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PCR去污試劑盒可去除PCR預(yù)混液中的污染DNA，而不會(huì)降低PCR靈敏度。
在不降低靈敏度的情況下對(duì)主混合物進(jìn)行凈化一直是一個(gè)挑戰(zhàn)。特別是當(dāng)靶向少量DNA時(shí)，污染DNA是一個(gè)主要問(wèn)題。由去污方案引起的qPCR測(cè)定中的任何靈敏度損失都是不可接受的。
圖1：PCR去污試劑盒方案
在DTT存在下，dsDNase在60℃時(shí)不可逆地失活，確保滅活后添加的任何模板都能安全地消化。
從20μl反應(yīng)中去除污染DNA的方案，但可以通過(guò)調(diào)整試劑盒內(nèi)容物的體積來(lái)放大或縮小。
500 pg untreated50 pg untreated5 pg untreated500 fg untreated50 fg untreatedNTC untreated500 pg decontaminated50 pg decontaminated5 pg decontaminated500 fg decontaminated50 fg decontaminatedNTC decontaminated5101520253035404502,5005,0007,50010,00012,50015,000Cycles14500 pg untreated:3450 pg untreated:-45 pg untreated:-5500 fg untreated:-1350 fg untreated:12NTC untreated:1500 pg decontaminated:1650 pg decontaminated:-125 pg decontaminated:-750 fg decontaminated:-9NTC decontaminated:22
| 周期 | 500 pg untreated | 50 pg untreated | 5 pg untreated | 500 fg untreated | 50 fg untreated | NTC untreated | 500 pg decontaminated | 50 pg decontaminated | 5 pg decontaminated | 500 fg decontaminated | 50 fg decontaminated | NTC decontaminated |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | -217 | -38 | -13 | -63 | -12 | -7 | -133 | 13 | -10 | 13 | 17 | 21 |
| 2 | -107 | -22 | 2 | -36 | -9 | -22 | -55 | 14 | -11 | -3 | 8 | -10 |
| 3 | -69 | -10 | -1 | -23 | -20 | -26 | -27 | 10 | -1 | -12 | 12 | -39 |
| 4 | -46 | -1 | 2 | -12 | -20 | -14 | -13 | 6 | -6 | 8 | -44 | |
| 5 | -29 | 7 | 4 | 3 | -7 | -4 | -4 | -2 | 5 | 5 | 4 | -63 |
| 6 | -11 | 9 | 6 | 8 | 11 | 1 | 5 | 1 | -6 | 9 | 4 | -63 |
| 7 | 6 | 5 | 11 | 12 | 9 | 15 | 2 | -4 | 11 | -7 | -44 | |
| 8 | 12 | -3 | 12 | 6 | -7 | 13 | 6 | 8 | 11 | 12 | 1 | -29 |
| 9 | 3 | -17 | 3 | 14 | -4 | 12 | -13 | 9 | 14 | -5 | 8 | -12 |
| 10 | -15 | -16 | -7 | 14 | 6 | 6 | -8 | -5 | 9 | -4 | 1 | 5 |
| 11 | -22 | 2 | -9 | 5 | 2 | 4 | -1 | -9 | 9 | 7 | 6 | 20 |
| 12 | -21 | 3 | -5 | -2 | -3 | 13 | -10 | 3 | 8 | 14 | 5 | 14 |
| 13 | -2 | -11 | -4 | 2 | 2 | 2 | -5 | -2 | -3 | 14 | -2 | 11 |
| 14 | 34 | -4 | -5 | -13 | 12 | 1 | 16 | -12 | -7 | -9 | 22 | |
| 15 | 89 | -3 | -16 | 8 | 7 | 60 | -3 | 2 | -9 | -5 | 19 | |
| 16 | 204 | 1 | -20 | -9 | -1 | 3 | 162 | 2 | -15 | -22 | -10 | 25 |
| 17 | 416 | 20 | -31 | -9 | -10 | 4 | 371 | 11 | -33 | -24 | -17 | 35 |
| 18 | 764 | 70 | -16 | -12 | -5 | -1 | 746 | 57 | -26 | -14 | -17 | 29 |
| 19 | 1,297 | 173 | 1 | -22 | -7 | -6 | 1,310 | 154 | -16 | -1 | -22 | 21 |
| 20 | 2,015 | 347 | 15 | -21 | -25 | -10 | 2,049 | 344 | 8 | 2 | -15 | 13 |
| 21 | 2,886 | 644 | 46 | -20 | -27 | -16 | 2,915 | 657 | 51 | -8 | -1 | 5 |
| 22 | 3,865 | 1,115 | 122 | -7 | -27 | -28 | 3,879 | 1,122 | 127 | -7 | 5 | 8 |
| 23 | 4,879 | 1,774 | 261 | 10 | -33 | -32 | 4,908 | 1,764 | 270 | 4 | -2 | 20 |
| 24 | 5,919 | 2,597 | 508 | 35 | -24 | -21 | 5,927 | 2,576 | 527 | 34 | -20 | 26 |
| 25 | 6,951 | 3,525 | 921 | 101 | -2 | -22 | 6,892 | 3,514 | 960 | 94 | -22 | 25 |
| 26 | 7,930 | 4,526 | 1,520 | 221 | 14 | -13 | 7,855 | 4,505 | 1,587 | 209 | 3 | 10 |
| 27 | 8,818 | 5,526 | 2,301 | 426 | 37 | -5 | 8,771 | 5,495 | 2,406 | 434 | 36 | 8 |
| 28 | 9,628 | 6,511 | 3,242 | 778 | 99 | 2 | 9,570 | 6,473 | 3,357 | 808 | 93 | 11 |
| 29 | 10,380 | 7,472 | 4,278 | 1,337 | 192 | 9 | 10,291 | 7,422 | 4,384 | 1,375 | 199 | 5 |
| 30 | 11,037 | 8,371 | 5,346 | 2,101 | 382 | 43 | 10,940 | 8,306 | 5,445 | 2,146 | 393 | 6 |
| 31 | 11,646 | 9,210 | 6,400 | 3,047 | 721 | 101 | 11,524 | 9,096 | 6,489 | 3,097 | 727 | 4 |
| 32 | 12,184 | 9,985 | 7,417 | 4,132 | 1,229 | 182 | 12,042 | 9,814 | 7,496 | 4,149 | 1,261 | -15 |
| 33 | 12,627 | 10,694 | 8,402 | 5,274 | 1,980 | 344 | 12,482 | 10,482 | 8,465 | 5,254 | 2,018 | -24 |
| 34 | 13,001 | 11,317 | 9,291 | 6,409 | 2,945 | 586 | 12,836 | 11,075 | 9,387 | 6,393 | 2,961 | -6 |
| 35 | 13,334 | 11,858 | 10,101 | 7,498 | 4,071 | 944 | 13,113 | 11,616 | 10,200 | 7,492 | 4,066 | 3 |
| 36 | 13,631 | 12,329 | 10,847 | 8,566 | 5,276 | 1,444 | 13,356 | 12,072 | 10,902 | 8,520 | 5,231 | 6 |
| 37 | 13,878 | 12,740 | 11,522 | 9,594 | 6,482 | 2,088 | 13,547 | 12,442 | 11,541 | 9,482 | 6,415 | 9 |
| 38 | 14,080 | 13,064 | 12,091 | 10,509 | 7,694 | 2,871 | 13,718 | 12,785 | 12,097 | 10,364 | 7,582 | -11 |
| 39 | 14,235 | 13,341 | 12,562 | 11,364 | 8,880 | 3,793 | 13,876 | 13,064 | 12,576 | 11,178 | 8,696 | -27 |
| 40 | 14,347 | 13,580 | 12,977 | 12,111 | 9,965 | 4,817 | 13,979 | 13,267 | 12,988 | 11,905 | 9,747 | -14 |
| 41 | 14,443 | 13,785 | 13,329 | 12,773 | 10,940 | 5,848 | 14,068 | 13,462 | 13,290 | 12,533 | 10,676 | -16 |
| 42 | 14,511 | 13,954 | 13,617 | 13,355 | 11,827 | 6,871 | 14,159 | 13,620 | 13,547 | 13,053 | 11,496 | -35 |
| 43 | 14,542 | 14,068 | 13,844 | 13,839 | 12,602 | 7,895 | 14,224 | 13,739 | 13,803 | 13,469 | 12,202 | -22 |
| 44 | 14,597 | 14,195 | 14,033 | 14,276 | 13,309 | 8,871 | 14,280 | 13,851 | 14,022 | 13,814 | 12,798 | |
| 45 | 14,664 | 14,332 | 14,217 | 14,689 | 14,022 | 9,824 | 14,347 | 13,952 | 14,234 | 14,147 | 13,385 | 13 |
圖2：未處理和去污染的qPCR 2x主混合物用于分析具有5個(gè)步驟的大腸桿菌gDNA 10倍系列稀釋。包括NTC樣品，并且連續(xù)稀釋的所有圖顯示平均三次重復(fù)。
使用PCR去污試劑盒進(jìn)行治療不會(huì)降低PCR的靈敏度。即使稀釋DNA，Cq水平也沒(méi)有顯著改變（圖2）。
儲(chǔ)存條件：儲(chǔ)存在-20?C。
樣品材料：該試劑盒可用于減少普通PCR和基于探針的qPCR混合物中的污染DNA。對(duì)于一些基于SYBR的qPCR混合物也是有效的。
質(zhì)量控制：測(cè)試試劑盒中是否存在RNase。
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