Human Loopamp 972000 Instructions For Use Manual

I nstruct i ons for Use
380236-D
English
L oopamp MTBC Detect i on K i t
I NTENDED USE
The L oopamp 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 and
hospitals by adequately trained personnel. The result can be
interpreted either by automated turbidimeter or visually under UV
irradiation.
TEST PR I NC I P L ES
Th i s product i s based on the nuc l e i c ac i d amp li f i cat i on method,
L AMP ( L oop-med i ated I sotherma l Amp li f i cat i on ), deve l oped by E i ken
Chem i ca l Co., L td.
The characteristics of the L AMP method are as follows: (1) on l y one
enzyme i s requ i red, and the amp li f i cat i on react i on proceeds under
1),2)
i sothermal cond i t i ons; (2) i t has extreme l y h i gh spec i f i c i ty because of
the use of four pr i mers recogn i z i ng s i x d i st i nct reg i ons on the target; (3)
i t has a h i gh amp li f i cat i on eff i c i ency and can produce a high
concentration of amplified product in a short time, which makes visual or
automated detection possible. 3),4)
The pr i mers prov i ded w i th th i s product are des i gned i n the DNA
gyrase subun i t B (gyrB) and I nsert i on sequence I S6110 ( I S) reg i on of
the MTBC genome DNA, which has been conf i rmed by the a li gnment
ana l ys i s of the se l ected base sequences of MTBC and nontubercu l ous
mycobacter i a to have a we ll -conserved base sequence i n MTBC.
The DNA from untreated sputum or NA L C-NaOH-treated sputum i s
extracted us i ng the L oopamp PURE DNA Extract i on K i t (sold
separately). Then, the DNA so l ut i on i s d i spensed i nto a react i on tube.
The strand d i sp l acement
tr i phosphates (dATP, dCTP, dGTP, and dTTP ) , ca l ce i n, reaction buffers,
and MTBC-spec i f i c pr i mers are stored i n the dr i ed form i n the cap of the
react i on tube. This dr i ed L AMP reagent (MTBC detect i on reagent
(dMTB)) is dissolved when the DNA solution is added. The react i on tube
i s then i ncubated at 67.0 C, and the DNA i s amp li f i ed through cata l ys i s
by the strand d i sp l acement DNA po l ymerase per the L AMP react i on.
The detect i on of amp li f i ed products i s based on the turb i d i ty
measurement of a by-product, magnes i um pyrophosphate ( wh i te
prec i p i tate). 3) Alternatively, a v i sua l judgment under UV irradiation can
be used i nstead of turb i d i ty measurement. Before react i on, ca l ce i n i n
the reagent i s i n the quenched state because of manganese i ons bound
to it; however, once the L AMP react i on i s started, pyrophosphate i ons
are generated and b i nd out the manganese i ons, and ca l ce i n becomes
4)
f l uorescent.
KIT CONTENTS
Reagents are stab l e unt il the date on the l abe l, assum i ng the
conta i ner rema i ns unopened at a storage temperature of 230 C. The
reagents are also confirmed stable after opening the container when
adhered to these procedural directions.
MTBC detect i on reagent ······························ 2 x 48 tubes
The fo ll ow i ng reagents i n the dr i ed form are conta i ned
i n each react i on tube.
Bst DNA po l ymerase
Deoxynuc l eot i de tr i phosphates
Magnes i um su l fate
Ca l ce i n
Manganese ch l or i de
Pr i mers b)
Pos i t i ve contro l MTB (PC MTB)
Negat i ve contro l MTB (NC MTB) ····················· 3 x 0.5 m L
30 µL dropper ········································ 1 x 18 droppers
a) Bst DNA po l ymerase der i ved from Bac ill us stearothermoph il us i s
a strand d i sp l acement DNA po l ymerase that l acks 53
exonuc l ease act i v i ty.
972000
DNA po l ymerase, deoxynuc l eot i de
a)
c) ···················· 1 x 0.4 m L
SSP available in EUDAMED
b) Pr i mers des i gned i n the gyrB and I S reg i on of the MTBC genome
DNA, pur i f i ed from synthes i zed o li gonuc l eot i des by HP L C.
c) The PC MTB conta i ns a product resu l t i ng from the i n v i tro
amp li f i cat i on of a temp l ate genome DNA of Mycobacter i um
tubercu l os i s H37Rv (GenBank No. NC_000962 ) or i g i n.
Abbrev i at i ons of the fo ll ow i ng reagents and L ot No., as well as
manufacturer (EKN), are pr i nted on the conta i ners as shown be l ow:
Reagents
Pos i t i ve contro l MTB
Negat i ve contro l MTB
*Metrological Traceability Information
The Positive Control is derived from the amplification product of
genomic 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. This
fragment is quantitated through photospectrometric analysis, and its
DNA concentration of PC MTB is adjusted at 1,000 copies/μL.
WARN I NGS AND PRECAUT I ONS
(1) For i n v i tro d i agnost i c use on l y.
(2) Th i s product i s des i gned on l y for c li n i ca l d i agnos i s of MTBC from
sputum samp l es of human or i g i n. Do not use it for other purposes.
(3) When us i ng th i s product, a l ways fo ll ow th ese Instructions for Use.
(4) Do not freeze the reagents.
(5) Do not use any exp i red reagent.
(6) Do not m i x d i fferent l ots.
(7) Do not rep l en i sh any reagent.
(8) The p erformance of the Loopamp MTBC Detection Kit depends
on the operator prof i c i ency and adherence to procedura l
d i rect i ons. Test i ng shou l d be done by adequate l y tra i ned
personne l strictly according to the instructions provided.
(9) Remove the requ i red number of react i on tubes from the packag i ng
before using them and re-sea l the a l um i n i um pouch i mmed i ate l y.
(10) Do not remove the des i ccant from the a l um i n i um pouch. H i gh-
l eve l hum i d i ty may deter i orate the dr i ed L AMP reagent i n the
react i on tubes.
(11) Exposure to heat, humidity, and li ght m i ght deter i orate the dMTB.
Thus, remove on l y the requ i red number of react i on tubes ( sum of
samp l es and contro l s ) 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 the
procedure.
(13) Sputum samp l es pose a potent i a l r i sk for i nfect i on. Take a ll
necessary prevent i ve measures to avo i d b i ohazard.
(14) The PC MTB and the NC MTB conta i n a sma ll amount of sod i um
az i de as a preservat i ve. As sod i um az i de i s c l ass i f i ed as tox i c,
avo i d any contact w i th eyes, mouth, or sk i n.
(15) I n case of acc i denta l contact of any reagent w i th eyes, mouth, or
sk i n, i mmed i ate l y r i nse the affected s i te w i th running water and
seek medical advice.
(16) Do not d il ute or add the PC MTB to the samp l es. Instead, use the
PC MTB on l y as descr i bed i n th i s package i nsert to avo i d DNA
contam i nat i on.
(17) Store the PC MTB and any pos i t i ve sputum samp l es separate l y
from the other k i t reagents.
(18) The cap of each react i on tube conta i ns the dMTB i n the dr i ed form.
Do not touch the i ns i de of the cap.
(19) Before us i ng the react i on tubes, check carefu ll y i f they have any
cracks or scratches. Damaged tubes m i ght g i ve fa l se resu l ts and
l ead to DNA contam i nat i on of the i ncubator and work area.
( 20 ) Do not expose react i on tubes to UV li ght before the end of the
L AMP react i on. Pro l onged exposure to UV li ght m i ght damage the
tubes and l ead to fa l se resu l ts.
( 21 ) When UV light i s used for v i sua l f l uorescence judgment, do not
stare d i rect l y at it. As UV li ght i s harmfu l to the eyes, even
watch i ng for a short per i od can i rr i tate eyes and cause symptoms
s i m il ar to conjunct i v i t i s. Instead, use a g l ass screen or wear
1
L abe lli ng on the tube Code on the cap
PC MTB PC
L ot No., EKN MTB
NC MTB NC
L ot No., EKN MTB
5)