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 PDFInfo
- Publication number
- 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
- Authority
- CN
- China
- Prior art keywords
- pna
- probe
- salmonella
- nucleic acid
- salmonellas
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000607142 Salmonella Species 0.000 title claims abstract description 49
- 239000000523 sample Substances 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 31
- 238000002509 fluorescent in situ hybridization Methods 0.000 title abstract description 14
- 108091093037 Peptide nucleic acid Proteins 0.000 title abstract 5
- 238000009396 hybridization Methods 0.000 claims abstract description 26
- 241000894006 Bacteria Species 0.000 claims description 19
- 238000001514 detection method Methods 0.000 claims description 17
- 238000005516 engineering process Methods 0.000 claims description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 230000001580 bacterial effect Effects 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000007983 Tris buffer Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 229920001917 Ficoll Polymers 0.000 claims description 3
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 claims description 3
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Natural products C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 claims description 3
- -1 methane amide Chemical class 0.000 claims description 3
- 229920000642 polymer Polymers 0.000 claims description 3
- 239000001488 sodium phosphate Substances 0.000 claims description 3
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 3
- 229910000406 trisodium phosphate Inorganic materials 0.000 claims description 3
- 235000019801 trisodium phosphate Nutrition 0.000 claims description 3
- 230000001717 pathogenic effect Effects 0.000 abstract description 5
- 230000009977 dual effect Effects 0.000 abstract description 2
- 238000003556 assay Methods 0.000 abstract 3
- 108020004414 DNA Proteins 0.000 abstract 2
- 102000053602 DNA Human genes 0.000 abstract 2
- 229920002477 rna polymer Polymers 0.000 abstract 2
- 230000035699 permeability Effects 0.000 abstract 1
- 235000013305 food Nutrition 0.000 description 15
- 241001354013 Salmonella enterica subsp. enterica serovar Enteritidis Species 0.000 description 4
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 206010016952 Food poisoning Diseases 0.000 description 3
- 208000019331 Foodborne disease Diseases 0.000 description 3
- 239000008186 active pharmaceutical agent Substances 0.000 description 3
- 244000052616 bacterial pathogen Species 0.000 description 3
- 235000013601 eggs Nutrition 0.000 description 3
- 235000013372 meat Nutrition 0.000 description 3
- 239000008267 milk Substances 0.000 description 3
- 235000013336 milk Nutrition 0.000 description 3
- 210000004080 milk Anatomy 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 206010004022 Bacterial food poisoning Diseases 0.000 description 2
- 241000588921 Enterobacteriaceae Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241001138501 Salmonella enterica Species 0.000 description 2
- 241000392514 Salmonella enterica subsp. enterica serovar Dublin Species 0.000 description 2
- 241000607272 Vibrio parahaemolyticus Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 125000000022 2-aminoethyl group Chemical group [H]C([*])([H])C([H])([H])N([H])[H] 0.000 description 1
- 241001464929 Acidithiobacillus caldus Species 0.000 description 1
- 241000272525 Anas platyrhynchos Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 241000115153 Bacteroides xylanisolvens Species 0.000 description 1
- 241000514942 Bacteroides xylanolyticus Species 0.000 description 1
- 241001135265 Cronobacter sakazakii Species 0.000 description 1
- 241000605800 Desulfobulbus propionicus Species 0.000 description 1
- 241001646719 Escherichia coli O157:H7 Species 0.000 description 1
- 206010017914 Gastroenteritis salmonella Diseases 0.000 description 1
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 1
- 201000008225 Klebsiella pneumonia Diseases 0.000 description 1
- 241000588747 Klebsiella pneumoniae Species 0.000 description 1
- 241000186779 Listeria monocytogenes Species 0.000 description 1
- 241001585714 Nola Species 0.000 description 1
- 206010035717 Pneumonia klebsiella Diseases 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 241000316848 Rhodococcus <scale insect> Species 0.000 description 1
- 241001524101 Rhodococcus opacus Species 0.000 description 1
- 241000533331 Salmonella bongori Species 0.000 description 1
- 241000592155 Salmonella enterica subsp. enterica serovar Agona Species 0.000 description 1
- 241000596091 Salmonella enterica subsp. enterica serovar Anatum Species 0.000 description 1
- 241000607132 Salmonella enterica subsp. enterica serovar Gallinarum Species 0.000 description 1
- 241000204914 Salmonella enterica subsp. enterica serovar Give Species 0.000 description 1
- 241000607726 Salmonella enterica subsp. enterica serovar Heidelberg Species 0.000 description 1
- 241000531795 Salmonella enterica subsp. enterica serovar Paratyphi A Species 0.000 description 1
- 241000607683 Salmonella enterica subsp. enterica serovar Pullorum Species 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 241000605118 Thiobacillus Species 0.000 description 1
- 208000037386 Typhoid Diseases 0.000 description 1
- 241000607253 Vibrio mimicus Species 0.000 description 1
- 241000607447 Yersinia enterocolitica Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000001848 dysentery Diseases 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000009432 framing Methods 0.000 description 1
- 235000013905 glycine and its sodium salt Nutrition 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 235000021049 nutrient content Nutrition 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000003375 selectivity assay Methods 0.000 description 1
- 229910021653 sulphate ion Inorganic materials 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 201000008297 typhoid fever Diseases 0.000 description 1
- 229940098232 yersinia enterocolitica Drugs 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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
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
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
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
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
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。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210079061.2A CN102586466B (zh) | 2012-03-23 | 2012-03-23 | 肽核酸原位荧光杂交技术检测沙门氏菌的方法及pna探针 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210079061.2A CN102586466B (zh) | 2012-03-23 | 2012-03-23 | 肽核酸原位荧光杂交技术检测沙门氏菌的方法及pna探针 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102586466A true CN102586466A (zh) | 2012-07-18 |
| CN102586466B CN102586466B (zh) | 2014-02-26 |
Family
ID=46475688
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210079061.2A Expired - Fee Related CN102586466B (zh) | 2012-03-23 | 2012-03-23 | 肽核酸原位荧光杂交技术检测沙门氏菌的方法及pna探针 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102586466B (zh) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104087654A (zh) * | 2013-04-01 | 2014-10-08 | 中国农业大学 | 沙门氏菌及其五种血清型的多重pcr鉴定试剂盒 |
| CN110283920A (zh) * | 2019-07-12 | 2019-09-27 | 中国计量大学 | 检测志贺氏菌属的肽核酸原位荧光杂交鉴定方法及肽核酸探针 |
| CN111575391A (zh) * | 2020-05-12 | 2020-08-25 | 浙江省检验检疫科学技术研究院 | 液相原位荧光杂交检测大肠杆菌o157:h7的鉴定方法、试剂盒和探针 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0950718A1 (en) * | 1997-07-09 | 1999-10-20 | Dai Nippon Printing Co., Ltd. | Method for detecting dna with probe pna |
| WO2002027036A2 (en) * | 2000-09-26 | 2002-04-04 | Boston Probes, Inc. | Probes, probe sets, methods and kits pertaining to the detection, identification and/or enumeration of bacteria |
| US6664045B1 (en) * | 1998-06-18 | 2003-12-16 | Boston Probes, Inc. | PNA probes, probe sets, methods and kits pertaining to the detection of microorganisms |
| WO2011121544A1 (en) * | 2010-03-29 | 2011-10-06 | Universidade Do Minho | Peptide nucleicacid probe, kit and method for detection and/or quantification of salmonella spp. and applications thereof |
| CN102329882A (zh) * | 2011-10-15 | 2012-01-25 | 浙江省质量技术监督检测研究院 | 三种食源性致病菌多重快速检测法及检测引物组和试剂盒 |
| CN102358908A (zh) * | 2011-10-28 | 2012-02-22 | 浙江省检验检疫科学技术研究院 | 单核细胞增多性李斯特菌的肽核酸原位荧光鉴定方法及pna探针 |
-
2012
- 2012-03-23 CN CN201210079061.2A patent/CN102586466B/zh not_active Expired - Fee Related
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0950718A1 (en) * | 1997-07-09 | 1999-10-20 | Dai Nippon Printing Co., Ltd. | Method for detecting dna with probe pna |
| US6664045B1 (en) * | 1998-06-18 | 2003-12-16 | Boston Probes, Inc. | PNA probes, probe sets, methods and kits pertaining to the detection of microorganisms |
| WO2002027036A2 (en) * | 2000-09-26 | 2002-04-04 | Boston Probes, Inc. | Probes, probe sets, methods and kits pertaining to the detection, identification and/or enumeration of bacteria |
| WO2002027036A3 (en) * | 2000-09-26 | 2003-08-07 | Boston Probes Inc | Probes, probe sets, methods and kits pertaining to the detection, identification and/or enumeration of bacteria |
| WO2011121544A1 (en) * | 2010-03-29 | 2011-10-06 | Universidade Do Minho | Peptide nucleicacid probe, kit and method for detection and/or quantification of salmonella spp. and applications thereof |
| CN102329882A (zh) * | 2011-10-15 | 2012-01-25 | 浙江省质量技术监督检测研究院 | 三种食源性致病菌多重快速检测法及检测引物组和试剂盒 |
| CN102358908A (zh) * | 2011-10-28 | 2012-02-22 | 浙江省检验检疫科学技术研究院 | 单核细胞增多性李斯特菌的肽核酸原位荧光鉴定方法及pna探针 |
Non-Patent Citations (1)
| Title |
|---|
| C. ALMEIDA: "Fluorescence In Situ Hybridization Method Using a Peptide Nucleic Acid Probe for Identification of Salmonella spp. in a BroadSpectrum of Samples", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》, vol. 76, no. 13, 31 July 2010 (2010-07-31) * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104087654A (zh) * | 2013-04-01 | 2014-10-08 | 中国农业大学 | 沙门氏菌及其五种血清型的多重pcr鉴定试剂盒 |
| CN104087654B (zh) * | 2013-04-01 | 2016-04-27 | 中国农业大学 | 沙门氏菌及其五种血清型的多重pcr鉴定试剂盒 |
| CN110283920A (zh) * | 2019-07-12 | 2019-09-27 | 中国计量大学 | 检测志贺氏菌属的肽核酸原位荧光杂交鉴定方法及肽核酸探针 |
| CN111575391A (zh) * | 2020-05-12 | 2020-08-25 | 浙江省检验检疫科学技术研究院 | 液相原位荧光杂交检测大肠杆菌o157:h7的鉴定方法、试剂盒和探针 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102586466B (zh) | 2014-02-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Czajkowski et al. | Detection, identification and differentiation of Pectobacterium and Dickeya species causing potato blackleg and tuber soft rot: a review | |
| Bhatt et al. | Milk microbiome signatures of subclinical mastitis‐affected cattle analysed by shotgun sequencing | |
| Hein et al. | Real-time PCR for the detection of Salmonella spp. in food: an alternative approach to a conventional PCR system suggested by the FOOD-PCR project | |
| Gorski | Selective enrichment media bias the types of Salmonella enterica strains isolated from mixed strain cultures and complex enrichment broths | |
| Koo et al. | Phylogenetic group distribution and prevalence of virulence genes in Escherichia coli isolates from food samples in South Korea | |
| CN103667498B (zh) | 副溶血性弧菌的检测方法 | |
| Soni et al. | Phylogenetic analysis of the Listeria monocytogenes based on sequencing of 16S rRNA and hlyA genes | |
| Wang et al. | Rapid and sensitive detection of Listeria monocytogenes by cross-priming amplification of lmo0733 gene | |
| Park et al. | The analytical comparison of phage-based magnetoelastic biosensor with TaqMan-based quantitative PCR method to detect Salmonella Typhimurium on cantaloupes | |
| Sahu et al. | Rapid detection of Salmonella contamination in seafoods using multiplex PCR | |
| CN110951898B (zh) | 克罗诺杆菌属内4个种的特异性新分子靶标及其快速检测方法 | |
| CN103898222B (zh) | 一种基于bcfD基因的沙门氏菌分子检测试剂盒及其非诊断性检测方法 | |
| CN101613745B (zh) | 金黄色葡萄球菌的多重pcr-mix快速检测试剂盒及检测方法 | |
| Babu et al. | Optimization and evaluation of a multiplex PCR for simultaneous detection of prominent foodborne pathogens of Enterobacteriaceae | |
| CN103131760A (zh) | 一种可以同时检测六种治病微生物的悬浮芯片检测方法 | |
| Huang et al. | Application of molecular biological techniques to analyze Salmonella seasonal distribution in stream water | |
| CN102586466B (zh) | 肽核酸原位荧光杂交技术检测沙门氏菌的方法及pna探针 | |
| CN101974644B (zh) | 金黄色葡萄球菌肠毒素基因pcr分型检测试剂盒及方法 | |
| Haigh et al. | A novel clinical syndrome and detection of Anaplasma ovis in Mongolian reindeer (Rangifer tarandus) | |
| CN108034740B (zh) | 用于检测印第安纳沙门菌的引物组、该引物组的用途、包含该引物组的试剂盒及其检测方法 | |
| CN102586465B (zh) | 肽核酸原位荧光杂交技术检测弧菌的方法及pna探针 | |
| CN102358908B (zh) | 单核细胞增多性李斯特菌的肽核酸原位荧光鉴定方法及pna探针 | |
| CN104031129A (zh) | 用于检测金黄色葡萄球菌和/或表皮葡萄球菌的肽核酸探针组及其试剂盒、使用方法、用途 | |
| Li et al. | PMAxx combined with recombinase aided amplification technique for specific and rapid detection of Salmonella in milk | |
| CN104726575A (zh) | 一种快速检测禽源样品中沙门菌的lamp试剂盒 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C53 | Correction of patent of invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Li Ke Inventor after: Zhang Xiaofeng Inventor after: Wu Pan Inventor after: Shuai Jiangbing Inventor after: He Yongqiang Inventor before: Li Ke Inventor before: Zhang Xiaofeng Inventor before: Wu Shan Inventor before: Shuai Jiangbing Inventor before: He Yongqiang |
|
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140226 |