2019. 9Code No. KN-T10C807

Instruction Manual
Kaneka Nucleic Acid Chromatography Food Poisoning Bacteria Detection Kit(Salmonella sp./O4 group / O7 group / O9 group) version 2Caution■This product is for research purposes only. Please do not use for medical care for humans or animals,clinical diagnosis, etc.
■Retain the various reagents of this product in strict accordance with the storage protocols described inthis instruction manual. Be careful of contamination when storing.
■The specifications of this product are subject to change without notice.
■Please use the equipment, devices, reagents (including culture medium) used for the preparation andreaction of PCR solution according to this instruction manual and the usage protocols specified by eachmanufacturer/distributor.
■Please be responsible for the diagnosis and use of the results obtained by this product. We will not beliable for any damages or losses incurred by you because of implementation.
■The customer should verify the operation procedure not described in this instruction manual and thevalidity of the detection result for each sample.

Features/Contents
This product is a kit for detecting Salmonella and O4, O7, and O9 groups of Salmonella, which are known
to cause food poisoning, quickly and easily. This product contains amplification enzyme mix, primer mix,
test strip (5 lines), and chase buffer. Please prepare media and DNA extraction reagent (if necessary)
separately.
Component
Amplification enzyme mix
Primer mix
Test strip (5 lines)
Chase bufferInstruction ManualContents (100 tests)
1 mL x 1
0.8 mL x 1
50 strips x 2 bottles
16 mL x 1
1 manual

Storage Conditions/Expiration◼ How to Store Date

Please avoid direct sunlight・Amplification enzyme mix: -20℃ storage
・Primer mix: -20℃ storage
・Test strip (5 lines): Store at room temperature
・Chase buffer: Store at room temperature【Precautions】
If the test strip (5 lines) contains moisture for a long time, the detection performance may deteriorate,
so put it in the container (including the desiccant) after opening and store it with the lid tightly closed
to prevent absorbing moisture.
For amplification enzyme mix and primer mix, detection performance may deteriorate if freeze-thawis repeated, so all is not used at one time, we recommend to store by dispensing and minimizing thefreeze-thaw cycle as much as possible.
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Expiration Date
Stated on the packaging of this product.

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How to Use◼

Equipment and device required in addition to this product1．Micropipette
2. Micropipette with filtered tip
3. Thermal cycler (Recommended device: LifeECO Thermal Cycler (Bioer Technology))
4. 1.5 mL tube
5. 0.2 mL tube（For PCR）
6. Small incubator (for 1.5 mL, capable of heating above 95℃)
7. Low speed centrifuge◼

CulturePlease perform the following culture method.
Sample Types
Culture method (1st phase)
Liquid egg

FeedRaw meat

Feces/meconium

Intestinal swab(broiler)

Add 25 g of liquid egg to 225 mL of buffered
peptone water (BPW) and incubate at 37 ℃
for 19 hours or longer.

Add 25 g of feed (feed ingredients, mixedfeed) to 250 mL of buffered peptone water(BPW) and incubate at 37°C for 19 hours orlonger.
Add 25 g of raw meat to 225 mL of buffered
peptone water (BPW) and incubate at 37°C
for 22 hours or longer.
Add 25 g of feces and meconium to 225 mLof buffered peptone water (BPW) andincubate at 37°C for 22 hours or longer.
Put 5 swabs swabbed in the intestine(rectal) in 10 ml Hana Tetrathione (HTT)
medium and incubate at 42°C for 22 hoursor longer.

Culture method (2nd phase)
Add 10 μL of BPW (supernatant) after
culturing to 500 μL of Brain Heart
Infusion (BHI) medium and incubate
at 37°C for 3-4 hours.

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DNA extraction from culture
Please extract DNA from the culture broth according to the following method.
Sample Types
DNA extraction method
Set the small incubator to 98°C and heat it before the inspection.
Dispense 100μL of Solution A of Kaneka Simple DNA Extraction Kit ver.2 in aLiquid egg
1.5 mL tube (for each sample). Add 50μL of culture solution (supernatant) to it.
Heat the 1.5mL tube containing the sample at 98°C for 8 minutes.
After heating, return the 1.5mL tube to room temperature, add 14μL of Solution
Feed
B of Kaneka Simple DNA Extraction Kit ver.2, and mix.
Use the supernatant as the DNA extract.
Raw meatPreheat the small incubator to 98°C before testing.
Dispense 900μL of 1 x TE buffer into a 1.5 mL tube (for each sample). AddFeces/meconium 
100μL of culture solution (supernatant).
Intestinal swabHeat the 1.5mL tube containing the sample at 98°C for 10 minutes.
Use the supernatant as the DNA extract.
(broiler)

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Preparation of PCR solutionThaw the frozen primer mix* and amplification enzyme mix* in advance.
Dispense and mix 8 L of primer mix and 10 L of amplification enzyme mix into a 0.2 mL tube
to prepare a master mix.
*Before dispensing, mix the primer mix and amplification enzyme mix thoroughly by pipetting. Ifthe amplified enzyme mix shows a white precipitate after thawing, completely dissolve it beforeuse.
Primer mix
Amplification enzyme mix◼

8 L10 L

Add 2 L of DNA extract to the 0.2 mL tube in which the master mix was prepared.
After preparation, place on ice, or something cold, to prevent enzyme inactivation and nonspecific amplification reaction.

PCRStart the thermal cycler, set the 0.2 mL tube, and then perform the amplification reaction with thefollowing program.
StepsTemp./Time# of Cycles
Step 125°C / 10 min
1
Step 294 °C / 2 min
194 °C / 5 sec →Step 361 °C / 35 sec
3572 °C / 20 sec
Step 44 °C / （※）
1【Precautions】⚫ Immediately after preparing the PCR solution, perform the reaction in the thermal cycler.
⚫ When preparing the reaction solution, use a micropipette with sterilized filtered tip.
⚫ (*) After completion of the reaction, remove the 0.2 mL tube on the day and perform detection with atest strip.
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Detection by test strip
After the PCR, remove the 0.2 mL tube from the thermal cycler, open the lid of the 0.2 mL tube,and add 160μL of chase buffer.
Insert the test strip into a 0.2 mL tube, allow the part with the sample of the test strip to soak in
the reaction solution, and leave it as it is.
After 10 minutes, visually analyze the control line and each detection line.

Test strip overview

3【Precautions】
【Precautions】
Determine by line coloring 10 minutes after insertion.
Wear gloves when handling the test strip and always hold the top part of absorbent pad. Be careful
not to touch the test strip insertion part (part with sample) and detection unit with your hands.
When inserting the test strip into the tube, be sure to insert it firmly until the part with the sample hits
the bottom of the 0.2 mL tube.

When inserting the test strip, be careful of contamination due to splashing of the reaction solution.
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Determining the Result
Judge the result according to the following criteria.

Example of results
Pattern A: Salmonella not detected
Pattern B: Detects Salmonella that do not belong to O4, O7, and O9 groups
Pattern C: Detects Salmonella belonging to O4 group
Pattern D: Salmonella belonging to O7 group is detected
Pattern E: Detects Salmonella belonging to O9 group
Patterns F to H (example): Unable to detect (retest required)【Precautions】
The control line is always colored when the amplification reaction is proceeding normally. If the control
line does not stain, the amplification reaction may not have proceeded normally, so please perform aretest.
Each detection line will only be colored if it is positive. However, if the O4, O7, and O9 group detectionlines are colored, the Salmonella detection line is also colored. If the O4, O7, and O9 detection lines
are colored but the Salmonella detection line is not colored, it has failed to detect so please perform
a retest. (Use the same DNA extract when retesting).

Notes on using this kit<Contamination measures>◼ To prevent misjudgment due to contamination, replace the used micropipette tip every time. Also, usea micropipette tip with a filter.
◼ We recommend that each step of DNA extraction, PCR solution preparation, and test strip detection
be performed in separate laboratories. If it is difficult to carry out in separate laboratories, separate
workbenches, or work areas within the same laboratory.
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Before opening the tubes for reagents, extracted DNA, etc., spin them down with a low speed
centrifuge. (To prevent the contents from scattering and reagents attached to the lid from sticking tofingers)
Regularly clean micropipettes and other equipment and devices used with 0.1% sodium hypochlorite
and commercially available DNA removers.
If contamination occurs, decontaminate with 0.55% sodium hypochlorite (or a commercially available
DNA remover) or UV irradiation.

＜Disposal precautions＞◼ When discarding the used test strip and amplification solution, be careful not to touch the detection
part and put them in a plastic bag, etc. before discarding.
◼ When discarding the sample after DNA extraction, please take into consideration the sanitary andenvironmental aspects in accordance with the local regulations on waste and the regulations of thefacility.
＜Performance＞◼ Proper detection results can be obtained when detection is performed on the genomic DNA (5 pg/testor more) extracted from Salmonella strains according to the method described in this instruction
manual.
◼ We recommend using this product with Life ECO Thermal Cycler (Bioer Technology). The detection
sensitivity of this product may differ depending on the thermal cycler used.
◼ Detection sensitivity may differ depending on the DNA extraction method.
◼ If the amount of DNA after DNA extraction is extremely large or if the amount of culture medium
brought in is large, the PCR amplification reaction may be blocked, and each detection line and controlline may not be detected. In that case, it may be improved by diluting the DNA extract for PCR.

Assurance◼

Our responsibility is limited to the replacement of the product with a defective product, and we will notbe liable for any other damage, whether direct or indirect. Please be aware of this before using thisproduct.

Contact Information
Kaneka Corporation Medical Solutions Vehicle
2-3-18, NAKANOSHIMA, KITA-KU, OSAKA, 530-8288, JAPANURL https://www.kaneka-labtest.com

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