TCNCYH Phu trwong 74 (3) - 2011
mG DUNG KY THUAT PCR DA M 6 | , DIEN DI POLYACRYLAMIDE, NHUOM BAC D^ PHAT HIEN CAC LOCUT STR TRONG XAC DINH
• • • ,
HUYET
T H 6 N GNguyin Dire Nhy Vien Phdp y Qudc gia
Nghien cwu nham hoan thien ky thu$t PCR da moi, aen di poiyacryiamide, nhudm bac trong xac anh huyit thong.
Doi tuvng va phwang phap: 154 ca thi tw 50 cap nghi nga bo - con, 18 gia dinh cd bo, me, con dwac phan tich ADN tir mau miu, mdng tay, tde, hoac te bao niem mac mi$ng di xac anh quan he huyit thing tai Vien Phap y Quoe gia. Kit qua nghien cwu: ADN tach chiit tw cac miu sinh pham khac nhau bang phwang phap^chelex diu cd ndng do twang doi cao. Mau mau cho ndng do ADN cao nhit, tiep din la mau ti bao niem mac mieng, miu mdng tay va mau tde. Phan ung PCR da mil theo tung bd ba locut STR, aen di gel poiyacryiamide va nhudm bac cho cac bang rd rang, sac net.
Qua phan tich 15 locut STR da xac anh duvc 55 cap bd - con cd quan he huyit thong vai dd chinh xac dat 99,97%
din 99,999% va 13 cap bo - con khdng cd quan he huyit thing. Kit luan: Da hoan thi$n dwac ky thuat, PCR da mdi, aen di gel poiyacryiamide va nhupm bac.
TO khoa: Phan tich ADN, xac djnh huyit thdng
I. DAT V A N O ^
Ci'ng dung eic ky thuat phin tich ADN l i mdt trong nhung nhiem vu vd eiing quan trpng trong cdng tic glim dinh tu phip phuc vu diiu tra hinh su, xic djnh huyit thing phuc vu nhu c i u thye tiin cua x i hdi. Tii- nim 1985, khi lin diu tien Peter Gill v i Alex Jeffreys Cpng dung ky thuit ADN vio ITnh vyc niy, cho din nay da cd rit nhiiu eic nhi khoa hpc trdn thi gidi di siu nghien CCPU v i phit triin nhanh chdng. Trpng glim djnh huyit thong, nhin dang c i thi, ngudi ta su- dung cie locut di truyin ChCpa eic doan ADN ed trinh ty lip lai ngin cdn gpi l i cie loeOt STR (Short tandem repeat) d i phin tich. Cie doan lip lai ngin niy thudng cd chiiu dii tip 2 din 6 nueleotit v i d moi c i t h i cd so lin lip lai khic nhau, do v i y eic locut niy cd tinh da hinh cao ndn rit ed g i i trj de phin bidt c i the. Vi du, locut D7S280 thudng su' dung trong giim djnh huyit thong cd trinh ty lap lai la "GATA". Cie alien khic nhau cua locut niy ed tCr 6 din 15 trinh ty lap Igi "GATA". Thdng thudng, cie doan gen sau khi dugc khuich dgi bing phuang phap PCR da mdi se dugc phit hidn bing phuang phip dien di, nhudm bac hoic g i n huynh quang [2].
Sy lya chpn v i Cpng dung c i e ky thuit phin tich ADN trpng x i c djnh huyit thong hit SCPC c i n thiit, ddi hdi tinh khoa hpc, chinh xic, nhanh chdng.
(y Vidt Nam, bdn canh phuang phip gin huynh quang di$n di mao quan trdn m i y giai trinh ty thi
phuang phip dien di gel poiyacryiamide, nhudhi bac d i phit hien cie locut STR thudng dugc nhiiu phdng xet nghiem lya chpn do chi phi thip han nhiiu, d i ding nhin biit bing mit thudng v i phii hgp vdi dieu kien trang thiit bj. Vi viy, chung tdi thye hien nghien CCPU niy vdi muc tieu: Hoin thien ky thuit PCR da moi, didn di poiyacryiamide v i nhudm bac trong x i c djnh huyit thong.
II. OQl TU'QNG vA PHU'QNG P H A P 1.{>di tu'ang
130 c i t h i , gom 50 cap nghi ngd bd - con, 10 gia dinh cd du bo, me, eon dugc xdt nghiem ADN d i xac djnh quan hd huyit thong bo - eon tai Khoa Y - Sinh hpc, Vien Phip y Qudc gia trong thdi gian tip nim 1/2009 din 3/2011. Trong dd: 90 c i t h i dugc liy mau miu tuai hoic luu miu khd trdn the liy miu chuyen dung, 14 c i t h i liy miu mdng tay, 16 c i the liy miu tde v i 10 c i t h i dugc liy t i bio niem mac mieng.
2. Phu'ang phap
Chuin bj miu: Mdt trong cie loai mau (miu tuai 5 -10 nl, miu khd 0,2 em^giiy thim, chin tde 3 - 5 sgi, mdng tay 0,2 - 0,5g dugc rCpa sach, 2 diu tim bdng phit t i bio nidm mgc midng) cho vio ing eppendorf 1.5ml. Tich chiit ADN tong so: Theo phuang phip dOng chelex ^0% [9].
Bo sung 1ml ddm PBS vio trong ing eppendorf, lie diu 3 - 5 giiy, d i 5 phut.
TCNCYH PhLi trwong 74 (3) - 2011
Ly t i m d nhiet dd phdng 12.000 vdng/phut, 5 phut. Hut bd djch nil, giLP lai can.
(Lip lai buoc 1^2tU'1 din 2 lin di Idm sach miu).
- Bo sung 150 - 200 dung djch chelex 10%, 10 - 15 ml dung djch protein K (lOmg/ml)
Lie manh 3 5 giiy. 0 d nhidt dd 56°C trong vdng 60 phCiL Dun sdi trong 8 phut.
Ly tim 12.000 vdng/phut trong 10 phut. Sau dd hut djch pha tren (100 - 200ml) sang cng eppendprf 1.5ml vd triing v i bao quan d - 20°C. Djch noi dugc SU' dung cho PCR.
Dd tinh sach v i nong dd ADN dugc do 0 0 260/280 nm v i dien di gel agarose 2%.
Phan Cpng PCR: Su' dung kit multiplex PCR gom 9 locut STR theo c i e bd ba (CFS1P0, TPOX, TH01), (D16S359, D7S820, D3S317), (F13A01, FESFPS, vWA) cua hang Promega (My); 6 Ipcut STR dugc thiit k i thep bd ba (D3S1358, D8S1179, D18S51), (D5S818, D21S11, FGA) va Amelogenin cua hing Invitrogen.
Phan Ci'ng PCR dugc tiin hanh vdi t h i tich 25 ml gdm eic thinh phin [6]:
Thanh phin STR buffer 10X
Primer set 10X
Taq DNA polymerase (5unit/ml) ADN khudn (1 - 50ng/ml)
Nude deion B6
Ndng do phan irng IX
IX 0.75 unit 1 lOng/phan Cpng
T h i tich (ml) 2,5ml 2,5ml 0.15ml 2 - 5 m l . sung nude dat tong t h i tich 25ml
2phut, lOehuky 1 phut, 70°C-1.5 Chu trinh nhiet dugc thye hidn diing cho miy PCR (Eppendorf - OCPC) nhu sau: 96°C -
diu [94°C - 1 phCit, 60°C - 1 phut, 70°C -1,5 phut], 20 chu ky tiip theo [90°C - 1 phut, 60°C - phut], 60°C - 30 phut, giti' lanh 4°C [8].
Dien di: Dien di san pham PCR (2,5 - 3 ml) trdn gel poiyacryiamide 7%, nguon dien de on djnh 250V (60 Watt) d 50°C trong 3 gid.
Nhudm ADN bing phuang phip nhudm bac cai tiin cua Halima Benbouza (2006) [4].
Cac bu'ac 1. Co djnh 2. Nhudm 3. Ru-a 4. Phat triin 5. Dipng phan Cpng
Dung djch C2H5OH 100%
CH3COOH AgNOs HCOH 37%
H2O NaOH HCOH 37%
C2H5OH 100%
CH3COOH
Tdng sd 1000 ml 100 ml
5 ml 1,5g 1,5 ml 1000 ml
15 g 2 ml 100 ml
5 ml
Thai gian 5 phCit 6 - 7 phut
1 giiy 3 - 5 phut
1 - 3 phut
Nhiet do 10-12°C 22 -24°C 22 -24°C 22 -24°C 22 -24°C
I. KiT QUA
1. Kit qua tach chiit ADN tip cac miu sinh phim khac nhau
Bang 1. Kit qua do tinh sach va ndng do ADN tach chiit du>ac tu> cac miu sinh pham khac nhau Loai miu (s6 lu'ang)
Miu tuai, khd (5-10 \i\, 0,2 cm^) Mdng tay (0,2 - 0,5g)
T i bio nidm mac midng (2 t i m bdng) Chin tde (3 - 5 sgi)
86 miu 90 28 20 16
Kit qua do OD 260/280nm 1.856±0.0529 1.620±0.0015 1.782±0.0034 1.600±0.0081
Ndng do ADN (ng) 900- 1.100
600 - 800 750 - 900 500 - 750
TCNCYH Phu trwong 74 (3) - 2011
ADN tich chiit tip cie m i u sinh pham khic nhau deu ed dp tinh sach tuang ddi cao. Mau miu cho nong dp ADN cao nhit, tiep den l i miu t i bio niem mac mieng, miu mdng tay va miu chin tde.
Dien di 5 ]i\ DNA sau khi tich chiit, k i t qua d hinh 1.
2 3 4 5 1
Ghi chu:
1. Miu ADN tieu chuin (K256. Promega) 2. Miu ADN tach chiit tw mau khd 3. Miu ADN tach chiit tw mdng tay
4. Miu ADN tach chiit tw que phit ti bdo niem mac midng 5. Mau ADN tach chiit tde
Hinh 1. Kit qua dien di ADN tren gel agarose 2%
a) Khdng cd quan he huyit thong b) Cd quan he huyet thdng Marker Bd Con Me CSF1P0
Marker Bd Con D16S359
D7S820
TPOX
i •
D13S317 15 14 13 12 11 10 9 8 7
Hinh 2 (a, b). Kit qua dien di san phim PCR tai bd ba lociit (D16S359, D7S820, D3S317) v i (CFS1P0, TPOX, TH01)
Ghi chu: (7 -^ 15) v i (5 ^ 11): S6 trinh ty lap lai cua mdi alen tren marker D3S317 v i TH01. Hinh 2a).
Trdn locut D3S317, ngudi b6 mang alen dj hgp tw 8 11, ngudi con mang alen dj hgp tu 9 -10. Nhu viy, con khdng nhin alen nio tip b6, khdng tuin theo qui luit di truyin, khdng ed quan hd huyet thdng; Hinh 2b). Trdn locut TH01. ca ngudi bo, con v i m? mang alen dj hgp tu 7 9. Nhu viy, theo qui luit di truyen ngudi con se nhin mdt alen 7 (hoic 9) tii- bo v i mdt alen 9 (hoic 7) ti. me, cd quan hd huyet thdng.
TCNCYH Phu trwong 74 (3) - 2011
3. Kit qua phan tich cac locut STR xac djnh mdi quan he huyit thdng
San pham PCR da mdi sau khi dugc dien di, nhudm bae se dugc phan tich tren tong so 15 Ipcut STR, tinh xic xuit ngudi cha dya tren cdng thupc Bayes, tan suit alen ngudi Viet Nam va kit hgp chisd PI [3, 7].
Bang 3. Kit qua phan tich xac djnh mdi quan he huyit thdng bd - con Cac cap du^c xet nghiem
Bo - con Bd - con - me Tong so
Cd quan he huyet thong 42
13 55
Khong co quan he huyit thong 8
5 13
IV. BAN LUAN
Kit qua thu miu va tach chiit ADN tir cie miu sinh phim khac nhau
Trong nghien CCPU niy, chung tdi d i thu thip cie miu sinh phim la miu tuai, m i u khd, tde ed chin, t i bio nidm mac mieng, hoac mdng tay cua 154 c i t h i til' 50 cap nghi ngd bd - con, 18 gia dinh qd du bo, me, con. Kit qua tich chiit ADN bing phuang phip chelex tCp cho thiy dp tinh sach v i .^idng dp ADN thu dugc tip cie loai mau ndi tren deu tuang ddi cao, du d i phin tich. Ndng dp ADN thu dugc tCp miu miu cao nhit v i ADN thu dugc tip chin tde cd nong dp thip sp vdi cie Ipai mau cdn lai. Kit qua niy phii hgp vdi k i t qua nghien CLPU eua Lee (2001) [5].
Kit qua nghien CCPU cdn cho thiy, sy da dang trong vide lya chpn eic ngudn mau khic nhau trpng xet nghiem ADN xac djnh quan hd huyit thing v i trpng diiu tra hinh sy. Mpntserrat (2001) cung che ring mau miu l i nguon cung c i p ADN tuydt vdi, tuy nhien vide thu lugng ADN tCr miu t i bio niem mac mieng, chin tde, hay mdng tay rit dan gian, rit phii hgp d cie nghien CCPU ed quy md Idn, sd lugng ADN vin du d i phin tich v i chit lugng tdt [6]. ADN tong sd thu dugc dung lim ADN khudn cua phan Cpng PCR da mdi d i u cho k i t qua tot. Theo Le Dinh Luang (2001), d cung mdt c i t h i kit qua cie bing ADN thu dugc sau phan Cpng PCR tip nguon l i miu, tde v i t i bio nidm mac mieng diu giong nhau, nhu v i y trong ITnh vyc hinh sy hay trong nghien CCPU khoa hpc, ADN thu dugc tCp mdt trpng cie mau sinh pham tren v i tich chiit ADN bing chelex d i u cd gia trj x i c djnh c i the nhu nhau [1]. Tip k i t qua nghidn CCPU, chung tdi da hpin thidn ky thuit tich chiit ADN bing su- dung chelex doi vdi tii-ng logi m i u m i u tuai, khd, t i b i o
nidm mac mieng, chin tde v i mdng tay vdi lugng mau nhd cung du lugng ADN de phin tich, phii hgp vdi giam djnh ADN trong phip y v i diiu tra hinh su.
Kit qua PCR da mdi, dien di ADN tren gel poiyacryiamide va nhupm bac
Thdng thudng, theo tieu chuan cua cie phdng xet nghidm ADN tren t h i gidi hidn nay, ngudi ta su dung tip 9 d i n 16 locut STR de phin tich, xic djnh c i t h i hay quan he huyit thing. So lugng locOt STR cing nhiiu thi dp chinh xac cing cao [2]. Tuy nhien, bd kit multiplex PCR diing cho phuang phap dien di poiyacryiamide, nhudm bac cua Promega hien nay chi cd 9 locut STR theo cie bd ba (CFS1P0, TPOX, TH01), (D16S359, D7S820, D3S317), (F13A01, FESFPS, vWA) [8]. Vi viy, de ting dd ehfnh x i c ehung tdi su- dung them 6 cap mdi cua hang Invitrogen v i thiit k i theo eic bp ba (D3S1358, D8S1179, D18S51), (D5S818, D21S11, FGA), va 1 locut Amelogenin d i xic djnh nhiim sic t h i gidi tinh. K i t qua nghien CCPU da tdi uu h6a chu trinh nhidt cho phan u-ng multiplex PCR nhu- nhau doi vdi 5 bd ba locut STR v i locut Amelogenin. Phan Cpng multiplex PCR theo tii-ng bp ba locut cho phep tiit kiem dugc hda chat, nhien lieu v i thdi gian xet nghiem.
Sau phan Cpng PCR, san pham ADN dugc dien di trdn gel polyacrylamid 7%. Khi dien di, chCing t6i diiu chinh lugng ADN cho phu hgp vdi dd sic net v i dp d i m m i u sau khi nhudm 2,5 - 3ml san phim PCR ddi vdi cac bd 3 locut STR diing trcng dien di d i che k i t qua tdt. Tai mdi locut STR diu cd su- dung marker tuang u-ng de x i c djnh alen eua tipng c i t h i dugc chinh xic. Dien di san pham PCR tren gel polyacrylamid cd kich thude 20 x 40 cm trong thdi gian 3 gid da cho phdp tich biet cie doan ADN cd dd dii ngin khic nhau. Cie y i u to lidn quan
TCNCYH Phu tmxyng 74 (3) -2011 dugc lya chpn va toi uu hda va tang dp phan giai
len hing trim lin giup phan biet rd cac doan ADN chi lech nhau 4 nueleotid.
Trong nghien CCPU nay, chung tdi Cpng dung thanh cdng qui trinh nhupm bac cai tiin cua Halima Benbouza (2006). Hinh 2(a,b) cho thiy tit ca cac bang va thang alen diu tach biet rd rang, sic net de dang phin biet bing mit thudng, Vdi qui trinh nhupm nay chi mat tip 17 20 phut, tiit kiem dugc 40 phut so vdi qui trinh nhupm bac cua Bassam (1991). Ngoai uu diem tiit kiem thdi gian, chung tdi thay phuang phap nhupm bac dugc su' dung cdn tiet kiem dugc nhiiu hda chat nhu NaOH, C2H5OH, CH3COOH, vi dung dich nhupm theo phuang phap nay cdn dugc tai su dung 5 - 6 lin neu so vdi cac qui trinh trude day dung NaaCOa dung 1 lin [3].
Kit qua phan tich tren cac locut STR xac djnh mdi quan he huyit thing
Tong sd 15 locut STR dugc su- dung trong nghien CLPU niy la nhu-ng locut STR cd tinh phan biet ca the cao, dang dugc su- dung pho biin tren hau hit cac phdng xet nghiem ADN phuc vu dieu tra hinh su, xac djnh huyet thong va xay dung tang thu ADN [2]. Sau khi xac djnh dugc alen cua tipng ca the tren moi locut STR. Trong nghien CCPU nay, chung tdi khdng gap trudng hgp nao bo va con chi CO mdt alen khdng trung nhau. Trudng hgp bo va con cd cac alen tren 2 locut khdng triing nhau trd nen se khing dinh khdng cd quan he huyit thong vdi xac xuit 100% [2]. Trudng hgp bo va con cd alen triing nhau tit ca 15/15 locut se tinh xac xuit ngudi cha (probability of paternity), tinh toan dua tren cdng thCpc Bayes va tan suit alen ngudi Viet Nam va kit hgp chi sd PI (paternity index) [3,7].
Kit qua trong sd 50 cap nghi ngd bd - con, cd 42 cap bd con cd quan he huyit thing va trong sd 18 gia dinh cd du bo, me, con, xic dinh dugc 13 cap bo con cd quan he huyit thing. Kit qua phan tich tren tong sd 15/15 locut STR trung nhau, vdi xac xuat ngudi bo dat tdi 99,97% din 99,995%, dat tieu chuin so vdi cac phdng xet nghiem hien dai tren thi gidi. Cd 13 cap khdng cd quan he huyet thong bo con.
V. K^T LUAN
Qua phin tich 154 ca the tii' 50 cap nghi ngd bo con, 18 gia dinh cd bo, me, eon, chung tdi d i hoan
thien dugc ky thuat tach chiit ADN bing phuang phap chelex, tdi uu hda dugc phan ipng PCR da mdi theo cac bp ba locut STR va dien di gel poiyacryiamide, nhupm bac. Nhupm bac theo phuang phap cua Halima Benbouza da tiit kiem dugc thdi gian, hda chit va cho kit qua tdt. Kit qua qua phan tich 15 locut STR da xac djnh dugc 55 cap bd - con cd quan he huyit thong vdi do tin cay tip 99,97% den 99,999% va 13 cap bo - con khdng cd quan he huyit thong.
TAI LIEU THAM KHAQ
1. Le Dinh Lu'ang va cdng sy (2001). Nghien CLPU tinh dac trung ca the va tin sd phan bd cac alen cua ba locut VNTR (D1S80, YNZ22 va ApoB) d Viet Nam bing ky thuat PCR. Tap chi Di truyin hpc va LPng dung. So 4/2001.
2. Butler J.M.(2001), Forensic DNA typing:
Biology and Technology behind STR marker, Academic Press, London.
3. Dragan Trimorac, Moses S.Schanfiled, Damir Trimorac (2000). Application of forensic DNA testing in the legal system. Croatian Medical Journal.41 (1). 32-46.
4. Halima Benbouza, Jean - Marie Jacquemine, Jean Pierre Baudoin, Guy Mergeai (2006).
Optimization of a reliable, fast, cheap and sensitive silver staining method to detect SSR marker in poiyacryiamide gels. Biotechnol. Agron. Soc.
Environ. 10(2), 7 7 - 8 1 .
5. Henry C. Lee, Carll Ladd (2001). Preservation and collection of biological evidence. Croatian Medical Journal, 42 (3). 225 - 228.
6. Montserrat Garcia Closas, et al (2001).
Collection of Genomic DNA from Adults in Epidemiological Studies by Buccal Cytobrush and Mouthwash. Cancer Epidemiology, Biomaker and prevention. Vol 10, June.. 2001.
7. Nguyen Due Nhu, Seung Beom Hong, Nam Ye Kim, Wook Kim, Muyn Soo Han (2008).
Vietnamese Population Data Analysis on 17 STR loci by using AmpFfSTR® Identifiler and Powerplex® 16 Kits. Korean J. of Forensic Sci. Vol.
9, No. 1, June, 2008.
8. Promega Corp (2006). GenePrint™ STR
TCNCYH Phu trwong 74 (3) - 2011
Systems (Silver stain detection). Technical Manual, USA, 7/06.
9. Walsh, P.S., Metzger, D.A. and Higuchi, R.
(1991). Chelex 100 as a medium for simple extrac- tipn of DNA of PCR based typing from forensic material. Bio techniques 10, 506 -13.
Summary
APPLICATION OF MULTIPLEX PCR, POLYACRYLAMIDE ELECTROPHORESIS, SILVER STAIN TECHNIQUE TO DETECT STR LOCI IN PATERNITY TEST
Objectives: To complete techniques of multiplex PCR, poiyacryiamide electrophoresis and silver stained in paternity test. Subjects and methods: 154 individuals from 50 pairs of father - child suspects, 18 families with father, mother, child were analyzed DNA samples from blood, nails, hairs, or buccal swabs in paternity test at the National Institute of Forensic Medicine. Results: DNA extraction from different biological samples have relatively high concentrations. Blood samples for DNA, the highest concentration, followed by the sample of buccal swabs, nail and hair samples. Multiplex PCR for each of the three STR loci, electrophore- sis on poiyacryiamide gel and silver stained for clear and sharp bands. After analyzing on 15 STR loci, we identified 55 pairs of parent - child have blood relationship with the probability of paternity from 99.97% to 99.999%, and 13 pairs have no blood relationship. Conclusion: We have completed techniques of multiplex PCR, poiyacryiamide electrophoresis and silver staining.
Keywords: DNA analysis, paternity test
NGHIEN CU'U CHAN DOAN SQM SOC NHIEM
K H U A N OTRE EM TAI KHOA H 6 I S l / C CAP CU'U BENH VIEN NHI TRUNG U'QNG
Pham Van Thang Trwong Dai hoc Y Hd Noi.
Xac anh mdt so trieu chCmg lam sang va can lam sang giup chin doan sam soc nhiem khuan. Ddi tuvng va phwang phap: md ta tiin cwu 67 b^nh nhi dwac chin doan sdc nhiim khuan (SNK) diiu tri t^i khoa Hdi siJc cap cCni (HSCC), b^nh vi$n Nhi Trung wang tw thang 6 nam 2003 din thang 11 nam 2006. Tieu chuan chin doan SNK theo ACCM/SCCM - 2002. Kit qua cho thay cac trieu chwng lam sang: thay doi y thwc vai PPV 76%, Sn 91%, Sp 73% va p
< 0,001; diu hieu m^ch nhanh vai PPV 68%, Sn 88%, Sp 58% va p < 0,001; trieu chwng bai ni$u it vai PPV 72%, Sn 76%, Sp 70% va p < 0,001; diu hieu refill keo dai vdi PPV 74%, Sn 91%, Sp 66% va p < 0,001; nong dp lactat mau tang vai PPV 62%, Sn 40%, Sp 76% va p < 0,05. Kit lu$n: cd thi dwa vao cac trieu chwng thay doi y thCec, m^cti nhanh, bai nieu it, rfill keo dai va lactate tang di chin doan sdc nhiem khuin tre em.
Tir khoa: sdc nhiim khuan tre em, chin doan so-m sdc nhiim khuin
I. DAT
VAN D ^Sdc nhiem khuan (SNK) l i hdi chu-ng lim sing ning thudng gap tai khoa Hdi SCPC cap CCPU. Thong kd 5 nim tai khoa HSCC benh vien Nhi Trung l/ang SNK chiim 62,5%), ty Id tCp vong cao 85,1%
[1]. Nim 2002, Hidp hdi Hoi su-c Hoa Ky (ACCM) dua ra chiin luge chin doin v i diiu trj sdm theo mye tidu, da cai thidn ty Id tu- vong do SNK tii- 97%) (1960) cdn 9 -13%) trong 3 nim qua [2]. Tai Vidt Nam,
d i cd mdt so nghien CCKU ve SNK, tuy nhien chua cd cdng trinh nghien cu-u ve chin doin sdm SNK d tre em. C^ng trinh niy nhim muc tieu: Nghi§n
CCPU mdt sd trieu chu-ng lim sang v i cin lim sing giup cho chin doin sdm SNK tai khoa Hoi sCfC cip cu-u bdnh vidn Nhi Trung LTang.
II.
D 6 ITU'QNG VA PHU'QNG
PHAP I.Odi tu'angGom 67 bdnh nhin (BN) dugc chin doin SNK vao
