p knowlesi adv

p knowlesi adv

Plasmodium knowlesi

plasmepsin gene

150 tests

For general laboratory and research use only

Quantification of Plasmodium knowlesi genomes.

Advanced kit handbook HB10.03.07

1

Introduction to Plasmodium knowlesi

Plasmodium is a genus of protozoa that cause Malaria. Plasmodium species use two hosts in their lifecycle. Plasmodium knowlesi, which infects humans, uses Anophele mosquitoes as a vector. This species is most commonly found in South-East Asia.

As the mosquito bites a human, the sporozoites of the parasite are injected into the individual with the mosquito saliva. The sporozoites target the host liver where they replicate asexually within 24 hours, creating merozoites which are subsequently released into the host circulatory system where they invade erythrocytes and undergo nuclear division causing these cells to burst. The parasite can also reproduce sexually producing gametocytes which are then able to infect the vector host when they bite the human. In the mid gut of the mosquito these mature into micro-gametes with flagella and macro-gametes that fuse together to produce a zygote which later develops into an oocyst in the space between the epithelial cells and the basal lamina. These oocysts undergo asexual replication to produce sporozoites of around 1µm in diameter which relocate to the salivary gland ready to infect another individual when the mosquito feeds again.

After an incubation period of around 10 to 12 days, infection with P. knowlesi presents initially with fever, muscle pain, nausea. Symptoms including anaemia and confusion can also occur. Treatment is based on identification of the specific species causing the infection, where in the world the infection was acquired and drug resistance.

Quantification of Plasmodium knowlesi genomes.

Advanced kit handbook HB10.03.07

2

Specificity

MAX MIN

The Techne qPCR Kit for Plasmodium knowlesi (P. knowlesi ) genomes is designed for the in vitro quantification of P. knowlesi genomes. The kit is designed to have the broadest detection profile possible whilst remaining specific to the P. knowlesi genome.

The primers and probe sequences in this kit have 100% homology with a broad range of

P. knowlesi sequences based on a comprehensive bioinformatics analysis.

The target sequence within the plasmepsin gene has very low homology with other species of plasmodia that cause malaria. The primers and probe sequences in this kit have 100% homology with a broad range of clinically relevant reference 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 [email protected] and our bioinformatics team will answer your question.

Quantification of Plasmodium knowlesi genomes.

Advanced kit handbook HB10.03.07

3

Kit Contents

• P. knowlesi specific primer/probe mix (150 reactions BROWN)

FAM labelled

• P. knowlesi 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 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 work well with all commercially available Mastermixes.

Pipettors and Tips

Vortex and centrifuge

Thin walled 1.5 ml PCR reaction tubes

Quantification of Plasmodium knowlesi genomes.

Advanced kit handbook HB10.03.07

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Kit storage and stability

This kit is stable at room temperature but should be stored at -20ºC on arrival.

Techne does not recommend using the kit after the expiry date stated on the pack.

Once the lyophilized components have been re-suspended, 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.

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 Techne P. knowlesi 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 Techne

® 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

Techne™ is a trademark of Bibby Scientific 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 Techne® Prime Pro reagents cannot be construed as an authorization or implicit license to practice PCR under any patents held by Hoffmann-LaRoche Inc.

Quantification of Plasmodium knowlesi genomes.

Advanced kit handbook HB10.03.07

5

Principles of the test

Real-time PCR

A P. knowlesi 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 P. knowlesi 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 P.

knowlesi copy number / CT 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 P. knowlesi 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.

Quantification of Plasmodium knowlesi genomes.

Advanced kit handbook HB10.03.07

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 P. knowlesi target DNA even when present at low copy number. The Internal control is detected through the VIC channel and gives a CT 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 P. knowlesi 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.

Techne 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.

Quantification of Plasmodium knowlesi genomes.

Advanced kit handbook HB10.03.07

7

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.

2.

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.

Reconstitute the kit components in the RNase/DNase-free water supplied, according to the table below:

To ensure complete resuspension, vortex each tube thoroughly.

Component - resuspend in water

Pre-PCR pack

P. knowlesi primer/probe mix (BROWN)

Internal extraction control primer/probe mix (BROWN)

Endogenous control primer/probe mix (BROWN)

Pre-PCR heat-sealed foil

Internal extraction control DNA (BLUE)

Volume

165 µl

165 µl

165 µl

600 µl

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.

Component - resuspend in template preparation buffer

Post-PCR heat-sealed foil

Positive Control Template (RED) *

Volume

500 µl

* 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.

2.

Add 4µl of the Internal extraction control DNA (BLUE) to each sample in DNA lysis/extraction buffer per sample.

Complete DNA extraction according to the manufacturers protocols.

Quantification of Plasmodium knowlesi genomes.

Advanced kit handbook HB10.03.07

8

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.

Component

2x qPCR MasterMix

P. knowlesi primer/probe mix (BROWN)

Internal extraction control primer/probe mix (BROWN)

RNAse/DNAse free water (WHITE)

Final Volume

Volume

10 µl

1 µl

1 µl

3 µl

15 µl

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.

Component

2x qPCR MasterMix

Endogenous control primer/probe mix (BROWN)

RNAse/DNAse free water (WHITE)

Final Volume

Volume

10 µl

1 µl

4 µl

15 µl

3.

4.

5.

Pipette 15µl of each mix into individual wells according to your real-time PCR experimental plate set up.

Prepare sample DNA templates for each of your samples.

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

2x qPCR MasterMix

P. knowlesi primer/probe mix (BROWN)

RNAse/DNAse free water (WHITE)

Final Volume

Volume

10 µl

1 µl

4 µl

15 µl

Quantification of Plasmodium knowlesi genomes.

Advanced kit handbook HB10.03.07

9

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

Standard Curve

Tube 1 Positive control (RED)

Tube 2

Tube 3

Tube 4

Tube 5

Tube 6

Copy Number

2 x 10

5

per µl

2 x 10

4

per µl

2 x 10 3 per µl

2 x 10

2

per µl

20 per µl

2 per µl

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.

50 Cycles

Step

UNG treatment (if required) **

Enzyme activation

Denaturation

DATA COLLECTION *

Time

15 mins

2 mins

10s

60s

Temp

37 o

C

95 o

C

95 o

C

60 o C

* 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

Quantification of Plasmodium knowlesi genomes.

Advanced kit handbook HB10.03.07

10

YES NO

Interpretation of Results

Target

+ive

+ive

+ive

+ive

-ive

-ive

-ive

Internal control

+ive

-ive

+ive

-ive

+ive

-ive

+ive

Negative control

-ive

-ive

+ive

+ive

-ive or +ive

-ive or +ive

-ive or +ive

Positive control

+ive

+ive

+ive

+ive

+ive

-ive

-ive

Interpretation

+ive

+ive

*

*

-ive

Experiment fail

Experiment fail

* Where the test sample is positive and the negative control is also positive the interpretation of the result depends on the relative signal strength of the two results. This is calculated using the delta CT method by subtracting the target CT value from the negative control CT value (NC CT value – sample CT value). Where the test sample is positive and the NC is detected much later (delta CT ≥ 5) then the positive test result is reliable. Where the NC detection is at a similar level to the test sample (delta CT<5) then the positive test result is invalidated and a negative call is the correct result.

Internal PCR control

The CT 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. CT values of 28±3 are within the normal range.

When amplifying a P.

knowlesi 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.

Quantification of Plasmodium knowlesi genomes.

Advanced kit handbook HB10.03.07

11

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