淋病奈瑟菌核酸的不同提取方法比較及評價_《華西醫學》

作者：

四川大學華西醫院實驗醫學科臨床分子診斷實驗室;

關鍵詞：

淋病奈瑟菌 DNA提取實時熒光定量聚合酶鏈反應

視頻：

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目的　比較評價磁珠法、柱子吸附法和煮沸裂解法3種不同的核酸提取方法對淋病奈瑟菌（淋球菌）DNA提取效率的差異。方法　對已確診的淋球菌培養基挑取20個小菌落制成懸液，做1︰1～1︰100 000稀釋度稀釋，分別使用磁珠法、柱子吸附法和煮沸裂解法進行DNA提取，用實時熒光定量PCR對其提取效率進行評價。　結果　在不同濃度淋球菌的DNA提取中，以磁珠法的敏感性和重復性最高，而柱子吸附法和煮沸裂解法次之，＜1×105 copies /mL以下的拷貝數，3種DNA提取方法之間重復性和敏感性差異明顯，磁珠法的提取效率最高，柱子吸附法次之，煮沸裂解法最差；＞1×105 copies/mL的細菌拷貝數的樣本，3種方法差異隨濃度增高不斷縮小。結論　磁珠法提取淋球菌DNA的效率、敏感性和重復性最高，在實際臨床科研工作中，各實驗室可根據自身實際情況選擇不同的DNA提取方法。

引用本文： 莊杰,劉顯中,葉遠馨,周易,宋興勃,陸小軍,王軍,丁柳,代波,應斌武. 淋病奈瑟菌核酸的不同提取方法比較及評價. 華西醫學, 2012, 27(4): 560-563. doi: 復制

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1. 　Mackay IM. Real-time PCR in themicrobiology laboratory[J]. Clin Microbiol Infect, 2004, 10(3): 190-212.
2. 　姚遠, 張國富, 劉玉英. PCR污染與對策及假陰性原因[J]. 實用醫技雜志, 2002, 9(4): 276-277.
3. 　Knepp JH., Geahr MA, Forman MS. Comparison of automated and manual nucleic acid extraction methods for detection of enterovirus RNA[J]. J Clin Microbiol, 2003, 41(8): 3532-3536.
4. 　Konomi N, Lebwohl E, Zhang D. Comparison of DNA and RNA extraction methods for mummified tissues[J]. Mol Cell Probes, 2002, 16(6): 445-451.
5. 　Sweet D, Lorente M, Valenzuela A, et al. Increasing DNA extraction yield from saliva stains with a modified Chelex method[J]. Forensic Sci Int, 1996, 83(3): 167-177.
6. 　Stein A, Raoult D. A simple method amplification of DNA from paraffin embedded tissues[J]. Nucleic Acids Res, 1992, 20(19): 5237-5238.
7. 　Dundas N, Leos NK, Mitui M, et al. Comparison of automated nucleic acid extraction methods with manual extraction[J]. J Mol Diagn, 2008, 10(4): 311-316.
8. 　Claassen M, Van zyl GU, Preiser W. Extraction buffer contaminated bacterially as a cause of invalid HIV-1 viral load results on the NucliSens EasyQ system[J]. J Virol Methods, 2008, 150(12): 80-81.
9. 　Stevens W, Horsfield P, Scott LE. Evaluation of the performance of the automated NucliSENS easyMAG and EasyQ systems versus the Roche AmpliPrep-AMPLICOR combination for high-throughput monitoring of human immunodeficiency virus load[J]. J Clin Microbiol, 2007, 45(4): 1244-1249.
10. 　Thakur R, Sarma S, Goyal R. Comparison of DNA extraction protocols for mycobacterium tuberculosis in diagnosis of tuberculous meningitis by real-time polymerase chain reaction[J]. J Glob Infect Dis, 2011, 3(4): 353-356.
11. 　Ursi D, Dirven K, Loens K, et al. Detection of mycoplasma pneumoniae in respiratory samples by real-time PCR using an inhibition control[J]. Microbiol Methods, 2003, 55(1): 149-153.
12. 　Bahl MI, Bergström A, Licht TR. Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis[J]. FEMS Microbiol Lett, 2012, 329(2): 193-197.
13. 　Kouduka M, Suko T, Morono Y, et al. A new DNA extraction method by controlled alkaline treatments from consolidated subsurface sediments[J]. FEMS Microbiol Lett, 2012, 326(1): 47-54.

《華西醫學》

淋病奈瑟菌核酸的不同提取方法比較及評價

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《華西醫學》

淋病奈瑟菌核酸的不同提取方法比較及評價

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

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