Chan doan nhanh vi khuan lao khang rifampicin bang ky thuat khuech dai gene da moi dac hieu alen
Rapid Detection of Rifampicin Resistance IVlutations in Mycobacterium Tuberculosis Using Multiplex Allele Specific Polymerase Chain Reaction Assay
Hoang Quoc Truong*, Nguyen Trgng Chinh' Tran Thi Thanh Huyen*, Tran Thj Huyen Trang*. Le Hiru Song*, Nguyen Van Hung**
\u Thi Kim l^ien***
Benh vien Trung uang Qudn doi 108*
Benh vien phoi Trung uang**
Vien Ve sinh dich te Trung uang***
Tom t i t
Dat van de: Rifampicin la khang sinh quan trpng de dieu tri lao, tuy nhien, hieu q u i dieu tri cua khang sinh nay ngay cang bj g i i m do sU xuat hien cac chung vi khul'n khing thuoc. Muc tieu: Tim phuong phap xac djnh nhanh vi khuan lao khang thuoc Rifampicin. PhUOng phap: Nuoi cay vi khuan lam khang sinh do de danh gia khang thuoc. SU dung ky thuat khuech dai gene da moi dac hieu alen (MAS-PCR) va so sanh ket qua vdi giai trinh tU gene de tlm dot bien gene khang thuoc. Ket q u i : 15 chung vi khuan lao dugc nuoi cay thanh cong, trong do co 3 chung da khang. Ket q u i MAS-PCR da phat hien dugc cle dot bien tai cac vj tri rpoB516, rpoB526 va rpoB531 vdi thdi gian chi trong 1 ngay. Gili trinh tu doan gene rpoB da k h i n g dinh ket qua xac djnh nhUng dot bien tren. So vdi gili trinh tU gene va nuoi cay vi khuan thi ky thuat MAS-PCR dugc thuc hien trong thdi gian n g i n hon (1 ngay so vdi 3 va 30 ngay).
TUkhda: Lao, MAS-PCR, dot bien, khang thuoc
Summary
Introduction: Rifampicin is an important antibiotics which was chosen to treat tuberculosis. However, anti-TB activity of rifampicin is now being reduced by the emergence of drug resistance of Mycobacterium tuberculosis strains. Objectives: To establish the method for rapid detection of rifampicin resistance mutations in Mycobacterium tuberculosis. Method: Bacteria culture was used to assess antibiotic resistance.
To detect TB resistant to rifampicin, we used multiplex allele specific polymerase chain reaction assay (MAS- PCR) and compared the results with DNA sequencing. Results: 15 strains of TB were cultured successfully, among those, three strains were multidrug-resistant. The results showed that point mutation in the specific position rpoB516, rpoB526 and rpoB531 was detected through MAS-PCR assay within a day. The results of MAS-PCR were confirmed by rpoB gene sequencing. Compared with sequencing and bacterial culture techniques, the MAS-PCR was peri'ormed in a shorter time (1 day compared with 3 and 30 days).
/Ceyivo/'ds; Tuberculosis, MAS-PCR, mutation, drug resistance.
Phin bien khoa hoc: PGS. TS. NGUY§N OlNH Tl^N
I.Datvande
! Theo Td chu-c Y te The gidi (WHO), Viet Nam xep thU 13 trong tdng sd 22 qudc gia cd sd benh nhan lao eao nhat toan cau vdi ty le lao da khing nguyen phit la 2,3%. Theo thdng ke cua ChUdng trinh phdng chdng lao I Qudc gia hing n i m c l nUde cd them 120.000 benh I nhin lao cle the, trong dd ed 55.000 - 60.000 benh nhin
ho khac ra ddm ed AFB(-i-) bing soi trUe tiep. IVldi ngay cd khoing 50 ngUdi chet vl benh lao. Mdi lo ngai hing dau chinh la tinh trang khang da thude cua vi khul'n lao.
Bdi vi kiem solt va phit hien khdng che lao da khing thudc trong cdng ddng II rl't khd khin, ben canh dd chi phi de dieu tri lao khing da thudc cao ban hang chuc lan so vdi kinh phi de dieu tri lao thdng thudng.
Rifampicin (RIP) la khang sinh chu lUc trong dieu trj lao nen khi RIF bj khing thi van de dieu trj trd nen rl't phUe tap, hieu q u i dieu tri kem. Theo djnh nghia cua WHO thi lao da khang la vi khuan lao khing it nhl't vdi Rifampicin va Isoniazid. Do dd, d c xet nghiem p h i t hien khang RIF dugc coi la b i t budc trong chan doan lao khang da thudc [3]. Vi khuan lao khing dugc RIF la do chung cd kha nang tU bien ddi gene rpoB, la gene ma hda cho tieu don vj p-ARN polymerase, trong he gene cua chung. Vung quyet djnh dot bien gene khing RIF cua rpoB ed chieu dai 81 nucleotide, tuong Ung vdi codon tU vj tri 509 den 533 eua gene nay. Ngudi ta p h i t hien thay ring 96%
sd chung vi khuan khang RIF dugc p h i n lap ed chUa cae bien dpi d vung gene n i y [9]. Cho den nay, chan doln vi khuan lao khing RIF deu dua tren viec p h i t hien cle dot bien trong doan gene chUa 81 nucleotide nay. Cac ky thuat sinh hge phan tU (SHPT) d l dugc sU dung de phat hien d c dot bien gene rpoB bao gdm: g i l i trinh tU gene [1], polymerase chain reaction single-stranded conformation polymorphism (PCR-SSCP) [2], PCR-ELISA allele- specific PCR assay [6] va DNA macroarray [11]. Tuy nhien, do mdi phuang phap cd mdt sd Uu, nhuge diem rieng nen chua dugc sCr dung rdng rai trong thUc hanh.
Ky thuat khuech dai gene da mdi (Multiplex PCR) la mdt trong nhQng ky thuat cua sinh hgc phan tCf vdi viec sCr dung han mdt cap mdi (primer) trong
cung mdt p h i n Ung khuech dai gene (PCR), vdi ky thuat nay nhieu doan gene khac nhau trong cung mdt he gene (genome) eua ddi tugng nghien eUu se cung dugc n h i n len. Ky thuat nay giup g i i m bdt thdi gian lam d e xet nghiem b i n g phin Ung PCR don 1^, tiet kiem cdng sUc va chi phi. Multiplex PCR d l dugc Ung dung rdng rli tai d e phdng thi nghiem sinh hge p h i n tU tren the gidi de chan d o l n nhanh, chinh xle d e mam benh vi sinh vlt, die biet I I trong ehl'n d o l n lao [8]. Ky thuat nhin die hieu alen (allele specific amplification ASA) II mdt ky thuat khic trong ehl'n d o l n SHPT dugc Ung dung de xle djnh d e dot bien diem die hieu tren trinh tU gene can nghien eUu.
Trong nghien eUu nay ehung tdi Ung dung cdng nghe ket hgp Igi the cua hai ky thuat SHPT hien dai:
(1) n h i n gene da mdi (multiplex PCR) v l (2) khuech dai d i e hieu alen (allele specific amplification) trong chan d o l n p h i t hien vi khul'n lao khing rifampicin.
2. Doi tugng va phUcrng phap nghien cUu
2.1. Chdng vi khuan lao va chCing vi khuan lao da khing thuoc
Cle Chung vi khuan lao MTB-103, N21, Al 1, N l , N i l , N12, S40, N66, N2, S48 v l S I I , dugc tien hlnh p h i n lap tai Khoa Mien djch p h i n tU - Vien Ve sinh dich te Trung Uang theo quy trinh chuan hien hanh.
4 chung lao da khing HRSE: MTB-182HN, MTB- 215HN, MTB-230HN va MTB-224HN, dUgc nudi cay va lam khing sinh do tai benh vien phdi Trung uong theo quy trinh chuan.
2.2. Tich chiet ADN tong so td cic chung vi khuan lao va vi khuan lao da khing thuoc
Chung vi khuan lao da khing thudc dugc hda trong dem Tris-HCI (pH 7.5) va bien tinh 30 phut d 100°C. Sau khi bien tinh, 200 pL dung djch bat boat vi khul'n lao dugc hda trong400 pL dung djch dem B va 64 pL dung djch C. Cic dung djch B, C dugc pha che tai Khoa SHPT, Benh vien TWQO108. ADN dUdc t i c h chiet vdi phenol-chloroform: isoamyl alcohol (25:24:1) va tua bdi 0.6 the tich isopropanol. Cae budc xCr ly mau va t i c h chiet ADN dugc tien hanh rieng biet trong tu cay an toan sinh hgc d p II.
2.3. Khuech dai gene da moi die hiiu alen vi giii trinh tUgene
Phin Ung khuech dai gene da mdi d i e hi^u alen (MAS-PCR) dugc tien hanh de xlc djnh d c diem dot bien tuong Ung tai d e codon 516, 526 v l 531 eua gene rpoB. Phin Ung dugc tien h l n h vdi t h ^ tieh 50 pi eho mdi mau vdi ti le c^c thanh phan nhu sau: 10X Taq buffer, 25 mM MgCl2, 2.5 mM dNTP, primer-516F: 5'-CAGCTGAGCCAATTCATGGA- 3' (5 pM), primer-526F: 5'-CTGTCGGGGnGACCCA-3' (10 pM), primer-531F: 5'-CACAAGCGCCGACTGTC-3' (45 pM) va rpoBR: 5'-TTGACCC GCGCGTACAC-3' (60 pM), 0.5 pi Taq DNA polymerase (Fermentas). 50 ng ADN v l 13.5 pi nude eho p h i n Ung PCR. Phin Ung khuech dai gene dugc thuc hien tren m i y PCR tU ddng GeneAmp PCR System 9700 vdi ehu ky nhiet nhu sau: 96°C/ 3 phut; 25 ehu ky (95°C/ 50 gily, 68°C/ 40 gily, 72°C/ 1 phut); 72°C trong 7 phut.
Oe khang djnh ket q u i , ky thuat g i l i trinh tU gene dugc tien hanh. Gene rpoB ed chUa d e diem dot bien tai d e codon 516, 526 va 531 dugc n h i n b i n g p h i n Ung PCR sCr dung cap mdi: TuberLabF: 5'- TACGGTCGGCGAGCTGATCC-3' va TuberLabR: 5'- TACGGCGTTTCGATG AAC-3'. Sin pham eua p h i n Ung PCR sau dd dugc lam sach va tien hanh dgc trinh t u tren may g i l i trinh tU CEQ8800 (Beeman Coulter, My) theo hudng dan.cua nha sin xuat.
2.4. Phin tfch kit qui khuech dai gene da moi die hiiu alen va giii trinh tUgene
20 pi sin pham PCR cua mdi mau thi nghiem dugc phan tich b i n g phuang phap dien di tren gel agarose 2.5 % vdi dien the khdng ddi (lOOV). Oe danh gia d c diem dot bien gene rpoB b i n g MAS- PCR thang chuan AON 100 bp dugc sd dung.
Agarose gel dugc nhudm vdi ethidium bromide (0.5 pg/ml, Sigma) va chup I n h tren may dgc gel UVP, My. Mdi lan xet nghiem dugc tien hanh song song vdi 1 chUng am va 1 chUng duong. Cle bang ADN cd kich thudc 218 bp [rpoBSld), 185 bp {rpoB526) va 170 bp {rpoB531) khdng xuat tren dien di dugc coi la p h i n ufng PCR dUdng tinh ddi vdi lao khang
rifampicin. Ket q u i g i l i trinh tU gene rpoB xac djnh dot bien dugc p h i n tich bdi phan mem BioEdit.
3. Ket q u i nghien cUu va binh luan
3.1. Ket qui xic djnh dot bien gene rpoB bing ky thuit khuech dai die hiiu alen (allele specific amplification):
3.1.1. Xdc dinh ddt bien diem 516
Ket q u i d Hinh IA cho thay, khdng phit hien dot bien tai vj tri 516 vdi c l 3 mau da khang:
isoniazid (H), rifampicin (R), streptomycin (S) va ethambutol (E): MTB-182HN (gieng 2), MTB-215HN (gieng 3) v l MTB-230HN (gieng 4) khi so sanh vdi mau ddi ehUng II chung vi khuan lao hoang dai (gieng 1 v l 5). Do thiet ke mdi dac hieu nen khi co dot bien se khdng ed sin pham PCR. Gieng 6 II mau ddi ehUng I m vdi thang ADN chuan (M).
Hinh 1 A: Hinh anh dien di san pham PCR phit hien dot bien diem 516.
3.1.2. Xdc dinh dot bien diem 526 B)
^W bp — K(X1 hf> — -l>11,,, — ftaibp —
^OLI by, — 4iX,i bp — J
300 b p - J
2 0 L ) bp —
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^ f
fr
—'poa.s:iS(i8?bpi
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Hinh 1 B: Hinh anh dien di san pham PCR phit hien dot bien diem 526
Ket q u i d Hlnh 1B phat hien thay cd 2 mau dot bien la MTB-215HN (gieng 3) va MTB-230HN (gieng 4). Khi so sanh vdi 3 mau da biet cd da khang thuoc
bing nudi cay (gieng 2, 3, 4). Do thiet ke mdi die hieu nen khi cd dot bien thl se khdng cd b i n g PCR.
3.1.3. Xdc dinh ddt bien diem 531
TVi? ' ' ( ' C ' l l r )
Hinh 1 C: Hinh anh dien di san pham PCR p h i t hien dot bien diem 531.
Ket q u i xlc djnh dot bien diem 531 d Hinh 1 C cho thay cd 1 mau dot bien la MTB-182HN (gieng 2).
NhUvay: bing ky thuat PCR die hieu alen ehung tdi d l xle djnh dugc 3 mau khing thude tai 2 vj tri khic nhau dd la vj tri 526 (2 mau) va 531 (1 mau).
3.2. Ket qui giii trinh tUvCing quyet djnh dot bien khing rifampicin
Ket q u i g i l i trinh tU gene cho thi'y khdng p h i t hien dot bien tai codon 516 vdi d 3 mau MTB- 182HN, MTB-215HN va MTB-230HN. So vdi mau hoang dai va mau chuan lay tU Ngin hang gene ehung tdi xle djnh mau MTB-182HN ed dot bien tai vj tri 531 v l 2 mau (MTB-215HN va MTB-230HN) ed dot bien tai vj tri 526. Ket q u i nay hoan toan phu hgp vdi ky thuat PCR d i e hieu alen.
3.3. Kit qui xic djnh dot bien gene rpoB bing ky thuit khuech dai gene da moi die hiiu alen (multiplex allele-specific PCR):
TU ket q u i thu dugc d tren v l vdi muc dich phit trien d e ky thuat SHPT trong chan doln nhanh vi khuan lao da khang thude, die biet ddi vdi RIF v l INH.
Chung tdi Ung dung ky thuat khuech dai gene da mdi die hieu alen (MAS-PCR), II td hgp eua ky thuat nhan gene da mdi (multiplex PCR) v l khuech dai die hieu alen (allele specific amplification) vdi muc tieu phit hien cung luc d e dot bien khing rifampicin tai 3 codon 516, 526, 531 cua gene rpoB trong cung mdt phin Ung. Ket q u i trong Hinh 3 eho thay bing ky thuat PCR da mdi die hieu alen chung tdi phit hien 3 mau cd dot bien tai cle vj tri khic nhau. Gieng 1 va 5:
mau chUng dUOng khdng dot bien ed sin pham II 3 bing. Mau 2 cd 2 bing, mat bing thU 3 tUong Ung vdi diem dot bien 531, mau 3 va 4 khdng cd bing thU 2 tuong Ung vdi dot bien tai vj trf 526. Gieng 6: mau ddi ehUng am. M, thang ADN chuan.
lOOOtp-J
6<X b., —1 Mx.hr-J 400bp—1
»nobr—1
:oobo—i
lc»b3—1
"S*
n>o8M6 :isbp:.
rDo3i:6 185 bp'
Hinh 3: Hinh I n h dien di sin pham PCR da mdi die hieu alen xle djnh dot bien gene rpoB.
Vung quyet il|nh df t bien khing rifampicin (81-bp Rff resistance dertennining r^ion)
Gly I k Ser Gin Leu Ser Gin Phe Met Asp Gin Asn Asn Pro Leu Ser Gly Leu Thr His Lys Arg Arg Leu Ser Ala Leu Gly 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 irrB-H37Ra(CP000611) GGCACCAGCCAGCTGAGCCAATTCATGGACCAGAACAACCCGCTGTCGGGGTTGACCCACAAGCGCCGACTGTCGGCGCTGGGG
MTB-224HN/Conlrol ,
^f^B•182HN/HRSE T MrB-215HN/HRSE G
MrB-230HN/HRSE <5 r • prlmer-Sier: CAGCTGACGCAATTCATC GA
prlmer-526F: CTGTCGGGGTTGACCCA
prlmer-531F: CACAAGCGCCGACTGTC
Hinh 2: Trinh tU doan gene rpoB tren d c mau nghien cUu.
3.4. Ket qui nghiin cdu trin cic mau binh phi'm
Sau khi hoan thien ky thuat PCR da mdi die hieu alen ehung tdi tien hlnh thU nghiem tren 11 mau dugc cung d p bdi Vien Ve sinh djch te Trung uang. Ket q u i dugc trinh bay tren hinh 4. Oung nhU ket q u i khing sinh do, khdng ed dot bien gene nao dugc tim thay tren 11 mau nghien eUu. Tl't el deu ed 3 bang. Gieng 12: mau ddi ehUng am. M, thang ADN chuan.
Hinh 4: Ket q u i dien di sin phi'm PCR da mdi d i e hieu alen tren mau benh pham
4. Ban luan
Rifampicin (RIF) cd t i c ddng true tiep len q u i trinh trao ddi chat cua vi khuan lao boat ddng d giai doan sdm, ddng thdi RIF cdn duy tri boat tinh khang khuan sau dd. Vdi d i e tinh duge hge nay, khi rifampicin ket hgp vdi pyrazinamide se rut n g i n phac do dieu tri benh lao phdi tU 1 n i m xudng cdn 6 t h i n g [4]. Tuy nhien, theo thdi gian tinh trang vi khuan lao khang RIF d n g trd nen nghiem trgng dan den ty le that bai trong dieu trj ngay d n g t i n g [5].
Theo cle nghien cUu trUde day eho thay lao don khang INH la phd bien cdn don khing RIF rl't hiem gap. Ngugc lai, khing RIF lai thudng gap d d e ehung vi khul'n lao da khang INH, do vly, khing RIF dugc coi la mdt "dau an" trong chan d o l n benh lao da khing thude MDR-TB (Multi Drug Resistance Tuberculosis) [10].
Rifampicin Ue che q u i trinh phien ma eua vi khuan b i n g each t i e ddng trUc tiep nen enzyme ARN polymerase phu thudc ADN. Vi khul'n lao phat trien cd che khang RIF qua viec thay ddi cau true gene rpoB, day la gene ma hda cho tieu phan p-ARN polymerase cd trong he gene cua chung. Cac nghien cUu cdng bd tren the gidi eho thay vung quyet djnh
dot bi^n khing RIF eua vi khul'n lao ed kich thudc 81 nucleotide (27eodon), dot bien d vung nay dugc tim t h i y d 96% chung vi khuan lao khang RIF Do do vung gene n i y dugc coi I I mdt dau an quan trpng trong ehl'n d o l n nhanh lao khang RIF/ MDR [7],
Phan Idn d c dot bien (65-86%) dugc phit hien d vj tri codon 526 hole codon 531 cua gene rpoB\id\
mUe do khing RIF tUdng ddi eao, ndng dp Uc che toi thieu (minimal inhibitory concentration [MIC] >32 mg/ml). Trong vung quyet djnh dot bien khing RIF edn tim thay d c bien ddi khic tai vj tri d e codon 511, 516, 518, v l 522 vdi mUc do khing RIF thap ban. Dua tren trinh t u nucleotide vung quyet djnh dot bien khing rifampicin eua ehung vi khuan lao chung tdi sU dung 3 cap mdi (primer-516F, rpoBR);
(primer-526F rpoBR) v l (primer-531F, rpoBR), tUOng Ung de p h i t hien elc alen bj bien ddi eua 3 codon niy. Khi khdng cd dot bien tai d c codon ke tren, cac doan gene d i e hieu alen vdi kich thudc 218-, 185-, v l 170 nucleotide se dugc nhan len. Nguge lai, cac chung vi khul'n lao khang rifampicin se khong khuech dai cle doan gene d i e hieu alen niy.
Ket q u i cho thi'y b i n g phuang phap nay chung tdi d l thanh cdng trong viec phat hien cac dot bien diem tai d e vi tri can xac djnh. Oly la ket q u i rat dang quan t l m vi chi phi cua phuong phap chan doln nay chi bing 1 /10 so vdi ky thuat gili trinh tU gene.
Oe khing djnh lai ket q u i xlc djnh dot bien khing RIF b i n g ky thuat khuech dai d i e hieu alen (allele specific amplification), ehung tdi tien hanh g i l i trinh tU doan gene rpoB ed chufa vung quyet djnh dot bien khang rifampicin. Ket q u i cua giai trinh t u gene la hoan toan phu hgp vdi ket q u i cua ky thuat khuech dai dac hieu alen. NhU vly, tinh chinh xle cua ky thuat khuech dai d i e hieu alen (allele specific amplification) dUdc khang djnh.
TU eac ket q u i thu dugc va vdi muc dich phat trien d c ky thuat SHPT trong chan doan nhanh vi khul'n lao da khang thudc, dac biet ddi vdi RIF va INH. Chijng tdi ufng dung ky thuat khuech dai gene da mdi dac hieu alen (multiplex allele-specific PCR), la td hgp cua ky thuat nhan gene da mdi (multiplex PCR) va khuech dai dac hieu alen (allele specific
amplification) vdi mue tieu phat hien d c dot bien khing rifampicin tai 3 codon 516, 526, 531 cua gene rpoB trong cijng m d t p h i n Ung. Ket q u i cho thi'y chung tdi da thanh cdng khdng chi vdi ky thuat d o n moi m l ngay d ky thuat da mdi cung eho d c ket q u i tuong tU.
Hien nay, de phat hien vi khul'n lao va lao khang da thude theo phUdng p h i p soi, p h i n lap nudi d y v l l l m k h i n g sinh dd can it nhl't tU 1 tuan den 1 t h i n g de biet ket q u i . Do d d , viee dieu trj se cham tre, hieu q u i kem, nguy co l l y nhiem nhUng chung lao k h i n g thude eho cdng d d n g t i n g eao. Vi v l y , viee p h i t trien d e ky thuat chan d o l n nhanh ed y nghia het sUe to Idn khdng chi t r o n g viee theo ddi dieu trj m l edn cd y nghia t r o n g viee khdng che kiem s o l t benh lao phdi. Mae du ky thuat khuech dai gene da mdi d i e hieu alen cung n h u d e ky thuat chan d o l n sinh hgc p h i n tU k h i c khdng the thay the h o l n t o i n ky thuat nudi e l y nhUng ky thuat n i y ed the sU durig de sang Ige, x l e djnh nhanh ddi vdi vi khul'n lao ndi chung va vi khul'n lao k h i n g thude ndi rieng. Ben eanh do, MAS-PCR I I t d hgp eua d e xet nghiem chan doan lao d o n khang thudc rieng biet do dd se giup ngUdi lam xet nghiem rut n g i n dugc thdi gian v l thao t i e khi l l m ehl'n d o l n cung nhu l l m g i i m ehi p h i xet nghiem eho benh n h i n . 5. Ket luan
B i ng ky thuat PCR da mdi d i e hieu alen (MAS- PCR) ehung tdi d l x l c djnh dUgc 3 mau khang thude tai 2 vj tri k h i c nhau d d la vj t r i 526 (2 mau) v l 531 (1 mau). Ket q u i g i l i trinh tU doan gene 81 nucleotide eua gene rpoB da khang d j n h ket q u i x l c djnh dot bien k h i n g RIF bang ky thuat khuech dai d i e hieu alen (allele specific amplification) la hoan t o i n chinh xle. Nhu v l y , MAS-PCR la phUong p h i p ehl'n doan nhanh, it tdn kem nen dugc dUa v i o Ung d u n g t r o n g thUe h l n h lam sang.
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