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首页 > 过刊浏览>2018年第28卷第2期 >90-97. DOI:10.3969. j. issn. 1671 -7856. 2018. 02. 016
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树鼩粪便中沙门氏菌LAMP 检测方法建立及应用
作者:
  • 王璇

    王璇

    中国医学科学院/ 北京协和医学院医学生物学研究所树鼩种质资源中心,中国医学科学院医学生物学研究所实验树鼩标准化与应用研究省创新团队,云南省眼科疾病防治研究重点实验室,昆明 650118
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  • 袁园

    袁园

    中国医学科学院/ 北京协和医学院医学生物学研究所树鼩种质资源中心,中国医学科学院医学生物学研究所实验树鼩标准化与应用研究省创新团队,云南省眼科疾病防治研究重点实验室,昆明 650118
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  • 张志成

    张志成

    中国医学科学院/ 北京协和医学院医学生物学研究所树鼩种质资源中心,中国医学科学院医学生物学研究所实验树鼩标准化与应用研究省创新团队,云南省眼科疾病防治研究重点实验室,昆明 650118
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  • 刘城秀

    刘城秀

    中国医学科学院/ 北京协和医学院医学生物学研究所树鼩种质资源中心,中国医学科学院医学生物学研究所实验树鼩标准化与应用研究省创新团队,云南省眼科疾病防治研究重点实验室,昆明 650118
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  • 宋庆凯

    宋庆凯

    中国医学科学院/ 北京协和医学院医学生物学研究所树鼩种质资源中心,中国医学科学院医学生物学研究所实验树鼩标准化与应用研究省创新团队,云南省眼科疾病防治研究重点实验室,昆明 650118
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  • 孙晓梅

    孙晓梅

    中国医学科学院/ 北京协和医学院医学生物学研究所树鼩种质资源中心,中国医学科学院医学生物学研究所实验树鼩标准化与应用研究省创新团队,云南省眼科疾病防治研究重点实验室,昆明 650118
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作者单位:

(中国医学科学院/ 北京协和医学院医学生物学研究所树鼩种质资源中心,中国医学科学院医学生物学研究所实验树鼩标准化与应用研究省创新团队,云南省眼科疾病防治研究重点实验室,昆明 650118)

中图分类号:

R-33

基金项目:

云南省科技人才和平台计划项目(2017HC019);重点实验室运行补助专项(2017DG008);云南省重大科技专项(2017ZF007)


Establishment and application of a loop-mediated isothermal amplification assay for the detection of Salmonella in feces of tree shrews
Author:
  • WANG Xuan

    WANG Xuan

    Institute of Medical Biology, Chinese Academy of Medical Sciences (CAMS)& Peking Union Medical College (PUMC) Institute of Medical Biology Tree Shrew Germplasm Resource Center, Institute of Medical Biology, Chinese Academy of Medicine Sciences Experimental Tree Shrew Standardization and Application Research Provincial Innovation Team, The Key Laboratory of Yunnan Province for Ophthalmic Research and Disease Control, Kunming 650118, China
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  • YUAN Yuan

    YUAN Yuan

    Institute of Medical Biology, Chinese Academy of Medical Sciences (CAMS)& Peking Union Medical College (PUMC) Institute of Medical Biology Tree Shrew Germplasm Resource Center, Institute of Medical Biology, Chinese Academy of Medicine Sciences Experimental Tree Shrew Standardization and Application Research Provincial Innovation Team, The Key Laboratory of Yunnan Province for Ophthalmic Research and Disease Control, Kunming 650118, China
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  • ZHANG Zhicheng

    ZHANG Zhicheng

    Institute of Medical Biology, Chinese Academy of Medical Sciences (CAMS)& Peking Union Medical College (PUMC) Institute of Medical Biology Tree Shrew Germplasm Resource Center, Institute of Medical Biology, Chinese Academy of Medicine Sciences Experimental Tree Shrew Standardization and Application Research Provincial Innovation Team, The Key Laboratory of Yunnan Province for Ophthalmic Research and Disease Control, Kunming 650118, China
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  • LIU Chengxiu

    LIU Chengxiu

    Institute of Medical Biology, Chinese Academy of Medical Sciences (CAMS)& Peking Union Medical College (PUMC) Institute of Medical Biology Tree Shrew Germplasm Resource Center, Institute of Medical Biology, Chinese Academy of Medicine Sciences Experimental Tree Shrew Standardization and Application Research Provincial Innovation Team, The Key Laboratory of Yunnan Province for Ophthalmic Research and Disease Control, Kunming 650118, China
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  • SONG Qingkai

    SONG Qingkai

    Institute of Medical Biology, Chinese Academy of Medical Sciences (CAMS)& Peking Union Medical College (PUMC) Institute of Medical Biology Tree Shrew Germplasm Resource Center, Institute of Medical Biology, Chinese Academy of Medicine Sciences Experimental Tree Shrew Standardization and Application Research Provincial Innovation Team, The Key Laboratory of Yunnan Province for Ophthalmic Research and Disease Control, Kunming 650118, China
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  • SUN Xiaomei

    SUN Xiaomei

    Institute of Medical Biology, Chinese Academy of Medical Sciences (CAMS)& Peking Union Medical College (PUMC) Institute of Medical Biology Tree Shrew Germplasm Resource Center, Institute of Medical Biology, Chinese Academy of Medicine Sciences Experimental Tree Shrew Standardization and Application Research Provincial Innovation Team, The Key Laboratory of Yunnan Province for Ophthalmic Research and Disease Control, Kunming 650118, China
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Affiliation:

(Institute of Medical Biology, Chinese Academy of Medical Sciences (CAMS)& Peking Union Medical College (PUMC) Institute of Medical Biology Tree Shrew Germplasm Resource Center, Institute of Medical Biology, Chinese Academy of Medicine Sciences Experimental Tree Shrew Standardization and Application Research Provincial Innovation Team, The Key Laboratory of Yunnan Province for Ophthalmic Research and Disease Control, Kunming 650118, China)

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    摘要:

    目的 建立树鼩粪便沙门氏菌的环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测方法,并对此方法进行初步应用?方法 根据沙门氏菌种属特异性基因invA(侵袭蛋白基因A)的保守序列,设计合成特异性LAMP 引物?分别设定十个反应温度即57℃ ~66℃和十个反应时间即24 ~42 min 进行优化,并测定本方法的特异性和灵敏度;同时进行普通PCR 实验,作为本方法的验证和比较?用建立的LAMP 法对91 份野生来源的树鼩稀便样品进行检测?结果 优化反应条件选出反应温度为62℃,反应时间34 min;该方法对沙门氏菌的灵敏度可达3.36 ×101 CFU/ mL,比普通PCR 方法高10 ~100 倍;对10 种树鼩来源的肠道细菌进行LAMP 检测,只有沙门氏菌属的肠炎沙门氏菌和乙型副伤寒沙门氏菌检测为阳性,其余为阴性;对91 份野生树鼩粪便样品进行LAMP检测,阳性检出率为20.88%;LAMP 法的所有反应可在40 min 内完成?结果 可通过肉眼观察颜色变化直接判定?结论 所建立的LAMP 方法具有便捷?快速?灵敏?特异等特点,可用于树鼩粪便中沙门氏菌的大规模快速检测?

    Abstract:

    Objective To Establish a loop-mediated isothermal amplification (LAMP) assay for detection of Salmonella in fecal samples of tree shrews, and report the result of preliminary application of this method. Methods LAMP primers were designed and synthesized according to the conserved sequence of Salmonella specific gene invA (invasive protein gene A). To optimize the reaction time and temperature by setting 10 reaction times (24 to 42 min) and temperature (57℃ to 66℃) and tested its specificity and sensitivity. At the same time, a conventional PCR test was performed to verify and compare with the LAMP assay. 91 fecal samples of wild-derived tree shrews were detected by the LAMP assay. Results The experimental condition was confirmed as 62℃ and 34 min. The sensitivity of Salmonella was 3.36 ×101 CFU/ mL, which was 10 to 100 times higher than that of conventional PCR assay. In 10 kinds of intestinal bacteria for LAMP amplification, Salmonella enteritidis and Salmonella paratyphi B were positive, the others were negative. Among the 91 samples of tree shrew fecal samples detected by the LAMP assay, the positive detection rate was 20.88%. The LAMP assay can be completed within 40 min, the result can be observed and judged visually by color changes. Conclusions The LAMP assay established in this study is convenient, rapid, sensitive and specific. It can be used as a rapid measure for large?scale detection of Salmonella in feces of tree shrews.

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王璇,袁园,张志成,刘城秀,宋庆凯,孙晓梅.树鼩粪便中沙门氏菌LAMP 检测方法建立及应用[J].中国比较医学杂志,2018,28(2):90~97.

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历史
  • 收稿日期:2017-09-20
  • 在线发布日期: 2018-03-14
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