User Manual (201409)

™
PetNAD
Canine Leishmaniasis Detection Kit
For Leishmania donovani complex
User Manual
For Research Use Only
Manufacturer:
GeneReach Biotechnology Corporation
TEL: 886-4-24639869
No. 19, Keyuan
2nd
FAX: 886-4-24638255
Road, Central Taiwan Science Park, Taichung City, Taiwan 407
Web Site: www.petnad.com
2014/09
PetNAD™ Canine Leishmaniasis Detection Kit
Content
INTENDED USE ................................................................................ 1
SCIENTIFIC MEANINGS ................................................................ 1
SUMMARY AND EXPLANATION................................................. 2
PRINCIPLES OF THE PROCEDURE ............................................ 3
PRODUCT DESCRIPTION.............................................................. 4
A.
Materials Provided .................................................................................4
B.
Materials and Equipments Required, but Not Provided .........................4
C.
Storage and Stability ..............................................................................5
D.
Sample Type...........................................................................................5
RECOMMENDED NUCLEIC ACID EXTRACTION METHODS
.............................................................................................................. 5
PRECAUTIONS ................................................................................. 6
LIMITATIONS .................................................................................. 7
PROCEDURE ..................................................................................... 8
A.
PetNAD™ Canine Leishmaniasis Detection Kit Quick Guide ..............8
B.
Procedure ...............................................................................................9
DATA INTERPRETATION ........................................................... 11
i
PetNAD™ Canine Leishmaniasis Detection Kit
ANALYTICAL SENSITIVITY ...................................................... 11
TROUBLESHOOTING ................................................................... 12
REFERENCE ................................................................................... 14
ii
PetNAD™ Canine Leishmaniasis Detection Kit
INTENDED USE
PetNAD™ Canine Leishmaniasis Detection Kit is intended for in vitro
detection of the Leishmania donovani complex DNA based on insulated
isothermal polymerase chain reaction (iiPCR) technology. This kit is
designed specially to be used with an insulated isothermal (iiPCR)compatible instrument, POCKIT™ Nucleic Acid Analyzer. The assay
is intended for use by veterinarians or technicians with basic laboratory
skills.
This kit is intended for research use only.
SCIENTIFIC MEANINGS
Antibody induced by vaccine or obtained from maternal immunity
could lead to false positive interpretation in antibody-based diagnostic
procedures. Detecting pathogen’s nucleic acids, not antibody, PCRbased methods can avoid the false positive results described above.
Furthermore, with higher analytical sensitivity, PCR can detect lower
levels of viral signals than most if not all diagnostic methods. It can
reduce the chance of false negative results at early infection stage and
shorten the window period between time of infection and detection.
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PetNAD™ Canine Leishmaniasis Detection Kit
SUMMARY AND EXPLANATION
Canine leishmaniasis (CanL) is a vector-borne disease caused by
Leishmania parasites (Dereure et al., 1999). CanL could be categorized
into three forms: cutaneous, visceral and mucocutaneous leishmaniasis,
based on clinical symptoms. Visceral leishmaniasis (VL), endemic in
61 countries, is caused by Leishmania donovani complex. VL is a
severe zoonotic disease and an important public health issue worldwide
(Alvar et al., 2006; Chia et al., 1999). CanL is transmitted among dogs
through the bites of infected female Phlebotomine sandflies. Clinical
diagnosis of CanL is difficult at the asymptomatic stage and the acute
stage, due to its wide range of symptoms. Therefore, a sensitive and
specific identification tool is especially important for canine
leishmaniaisis detection.
PCR is one of the most commonly accepted methods that provide high
sensitivity and specificity for canine leishmaniasis detection. However,
conventional PCR assays could take three to four hours and require
sophisticated thermocyclers and well-trained technicians to perform.
GeneReach has developed PetNAD™ Canine Leishmaniasis Detection
Kit based on iiPCR technology, which significantly reduces reaction
time and offers sensitivity and specificity comparable to those of
conventional nested PCR (Tsai, 2012; Chang, 2012). Furthermore, this
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PetNAD™ Canine Leishmaniasis Detection Kit
simple and easy assay is completed rapidly in a portable POCKIT™
Nucleic Acid Analyzer.
PRINCIPLES OF THE PROCEDURE
In iiPCR, hydrolysis probe-based chemistry is used to generate
fluorescent signal during amplification of target DNA. The primers and
probe target the internal transcribed spacer 1 (ITS1) and do not crossreact with nucleic acid from host and other canine pathogens.
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PetNAD™ Canine Leishmaniasis Detection Kit
PRODUCT DESCRIPTION
A. Materials Provided (24 tests/kit)
Component
Premix Pack
Premix Buffer
Contents or Purpose

Canine leishmaniasis Premix
24 bags (1 canine
(lyophilized pellet) containing
leishmaniasis Premix
dNTPs, primers, probe, and
vial and 1 desiccating
enzyme for amplification.
agent/bag)

Desiccating agent pack.

Reaction buffer to re-dissolve
A
Amount
2 vials (1.3 ml/vial)
the lyophilized pellet.
P(+) Control

Dried plasmid containing
1 vial
canine leishmaniasis partial
sequence as positive control.
P(+) Control
Buffer

Reaction buffer to re-dissolve
1 vial (110 μl/vial)
P(+) Control.
R-tube
1 bag (24 pieces/bag)
Cap
1 bag (24 pieces/bag)
User Manual
1 copy
B. Materials and Equipment Required, but Not Provided
1)
PetNAD™ Nucleic Acid Co-prep Kit or taco™ Automatic
Nucleic Acid Extraction System.
2)
POCKIT™ Nucleic Acid Analyzer (POCKIT™):
PetNAD™-compatible instrument.
3)
cubee™ Mini-Centrifuge (cubee™).
4
PetNAD™ Canine Leishmaniasis Detection Kit
4)
Micropipette and filter tips.
C. Storage and Stability
1) The kit should be stored at 4°C and is stable until the
expiration date stated on the label.
2) Store Premix vials in sealed Premix Pack to avoid hydration
of lyophilized components.
3) Reconstituted P(+) Control is stable for 6 months at 4°C.
Aliquot reconstituted P(+) Control to avoid degradation of
nucleic acid.
D. Sample Type
DNA extracted from whole blood or conjunctival swab.
RECOMMENDED NUCLEIC ACID EXTRACTION
METHODS
A. PetNAD™ Nucleic Acid Co-prep Kit.
B. taco™ DNA/RNA Extraction Kit, compatible instrument—
taco™ Automatic Nucleic Acid Extraction System.
Note: Please follow the instruction manual of above extraction
methods to obtain optimal results. It is the user’s responsibility
to validate the combination of this reagent set with nucleic acids
extracted by other methods for any particular application.
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PetNAD™ Canine Leishmaniasis Detection Kit
PRECAUTIONS
A.
Do not open R-tube(s) after reaction to prevent any carryover
contamination.
B.
Perform extraction and amplification in two independent spaces
to minimize contamination.
C.
Do not reuse R-tube and Premix.
D.
Include the P(+) Control to:
E.
1)
Ensure POCKIT™ is working normally.
2)
Ensure detection kit performance after storage.
To get optimal fluorescence detection.
1)
Wear
powder-free
gloves
to
Labeling area
handle R-tubes.
2)
Do not label in the detection area
of R-tube.
6
Detection area
PetNAD™ Canine Leishmaniasis Detection Kit
LIMITATIONS
A. PetNAD Canine Leishmaniasis Detection Kit targets Leishmania
donovani complex sequence, and may also detect Leishmania
aethiopica, Leishmania major, and Leishmania tropica.
B. The test should be used only for testing nucleic acid extracted from
animal specimens. Do not add specimens (e.g. whole blood) directly
into Premix.
C. PetNAD™ Nucleic Acid Co-prep Kit and taco™ mini Automatic
Nucleic Acid Extraction System are recommended for nucleic acid
extraction.
D. Any deviations from the recommended procedure may lead to
suboptimal results. Quality of the extracts should be validated by the
users.
E. For PetNAD™ Canine Leishmaniasis Detection Kit, it is strongly
recommended to use freshly prepared nucleic acid (within 1 hour
after extraction) to achieve optimal results.
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PetNAD™ Canine Leishmaniasis Detection Kit
PROCEDURE
A. PetNAD™ Canine Leishmaniasis Detection Kit Quick Guide
8
PetNAD™ Canine Leishmaniasis Detection Kit
B.
Procedure
Note: Before preparing the reactions for iiPCR testing, turn
on POCKIT™ to initiate the calibration for the instrument.
The device will complete self-test within 5 minutes. Please
refer to the user manual of POCKIT™ for further details.
Note: Before using for the first time, add 100 μl P(+) Control
Buffer to P(+) Control. Store reconstituted P(+) Control at
4°C.
1) Label R-tube(s) in the label area.
2) Prepare one Premix for each sample. (Premix tube is in
Premix Pack. Each Premix Pack contains one Premix tube.)
Note: When the pellet is not found at the bottom of the tube,
spin tube briefly to bring it down.
3) Add 50 μl Premix Buffer A to each Premix tube.
4) Add 5 μl nucleic acid extract or P(+) Control to each Premix
tube. Spin Premix tube for 10 seconds in a mini centrifuge
(such as cubee™).
5) Transfer 50 μl Premix/sample mixture into R-tube.
6) Seal top of each R-tube with a cap. Make sure R-tube is
capped tightly.
7) Place R-tube into the holder of POCKIT™.
8) Spin tube briefly in cubee™ to make sure all solution is
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PetNAD™ Canine Leishmaniasis Detection Kit
collected at the bottom of R-tube.
Note: Make sure there are no bubbles in the solution.
Note: Start reaction within 1 hour (to prevent nucleic acid
degradation and non-specific reaction).
9) POCKIT™ reaction:
a)
Select ″520 nm″.
b)
When ″System READY″ is displayed, place the holder
with R-tube(s) into the reaction chamber.
c)
Tap cap of each R-tube to make sure the tube is
positioned properly.
10) Close lid and press ″Run″ to start reaction program.
11) Test results are shown on the monitor after reaction is
completed.
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PetNAD™ Canine Leishmaniasis Detection Kit
DATA INTERPRETATION
* One example of results shown on the monitor.
520 nm
Interpretation
Canine Leishmaniasis Positive
Canine Leishmaniasis Negative
Repeat reaction with freshly prepared nucleic
acid.
ANYLYTICAL SENSITIVITY
The detection limit of PetNAD™ Canine Leishmaniasis Detection Kit
is about 10 copies/reaction.
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PetNAD™ Canine Leishmaniasis Detection Kit
TROUBLESHOOTING
Problems
Possible causes
Solutions
False Positive
1) Reuse of micro-
 Micro-centrifuge tubes, tips, R-
centrifuge tubes,
tubes and Premix are for single-use
tips, R-tubes and
only. Reusing these accessories
Premix.
would cause cross-contamination,
and therefore false positive results.
 Used micro-centrifuge tubes, tips,
R-tubes and Premix should be
collected and discarded according
to local regulation. Do not place the
waste close to the working area to
prevent cross-contamination.
 Use aerosol-free tips.
2) Contaminated
micropipette
 Consult with a GeneReach
3) Contaminated
reagent
technical support representative or
local distributor.
 Consult with a GeneReach
4) Contaminated
working area
technical support representative on
how to clean up working area.
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PetNAD™ Canine Leishmaniasis Detection Kit
Problems
Possible causes
Solutions
False
1) Nucleic acid
 Consult manual of nucleic acid
Negative
extraction failed.
extraction kit.
 Do not overload PCR with too
2) PCR inhibition
much nucleic acid.
 Spike nucleic acid sample (5 μl)
into a P(+) Control reaction for a
parallel PCR reaction. Negative
results indicate the presence of
inhibitors in the nucleic acid. In
that case, prepare another nucleic
acid extract.
Heavy
 Consult with a GeneReach
1) Leakage or spill of
contamination
reaction from R-
technical support representative or
of amplicons
tube into reaction
local distributor.
in reaction
chamber of
chamber of
POCKIT™.
POCKIT™.
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PetNAD™ Canine Leishmaniasis Detection Kit
REFERENCE
1.
Alvar J., Yactayo S., Bern C., (2006). Leishmaniasis and poverty.
Trends Parasitol 2006, 22:552-557.
2.
Chai J.J., Zuo X.P., Zhang S., Zhao J.P., Zhang J.M., (1999). The
Desert-type kala-azar in Xinjiang, China. In Epidemiology and Control
of Leishmaniasis in Central Eurasia. Edited by Matsumoto Y. Tokyo.
International Press Editing Centre Incorporation; 1999:15-19.
3.
Chang, H.F.G., Tsai, Y.L., Tsai, C.F., Lin, C.K., Lee, P.Y., Teng, P.H.,
Su, C. and Jeng, C.C., (2012). A thermally baffled device for highly
stabilized convective PCR. Biotechnology Journal 7(5): 662-666, doi:
10.1002/ biot.201100453
4.
Companion Animal Parasite Council. Capcvet.org. Retrieved 2012-1004.
5.
Dereure J., Pratlong F., Dedet, J.P (1999) Geographical distribution and
the identification of parasites causing canine leishmaniasis in the
Mediterranean Basin. Canine leishmaniasis: an update. Proceedings of
the International Canine Leishmaniasis Forum. Barcelona, Spain
6.
Tsai Y.L., Wang H.T.T., Chang H.F. G., Tsai C.F., Lin C.K., Teng P.H.,
Su C. and Jeng C.C., (2012). Development of TaqMan probe-based
insulated isothermal PCR (iiPCR) for sensitive and specific on-site
pathogen detection. PLoS ONE 7(9): e45278.
doi:10.1371/journal.pone.0045278.
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