Luận văn nghiên cứu phát triển và hoàn thiện quy trình PCR đa mồi để phát hiện trực tiếp streptococcus suis từ dịch não tủy của người

I HC QUC GIA H NI TRNG I HC KHOA HC T NHIấN - ng Th Kiu Oanh NGHIấN CU PHT TRIN V HON THIN QUY TRèNH PCR A MI PHT HIN TRC TIP Streptococcus suis T DCH NO TY NGI LUN VN THC S KHOA HC H Ni 2013 I HC QUC GIA H NI TRNG I HC KHOA HC T NHIấN - ng Th Kiu Oanh NGHIấN CU PHT TRIN V HON THIN QUY TRèNH PCR A MI PHT HIN TRC TIP Streptococcus suis T DCH NO TY NGI Chuyờn ngnh: Vi sinh vt hc Mó s: 60 42 40 LUN VN THC S KHOA HC NGI HNG DN KHOA HC: PGS.TS PHAN Lấ THANH HNG H Ni - 2013 Lun thc s hc Vi sinh vt LI CM N hon thnh lun ny, tụi xin by t lũng bit n sõu sc ti PGS.TS Phan Lờ Thanh Hng, Trng Khoa Vi Khun, Trng Phũng Vi khun hụ hp, Vin V sinh dch t Trung ng ó tn tỡnh hng dn, ng viờn v to mi iu kin tụi hon thnh ti Tụi cng xin by t lũng cm n sõu sc ti TS Nguyn Thanh Thy Trng Khoa An ton sinh hc - Qun lý cht lng, Vin V sinh dch t Trung ng v th Khoa ó to mi iu kin v thi gian cho tụi thc hin ti Tụi xin cm n th Phũng Vi khun hụ hp, Vin V sinh dch t Trung ng hc ó ch bo, giỳp , chia s tn tỡnh cho tụi nhng kinh nghim chuyờn mụn quỏ trỡnh lm thc nghim Tụi xin gi li cm n chõn thnh n cỏc thy, cỏc cụ Khoa sinh hc, c bit l cỏc thy cụ B mụn Vi sinh vt hc, trng i hc Khoa hc T nhiờn, i hc Quc gia H Ni ó to iu kin cho tụi quỏ trỡnh hc Tụi xin cm n gia ỡnh v bn bố luụn luụn ng viờn tinh thn tụi hon thnh lun ny H Ni, ngy thỏng nm 2013 Hc viờn ng Th Kiu Oanh ng Th Kiu Oanh Cao hc K18 Lun thc s hc Vi sinh vt MC LC Capsular polysaccharide Deoxyribonucleic acid Polymerase chain reaction T VN 1.1 GII THIU V Streptococcus suis.suis 1.1.1 Gii thiu chung 1.1.2 Mt s yu t c lc chớnh ca vi khun liờn quan n chn oỏn 1.1.3 C ch gõy bnh ca S.suis 1.2 S LY NHIM TRấN NGI CA S.suis .15 1.3 BNH V TRIU CHNG 19 1.3.1 ng lõy truyn 19 1.3.2 Triu chng 20 1.3.3 Bin phỏp phũng bnh 21 1.3.4 Bin phỏp chng dch 22 1.3.5 Nguyờn tc iu tr .22 1.4 CC PHNG PHP CHN ON PHềNG TH NGHIM 23 1.4.1 Nuụi cy phõn lp 23 1.4.2 Nhum Gram 23 1.4.3 Phn ng catalase 23 1.4.4 Xột nghim nh danh, nh typ: 23 1.4.5 Phỏt hin vi khun S suis bng phn ng Realtime PCR 24 1.5 PHNG PHP PCR 25 1.5.1 Gii thiu v phng phỏp khuch i gen (polymerase chain reaction PCR) 25 1.5.2 Nguyờn lý c bn ca k thut PCR 25 1.5.3 Gii thiu v phn ng PCR a mi 30 CHNG VT LIU V PHNG PHP NGHIấN CU 36 2.1 VT LIU NGHIấN CU 36 2.1.1 Chng vi khun s dng nghiờn cu 36 2.1.2 Dch nóo ty nn 36 2.1.3 Dch nóo ty ca bnh nhõn c chn oỏn lõm sng viờm mng nóo cp tớnh S suis ( ỏnh giỏ hiu qu ca quy trỡnh PCR a mi c xõy dng ti): 37 ng Th Kiu Oanh Cao hc K18 Lun thc s hc Vi sinh vt 2.1.4 Sinh phm nghiờn cu: 37 2.1.5 Trang thit b, dng c 39 2.2 PHNG PHP NGHIấN CU: 40 2.2.1 Nghiờn cu to bnh phm mụ phng 40 2.2.2 Nghiờn cu la chn t hp mi i din cho S suis v mt s yu t ch yu liờn quan n c lc ca vi khun 46 2.2.3 Nghiờn cu ti u chu trỡnh nhit 47 2.2.4 Nghiờn cu ti u cỏc thnh phn tham gia phn ng 49 2.2.5 Nghiờn cu mc phỏt hin vi khun S suis v mt s yu t c lc ca vi khun bi quy trỡnh PCR a mi c xõy dng nghiờn cu Tớnh n nh ca PCR a mi 49 2.2.6 ỏnh giỏ tớnh c hiu ca cỏc cp mi (kh nng bt cp chộo) vi nhng vi khun ph bin gõy hi chng lõm sng viờm mng nóo ging S suis 50 2.2.7 Xõy dng quy trỡnh x lý bnh phm tỏch chit DNA ca S suis 50 2.3 NH GI CC GI TR HU CH CA PHNG PHP 52 2.3.1 Tiờu chun xỏc nh chn oỏn dng tớnh v õm tớnh .52 2.3.2 Cỏc ch s tớnh toỏn tin cy v giỏ tr ca phng phỏp [3, 4] 52 CHNG KT QU V THO LUN 55 3.1 XY DNG QUY TRèNH PCR A MI PHT HIN TRC TIP Streptococcus suis V MT S C LC PH BIN CA VI KHUN .55 3.1.1 To bnh phm mụ phng 55 3.1.2 La chn cỏc cp mi v t hp mi phự hp: 56 3.1.3 Xỏc nh iu kin ti u ca phn ng PCR a mi phỏt hin S suis v mt s yu t c lc ph bin 58 59 59 3.1.4 Ti u húa cỏc thnh phn tham gia phn ng PCR a mi phỏt hin vi khun S suis v mt s yu t c lc ph bin 60 62 62 3.1.5 nhy v tớnh n nh ca quy trỡnh PCR c xõy dng (kh nng lp li kt qu) 64 lp li ca k thut 64 (10 ln th nghim) .64 ng Th Kiu Oanh Cao hc K18 Lun thc s hc Vi sinh vt .65 0/10 65 3.1.6 ỏnh giỏ kh nng bt cp chộo ca cỏc cp mi (tớnh c hiu) vi nhng vi khun ph bin gõy bnh cnh lõm sng ging S suis 65 3.2 XY DNG QUY TRèNH X Lí BNH PHM N GIN, D THC HIN BC L DNA CA VI KHUN Streptococcus suis T HIU QU (NH GI BNG KT QU PHT HIN CA PCR A MI) 66 3.3 NH GI CC GI TR HU CH CA PHNG PHP PCR A MI KHI P DNG VI BNH PHM LM SNG 70 3.3.1 Kt qu nuụi cy phõn lp/xỏc nh c vi khun t bnh phm: 71 3.3.2 Cỏc chi s ỏnh giỏ chớnh xỏc v giỏ tr hu ớch ca cỏc phng phỏp 71 KT LUN 75 TI LIU THAM KHO 78 ng Th Kiu Oanh Cao hc K18 Lun thc s hc Vi sinh vt DANH MC HèNH Hỡnh 1.1: Vi khun liờn cu lnliờn cu khun ln qua kớnh hin vi in t (http://genome.jgi-psf.org/strsu/strsu.home.html) Hỡnh 1.2: Khun lc S suis trờn mụi trng thch mỏu cu v thch mỏu nga Hỡnh 1.3 Bnh nhõn b nhim S suis (http://www.pig333.com/what_the_experts_say/streptococcus-suiszoonotic-epidemic-in-asia_4091/) 21 Hỡnh 1.4 S phn ng PCR 27 (http://fmel.ifas.ufl.edu/buzz/csPCR.shtml) 27 Hỡnh 3.1 Th nghim cỏc t hp mi phự hp (t t hp n t hp 8) 56 Hỡnh 3.2.Th nghim cỏc t hp mi phự hp (t t hp n t hp 16) 57 Hỡnh 3.3 PCR a mi vi cỏc iu kin phn ng ti u phỏt hin trc tip S suis bnh phm 60 Hỡnh 3.4 Th nghim hm lng cỏc mi vi nng mi mi l 20pmol/àl 61 Hỡnh 3.5 Th nghim hm lng mi vi nng mi khỏc 62 Hỡnh 3.6 Th nghim Quy trỡnh PCR ó xõy dng trờn mu bnh phm 62 vi cỏc th tớch mu khỏc 63 Hỡnh 3.7 Mc phỏt hin vi khun S suis v yu t c lc ca vi khun bi PCR a mi ó hon thin (kt qu vi bnh phm mụ phng) 64 Hỡnh 3.8 Kim tra tớnh c hiu ca t hp gen mimi & quy trỡnh PCR a mi 65 Hỡnh 3.9 X lý mu bnh phm dch nóo ty nhit 850C v 900C ng Th Kiu Oanh Cao hc K18 Lun thc s hc Vi sinh vt 60 phỳt 67 Hỡnh 3.10 Hiu qu x lý dch nóo ty nhit 800C vi thi gian khỏc 68 Hỡnh 3.11 Hiu qu x lý dch nóo ty bng Kit v bng nhit 800C/60 69 Hỡnh 3.12 Hiu qu x lý dch nóo ty ca bnh nhõn 800C/60(ỏnh giỏ bi PCR n mi) 69 Hỡnh 3.13 ng dng quy trỡnh PCR a mi ó c xõy dng trờn mu bnh phm 70 ng Th Kiu Oanh Cao hc K18 Lun thc s hc Vi sinh vt DANH MC BNG Bng 1.1 H gen ca mt s chng Sreptococcus suis .4 Bng 2.1 Mt s trỡnh t mi ch yu (primer) c s dng nghiờn cu [11,25,39,41] .39 Bng 2.2 Cỏc t hp mi c s dng th nghim .46 Bng Cỏc chu trỡnh PCR a mi c th nghim 47 Bng 2.3 Bng s liu c bn tớnh toỏn cỏc ch s 53 Bng 2.4 Mc LR+, LR - liờn quan n kh nng mc bnh v khụng mc bnh 53 Bng 3.1 Kt qu khng nh li c tớnh ca vi khun dựng lm mu 55 Bng 3.2 Cỏc t hp mi thớch hp c la chn thớ nghim 57 Bng 3.3 nh hng ca cỏc chu trỡnh PCR n kh nng phỏt hin S.suis .58 Bng 3.4 Kh nng phỏt hin vi khun v cỏc yu t c lc thay i iu kin quy trỡnh PCR a mi 59 Bng 3.5 Thnh phn tham gia phn ng PCR a mi c kho sỏt 60 Bng 3.6 Tớnh n nh ca Quy trỡnh PCR thc hin trờn mu bnh phm .64 Bng 3.7 nh hng ca cỏc phng phỏp x lý bnh phm dựng/khụng dựng kit 66 Bng 3.8 nhy v c hiu ca phng phỏp PCR a mi so vi phng phỏp nuụi cy phõn lp (chun vng) vi n = 144 71 Bng 3.9 So sỏnh kt qu ba hai phng phỏp: PCR n mi, a mi v nuụi cy phõn lpNCPL 72 Bng 3.10 Cỏc ch s ỏnh giỏ chớnh xỏc v s hu ớch ca phng phỏp PCR a mi v nuụi cy phõn lp (n=153) 72 ng Th Kiu Oanh Cao hc K18 Lun thc s hc ng Th Kiu Oanh Vi sinh vt Cao hc K18 Lun thc s hc Vi sinh vt giỏ bi PCR n mi) M: thang chun DNA; (+): chng dng; (-): chng õm; Sp1-Sp5: mu bnh phm lõm sng (DNT) c x lý 800C/60 c mu bnh phm, vch DNA v trớ kớch thc 498 bp ca cp mi c hiu cho typ ca vi khun S.suis u xut hin rt rừ nột Nh vy phng phỏp x lý mu bnh phm 800C 60 phỳt thớch hp x lý mu bnh phm dch nóo ty bc l DNA ca vi khun S.suis M (+) 10 11 12 13 14 15 16 17 18 Hỡnh 3.13 19 20 21 22 23 24 25 26 27 M 28 29 30 31 32 33 34 35 101 (-) ng dng quy trỡnh PCR a mi ó c xõy dng trờn mu bnh phm M: thang chun DNA; 1, 2, 35: 35 mu bnh phm (+): chng dng; (-): chng õm 101: mu bnh phm mụ phng vi nng 10 vk/p Hỡnh 3.13 cho thy kt qu chy PCR a mi trờn 35 mu bnh phm, ú cú cỏc mu bnh phm 16, 17, 24, 25, 26, 31,32 khụng thy xut hin sn phm ca PCR a mi Trong s cỏc mu cũn li ch yu hin hoc vch sn phm, ch cú mu 8, 23, 27 v 29 l cú vch sn phm khng nh li kt qu phõn tớch, i vi nhng mu ny cú th kim tra li bng phng phỏp PCR n mi 3.3 NH GI CC GI TR HU CH CA PHNG PHP PCR A MI KHI P DNG VI BNH PHM LM SNG ng Th Kiu Oanh 70 Cao hc K18 Lun thc s hc Vi sinh vt 3.3.1 Kt qu nuụi cy phõn lp/xỏc nh c vi khun t bnh phm: S bnh phm ca bnh nhõn c chn oỏn VMN m nghi S suis: 53 S bnh phm nuụi cy phõn lp xỏc nh l S suis : 44 mu (44/53 trng hp nhim S suis) S bnh phm xỏc nh l S suis bng PCR a mi: 53 mu (53/53 trng hp nhim S suis ) 3.3.2 Cỏc chi s ỏnh giỏ chớnh xỏc v giỏ tr hu ớch ca cỏc phng phỏp Trong trng hp tiờu chun xỏc nh viờm mng nóo S suis l phi dng tớnh phng phỏp nuụi cy phõn lp (c coi nh chun vng) S mu c phõn tớch bao gm: tng s mu bnh phm c chn oỏn lõm sng VMN m nghi vi khun S suis l 44 mu v s mu õm tớnh thc s (100 mu): n = 44 +100 = 144 Bng 3.8 nhy v c hiu ca phng phỏp PCR a mi so vi phng phỏp nuụi cy phõn lp (chun vng) vi n = 144 Kt qu chn oỏn S suis bng nuụi cy phõn lp Dng tớnh m tớnh Phng phỏp Dng tớnh a mi PCR (phng phỏp th m tớnh nghim) Tng Ch s ỏnh giỏ chớnh xỏc Tng 44 144 100 100 44 100 144 Phng phỏp nuụi cy nhy c hiu 100% 100% Phng phỏp PCR a mi 100% 100% Kt qu Bng 3.8 cho thy, phng phỏp PCR a mi phỏt hin liờn cu lnliờn cu khun ln so vi nuụi cy phõn lp cú nhy t 100% v c hiu t 100% Trong trng hp ny tiờu chun xỏc nh viờm mng nóo S suis l dng ng Th Kiu Oanh 71 Cao hc K18 Lun thc s hc Vi sinh vt tớnh vi hai phng phỏp l nuụi cy phõn lp hoc PCR a mi S mu c phõn tớch bao gm: tng s mu bnh phm c chn oỏn lõm sng VMN m nghi vi khun S suis l 53 mu v s mu õm tớnh thc s (100 mu): n = 53 +100 = 153 Bng 3.9 So sỏnh kt qu ba hai phng phỏp: PCR n mi, a mi v nuụi cy phõn lpNCPL K thut chn oỏn Kt qu phỏt hin vi khun S suis tng s mu bnh phm nghi ng (n = 53) Dng tớnh (+) m tớnh (-) (%) (%) Nuụi cy phõn lp PCR a mi PCR a mi Bnh nhõn c chn oỏn lõm sng VMN nghi S suis 44 (83,02%) (16,98%) 53 53 (100%) 53 (100%) (0%) (0%) 53 53 Bng 3.9 cho thy kt qu phỏt hin ca PCR n mi tng t PCR a mi (cú cp mi khỏc nhau) v nhiu hn phng phỏp NCPL l 16,98%, núi cỏch khỏc t l õm tớnh gi ca NCPL l 16,98% Bng 3.10 Cỏc ch s ỏnh giỏ chớnh xỏc v s hu ớch ca phng phỏp PCR a mi v nuụi cy phõn lp (n=153) Kt qu chn oỏn S suis Tng Dng tớnh m tớnh Phng phỏp Dng tớnh 53 53 a mi PCR m tớnh 100 100 Tng 53 100 153 Phng phỏp nuụi Dng tớnh 44 44 cy phõn lp m tớnh 100 109 Tng 53 100 153 So sỏnh cỏc giỏ tr ca hai phng phỏp vi tng s mu = 153 Phng phỏp nuụi cy Phng phỏp PCR a mi Ch s ỏnh giỏ chớnh xỏc: nhy 83,02% 100% c hiu 100% 100% T l dng tớnh gi 0 T l õm tớnh gi 16,98% Ch s ỏnh giỏ s hu ớch: ng Th Kiu Oanh 72 Cao hc K18 Lun thc s hc Vi sinh vt LR+ LR- - - 0,17 Giỏ tr d oỏn dng tớnh (PPV) Giỏ tr d oỏn õm tớnh (NPV) 1.00 0.917 1.00 1.00 Kt qu Bng 3.10 cho thy, phng phỏp PCR a mi phỏt hin liờn cu lnliờn cu khun ln cú nhy rt cao vi 100%; ú phng phỏp nuụi cy cú nhy l 83,02% T l õm tớnh gi ca phng phỏp PCR a mi l 0%, ú t l õm tớnh gi ca phng phỏp nuụi cy gn 17% (16,98%) C hai phng phỏp u cú c hiu 100% v t l dng tớnh gi l 0% Nu xột nghim PCR a mi dng tớnh vi vi khun S suis, xỏc xut mc bnh l 100%, ú nu nuụi cy õm tớnh vi S suis, xỏc xut khụng mc bnh S suis l 91,7% Núi cỏch khỏc, giỏ tr d oỏn dng tớnh ca PCR a mi v nuụi cy l 100% v giỏ tr d oỏn õm tớnh ca PCR a mi l 100% v ca nuụi cy l 91,7% Vi iu kin Vit Nam hin nay, cú nhiu lý giỏ tr õm tớnh nuụi cy cú nhiu kh nng l õm tớnh gi (do dựng khỏng sinh trc vo bnh vin, vi khun b khỏng sinh c ch khụng phỏt trin c trờn mụi trng nuụi cy, k nng nuụi cy khụng chun, mụi trng nuụi cy khụng m bo cht lng), nhng bnh phm cha DNA ca vi khun nờn cú kt qu dng tớnh s dng phng phỏp PCR, vúi lý nh vy nờn cỏc mu õm tớnh nuụi cy nhng dng tớnh PCR khụng th coi l cỏc mu õm tớnh thc ( tớnh theo cụng thc) c Cng nh vy, s khụng chớnh xỏc nu ch coi cỏc mu bnh phm phỏt hin c vi khun bng nuụi cy phõn lp l t l dng tớnh thc Chỳng tụi ó tớnh toỏn nhy, c hiu v cỏc giỏ tr hu ớch khỏc ỏp dng PCR a mi trờn 153 mu bnh phm (bao gm 53 mu ca bnh nhõn cú biu hin viờm mng nóo m nghi vi khun S suis v 100 mu DNT ca bnh nhõn viờm nóo vi rut (da trờn lõm sng v xột nghim sinh húa - t bo ca DNT) da trờn nguyờn tc nh sau: - ỏnh giỏ hiu qu phỏt hin ca PCR a mi vi qui nh l dng tớnh kt qu phỏt hin vi khun dng tớnh phng phỏp ó c coi l chun ng Th Kiu Oanh 73 Cao hc K18 Lun thc s hc Vi sinh vt vng (nuụi cy phõn lp), khụng tớnh cỏc trng hp ch dng tớnh PCR a mi (l phng phỏp ang c ỏnh giỏ) (n=144) nhy ca mi phng phỏp xột nghim so sỏnh vi phng phỏp c coi l chun vng l t l dng tớnh thc ca mu bnh phm m xột nghim cn c so sỏnh cú th phỏt hin c Vi c mu l 144, phng phỏp PCR a mi phỏt hin liờn cu lnliờn cu khun ln so vi nuụi cy phõn lp cú nhy t 100% v c hiu t 100% Ch s LR + cho thy t l dng tớnh thc ca c hai phng phỏp nh v nu ó phỏt hin c vi khun hay yu t di truyn ca vi khun thỡ chc chn l dng tớnh, ch s LR - cho thy t l õm tớnh gi ca PCR a mi ỏp dng vi S suis l 0% - So sỏnh hiu qu phỏt hin vi khun ca phng phỏp PCR a mi vi PCR n mi v nuụi cy phõn lp.Trong trng hp ny tiờu chun xỏc nh viờm mng nóo S suis l dng tớnh vi hai phng phỏp: nuụi cy phõn lp hoc PCR (n=153) phn phõn tớch ny, c s thit lp cỏc bng tớnh toỏn l: cỏc mu c coi l dng tớnh cú ớt nht mt hai phng phỏp dng tớnh v cỏc mu õm tớnh c hai phng phỏp u õm tớnh Trong 53 mu bnh phm t 53 bnh nhõn c chn oỏn lõm sng l VMN nghi S suis cú 44 mu nuụi cy dng tớnh, 09 mu nuụi cy õm tớnh Nu phỏt hin bng PCR a mi cú 100% s mu dng tớnh (53/53) Nu tớnh theo quy nh viờm mng nóo xỏc nh S suis l dng tớnh vi hai phng phỏp: nuụi cy phõn lp hoc PCR thỡ nhy ca phng phỏp nuụi cy l 83,02% v nhy ca PCR a mi l 100% (Bng 3.10), tớnh xỏc thc ca PCR a mi c kim chng vi PCR n mi (quy trỡnh chun ca phũng thớ nghim Liờn cu, Vin Quc gia cỏc Bnh nhim trựng, Tokyo, Nht Bn) Trong trng hp ny, c hiu ca phng phỏp PCR a mi so sỏnh vi nuụi cy phõn lp l 100% hay t l õm tớnh gi ca phng phỏp PCR a mi l 0%, ú t l õm tớnh gi phng phỏp nuụi cy xp x 17% (Bng 3.10) Ngoi ra, chỳng ng Th Kiu Oanh 74 Cao hc K18 Lun thc s hc Vi sinh vt tụi s dng mi tham kho t ti liu khoa hc ó cụng b cho k thut PCR a mi th nghim ny nờn t hp mi ó la chn cng ó c kho sỏt tớnh c hiu KT LUN Xõy dng c quy trỡnh PCR a mi phỏt hin trc tip vi khun S suis (liờn cu lnliờn cu khun ln) v mt s yu t c lc c bn ca vi khun bnh phm ngi t cỏc yờu cu v gii hn phỏt hin/ nhy v tớnh c hiu/ c hiu v tớnh lp li kt qu nh sau: - Vi bnh phm mụ phng (m bo ging bnh phm tht bao gm vi khun c thun nht v chun dch nóo ty ca ngi (t bnh nhõn b hi chng nóo cp): + Gii hn phỏt hin t vi khun/1 ng phn ng + Tớnh c hiu: khụng cú hin tng bt cp nhm vi vi khun khỏc loi - Vi bnh phm lõm sng: + nhy: t 100% + c hiu so vi cỏc phng phỏp phỏt hin khỏc (nuụi cy phõn lp, PCR n mi): t 100% - Kh nng lp li kt qu ca PCR a mi: t 100% vi bnh phm mụ phng v bnh phm lõm sng Xõy dng c quy trỡnh x lý mu bnh phm dch nóo ty n gin khụng dựng sinh phm thng mi m t hiu qu phỏt hin bng PCR a mi: - X lý bng nhit 800C/60 phỳt - m bo cht lng (bc l DNA ca vi khun, kh cht c ch) tng ng Th Kiu Oanh 75 Cao hc K18 Lun thc s hc Vi sinh vt ng nh tỏch chit bng b sinh phm ng Th Kiu Oanh 76 Cao hc K18 Lun thc s hc Vi sinh vt KIN NGH Phng phỏp PCR a mi l mt phng phỏp sinh hc phõn t chn oỏn nhanh Streptococcus suis t dch nóo ty ngi Phng phỏp PCR a mi tit kim thi gian, cụng sc cng nh lm gim giỏ thnh xột nghim ca mu bnh phm Vỡ th, nu 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