华西口腔医学杂志 ›› 2016, Vol. 34 ›› Issue (3): 307-311.doi: 10.7518/hxkq.2016.03.018

• 口腔微生物学专栏 • 上一篇    下一篇

牙龈卟啉单胞菌合成环二腺苷酸的高效液相色谱-串联质谱法定性分析

谭咏梅1,杨小军1,杜娟2,赵望泓1,陈晓丹3,侯晋1   

  1. 1.南方医科大学南方医院口腔科;2.南方医科大学公共卫生与热带医学学院卫生监测中心,广州510515;3.汕头大学医学院第二附属医院口腔科,汕头515041
  • 收稿日期:2015-10-16 修回日期:2016-02-18 出版日期:2016-06-01 发布日期:2016-06-01
  • 通讯作者: 侯晋,讲师,博士,E-mail:houjin@smu.edu.cn
  • 作者简介:谭咏梅,硕士,E-mail:yobecute@126.com
  • 基金资助:
    国家自然科学基金(81500870);南方医科大学南方医院院长基金(2013Z006,2012C005)

Qualitative analysis of bis-(3’-5’)-cyclic dimeric adenosine monophosphate of Porphyromonas gingivalis by high performance liquid chromatography coupled with mass spectrometry

Tan Yongmei1, Yang Xiaojun1, Du Juan2, Zhao Wanghong1, Chen Xiaodan3, Hou Jin1   

  1. 1. Dept. of Stomatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China; 2. Hygiene Detection Center, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China; 3. Dept. of Stomatology, The Second Affiliated Hospital of Shantou University Medical College, Shantou 515041, China) Supported by: National Natural Science Foundation of China(81500870); President Foundation of Nanfang Hospital, Southern Medical University (2013Z006, 2012C005). Correspondence: Hou Jin, E-mail: houjin@smu.edu.cn.
  • Received:2015-10-16 Revised:2016-02-18 Online:2016-06-01 Published:2016-06-01

摘要: 目的 定性检测牙龈卟啉单胞菌(P. gingivalis)是否能产生细菌信号分子环二腺苷酸(c-di-AMP),为探索其在P. gingivalis生命代谢以及牙周炎免疫中的作用奠定基础。方法 以P. gingivalis标准菌株ATCC33277为实验菌株,抽提细菌内核酸物质作为样品,配置c-di-AMP标准品,通过高效液相色谱-串联质谱法(HPLC-MS/MS)和高效液相色谱法(HPLC)对样品进行验证。结果 HPLC-MS/MS检出限按照信噪比(S/N)3∶1计算,c-di-AMP标准品出峰的保留时间为7.49 min,P. gingivalis提取物样品在保留时间为8.82 min时有目标峰出现(大于3 S/N)。HPLC检测结果表明,P. gingivalis核酸提取物样品及c-di-AMP标准品均在15.7 min处出现目标峰,且二者的紫外吸收光谱相同。结论 牙龈卟啉单胞菌核酸提取物中含有c-di-AMP,牙龈卟啉单胞菌可以合成产生c-di-AMP。

关键词: 环二腺苷酸, 牙龈卟啉单胞菌, 高效液相色谱-串联质谱法

Abstract: Objective To test whether Porphyromonas gingivalis (P. gingivalis) could produce bacterial signal molecule, bis-(3’-5’)-cyclic dimeric adenosine monophosphate (c-di-AMP) and lay the foundation for explorations of its roles in life metabolism and periodontitis immunity of P. gingivalis.Methods P. gingivalis standard strain ATCC33277 was used as the experimental strain to extract nucleic acids from the bacteria. Then, c-di-AMP was detected using high performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS). Subsequently, HPLC was used to validate the sample further. Results Based on the signal/noise (S/N) for 3∶1, the limit of determination of HPLC-MS/MS for peak time of c-di-AMP standard substances was 7.49 min and nucleic acid extractions from P. gingivalis was 8.82 min (S/N>3). Further confirmation of HPLC showed that nucleic acid extractions from both P. gingivalis and c-di-AMP standard substances presented goal absorbent peaks at 15.7 min, with the same ultraviolet absorbent spectrum. Conclusion The nucleic acid extractions from P. gingivalis contained c-di-AMP, which shows that P. gingivalis could produce c-di-AMP.

Key words: bis-(3’-5’)-cyclic dimeric adenosine monophosphate, Porphyromonas gingivalis, high performance liquid chromatography coupled with mass spectrometry

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