Internal extraction control primer/probe mix (150 reactions BROWN) Choice of VIC channel or CY5 channel

• Endogenous ACTB primer/probe mix (150 reactions BROWN) FAM labeled, BHQ quenched

• RNAse/DNAse free water

Reagents and equipment to be supplied by the user

Real-Time PCR Instrument

DNA extraction kitThis kit is designed to work well with all processes that yield high quality DNA with minimalPCR inhibitors.

oasigTM Lyophilised 2 x qPCR MastermixThis kit is designed to work well with all commercially available Mastermixes. However, werecommend the use of oasigTM Lyophilsed 2x qPCR MasterMix.

Pipettors and Tips

Vortex and centrifuge

Thin walled 1.5 ml PCR reaction tubes


Kit storage and stabilityThis kit is stable at room temperature but should be stored at -20ºC on arrival.PrimerDesign does not recommend using the kit after the expiry date stated on the pack.Once the lyophilized components have been re-suspended, unnecessary repeatedfreeze/thawing should be avoided. The kit is stable for six months from the date ofresuspension under these circumstances.

Suitable sample materialAll kinds of sample material suited for PCR amplification can be used. Please ensure thesamples are suitable in terms of purity, concentration, and RNA/DNA integrity (An internalPCR control is supplied to test for non specific PCR inhibitors). Always run at least onenegative control with the samples. To prepare a negative-control, replace the template RNAsample with RNAse/DNAse free water.

Dynamic range of testUnder optimal PCR conditions PrimerDesign genesig detection kits have very high primingefficiencies of >95% and can detect less than 100 copies of target template.

Notices and disclaimersDuring the warranty period PrimerDesign genesig detection kits allow precise and reproducible data recovery combined with excellentsensitivity. For data obtained by violation to the general GLP guidelines and the manufacturer’s recommendations the right to claim underguarantee is expired. Black Hole Quencher”, “BHQ”, “CAL Fluor, “Quasar” and “Pulsar” are registered trademarks of BiosearchTechnologies, Inc., Novato, CA. This technology is protected by U.S. and World-wide patents either issued or in application and is licensedand sold under agreement with Biosearch Technologies, Inc. These products are sold exclusively for R&D use by the purchaser. They maynot be used for human or veterinary in vitro diagnostic (IVD) applications and they may not be re-sold, distributed or re-packaged withoutexpress written authorization from Biosearch Technologies Inc. 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 onyour specific application you may need a license from Roche or PE to practice PCR. Additional information on purchasing licenses to practicethe PCR process may be obtained by contacting the Director of Licensing at Roche Molecular Systems, 1145 Atlantic Avenue, Alameda, CA94501 or Applied Biosystems business group of the Applera Corporation, 850 Lincoln Centre Drive, Foster City, CA 94404. In addition, the5' nuclease assay and other homogeneous amplification methods used in connection with the PCR process may be covered by U.S. Patents5,210,015 and 5,487,972, owned by Roche Molecular Systems, Inc, and by U.S. Patent 5,538,848, owned by The Perkin-Elmer Corporation.The purchase of Biosearch Technologies products does not, either expressly or by implication, provide a license to use this or otherpatented technology. Licensing information can be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln CentreDrive, Foster City, CA 94404 or the Licensing Department at Roche Molecular Systems Inc., 1145 Atlantic Avenue, Alameda, CA 94501.”

TrademarksPrimerDesign ™ is a trademark of PrimerDesign 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.ABI, 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-Geneis 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 PrimerDesign ™ reagents cannot be construed as an authorization or implicit license to practice PCR under anypatents held by Hoffmann-LaRoche Inc.


Principles of the test

Real-time PCRA A. baumannii specific primer and probe mix is provided and this can be detected throughthe FAM channel.

The primer and probe mix provided exploits the so-called TaqMan® principle. During PCRamplification, forward and reverse primers hybridize to the A. baumannii DNA/cDNA. Afluorogenic probe is included in the same reaction mixture which consists of a DNA probelabeled with a 5`-dye and a 3`-quencher. During PCR amplification, the probe is cleaved andthe reporter dye and quencher are separated. The resulting increase in fluorescence can bedetected on a range of real-time PCR platforms.

Positive controlFor copy number determination and as a positive control for the PCR set up, the kit containsa positive control template. This can be used to generate a standard curve of A. baumanniicopy number / CT value. Alternatively the positive control can be used at a single dilutionwhere full quantitative analysis of the samples is not required. Each time the kit is used, atleast one positive control reaction must be included in the run. A positive result indicates thatthe primers and probes for detecting the target A. baumannii gene worked properly in thatparticular experimental scenario. If a negative result is obtained the test results are invalidand must be repeated. Care should be taken to ensure that the positive control does notcontaminate any other kit component which would lead to false-positive results. This can beachieved by handling this component in a Post PCR environment. Care should also be takento 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 thepositive control into the positive control well.

Negative controlTo confirm the absence of contamination, a negative control reaction should be includedevery time the kit is used. For this reaction, the RNAse/DNAse free water should be usedinstead of template. A negative result indicates that the reagents have not becomecontaminated while setting up the run. If a positive result is obtained the results should beignored and the test samples repeated. Possible sources of contamination should first beexplored and removed.


Internal DNA extraction controlWhen performing DNA extraction, it is often advantageous to have an exogenous source ofDNA template that is spiked into the lysis buffer. This control DNA is then co-purified withthe 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 PCRinhibitors are not present at a high concentration.

A separate primer and probe mix are supplied with this kit to detect the exogenous DNAusing real-time PCR. The primers are present at PCR limiting concentrations which allowsmultiplexing with the target sequence primers. Amplification of the control DNA does notinterfere with detection of the A. baumannii target DNA even when present at low copynumber. The Internal control is detected through the VIC channel and gives a CT value of 26+/-3.

Endogenous ACTB controlTo confirm extraction of a valid biological template, a primer and probe mix is included todetect the Actin Beta (ACTB) gene. Detection of ACTB is through the FAM channel and it isNOT therefore possible to perform a multiplex for ACTB and the A. baumannii primers. Apoor ACTB 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-N-glycosylase (UNG) in the reaction mix. Some commercial mastermix preparations containUNG or alternatively it can be added as a separate component. UNG can only prevent carryover from PCR reactions that include deoxyuridine triphosphate (dUTP) in the original PCRreaction. PrimerDesign recommend the application of 0.2U UNG per assay with a 15 minuteincubation step at 37°C prior to amplification. The heat-labile UNG is then inactivated duringthe Taq polymerase activation step (95°C for 10 minutes).


Component Volume

A. baumannii Primer/Probe mix (BROWN) 165 µl

Internal extraction control DNA (BLUE) 600 µl

Internal extraction control primer/probe mix (BROWN)

Endogenous ACTB primer/probe mix (BROWN)

Positive Control Template (RED) * 500 µl

Pre-PCR transparent envelope

Pre-PCR heat-sealed foil

Post-PCR heat-sealed foil

165 µl165 µl

Bench side ProtocolTo minimize the risk of contamination with foreign DNA, we recommend that all pipetting beperformed in a PCR clean environment. Ideally this would be a designated PCR lab or PCRcabinet. 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 notspilt upon opening the tube.

2. Reconstitute the kit components in the RNase/DNase-free water supplied, according tothe 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 beopened and handled in a separate laboratory environment, away from the other components.

DNA extractionThe internal extraction control DNA can be added either to the DNA lysis/extraction bufferor to the RNA sample once it has been resuspended in lysis buffer.

DO NOT add the internal extraction control DNA directly to the unprocessed biologicalsample 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 DNAlysis/extraction buffer per sample.

2. Complete DNA extraction according to the manufacturers protocols.


Component Volume

oasigTM 2 x qPCR MasterMix1 µlA. baumannii 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

oasigTM 2 x qPCR MasterMix1 µlEndogenous ACTB Primer/Probe mix (BROWN)

Final Volume 15 µl

10 µl

RNAse/DNAse free water (WHITE) 4 ͮ

µl

Real-time PCR detection

1. Prepare a reaction mix according to the tables below:Include sufficient reactions for the standard curve wells (6 samples in duplicate)and also the negative control.

A. baumannii detection mix

Endogenous ACTB detection mix (Optional)

2. Pipette 15µl of this mix into each well according to your real-time PCR experimentalplate set up.

3. Prepare sample DNA templates for each of your samples (suggested concentration5ng/µl) in RNAse/DNAse free water.If the concentration of DNA is not known, then dilute your DNA sample reactions 1:20(10µl of sample DNA and 190µl of water).

4. Pipette 5µl of diluted DNA template into each well, according to your experimentalplate set up.For negative control wells use 5µl of RNAse/DNAse free water. The final volume ineach well is 20µl.


Standard Curve Copy NumberTube 1 Positive control (RED) 2 x 105 per µlTube 2

Tube 3

Tube 4

Tube 5Tube 6

2 x 104 per µl

2 x 103 per µl2 x 102 per µl

20 per µl

2 per µl

Standard Curve Copy NumberStep

UNG treatment (if required) **

Enzyme activation (if required)

Denaturation

DATA COLLECTION *

Time Temp

15 mins

15 mins

10s60s

37 oC

95 oC

95 oC60 oC

50 Cycles

5. Preparation of standard curve dilution series.

1) Pipette 900µl of RNAse/DNAse free water into 5 tubes and label 2-62) Pipette 100µl of Positive Control Template (RED) into tube 23) Vortex thoroughly4) Change pipette tip and pipette 100µl from tube 2 into tube 35) Vortex thoroughly

Repeat steps 4 and 5 to complete the dilution series

6. Pipette 5µl of standard template into each well, according to your experimental plateset up.The final volume in each well is 20µl.

Amplification Protocol

Amplification conditions using oasigTM 2 x qPCR MasterMix.

* Fluorogenic data for the control DNA should be collected during this step through the FAM and VIC channels** Required if your Mastermix includes UNG to prevent PCR carryover contamination


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Positivecontrol

Negativecontrol

Internalcontrol

Target Interpretation

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Experiment fail

Experiment fail

Interpretation of Results

Internal PCR controlWhen used according to the above protocols, (assuming a 100% extraction efficiency) andthat 1:50 of extracted DNA is used in the reaction a CT value of 26 is expected. However,this can vary significantly depending on the extraction efficiency, the quantity of DNA addedto the PCR reaction and the individual machine settings. CT values of 26±3 are within thenormal range. When amplifying a A. baumannii sample with a high genome copy number,the internal extraction control may not produce an amplification plot. This does notinvalidate the test and should be interpreted as a positive experimental result.

Endogenous ACTB controlThe signal obtained from the ACTB primer and probe set will vary according to the amountof biological material present in a given sample. An early signal indicates the presence of agood yield of biological material. A late signal suggests that little biological material is presentin the sample.


A Baumannii

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