CN102586466A - 肽核酸原位荧光杂交技术检测沙门氏菌的方法及pna探针 - Google Patents

肽核酸原位荧光杂交技术检测沙门氏菌的方法及pna探针 Download PDF

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CN102586466A
CN102586466A CN2012100790612A CN201210079061A CN102586466A CN 102586466 A CN102586466 A CN 102586466A CN 2012100790612 A CN2012100790612 A CN 2012100790612A CN 201210079061 A CN201210079061 A CN 201210079061A CN 102586466 A CN102586466 A CN 102586466A
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李可
张晓峰
吴珊
帅江冰
何永强
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ZHEJIANG ENTRY-EXIT INSPECTION AND QUARANTINE BUREAU
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Abstract

本发明涉及PNA-FISH法鉴定沙门氏菌属的方法,尤其涉及用于鉴定主要食源致病性沙门氏菌(Salmonella)的方法和PNA探针。肽核酸原位荧光杂交技术检测沙门氏菌用PNA探针Sal-invA-1,所述的PNA探针的序列为TCTGGATGGTATGCC。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

肽核酸原位荧光杂交技术检测沙门氏菌的方法及PNA探针
技术领域
本发明涉及PNA-FISH法鉴定沙门氏菌属的方法,尤其涉及用于鉴定主要食源致病性沙门氏菌(Salmonella)的方法和PNA探针。
背景技术
沙门氏菌属是一群形态和培养特性都类似的肠杆菌科中的一个大属,也是肠杆菌科中最重要的一个属。沙门氏菌是革兰氏阴性致病菌,在自然界分布极广,且株型繁多,目前全世界已分离出2,523个血清型。据资料统计,我国每年发生的细菌性食物中毒事件占食物中毒事件总数的30%~90%,人数占食物中毒总人数的60%~90%(陈建林等,1996),而在细菌性食物中毒的病原菌中,约40%为沙门氏菌。而在引起沙门氏菌中毒的食品中,约90%是肉、蛋、奶等动物性食品,这些食品中含有多种丰富的营养成分,沙门氏菌会非常迅速地繁殖。人们一旦摄入含有大量沙门氏菌(105~106cfu/g)的食品,就会引起细菌性感染,进而在毒素的作用下发生食物中毒。沙门氏菌等微生物污染已对我们的食品安全构成了主要的威胁。不过与人类疾病有关的血清型主要集中于A~E群,其中以鼠伤寒沙门氏菌(S.Typhimurium)、肠炎沙门氏菌(S.Enteritidis)及猪霍乱沙门氏菌(S.Choleraesuis)最为常见。
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主要食源致病性沙门氏菌特异性的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沙门氏菌的方法,实现对主要食源致病性沙门氏菌的快速检测。
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沙门氏菌用PNA探针Sal-invA-1,所述的PNA探针的序列为TCTGGATGGTATGCC。
为了验证探针的敏感度和特异性,用BLAST(http://blast.ncbi.nlm.nih.gov/)对探针进行了验证。BLAST搜索发现,所设计的探针Sal-invA-1对主要食源性沙门氏菌具有较高的特异性,包括鼠伤寒沙门氏菌(S.Typhimurium)、丙型副伤寒沙门氏菌(S.Paratyphi)、肠炎沙门氏菌(S.Enteritidis)、鸡伤寒沙门氏菌(S.Gallinarum)、阿哥纳沙门氏菌(S.Agona)、海德堡沙门氏菌(S.Heidelberg)、纽波特沙门氏菌(S.Newport)、鸡白痢沙门氏菌(S.Pullorum)、都柏林沙门氏菌(S.Dublin)、猪霍乱沙门氏菌(S.Choleraesuis)等常见沙门氏菌。但也有一些非目标细菌如:喜温硫杆菌(Acidithiobacillus caldus)、硫酸盐还原菌(Desulfobulbus propionicus)、解木聚糖拟杆菌(Bacteroides xylanisolvens)、不透明红球菌(Rhodococcus opacus)等与探针Sal-invA-1有重合区域,但上述菌株均与沙门氏菌关系较为疏远,也并非常见食品中的致病菌。因此,从理论角度分析,Sal-invA-1探针有较好的灵敏度和特异性,可用于沙门氏菌PNA检测分析方法的建立。
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沙门氏菌的方法,该方法包括以下的步骤:
1)离心收集对数生长期的细菌,PBS调整细菌浓度至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)将25μl含500pmole/mlPNA的杂交缓冲液滴加于玻片上,所述的PNA探针的序列为TCTGGATGGTATGCC,55℃作用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,风干后荧光显微镜观察荧光亮度及细菌的形态。
作为优选,所述的杂交缓冲液pH7.5,包括:10%(w/v)硫酸葡聚糖,10mM 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,5mM Na2EDTA,0.2%(v/v)TritonX-100,50mM Tris/HCl。
作为优选,所述的洗涤缓冲液pH10,包括:15mM Tris,15mM NaCl和0.1%(v/v)TritonX-100。
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沙门氏菌用PNA探针Sal-invA-1,所述的PNA探针的序列如表1。
表1 PNA探针序列
Figure BDA0000146275340000031
aBacUin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 hybridizationfor rapid detection,identification and enumeration of specific micro-organisms.JAppl Microbiol 90(2):180-189。
探针合成和标记由韩国Panagene完成。
我们选取了包括沙门氏菌两个种(Salmonella entrica和Salmonella bongori)的19个血清型,共计25株沙门氏菌进行了探针的灵敏度实验。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,10mMNaCl,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,5mMNa2EDTA,0.2%(v/v)TritonX-100,50mM Tris/HCl)滴加于玻片上,55℃作用1.5小时,杂交后玻片于55℃预热的洗涤缓冲液(pH10,5mM Tris,15mMNaCl,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。
如表2所示,除了鸭沙门沙门氏菌(S.Anatum)、斯坦利沙门氏菌(S.Stanley)、康德沙门氏菌(S.Kande)、诺拉沙门氏菌(S.Nola)、巴基斯坦沙门氏菌(S.Pakistan)、吉伟沙门氏菌(S.Give)、维肖沙门氏菌(S.Virchow)8株菌株之外,其余17株沙门氏菌全部与Sal-invA-1探针呈杂交阳性。此外,所有的菌株都能和阳性对照探针BacUin结合。上述结果和预设结果基本一致,探针Sal-invA-1能检测到自己的目标菌株,有较高的敏感度。
表2  沙门氏菌PNA探针灵敏度验证
Figure BDA0000146275340000041
Figure BDA0000146275340000051
Annotate:+:positive;-:negative.
a无菌株描述
实施例2  PNA-FI SH特异性验证
选取12株有代表性的革兰氏阴性细菌和阳性细菌被用于和探针杂交,包括肺炎克雷伯氏菌、宋氏志贺氏菌、阪崎肠杆菌、铜绿假单胞菌、单核细胞增生李斯特菌、副溶血弧菌、副溶血弧菌、拟态弧菌、大肠杆菌、大肠杆菌O157:H7、小肠结肠炎耶尔森氏菌、金黄色葡萄球菌(2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Sal-invA-1不能与这些非目标菌株结合(表3)。与预期结果一致,Sal-invA-1有很好的特异性。
表3  沙门氏菌PNA探针特异性验证
Figure BDA0000146275340000052
Figure BDA0000146275340000061
a无菌株描述
实施例3  食品和环境来源沙门氏菌分离株PNA-FISH法和API法鉴定比对
就目前所发生沙门氏菌食品污染事件和相关研究报道称,绝大多数是由肉、蛋、奶等动物性食品污染所致,因此,本研究也主要就肉、蛋、奶三大类食品和若干环境样品进行了PNA-FISH检测。在300份食品及食品相关的样品中,Sal-invA-1探针检测到了16份阳性样品,而用API方法鉴定得出17份阳性样品(表4),结果表明用Sal-invA-1探针杂交的PNA-FISH方法对沙门氏菌有良好的检出率,与传统经典方法相比,检测结果基本吻合,可用于主要食源性致病沙门氏菌的检测。
表4  PAN-FISH法和API法在实际样品中的检测结果比较
Figure BDA0000146275340000071
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沙门氏菌的方法及PNA探针
 
<160>1
 
<210>1
<211>15
<212>DNA
<213>人工序列
<400>1
TCTGGATGGT ATGCC 15

Claims (4)

1.肽核酸原位荧光杂交技术检测沙门氏菌用PNA探针,其特征在于:PNA探针的序列为TCTGGATGGTATGCC。
2.肽核酸原位荧光杂交技术检测沙门氏菌的方法,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,PBS调整细菌浓度至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)将25μl含500pmole/mlPNA的杂交缓冲液滴加于玻片上,所述的PNA探针的序列为TCTGGATGGTATGCC,55℃作用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,风干后荧光显微镜观察荧光亮度及细菌的形态。
3.根据权利要求2所述的肽核酸原位荧光杂交技术检测沙门氏菌的方法,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沙门氏菌的方法,其特征在于洗涤缓冲液pH10,包括:15 mM Tris,15mM NaCl和0.1% (v/v) TritonX-100。
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