黴漿菌之污染測試--PCR法

1.0 原理:
利用具特殊專一性之primers,經由PCR反應來複製mycoplasma DNA。所用之primers來自mycoplasma之conserved 16S-23S rRNA序列,由於此段spacer之序列依mycoplsma種類不同而不同,因此可依所複製之DNA大小及其restriction fragment大小差異來作偵測與鑑定。
  1.1 特點:
1.1.1 靈敏(e.g. 0.1~1.6 CFU / 5 ul sample) 與快速(一天)。
1.1.2 可偵測不易培養之mycoplasma (e.g. M. hyorhinis)。
1.1.3 不需培養mycoplasma作為正反應對照組,避免可能之污染。
1.2 缺點︰
1.2.1 PCR反應很靈敏,易有偽陽性結果。
1.2.2 此方法尚在評估中,故結果僅作為參考和內部品管之一部份。
2.0 材料與設備:
 

2.1 ATCC mycoplasma detection kit:
2.1.1 可作50~100 reactions.
2.1.2 提供1st stage primer mixture與2nd stage primer mixture
2.1.3 positive control DNA (A. laidlawii ; M. pirum)

2.2 PCR reagents :
2.2.1 Taq polymerase ( 5 U / ul )
2.2.2 dNTP( dGTP, dCTP, dTTP, dATP, 1.25 mM each )
2.2.3 10x PCR buffer (with 1.5mM MgCl2)
2.2.4 25mM MgCl2
2.2.5 sterile ddH2O

2.3 Machine / Equipment :
2.3.1 PCR thermal cycler
2.3.2 agarose電泳設備
2.3.3 DNA電泳膠體觀察設備
2.3.4 手套
2.3.5 無菌PCR反應管
2.3.6 無菌1.5 ml微量離心管
2.3.7 無菌2ul, 20ul, 200ul, 1000ul Tips/ Pipetmans

3.0 方法:
此PCR反應是一種nested PCR,包括2階段PCR反應,1 st stage PCR結束後,取其反應物作2nd stage PCR,然後取2 nd PCR產物作agarose gel electrophoresis,依DNA band之有無及片段大小來分析結果。
  3.1 1st stage PCR reaction(total volume 25 ul):
3.1.1 測試樣品 ( 2 ul / each )
3.1.1.1 直接取樣測試細胞之培養液。
3.1.1.2 Positive control : mycoplasma DNA ( A. laidlawii ; M. pirum )
3.1.1.3 Negative control : ddH2O
3.1.2 Reaction mixture ( 23 ul / each )
3.1.2.1 10x PCR buffer (含1.5 mM MgCl2 ) 2.5 ul
3.1.2.2 1st stage primer mixture 0.5 ul
3.1.2.3 dNTP ( 1.25 mM each ) 1.0 l
3.1.2.4 MgCl2 ( 25 mM ) 0.5 ul
3.1.2.5 Taq DNA polymerase ( 5 U/ul ) 0.1 ul
3.1.2.6 ddH2O 18.4 ul
3.1.3 PCR program ( 1st PCR與2 nd PCR相同):使用PCR thermal cycler
3.1.3.1 step 1 : denaturation: 94 ℃ 30 sec
3.1.3.2 step 2 : denaturation: 94 ℃ 30 sec
3.1.3.3 step 3 : annealing: 55 ℃ 2 min
3.1.3.4 step 4 : extension: 72 ℃ 2 min
3.1.3.5 重複3.1.3.2至3.1.3.4步驟30 cycles
3.1.3.6 step 5 : final extension 72 ℃ 5 min

3.2 2 nd stage PCR reaction(total volume 25 ul)
3.2.1 測試樣品:1st stage PCR product(1 ul / each)。
3.2.2 Reaction mixture ( 24 ul / each )
3.3.2.1 10x PCR buffer (含1.5 mM MgCl2 ) 2.5 ul
3.3.2.2 2 nd stage primer mixture 0.5 ul
3.3.2.3 dNTP ( 1.25 mM each ) 1.0 ul
3.3.2.4 MgCl2 ( 25 mM ) 0.5 ul
3.3.2.5 Taq DNA polymerase ( 5U/ul ) 0.1 ul
3.3.2.6 ddH2O 19.4 ul
3.2.3 PCR program同3.1.3;使用PCR thermal cycler

4.0 膠片電泳分析
  4.1 膠片 (2.0%) 置備: 將2 g agarose溶於100 ml ( 1x ) TAE buffer。
4.2 電泳液:(1x) TAE buffer(Tris-acetate/EDTA electropheresis buffer)
4.3 選擇100~500 bp DNA size Marker:取100 bp ladder Marker 5 ul ( 25 ng/ul )
4.4 取10 ul 2 nd stage PCR產物分析,各加入2 ul ( 6x ) loading dye。
4.5 進行電泳分離100V, 25 min。
4.6 膠片染色:ethidium bromide染色10 min,H2O退染10mim。(EtBr為致癌物質,請戴手套並小心操作)
4.7 結果:使用UV light觀察,並照相記錄。
 
  Figure 1 : Agarose gel electrophoresis of the 2nd -stage PCR Products from eight commonly encountered Mycoplasma and A. laidlawii Species. ( from ATCC mycoplasma detection kit)
   
  Table 1 : Variations of restriction fragment lengths of the 16S-23S rRNA intergenic spacer regions of commonly encountered species of mycoplasma. (from ATCC mycoplasma detection kit)
 
Mycoplasma species Size of 2nd stage PCR product Size of restriction Fragments (bp)
Vsp I Hind III Cla I
M. arginini 236 bp 134, 102 - -
M. fermentans 365 bp 270, 95 241, 124 -
M. hominis** 236 bp 123, 113 - 253, 62
M. hyorhinis 315 bp - - -
M. oral 290 bp 151, 139 - -
M. pirum 323 bp 169, 154 285, 38 -
M. salivarium 269 bp - - -
A. laidlawii 426 bp 219, 198 - -
  219 bp 189, 39 - -
  * No restriction site
** When a polyacrylamide gel is used for the resolution of amplified DNA products, a double-band product with one bp difference may be observed for M. hominis. This feature can serve as a good indicator for differentiated M. hominis from M. arginini, which only produces a single-band DNA product.
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