WO2021115077A1 - 一种基于RsaI单酶切gluA基因区分克罗诺杆菌属不同种的分子分型方法 - Google Patents

一种基于RsaI单酶切gluA基因区分克罗诺杆菌属不同种的分子分型方法 Download PDF

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WO2021115077A1
WO2021115077A1 PCT/CN2020/129687 CN2020129687W WO2021115077A1 WO 2021115077 A1 WO2021115077 A1 WO 2021115077A1 CN 2020129687 W CN2020129687 W CN 2020129687W WO 2021115077 A1 WO2021115077 A1 WO 2021115077A1
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cronobacter
rsai
glua
gene
glua gene
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叶应旺
汪馨
沈益忠
张丹凤
蒋秀婷
董晶晶
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合肥工业大学
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  • the invention belongs to the technical field of food microbial inspection, and specifically relates to a molecular typing method for distinguishing different species of Cronobacterium genus based on RsaI single enzyme digestion of gluA gene.
  • Cronobacter is a food-borne pathogen that can cause serious infections.
  • the survey of food contamination data indicates that infant formula milk powder is the main transmission vector of its infection.
  • the typing techniques of Cronobacter mainly include antibiotic typing, phage typing, Enterobabcterial Repetitive Intergenic Consensus-PCR (ERIC-PCR), pulsed field gel electrophoresis (Pulsed Field Gel Electrophoresis, PFGE), random amplification Polymorphism (Random Applied Polymorphic DNA-PCR) and other technologies, these methods cannot effectively distinguish the different species of Kronobacter, and multi-site sequence analysis (Multil°Cus Sequenching Typing, MLST) can be Different species of the genus are distinguished, but the operation is cumbersome and the technical operation requirements are high (nucleic acid purification, sequencing and sequence comparison analysis), and it takes a long time (at least 2 days for ordinary laboratories), which is not conducive to the contamination of the bacteria in the food processing process Rapid identification of the source. Therefore, the
  • the present invention provides a method for distinguishing different spp kind of molecular typing method.
  • a molecular typing method based on RsaI single restriction digestion of gluA gene to distinguish different species of Kronobacter has the following steps:
  • gluA gene is Cronobacterium alpha-glucosidase gene
  • the specific gluA gene of Cronobacterium was amplified, and gluA gene fragments of six different bacteria of Cronobacterium were obtained.
  • the amplified products of the gluA gene fragments of the six different bacteria were digested with RsaI restriction enzymes, and the differential PCR-RFLP fingerprints of the six different bacteria of the genus Kronobacterium were obtained.
  • the six different species of Norobacterium are: 1. Cronobacter sakazakii, 2. Cronobacter turicensis (Cronobacterturicensis); 3 Cronobacter muytjensii, 4. Berling Cronobacter dublinnensis, 5. Cronobacter malonaticus, 6. Cronobacter condimenti.
  • step (1) The specific operation of step (1) is as follows: The size of the amplified fragment of Cronobacterium-specific gluA gene is 1680bp, and the amplification conditions are: PCR amplification is carried out on a PCR machine, reaction system: final concentration of 10mmol/L primer 0.25- 1.0 ⁇ L, DNA template 4-10ng, 15-30mmol/L Tris-HCl, 60-120mmol/L KCl, 2.0-3.0mmol/L MgCl 2 , add sterile double distilled water to 50 ⁇ L; reaction conditions: 94°C pre-denaturation 2min; 94°C denaturation for 30s, 54-58°C annealing for 45-60s, 72°C extension for 50-60s, 30 cycles; 72°C for 10min.
  • step (2) restriction conditions: 10-15 ⁇ L of gluA gene PCR amplification product, 10-15 ⁇ L of nuclease-free sterile water, 1 ⁇ buffer 2.5 ⁇ L: 33mM Tris-acetate, 10mM magnesium acetate, 66mM potassium acetate, 0.1 mg/ml BSA; RsaI restriction endonuclease 1-2 ⁇ L, incubate at 37°C for 0.5-3.0h.
  • the present invention realizes the rapid and stable identification of six different species of Cronobacterium, only 3h-4h; the existing RAPD-PCR and ERIC-PCR technologies are unstable and have poor reproducibility; the PFGE technology operation is extremely cumbersome and consumes Time is at least 10h; MLST technology is cumbersome to operate and has high technical requirements, which takes more than 2 days.
  • the present invention only needs to use one restriction enzyme RsaI to cut the gluA gene, avoiding In the RFLP typing technology based on other genes, the differences in the conditions of double digestion or even multiple digestion caused problems such as weakening of enzyme activity and mutual interference.
  • Figure 1 is a diagram of gluA gene fragments of six different species of Cronobacter.
  • Figure 2 shows the RFLP polymorphism map of the gluA gene digested by RsaI from six different species of Cronobacterium.
  • a molecular typing method based on RsaI single enzyme digestion of gluA gene to distinguish different species of Cronobacterium spp. consists of two experimental stages. The first stage is shown in Figure 1 to amplify the specific gluA gene fragment of Cronobacterium. In the second stage, as shown in Figure 2, the Kronobacter-specific gluA gene fragment amplified in the first stage was digested with RsaI to obtain different restriction patterns, and then to distinguish six different strains of Kronobacter.
  • the gluA group encodes Cronobacterium alpha-glucosidase.
  • gluA gene Amplify the gluA gene with the amplification primer F: 5'-AGGGATCCTGAAAGCAATCGACAAGAA-3'; R: 5'-CCGAAGCTTACTCATTACCCCTCCTGATG-3' to obtain the gluA gene fragments of six different species of Cronobacterium.
  • turicensis lane 3 (channel 3): C. muytjensii (Scronobacter moginensis) ATCC51329; lane4 (channel 4): C. dublinnensis (Cronobacter dublin); lane5, 6 (channel 5, 6): C.malonaticus (Cronobacter malonate); lane 12 (channel 12): C.condimenti (Kantii Montekronobacter); Other lanes (other channels): C.sakazakii (Kronobacter sakazaki).
  • the size of the amplified fragment is 1680bp.
  • the amplification conditions of the gluA gene are: PCR amplification is carried out on a PCR machine, reaction system: primer (10mmol/L) 0.5-1 ⁇ L, DNA template 5-10ng, 20-25mmol/L Tris- HCl, 80-100mmol/L KCl, 2.5-3.0mmol/L MgCl 2 ), add sterile double distilled water to 50 ⁇ L. Reaction conditions: 94°C pre-denaturation for 5min; 94°C denaturation for 30s, 56-58°C annealing for 45-60s, 72°C extension for 50-60s, 35 cycles; 72°C for 10min.
  • the amplified gluA gene fragments of the four different bacteria were digested with RsaI restriction enzymes to distinguish the RsaI digestion conditions of the six different species of Kronobacter RFLP digestion polymorphism: gluA gene PCR Amplification product 10 ⁇ L, 1 ⁇ buffer 3 ⁇ L (33mM Tris-acetate, 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA), RsaI restriction endonuclease 1-2 ⁇ L, nuclease-free sterile water 14-16 ⁇ L, Incubate at 37°C for 0.5-3.0h.
  • muytjensii ATCC51329 (Scronobacter morginii ATCC51329); lane4 (channel 4): C. dublinnensis (Cronobacter dublin); lane5, 6 (channel 5, 6): C. malonaticus (malonate Cronobacter); lane 12 (channel 12): C.condimenti (Condimenti Montecronobacterium); Other lanes: C.sakazakii (Cronobacter sakazakii).

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Abstract

本申请公开一种基于RsaI单酶切gluA基因区分克罗诺杆菌属不同种的分型方法。本申请的方法分为两个阶段，1.第一阶段，通过克罗诺杆菌特异性引物扩增得到克罗诺菌属六种不同菌的gluA基因片段，2.第二阶段，采用RsaI酶切第一阶段扩增的克罗诺杆菌特异性gluA基因片段，获得克罗诺菌属六种不同菌的差异性PCR-RFLP指纹图谱，进而区分克罗诺菌属不同菌种。通过大量克罗诺杆菌属的菌株事例验证，该分子分型方法能有效获得不同种差异性的酶切多态性图谱，区分克罗诺菌杆属六个不同菌种。

Description

一种基于RsaI单酶切gluA基因区分克罗诺杆菌属不同种的分子分型方法 技术领域
本发明属于食品微生物检验技术领域，具体涉及基于RsaI单酶切gluA基因区分克罗诺杆菌属不同种的分子分型方法。
背景技术
克罗诺杆菌属(Cronobacter)，是能引起严重感染的食源性致病菌。食品污染数据调查表明婴幼儿配方奶粉是其感染的主要传播媒介。目前，克罗诺杆菌属的分型技术主要包括抗生素分型、噬菌体分型、Enterobabcterial Repetitive Intergenic Consensus-PCR(ERIC-PCR),脉冲场凝胶电泳(Pulsed Field Gel Electrophoresis,PFGE)、随机扩增多态性(Random Applied Polymorphic DNA-PCR)等技术，这些方法无法有效区分克罗诺菌属不同的菌种，而多位点序列分析(Multil℃us Sequenching Typing,MLST)能对克罗诺菌属的不同种进行区分，但操作繁琐且技术操作要求高(核酸纯化、测序与序列比较分析)，耗时较长(普通实验室需至少2天以上)，不利于食品加工过程中该菌污染源头的快速鉴定。因而，操作方便、快速地区分克罗诺菌属不同种的分子分型方法对食品中克罗诺杆菌污染的精准溯源显得极为迫切。
发明内容
为了方便、准确、快速鉴定克罗诺杆菌属的不同菌种，达到对食品中克罗诺杆菌污染源头的快速鉴定，本发明提供一种基于RsaI单酶切gluA基因区分克罗诺杆菌属不同种的分子分型方法。
一种基于RsaI单酶切gluA基因区分克罗诺杆菌属不同种的分子分型方法操作步骤如下：
(1)扩增基因片段
选用克罗诺杆菌特异性gluA基因，所述gluA基因为克罗诺杆菌α-葡萄糖苷酶基因；
用一对扩增引物F:5’-AGGGATCCTGAAAGCAATCGACAAGAA-3’；
R：5’-CCGAAGCTTACTCATTACCCCTCCTGATG-3’；
对克罗诺杆菌特异性gluA基因扩增，得到克罗诺菌属六种不同菌的gluA基因片段，
(2)酶切扩增产物
采用RsaI限制性内切酶对扩增得到的六种不同菌的gluA基因片段的扩增产物分别进行酶 切，获得克罗诺菌属六种不同菌的差异性PCR-RFLP指纹图谱，克罗诺菌属六种不同菌分别为：1.阪崎克罗诺杆菌(Cronobacter sakazakii)，2.苏黎世克罗诺杆菌(Cronobacterturicensis)；3莫金斯克罗诺杆菌(Cronobacter muytjensii)，4.柏林克罗诺杆菌(Cronobacter dublinnensis)，5.丙二酸盐克罗诺杆菌(Cronobacter malonaticus)，6.康帝蒙提克罗诺杆菌(Cronobacter condimenti)。
进一步限定的技术方案如下：
步骤(1)的具体操作如下：克罗诺杆菌特异性gluA基因扩增片段大小为1680bp，扩增条件是：PCR扩增在PCR仪上进行，反应体系：终浓度为10mmol/L引物0.25-1.0μL，DNA模板4-10ng，15-30mmol/L Tris-HCl、60-120mmol/L KCl、2.0-3.0mmol/L MgCl 2，加无菌双蒸水至50μL；反应条件：94℃预变性2min；94℃变性30s，54-58℃退火45-60s，72℃延伸50-60s，30个循环；72℃10min。
步骤(2)中，酶切条件：gluA基因PCR扩增产物10-15μL，无核酸酶无菌水10-15μL，1×buffer 2.5μL：33mM Tris-acetate，10mM magnesium acetate，66mM potassium acetate，0.1mg/ml BSA；RsaI限制性内切酶1-2μL,37℃孵育0.5-3.0h。
本发明的有益技术效果体现在以下方面：
1.本发明实现快速、稳定鉴定克罗诺杆菌属的六种不同菌种，只需要3h-4h；现有RAPD-PCR和ERIC-PCR技术不稳定、重复性差；PFGE技术操作极为繁琐，耗时至少10h以上；MLST技术操作繁琐且技术要求高，耗时在2天以上。
2.本发明通过对gluA基因的序列分析及限制性核酸内切酶的在线筛选(RsaI准确切断序列中GT AC)，只需要使用一种限制性内切酶RsaI对gluA基因进行酶切，避免基于其他基因的RFLP分型技术中双酶切甚至多酶切带来的酶切条件差异导致酶活性减弱及相互干扰等问题。
3.采用RsaI单酶切反应，只需要通过发明中的一对特异性引物对克罗诺杆菌进行PCR扩增，然后对扩增产物进行RsaI单酶切，不仅实验操作方便、快速，而且酶切反应具有彻底、干扰少，酶切条带清晰等优点。
附图说明
图1为克罗诺杆菌属六种不同种的gluA基因片段图。
图2为克罗诺杆菌属六种不同种的RsaI酶切gluA基因的RFLP多态性图谱。
具体实施方式
下面结合实施例，对本发明作进一步地详细说明。
实施例
一种基于RsaI单酶切gluA基因区分克罗诺杆菌属不同种的分子分型方法为两个实验阶段，第一阶段如图1所示，扩增克罗诺杆菌特异性gluA基因片段，第二阶段如图2所示，采用RsaI酶切第一阶段扩增的克罗诺杆菌特异性gluA基因片段，获得不同的酶切图谱，进而区分克罗诺杆菌属六种不同菌种。
具体操作步骤如下：
(1)选用gluA基因
gluA基编码克罗诺杆菌α-葡萄糖苷酶。
(2)对gluA基因扩增
用扩增引物F:5’-AGGGATCCTGAAAGCAATCGACAAGAA-3’；R：5’-CCGAAGCTTACTCATTACCCCTCCTGATG-3’对gluA基因扩增，得到克罗杆菌属六种不同菌的gluA基因片段，见图1：克罗诺杆菌属六种不同菌种的菌株gluA扩增结果；图1中M(孔道M)：DL2000分子量标记物(100bp，250bp，500bp，750bp，1000bp，2000bp)；lane1(孔道1):C.sakazakii ATCC29544(阪崎克罗诺杆菌ATCC29544)；lane 2，11(孔道2,11)：苏黎世克罗诺杆菌(C.turicensis)；lane3(孔道3):C.muytjensii(莫金斯克罗诺杆菌)ATCC51329；lane4(孔道4):C.dublinnensis(都柏林克罗诺杆菌)；lane5,6(孔道5，6):C.malonaticus(丙二酸盐克罗诺杆菌)；lane 12(孔道12)：C.condimenti(康帝蒙提克罗诺杆菌)；Other lanes(其他孔道):C.sakazakii(阪崎克罗诺杆菌)。
扩增片段大小为1680bp，gluA基因的扩增条件是：PCR扩增在PCR仪上进行，反应体系：引物(10mmol/L)0.5-1μL，DNA模板5-10ng，20-25mmol/L Tris-HCl、80-100mmol/L KCl、2.5-3.0mmol/L MgCl 2)，加无菌双蒸水至50μL。反应条件：94℃预变性5min；94℃变性30s，56-58℃退火45-60s，72℃延伸50-60s，35个循环；72℃10min。
(3)RsaI单酶切gluA基因扩增产物反应
采用RsaI限制性内切酶对扩增的四种不同菌的gluA基因片段分别进行单酶切，区分克罗诺杆菌属六种不同种RFLP酶切多态性的RsaI酶切条件：gluA基因PCR扩增产物10μL，1×buffer 3μL(33mM Tris-acetate，10mM magnesium acetate，66mM potassium acetate，0.1mg/ml BSA),RsaI限制性内切酶1-2μL，无核酸酶无菌水14-16μL，37℃孵育0.5-3.0h。
获得克罗诺菌属六种不同菌的差异性分子指纹图谱，进而为比对区分克罗杆菌属的不同菌种提供技术基础。见图2：克罗诺杆菌属六种不同菌种gluA基因的RsaI酶切结果， 图2中：图1中M(孔道M)：DL2000分子量标记物(100bp，250bp，500bp，750bp，1000bp，2000bp)；lane1(孔道1)：C.sakazakii ATCC29544(阪崎克罗诺杆菌ATCC29544)；lane 2，11(孔道2,11)：苏黎世克罗诺杆菌(C.turicensis)；lane3(孔道3):C.muytjensii ATCC51329(莫金斯克罗诺杆菌ATCC51329)；lane4(孔道4):C.dublinnensis(都柏林克罗诺杆菌)；lane5,6(孔道5，6)：C.malonaticus(丙二酸盐克罗诺杆菌)；lane 12(孔道12)：C.condimenti(康帝蒙提克罗诺杆菌)；Other lanes(其他孔道):C.sakazakii(阪崎克罗诺杆菌)。

Claims (3)

  1. 一种基于RsaI单酶切gluA基因区分克罗诺杆菌属不同种的分子分型方法，其特征在于操作步骤如下：
    (1)扩增基因片段
    选用克罗诺杆菌特异性gluA基因，所述gluA基因为克罗诺杆菌α-葡萄糖苷酶基因；
    用一对扩增引物F:5’-AGGGATCCTGAAAGCAATCGACAAGAA-3’；
    R：5’-CCGAAGCTTACTCATTACCCCTCCTGATG-3’；
    对克罗诺杆菌特异性gluA基因扩增，得到克罗诺菌属六种不同菌的gluA基因片段，
    (2)酶切扩增产物
    采用RsaI限制性内切酶对扩增得到的六种不同菌的gluA基因片段的扩增产物分别进行酶切，获得克罗诺菌属六种不同菌的差异性PCR-RFLP指纹图谱，克罗诺菌属六种不同菌分别为：1.阪崎克罗诺杆菌(Cronobacter sakazakii)，2.苏黎世克罗诺杆菌(Cronobacterturicensis)；3莫金斯克罗诺杆菌(Cronobacter muytjensii)，4.柏林克罗诺杆菌(Cronobacter dublinnensis)，5.丙二酸盐克罗诺杆菌(Cronobacter malonaticus)，6.康帝蒙提克罗诺杆菌(Cronobacter condimenti)。
  2. 根据权利要求1所述的一种基于RsaI单酶切gluA基因区分克罗诺杆菌属不同种的分子分型方法，其特征在于：步骤(1)的具体操作如下：
    克罗诺杆菌特异性gluA基因扩增片段大小为1680bp，扩增条件是：PCR扩增在PCR仪上进行，反应体系：终浓度为10mmol/L引物0.25-1.0μL，DNA模板4-10ng，15-30mmol/L Tris-HCl、60-120mmol/L KCl、2.0-3.0mmol/L MgCl 2，加无菌双蒸水至50μL；反应条件：94℃预变性2min；94℃变性30s，54-58℃退火45-60s，72℃延伸50-60s，30个循环；72℃10min。
  3. 根据权利要求1所述的一种基于RsaI单酶切gluA基因区分克罗诺杆菌属不同种的分子分型方法，其特征在于：步骤(2)中，酶切条件：gluA基因PCR扩增产物10-15μL，无核酸酶无菌水10-15μL，1×buffer 2.5μL：33mM Tris-acetate，10mM magnesium acetate，66mM potassium acetate，0.1mg/ml BSA；RsaI限制性内切酶1-2μL,37℃孵育0.5-3.0h。
PCT/CN2020/129687 2019-12-09 2020-11-18 一种基于RsaI单酶切gluA基因区分克罗诺杆菌属不同种的分子分型方法 WO2021115077A1 (zh)

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