Code No. KN-T10C815
1st edition (April 2021)

Instructions for use: KANEKA DNA Chromatography MABC/erm (41)Precautions for use⚫

Wear personal protective equipment (such as rubber gloves, safety glasses, and masks) before
using KANEKA DNA Chromatography MABC/erm (41).

⚫

The results obtained from KANEKA DNA Chromatography MABC/erm (41) should be evaluated andused at your own discretion. Kaneka Corporation is not responsible, whether directly or indirectly,
for any damages or loss which may be caused by the evaluation or use of the obtained results.

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It is your responsibility to verify the validity of any detection results obtained using an operatingprocedure that is not indicated in these instructions for use.

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KANEKA DNA Chromatography MABC/erm (41) is a set of reagents intended for research
applications and is not to be used to diagnose, or help to diagnose, a disease.

1. Product description
KANEKA DNA Chromatography MABC/erm (41) (research reagents) is intended to identify three subspecies ofMycobacteroides abscessus complex (MABC) (M. abscessus subsp. abscessus [M. abscessus], M. abscessus
subsp. bolletii [M. bolletii], and M. abscessus subsp. massiliense [M. massiliense]) and two genetic types (T28C,full-length*1) related to erm (41) in bacterial suspension or bacterial broth medium based on the multiplex PCR
and nucleic acid chromatography techniques.

*1. T28C strain: A strain in which nucleotide 28 of erm (41) is C. Full-length: A strain that keeps the fulllength of the erm (41) gene intact without any partial deletions (nucleotides 64–65 and 159–432 are
typically deleted). KANEKA DNA Chromatography MABC/erm (41) was co-developed by Dr. Yoshihiko
Hoshino, the National Institute of Infectious Diseases, and Kaneka Corporation. Its detection capability
was published in EBioMedicine (2021 Feb; 64:103187).
2. Kit structure/storage conditions
Component
Test strips
Detection buffer

Volume (20 tests)

Storage temperature

Expiration date

20 strips
3.2 mL x 1 vial

2 to 30˚C
Indicated on thelabel

Results card

20 pcs.

PCR mix*2

200 µL x 1 vial

Primer mix

100 µL x 1 vial

-20˚C or less

*2. The PCR mix contains heat-resistant DNA polymerase, dNTPs, dUTP, and uracil DNA
glycosylase (UNG).

－ 1 －3. Detection principle
DNA regions that have been specifically stored in each item are amplified with multiplex PCR by using
primers that specifically amplify each item. The amplified product is spread on a test strip, and MABC
subspecies and erm (41) genetic types are determined based on the colored line pattern. The coloredlines are checked visually, so no equipment is needed to evaluate the result (such as a gel imaging
equipment or real-time PCR system).
<Names of test strip parts>
Enlarged conjugate portion(Dotted lines: Areas wherelines are colored)

Overall view(Top)
Absorption pad

C: Control line
Evaluation
aid lines

Conjugate portion

Sample pad(Bottom)

4. How to use
Instruments and equipment that are necessary but not included in KANEKA DNA

4.1.

Chromatography MABC/erm (41)

• Micropipette and micropipette tips with filters• Thermal cycler (recommended: LifeECO Thermal Cycler/Bioer Technology)• Heat block (can be substituted with a thermal cycler)• 0.2 mL tubes• Desktop centrifuge• KANEKA Microbial DNA Extraction Reagent (sold separately)Preparation of DNA extract

4.2.

Applicable specimens
Suspension of culture grown on a solid medium or in a liquid medium or broth medium.

•Equivalent to McFarland No. 1 (approximately 108 cfu/mL) or culture positive liquid medium•

Bacterial suspension prepared with colonies in sterile distilled water so that they are equivalent
to McFarland No. 1

DNA extract reagent"KANEKA Microbial DNA Extraction Reagent" (sold separately)<For liquid medium>(1) Transfer 20 µL of liquid medium equivalent to McFarland No. 1 to a 0.2 mL tube, mix with20 µL of solution A of KANEKA Microbial DNA Extraction Reagent, then heat the mixture at－ 2 －98˚C for 10 minutes on a heat block or thermal cycler.
(2) After cooling the mixture to room temperature, add 80 µL of solution B of KANEKA Microbi
al DNA Extraction Reagent, then pipette the tube.
<For colonies>(1) Suspend colonies in sterile distilled water*3 so that they are equivalent to McFarland No. 1.
(2) Transfer 20 µL of bacterial suspension to a 0.2 mL tube, mix with 20 µL of solution A of KANEKA
Microbial DNA Extraction Reagent, then heat the mixture at 98˚C for 10 minutes on a heat blockor thermal cycler.
(3) After cooling the mixture to room temperature, add 80 µL of solution B of KANEKA Microbial DNA
Extraction Reagent, then pipette the tube.
*3. When collecting colonies, be careful not to scrape up the solid medium with the colonies.

4.3 Preparation of PCR reaction solution(1) Thaw the PCR mix and primer mix beforehand. *4(2) Dispense and mix the PCR mix and primer mix at a ratio of 10 µL and 5 µL, respectively, into
0.2 mL tubes to prepare the master mix for the number of tubes necessary.
(3) Add 5 µL of the DNA extract prepared in 4.2 into a new 0.2 mL tube.
(4) Add 15 μL of the master mix into the 0.2 mL tube in (3) and mix the mixture by pipetting thetube. *5*4. The PCR mix and primer mix should be mixed thoroughly before dispensing. In particular, thePCR mix may show a white precipitate after thawing. In such a case, completely dissolve theprecipitate before use.
*5. Once prepared, the mixture should be kept in a place where the temperature does not rise,such as on ice, in order to prevent the deactivation of enzyme and non-specific amplification.
4.4. PCR reaction
Load the 0.2 mL tube on the thermal cycler and perform an amplification reaction under the conditions
listed below.
<PCR conditions>Temperature (˚C)
2594
94
65
72

Time (sec)
300
60
51015－ 3 －Cycle1135

4.5. Detection with test strips*6(1) Place a test strip on a results card (optional). *7(2) After the reaction is complete, remove the PCR tube, open the lid, separate 5 µL of the amplified
product, and add the product onto the sample pad of the test strip. *8(3) Similarly, add 70 µL of detection buffer on the sample pad.
(4) At 10 minutes after spreading, visually check the pattern of the colored lines and evaluate theresult.
*6. Wear rubber gloves or similar protective gloves when handling test strips, and always hold theabsorption pad (the upper part of the test strip). Do not touch the sample pad or conjugate portionof the test strips.
*7. This step may be omitted. Placing a test strip on a results card for detection makes it easier toevaluate the pattern of the colored lines. Place a test strip if necessary, as shown in the figure below.
Place the absorption padof the test strip into thedepressed portion of theresults card, with theprinted face up. (Optional)*8. When applying the amplified product to a test strip, use caution to prevent contamination caused bydispersed PCR reaction solution. Use the test strips indoor where the temperature is controlled to15˚C or higher because the colored lines may appear light if the environmental temperature issignificantly low (such as outdoors in the winter) while the amplified product is being spread.

5. Evaluation of result
Negative: Only the control line (C) is colored.
Positive: In addition to the control line (C), a test line or test lines (T1–T5) are colored.
Invalid: The control line (C) is not colored. *9－ 4 －Positive*10
Example ofresult

Negative

Invalid(example)

C onlyNo colored No coloredline on Cline on C

Coloration
pattern*9. The control line is always colored. If it is not colored, the control may have not been amplified
properly. Retest the specimen.
*10. These are examples of typical coloration patterns. erm (41) [full]: A strain that keeps the erm(41) gene intact without any partial deletions. erm (41) [truncation]: A strain with erm (41) thathas partial deletions (nucleotides 64-65 and 159-432 are typically deleted).
6. Precautions for use⚫

Always follow the storage conditions and expiration date indicated in these instructions for use.

⚫

Wear personal protective equipment (such as protective gloves, safety glasses, and masks) beforeusing KANEKA DNA Chromatography MABC/erm (41).

⚫

The specifications of KANEKA DNA Chromatography MABC/erm (41) are subject to change withoutprior notice.

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Follow the instructions specified by the manufacturers of the instruments, equipment, and reagents.

⚫

The results obtained from KANEKA DNA Chromatography MABC/erm (41) should be evaluated andused at your own discretion. Kaneka Corporation is not responsible, whether directly or indirectly, forany damages or loss which may be caused by the evaluation or use of the obtained results.

⚫

You are responsible for verifying the validity of a procedure that is not indicated in these instructions
for use and of any detection results obtained from specimens.

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Exposing the test strips to humidity for a long period of time may lower their detection capability. Storethe open container with the lid closed tightly, away from humidity.

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Replace the micropipette tips after each use in order to prevent false results due to contamination.
Use micropipette tips with filters.

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It is recommended that the DNA extraction, preparation of the PCR reaction solution, and detection
with test strips be done in different laboratories. If it is not possible to do so, separate the work benches－ 5 －or work areas within one laboratory.
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Before opening tubes containing reagents, DNA extract, etc., spin down the tubes in a desktop
centrifuge or other equipment (to prevent the content from dispersing or the reagents on the lid fromtransferring to the operator's fingers).

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Clean the instruments and equipment being used, such as the micropipette, periodically with 0.1%
sodium hypochlorite or a commercially available DNA removal agent.

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If contamination has occurred, decontaminate the work environment with 0.55% sodium hypochlorite(or a commercially available DNA removal agent), ultraviolet (UV) light, or other methods.

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To dispose of used test strips and amplification reaction solution, place them in a plastic bag or similar
container, making sure not to touch the conjugate portion.

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Dispose of the specimens after DNA extraction in accordance with the regulations concerning waste
in your region and institution while considering hygiene and environmental factors.

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KANEKA DNA Chromatography MABC/erm (41) is optimized for LifeECO Thermal Cycler of Bioer
Technology. Using other models may render unclear results.

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KANEKA DNA Chromatography MABC/erm (41) is a set of reagents intended for research applications
and is not to be used to diagnose, or help to diagnose, a disease.

<Contact information>
For inquiries, contact Kaneka Corporation.
TEL: 81-79-445-2406 (Business hours: Monday through Friday 9:00 to 17:00, excluding holidays)
URL: https://www.kaneka-labtest.com－ 6 －