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¡¡ | 1. EMA, PMA, PMAxxÀÇ Â÷ÀÌÁ¡Àº ¹«¾ùÀԴϱî? |
¡¡ | Viability PCRÀº ¿ø·¡ EMA (ethidium monoazide)¸¦ ÀÌ¿ëÇÏ¿© dead cell DNA¸¦ ºÒȰ¼ºÈ½ÃÄ×½À´Ï´Ù. BiotiumÀº Montana State University¿Í Çù·ÂÇÏ¿© PMA (propidium monoazide)¸¦ °³¹ßÇß½À´Ï´Ù. PMA´Â EMA¿¡ ºñÇØ dead cell¿¡ ´ëÇØ ´õ ¼±ÅÃÀûÀ̸ç viable microbe¿Í virus °ËÃâ¿¡ ³Î¸® »ç¿ëµÇ¾îÁ® ¿Ô½À´Ï´Ù. PMAxx´Â Biotium¿¡¼ Ãâ½ÃµÈ »õ·Î¿î viability PCR dye·Î, dead cell DNA°¡ PCR ÁõÆøÀÌ µÇÁö ¾Êµµ·Ï Çϴµ¥ ´õ È¿°úÀûÀÔ´Ï´Ù. ±×·¡¼ live & dead bacteria¸¦ °¡Àå Àß ±¸º°ÇÒ ¼ö ÀÖ½À´Ï´Ù. |
¡¡ | 2. PMA stockÀº DMSO¿¡ ÁغñÇØ¾ß Çϳª¿ä? |
¡¡ | ºñ¼ö¿ë¼ºÀÎ EMA¿Í´Â ´Þ¸® PMA³ª PMAxx´Â ¼ö¿ë¼ºÀ̶ó 20mM solution in water·Î Á¦°øµË´Ï´Ù. °íü·Î Á¦°øµÉ °æ¿ì¿¡µµ DMSO¿¡ ³ìÀÏ ÇÊ¿ä´Â ¾ø½À´Ï´Ù. |
¡¡ | 3. PMA³ª PMAxxÀÇ phytolysis¸¦ À§ÇØ ¾î¶² ±¤¿øÀ» ÀÌ¿ëÇØ¾ß Çϳª¿ä? |
¡¡ | BiotiumÀº PMA-Lite LED Photolysis Device¸¦ Á¦°øÇÕ´Ï´Ù. ±¤¿øÀ» ÅëÇØ PMAxx³ª PMA¸¦ dsDNA·Î cross-linking ½Ãŵ´Ï´Ù. |
¡¡ | 4. PMA¸¦ »ç¿ëÇϰí Àִµ¥ PMAxx·Î ¹Ù²Ù¾î¾ß Çϳª¿ä? |
¡¡ | Bacteria¸¦ ÀÌ¿ëÇÏ¿© viability PCR °á°ú¸¦ ºñ±³ÇØ º¸¾ÒÀ»¶§ PMAxx°¡ ´õ Ȱ¼ºÀÌ ÁÁ¾Ò½À´Ï´Ù. ÃÖ±Ù ³ë·Î¹ÙÀÌ·¯½º¸¦ ÀÌ¿ëÇÑ viability PCR ³í¹®¿¡¼µµ PMAxx°¡ ´õ Àß ÀÛ¿ëÇßÀ½À» È®ÀÎÇÒ ¼ö ÀÖ½À´Ï´Ù. YeastÀÇ °æ¿ì¿¡´Â PMA°¡ PMAxx¿Í ºñ½ÁÇϰųª Á¶±Ý ´õ Ȱ¼ºÀÌ ÁÁ¾Ò½À´Ï´Ù. µû¶ó¼ baceria³ª virus¿¡´Â PMAxx, yeast³ª fungusÀÇ viability PCR¿¡´Â PMA¸¦ ÃßõÇÕ´Ï´Ù. |
¡¡ | 5. PMA³ª PMAxx¸¦ ȯ°æ »ùÇÿ¡ »ç¿ëÇÒ ¼ö ÀÖ³ª¿ä? |
¡¡ | ³×, ¸¹Àº ¿¬±¸ÀÚµéÀÌ ÀÌ¹Ì È¯°æ »ùÇÿ¡ »ç¿ëÇϰí ÀÖÀ¸³ª pure culture º¸´Ù´Â °í·ÁÇÒ »çÇ×µéÀÌ ÀÖ½À´Ï´Ù. ȯ°æ»ùÇÿ¡ ÀÌ¿ëÇÑ ³í¹®À» Âü°íÇØ ÁÖ¼¼¿ä. Water samples In order to perform viability PCR on water samples, some users have developed methods to concentrate the microorganisms onto a 0.45 um filter, and then treat the filter with PMA. An example protocol can be found in this publication: Ditommasso S., et al. (2015) Viability-qPCR for detecting Legionella: Comparison of two assays based on different amplicon lengths. Mol. Cell Probes. doi: 10.1016/j.mcp.2015.05.011. Opaque or complex samples Viability PCR with PMA has been successfully reported in various complex sample types, such as sewage or soil. No reports have been made yet with PMAxx™ in complex samples, but we would expect it to work as well or better than PMA. The main considerations for complex samples are dye concentration and light penetration. Typically a higher dye concentration (100 uM or more) is needed because some dye may bind to contaminants in the sample. Longer light treatment and more mixing during light treatment may be required, since it will be harder for the light to penetrate through the sample. An example protocol can be found in this publication: Guo F. and Zhang T. (2014) Detecting the nonviable and heat-tolerant bacteria in activated sludge by minimizing DNA from dead cells. Microb. Ecol. dio 10.1007/s00248-014-0389-2. |
¡¡ | 6. PMA³ª PMAxx¸¦ ½Ç¼ö·Î ½Ç¿Â¿¡ ¹æÄ¡ÇßÀ»¶§ ¾ÈÁ¤¼ºÀÌ ±¦ÂúÀ»±î¿ä? |
¡¡ | ºû¿¡ ³ëÃâµÇÁö ¾Ê´Â´Ù¸é chemically stable ÇϹǷΠ±¦Âú½À´Ï´Ù. ¿Èê°£ ½Ç¿Â¿¡¼ ¹æÄ¡ÇÑ ÈÄ HPLC·Î ºÐ¼®ÇßÀ»¶§ ¹®Á¦°¡ ¾øÀ½À» ³»ºÎ½ÇÇèÀ» ÅëÇØ È®ÀÎÇÏ¿´½À´Ï´Ù. ºû¿¡ ¸Å¿ì ¹Î°¨ÇÏ¿© room light ¿¡µµ ¿µÇâÀ» ¹ÞÀ» ¼ö ÀÖÀ¸¸ç chemical change¸¦ ÃÊ·¡ÇÕ´Ï´Ù. µû¶ó¼ Ç×»ó ¾îµÎ¿î °÷¿¡¼ Çڵ鸵 ÇϽñ⸦ Á¶¾ðµå¸³´Ï´Ù. |
¡¡ | 7. PMA-Lite¿¡¼ LED Á¶¸íÀÇ ÆÄÀå°ú ¹à±â´Â ¾î´ÀÁ¤µµ ÀԴϱî? |
¡¡ | PMA-LiteÀÇ LED´Â 600-800 ¹Ð¸® ĵ¨¶ó (mcd)ÀÇ ¹à±â¸¦ °®½À´Ï´Ù. °¢ Æ©ºê ¿·¿¡ 3 °³ÀÇ LED°¡ ÀÖ°í (ÇÑ ÂÊ ÇÏ´Ü, µÎ ÂÊ) ÆÄÀåÀº 465-475nmÀÔ´Ï´Ù. |
¡¡ | 8. PMA ¶Ç´Â PMAxx ó¸® ÈÄ DNA¸¦ Á¤Á¦ÇØ¾ß Çϳª¿ä? ¾Æ´Ï¸é ½Ã·á¿¡¼ Á÷Á¢ PCRÀ» ¼öÇà ÇÒ ¼ö ÀÖ½À´Ï±î? |
¡¡ | ÀϹÝÀûÀ¸·Î´Â PMA ó¸® ÈÄ¿¡ ¼¼Æ÷¿¡¼ DNA¸¦ Á¤Á¦ÇÏ¿© PCR template·Î »ç¿ëÇÏ´Â °ÍÀ¸·Î ¾Ë·ÁÁ® ÀÖ½À´Ï´Ù. ±×·¸Áö¸¸ DNA Á¤Á¦¾øÀÌ, PMA¸¦ ó¸®ÇÑ cell lysate·Îµµ viability PCRÀÌ Àß ¼öÇàµÈ »ç·ÊµéÀÌ ¸¹ÀÌ ÀÖ½À´Ï´Ù. °¡Àå Áß¿äÇÑ °ÍÀº lysate ÁغñÇϱâ Àü¿¡ ¸ðµç PMA dyeµéÀÌ Á¦´ë·Î photolysis°¡ µÇ¾ú´ÂÁö¸¦ È®ÀÎÇÏ´Â °ÍÀÔ´Ï´Ù. ºÐÇØµÇÁö ¾Ê°í ÀÜÁ¸ÇÏ´Â PMA°¡ PCR °úÁ¤¿¡ ¹æÇظ¦ ÁÙ ¼ö ÀÖ½À´Ï´Ù. ¸¸¾à ÀÌ ¹æ¹ýÀ» ¿øÇϽŴٸé, dye ¹× light 󸮸¦ ÇÑ ÀÌÈÄ¿¡ ¿ë¾×¿¡ ³²Àº dye¸¦ Á¦°ÅÇϱâ À§ÇØ cellÀ» pelletÈ Çϴ°ÍÀÌ ÁÁ½À´Ï´Ù. ÀÌ»óÀûÀÎ PMA ¹× light ó¸® Á¶°ÇÀ» °áÁ¤Çϱâ À§ÇØ control ½ÇÇèÀ» °°ÀÌ ¼öÇàÇÏ¼Å¾ß ÇÕ´Ï´Ù. |
¡¡ | 9. PMA³ª PMAxx ó¸® Àü ÈÄ¿¡ sampleÀ» ¾ó·Áµµ µÇ³ª¿ä? |
¡¡ | ÀÌ dyeµéÀº membrane-impermeable dyeÀ̸ç, membraneÀÌ ¼Õ»óµÈ dead cell¿¡ ÀÛ¿ëÇÏ¿© DNA¸¦ º¯Çü½ÃŰ´Â ¿ø¸®¸¦ ÀÌ¿ëÇÕ´Ï´Ù. ¸¸ÀÏ »ùÇÃÀÌ PMA ó¸®Àü¿¡ ¾ó·ÁÁ® ÀÖ´Ù¸é live cell membarneµµ Åõ°úÇÏ¿© live/dead cell ±¸ºÐÀ» ÇÏÁö ¸øÇÏ°Ô µË´Ï´Ù. PMA ó¸®ÇÏ¿© photolysis±îÁö ÁøÇàµÈ ÈÄ¿¡´Â DNA extraction°ú PCR Àü¿¡ º¸°üÀ» À§ÇØ ¾ó·Áµµ µË´Ï´Ù. |
¡¡ | 10. viability PCRÀ» À§ÇØ ¾î¶² primer¸¦ ÀÌ¿ëÇØ¾ß Çϳª¿ä? |
¡¡ | Biotium¿¡¼´Â ÀϺΠ±ÕÁÖ¿ë¿¡ ´ëÇÑ viability PCR kit¸¦ Á¦°øÇÕ´Ï´Ù. ¿©±â¿¡´Â strain-specific primer set°¡ Á¦°øµË´Ï´Ù. amplicon length°¡ viability PCR¿¡ ¿µÇâÀ» ¸¹ÀÌ ¹ÌĨ´Ï´Ù. ±ä ±æÀÌÀÇ ampliconÀϼö·Ï ´õ ÁÁÀº °á°ú¸¦ Á¦°øÇϹǷΠ±æ°Ô µðÀÚÀÎÇϼ¼¿ä. |
¡¡ | 11. ±×¶÷¾ç¼º°ú ±×¶÷À½¼º ¹ÚÅ׸®¾Æ°¡ È¥ÇÕµÈ unknown sampleÀ» ó¸®ÇÒ ¶§, PMA Enhancer¸¦ »ç¿ëÇØµµ µÇ³ª¿ä? |
¡¡ | ¾Æ´Ï¿ä, PMA Enhancer´Â ±×¶÷À½¼º ¹ÚÅ׸®¾Æ¿¡¸¸ »ç¿ëÇØ¾ß ÇÕ´Ï´Ù. ±×¶÷¾ç¼º ¼¼±Õ¿¡´Â À¯ÇØÇÑ ¿µÇâÀ» ³¢Ä¨´Ï´Ù. |
¡¡ | °ü·Ã Á¦Ç° |
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