For research use

Loopamp TM

Cryptosporidium Detection Kit[Characteristics]
LAMP (Loop-mediated Isothermal Amplification)
method is a novel
gene amplification method capturing the following characteristics: (1) Only
one enzyme is required and the amplification reaction proceeds underisothermal conditions 1) 2). (2) 4 primers recognizing 6 distinct regions on
the target achieve high specificity. (3) High amplification efficiency
allows amplification within a short time. (4) It produces tremendous
amount of amplified products which makes simple detection possible 3) 4) 5)
6). Amplification of nucleic acids with this kit is conducted by the LAMP
method using Cryprosporidium 18S rRNA sequence as the primers. From
whether the amplification occurred or not, the existence of Cryptosporidium
spp. can be determined. By using the specifically designed Loopamp
Realtime Turbidimeters, detection does not need electrophoresis and all
steps from amplification to detection are done within one reaction tube.

[Contents of the kit]
48 tests(1) 2 × Reaction Mix. (RM) *1
0.6 ml × 1 tube
0.12 ml ×1 tube
(2) Primer Mix. Cry (PM Cry)*1
60 µl × 1 tube
(3) Bst DNA Polymerase (Bst DNA Polymerase)*1(4) Distilled Water (DW)*1
1.0 ml × 1 tube
0.1 ml × 1 tube
(5) Positive Control Cry (PC Cry)*1*1: The notation on each reagent tube is shown in ( ).

[Intended Use]
Detection of Cryptosporidium spp. in environmental specimens.

[Assay principle]
This kit uses LAMP method as assay principle. First, concentrateenvironmental water sample, then extract DNA and use it as sample
solution. Mix the sample solution with 2 x Reaction Mix (RM), Primer
Mix. Cry (PM Cry), Distilled Water (DW) and Bst DNA Polymerase andincubate it.
When Cryptosporidium 18S rRNA sequence can berecognized by the primer, its DNA will be amplified with the activity of Bst
DNA polymerase. The detection of the nucleic acid is done by detecting
the turbidity change3) caused by the amplification by-product magnesium
pyrophosphate (white precipitate) and then determines whether there is
Cryptosporidium spp. For further details of the LAMP principle, refer toEiken GENOME SITE (URL: http://loopamp.eiken.co.jp/e/).
This kit is developed for the purpose of qualitative analysis, not forquantitative analysis.

immediately.
(2) Preparation of master mix (Operate on ice)
Dispense the appropriate amount according to the number of tests to be
conducted into the separately prepared sterilized tube under thefollowing proportion per test:<for 1 test> <for 10 tests>
2 × Reaction Mix. (RM)
12.5µl
125µl
Primer Mix. Cry (PM Cry)
2.5µl
25µl
Bst DNA Polymerase
1.0µl
10µl
Distilled Water (DW)
4.0µl
40µl
Total
20µl
200µl
The prepared master mix should be used as soon as possible.
4. Operation procedure1) Mixing Master Mix with sample solution (Operate on ice)
(1) Dispense 20µl of Master Mix into each Loopamp Reaction Tube.
(2) Add 5µl of sample solution to the master mix, and the volume of thesolution should be 25µl in total. Mix the solution well by pipetting or
tapping the tube with the cap closed and then spin down.
(3) For control reactions, use 5µl of Positive Control Cry (PC Cry) as
positive control and 5µl of Distilled Water for as negative control.
Be careful not to cause air-bubbles when mixing.
By comparing the solution volume in all tubes, check visually if properamount of sample solution/master mix has been dispensed into thereaction tube.
2) The amplification reaction and realtime turbidity detection(1) Loopamp Realtime Turbidimeters are applicable to this kit. Turn on thedevice about 20 minutes before use. Set the parameters as follows:[Temperature]: Reaction Block 63ºC, Hot Bonnet 73ºC
[Measurement Time]: 60 min[Inactivation]: 80ºC, 5 min(2) Confirm that the temperature has reached 63ºC
(3) Set the prepared reaction tubes and immediately start reaction.
(4) Check whether the positive and negative controls’ turbidity rose from thedisplay screen of the device. If the positive control’s turbidity rose andthe negative control’s turbidity didn’t, then LAMP reaction proceeded
properly.(Fig.1) If not, there might have been error in the process.
Restart from reagent preparation and check again.
(5) If the increase in turbidity is confirmed within 60 minutes, then it isjudged as positive, if not, negative.
(6) The rising timing or turbidity of samples might be different from thepositive control.
(7) After enzyme inactivation (80ºC, 5 min), done automatically by
Loopamp Realtime Turbidimeter, is confirmed to have ended, remove
the used reaction tubes and dispose them with caps closed.

■Amplification Curve Pattern[How to use]

DWPC Cry

0.7
0.6
0.5

Turbidity

1. Materials required but not provided○ Sterilized tubes for master mix preparation (0.5ml or 1.5ml)○ Micropipette (0.5~10µl, 10~100µl, 100~1000µl)○ Pipette tips with filter○ Heat block (Capable of heating up to 95 ºC )○ Aluminum rack for cooling tubes○ Crushed ice and ice box○ Centrifuge for microtube○ Centrifuge for 8-strip tube
○ Vortex mixer○ Ultrasonic Cleaner○ Loopamp Reaction Tube
○ Loopamp Realtime Turbidimeter○ Proteinase K solution*2*2: Regarding preparation of this solution, refer to “Loopamp
Cryptosporidium/Giardia Detection Kit Operation Manual”.
2. Preparing sample solution
1) Prepare the beads-oocyst and/or -cyst complex from environmental water(Beforesample by immunomagnetic beads separation method7).
dissociation of beads-oocysts/-cysts complex)
2) DNA Extraction8)
Regarding DNA extraction, refer to “Loopamp Cryptosporidium /Giardia
Detection Kit Operation Manual.
3) Before the measurement, heat the sample solution at 95ºC for 5 minutes,
and then immediately place them on the ice for more than 5 minutes.
3. Preparing the reagent(1) Take out the reagents stored at -20ºC, and thaw them at room
temperature. Once the reagents are thawed, keep them on ice

0.4
0.3
0.2
0.1
0.0
0102030405060Time ( m i n . ）Fig.1 The amplification curve pattern of Positive Control Cry (PC Cry) *3(Loopamp Realtime Turbidimeter LA-320C is used)*3: The copy numbers do not necessarily correlate with rising timing.

■ Protocol<Pre-treatment; DNA extraction solution>
Prepare the beads-oocyst and/or -cyst complex from environmental water sample byimmunomagnetic separation method. (before dissociation of beads-cysts/-oocyctscomplex)*4

Extract DNA

Incubate at 95ºC for 5 minutes, then immediately cool down them on ice. .

<Preparation of master mix.> (Operate on ice)
Prepare the necessary volume of master mix. and mix well,(including positive, negative control)
Preparation of master mix. (per test)
2 × Reaction Mix. (RM)
12.5µlPrimer Mix. Cry (PM Cry)
2.5µl
Bst DNA Polymerase
1.0µl
Distilled Water
4.0µl
Total
20.0µlMix well*5and spin down the master mix, then keep it on ice<Procedure> (Operate on ice)
Dispense 20µl of master mix. into each reaction tube

Add 5µl of sample solution, or control (Total of 25µl as LAMP reaction)

Add 5µl of Positive Control Cry (PC Cry) for positive control and add 5µl ofDistilled Water (DW) for negative control
Mix by tapping or pipetting then spin down
Be careful not to cause air bubbles<LAMP reaction>
Set the samples in turbidimeter reaction block and start reaction
63ºC, 60 min
Inactivation (80ºC, 5 min)(Loopamp Realtime Turbidimeter will perform inactivation automatically)
Turbidity detection, judgment*4: Regarding DNA extraction, please refer to “Loopamp Cryptosporidium/Giardia Detection Kit Operation manual.
*5: Mix by tapping or invert them several times or vortex them for 1 second
× 3 times.

[Sensitivity]
Minimum detection limit: 60 copies/test*5*6: The sensitivity is determined by using plasmid DNA which target genesequence of Cryptosporidium parvum is inserted.

[Cautions for operation]
1. Sample handling
1) The sample (DNA extraction solution) should be used immediately.
However, if it is to be stored for long period of time, it should be stored
under -80ºC and repeated freezing and thawing should be avoided.
2. Reagent handling
1) The reagent kit should be stored at -20ºC. To prevent the reagents fromdeterioration, only take out the necessary amount of reagents from thefreezer before use. No decline was observed in the kit performanceeven after repeated freezing and thawing for 20 times in the quality
control test. But, in order to maintain the reagent performance, avoid
unnecessary freezing and thawing.
2) Thaw the reagent at room temperature, and keep them on ice for reagents
preparation. Before use, spin down the tubes to drop down the solution
staying on the tube wall or on the cap. Mix well the solution and spin
down again before use. Notice that fierce mixing should be avoided as it
can inactivate the Bst DNA Polymerase.
3) Positive Control Cry (PC Cry) contains high number of target DNA. Inorder to prevent Positive Control Cry from contaminating other samples orreagents, always spin down the tube, and open the cap as shortly aspossible. Moreover, to avoid contamination, do not use Positive Control
Cry in any other way not written in this instruction.
4) Keep positive and suspected positive samples away from the reagent
when handling5) Use the kit before the expiration date, which is labeled on the outer box(Exp. Date).
6) If there is any reagent left, do not mix or use it kits even if they are in thesame lot.
3. Handling the reaction tube
1) Only use the specified Loopamp Reaction Tube for turbidity detection.
Other reaction tubes might have different optical transparency and cancause misjudgment.
2) Take full care when handling reaction tubes, as they are vulnerable toscratches or damages.
3) Check carefully to see if the reaction tubes have any crack or scratch

before use. Crack or scratch on the tube might not only cause false
judgment but also contaminate the equipment. If the tubes are broken
inside the reaction block of Loopamp Realtime Turbidimeter, the reaction
mixture can spill inside the equipment and cause unrecoverable
contamination and malfunction.
4) By comparing the solution volume in all tubes, check visually if properamount of sample solution and master mix has been dispensed into thereaction tube.
4. Cautions for amplification reaction
Since bubbles in the solution will interfere the turbidity measurement andcause false judgment, try not to cause any bubble when mixing themaster mix and the sample solution. If bubbles appear, spin down toremove them.
5. Cautions for detection and judgment
1) Use only Loopamp Realtime Turbidimeter and Loopamp Reaction Tubefor the assay.
2) Start up Loopamp Realtime Turbidimeter at least about 20 minutes
before use it.
3) For judgment, check whether the turbidity of Positive Control Cry (PCCry) has risen to determine whether the reagent is performing properly (ifthe nucleic acid amplification reaction is performed properly, the turbidity
will start to rise around 20 minutes after the reaction started). There arecases where the turbidity of the sample starts rising later than the positivecontrol.
6. Handling reaction tubes after use
1) The caps of the used reaction tube should not be opened. Pay special
attention not to accidentally open the cap when taking the tubes out of
the turbidimeter. Contamination of amplified products on other samples
may not only cause false judgment of the test result but also pollute
testing area. In this case, a correct test result might not be obtained until
the contamination is removed completely.
2) . Keep the cap of the used tube completely closed and dispose it
according to the relevant regulations and instructions by incineration orafter double bagging it with sealable vinyl bag. To prevent the amplified
products from dispensing, do not conduct autoclave sterilization
treatment for disposal.

[Cautions for handling]
1. This kit is for the purpose of environment detection of Cryptosporidium
spp., not for medical or clinical diagnostic purposes on human or animal
samples.
2. Although the test result is negative, this test is incapable of determining
the complete absence of Cryptosporidium spp. Combined interpretations
with other test results are recommended.
3. Abide by the necessary counter biohazard regulations for sample taking
and handling9).
4. LAMP reaction is extremely sensitive and even the slightest amount ofamplified products tainted into the reaction might cause the false result.
Therefore, avoid this type of contamination by carrying out the sample
and reagent preparation in different clean benches. Avoid electrophoresis
or operations that need to handle amplified products.
5. Do not expose the Loopamp Reaction Tube, master mix. preparation
tubes to UV light. A change in color or degeneration caused byultraviolet lamp sometimes results in misjudgment.
6. If there is DNase in the reaction solution, the reaction might cause thefalse result. Pay attention to handle the operation under proper
environment with clean equipments and instruments.
7. If the operator does not have the knowledge or experience in genetic test,
there is a possibility of false judgment. Therefore, be sure to use the kit
under the supervision of the genetic testing experienced andknowledgeable technicians.
8. EIKEN CHEMICAL CO., LTD. does not bear responsibility for false
judgment or any consequential damage from the misuse or mishandling.
9. The reagent tube is made of polypropylene and the main material for kitcase is paper. The institution disposing the reagents, reagent tubes,container, and lab-ware and case should bear the responsibility and abideby the clinical waste disposal regulations, water pollution prevention law,and any local regulation related.

[Unit, Storage, Expiration and code No.]
Product
LoopampTM
Cryptosporidium Detection Kit

Unit

Storage

Expiration

Code No.

48 tests-20°C

1 year

LMP741[Reference]
1）Notomi T. et al..: Nucleic Acids Research 28, No.12, e63（2000）
2）Nagamine K. et al.: Clin. Chem. 47, No 9, 1742－1743（2001）
3）Mori Y. et al.: Biochem. Biophys. Res. Commun. 289, No.1, 150－154（2001）

4 ） Tomita N. et al..: Abstract for the 73rd Annual Meeting of theBiochemistry Society of Japan（2000）
5）Mori Y. et al.: Abstract for the 23rd Annual Meeting of the Molecular
Biology Society of Japan（2000）
6）Tomita N. et al.: Abstract for the 26th Annual Meeting of the Molecular
Biology Society of Japan（2003）
7）Japan Water Works Association: Cryptosporidium-Commentary and test
procedure-（in Japanese, 2003）
8）Endo. T. et al; Study report (in Japanese, 2006).
9）National Institute of Infectious Disease: The guideline for the bio-safety
and bio-hazard.(in Japanese, 2007).

Manufacturer

8092 ＡＭＫ
Rev. Mar.2012