本文關(guān)鍵詞： 結(jié)核分枝桿菌實(shí)驗(yàn)室 終末消毒效果評(píng)價(jià) 方法建立　出處：《中國(guó)公共衛(wèi)生》2016年11期 　論文類(lèi)型：期刊論文

【摘要】：目的建立一種便捷可靠的輔助評(píng)價(jià)結(jié)核桿菌實(shí)驗(yàn)室終末消毒效果的新方法。方法首先以結(jié)核分枝桿菌復(fù)合群特異性插入序列IS6110為靶標(biāo)自主設(shè)計(jì)引物IS6110P,并經(jīng)結(jié)核分枝桿菌標(biāo)準(zhǔn)株H37Rv的培養(yǎng)物驗(yàn)證其特異性和靈敏度;其次,采用H37Rv培養(yǎng)物抽濾膜經(jīng)甲醛終末消毒后分別采用普通核酸提取法和完整基因組提取核酸法提取核酸,再經(jīng)IS6110P引物進(jìn)行特異性PCR擴(kuò)增驗(yàn)證其輔助評(píng)價(jià)甲醛終末消毒效果的情況,同時(shí)對(duì)抽濾膜進(jìn)行分離培養(yǎng)以驗(yàn)證其評(píng)價(jià)結(jié)果;最后,采集經(jīng)甲醛終末消毒結(jié)核桿菌實(shí)驗(yàn)室內(nèi)的拭子和大氣樣本采用上述方法檢測(cè),并與分離培養(yǎng)結(jié)果相驗(yàn)證,以檢測(cè)該方法的評(píng)價(jià)效果。結(jié)果 IS6110P可靈敏特異地對(duì)結(jié)核分枝桿菌進(jìn)行擴(kuò)增,檢測(cè)限可達(dá)3.5 cfu/R;H37Rv培養(yǎng)物抽濾膜甲醛終末消毒后經(jīng)普通核酸提取擴(kuò)增仍有特異性核酸片段檢出,而經(jīng)完整基因組提取核酸擴(kuò)增無(wú)特異性核酸片段檢出;終末消毒后的結(jié)核分枝桿菌實(shí)驗(yàn)室采用普通核酸提取仍可擴(kuò)增出結(jié)核分枝桿菌特異性核酸片段,而完整基因組提取核酸無(wú)特異性核酸擴(kuò)增片段檢出;平行的結(jié)核分枝桿菌分離培養(yǎng)實(shí)驗(yàn)結(jié)果均為陰性。結(jié)論采用IS6110P擴(kuò)增結(jié)核分枝桿菌完整基因組的核酸提取物為輔助評(píng)價(jià)結(jié)核分枝桿菌實(shí)驗(yàn)室終末消毒效果的靈敏、便捷和可靠的新方法。
[Abstract]:Objective to establish a convenient and reliable method to evaluate the efficacy of terminal disinfection of Mycobacterium tuberculosis in laboratory. Methods the primer IS6110Pwas designed with the specific insertion sequence IS6110 of Mycobacterium tuberculosis as the target. The specificity and sensitivity of the culture of the standard strain H37Rv were verified. Secondly, H37Rv culture was used to extract the nucleic acid from the filter membrane by formaldehyde terminal disinfection, and then the nucleic acid was extracted by ordinary nucleic acid extraction method and complete genome extraction method, respectively. Then the specific PCR amplification with IS6110P primer was used to verify the effect of formaldehyde terminal disinfection. At the same time, the filter membrane was separated and cultured to verify the evaluation results. The swabs and atmospheric samples collected from the laboratory of formalin terminal disinfection of Mycobacterium tuberculosis were detected by the above method and verified with the results of isolation and culture. Results IS6110P could sensitively and specifically amplify Mycobacterium tuberculosis, and the detection limit could be up to 3.5 cfur / r ~ (37) Rv culture after the final disinfection of formaldehyde in the filter membrane, and the specific nucleic acid fragments could still be detected by ordinary nucleic acid extraction and amplification. However, the nucleic acid fragments of Mycobacterium tuberculosis were amplified by complete genome extraction, and the isolated fragments of mycobacterium tuberculosis could be amplified by ordinary nucleic acid extraction in the laboratory of Mycobacterium tuberculosis after final disinfection. However, the complete genome extracted nucleic acid with no specific nucleic acid amplification fragment was detected. Conclusion the nucleic acid extract from the complete genome of Mycobacterium tuberculosis amplified by IS6110P is a sensitive method to evaluate the efficacy of terminal disinfection of Mycobacterium tuberculosis in laboratory. Convenient and reliable new method.
【作者單位】： 遼寧省疾病預(yù)防控制中心結(jié)核病防制所;
【基金】：遼寧省科技廳計(jì)劃項(xiàng)目(2013225079);遼寧省科技廳衛(wèi)生安全重點(diǎn)實(shí)驗(yàn)室項(xiàng)目(2007403016)
【分類(lèi)號(hào)】：R378.911;R440

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