{"id":49,"date":"2022-09-20T04:59:02","date_gmt":"2022-09-20T04:59:02","guid":{"rendered":"https:\/\/bioamipharm.com\/?page_id=49"},"modified":"2022-12-07T10:08:41","modified_gmt":"2022-12-07T10:08:41","slug":"nguon-goc-ky-thuat","status":"publish","type":"page","link":"https:\/\/bioamipharm.com\/index.php\/nguon-goc-ky-thuat\/","title":{"rendered":"Ngu\u1ed3n G\u1ed1c K\u1ef9 Thu\u1eadt"},"content":{"rendered":"\t\t
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Ngu\u1ed3n G\u1ed1c K\u1ef9 Thu\u1eadt<\/h2>\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t\t<\/div>\n\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t\t<\/div>\n\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t
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Real-time PCR l\u00e0 g\u00ec?<\/h1>

\u00a0<\/strong><\/p>

Trong k\u1ef9 thu\u1eadt PCR, sau khi ho\u00e0n t\u1ea5t khu\u1ebfch \u0111\u1ea1i \u0111o\u1ea1n DNA \u0111\u00edch, ng\u01b0\u1eddi l\u00e0m th\u00ed nghi\u1ec7m ph\u1ea3i ti\u1ebfp t\u1ee5c l\u00e0m m\u1ed9t s\u1ed1 b\u01b0\u1edbc th\u00ed nghi\u1ec7m \u0111\u1ec3 \u0111\u1ecdc k\u1ebft qu\u1ea3 x\u00e1c \u0111\u1ecbnh c\u00f3 s\u1ea3n ph\u1ea9m khu\u1ebfch \u0111\u1ea1i mong mu\u1ed1n trong \u1ed1ng ph\u1ea3n \u1ee9ng hay kh\u00f4ng, v\u00e0 giai \u0111o\u1ea1n n\u00e0y g\u1ecdi l\u00e0 giai \u0111o\u1ea1n th\u00ed nghi\u1ec7m sau PCR. Trong giai \u0111o\u1ea1n n\u00e0y, ng\u01b0\u1eddi l\u00e0m th\u00ed nghi\u1ec7m c\u00f3 th\u1ec3 th\u1ef1c hi\u1ec7n \u0111i\u1ec7n di s\u1ea3n ph\u1ea9m PCR tr\u00ean gel agarose \u0111\u1ec3 xem c\u00f3 v\u1ea1ch s\u1ea3n ph\u1ea9m khu\u1ebfch \u0111\u1ea1i \u0111\u00fang k\u00edch th\u01b0\u1edbc mong mu\u1ed1n hay kh\u00f4ng, c\u0169ng c\u00f3 th\u1ec3 th\u1ef1c hi\u1ec7n th\u00ed nghi\u1ec7m lai v\u1edbi c\u00e1c \u0111o\u1ea1n d\u00f2 \u0111\u1eb7c hi\u1ec7u (tr\u00ean m\u00e0ng, tr\u00ean gi\u1ebfng hay phi\u1ebfn nh\u1ef1a…) \u0111\u1ec3 xem s\u1ea3n ph\u1ea9m khu\u1ebfch \u0111\u1ea1i c\u00f3 tr\u00ecnh t\u1ef1 mong mu\u1ed1n hay kh\u00f4ng. K\u1ef9 thu\u1eadt PCR m\u00e0 c\u1ea7n ph\u1ea3i c\u00f3 giai \u0111o\u1ea1n th\u00ed nghi\u1ec7m \u0111\u1ec3 \u0111\u1ecdc v\u00e0 ph\u00e2n t\u00edch sau khi ho\u00e0n t\u1ea5t ph\u1ea3n \u1ee9ng khu\u1ebfch \u0111\u1ea1i, ng\u00e0y h\u00f4m nay \u0111\u01b0\u1ee3c g\u1ecdi l\u00e0 PCR c\u1ed5 \u0111i\u1ec3n (classical PCR).<\/p>

Real-time PCR l\u00e0 k\u1ef9 thu\u1eadt PCR m\u00e0 k\u1ebft qu\u1ea3 khu\u1ebfch \u0111\u1ea1i DNA \u0111\u00edch hi\u1ec3n th\u1ecb \u0111\u01b0\u1ee3c ngay sau m\u1ed7i chu k\u1ef3 nhi\u1ec7t c\u1ee7a ph\u1ea3n \u1ee9ng, ch\u00ednh v\u00ec v\u1eady n\u00ean \u0111\u01b0\u1ee3c g\u1ecdi l\u00e0 real-time; v\u00e0 do \u0111\u1eb7c \u0111i\u1ec3m n\u00e0y n\u00ean v\u1edbi real-time PCR ng\u01b0\u1eddi l\u00e0m th\u00ed nghi\u1ec7m kh\u00f4ng c\u1ea7n thi\u1ebft ph\u1ea3i l\u00e0m ti\u1ebfp c\u00e1c th\u00ed nghi\u1ec7m \u0111\u1ec3 \u0111\u1ecdc v\u00e0 ph\u00e2n t\u00edch k\u1ebft qu\u1ea3 \u0111\u1ec3 x\u00e1c \u0111\u1ecbnh c\u00f3 s\u1ea3n ph\u1ea9m khu\u1ebfch \u0111\u1ea1i \u0111\u00edch hay kh\u00f4ng v\u00ec k\u1ebft qu\u1ea3 cu\u1ed1i c\u00f9ng c\u1ee7a ph\u1ea3n \u1ee9ng khu\u1ebfch \u0111\u1ea1i c\u0169ng \u0111\u01b0\u1ee3c hi\u1ec3n th\u1ecb ngay sau khi ho\u00e0n t\u1ea5t ph\u1ea3n \u1ee9ng khu\u1ebfch \u0111\u1ea1i. Nh\u01b0 v\u1eady, n\u00ean c\u00f3 th\u1ec3 n\u00f3i real-time PCR l\u00e0 k\u1ef9 thu\u1eadt nh\u00e2n b\u1ea3n DNA \u0111\u00edch trong \u1ed1ng nghi\u1ec7m th\u00e0nh h\u00e0ng t\u1ef7 b\u1ea3n sao d\u1ef1a v\u00e0o c\u00e1c chu k\u1ef3 nhi\u1ec7t v\u00e0 k\u1ebft qu\u1ea3 khu\u1ebfch \u0111\u1ea1i trong \u1ed1ng ph\u1ea3n \u1ee9ng \u0111\u01b0\u1ee3c hi\u1ec3n th\u1ecb c\u00f9ng l\u00fac v\u1edbi ph\u1ea3n \u1ee9ng khu\u1ebfch \u0111\u1ea1i x\u1ea3y ra \u0111\u1ec3 ng\u01b0\u1eddi l\u00e0m th\u00ed nghi\u1ec7m c\u00f3 th\u1ec3 th\u1ea5y \u0111\u01b0\u1ee3c.<\/p><\/div>\n\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

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C\u00e1c v\u1ea5n \u0111\u1ec1 k\u1ef9 thu\u1eadt c\u01a1 b\u1ea3n c\u1ea7n bi\u1ebft c\u1ee7a real-time PCR<\/h1>

\u00a0<\/strong><\/p>

1.\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Bi\u1ec3u \u0111\u1ed3 khu\u1ebfch \u0111\u1ea1i c\u1ee7a real-time PCR<\/u><\/h2>

\u00a0<\/em><\/strong><\/p>

Trong real-time PCR, hi\u1ec3n th\u1ecb c\u01a1 b\u1ea3n \u0111\u1ec3 ng\u01b0\u1eddi l\u00e0m th\u00ed nghi\u1ec7m c\u00f3 th\u1ec3 quan s\u00e1t \u0111\u01b0\u1ee3c trong qu\u00e1 tr\u00ecnh nh\u00e2n b\u1ea3n DNA c\u1ee7a c\u00e1c \u1ed1ng ph\u1ea3n \u1ee9ng l\u00e0 m\u1ed9t bi\u1ec3u \u0111\u1ed3 khu\u1ebfch \u0111\u1ea1i (amplification graph). Bi\u1ec3u \u0111\u1ed3 n\u00e0y c\u00f3 tr\u1ee5c tung (Y) l\u00e0 c\u01b0\u1eddng \u0111\u1ed9 hu\u1ef3nh quang ph\u00e1t ra t\u1eeb c\u00e1c \u1ed1ng ph\u1ea3n \u1ee9ng khi nh\u1eadn \u00e1nh s\u00e1ng k\u00edch th\u00edch, c\u00f2n tr\u1ee5c ho\u00e0nh (X) l\u00e0 c\u00e1c chu k\u1ef3 nhi\u1ec7t. Tr\u00ean bi\u1ec3u \u0111\u1ed3 khu\u1ebfch \u0111\u1ea1i n\u00e0y (bi\u1ec3u \u0111\u1ed3 1<\/em><\/strong>), ng\u01b0\u1eddi l\u00e0m th\u00ed nghi\u1ec7m s\u1ebd th\u1ea5y \u0111\u1ed1i v\u1edbi t\u1eebng<\/p>

\u1ed1ng ph\u1ea3n \u1ee9ng, c\u01b0\u1eddng \u0111\u1ed9 hu\u1ef3nh quang m\u00e0 m\u00e1y ghi nh\u1eadn \u0111\u01b0\u1ee3c trong nh\u1eefng chu k\u1ef3 \u0111\u1ea7u s\u1ebd r\u1ea5t th\u1ea5p v\u00e0 h\u1ea7u nh\u01b0 kh\u00f4ng thay \u0111\u1ed5i, hi\u1ec3n th\u1ecb b\u1eb1ng m\u1ed9t \u0111\u01b0\u1eddng th\u1eb3ng n\u1eb1m ngang, ch\u00fang ta c\u00f3 th\u1ec3 g\u1ecdi \u0111\u00e2y l\u00e0 \u201cgiai \u0111o\u1ea1n \u1ee7\u201d hay \u201cgiai \u0111o\u1ea1n ti\u1ec1m ph\u1ee5c\u201d, v\u00ec trong giai \u0111o\u1ea1n n\u00e0y d\u00f9 DNA \u0111\u00edch \u0111\u00e3 c\u00f3 th\u1ec3 \u0111\u01b0\u1ee3c nh\u00e2n b\u1ea3n th\u00e0nh c\u00e1c b\u1ea3n sao nh\u01b0ng do s\u1ed1 l\u01b0\u1ee3ng ch\u01b0a \u0111\u1ee7 \u0111\u1ec3 gi\u00fap cho ch\u1ea5t ph\u00e1t hu\u1ef3nh quang nh\u1eadn \u0111\u01b0\u1ee3c \u00e1nh s\u00e1ng k\u00edch th\u00edch ph\u00e1t ra \u00e1nh s\u00e1ng hu\u1ef3nh quang \u0111\u1ee7 c\u01b0\u1eddng \u0111\u1ed9 \u0111\u1ec3 m\u00e1y ghi nh\u1eadn. Nh\u01b0ng m\u1ed9t khi s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n sao c\u1ee7a DNA \u0111\u00edch \u0111\u1ea1t \u0111\u1ebfn m\u1ed9t ng\u01b0\u1ee1ng nh\u1ea5t \u0111\u1ecbnh th\u00ec \u00e1nh s\u00e1ng hu\u1ef3nh quang ph\u00e1t ra s\u1ebd \u0111\u1ee7 c\u01b0\u1eddng \u0111\u1ed9\u00a0 \u0111\u1ec3 \u0111\u01b0\u1ee3c m\u00e1y ghi nh\u1eadn v\u00e0 l\u00fac n\u00e0y ch\u00fang ta s\u1ebd th\u1ea5y \u0111\u01b0\u1eddng bi\u1ec3u di\u1ec5n khu\u1ebfch \u0111\u1ea1i b\u1eaft \u0111\u1ea7u ng\u00f3c l\u00ean. C\u01b0\u1eddng \u0111\u1ed9 hu\u1ef3nh quang trong \u1ed1ng ph\u1ea3n \u1ee9ng t\u1eeb l\u00fac n\u00e0y tr\u1edf \u0111i s\u1ebd t\u0103ng g\u1ea5p \u0111\u00f4i sau m\u1ed7i chu k\u1ef3 nhi\u1ec7t do s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n sao c\u1ee7a DNA \u0111\u00edch t\u0103ng g\u1ea5p \u0111\u00f4i sau m\u1ed7i chu k\u1ef3. Ch\u00fang ta g\u1ecdi giai \u0111o\u1ea1n n\u00e0y l\u00e0 \u201cgiai \u0111o\u1ea1n l\u0169y th\u1eeba\u201d v\u1ec1 c\u01b0\u1eddng \u0111\u1ed9 hu\u1ef3nh quang, kh\u00f4ng ph\u1ea3i l\u00e0 giai \u0111o\u1ea1n t\u0103ng tr\u01b0\u1edfng l\u0169y th\u1eeba v\u1ec1 s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n sao c\u1ee7a DNA \u0111\u00edch. C\u01b0\u1eddng \u0111\u1ed9 hu\u1ef3nh quang trong \u1ed1ng ph\u1ea3n \u1ee9ng s\u1ebd t\u0103ng tr\u01b0\u1edfng \u0111\u1ebfn m\u1ed9t m\u1ee9c n\u00e0o \u0111\u00f3 th\u00ec \u0111\u1ed9 t\u0103ng tr\u01b0\u1edfng s\u1ebd ch\u1eadm d\u1ea7n v\u00e0 \u0111\u1ea1t \u0111\u1ebfn b\u00ecnh nguy\u00ean v\u00ec c\u00e1c b\u1ea3n sao c\u1ee7a DNA \u0111\u00edch, do ph\u1ea3n \u1ee9ng \u0111\u00e3 c\u1ea1n d\u1ea7n dNTP v\u00e0 enzyme Taq polymerase <\/em>kh\u00f4ng ho\u1ea1t \u0111\u1ed9ng hi\u1ec7u qu\u1ea3 n\u1eefa, n\u00ean s\u1ebd kh\u00f4ng c\u00f2n gia t\u0103ng s\u1ed1 l\u01b0\u1ee3ng theo c\u1ea5p s\u1ed1 2 n\u1eefa. Ch\u00fang ta g\u1ecdi giai \u0111o\u1ea1n n\u00e0y l\u00e0 \u201cgiai \u0111o\u1ea1n b\u00ecnh nguy\u00ean\u201d.<\/p>

Ph\u00e2n t\u00edch m\u1ed9t \u0111\u01b0\u1eddng bi\u1ec3u di\u1ec5n khu\u1ebfch \u0111\u1ea1i (amplification curve) c\u1ee7a m\u1ed9t \u1ed1ng ph\u1ea3n \u1ee9ng sau khi ho\u00e0n t\u1ea5t \u0111\u01b0\u1ee3c c\u00e1c chu k\u1ef3 nhi\u1ec7t, ch\u00fang ta s\u1ebd th\u1ea5y m\u1ed9t th\u00f4ng s\u1ed1 h\u1ebft s\u1ee9c quan tr\u1ecdng lu\u00f4n \u0111i k\u00e8m v\u1edbi n\u00f3, \u0111\u00f3 l\u00e0 chu k\u1ef3 ng\u01b0\u1ee1ng (Ct, threshold cycle). Chu k\u1ef3 ng\u01b0\u1ee1ng hay Ct l\u00e0 chu k\u1ef3 nhi\u1ec7t m\u00e0 \u1edf t\u1ea1i th\u1eddi \u0111i\u1ec3m n\u00e0y thi\u1ebft b\u1ecb real-time ghi nh\u1eadn \u0111\u01b0\u1ee3c t\u00edn hi\u1ec7u hu\u1ef3nh quang ph\u00e1t ra t\u1eeb \u1ed1ng ph\u1ea3n \u1ee9ng b\u1eaft \u0111\u1ea7u v\u01b0\u1ee3t qua c\u01b0\u1eddng \u0111\u1ed9 hu\u1ef3nh quang n\u1ec1n. \u0110\u1ec3 c\u00f3 th\u1ec3 x\u00e1c \u0111\u1ecbnh \u0111\u01b0\u1ee3c c\u01b0\u1eddng \u0111\u1ed9 hu\u1ef3nh quang n\u1ec1n, thi\u1ebft b\u1ecb real-time th\u01b0\u1eddng ghi nh\u1eadn c\u01b0\u1eddng \u0111\u1ed9 t\u00edn hi\u1ec7u hu\u1ef3nh quang xu\u1ea5t hi\u1ec7n trong \u1ed1ng ph\u1ea3n \u1ee9ng trong m\u1ed9t s\u1ed1 chu k\u1ef3 \u0111\u1ea7u, ch\u00fang ta g\u1ecdi l\u00e0 c\u00e1c chu k\u1ef3 n\u1ec1n (basal cycle), v\u00e0 l\u1ea5y trung b\u00ecnh c\u1ed9ng c\u1ee7a c\u00e1c c\u01b0\u1eddng \u0111\u1ed9 hu\u1ef3nh quang n\u00e0y l\u00e0m c\u01b0\u1eddng \u0111\u1ed9 hu\u1ef3nh quang n\u1ec1n. \u0110\u01b0\u1eddng c\u1eaft ngang \u0111i qua c\u01b0\u1eddng \u0111\u1ed9 hu\u1ef3nh quang n\u1ec1n n\u00e0y \u0111\u01b0\u1ee3c g\u1ecdi l\u00e0 \u0111\u01b0\u1eddng n\u1ec1n (base line). Chu k\u1ef3 ng\u01b0\u1ee1ng l\u00e0 tr\u1ecb s\u1ed1 \u0111\u01b0\u1ee3c x\u00e1c \u0111\u1ecbnh b\u1eb1ng s\u1ed1 chu k\u1ef3 m\u00e0 \u1edf \u0111\u00f3 \u0111\u01b0\u1eddng n\u1ec1n c\u1eaft \u0111\u01b0\u1ee3c \u0111\u01b0\u1eddng bi\u1ec3n di\u1ec5n khu\u1ebfch \u0111\u1ea1i. Do \u0111\u01b0\u1ee3c t\u00ednh to\u00e1n nh\u01b0 v\u1eady n\u00ean chu k\u1ef3 ng\u01b0\u1ee1ng th\u01b0\u1eddng l\u00e0 m\u1ed9t s\u1ed1 l\u1ebb (v\u00ed d\u1ee5: Ct = 28.35) ch\u1ee9 \u00edt khi l\u00e0 m\u1ed9t s\u1ed1 ch\u1eb5n.<\/p><\/div>\n\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

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C\u00f3 nh\u1eefng \u1ed1ng ph\u1ea3n \u1ee9ng c\u00f3 Ct s\u1edbm v\u00e0 c\u0169ng c\u00f3 nh\u1eefng \u1ed1ng ph\u1ea3n \u1ee9ng c\u00f3 Ct xu\u1ea5t hi\u1ec7n mu\u1ed9n h\u01a1n, v\u00e0 c\u00e2u h\u1ecfi \u0111\u01b0\u1ee3c \u0111\u1eb7t ra l\u00e0 l\u00fd do n\u00e0o \u0111\u00e3 l\u00e0m \u0111\u01b0\u1ee3c nh\u01b0 v\u1eady? C\u00e2u tr\u1ea3 l\u1eddi ch\u00ednh x\u00e1c l\u00e0 do s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n DNA \u0111\u00edch ban \u0111\u1ea7u (starting quantity, Sq) c\u00f3 trong \u1ed1ng ph\u1ea3n \u1ee9ng nhi\u1ec1u hay \u00edt. N\u1ebfu trong \u1ed1ng ph\u1ea3n \u1ee9ng s\u1ed1 l\u01b0\u1ee3ng DNA \u0111\u00edch nhi\u1ec1u th\u00ec s\u1ebd c\u1ea7n \u00edt chu k\u1ef3 nhi\u1ec7t h\u01a1n \u0111\u1ec3 \u0111\u1ea1t \u0111\u1ebfn s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n sao \u0111\u1ee7 \u0111\u1ec3 \u1ed1ng ph\u1ea3n \u1ee9ng cho \u0111\u01b0\u1ee3c t\u00edn hi\u1ec7u hu\u1ef3nh quang m\u00e0 m\u00e1y s\u1ebd ghi nh\u1eadn \u0111\u01b0\u1ee3c, c\u00f2n n\u1ebfu s\u1ed1 l\u01b0\u1ee3ng DNA \u0111\u00edch \u00edt h\u01a1n th\u00ec c\u1ea7n nhi\u1ec1u chu k\u1ef3 nhi\u1ec7t h\u01a1n.<\/p>

2.\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Bi\u1ec3u \u0111\u1ed3 chu\u1ea9n c\u1ee7a real-time PCR<\/u><\/h2>

\u00a0<\/em><\/strong><\/p>

Nh\u01b0 \u0111\u00e3 n\u00f3i Ct c\u1ee7a m\u1ed9t \u1ed1ng ph\u1ea3n \u1ee9ng real-time PCR s\u1edbm hay mu\u1ed9n (Ct th\u1ea5p hay Ct cao) l\u00e0 t\u00f9y thu\u1ed9c v\u00e0o s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n DNA \u0111\u00edch ban \u0111\u1ea7u c\u00f3 trong \u1ed1ng ph\u1ea3n \u1ee9ng. \u0110\u00e2y ch\u00ednh l\u00e0 m\u1ed9t \u0111\u1eb7c \u0111i\u1ec3m v\u01b0\u1ee3t tr\u1ed9i c\u1ee7a real-time PCR so v\u1edbi PCR c\u1ed5 \u0111i\u1ec3n, v\u00ec nh\u1edd d\u1ef1a v\u00e0o \u0111\u1eb7c \u0111i\u1ec3m n\u00e0y m\u00e0 ng\u01b0\u1eddi l\u00e0m th\u00ed nghi\u1ec7m x\u00e1c \u0111\u1ecbnh \u0111\u01b0\u1ee3c s\u1ed1 copies c\u1ee7a t\u00e1c nh\u00e2n \u0111\u00edch c\u00f3 trong m\u1eabu th\u1eed, m\u1ed9t th\u00f4ng s\u1ed1 m\u00e0 PCR c\u1ed5 \u0111i\u1ec3n kh\u00f4ng th\u1ec3 n\u00e0o l\u00e0m \u0111\u1ec3 c\u00f3 \u0111\u01b0\u1ee3c k\u1ebft qu\u1ea3 ch\u00ednh x\u00e1c. Ng\u01b0\u1eddi l\u00e0m th\u00ed nghi\u1ec7m ch\u1ec9 \u0111\u1ecdc k\u1ebft qu\u1ea3 PCR c\u1ed5 \u0111i\u1ec3n sau khi ho\u00e0n t\u1ea5t ph\u1ea3n \u1ee9ng khu\u1ebfch \u0111\u1ea1i, v\u00e0 k\u1ebft qu\u1ea3 n\u00e0y n\u1ebfu \u0111\u1ecbnh l\u01b0\u1ee3ng \u0111\u01b0\u1ee3c th\u00ec c\u0169ng ch\u1ec9 l\u00e0 k\u1ebft qu\u1ea3 \u0111\u1ecbnh l\u01b0\u1ee3ng<\/p>

s\u1ed1 b\u1ea3n sao c\u1ee7a DNA \u0111\u00edch sau khi ho\u00e0n t\u1ea5t khu\u1ebfch \u0111\u1ea1i. M\u00e0 trong PCR, s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n sao cu\u1ed1i c\u00f9ng kh\u00f4ng ph\u1ea3n \u1ea3nh \u0111\u01b0\u1ee3c m\u1ed9t c\u00e1ch ch\u00ednh x\u00e1c s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n DNA \u0111\u00edch ban \u0111\u1ea7u c\u00f3 trong m\u1eabu th\u1eed v\u00ec trong \u0111a s\u1ed1 c\u00e1c tr\u01b0\u1eddng h\u1ee3p s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n sao c\u00f3 trong<\/p><\/div>\n\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

\n\t\t\t\t
\n\t\t\t\t\t
\n\t\t\t\t\t\t\t\t\t\t\"\"\t\t\t\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
\n\t\t\t\t
\n\t\t\t\t\t

\u1ed1ng ph\u1ea3n \u1ee9ng PCR l\u00e0 s\u1ed1 l\u01b0\u1ee3ng c\u1ef1c \u0111\u1ea1i sau khi PCR \u0111\u1ea1t \u0111\u01b0\u1ee3c giai \u0111o\u1ea1n b\u00ecnh nguy\u00ean.<\/p>

Tuy nhi\u00ean \u0111\u1ec3 real-time PCR x\u00e1c \u0111\u1ecbnh \u0111\u01b0\u1ee3c ch\u00ednh x\u00e1c s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n \u0111\u00edch ban \u0111\u1ea7u c\u00f3 trong m\u1eabu th\u1eed, ng\u01b0\u1eddi l\u00e0m th\u00ed nghi\u1ec7m ph\u1ea3i th\u1ef1c hi\u1ec7n real-time PCR c\u00e1c m\u1eabu th\u1eed ch\u1ee9a c\u00e1c s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n DNA \u0111\u00edch ban \u0111\u1ea7u c\u1ea7n x\u00e1c \u0111\u1ecbnh s\u1ed1 l\u01b0\u1ee3ng c\u00f9ng l\u00fac v\u1edbi c\u00e1c m\u1eabu chu\u1ea9n ch\u1ee9a s\u1ed1 l\u01b0\u1ee3ng DNA \u0111\u00edch ban \u0111\u1ea7u \u0111\u00e3 bi\u1ebft r\u00f5 s\u1ed1 l\u01b0\u1ee3ng, v\u00e0 th\u01b0\u1eddng th\u00ec c\u00e1c m\u1eabu chu\u1ea9n l\u00e0 c\u00e1c m\u1eabu pha lo\u00e3ng theo h\u1ec7 s\u1ed1 pha lo\u00e3ng 10 ch\u1ee9a s\u1ed1 l\u01b0\u1ee3ng DNA \u0111\u00edch ban \u0111\u1ea7u. Sau khi ho\u00e0n t\u1ea5t c\u00e1c chu k\u1ef3 nhi\u1ec7t c\u1ee7a real-time PCR, tr\u00ean bi\u1ec3u \u0111\u1ed3 khu\u1ebfch \u0111\u1ea1i (bi\u1ec3u \u0111\u1ed3 2<\/em><\/strong>), c\u00e1c m\u1eabu chu\u1ea9n v\u00e0 c\u00e1c m\u1eabu th\u1eed s\u1ebd c\u00f3 c\u00e1c \u0111\u01b0\u1eddng bi\u1ec3u di\u1ec5n khu\u1ebfch \u0111\u1ea1i c\u1ee7a n\u00f3 v\u00e0 t\u01b0\u01a1ng \u1ee9ng v\u1edbi c\u00e1c \u0111\u01b0\u1eddng bi\u1ec3u di\u1ec5n<\/p>

khu\u1ebfch \u0111\u1ea1i n\u00e0y, c\u00e1c m\u1eabu chu\u1ea9n v\u00e0 m\u1eabu th\u1eed \u0111\u1ec1u c\u00f3 m\u1ed9t chu k\u1ef3 ng\u01b0\u1ee1ng (Ct).<\/p>

\u0110\u01b0\u1eddng bi\u1ec3u di\u1ec5n v\u1ebd l\u00ean m\u1ed1i quan h\u1ec7 gi\u1eefa chu k\u1ef3 ng\u01b0\u1ee1ng v\u1edbi s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n DNA \u0111\u00edch ban \u0111\u1ea7u c\u00f3 trong c\u00e1c m\u1eabu chu\u1ea9n \u0111\u01b0\u1ee3c g\u1ecdi l\u00e0 \u0111\u01b0\u1eddng\u00a0 bi\u1ec3u\u00a0 di\u1ec5n\u00a0 chu\u1ea9n \u00a0(standard<\/p>

curve).\u00a0 \u0110\u00e2y\u00a0 l\u00e0\u00a0 m\u1ed9t\u00a0 \u0111\u01b0\u1eddng \u00a0th\u1eb3ng<\/p>

tuy\u1ebfn t\u00ednh \u0111i qua c\u00e1c \u0111i\u1ec3m t\u1ecda \u0111\u1ed9 x\u00e1c \u0111\u1ecbnh b\u1edfi s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n DNA \u0111\u00edch ban \u0111\u1ea7u c\u1ee7a t\u1eebng m\u1eabu chu\u1ea9n<\/p><\/div>\n\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

\n\t\t\t\t
\n\t\t\t\t\t
\n\t\t\t\t\t\t\t\t\t\t\"\"\t\t\t\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
\n\t\t\t\t
\n\t\t\t\t\t
<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
\n\t\t\t\t
\n\t\t\t\t\t

Bi\u1ec3u \u0111\u1ed3 3<\/em><\/strong>: Bi\u1ec3u \u0111\u1ed3 chu\u1ea9n v\u1ebd l\u00ean \u0111\u01b0\u1eddng bi\u1ec3u di\u1ec5n chu\u1ea9n v\u1ec1 m\u1ed1i quan h\u1ec7 gi\u1eefa s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n DNA \u0111\u00edch c\u00f3 trong c\u00e1c m\u1eabu chu\u1ea9n v\u00e0 chu k\u1ef3 ng\u01b0\u1ee1ng t\u01b0\u01a1ng \u1ee9ng. Trong bi\u1ec3u \u0111\u1ed3 \u1edf tr\u00ean n\u00e0y, ch\u00fang ta E2-E4-E7 l\u00e0 c\u00e1c \u1ed1ng ph\u1ea3n \u1ee9ng ch\u1ee9a c\u00e1c m\u1eabu chu\u1ea9n c\u00f2n B4 l\u00e0 \u1ed1ng ph\u1ea3n \u1ee9ng ch\u1ee9a m\u1eabu th\u1eed.<\/p>

(tr\u00ean tr\u1ee5c tung) v\u00e0 chu k\u1ef3 ng\u01b0\u1ee1ng c\u1ee7a \u1ed1ng ph\u1ea3n \u1ee9ng ch\u1ee9a s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n DNA \u0111\u00edch n\u00e0y (bi\u1ec3u \u0111\u1ed3 3<\/em><\/strong>). Tr\u00ean bi\u1ec3u \u0111\u1ed3 chu\u1ea9n n\u00e0y, tr\u1ee5c tung (Y) l\u00e0 Ct c\u00f2n tr\u1ee5c ho\u00e0nh (X) l\u00e0 s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n DNA \u0111\u00edch ban \u0111\u1ea7u c\u00f3 trong c\u00e1c m\u1eabu chu\u1ea9n. Do c\u00e1c m\u1eabu chu\u1ea9n th\u01b0\u1eddng \u0111\u01b0\u1ee3c pha c\u00e1ch nhau theo h\u1ec7 s\u1ed1 pha lo\u00e3ng 10, n\u00ean s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n DNA \u0111\u00edch trong c\u00e1c m\u1eabu chu\u1ea9n \u0111\u01b0\u1ee3c bi\u1ec3u th\u1ecb b\u1eb1ng logarith c\u01a1 s\u1ed1 10 (log10<\/sub>) c\u1ee7a s\u1ed1 l\u01b0\u1ee3ng n\u00e0y. Do v\u1eady tr\u1ecb 1.5, 2, 2.5, 3, 3.5, 4, 4.5 tr\u00ean tr\u1ee5c X l\u00e0 t\u01b0\u01a1ng \u1ee9ng v\u1edbi c\u00e1c s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n \u0111\u00edch l\u00e0 101.5<\/sup>, 102<\/sup>, 102.5<\/sup>, 103<\/sup>,<\/p>

103.5<\/sup>, 104<\/sup>,\u00a0 104.5<\/sup> t\u1ee9c\u00a0 l\u00e0 32,\u00a0 100, 316, 1000, 3162,\u00a0 10000, 31623 copies\u00a0 trong \u1ed1ng ph\u1ea3n<\/p>

\u1ee9ng.<\/p>

C\u00f3 hai th\u00f4ng s\u1ed1 \u0111\u01b0\u1ee3c m\u00e1y t\u00ednh to\u00e1n v\u00e0 hi\u1ec3n th\u1ecb tr\u00ean m\u1ed9t \u0111\u01b0\u1eddng bi\u1ec3u di\u1ec5n chu\u1ea9n. Hai th\u00f4ng s\u1ed1 n\u00e0y r\u1ea5t c\u00f3 gi\u00e1 tr\u1ecb \u0111\u1ec3 ng\u01b0\u1eddi l\u00e0m th\u1eed nghi\u1ec7m c\u00f3 th\u1ec3 \u0111\u00e1nh gi\u00e1 \u0111\u01b0\u1ee3c thao t\u00e1c pipetting c\u1ee7a m\u00ecnh trong qu\u00e1 tr\u00ecnh l\u00e0m th\u1eed nghi\u1ec7m.<\/p>

Th\u00f4ng s\u1ed1 \u0111\u1ea7u ti\u00ean l\u00e0 h\u1ec7 s\u1ed1 t\u01b0\u01a1ng quan R2<\/sup><\/em>, \u0111\u00e1nh gi\u00e1 \u0111\u1ed9 ch\u00ednh x\u00e1c c\u1ee7a thao t\u00e1c pipetting l\u1ea5y c\u00f3 \u0111\u00fang th\u1ec3 t\u00edch mong mu\u1ed1n hay kh\u00f4ng. Tr\u1ecb s\u1ed1 R2<\/sup> ph\u1ea3i \u0111\u1ea1t l\u00e0 tr\u00ean hay b\u1eb1ng (\u2265) 0.99 c\u00f3 ngh\u0129a l\u00e0 \u0111\u01b0\u1eddng bi\u1ec3u di\u1ec5n chu\u1ea9n ph\u1ea3i \u0111\u1ea1t tuy\u1ebfn t\u00ednh cao. Khi ng\u01b0\u1eddi l\u00e0m th\u1eed nghi\u1ec7m kh\u00f4ng l\u1ea5y \u0111\u01b0\u1ee3c c\u00e1c th\u1ec3 t\u00edch ch\u00ednh x\u00e1c th\u00ec ch\u1eafc ch\u1eafn c\u00e1c th\u1ec3 t\u00edch m\u1eabu chu\u1ea9n cho v\u00e0o real-time PCR mix s\u1ebd kh\u00f4ng th\u1ec3 n\u00e0o ch\u00ednh x\u00e1c \u0111\u01b0\u1ee3c, n\u00ean R2<\/sup> ch\u1eafc ch\u1eafn s\u1ebd kh\u00f4ng th\u1ec3 n\u00e0o \u0111\u1ea1t \u2265 0.99.<\/p>

M\u1ed9t tr\u1ecb s\u1ed1 n\u1eefa r\u1ea5t c\u00f3 gi\u00e1 tr\u1ecb m\u00e0 ng\u01b0\u1eddi l\u00e0m th\u1eed nghi\u1ec7m ph\u1ea3i bi\u1ebft r\u00f5 \u0111\u00f3 l\u00e0 hi\u1ec7u qu\u1ea3<\/u> PCR<\/u><\/em>, \u0111\u01b0\u1ee3c g\u1ecdi l\u00e0 E% (PCR efficiency). M\u1ed9t khi PCR \u0111\u1ea1t hi\u1ec7u qu\u1ea3 l\u00fd t\u01b0\u1edfng, th\u00ec c\u1ee9 sau m\u1ed7i chu k\u1ef3 nhi\u1ec7t c\u01b0\u1eddng \u0111\u1ed9 hu\u1ef3nh quang trong m\u1ed9t \u1ed1ng ph\u1ea3n \u1ee9ng ph\u1ea3i t\u0103ng g\u1ea5p \u0111\u00f4i, c\u00f2n n\u1ebfu \u1edf hai \u1ed1ng ph\u1ea3n \u1ee9ng c\u00f3 s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n DNA \u0111\u00edch ban \u0111\u1ea7u c\u00e1ch nhau m\u1ed9t h\u1ec7 s\u1ed1 pha lo\u00e3ng A<\/strong>, th\u00ec hai \u0111\u01b0\u1eddng bi\u1ec3u di\u1ec5n khu\u1ebfch \u0111\u1ea1i s\u1ebd c\u00e1ch nhau n <\/strong>chu k\u1ef3 v\u1edbi c\u01b0\u1eddng \u0111\u1ed9 hu\u1ef3nh quang c\u1ee7a hai \u1ed1ng ph\u1ea3n \u1ee9ng c\u0169ng s\u1ebd c\u00e1ch nhau m\u1ed9t h\u1ec7 s\u1ed1 pha lo\u00e3ng A = 2n<\/sup>. Nh\u01b0 v\u1eady, n\u1ebfu \u0111\u1ed9 pha lo\u00e3ng l\u00e0 10 l\u1ea7n th\u00ec Ct c\u1ee7a c\u00e1c \u0111\u1ed9 pha lo\u00e3ng DNA \u0111\u00edch c\u1ee7a m\u1eabu chu\u1ea9n li\u00ean ti\u1ebfp nhau s\u1ebd c\u00e1ch nhau l\u00e0 3.32 chu k\u1ef3 (t\u00ednh to\u00e1n t\u1eeb c\u00f4ng th\u1ee9c tr\u00ean v\u1edbi A = 10, s\u1ebd t\u00ednh \u0111\u01b0\u1ee3c n = 3.32 chu k\u1ef3). Trong bi\u1ec3u \u0111\u1ed3 chu\u1ea9n v\u1edbi c\u00e1c m\u1eabu chu\u1ea9n c\u00e1ch nhau qua \u0111\u1ed9 pha lo\u00e3ng h\u1ec7 s\u1ed1 10 th\u00ec hi\u1ec7u qu\u1ea3 khu\u1ebfch \u0111\u1ea1i E \u0111\u01b0\u1ee3c t\u00ednh l\u00e0 b\u1eb1ng c\u00f4ng th\u1ee9c 10-1\/slope<\/sup> v\u1edbi slope ch\u00ednh l\u00e0 \u0111\u1ed9 \u0111\u1ed1c c\u1ee7a \u0111\u01b0\u1eddng bi\u1ec3u di\u1ec5n chu\u1ea9n. M\u1ed9t c\u00e1ch l\u00fd t\u01b0\u1edfng, hi\u1ec7u qu\u1ea3 khu\u1ebfch \u0111\u1ea1i E c\u1ee7a PCR ph\u1ea3i \u0111\u1ea1t \u0111\u01b0\u1ee3c l\u00e0 2 ngh\u0129a l\u00e0 s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n sao nh\u00e2n b\u1ea3n t\u1eeb b\u1ea3n DNA \u0111\u00edch ph\u1ea3i t\u0103ng g\u1ea5p \u0111\u00f4i sau m\u1ed7i chu k\u1ef3 nhi\u1ec7t trong giai \u0111o\u1ea1n t\u0103ng tr\u01b0\u1edfng l\u0169y th\u1eeba, t\u1ee9c l\u00e0 2 = 10-1\/slope<\/sup>. T\u1eeb c\u00f4ng th\u1ee9c n\u00e0y, ch\u00fang ta s\u1ebd t\u00ednh ra \u0111\u01b0\u1ee3c \u0111\u01b0\u1eddng bi\u1ec3u di\u1ec5n chu\u1ea9n l\u00fd t\u01b0\u1edfng<\/p>

ph\u1ea3i c\u00f3 \u0111\u1ed9 \u0111\u1ed1c (slope) l\u00e0 -3.32. Nh\u01b0 v\u1eady, ch\u00fang ta th\u1ea5y \u0111\u1ed9 d\u1ed1c ch\u00ednh l\u00e0 s\u1ef1 c\u00e1ch bi\u1ec7t nhau v\u1ec1 chu k\u1ef3 ng\u01b0\u1ee1ng gi\u1eefa c\u00e1c \u1ed1ng chu\u1ea9n c\u00f3 s\u1ed1 l\u01b0\u1ee3ng gi\u1ea3m d\u1ea7n theo h\u1ec7 s\u1ed1 pha lo\u00e3ng<\/p>

  1. Hi\u1ec7u qu\u1ea3 khu\u1ebfch \u0111\u1ea1i c\u0169ng c\u00f3 th\u1ec3 t\u00ednh b\u1eb1ng ph\u1ea7n tr\u0103m (%) v\u1edbi c\u00f4ng th\u1ee9c t\u00ednh t\u1eeb E l\u00e0: E% = (E-1) x 100%. V\u1edbi E \u0111\u1ea1t l\u00fd t\u01b0\u1edfng l\u00e0 2 th\u00ec E% = 100 %. \u0110\u1ed1i v\u1edbi b\u1ea5t c\u1ee9 m\u1ed9t \u0111\u01b0\u1eddng bi\u1ec3u di\u1ec3n chu\u1ea9n n\u00e0o, \u0111\u1ec1u c\u00f3 th\u1ec3 t\u00ednh \u0111\u01b0\u1ee3c hi\u1ec7u qu\u1ea3 khu\u1ebfch \u0111\u1ea1i E % = (10-1\/slope<\/sup><\/li><\/ol>

    \u2013 1) x 100 %. Do v\u1eady n\u1ebfu \u0111\u1ed9 d\u1ed1c c\u1ee7a m\u1ed9t \u0111\u01b0\u1eddng bi\u1ec3u di\u1ec5n chu\u1ea9n l\u00e0 -3.436 th\u00ec E s\u1ebd l\u00e0 10-(1\/-3.436)<\/sup> = 1.954, v\u1eady th\u00ec hi\u1ec7u qu\u1ea3 % c\u1ee7a khu\u1ebfch \u0111\u1ea1i (hi\u1ec7u qu\u1ea3 PCR) s\u1ebd l\u00e0: E % = (1.954 \u2013 1) x 100% = 95.4 %, c\u00f3 ngh\u0129a l\u00e0 trong giai \u0111o\u1ea1n t\u0103ng tr\u01b0\u1edfng l\u0169y th\u1eeba s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n sao sau m\u1ed7i chu k\u1ef3 nhi\u1ec7t s\u1ebd \u0111\u01b0\u1ee3c nh\u00e2n l\u00ean 1.954 l\u1ea7n hay hi\u1ec7u qu\u1ea3 PCR l\u00e0 95.4 %. D\u0129 nhi\u00ean hi\u1ec7u qu\u1ea3 PCR l\u00fd t\u01b0\u1edfng l\u00e0 100 %, nh\u01b0ng trong th\u1ef1c t\u1ebf th\u00ec hi\u1ec7u qu\u1ea3 PCR ch\u1ea5p nh\u1eadn \u0111\u01b0\u1ee3c ph\u1ea3i trong kho\u1ea3ng 90 % – 105 %. Ng\u01b0\u1eddi l\u00e0m th\u00ed nghi\u1ec7m ph\u1ea3i bi\u1ebft d\u00f9ng hi\u1ec7u qu\u1ea3 PCR hi\u1ec3n th\u1ecb trong bi\u1ec3u \u0111\u1ed3 chu\u1ea9n \u0111\u1ec3 \u0111\u00e1nh gi\u00e1 thao t\u00e1c pipetting \u0111\u1ec3 pha lo\u00e3ng m\u1eabu c\u1ee7a m\u00ecnh. Sai l\u1ea7m \u0111\u1ea7u ti\u00ean c\u00f3 th\u1ec3 g\u1eb7p trong thao t\u00e1c pha lo\u00e3ng m\u1eabu l\u00e0 mang l\u01b0\u1ee3ng DNA \u0111\u00edch t\u1eeb m\u1eabu n\u1ed3ng \u0111\u1ed9 cao xu\u1ed1ng m\u1eabu c\u00f3 n\u1ed3ng \u0111\u1ed9 th\u1ea5p h\u01a1n, m\u00e0 th\u01b0\u1eddng l\u00e0 do kh\u00f4ng thay \u0111\u1ea7u pipette sau khi mang th\u1ec3 t\u00edch t\u1eeb n\u1ed3ng \u0111\u1ed9 cao xu\u1ed1ng n\u1ed3ng \u0111\u1ed9 th\u1ea5p, v\u00ec v\u1eady m\u00e0 s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n DNA \u0111\u00edch c\u00f3 trong c\u00e1c m\u1eabu chu\u1ea9n \u0111\u01b0\u1ee3c pha lo\u00e3ng s\u1ebd nhi\u1ec1u h\u01a1n t\u00ednh to\u00e1n. V\u00ed d\u1ee5 n\u1ebfu thao t\u00e1c pha lo\u00e3ng ch\u00ednh x\u00e1c, th\u00ec t\u1eeb 10.000 copies xu\u1ed1ng 1.000 copies r\u1ed3i 100 copies s\u1ebd ch\u00ednh x\u00e1c nh\u01b0 v\u1eady; nh\u01b0ng n\u1ebfu kh\u00f4ng thay \u0111\u1ea7u pipette sau m\u1ed7i l\u1ea7n h\u00fat m\u1eabu v\u00e0 l\u01b0\u1ee3ng b\u1ea3n \u0111\u00edch mang theo \u1edf \u0111\u1ea7u pipette l\u00e0 40% th\u00ec ch\u00fang ta s\u1ebd c\u00f3 c\u00e1c\u00a0 s\u1ed1 l\u01b0\u1ee3ng s\u1ebd l\u00e0 10.000 copies xu\u1ed1ng 4.000 copies r\u1ed3i 1.600 copies. V\u00ec v\u1eady n\u00ean hi\u1ec7u qu\u1ea3 PCR \u0111\u01b0\u1ee3c t\u00ednh to\u00e1n b\u1eb1ng gi\u00e1 tr\u1ecb E s\u1ebd kh\u00f4ng \u0111\u1ea1t \u0111\u01b0\u1ee3c l\u00fd t\u01b0\u1edfng 90 % \u0111\u1ebfn 105 % m\u00e0 s\u1ebd cao h\u01a1n 105 %. L\u00fd do l\u00e0 chu k\u1ef3 ng\u01b0\u1ee1ng c\u1ee7a c\u00e1c m\u1eabu chu\u1ea9n s\u1ebd b\u1ecb g\u1ea7n nhau h\u01a1n l\u00e0m\u00a0 cho \u0111\u1ed9 d\u1ed1c c\u1ee7a \u0111\u01b0\u1eddng bi\u1ec3u di\u1ec5n chu\u1ea9n th\u1ea5p \u0111i. Ng\u01b0\u1ee3c l\u1ea1i n\u1ebfu thao t\u00e1c pha lo\u00e3ng m\u00e0 kh\u00f4ng cho \u0111\u1ee7 th\u1ec3 t\u00edch m\u1eabu chu\u1ea9n h\u00fat t\u1eeb m\u1eabu c\u00f3 s\u1ed1 l\u01b0\u1ee3ng DNA chu\u1ea9n cao \u0111\u1ec3 pha m\u1eabu chu\u1ea9n l\u01b0\u1ee3ng th\u1ea5p h\u01a1n (sai l\u1ea7m n\u00e0y c\u00f3 th\u1ec3 g\u1eb7p khi d\u00f9ng c\u00e1c \u0111\u1ea7u pipette b\u1ecb d\u00ednh dung d\u1ecbch tr\u00ean th\u00e0nh n\u00ean kh\u00f4ng \u0111\u1ea9y \u0111\u01b0\u1ee3c h\u1ebft l\u01b0\u1ee3ng m\u1eabu t\u1eeb \u0111\u1ea7u pipette xu\u1ed1ng m\u1eabu k\u1ebf), v\u00e0 ch\u00ednh nh\u01b0 v\u1eady n\u00ean chu k\u1ef3 ng\u01b0\u1ee1ng c\u1ee7a c\u00e1c m\u1eabu chu\u1ea9n li\u00ean ti\u1ebfp nhau s\u1ebd c\u00e1ch xa nhau l\u00e0m cho \u0111\u1ed9 d\u1ed1c c\u1ee7a \u0111\u01b0\u1eddng bi\u1ec3u di\u1ec5n chu\u1ea9n th\u1ea5p \u0111i, v\u00e0 hi\u1ec7u qu\u1ea3 PCR s\u1ebd d\u01b0\u1edbi 95 %. Hi\u1ec7u qu\u1ea3 PCR c\u0169ng l\u00e0 m\u1ed9t th\u00f4ng s\u1ed1 r\u1ea5t c\u00f3 gi\u00e1 tr\u1ecb \u0111\u1ec3 nh\u00e0 nghi\u00ean c\u1ee9u t\u1ed1i \u01b0u \u0111\u01b0\u1ee3c k\u1ef9 thu\u1eadt real-time PCR m\u00e0 m\u00ecnh. N\u1ebfu hi\u1ec7u qu\u1ea3 PCR th\u1ea5p th\u00ec nguy\u00ean do c\u00f3 th\u1ec3 l\u00e0 thi\u1ebft k\u1ebf m\u1ed3i ch\u01b0a \u0111\u1ea1t \u0111\u1ed9 nh\u1ea1y, do t\u00e1ch chi\u1ebft DNA \u0111\u00edch c\u00f2n c\u00e1c \u1ee9c ch\u1ebf ng\u0103n c\u1ea3n ph\u1ea3n \u1ee9ng<\/p>

    khu\u1ebfch \u0111\u1ea1i. N\u1ebfu hi\u1ec7u qu\u1ea3 PCR cao th\u00ec nguy\u00ean do l\u1ea1i c\u00f3 th\u1ec3 l\u00e0 do s\u1ef1 b\u1eaft c\u1eb7p kh\u00f4ng \u0111\u1eb7c hi\u1ec7u c\u1ee7a m\u1ed3i hay c\u1ee7a \u0111o\u1ea1n d\u00f2.<\/p>

    Nh\u01b0 v\u1eady, vi\u1ec7c thi\u1ebft l\u1eadp bi\u1ec3u \u0111\u1ed3 chu\u1ea9n l\u00e0 r\u1ea5t c\u1ea7n thi\u1ebft khi l\u00e0m real-time PCR ch\u1ea9n \u0111o\u00e1n, \u0111\u1eb7c bi\u1ec7t khi \u0111\u1ec3 x\u00e1c \u0111\u1ecbnh ch\u00ednh x\u00e1c s\u1ed1 l\u01b0\u1ee3ng t\u00e1c nh\u00e2n \u0111\u00edch ban \u0111\u1ea7u c\u00f3 trong m\u1eabu th\u1eed. Bi\u1ec3u \u0111\u1ed3 chu\u1ea9n c\u00f3 c\u00f4ng d\u1ee5ng tr\u01b0\u1edbc ti\u00ean l\u00e0 \u0111\u00e1nh gi\u00e1 \u0111\u01b0\u1ee3c thao t\u00e1c pipetting c\u1ee7a ng\u01b0\u1eddi l\u00e0m th\u1eed nghi\u1ec7m c\u00f3 \u0111\u1ea1t kh\u00f4ng. \u0110\u00e2y l\u00e0 m\u1ed9t vi\u1ec7c l\u00e0 h\u1ebft s\u1ee9c quan tr\u1ecdng v\u00ec n\u1ebfu thao t\u00e1c pipetting m\u00e0 kh\u00f4ng ch\u00ednh x\u00e1c th\u00ec s\u1ebd kh\u00f4ng th\u1ec3 n\u00e0o c\u00f3 \u0111\u01b0\u1ee3c k\u1ebft qu\u1ea3 \u0111\u1ecbnh l\u01b0\u1ee3ng ch\u00ednh x\u00e1c \u0111\u01b0\u1ee3c. Do v\u1eady s\u1ebd th\u1eadt l\u00e0 sai l\u1ea7m v\u00e0 thi\u1ebfu hi\u1ec3u bi\u1ebft <\/u><\/em>n\u1ebfu nh\u00e0 s\u1ea3n xu\u1ea5t b\u1ed9 thu\u1ed1c th\u1eed real-time PCR d\u00e0nh \u0111\u1ec3 \u0111\u1ecbnh l\u01b0\u1ee3ng t\u00e1c nh\u00e2n \u0111\u00edch l\u1ea1i cung c\u1ea5p s\u1eb5n c\u00e1c PCR mix c\u00f3<\/u> cho tr\u01b0\u1edbc c\u00e1c l\u01b0\u1ee3ng chu\u1ea9n c\u1ee7a DNA \u0111\u00edch<\/u><\/em>, g\u1ecdi l\u00e0 c\u00e1c b\u1ed9 chu\u1ea9n, m\u00e0 kh\u00f4ng \u0111\u1ec3 cho ng\u01b0\u1eddi l\u00e0m th\u00ed nghi\u1ec7m t\u1ef1 pha lo\u00e3ng c\u00e1c m\u1eabu chu\u1ea9n \u0111\u1ec3 cho v\u00e0o real-time PCR mix.<\/p>

    Sau khi \u0111\u00e3 \u0111\u00e1nh gi\u00e1 \u0111\u01b0\u1ee3c thao t\u00e1c pipetting c\u1ee7a ng\u01b0\u1eddi l\u00e0m th\u1eed nghi\u1ec7m l\u00e0 \u0111\u1ea1t, ng\u01b0\u1eddi l\u00e0m th\u1eed nghi\u1ec7m s\u1ebd s\u1eed d\u1ee5ng c\u00f4ng d\u1ee5ng th\u1ee9 hai c\u1ee7a bi\u1ec3u \u0111\u1ed3 chu\u1ea9n, \u0111\u00f3 l\u00e0 x\u00e1c \u0111\u1ecbnh \u0111\u01b0\u1ee3c s\u1ed1 l\u01b0\u1ee3ng t\u00e1c nh\u00e2n \u0111\u00edch ban \u0111\u1ea7u c\u00f3 trong m\u1eabu th\u1eed. Gi\u00e1 tr\u1ecb n\u00e0y \u0111\u01b0\u1ee3c t\u00ednh to\u00e1n nh\u1edd v\u00e0o h\u00e0m s\u1ed1 bi\u1ec3u th\u1ecb m\u1ed1i t\u01b0\u01a1ng quan gi\u1eefa chu k\u1ef3 ng\u01b0\u1ee1ng (Y = Ct) v\u1edbi log10<\/sub> c\u1ee7a s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n DNA \u0111\u00edch ban \u0111\u1ea7u c\u00f3 trong \u1ed1ng ph\u1ea3n \u1ee9ng (X = log10<\/sub> Sq). H\u00e0m s\u1ed1 \u0111\u00f3 l\u00e0: Y = [slope (X)] + intercept, v\u00e0 c\u00e1c th\u00f4ng s\u1ed1 slope v\u00e0 intercept \u0111\u1ec1u hi\u1ec3n th\u1ecb tr\u00ean bi\u1ec3u \u0111\u1ed3 chu\u1ea9n. T\u1eeb h\u00e0m s\u1ed1 n\u00e0y, ng\u01b0\u1eddi l\u00e0m th\u00ed nghi\u1ec7m s\u1ebd hi\u1ec3u \u0111\u01b0\u1ee3c t\u1ea1i sao m\u00e1y t\u00ednh hi\u1ec3n th\u1ecb \u0111\u01b0\u1ee3c Sq, \u0111\u00f3 l\u00e0 nh\u1edd t\u00ednh to\u00e1n t\u1eeb Sq = 10[(Ct<\/sup> \u2013<\/sup> intercept)\/slope]<\/sup>. T\u1eeb Sq n\u00e0y, ng\u01b0\u1eddi l\u00e0m th\u00ed nghi\u1ec7m s\u1ebd t\u00ednh \u0111\u01b0\u1ee3c s\u1ed1 l\u01b0\u1ee3ng t\u00e1c nh\u00e2n \u0111\u00edch ban \u0111\u1ea7u c\u00f3 trong m\u1eabu th\u1eed d\u1ef1a v\u00e0o hi\u1ec7u qu\u1ea3 t\u00e1ch chi\u1ebft nucleic acid \u0111\u00edch t\u1eeb m\u1eabu th\u1eed v\u00e0 c\u00e1c pha lo\u00e3ng n\u1ebfu c\u00f3 trong qu\u00e1 tr\u00ecnh t\u00e1ch chi\u1ebft c\u1ee7ng nh\u01b0 th\u1ef1c hi\u1ec7n real-time PCR. V\u00ed d\u1ee5: khi s\u1eed d\u1ee5ng c\u00e1c b\u1ed9 thu\u1ed1c th\u1eed ph\u00e1t hi\u1ec7n v\u00e0 \u0111\u1ecbnh l\u01b0\u1ee3ng HCV do c\u00f4ng ty Nam Khoa s\u1ea3n xu\u1ea5t th\u00ec s\u1ed1 copies HCV c\u00f3 trong 1 ml huy\u1ebft thanh th\u1eed nghi\u1ec7m s\u1ebd b\u1eb1ng N x 71 v\u1edbi N l\u00e0 s\u1ed1 copies \u0111\u1ecbnh l\u01b0\u1ee3ng \u0111\u01b0\u1ee3c trong \u1ed1ng ph\u1ea3n \u1ee9ng, hay khi khi s\u1eed d\u1ee5ng c\u00e1c b\u1ed9 thu\u1ed1c th\u1eed ph\u00e1t hi\u1ec7n v\u00e0 \u0111\u1ecbnh l\u01b0\u1ee3ng HBV do c\u00f4ng ty Nam Khoa s\u1ea3n xu\u1ea5t th\u00ec s\u1ed1 copies HBV c\u00f3 trong 1 ml huy\u1ebft thanh th\u1eed nghi\u1ec7m s\u1ebd b\u1eb1ng N x 50 v\u1edbi N l\u00e0 s\u1ed1 copies \u0111\u1ecbnh l\u01b0\u1ee3ng \u0111\u01b0\u1ee3c trong \u1ed1ng ph\u1ea3n \u1ee9ng (xem ph\u1ee5 l\u1ee5c \u0111\u1ec3 bi\u1ebft c\u00e1ch t\u00ednh to\u00e1n \u0111\u1ec3 c\u00f3 c\u00e1c h\u1ec7 s\u1ed1 n\u00e0y).<\/p><\/div>\n\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

    \n\t\t\t\t
    \n\t\t\t\t\t

    M\u00e1y real-time PCR<\/h1>

    \u00a0<\/strong><\/p>

    \u0110i\u1ec1u ki\u1ec7n \u0111\u1ea7u ti\u00ean \u0111\u1ec3 c\u00f3 th\u1ec3 th\u1ef1c hi\u1ec7n \u0111\u01b0\u1ee3c real-time PCR l\u00e0 ph\u1ea3i c\u00f3 m\u00e1y real-time PCR. M\u00e1y n\u00e0y c\u0169ng c\u00f3 m\u1ed9t bu\u1ed3ng \u1ee7 nhi\u1ec7t ch\u1ea1y \u0111\u01b0\u1ee3c ch\u01b0\u01a1ng tr\u00ecnh lu\u00e2n nhi\u1ec7t nh\u01b0 m\u00e1y PCR b\u00ecnh th\u01b0\u1eddng, nh\u01b0ng c\u00f3 th\u00eam m\u1ed9t thi\u1ebft b\u1ecb \u0111\u01b0\u1ee3c g\u1ecdi l\u00e0 thi\u1ebft b\u1ecb real-time. \u0110\u00e2y l\u00e0 m\u1ed9t thi\u1ebft b\u1ecb quang h\u1ecdc c\u00f3 hai ch\u1ee9c n\u0103ng: (1) C\u00f3 c\u00e1c ngu\u1ed3n s\u00e1ng ph\u00e1t ra \u0111\u01b0\u1ee3c c\u00e1c tia s\u00e1ng\u00a0 k\u00edch th\u00edch (excitation light) c\u00f3 b\u01b0\u1edbc s\u00f3ng x\u00e1c \u0111\u1ecbnh l\u00ean c\u00e1c tube ph\u1ea3n \u1ee9ng real-time PCR;<\/p>