CN102586465B - 肽核酸原位荧光杂交技术检测弧菌的方法及pna探针 - Google Patents
肽核酸原位荧光杂交技术检测弧菌的方法及pna探针 Download PDFInfo
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Abstract
本发明涉及应用PNA-FISH法鉴定弧菌属的方法，尤其涉及用于鉴定重要致病性弧菌（Vibrio）的方法和PNA探针。肽核酸原位荧光杂交技术检测弧菌用PNA探针，PNA探针的序列为TACCCCCCTCTACAG。本发明的PNA具有与DNA和RNA特异性结合的高稳定性、杂交快速性、及其良好的细胞穿透性，使本发明的检测方法具有快速，准确、灵敏的特性。同时结合原位荧光杂交技术使检测具有分子生物学和形态学的双重保证，更加提高了鉴定的准确率。
Description
技术领域
本发明涉及应用PNA-FISH法鉴定弧菌属的方法，尤其涉及用于鉴定重要致病性弧菌（Vibrio）的方法和PNA探针。
背景技术
我国水产动物的病害种类达200余种，常年养殖病害发病率达50%以上，损失率20%左右，估计每年因水产养殖病害问题而造成的直接经济损失就达百亿元之巨，并且还有上升的趋势。从近年来我国海水养殖病害监测报告来看，病害以生物源性疾病为主，其中又以弧菌（Vibrio）疾病为重，霍乱弧菌（V. cholerae）、副溶血弧菌（V. parahaemolyticus）、哈维氏弧菌（V. harveyi）、创伤弧菌（V. vulnificus）、溶藻弧菌（V. alginolyticus）、鳗弧菌（V. anguillarum）、梅氏弧菌（V. metschnikovi）等都是重要的细菌病原。因此，发展对弧菌病原菌的快速检测和鉴定技术对于食品安全、水产养殖等均具有十分重要的意义。
肽核酸是1991年由丹麦科学家Nielsen等人设计的一种以中性酰胺键为骨架的全新的DNA模拟物，其骨架结构单元为（2-氨乙基）甘氨酸，碱基部分通过亚甲基羰基连接与主骨架的氨基N上，可以序列特异地与DNA、RNA结合。其骨架为电中性，与DNA-DNA或RNA-DNA互补链相比，PNA-DNA和PNA-RNA互补不存在静电排斥作用，因此具有很高的DNA或RNA亲和性，在无错配的情况下其结合具有高稳定性，且杂交速度快，具有良好的细胞穿透性，是核酸探针的良好选择。同时PNA探针结合原位荧光杂交技术（FISH），与传统生化鉴定比较，PNA-FISH法可节约大量时间，若不计增菌时间，一般耗时不超过4个小时。与PCR等方法比较，PNA-FISH法的结果判断基于荧光检测和形态检测两部分，较PCR法等假阳性较低，且PNA-FISH法可省去核酸提取的步骤。
发明内容
本发明的一个目的是设计了具弧菌特异性的PNA探针，本发明的另外一个目的是提供肽核酸原位荧光杂交技术检测弧菌的方法，实现对弧菌属的快速检测。
为了实现上述第一个目的，本发明采用以下技术方案：
肽核酸原位荧光杂交技术检测弧菌用PNA探针， PNA探针的序列为TAC CCC CCT CTA CAG。
为了验证探针的灵敏度和特异性，用BLAST（http://blast.ncbi.nlm.nih.gov/）和ProbeCheck（http://www.microbial-ecology.net/probecheck）对探针进行了验证。BLAST 搜索发现，所设计的探针具有较高的特异性。但也有一些的非目标细菌与探针Vib-16S-1有重合区域，如Aliivibrio wodanis，Pantoea agglomerans，Listonella pelagia，Allomonas enterica，Listonella anguillarum，Exiguobacterium aurantiacum，Thorsellia anophelis，Antarctic bacterium，Rhodobacter capsulatus，但以上非目标细菌与弧菌关系都较疏远，也并非常见食品中的致病菌，因此一般不会导致假阳性结果。
ProbeCheck验证结果显示，探针Vib-16S-1能检测到ProbeCheck中小亚基（16S/18S）数据库中所有的2000株弧菌，但同时也能检测到635株非目标菌，其中包括352个非培养细菌，对弧菌属的特异性为99.86%，灵敏度为100%。又将PNA探针与大亚基(23S/28S)数据库匹配检测，发现仅有一株Arthonia didyma与之匹配，也不会与我们的目标菌混淆。
为了实现上述第二个目的，本发明采用以下技术方案：
肽核酸原位荧光杂交技术检测弧菌的方法，该方法包括以下的步骤：
1）离心收集对数生长期的细菌，用PBS洗涤一次，再用PBS调整菌液浓度至OD600=0.5-2.0,取10μl 菌液于98%酒精清洗过的玻片上涂匀，火焰固定，将玻片于80%的酒精中浸泡15分钟，风干；
2）25μl含500pmole/mlPNA的杂交缓冲液滴加于玻片上， PNA探针的序列为TAC CCC CCT CTA CAG，55℃作用1-1.5小时，杂交后玻片于55℃预热的洗涤缓冲液中洗涤2次，每次10分钟，取2～5μl菌液涂片，风干后显微镜观察其荧光亮度及细菌的形态。
作为优选，上述的杂交缓冲液pH7.5，包括：10%（w/v） 硫酸葡聚糖，10 mM NaCl，30% (v/v)甲酰胺，0.1%（w/v）焦磷酸钠，0.2%（w/v）聚乙烯吡咯烷酮，0.2%（w/v）聚蔗糖，5 mM Na2EDTA，0.2% (v/v) TritonX-100，50 mM Tris/HCl。
作为优选，上述的洗涤缓冲液pH10，包括：5 mM Tris，15mM NaCl，0.1% (v/v) TritonX-100。
2. 固相PNA荧光原位杂交
本发明由于采用上述的技术方案，PNA具有与DNA和RNA特异性结合的高稳定性、杂交快速性、及其良好的细胞穿透性，使本发明的检测方法具有快速，准确、灵敏的特性。同时结合原位荧光杂交技术使检测具有分子生物学和形态学的双重保证，更加提高了鉴定的准确率。
具体实施方式
实施例 1 PNA-FISH敏感度验证
肽核酸原位荧光杂交技术检测弧菌用PNA探针， PNA探针的序列为TAC CCC CCT CTA CAG。
经过BLAST和ProbeCheck验证，所设计的探针Vib-16S-1理论上具有很高的灵敏度和特异性。与阳性对照探针BacUin一起被用于后续检测。
表1 PNA 探针序列
| 探针 | 序列 (5’-3’)荧光标记 | 探针位置碱基序列 ; GenBank 号 |
| BacUina | CTGCCTCCCGTAGGA-FAM | － |
| Vib-16S -1 | TAC CCC CCT CTA CAG | 106-120; HM590221.1 |
a BacUin为阳性对照探针；引用自Perry-O'Keefe, H., Stender, H., Broomer, A., Oliveira, K., Coull, J., Hyldig-Nielsen, J.J., 2001. Filter-based PNA in situ hybridization for rapid detection, identification and enumeration of specific micro-organisms. J Appl Microbiol 90(2): 180-189.
探针合成和标记由韩国Panagene完成。
本发明选取了7种弧菌共12株细菌进行了灵敏度验证试验方法如下所述：
离心（2000g，5min）收集对数生长期的细菌，用PBS洗涤一次，再用PBS调整菌液浓度至OD600=0.5-2.0,取10μl 菌液于98%酒精清洗过的玻片上涂匀，火焰固定，将玻片于80%的酒精中浸泡15分钟，风干；25μl含500pmole/mlPNA的杂交缓冲液（pH7.5，10%（w/v） 硫酸葡聚糖，10 mM NaCl，30% (v/v)甲酰胺，0.1%（w/v）焦磷酸钠，0.2%（w/v）聚乙烯吡咯烷酮，0.2%（w/v）聚蔗糖，5 mM Na2EDTA，0.2% (v/v) TritonX-100，50 mM Tris/HCl）滴加于玻片上，55℃作用1-1.5小时，杂交后玻片于55℃预热的洗涤缓冲液（pH10， 5 mM Tris，15mM NaCl，0.1% (v/v) TritonX-100）中洗涤2次，每次10分钟，取2～5μl菌液涂片，风干后显微镜观察其荧光亮度及细菌的形态。
每次试验（包括以下实例2和3）都同步进行阳性对照和阴性对照试验，阳性对照试验，用探针BacUin替代其他探针，而阴性对照试验中，用空白替代其他探针。
结果显示，所有实验的弧菌菌株都与阳性对照探针BacUin和探针Vib-16S-1呈杂交阳性（见表2）。上述结果和预设结果一致，探针Vib-16S-1能检测到自己的目标菌株，有较高的敏感度。
杂交条件被优化确立后，我们进一步研究了Vib-16S-1探针的灵敏度和特异性。我们选取了7种弧菌共12株细菌进行了灵敏度验证。如表2所示，
表2 弧菌PNA探针灵敏度验证
a无菌株描述。
实施例 2 PNA-FISH特异性验证
选取14株具有代表性的革兰氏阴性细菌和阳性细菌菌株，分别为2株鼠伤寒沙门氏菌、2株金黄色葡萄球菌、肺炎克雷伯氏菌、宋氏志贺氏菌、阪崎肠杆菌、铜绿假单胞菌、嗜水气单胞菌、大肠杆菌、大肠杆菌O157:H7、肠炎沙门氏菌、小肠结肠炎耶尔森氏菌、单核细胞增生李斯特氏菌各1株。试验方法如上所述。结果显示只有阳性对照探针BacUin与所有细菌都能结合，而探针Vib-16S-1不能与这些非目标菌株结合（表3）。与预期结果一致，Vib-16S-1有很好的特异性。
表3 弧菌PNA探针特异性验证
a无菌株描述。
实施例 3 食品和环境来源弧菌分离株PNA-FISH法和API法鉴定比对
因水产品和水体是弧菌的高发区，所以采用来自于食品加工企业和超市的鱼虾类等水产品的食品样本，和来自养殖水体和附近水域的环境样品，经过样品收集、增菌、分离等步骤后，用PNA-FISH法参照前述的方法进行鉴定，同时使用API法参照试剂盒说明书进行鉴定比对。
结果显示（表4），在380个食品及食品相关的样品中，探针Vib-16S -1检测到了38个弧菌阳性样品，46株弧菌。与用结合API试剂条的生化方法得到的结果一致，鉴定结果显示有15株霍乱弧菌，11株副溶血弧菌，11株溶藻弧菌，8株创伤弧菌，1株河流弧菌。表明Vib-16S -1探针能够有效检测食品和环境样品中的弧菌。
表 4 PAN-FISH法和API法在实际样品中的检测结果比较
序列表
<110>浙江省检验检疫科学技术研究院
<120>肽核酸原位荧光杂交技术检测弧菌的方法及PNA探针
<160>1
<210>1
<211>15
<212>DNA
<213>人工序列
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TACCCCCCTC TACAG 15
Claims (4)
1.肽核酸原位荧光杂交技术检测弧菌（Vibrio）用PNA探针，其特征在于：PNA探针的序列为TAC CCC CCT CTA CAG。
2.肽核酸原位荧光杂交技术检测弧菌的方法，其特征在于该方法包括以下的步骤：
1）离心收集对数生长期的细菌，用PBS洗涤一次，再用PBS调整菌液浓度至OD600=0.5-2.0,取10μl 菌液于98%酒精清洗过的玻片上涂匀，火焰固定，将玻片于80%的酒精中浸泡15分钟，风干；
2）25μl含500pmole/mlPNA的杂交缓冲液滴加于玻片上， PNA探针的序列为TAC CCC CCT CTA CAG，55℃作用1-1.5小时，杂交后玻片于55℃预热的洗涤缓冲液中洗涤2次，每次10分钟，取2～5μl菌液涂片，风干后显微镜观察其荧光亮度及细菌的形态；
上述的方法不应用于疾病的诊断。
3.根据权利要求2所述的肽核酸原位荧光杂交技术检测弧菌的方法，其特征在于杂交缓冲液pH7.5，包括：10%（w/v） 硫酸葡聚糖，10 mM NaCl，30% (v/v)甲酰胺，0.1%（w/v）焦磷酸钠，0.2%（w/v）聚乙烯吡咯烷酮，0.2%（w/v）聚蔗糖，5 mM Na2EDTA，0.2% (v/v) TritonX-100，50 mM Tris-HCl。
4.根据权利要求2所述的肽核酸原位荧光杂交技术检测弧菌的方法，其特征在于洗涤缓冲液pH10，包括：5 mM Tris，15mM NaCl，0.1% (v/v) TritonX-100。
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