{"id":3639,"date":"2025-05-27T07:45:09","date_gmt":"2025-05-27T07:45:09","guid":{"rendered":"https:\/\/oligox.abtvn.com\/?p=3639"},"modified":"2025-08-01T04:14:02","modified_gmt":"2025-08-01T04:14:02","slug":"taqman-probe-trong-realtime-pcr","status":"publish","type":"post","link":"https:\/\/oligox.abtvn.com\/en\/taqman-probe-trong-realtime-pcr\/","title":{"rendered":"\u1ea2nh h\u01b0\u1edfng c\u1ee7a TaqMan probe \u0111\u1ebfn \u0111\u1ed9 \u0111\u1eb7c hi\u1ec7u v\u00e0 \u0111\u1ed9 nh\u1ea1y trong Realtime PCR"},"content":{"rendered":"

Trong th\u1ebf gi\u1edbi c\u1ee7a c\u00f4ng ngh\u1ec7 sinh h\u1ecdc ph\u00e2n t\u1eed hi\u1ec7n \u0111\u1ea1i, \u0111\u1ed9 ch\u00ednh x\u00e1c v\u00e0 \u0111\u1ed9 \u0111\u1eb7c hi\u1ec7u<\/strong> l\u00e0 nh\u1eefng y\u1ebfu t\u1ed1 s\u1ed1ng c\u00f2n c\u1ee7a m\u1ed9t x\u00e9t nghi\u1ec7m. V\u1edbi vai tr\u00f2 trung t\u00e2m trong ph\u1ea3n \u1ee9ng Real-time PCR (qPCR)<\/strong>, TaqMan Probe<\/strong> kh\u00f4ng ch\u1ec9 gi\u00fap ph\u00e1t hi\u1ec7n tr\u00ecnh t\u1ef1 m\u1ee5c ti\u00eau m\u1ed9t c\u00e1ch ch\u1ecdn l\u1ecdc, m\u00e0 c\u00f2n g\u00f3p ph\u1ea7n t\u1ed1i \u01b0u h\u00f3a to\u00e0n b\u1ed9 hi\u1ec7u su\u1ea5t ph\u1ea3n \u1ee9ng. V\u1eady <\/strong>c\u01a1 ch\u1ebf ho\u1ea1t \u0111\u1ed9ng c\u1ee7a TaqMan Probe l\u00e0 g\u00ec? L\u00e0m th\u1ebf n\u00e0o \u0111\u1ec3 thi\u1ebft k\u1ebf probe \u0111\u1ea1t hi\u1ec7u qu\u1ea3 cao nh\u1ea5t? V\u00e0 \u0111\u00e2u l\u00e0 nh\u1eefng \u0111\u1ed9t ph\u00e1 m\u1edbi trong c\u00f4ng ngh\u1ec7 t\u1ed5ng h\u1ee3p \u0111\u1ea7u d\u00f2 gi\u00fap n\u00e2ng cao \u0111\u1ed9 nh\u1ea1y v\u00e0 lo\u1ea1i b\u1ecf sai s\u1ed1? B\u00e0i vi\u1ebft sau s\u1ebd gi\u1ea3i m\u00e3 nh\u1eefng c\u00e2u h\u1ecfi tr\u00ean – t\u1eeb nguy\u00ean l\u00fd c\u01a1 b\u1ea3n \u0111\u1ebfn c\u00e1c c\u1ea3i ti\u1ebfn ti\u00ean ti\u1ebfn – \u0111\u1ec3 mang \u0111\u1ebfn c\u00e1i nh\u00ecn to\u00e0n di\u1ec7n v\u1ec1 vai tr\u00f2 thi\u1ebft y\u1ebfu c\u1ee7a TaqMan Probe trong \u0111\u1ed9 ch\u00ednh x\u00e1c c\u1ee7a qPCR.<\/p>\n\n\n\n

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Real-time PCR v\u00e0 Taqman Probe<\/strong><\/h2>\n\n\n\n

Real-time PCR<\/a><\/strong> (Ph\u1ea3n \u1ee9ng chu\u1ed7i Polymerase th\u1eddi gian th\u1ef1c), hay c\u00f2n g\u1ecdi l\u00e0 qPCR, l\u00e0 m\u1ed9t k\u1ef9 thu\u1eadt sinh h\u1ecdc ph\u00e2n t\u1eed cho ph\u00e9p ph\u00e1t hi\u1ec7n v\u00e0 \u0111\u1ecbnh l\u01b0\u1ee3ng c\u00e1c \u0111o\u1ea1n DNA ho\u1eb7c RNA m\u1ee5c ti\u00eau m\u1ed9t c\u00e1ch ch\u00ednh x\u00e1c ngay trong qu\u00e1 tr\u00ecnh khu\u1ebfch \u0111\u1ea1i, thay v\u00ec ph\u1ea3i \u0111\u1ee3i \u0111\u1ebfn khi ph\u1ea3n \u1ee9ng k\u1ebft th\u00fac nh\u01b0 PCR truy\u1ec1n th\u1ed1ng. Nh\u1edd kh\u1ea3 n\u0103ng theo d\u00f5i t\u00edn hi\u1ec7u hu\u1ef3nh quang t\u0103ng l\u00ean sau m\u1ed7i chu k\u1ef3 nhi\u1ec7t, qPCR \u0111\u00e3 tr\u1edf th\u00e0nh m\u1ed9t c\u00f4ng c\u1ee5 v\u00f4 gi\u00e1 trong nhi\u1ec1u l\u0129nh v\u1ef1c, t\u1eeb nghi\u00ean c\u1ee9u c\u01a1 b\u1ea3n nh\u01b0 ph\u00e2n t\u00edch bi\u1ec3u hi\u1ec7n gene, \u0111\u1ebfn c\u00e1c \u1ee9ng d\u1ee5ng th\u1ef1c ti\u1ec5n nh\u01b0 ch\u1ea9n \u0111o\u00e1n b\u1ec7nh truy\u1ec1n nhi\u1ec5m do virus, vi khu\u1ea9n, ph\u00e1t hi\u1ec7n c\u00e1c y\u1ebfu t\u1ed1 di truy\u1ec1n, v\u00e0 ki\u1ec3m nghi\u1ec7m an to\u00e0n th\u1ef1c ph\u1ea9m. Kh\u1ea3 n\u0103ng \u0111\u1ecbnh t\u00ednh v\u00e0 \u0111\u1ecbnh \u0111\u00e3 \u0111\u01b0a qPCR tr\u1edf th\u00e0nh “ti\u00eau chu\u1ea9n v\u00e0ng” trong nhi\u1ec1u \u1ee9ng d\u1ee5ng ch\u1ea9n \u0111o\u00e1n, mang l\u1ea1i \u0111\u1ed9 tin c\u1eady v\u01b0\u1ee3t tr\u1ed9i, \u0111\u1eb7c bi\u1ec7t khi c\u00f3 s\u1ef1 k\u1ebft h\u1ee3p v\u1edbi c\u00e1c c\u00f4ng ngh\u1ec7 \u0111\u1ea7u d\u00f2 (probe) nh\u01b0 TaqMan<\/strong>.<\/p>\n\n\n\n

Trong s\u1ed1 c\u00e1c c\u00f4ng ngh\u1ec7 \u0111\u1ea7u d\u00f2, TaqMan probe<\/a><\/strong> n\u1ed5i b\u1eadt v\u1edbi c\u01a1 ch\u1ebf ho\u1ea1t \u0111\u1ed9ng \u0111\u1ed9c \u0111\u00e1o v\u00e0 vai tr\u00f2 quan tr\u1ecdng trong vi\u1ec7c n\u00e2ng cao \u0111\u1ed9 ch\u00ednh x\u00e1c c\u1ee7a x\u00e9t nghi\u1ec7m qPCR. TaqMan probe l\u00e0 m\u1ed9t oligonucleotide s\u1ee3i \u0111\u01a1n, th\u01b0\u1eddng d\u00e0i t\u1eeb 18 \u0111\u1ebfn 30 nucleotide, \u0111\u01b0\u1ee3c thi\u1ebft k\u1ebf \u0111\u1ec3 lai \u0111\u1eb7c hi\u1ec7u v\u00e0o m\u1ed9t tr\u00ecnh t\u1ef1 DNA m\u1ee5c ti\u00eau n\u1eb1m gi\u1eefa c\u1eb7p m\u1ed3i xu\u00f4i (forward primer) v\u00e0 m\u1ed3i ng\u01b0\u1ee3c (reverse primer). \u0110i\u1ec3m \u0111\u1eb7c tr\u01b0ng c\u1ee7a TaqMan probe l\u00e0 n\u00f3 \u0111\u01b0\u1ee3c \u0111\u00e1nh d\u1ea5u k\u00e9p: m\u1ed9t ph\u00e2n t\u1eed ph\u00e1t hu\u1ef3nh quang (reporter) \u1edf \u0111\u1ea7u 5′ v\u00e0 m\u1ed9t ph\u00e2n t\u1eed d\u1eadp t\u1eaft hu\u1ef3nh quang (quencher) \u1edf \u0111\u1ea7u 3′. <\/p>\n\n\n\n

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C\u01a1 ch\u1ebf ho\u1ea1t \u0111\u1ed9ng c\u1ee7a TaqMan probe d\u1ef1a tr\u00ean hai y\u1ebfu t\u1ed1 ch\u00ednh: hi\u1ec7n t\u01b0\u1ee3ng truy\u1ec1n n\u0103ng l\u01b0\u1ee3ng c\u1ed9ng h\u01b0\u1edfng Forster (FRET) v\u00e0 ho\u1ea1t t\u00ednh 5′-3′ exonuclease c\u1ee7a enzyme Taq polymerase.<\/p>\n\n\n\n

    \n
  1. Khi probe c\u00f2n nguy\u00ean v\u1eb9n v\u00e0 t\u1ef1 do trong dung d\u1ecbch ph\u1ea3n \u1ee9ng ho\u1eb7c ch\u01b0a b\u1ecb ph\u00e2n c\u1eaft, ph\u00e2n t\u1eed reporter v\u00e0 quencher \u1edf g\u1ea7n nhau. Do \u0111\u00f3, n\u0103ng l\u01b0\u1ee3ng t\u1eeb reporter khi b\u1ecb k\u00edch th\u00edch s\u1ebd \u0111\u01b0\u1ee3c truy\u1ec1n sang quencher, l\u00e0m d\u1eadp t\u1eaft t\u00edn hi\u1ec7u hu\u1ef3nh quang c\u1ee7a reporter (hi\u1ec7u \u1ee9ng FRET).  <\/li>\n\n\n\n
  2. Trong giai \u0111o\u1ea1n b\u1eaft c\u1eb7p (annealing) c\u1ee7a m\u1ed7i chu k\u1ef3 PCR, TaqMan probe s\u1ebd b\u1eaft c\u1eb7p \u0111\u1eb7c hi\u1ec7u v\u00e0o tr\u00ecnh t\u1ef1 DNA m\u1ee5c ti\u00eau (n\u1ebfu c\u00f3 s\u1ef1 hi\u1ec7n c\u1ee7a DNA m\u1ee5c ti\u00eau).<\/li>\n\n\n\n
  3. \u1ede giai \u0111o\u1ea1n k\u00e9o d\u00e0i (extension), Taq polymerase b\u1eaft \u0111\u1ea7u t\u1ed5ng h\u1ee3p s\u1ee3i DNA m\u1edbi t\u1eeb m\u1ed3i. Khi enzyme di chuy\u1ec3n d\u1ecdc theo s\u1ee3i khu\u00f4n v\u00e0 g\u1eb7p ph\u1ee9c h\u1ee3p probe-DNA m\u1ee5c ti\u00eau, ho\u1ea1t t\u00ednh 5′-3′ exonuclease v\u1ed1n c\u00f3 c\u1ee7a Taq polymerase s\u1ebd th\u1ee7y ph\u00e2n (ph\u00e2n c\u1eaft) probe.  <\/li>\n\n\n\n
  4. S\u1ef1 ph\u00e2n c\u1eaft n\u00e0y gi\u1ea3i ph\u00f3ng ph\u00e2n t\u1eed reporter ra xa kh\u1ecfi quencher, ph\u00e1 v\u1ee1 hi\u1ec7u \u1ee9ng FRET. L\u00fac n\u00e0y, reporter kh\u00f4ng c\u00f2n b\u1ecb d\u1eadp t\u1eaft v\u00e0 s\u1ebd ph\u00e1t ra t\u00edn hi\u1ec7u hu\u1ef3nh quang khi \u0111\u01b0\u1ee3c k\u00edch th\u00edch b\u1edfi ngu\u1ed3n s\u00e1ng t\u1eeb m\u00e1y real-time PCR.  <\/li>\n\n\n\n
  5. C\u01b0\u1eddng \u0111\u1ed9 t\u00edn hi\u1ec7u hu\u1ef3nh quang t\u0103ng l\u00ean sau m\u1ed7i chu k\u1ef3 PCR, t\u1ef7 l\u1ec7 thu\u1eadn v\u1edbi l\u01b0\u1ee3ng s\u1ea3n ph\u1ea9m PCR \u0111\u01b0\u1ee3c t\u1ea1o ra, v\u00e0 do \u0111\u00f3, t\u1ef7 l\u1ec7 thu\u1eadn v\u1edbi l\u01b0\u1ee3ng DNA m\u1ee5c ti\u00eau ban \u0111\u1ea7u trong m\u1eabu. M\u00e1y real-time PCR s\u1ebd ghi nh\u1eadn s\u1ef1 gia t\u0103ng t\u00edn hi\u1ec7u n\u00e0y theo th\u1eddi gian th\u1ef1c.  <\/li>\n<\/ol>\n\n\n\n
    \"H\u00ecnh
    H\u00ecnh 1.<\/strong> Nguy\u00ean l\u00fd ho\u1ea1t \u0111\u1ed9ng c\u1ee7a Taqman probe trong Real Time PCR<\/em><\/figcaption><\/figure>\n\n\n\n

    Ho\u1ea1t t\u00ednh 5′-3′ exonuclease c\u1ee7a Taq polymerase \u0111\u00f3ng vai tr\u00f2 then ch\u1ed1t trong c\u01a1 ch\u1ebf n\u00e0y. N\u1ebfu enzyme s\u1eed d\u1ee5ng kh\u00f4ng c\u00f3 ho\u1eb7c c\u00f3 ho\u1ea1t t\u00ednh n\u00e0y y\u1ebfu, ho\u1eb7c n\u1ebfu ho\u1ea1t t\u00ednh enzyme b\u1ecb \u1ee9c ch\u1ebf b\u1edfi c\u00e1c y\u1ebfu t\u1ed1 trong m\u1eabu ph\u1ea3n \u1ee9ng, probe s\u1ebd kh\u00f4ng \u0111\u01b0\u1ee3c ph\u00e2n c\u1eaft m\u1ed9t c\u00e1ch hi\u1ec7u qu\u1ea3. \u0110i\u1ec1u n\u00e0y d\u1eabn \u0111\u1ebfn t\u00edn hi\u1ec7u hu\u1ef3nh quang y\u1ebfu, kh\u00f4ng \u0111\u1eb7c hi\u1ec7u, ho\u1eb7c kh\u00f4ng t\u01b0\u01a1ng quan ch\u00ednh x\u00e1c v\u1edbi l\u01b0\u1ee3ng s\u1ea3n ph\u1ea9m PCR, t\u1eeb \u0111\u00f3 \u1ea3nh h\u01b0\u1edfng tr\u1ef1c ti\u1ebfp \u0111\u1ebfn \u0111\u1ed9 ch\u00ednh x\u00e1c v\u00e0 \u0111\u1ed9 tin c\u1eady c\u1ee7a to\u00e0n b\u1ed9 x\u00e9t nghi\u1ec7m. Do \u0111\u00f3, ch\u1ea5t l\u01b0\u1ee3ng v\u00e0 lo\u1ea1i Taq polymerase, c\u00f9ng v\u1edbi c\u00e1c \u0111i\u1ec1u ki\u1ec7n ph\u1ea3n \u1ee9ng t\u1ed1i \u01b0u cho ho\u1ea1t \u0111\u1ed9ng c\u1ee7a enzyme, l\u00e0 nh\u1eefng y\u1ebfu t\u1ed1 n\u1ec1n t\u1ea3ng \u0111\u1ea3m b\u1ea3o s\u1ef1 th\u00e0nh c\u00f4ng c\u1ee7a x\u00e9t nghi\u1ec7m s\u1eed d\u1ee5ng TaqMan probe.<\/p>\n\n\n\n

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    C\u00e1c th\u00f4ng s\u1ed1 \u1ea3nh h\u01b0\u1edfng \u0111\u1ebfn \u0111\u1ed9 ch\u00ednh x\u00e1c c\u1ee7a m\u1ed9t x\u00e9t nghi\u1ec7m Real-time PCR<\/strong><\/h2>\n\n\n\n

    \u0110\u1ed9 ch\u00ednh x\u00e1c c\u1ee7a m\u1ed9t x\u00e9t nghi\u1ec7m real-time PCR \u0111\u01b0\u1ee3c \u0111\u00e1nh gi\u00e1 qua nhi\u1ec1u th\u00f4ng s\u1ed1 quan tr\u1ecdng, v\u00e0 TaqMan probe \u0111\u00f3ng vai tr\u00f2 trung t\u00e2m trong vi\u1ec7c t\u1ed1i \u01b0u h\u00f3a c\u00e1c th\u00f4ng s\u1ed1 n\u00e0y. C\u00e1c th\u00f4ng s\u1ed1 \u0111\u00f3 bao g\u1ed3m:<\/p>\n\n\n\n

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    1. \u0110\u1ed9 nh\u1ea1y (Sensitivity):<\/strong> L\u00e0 kh\u1ea3 n\u0103ng ph\u00e1t hi\u1ec7n \u0111\u01b0\u1ee3c m\u1ed9t l\u01b0\u1ee3ng r\u1ea5t nh\u1ecf tr\u00ecnh t\u1ef1 DNA\/RNA m\u1ee5c ti\u00eau trong m\u1eabu. X\u00e9t nghi\u1ec7m qPCR s\u1eed d\u1ee5ng TaqMan probe c\u00f3 \u0111\u1ed9 nh\u1ea1y r\u1ea5t cao, c\u00f3 th\u1ec3 ph\u00e1t hi\u1ec7n ch\u1ec9 v\u00e0i b\u1ea3n sao c\u1ee7a tr\u00ecnh t\u1ef1 \u0111\u00edch. \u0110\u1ed9 nh\u1ea1y th\u01b0\u1eddng \u0111\u01b0\u1ee3c \u0111\u00e1nh gi\u00e1 d\u1ef1a tr\u00ean gi\u00e1 tr\u1ecb gi\u1edbi h\u1ea1n ph\u00e1t hi\u1ec7n (Limit of Detection – LOD):<\/strong> L\u00e0 n\u1ed3ng \u0111\u1ed9 th\u1ea5p nh\u1ea5t c\u1ee7a l\u01b0\u1ee3ng ph\u00e2n t\u00edch m\u00e0 x\u00e9t nghi\u1ec7m c\u00f3 th\u1ec3 ph\u00e1t hi\u1ec7n \u0111\u01b0\u1ee3c m\u1ed9t c\u00e1ch \u0111\u00e1ng tin c\u1eady, th\u01b0\u1eddng v\u1edbi \u0111\u1ed9 tin c\u1eady 95%.<\/li>\n\n\n\n
    2. \u0110\u1ed9 \u0111\u1eb7c hi\u1ec7u (Specificity): <\/strong>L\u00e0 kh\u1ea3 n\u0103ng c\u1ee7a x\u00e9t nghi\u1ec7m ch\u1ec9 ph\u00e1t hi\u1ec7n \u0111\u00fang tr\u00ecnh t\u1ef1 m\u1ee5c ti\u00eau mong mu\u1ed1n m\u00e0 kh\u00f4ng c\u00f3 ph\u1ea3n \u1ee9ng ch\u00e9o v\u1edbi c\u00e1c tr\u00ecnh t\u1ef1 kh\u00f4ng li\u00ean quan ho\u1eb7c c\u00e1c s\u1ea3n ph\u1ea9m ph\u1ee5 kh\u00f4ng mong mu\u1ed1n. TaqMan probe b\u1ed5 sung m\u1ed9t l\u1edbp ki\u1ec3m so\u00e1t \u0111\u1eb7c hi\u1ec7u th\u1ee9 ba, sau c\u1eb7p m\u1ed3i xu\u00f4i v\u00e0 m\u1ed3i ng\u01b0\u1ee3c, l\u00e0m t\u0103ng \u0111\u00e1ng k\u1ec3 \u0111\u1ed9 tin c\u1eady c\u1ee7a k\u1ebft qu\u1ea3.<\/li>\n\n\n\n
    3. \u0110\u1ed9 ch\u1ee5m (Precision):<\/strong> \u0110\u1ecbnh ngh\u0129a: M\u1ee9c \u0111\u1ed9 g\u1ea7n g\u0169i gi\u1eefa c\u00e1c k\u1ebft qu\u1ea3 th\u1eed nghi\u1ec7m \u0111\u1ed9c l\u1eadp thu \u0111\u01b0\u1ee3c trong nh\u1eefng \u0111i\u1ec1u ki\u1ec7n quy \u0111\u1ecbnh. \u0110\u1ed9 ch\u1ee5m th\u01b0\u1eddng \u0111\u01b0\u1ee3c th\u1ec3 hi\u1ec7n d\u01b0\u1edbi d\u1ea1ng \u0111\u1ed9 l\u1ec7ch chu\u1ea9n (SD) ho\u1eb7c h\u1ec7 s\u1ed1 bi\u1ebfn thi\u00ean (%CV). \u0110\u1ed9 ch\u1ee5m bao g\u1ed3m:\n