PCR Technology

1. PCR Technology Use of a Liposomal Internal Control Vehicle for Whole-Process Quality Assurance of Nucleic Acid Amplification 黃慶輝 2014/08/19

2. 伊波拉病毒 (Ebola Virus) 病毒呈長條形，外有病毒套膜，內有非分段的負股ＲＮＡ（enveloped, nonsegmented, negative-strand RNA） 致死率高達 80~90% 症狀沒有特異性，發高燒、嚴重倦怠、肌肉痛、頭痛、嘔吐、和腹瀉等等 目前常用的診斷方法為: RT-PCR、ELISE 如何精準判斷呢 ? http://liuchienyi.pixnet.net/blog/post/58030995-%E5%8D%B1%E6%A9%9F%E7%B8%BD%E5%8B%95%E5%93%A1%28%E6%B7%BA%E8%AB%87%E4%BC%8A%E6%B3%A2%E6%8B%89%E7%97%85%E6%AF%92%29

3. Is this data true? False-negative result ? https://norgenbiotek.com/display-product.php?ID=426

4. The principal sources of false-negative results • I. Inhibition PCR inhibitors generally exert their effects through direct interaction with DNA or interference with thermostableDNA polymerases. EX: Heme、phenol • II. Human and Technical Operational Errors • III. Presence of Nucleases Active nucleases lead to sample nucleic acid degradation and removal of the target sequences. EX: DNases、RNases To validate and improve the interpretation of data, it is critical to include the appropriate internal controls.

5. The difference of internal control Naked nucleic acid Endogenous Internal Controls Exogenous Internal Controls

6. Use of naked nucleic acid internal controls Advantages Similar amplification efficiency Easy operator Disadvantages Only be spiked into the sample at the lysis stage. Sample storage and transportation Real-time PCR analysis Nucleic acid extraction

7. Endogenous Internal Controls EX: Inhibition or preferential amplification Advantages Confirmation of the integrity of the nucleic acid Quality assurance of the early stages. Disadvantages Dissimilar amplification efficiencies Real-time PCR analysis Nucleic acid extraction

8. The difference of exogenous Internal Controls Cell or virus types: Unrelated Related Recombinant DNA technology Synthetic Vehicles Assembled In Vitro Liposome material

9. Internal Controls Retrieved from Unrelated Bacteria or Viruses Dissimilar amplification efficiencies • Disadvantages • Involves the use of several primer sets and a multiplex amplification. • This approach is overall considered to be nonideal. Real-time PCR analysis Nucleic acid extraction

10. Internal Controls Retrieved from Related Bacteria or Viruses Interest and the internal control must be distinguishable. • Advantages • Amplified with equal efficiency to the analysis targets. • Similar physical and chemical properties to the organism Real-time PCR analysis Nucleic acid extraction

11. Internal Controls Cloned into Genetically Modified Cells or Viruses • Carrying suitable internal controls • Similar stability and disruption during the lysis process • Similar amplification efficiency Cell Virus

12. Liposome -based internal control particle technology • Liposomes can be regarded as the simplest cell membrane and the space available inside liposomes is very large. • Similar stability and disruption during the lysis process • Similar amplification efficiency • It must be nonpermeable to nucleases Bacteria Cell Mimics or Leukocyte Cell Mimics

13. SYNTHESIS OF IC PARTICLES • I. Preparation of naked internal control nucleic acid Internal controls can be made by straightforward PCR • II. Preparation of Liposomal IC Particles Film method Reverse -phase evaporation method(oil in water method)

14. Preparation of stable positively charged icParticles by film method http://www.hindawi.com/journals/jnt/2012/748909/fig3/

15. Preparation of negatively charged icparticles with increased density by reverse -phase evaporation method http://myfunnyvalentineblog.com/2011/05/skin-care-saturday-dr-spiller-biocosmetics.html Biophysical journal, 2014, 15, 346-354