Instructions for Use

SSP available in EUDAMED

380236-D
English972000b) Primers designed in the gyrB and IS region of the MTBC genome
DNA, purified from synthesized oligonucleotides by HPLC.
c) The PC MTB contains a product resulting from the in vitro
amplification of a template genome DNA of Mycobacterium
tuberculosis H37Rv (GenBank No. NC_000962 ) origin.
Abbreviations of the following reagents and Lot No., as well as
manufacturer (EKN), are printed on the containers as shown below:

Loopamp MTBC Detection Kit
INTENDED USE

Reagents

The Loopamp MTBC Detection Kit is a qualitative in vitro
diagnostic test to detect Mycobacterium tuberculosis complex (MTBC)
DNA extracted from the sputum in patients with any symptom
indicative of MTBC infection. The kit aids in the diagnosis of MTBC
infection and is intended to be used in professional laboratories andhospitals by adequately trained personnel. The result can beinterpreted either by automated turbidimeter or visually under UV
irradiation.

Positive control MTB
Negative control MTB

Labelling on the tube
PC MTB
Lot No., EKN
NC MTB
Lot No., EKN

Code on the cap
PC
MTB
NC
MTB

*Metrological Traceability Information
The Positive Control is derived from the amplification product ofgenomic DNA originating from Mycobacterium tuberculosis H37Rv(GenBank No. NC_000962). The specific region of Mycobacterium
tuberculosis H37Rv, which is the target of interest, serves as the
template for the amplification of the PC MTB DNA fragment. Thisfragment is quantitated through photospectrometric analysis, and its
DNA concentration of PC MTB is adjusted at 1,000 copies/μL.

TEST PRINCIPLESThis product is based on the nucleic acid amplification method,
LAMP ( Loop-mediated Isothermal Amplification ), developed by Eiken
Chemical Co., Ltd.
The characteristics of the LAMP method are as follows: (1) only one
enzyme is required, and the amplification reaction proceeds underisothermal conditions;1),2) (2) it has extremely high specificity because of
the use of four primers recognizing six distinct regions on the target; (3)it has a high amplification efficiency and can produce a highconcentration of amplified product in a short time, which makes visual or
automated detection possible.3),4)
The primers provided with this product are designed in the DNA
gyrase subunit B (gyrB) and Insertion sequence IS6110 (IS) region of
the MTBC genome DNA, which has been confirmed by the alignment
analysis of the selected base sequences of MTBC and nontuberculous
mycobacteria to have a well-conserved base sequence in MTBC.
The DNA from untreated sputum or NALC-NaOH-treated sputum is
extracted using the Loopamp PURE DNA Extraction Kit (soldseparately). Then, the DNA solution is dispensed into a reaction tube.
The strand displacement DNA polymerase, deoxynucleotide
triphosphates (dATP, dCTP, dGTP, and dTTP ) , calcein, reaction buffers,
and MTBC-specific primers are stored in the dried form in the cap of thereaction tube. This dried LAMP reagent (MTBC detection reagent(dMTB)) is dissolved when the DNA solution is added. The reaction tube
is then incubated at 67.0 C, and the DNA is amplified through catalysis
by the strand displacement DNA polymerase per the LAMP reaction.
The detection of amplified products is based on the turbidity
measurement of a by-product, magnesium pyrophosphate ( white
precipitate).3) Alternatively, a visual judgment under UV irradiation canbe used instead of turbidity measurement. Before reaction, calcein in
the reagent is in the quenched state because of manganese ions boundto it; however, once the LAMP reaction is started, pyrophosphate ions
are generated and bind out the manganese ions, and calcein becomes
fluorescent.4)

WARNINGS AND PRECAUTIONS(1) For in vitro diagnostic use only.
(2) This product is designed only for clinical diagnosis of MTBC fromsputum samples of human origin. Do not use it for other purposes.
(3) When using this product, always follow these Instructions for Use.
(4) Do not freeze the reagents.
(5) Do not use any expired reagent.
(6) Do not mix different lots.
(7) Do not replenish any reagent.
(8) The performance of the Loopamp MTBC Detection Kit depends
on the operator proficiency and adherence to procedural
directions. Testing should be done by adequately trained
personnel strictly according to the instructions provided.
(9) Remove the required number of reaction tubes from the packaging
before using them and re-seal the aluminium pouch immediately.
(10) Do not remove the desiccant from the aluminium pouch. Highlevel humidity may deteriorate the dried LAMP reagent in thereaction tubes.
(11) Exposure to heat, humidity, and light might deteriorate the dMTB.
Thus, remove only the required number of reaction tubes ( sum ofsamples and controls ) and re-seal the aluminium pouch
immediately.
(12) Read the instruction manual and ensure that the required equipment(turbidimeter or incubator) is available before commencing theprocedure.
(13) Sputum samples pose a potential risk for infection. Take allnecessary preventive measures to avoid biohazard.5)(14) The PC MTB and the NC MTB contain a small amount of sodium
azide as a preservative. As sodium azide is classified as toxic,
avoid any contact with eyes, mouth, or skin.
(15) In case of accidental contact of any reagent with eyes, mouth, or
skin, immediately rinse the affected site with running water andseek medical advice.
(16) Do not dilute or add the PC MTB to the samples. Instead, use the
PC MTB only as described in this package insert to avoid DNA
contamination.
(17) Store the PC MTB and any positive sputum samples separatelyfrom the other kit reagents.
(18) The cap of each reaction tube contains the dMTB in the dried form.
Do not touch the inside of the cap.
(19) Before using the reaction tubes, check carefully if they have any
cracks or scratches. Damaged tubes might give false results andlead to DNA contamination of the incubator and work area.
(20) Do not expose reaction tubes to UV light before the end of the
LAMP reaction. Prolonged exposure to UV light might damage thetubes and lead to false results.
(21) When UV light is used for visual fluorescence judgment, do notstare directly at it. As UV light is harmful to the eyes, evenwatching for a short period can irritate eyes and cause symptoms
similar to conjunctivitis. Instead, use a glass screen or wear

KIT CONTENTS
Reagents are stable until the date on the label, assuming thecontainer remains unopened at a storage temperature of 230 C. The
reagents are also confirmed stable after opening the container whenadhered to these procedural directions.
MTBC detection reagent ······························ 2 x 48 tubes
The following reagents in the dried form are containedin each reaction tube.
Bst DNA polymerase a)
Deoxynucleotide triphosphates
Magnesium sulfate
Calcein
Manganese chloride
Primers b)
Positive control MTB (PC MTB) c) ···················· 1 x 0.4 mL
Negative control MTB (NC MTB) ····················· 3 x 0.5 mL
30 µL dropper ········································ 1 x 18 droppersa) Bst DNA polymerase derived from Bacillus stearothermophilus isa strand displacement DNA polymerase that lacks 53
exonuclease activity.

1

corresponds to approximately 30 µL.

protective goggles/glasses or a UV-blocking eye mask wheneverlooking directly at UV light.
(22) Refer to the manual of the incubator. When the HumaLoop T or
the Real-Time Turbidimeter HumaTurb C+A is used, remove thereaction tubes from the incubator carefully to avoid burns.

(3) Dispense 30 µL of the negative control solution into a reaction tube
using the Loopamp PURE DNA Extraction Kit and close the cap.

Note: The volume between the two lines on the reaction tube
corresponds to approximately 30 µL.
(4) Dispense 30 µL of the PC MTB into a reaction tube using theprovided dropper and close the cap.
(5) Flick (or spin) down all tubes to collect the solution to the bottomof the tubes.

WASTE DISPOSAL(1) Do not open the tubes after DNA amplification. Leave the cap
closed and dispose of the used tubes as medical waste byincineration or after double bagging with sealable plastic bags.
(2) Never autoclave or reuse the reaction tubes; else, amplified
products will disperse and cause contamination.
(3) The main material for the reaction tubes and reagent tubes is PP; forthe reaction tube tray, PET; for the aluminium pouch, aluminium; andfor the kit case, paper.
(4) Dispose of any used reagent, container, or labware per local
regulations.

Note: Ensure that the liquid level is in the middle of the twolines on a reaction tube to ensure 30 µL of pipetting.
(6) Reconstitute the dried reagents in the cap by inverting thereaction tubes and collecting the DNA solution in the cap. Leave
the tubes standing upside down for 2 minutes to reconstitute thedried reagents.
(7) Invert the reaction tubes five times to mix the content. Ensure thatthe dried reagents in the cap are fully dissolved.
(8) Flick (or spin) down all tubes to collect the solution to the bottomof the tubes.

SPECIMEN COLLECTION(1) Use the most purulent part of the sputum sample.
(2) Sputum samples should be used immediately after collection.
(3) Collect sputum in a separate room from the LAMP amplification
room. Aerosols containing the MTBC DNA can be generated
during sputum collection and may cause contamination.

Amplification
For visual fluorescence detection（ For HumaLoop T）(1) Check that the temperature on the HumaLoop T is 67.0 C.
(2) Place the reaction tubes in the HumaLoop T and press the green
button to start the LAMP reaction (40 minutes at 67.0 C). Referto the HumaLoop T instruction manual for details on how to
operate the incubator.
(3) Confirm the completion of polymerase inactivation (automaticallycompleted by the HumaLoop T). Take all reaction tubes out of the
HumaLoop T.
（ For other incubator using UV light）(1) Set the incubator temperature to 67.0 C (with hot bonnet
temperature set to 10 C above the reaction temperature or asnear to this figure as possible – temperature accuracy: 0.5 C).
(2) Place the reaction tubes, and then start amplification reaction ( for40 minutes at 67.0 C).
(3) After 40 minutes, inactivate the polymerase using the HumaHeator heating block (f or 5 minutes at 80 C or 2 minutes at 95 C) toterminate the reaction.

MATERIALS REQUIRED BUT NOT PROVIDED
・ Loopamp PURE DNA Extraction K it
・ Pipette-60 Set
For visual fluorescence detection(For HumaLoop T)
・ HumaLoop T(For other incubator using UV light)
・ Incubator (temperature accuracy :  0.5 C; with hot bonnet )・ HumaHeat or other heating block
・ UV light or Blue LED light (wavelength : 240260 nm and 350370
nm )・ Goggles/glasses or a UV-blocking eye mask (optional)
For real-time turbidity detection
・ HumaTurb C+A・ HumaHeat or other heating block
For reagent and sample mixing
・ Centrifuge for microtubes (optional)
・ Centrifuge for eight connected tubes (optional)・ HuMax ITA, Micro Centrifuge (optional)

For real-time turbidity detection with HumaTurb C+A （ see theflowchart of the procedure）(1) Configure the Real-Time Turbidimeter HumaTurb C+A for
detection with this product.
(2) Check whether the temperature displayed reaches 67.0 C( allow the turbidimeter to warm up for 20 minutes before use ).
(3) Place the reaction tubes and start measurement.
(4) Watch the turbidimeter display to check the positive and negative
controls for any increase in the turbidity. If the turbidity increases
in the positive control but does not in the negative control, theamplification reaction is proceeding correctly (Fig. 1 ). If any othersituation occurs, however, the amplification reaction might beproceeding in the wrong way. In such a case, retest the affected
samples.
(5) Confirm the completion of polymerase inactivation (automaticallycompleted by the turbidimeter ). Take all reaction tubes out of theReal-Time Turbidimeter HumaTurb C+A and discard them without
opening.

PREPARATION OF REAGENTS（1）MTBC detection reagent
Remove the required number of tubes from the aluminium pouchand place them on the rack. (sum of samples and controls).

Note: After removing the required tubes, re-seal the aluminium
pouch with any unused tubes immediately.
（2）Negative control MTB（NC MTB）
Flick (or spin) down the tube to collect the content to the bottomof the tube. Pipette 60 µL of the NC MTB into the Heating Tube
provided in the Loopamp PURE DNA Extraction Kit. Follow the
instructions for use to process the NC MTB (hereinafter extracted
NC MTB is called “negative control solution”).

Note: A negative control should be included every time.
（3）Positive control MTB（PC MTB）
Flick (or spin) down the tube to collect the content to the bottomof the tube.

Amplification plots

Note: The PC MTB should be measured every time.

Negative sample
Positive sample 1
Positive sample 2

Negative control
Positive control

Turbidity

DNA Extraction
To extract the DNA from 60 µL sputum sample, follow the
instructions for the Loopamp PURE DNA Extraction Kit ( soldseparately ). Dispense 60 µL of the sputum sample into the Heating
Tube and load it into the HumaHeat or heating block preheated at
90 C. Use the most purulent sputum whenever possible.
Reagent and Sample Mixing(1) Turn on the HumaLoop T or the Real-Time Turbidimeter
HumaTurb C+A.
(2) Dispense 30 µL of the sample solution into a reaction tube using the
Loopamp PURE DNA Extraction Kit and close the cap.

0.7

0.7

0.6

0.6

0.5

0.5

Turbidity

MEASUREMENT PROCEDURE

0.4
0.3

0.2

0.1

0.1
0
0

5

10

15

20

25

30 35 40

Ti me ( mi n.)

Fig. 1: Amplification plots for controls.

2

0.3

0.2

0

Note: The volume between the two lines on the reaction tube

0.4

0

5

10

15

20

25

30 35 40

Ti me ( mi n.)

Fig. 2: Amplification plots for samples.

as invalid, and the test should be repeated.
After confirming that the run is valid, evaluate samples as follows: Positive sample: green fluorescent light is emitted.
 Negative sample: no fluorescent light is emitted.
For real-time turbidity detection with HumaTurb C+A
After confirming that the turbidity increases in the positive control
but not in the negative control, evaluate samples per the following
criteria (Figs. 1 and 2 ).

PROCEDURAL NOTES(1) The LAMP reaction is very sensitive, and any contamination witheven small amounts of the amplified product could lead to falsepositive results.
(2) Separate the sputum collection and LAMP testing areas.
(3) Clean benches with over 0.5% sodium hypochlorite before andafter performing the test.
(4) Take all necessary measures to avoid contamination, in particular,change gloves after transferring the sputum or if the gloves comeinto contact with the DNA solution.
(5) When handling this product, avoid microbial contamination andnuclease contamination. Even a small amount of DNasetransmitted from sweat or saliva to the reaction tube might
decompose DNA and cause a false result.
(6) Do not use sputum samples containing a large amount of blood,as this may affect measurements. Refer to the “Manual of ClinicalMicrobiology, 12th edition”6) in a case requiring sample
pretreatment.
(7) The DNA solution should ideally be used immediately after
preparation; if it is impossible, the DNA solut ion can be stored atroom temperature and used within 72 hours.
(8) When dispensing the solution into the reaction tube, avoid contact
between the Injection Cap provided in the Loopamp PURE DNA
Extraction Kit and the inner wall of the reaction tubes. Hold thetube rack upright and fill the tube until the level of the DNA solution
is between the two lines (30 µL). It may influence the performance
if it is inappropriate (over the upper line or under the lower line).
(9)（For HumaLoop T or other incubator using UV light）
If bubbles are present, f lick (or spin) down the tubes to remove
them.
（For Real-Time Turbidimeter HumaTurb C+A）
As bubbles in the reaction solution interfere with the turbidity
measurement and cause false judgment, avoid causing any
bubble when mixing reagent and sample solution. If bubbles arepresent, spin down or flick to remove the bubbles.
(10) The dMTB should be fully dissolved. Any undissolved portion
could influence the performance, such as decreasing thesensitivity. In particular, keep the tubes standing upside down for 2
minutes.
(11) The PC MTB contains a high copy number of the control DNA.
Avoid any contamination of other samples with the PC MTB.
Dispense the samples and the negative control solution and close
all reaction tubes before dispensing the PC MTB.
(12) Flick (or spin) down the PC MTB tube before opening it to collectthe content to the bottom of the tube. Close the tube immediately
after dispensing the PC MTB.
(13) When the HumaLoop T or the Real-Time Turbidimeter HumaTurb
C+A is used, polymerase inactivation is automatically performed.
(14) For other incubators, when visual f luorescence judgment ischosen, inactivate the polymerase (for 5 minutes at 80 C or 2
minutes at 95 C) before judgment, or false judgment will becaused.
(15) For other incubators, when UV light is used, do not stare directly
at it. Instead, use a glass board or wear goggles or a protective
eye mask whenever looking at UV light.
(16) Never open the reaction tubes once the LAMP reaction has
started or after completion. Be particularly careful when taking thereaction tubes out of the incubator to avoid opening the tubes
accidentally.
(17) Do not reuse any amplif ied product in the tubes forelectrophoresis or other applications.

 Positive: some increase is observed in turbidity.
 Negative: no increase is observed in turbidity.

Notes:(1) The minimum detectable sensitivity of this product is 0.38
genomes equivalent per test. Even with a negative test, patientswith any persisting symptom indicative of infection by MTBC
should undergo re-examination.
(2) Al though the primers have been designed to target a region
containing a relatively small number of variations, MTBC might
acquire further variations in this region and become less sensitive
to this product. Hence, a negative test does not always rule out

infection by MTBC.
(3) Test resul ts could be affected by specimen collection andtransport, specimen preparation, inhibitors, and other laboratory
procedural errors. A negative test does not exclude the presenceof MTBC from the specimen. In making a clinical diagnosis,
consider the patient’s clinical condition and all other available
laboratory results.
(4) This product is a k it for qualitative detection; it is not designed forquantitative measurement. The intensity of f luorescent li ght observedor the rise time of turbidity measured by the Real-Time Turbidimeter
HumaTurb C+A does not correlate with the template DNA
concentration.

INTERFERING SUBSTANCES
Our in-house studies have revealed that measurement was notaffected by the presence of free bilirubin ( 91.0 mg/dL ), conjugatedbilirubin (101.0 mg/dL ), chyle ( formazine turbidity : 7,300 ), andhaemolytic haemoglobin ( 2,475 mg/dL ).
Regarding drugs, our in-house studies have revealed thatmeasurement was not affected by the presence of isoniazid(100 µg/mL), ethambutol (20 µg/mL), rifampicin (100 µg/mL),pyrazinamide (500 µg/mL), kanamycin (20 µg/mL), and streptomycin(500 µg/mL).

PERFORMANCE CHARACTERISTICS
1. Accuracy
In testing the following samples: negative sample (concentration: 0 genome equivalent/test ) positive sample 1 (1.875 genomes equivalent/test ) positive sample 2 (125 genomes equivalent/test )The negative sample should test negative, while the positive
samples 1 and 2 should test positive.
2. Within-run Reproducibility
In testing f ive negative and positive samples simultaneously, thenegative sample should test negative throughout, while the positive
sample should test positive throughout.
3. Limit of Detection
0.38 genomes equivalent / test
4. Cross-reactivity
Regarding nontuberculous mycobacteria (Mycobacterium
asiaticum, Mycobacterium kansasii, Mycobacterium marinum,
Mycobacterium simiae, Mycobacterium scrofulaceum, Mycobacterium
szulgai,
Mycobacterium
gordonae,
Mycobacterium
xenopi,
Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium
gastri, Mycobacterium haemophilum, Mycobacterium malmoense,
Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium
flavescens) (1.0 x 104 genomes equivalent per test) and respiratory
disease bacteria (Streptococcus pneumoniae, Staphylococcus aureus,
Haemophilus influenzae, Moraxella catarrhalis, Klebsiella pneumoniae,
Escherichia coli, Legionella pneumophila, Pseudomonas aeruginosa,
Mycoplasma pneumoniae（I）, Mycoplasma pneumoniae（II）) (1.0 x

INTERPRETATION OF RESULTS
For visual fluorescence detection(For HumaLoop T)
Place each reaction tube in the Fluorescence Detection Unit,
irradiate, and observe the tube from the side.
(For other incubator using UV light)
Irradiate the bottom of each reaction tube and observe from theside through goggles/glasses or a UV-blocking eye mask.
For a valid run, the following resu lts must be obtained: Positive Control: green fluorescent li ght is emitted
 Negative Control: no fluorescent light is emitted
I f any control is invalid, all samples in the run should be reported

105 genomes equivalent per test), the measurement system tested
negative for all bacterial species; no cross-reaction occurred.
5. Reactivity against MTBC
Reactivity against Mycobacterium tuberculosis, Mycobacterium3

bovis, and Mycobacterium africanum was confirmed.
6. Information about a Calibrator
The performance test for this product used the plasmid DNA
containing the gyrB and IS region of the genome DNA of
Mycobacterium tuberculosis H37Rv (GenBank No. NC_000962) asa calibrator.
7. Clinical performance
To date, tuberculosis remains one of the world’s largest infections.
Globally, the total estimated number of new tuberculosis cases isabout 10 m illion, and that of sputum smear-positive is about 4.3
m illion.7) In Japan, 24,760 patients were newly registered asaffected with tuberculosis in 2008, of whom 2,216 died.8)
This product was examined in two medical institutions
designated for Class-2 infectious diseases (designated medical
institutions that had beds exclusive for patients with tuberculosis)
for performance in diagnosing patients suspected of tuberculosis.
Along with PCR and TRC, both of which had already been approvedin Japan, this kit was used to test sputum samples collected for 2days (320 samples from 160 subjects), and the results werecompared between kits. Notably, testing based on this productincluded evaluations for the DNA extract solution obtained from theuntreated sputum (hereinafter LAMP for untreated sputum) andthose for the DNA extract solution obtained from the sputum
pretreated by NALC-NaOH and some other processes (hereinafter
LAMP for pretreated sputum).
As shown in the tables below, the overall concordance rate wasfavorable as follows: LAMP for untreated sputum vs. PCR: 91.5%(291/318 subjects); LAMP for pretreated sputum vs. PCR: 92.1%(293/318); LAMP for untreated sputum vs. TRC: 94.3% (230/244);
LAMP for pretreated sputum vs. TRC: 93.0% (227/244). In addition,
the evaluations made by real-time turbidity detection and visualf luorescence detection completely agreed, except for one sample(this sample was bloody sputum and tested false negative in visualf l uorescence detection because of the disturbance by blood). Thus,
we regard the two types of evaluations as identical to each other.
The tables below show test resu l ts obtained by real-time turbidity
detection. Of note, the resu l ts of visual f l uorescence detection by
the HumaLoop T were the same as those by the Real-Time
Turbidimeter HumaTurb C+A.

LAMP

Positive
Negative
Overall concordance rate
Diagnostic sensitivity
Diagnostic specificity
Positive predictive value
Negative predictive value
Likelihood ratio +(sensitivity/(1-specificity))

Untreated sputum

Likelihood ratio ((1-sensitivity)/specificity)
LAMP
Positive
Negative
Overall concordance rate
Diagnostic sensitivity
Diagnostic specificity
Positive predictive value
Negative predictive value
Likelihood ratio +(sensitivity/(1-specificity))
Likelihood ratio ((1-sensitivity)/specificity)

Pretreated sputum

LAMP

Positive
UntreatedsputumNegative
Overall concordance rate
Diagnostic sensitivity
Diagnostic specificity
Positive predictive value
Negative predictive value
Likelihood ratio +(sensitivity/(1-specificity))
Likelihood ratio ((1-sensitivity)/specificity)

LAMP

Positive
PretreatedsputumNegative
Overall concordance rate
Diagnostic sensitivity
Diagnostic specificity
Positive predictive value
Negative predictive value
Likelihood ratio +(sensitivity/(1-specificity))
Likelihood ratio ((1-sensitivity)/specificity)

TRC
Positive
Negative163
5967
94.3% (230/244)
94.8% (163/172)
93.1% (67/72)
97.0% (163/168)
88.2% (67/76)
13.9
0.0451TRC
Positive
Negative
157
2
15
70
93.0% (227/244)
91.3% (157/172)
97.2% (70/72)
98.7% (157/159)
82.4% (70/85)
32.6
0.0895ORDERING INFORMATION
Product
CodeContents

972000980000

HuMax ITA

964000Loopamp PUREDNA Extraction Kit
HumaHeat
Incubator

971000

Pipette-60 Set

961000

HumaLoop T

963200

HumaTurb C+A

970000PCR
Positive
Negative
196
7
20
95
91.5% (291/318)
90.7% (196/216)
93.1% (95/102)
96.6% (196/203)
82.6% (95/115)

Product Name

Loopamp MTBC
96 tests
Detection Kit

Micro Centrifuge
90 tests
Heating Block
1 pipette; 4 x 96 tips
1 Main unit
1 Fluorescence Detection Unit
1 Control unit
1 Amplification Unit

NOTICEIn case of occurrence of any serious incident that has occurred inrelation to the device shall be reported to the authorised representative,
the manufacturer and the competent authority of the Member State inwhich the user and/or the patient is established.

13.1

REFERENCES
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) Tom ita N., et al.: Nat. Protoc. 3, No. 5, 877–882 (2008)
5) The guideline for the bio-safety and bio-hazard (by the JapaneseSociety for Bacteriology): Japanese Journal of Bacteriology 54,
No. 3, 667–715 (1999)
6) Isabella MGaby EP and Nicole P., et al.: Manual of ClinicalMicrobiology, 12th edition, ASM PRESS, 558–575 (2019)
7) WHO global TB database:https://www.who.int/teams/global-tuberculosis-programme/data
https://apps.who.int/iris/bitstream/handle/10665/336069/9789240
013131-eng.pdf
8) Kekkaku No Tokei 2009 (Statistics of TB 2009): Japan AntiTuberculosis Association

0.0999PCR
Positive
Negative
194
3
22
99
92.1% (293/318)
89.8% (194/216)
97.1% (99/102)
98.5% (194/197)
81.8% (99/121)
31.0
0.105

4

Flow chart
Operation procedure for real-time turbidity detection
Preparation of the sample solution and the negative control solution
Pipette 60 µL of sample and the NC MTB.
With the Loopamp PURE DNA Extraction Kit,
extract DNA from the collected sputum and the NC
MTB. The obtained solutions are used as the sample
solution and the negative control solution, respectively.
Reagent preparation
Mixing of reagent andsample solution

Take a required number of reaction
tubes of the dMTB.
( For samples, negative andpositive controls )Transfer 30 µL of sample or control solution into eachreaction tube.
Use the negative control solution as negative control.
Use the PC MTB as positive control.
(The positive control should be prepared at last.)
Invert the reaction tubes to collect the solution on thecap. Wait 2 minutes.
Wait two minutes with the reaction tubes inverted.
Invert the reaction tubes five times to mix the contents,
and then spin them down.
( Avoid any bubbles)
Amplification

Place the reaction tubes in the reaction block of theturbidimeter.
As directed in the instruction for use of the turbidimeter,
start reaction and measure and evaluate the turbidity (for40 minutes at 67.0 C.)
Confirm the complet ion of polymerase inactivation (for 5 mi nutes at
80 C or 2 minutes at 95 C). Take all reaction tubes out of theturbidimeter and discard them without opening. Be careful not todamage the tubes.

TABLE OF SYMBOLS
Catalog number

Consult instructions
for useExpiration dateIn vitro diagnostic
medical device

Manufacturer

Temperature limitation

Batch code

Contains sufficientfor <n> tests

Authorized
Representative in
the European
Community

Importer

HUMAN Gesellschaft für Biochemica
und Diagnostica mbH
Max-Planck-Ring 21, 65205 Wiesbaden, Germany

https://www.eiken.co.jp/en*Date of Revision: 2023-10-31

5