Transcript polimerazuli jaWvuri reaqcia realur droSi (raodenobrivi

polimerazuli jaWvuri reaqcia realur droSi
(Real-Time PCR)
m. axalaia
tuberkulozisa da filtvis daavadebaTa
erovnuli centri, erovnuli centraluri
mikobaqteriologiuri laboratoria
Georgia - Vanderbilt University, Nashville, Tennessee, USA,
2006-2008 CRDF project:
"Monitoring Chemotherapy Regimen Efficacy by
Mycobacterium Tuberculosis mRNA Detection in Sputum
Specimens”
“mkurnalobis monitoringis efqturobis
Sefaseba M. Tuberculosis rnm-is gamovleniT naxvelSi”
saqarTvelo-vanderbildis universiteti, neSvili, tenisi, aSS, 20062008, CRDF
polimerazuli jaWvuri reaqciis komponentebi
dnm matrica
 ori praimeri
 Termostabiluri dnm-polimeraza
 dezoqsinukleozidtrifosfatebi

Mg 2+ ionebi
 buferis xsnari

polimerazuli jaWvuri reaqcia

denaturacia – dnm-s orjaWviani molekulis
gaxleCva
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anilizacia – temperaturis swrafi dawevis
fonze praimerebis dakavSireba erTjaWvian
matricasTan
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elongacia – dnm-polimeraza axdens
matriculi jaWvis replikacias
polimerazuli jaWvuri reaqciis saxeobebi

ukutranskripciuli pjr

asimetriuli pjr
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pjr dnm-s Tavisufali boloebis
swrafi amplifikaciiT
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raodenobrivi pjr

raodenobrivi pjr realur droSi
raodenobrivi pjr realur droSi
principi: Cveulebrivi pjr
magram: dnm/rnm molekulebis
amplifikaciasTan erTad xdeba maTi raodenobis
gansazRvra realur
droSi TiToeuli amplifikaciis (ciklis)
Semdeg
amplificirebuli dnm/rnm molekulebis raodenobis gansazRvrisaTvis
gamoiyeneba:

fluoresenciuri saRebavebi

interkalirirebuli (ukuCarTviTi)
orjaWviani dnm
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modificirebuli dezoqsinukleotidebi,
romlebic fluoresencireben dnm
komplementarul fragmentebTan
hibridizaciis Semdeg
pjr realur droSi

multirezistentuli tuberkulozis
swrafi diagnostikisaTvis myar niadagze
miRebuli M.Tuberculosis kulturidan;
tuberkulozis swrafi diagnostikisaTvis
uSualod masalidan (naxveli);
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
tuberkulozis mkurnalobis
monitoringis efeqturobis Sesafaseblad.
pjr realur droSi
multirezistentuli tuberkulozis swrafi diagnostika

samizne jgufi: myar niadagze (LJ) miRebuli
196 M.Tuberculosis kultura

erTi testiT gamovlinda M.Tuberculosis 25
genuri mutacia, romelbic pasuxismgebelni
arian INH, RIF, STM, ETM rezistentobaze
multirezistentuli tuberkulozis swrafi diagnostika
Sedegebi
gamoyofili Stamebis raodenoba da Sedegbi
INH
RIF
SM
EMB
fenotipuri (lJ, DST)
R
R
S
S
genotipuri (Target-Enriched
Multiplex PCR)
R
S
R
S
INH
35
6
6
RIF
17
1
SM
48
EMB
3
antituberkulozuri preparatebi
sensitiuroba
specifiuroba
149
85.40%
96.30%
1
117
94.50%
99.40%
21
1
126
89.60%
99.20%
3
2
188
50%
98.90%
M.Tuberculosis swrafi diagnostika uSualod masalidan

100 axali sadiagnozo SemTxveva

kulturalurad dadebiTi 59 SemTxveva
M.Tuberculosis diagnostikis meTodebis
Sedareba
98.30
%
98.30
%
86.40
%
LJ
PCR
AFB
tuberkulozis mkurnalobis monitoringis efeqturobis Sefaseba



samizne jgufi: 65 axali sadiagnozo SemTxveva
masala Segrovda mkurnalobis dawyebidan
2 kviris, 1, 2 da 4 Tvis Semdeg
mgb baqterioskopuli gamokvleva (cil-nilseni), daTesva myar niadagze
(LJ), pjr realur droSi
M.Tuberculosis diagnostikis meTodebis
Sedareba
98%
100.00%
95.00%
90.00%
85.00%
83.10%
80.00%
75.00%
AFB
PCR
pjr realur droSi da daTesva myar niadagze (LJ)
(meTodebis Tanxmoba)
120
120
100
77
80
81
60
40
20
8
0
C+ P+
C+ P-
C-P+
C-P-
dadebiTobis maCveneblis Semcireba mkurnalobis monitoringis dros
80.00%
70.00%
60.00%
50.00%
Culture
PCR
40.00%
30.00%
20.00%
10.00%
0.00%
2 weeks
4 weeks
8 weeks 16 weeks
uaryofiTobis maCveneblis zrda MDR da Non MDR SemTxvevebSi
mkurnalobis monitoringis dros (pjr da daTesva LJ)
120.00%
100.00%
80.00%
Non MDR
MDR
60.00%
40.00%
20.00%
0.00%
≤ 1 Month
≥2 Month
daskvna

tuberkulozis swrafi diagnostika uSualod masalidan

multirezistentuli tuberkulozis swrafi diagnostika

maRali specifiuroba da sensitiuroba RIF da INH rezistentobis ganmsazRvreli genis
mimarT
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analizis Sedegs sWirdeba mxolod 2-4 saaTi rogorc gamoyofili kulturidan aseve
uSualod naxvelidan
Georgia - Vanderbilt University, (Nashville, Tennessee, USA), 2006-2008
CRDF project:
"Monitoring Chemotherapy Regimen Efficacy by Mycobacterium
Tuberculosis mRNA Detection in Sputum Specimens”.




"Simultaneous Detection of Tuberculosis Drug Resistance Mutations by a TargetEnriched Multiplex PCR" - Abstract for the 107th General Meeting of the American
Society for microbiology, May 21-25, 2007, Toronto, Canada.
“Prevalence of and Molecular Basis for Tuberculosis Drug Resistance in the Republic of
Georgia: Validation of a Templex System for the Detection of Drug Resistance-Related
Mutations" - Antimicrobial Agents and Chemotherapy. 52:725-9.USA.
“Monitoring Therapy Efficacy by Real-Time Mycobacterium tuberculosis mRNA
Detection in Sputum Specimens” - Abstract for the 108th General Meeting of the
American Society for Microbiology (June 1-5, 2008, Boston, MA), American Association
for Clinical Chemistry (January 23, 2009, Washington, DC).
”DNA and mRNA-Targeted Real-Time TaqMan PCR Assays for Rapid
Detection of Mycobacterium tuberculosis from Sputum Specimens” - Abstract
for 39th Union World Conference on Lung Health (16-20 October 2008, Paris,
France).
gmadlobT yuradRebisTvis