CN103215358A - Diarrheogenic escherichia coli detection kit and detection and typing method thereof - Google Patents
Diarrheogenic escherichia coli detection kit and detection and typing method thereof Download PDFInfo
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Abstract
The invention provides a diarrheogenic escherichia coli detection kit and a detection and typing method thereof, which are suitable for the technical field of molecular biology and microbiological detection. The detection kit comprises a PCR (Polymerase Chain Reaction) buffer solution, MgC12, dNTP, DNA (Deoxyribonucleic Acid) polymerase, primers of stp, sth, lt, aggR, eaeA, escV, stx1, stx2 and ipaH genes, probes SEQ ID NO:1-SEQ ID NO:27 and a Homo-tag the sequence of which is SEQ ID NO:28. The detection and typing method comprises the following steps of: obtaining and processing a sample, and adding the processed sample to a detection kit; carrying out fluorescent quantitative PCR reaction; and obtaining a detection result. The detection and genotyping method provided by the invention has the advantages of good specificity, high sensitivity, simple and convenient operation, easiness in identification of the result and is suitable for rapid screening and genotyping of diarrheogenic escherichia coli.
Description
Technical field
The invention belongs to molecular biology and microorganism detection technical field, relate in particular to a kind of diarrheagenic E. coli detection kit and detect classifying method.
Background technology
Intestinal bacteria behave and animal intestinal in often occupy bacterium, generally how not pathogenic, can cause that under certain condition enteron aisle infects outward.And that some serological type strain has is stronger pathogenic, can cause diarrhoea, and this kind bacterial strain can cause diarrhoea by diet.Diarrheagenic E. coli (DEC) is the encountered pathogenic bacteria of bacterial diarrhea, it comprises enteropathogenic Escherichia coli (enteropathogenie E.coli, EPEC), enterotoxigenic E.Coli (enterotoxigenic E.coli, ETEC), enteroinvasive E.Coli (enteroinvasive E.coli, EIEC), enterohemorrhagic Escherichia coli (enterohemorrhagic E.coli, EHEC) and EAEC (enteroaggregative E.coli, EAEC).DEC infects and is mainly seen in the infant, and its recall rate in the adult diarrhea patient increases to some extent in recent years, and becomes the The main pathogenic fungi that some areas cause diarrhoea, and higher resistance has been appearred in common drug, therefore has been subjected to paying close attention to widely.Traditional separation and Culture technology is the standard method that DEC identifies, detects, but conventional microbial culture and authentication method be not enough to various DEC are distinguished, because a lot of serotype and genotype are inconsistent.And traditional method need be through the experimental procedure of a series of complexity, and sense cycle is long, complex operation, and accuracy is low, and time and effort consuming can't satisfy clinical and needs real work.The clinical symptom that DEC causes mostly is watery diarrhea, can't instruct clinical application from symptom interpretation type, so this needs and can rapid detection can realize multiple detection again.
Along with the fast development of Protocols in Molecular Biology, round pcr has been widely used in detection and the classification of bacterium.Yet the rear electrophoresis of conventional round pcr causes polluting easily, easily produces false positive, makes detected result inaccurate.Real-time quantitative PCR is widely used with outstanding advantage such as quick, accurate, but currently used real time quantitative PCR method mostly is single fluorescence quantifying PCR method, can only detect a kind of pathogenic bacterium, can not satisfy the requirement of multiple detection, so efficient has much room for improvement.
Summary of the invention
The object of the present invention is to provide a kind of diarrheagenic E. coli detection kit, it is single to be intended to solve in the existing detection technique detected object, and detection efficiency is low, the problem that accuracy is not high.
The embodiment of the invention is achieved in that a kind of diarrheagenic E. coli detection kit, comprises PCR reaction buffer, MgCl
2, dNTP, archaeal dna polymerase, also comprise following primer and probe:
Primer and probe at the stp gene:
stp-F：5’-GCAAGCCCTCACGTAGCGAAAAAAGCGAGTGCACCTCGACA-3’(SEQ ID NO:1)，
stp-R：5’-GCAAGCCCTCACGTAGCGAACAGTTGACTGACTAAAAGAGGGG-3’(SEQ ID NO:2)，
stp-probe：5’-CGCGTCTCAAATATCCGTGAAACAACATGACGCG-3’(SEQ ID NO:3)；
Primer and probe at the sth gene:
sth-F:5’-GCAAGCCCTCACGTAGCGAAGTGGTCCTGAAAGCATGAATAG-3’(SEQ ID NO:4)，
sth-R:5’-GCAAGCCCTCACGTAGCGAACAACAAAGCAACAGGTACATACG-3’(SEQ ID NO:5)，
sth-probe:5’-CGCGGTGAATTGTGTTGTAATCCTGCTTGTACCGCG-3’(SEQ ID NO:6)；
Primer and probe at the lt gene:
lt-F:5’-GCAAGCCCTCACGTAGCGAAACAGGAGGTTTCTGCGTTAG-3’(SEQ ID NO:7)，
lt-R:5’-GCAAGCCCTCACGTAGCGAAGGTGGGAAACCTGCTAATCT-3’(SEQ ID NO:8)，
lt-probe:5’-CGCCGGTATTACAGAAATCTGAATATAGCTCCGGCG-3’(SEQ ID NO:9)；
Primer and probe at the aggR gene:
aggR-F:5’-GCAAGCCCTCACGTAGCGAATGCAAAAGAAGAAATCAACAGT-3’(SEQ ID NO:10)，
aggR-R:5’-GCAAGCCCTCACGTAGCGAACAGAATCGTCAGCATCAGCTAC-3’(SEQ ID NO:11)，
aggR-probe:5’-CGGACAAAAGTAGATGCTTGCAGTTGTCCG-3’(SEQ ID NO:12)；
Primer and probe at the eaeA gene:
eaeA-F:5’-GCAAGCCCTCACGTAGCGAAGTAACCAGGCTTCGTCACA-3’(SEQ ID NO:13)，
eaeA-R:5’-GCAAGCCCTCACGTAGCGAAGGAAAAAACGCTGACCCG-3’(SEQ ID NO:14)，
eaeA-probe:5’-CCCAGTGGTAATAACTTTGACGGTAGTTCACTGGG-3’(SEQ ID NO:15)；
Primer and probe at the escV gene:
escV-F:5’-GCAAGCCCTCACGTAGCGAAGGCTCTCTTCTTCTTTATGGCTG-3’(SEQ ID NO:16)，
escV-R:5’-GCAAGCCCTCACGTAGCGAAGGGAAAGAAGTTAGTTCAAGAGGAT-3’(SEQ ID NO:17)，
escV-probe:5’-CCCGCGCAACAGTTGTGGTGGATATCATTATCGCGGG-3’(SEQ ID NO:18)；
Primer and probe at the stx1 gene:
stx1-F:5’-GCAAGCCCTCACGTAGCGAAASAGCGGTTACATTGTCTGGT-3’(SEQ ID NO:19)，
stx1-R:5’-GCAAGCCCTCACGTAGCGAACTGCGTCAGTGAGGTTCCA-3’(SEQ ID NO:20)，
stx1-probe:5’-CCGCGTACGGGGATGCAGATAAATCGCGG-3’(SEQ ID NO:21)；
Primer and probe at the stx2 gene:
stx2-F:5’-GCAAGCCCTCACGTAGCGAACATGACAACGGACAGCAGTTA-3’(SEQ ID NO:22)，
stx2-R:5’-GCAAGCCCTCACGTAGCGAATCTGGTCATTGTATTACCACTGAA-3’(SEQ ID NO:23)，
stx2-probe:5’-CCGCCACTCACTGGTTTCATCATATCTGGCGG-3’(SEQ ID NO:24)；
Primer and probe at the ipaH gene:
ipaH-F:5’-GCAAGCCCTCACGTAGCGAAGAAAACCCTCCTGGTCCATC-3’(SEQ ID NO:25)，
ipaH-R:5’-GCAAGCCCTCACGTAGCGAAGTCTGGAAGGCCAGGTAGACTT-3’(SEQ ID NO:26)，
ipaH-probe:5’-CCCGGCTGGAGGACATTGCCCGGG-3’(SEQ ID NO:27)；
Wherein each bar probe sequence 5 ' end is connected with fluorophor, and 3 ' end is connected with quenching group;
This detection kit also comprises following Homo-tag:
5’-GCAAGCCCTCACGTAGCGAA-3’(SEQ ID NO:28)。
Another purpose of the embodiment of the invention is to provide a kind of diarrheagenic E. coli to detect and classifying method, and this detection and classifying method utilize diarrheagenic E. coli detection kit of the present invention, and step is as follows:
Obtain and the processing sample, be added in the diarrheagenic E. coli detection kit,
Carry out the quantitative fluorescent PCR reaction,
Obtain detected result.
Diarrheagenic E. coli detection kit provided by the invention and detection method thereof, according at present in the world to five kinds of classification (enterotoxigenic E.Colis (ETEC) of diarrheagenic E. coli, EAEC (EAEC), enteroinvasive E.Coli (EIEC), enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC)) design homology tailed primer and improvement molecular beacon probe, utilize the real-time fluorescence quantitative PCR technology, five kinds of diarrheagenic E. colis are detected and somatotype simultaneously, detection method specificity of the present invention is good, highly sensitive, easy and simple to handle, the result is easy to interpretation, avoided false-positive appearance, be applicable to rapid screening and somatotype diarrheagenic E. coli.
Embodiment
In order to make the technical problem to be solved in the present invention, technical scheme and beneficial effect clearer,, the present invention is further elaborated below in conjunction with embodiment.Should be appreciated that specific embodiment described herein only in order to explanation the present invention, and be not used in qualification the present invention.
The embodiment of the invention provides a kind of diarrheagenic E. coli detection kit based on improved fluorescent quantitative PCR technique, comprises PCR reaction buffer, MgCl
2, dNTP, archaeal dna polymerase, this detection kit also comprises following primer and probe simultaneously:
Primer and probe at the stp gene:
stp-F：5’-GCAAGCCCTCACGTAGCGAAAAAAGCGAGTGCACCTCGACA-3’(SEQ ID NO:1)，
stp-R：5’-GCAAGCCCTCACGTAGCGAACAGTTGACTGACTAAAAGAGGGG-3’(SEQ ID NO:2)，
stp-probe：5’-CGCGTCTCAAATATCCGTGAAACAACATGACGCG-3’(SEQ ID NO:3)；
Primer and probe at the sth gene:
sth-F:5’-GCAAGCCCTCACGTAGCGAAGTGGTCCTGAAAGCATGAATAG-3’(SEQ ID NO:4)，
sth-R:5’-GCAAGCCCTCACGTAGCGAACAACAAAGCAACAGGTACATACG-3’(SEQ ID NO:5)，
sth-probe:5’-CGCGGTGAATTGTGTTGTAATCCTGCTTGTACCGCG-3’(SEQ ID NO:6)；
Primer and probe at the lt gene:
lt-F:5’-GCAAGCCCTCACGTAGCGAAACAGGAGGTTTCTGCGTTAG-3’(SEQ ID NO:7)，
lt-R:5’-GCAAGCCCTCACGTAGCGAAGGTGGGAAACCTGCTAATCT-3’(SEQ ID NO:8)，
lt-probe:5’-CGCCGGTATTACAGAAATCTGAATATAGCTCCGGCG-3’(SEQ ID NO:9)；
Primer and probe at the aggR gene:
aggR-F:5’-GCAAGCCCTCACGTAGCGAATGCAAAAGAAGAAATCAACAGT-3’(SEQ ID NO:10)，
aggR-R:5’-GCAAGCCCTCACGTAGCGAACAGAATCGTCAGCATCAGCTAC-3’(SEQ ID NO:11)，
aggR-probe:5’-CGGACAAAAGTAGATGCTTGCAGTTGTCCG-3’(SEQ ID NO:12)；
Primer and probe at the eaeA gene:
eaeA-F:5’-GCAAGCCCTCACGTAGCGAAGTAACCAGGCTTCGTCACA-3’(SEQ ID NO:13)，
eaeA-R:5’-GCAAGCCCTCACGTAGCGAAGGAAAAAACGCTGACCCG-3’(SEQ ID NO:14)，
eaeA-probe:5’-CCCAGTGGTAATAACTTTGACGGTAGTTCACTGGG-3’(SEQ ID NO:15)；
Primer and probe at the escV gene:
escV-F:5’-GCAAGCCCTCACGTAGCGAAGGCTCTCTTCTTCTTTATGGCTG-3’(SEQ ID NO:16)，
escV-R:5’-GCAAGCCCTCACGTAGCGAAGGGAAAGAAGTTAGTTCAAGAGGAT-3’(SEQ ID NO:17)，
escV-probe:5’-CCCGCGCAACAGTTGTGGTGGATATCATTATCGCGGG-3’(SEQ ID NO:18)；
Primer and probe at the stx1 gene:
stx1-F:5’-GCAAGCCCTCACGTAGCGAAASAGCGGTTACATTGTCTGGT-3’(SEQ ID NO:19)，
stx1-R:5’-GCAAGCCCTCACGTAGCGAACTGCGTCAGTGAGGTTCCA-3’(SEQ ID NO:20)，
stx1-probe:5’-CCGCGTACGGGGATGCAGATAAATCGCGG-3’(SEQ ID NO:21)；
Primer and probe at the stx2 gene:
stx2-F:5’-GCAAGCCCTCACGTAGCGAACATGACAACGGACAGCAGTTA-3’(SEQ ID NO:22)，
stx2-R:5’-GCAAGCCCTCACGTAGCGAATCTGGTCATTGTATTACCACTGAA-3’(SEQ ID NO:23)，
stx2-probe:5’-CCGCCACTCACTGGTTTCATCATATCTGGCGG-3’(SEQ ID NO:24)；
Primer and probe at the ipaH gene:
ipaH-F:5’-GCAAGCCCTCACGTAGCGAAGAAAACCCTCCTGGTCCATC-3’(SEQ ID NO:25)，
ipaH-R:5’-GCAAGCCCTCACGTAGCGAAGTCTGGAAGGCCAGGTAGACTT-3’(SEQ ID NO:26)，
ipaH-probe:5’-CCCGGCTGGAGGACATTGCCCGGG-3’(SEQ ID NO:27)；
And Homo-tag:
5’-GCAAGCCCTCACGTAGCGAA-3’(SEQ ID NO:28)，
5 ' the end of wherein above-mentioned probe sequence SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27 is connected with fluorophor, and 3 ' end is connected with quenching group.
Particularly, the fluorophor that is connected to probe 5 ' end can send fluorescent signal, the quenching group that is connected to probe 3 ' end can suppress the fluorescent signal that fluorophor sends, in the quantitative fluorescent PCR reaction process, quenching group separates with fluorophor, fluorophor can send fluorescent signal, can carry out quantitative analysis to unknown template by the accumulation that detects fluorescent signal.Wherein, the above-mentioned fluorophor that is connected in 5 ' end can be FAM, HEX, ROX or Cy5, and the above-mentioned quenching group that is connected in 3 ' end can be preferably DABCYL for DABCYL or BHQ.
Particularly, in the detection kit of the embodiment of the invention, above-mentioned 9 groups of primers and probe branch are filled in two reaction tubes systems, wherein comprise at stp in the first reaction tubes system, sth, lt, the primer and the probe of four kinds of genes of aggR, be SEQ ID NO:1-SEQ ID NO:12, this reaction tubes system is used for the detection somatotype of enterotoxigenic E.Coli (ETEC) and EAEC (EAEC), and four kinds of probe sequence SEQ ID NO:3 wherein, SEQ ID NO:6, SEQ ID NO:9, the fluorophor of the 5 ' end of SEQ ID NO:12 is respectively FAM, HEX, ROX and Cy5; The second reaction tubes system comprises at eaeA, escV, stx1, the primer and the probe of stx2 and five kinds of genes of ipaH, be SEQ ID NO:13-SEQ ID NO:27, be used for enteroinvasive E.Coli (EIEC), the detection somatotype of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC), five kinds of probe sequence SEQ ID NO:15 wherein, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, the fluorophor of the 5 ' end of SEQ ID NO:27 is selected from FAM, HEX, ROX and Cy5, wherein have two probes to use with a kind of fluorophor, other three corresponding to other three kinds of fluorophors.Pass through aforesaid combination, make two kinds of reaction tubes systems constitute quadruple reaction system and five heavy reaction systems respectively, promptly comprise four groups of primer probes in the first reaction tubes system, comprise five groups of primer probes in the second reaction tubes system, being divided into two reaction tubes systems makes detected result more easy to identify, reduce the phase mutual interference, improved accuracy in detection.
More specifically, the primer of the above-mentioned first reaction tubes system and probe at gene in, stp, sth, lt are used to detect ETEC, aggR is used to detect EAEC; The primer of the above-mentioned second reaction tubes system and probe at gene in, eaeA and escV are used to detect EPEC and EHEC, stx1 and stx2 are used to detect EHEC simultaneously, detected result in conjunction with above-mentioned EPEC and EHEC can detect somatotype to EPEC and EHEC thus, and ipaH is used to detect EIEC.
Particularly, all comprise Homo-tag sequence (SEQ ID NO:28) in two reaction tubes systems of diarrheagenic E. coli detection kit of the present invention, this Home-tag sequence and above-mentioned stp, sth, lt, aggR, eaeA, escV, stx1, each of nine kinds of genes of stx2 and ipaH is identical to the label segment sequence of primer (being tailed primer).This Homo-tag sequential system can suppress the generation of primer dimer effectively, reduces in the multiplex PCR system difference of amplification efficiency between each gene, improves quantitative tolerance range.
Particularly, all comprise PCR reaction buffer, MgCl in the first and second reaction tubes systems of the diarrheagenic E. coli detection kit of the embodiment of the invention
2, dNTP, archaeal dna polymerase, wherein MgCl
2Working concentration be 2-4mmol, be preferably 3mmol; The concentration of dNTP is 0.1-0.3mmol, is preferably 0.2mmol; Archaeal dna polymerase concentration is 0.8-1.2U, is preferably 1U.
The archaeal dna polymerase of Shi Yonging can use any archaeal dna polymerase that is applicable to the quantitative fluorescent PCR reaction in embodiments of the present invention, is preferably the rTaq enzyme.
In the diarrheagenic E. coli detection kit of the embodiment of the invention, each is to primer in the first reaction tubes system, the working concentration scope that is SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:11 all is preferably 0.2-0.3 μ mol, each bar probe, promptly the concentration range of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO:12 all is preferably 0.2-0.25 μ mol.
In the diarrheagenic E. coli detection kit of the embodiment of the invention, each is to primer in the second reaction tubes system, be SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:17, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:22 and SEQ ID NO:23, the concentration range of SEQ ID NO:25 and SEQ ID NO:26 all is preferably 0.3-0.5 μ mol, each bar probe, i.e. SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, the concentration range of SEQ ID NO:24 and SEQ ID NO:27 all is preferably 0.2-0.24 μ mol.
In the diarrheagenic E. coli detection kit of the embodiment of the invention, all comprise the Home-tag sequence in the first and second reaction tubes systems, this Home-tag sequence concentration is preferably 0.5-1.0 μ mol, more preferably 0.5 μ mol.
The embodiment of the invention also provides a kind of diarrheagenic E. coli to detect and classifying method, and this detection and classifying method utilize diarrheagenic E. coli detection kit of the present invention to carry out, and this method may further comprise the steps:
S01 obtains sample and extracts nucleic acid and handle, and is added to then in the diarrheagenic E. coli detection kit of the present invention;
S02 carries out the quantitative fluorescent PCR reaction on the fluorescent PCR instrument;
S03 obtains detected result and analyzes.
Particularly, detection of the diarrheagenic E. coli of the embodiment of the invention and classifying method can use the reaction system of 25 μ L reaction systems or other volumes to detect and somatotype.Wherein, for 25 μ LPCR reaction systems, template (being detected sample) add-on is 5 μ L, for the PCR reaction system of other volumes, can carry out equal proportion to the template add-on and regulate.
Particularly, for above-mentioned steps S01, wherein said sample is not limited to any test sample, can detect and somatotype as long as can extract nucleic acid wherein.And wherein extract nucleic acid and can adopt water-boiling method that thalline is broken and obtain DNA, but be not limited thereto.
In step S02, the PCR response procedures is:
95 ℃ of pre-sex change 3min;
95 ℃ of sex change 10s, 58 ℃ of annealing 20s, 72 ℃ are extended 15s, totally 5 circulations;
95 ℃ of sex change 10s, 55 ℃ of annealing 20s, 72 ℃ are extended 15s, totally 40 circulations.
In step S03, fluorescence intensity to the quantitative fluorescent PCR reaction system is carried out Measurement and analysis, the standard that required cycle index Ct value is as a result of judged when reaching preset threshold, wherein 0＜Ct≤35 are positive, Ct 〉=38 or be to be judged as feminine gender at 0 o'clock, the Ct value is 35 between 38 the time, in 35＜Ct＜38 and there is obvious index amplification to be judged as the positive, otherwise negative.The fluorescence quantitative PCR detection of the embodiment of the invention and classifying method can detect the existence of the diarrheagenic E. coli in the sample to be detected and to its somatotype, utilize this detection and somatotype result, also can be widely used in clinical and the laboratory analysis and research for diarrheagenic E. coli, also can be used for detection for the diarrheagenic E. coli of food etc., to prevent food contamination, avoid the diarrhoea that causes thus.The fluorescence quantitative PCR detection of the embodiment of the invention and classifying method specificity are good, highly sensitive, easy and simple to handle, and the result is easy to interpretation.
Below in conjunction with embodiment diarrheagenic E. coli detection kit of the present invention and detection method are described in detail.
The preparation of embodiment one diarrheagenic E. coli detection kit
1. synthetic at stp, sth, lt, aggR, eaeA, escV, stx1, the primer and the probe of stx2 and nine kinds of genes of ipaH, be SEQ ID NO:1-SEQ ID NO:27, wherein each probe sequence 3 ' end all is connected with DABCYL, and the fluorophor of each bar probe sequence correspondence is as follows:
SEQ ID NO:3 is corresponding to HEX, and SEQ ID NO:6 is corresponding to FAM, and SEQ ID NO:9 is corresponding to ROX, and SEQ ID NO:12 is corresponding to Cy5,
SEQ ID NO:15 is corresponding to Cy5, and SEQ ID NO:18 is corresponding to HEX, and SEQ ID NO:21 is corresponding to ROX, and SEQ ID NO:24 is corresponding to ROX, and SEQ ID NO:27 is corresponding to FAM;
Synthesize Homo-tag sequence (SEQ ID NO:28), all be prepared into the storage liquid of 50 μ M concentration;
2. prepare PCR damping fluid, MgCl respectively
2Solution, dNTP solution, rTaq enzyme solution;
3. get PCR and react eight pipes, to wherein adding PCR damping fluid, MgCl
2Solution, dNTP solution, the rTaq enzyme solution makes it meet following ultimate density in 25 μ L systems and requires:
MgCl
2 3mmol
dNTP 0.2mmol
RTaq enzyme 1U,
And to the Homo-tag(50 μ M that wherein adds 0.25 μ L respectively), this Homo-tag ultimate density in 25 μ L reaction systems is 0.5 μ mol;
4. eight pipes that step 3 is obtained are divided into two groups: A group and B group, wherein add at stp in the A group, sth, lt, the primer and the probe of four kinds of genes of aggR, be SEQ ID NO:1-SEQ ID NO:12, wherein each is to primer, be SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:10 and the SEQ ID NO:11 ultimate density in 25 μ l systems is 0.3 μ mol, each bar probe, i.e. SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9 and the SEQ ID NO:12 ultimate density in 25 μ l systems is 0.2 μ mol; In the B group, add at eaeA, escV, stx1, the primer and the probe of stx2 and five kinds of genes of ipaH, be SEQ ID NO:13-SEQ ID NO:27, wherein each is to primer, be SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:17, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:22 and SEQ ID NO:23, SEQ ID NO:25 and the SEQ ID NO:26 ultimate density in 25 μ l systems is 0.5 μ mol, each bar probe, i.e. SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24 and the SEQ ID NO:27 ultimate density in 25 μ l systems is 0.2 μ mol;
5. A group pipe that obtains in above-mentioned steps 4 and B organize and add deionized water in the pipe respectively to make the reaction mixture volume be 20 μ l;
6. assembling makes the diarrheagenic E. coli detection kit.
Embodiment two
1. synthetic at stp, sth, lt, aggR, eaeA, escV, stx1, the primer and the probe of stx2 and nine kinds of genes of ipaH, be SEQ ID NO:1-SEQ ID NO:27, wherein each probe sequence 3 ' end all is connected with BHQ, and the fluorophor of each bar probe sequence correspondence is as follows:
SEQ ID NO:3 is corresponding to FAM, and SEQ ID NO:6 is corresponding to ROX, and SEQ ID NO:9 is corresponding to Cy5, and SEQ ID NO:12 is corresponding to HEX,
SEQ ID NO:15 is corresponding to FAM, and SEQ ID NO:18 is corresponding to FAM, and SEQ ID NO:21 is corresponding to Cy5, and SEQ ID NO:24 is corresponding to HEX, and SEQ ID NO:27 is corresponding to ROX;
Synthesize Homo-tag sequence (SEQ ID NO:28), all be prepared into the storage liquid of 50 μ M concentration;
2. prepare PCR damping fluid, MgCl respectively
2Solution, dNTP solution, rTaq enzyme solution;
3. get PCR and react eight pipes, to wherein adding PCR damping fluid, MgCl
2Solution, dNTP solution, the rTaq enzyme solution makes it meet following ultimate density in 25 μ L systems and requires:
MgCl
2 2mmol
dNTP 0.3mmol
RTaq enzyme 1.2U,
And to the Homo-tag(50 μ M that wherein adds 0.3 μ L respectively), this Homo-tag ultimate density in 25 μ L reaction systems is 0.6 μ mol;
4. eight pipes that step 3 is obtained are divided into two groups: A group and B group, wherein add at stp in the A group, sth, lt, the primer and the probe of four kinds of genes of aggR, be SEQ ID NO:1-SEQ ID NO:12, wherein each is to primer, be SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:10 and the SEQ ID NO:11 ultimate density in 25 μ l systems is 0.2 μ mol, each bar probe, i.e. SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9 and the SEQ ID NO:12 ultimate density in 25 μ l systems is 0.25 μ mol; In the B group, add at eaeA, escV, stx1, the primer and the probe of stx2 and five kinds of genes of ipaH, be SEQ ID NO:13-SEQ ID NO:27, wherein each is to primer, be SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:17, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:22 and SEQ ID NO:23, SEQ ID NO:25 and the SEQ ID NO:26 ultimate density in 25 μ l systems is 0.3 μ mol, each bar probe, i.e. SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24 and the SEQ ID NO:27 ultimate density in 25 μ l systems is 0.24 μ mol;
5. A group pipe that obtains in above-mentioned steps 4 and B organize and add deionized water in the pipe respectively to make the reaction mixture volume be 20 μ l;
6. assembling makes the diarrheagenic E. coli detection kit.
The embodiment tri-specific is analyzed
Obtain 204 strains and relate to bacteriums such as enterobacteria, coccus, vibrios, utilize the detection kit of embodiment one to carry out specificity experiment and with positive strain in contrast, the situation that detects bacterial strain sees Table 1, and wherein detected result is all negative.
Table 1 specificity analyses result
The result of table 1 shows that the detection kit detection specificity of utilizing embodiment one is good, non-specific amplification do not occur, has avoided the non-specific false-positive appearance that causes.
The detection somatotype of four pairs of diarrheagenic E. colis of embodiment
1. sample nucleic acid is handled: clinical fecal sample is got about 0.2g or 200ul is added in the 1.5mlEP pipe, and adding sample disposal liquid (PBS damping fluid or physiological saline) shakes each 10 seconds 3 times.Left standstill 10 minutes, centrifugal 5 minutes again with 8000rpm, draw supernatant and directly carry out nucleic acid extraction with water-boiling method, obtain nucleic acid-templated.
2. quantitative fluorescent PCR reaction:
Get the detection kit of embodiment one preparation, add nucleic acid-templated that 5 μ l steps 1 obtain in each PCR reaction tubes, composition is as follows in each PCR reaction tubes:
Then PCR is reacted eight pipes adding fluorescent PCR instrument (staragene-3005) and detect, the PCR response procedures is:
95 ℃ of pre-sex change 3min;
95 ℃ of sex change 10s, 58 ℃ of annealing 20s, 72 ℃ are extended 15s(totally 5 circulations);
95 ℃ of sex change 10s, 55 ℃ of annealing 20s, 72 ℃ are extended 15s(totally 40 circulations), and carry out fluorescent signal at the annealing stage of second loop cycle and detect, this program is automatically performed by instrument.
Carry out the serotype analysis to detecting sample simultaneously, obtain positive strain 1270 strains altogether.The serotype detected result of this 1270 strain bacterial strain is as shown in table 2 with the result's that the detection kit of utilizing embodiment one detects contrast.
Table 2 serotype and PCR method results of comparison
As shown in Table 2, there are situations such as detected result intersection, omission in the serotype analysis, and utilizes the detection kit of the embodiment of the invention effectively to detect somatotype to diarrheagenic E. coli, has avoided false-positive appearance, and has improved detection efficiency.
Embodiment five
Get 100 parts of food inspection samples, utilize the detection kit of embodiment two to detect, the concrete operations step is with embodiment four, and detected result slightly.The above only is preferred embodiment of the present invention, not in order to restriction the present invention, all any modifications of being done within the spirit and principles in the present invention, is equal to and replaces and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. a diarrheagenic E. coli detection kit comprises PCR reaction buffer, MgCl
2, dNTP, archaeal dna polymerase, it is characterized in that, also comprise following 9 groups of primers and probe:
At the primer of stp gene to SEQ ID NO:1 and SEQ ID NO:2 and probe SEQ ID NO:3,
At the primer of sth gene to SEQ ID NO:4 and SEQ ID NO:5 and probe SEQ ID NO:6,
At the primer of lt gene to SEQ ID NO:7 and SEQ ID NO:8 and probe SEQ ID NO:9,
At the primer of aggR gene to SEQ ID NO:10 and SEQ ID NO:11 and probe SEQ ID NO:12,
At the primer of eaeA gene to SEQ ID NO:13 and SEQ ID NO:14 and probe SEQ ID NO:15,
At the primer of escV gene to SEQ ID NO:16 and SEQ ID NO:17 and probe SEQ ID NO:18,
At the primer of stx1 gene to SEQ ID NO:19 and SEQ ID NO:20 and probe SEQ ID NO:21,
At the primer of stx2 gene to SEQ ID NO:22 and SEQ ID NO:23 and probe SEQ ID NO:24,
At the primer of ipaH gene to SEQ ID NO:25 and SEQ ID NO:26 and probe SEQ ID NO:27,
Wherein the described probe 5 ' end of each bar is connected with fluorophor, and 3 ' end is connected with quenching group;
This detection kit comprises that also sequence is the Homo-tag of SEQ ID NO:28.
2. diarrheagenic E. coli detection kit as claimed in claim 1 is characterized in that, described 9 groups of primers and probe branch are filled in two reaction tubes systems, and SEQ ID NO:1-SEQ ID NO:12 and SEQ ID NO:13-SEQ ID NO:27 are separated.
3. diarrheagenic E. coli detection kit as claimed in claim 1 is characterized in that, described archaeal dna polymerase is the rTaq enzyme.
4. diarrheagenic E. coli detection kit as claimed in claim 1 is characterized in that, described MgCl
2Working concentration be 2-4mmol, the working concentration of described dNTP is 0.1-0.3mmol, described archaeal dna polymerase working concentration is 0.8-1.2U.
5. diarrheagenic E. coli detection kit as claimed in claim 1, it is characterized in that, the working concentration scope of described primer sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:11 is 0.2-0.3 μ mol, and the working concentration scope of described probe sequence SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO:12 is 0.2-0.25 μ mol; The working concentration scope of described primer sequence SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25 and SEQ ID NO:26 is 0.3-0.5 μ mol, and the working concentration scope of described probe sequence SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24 and SEQ ID NO:27 is 0.2-0.24 μ mol.
6. diarrheagenic E. coli detection kit as claimed in claim 1 is characterized in that, described Home-tag sequence operative concentration is 0.5-1.0 μ mol.
7. diarrheagenic E. coli detection kit as claimed in claim 1 is characterized in that described fluorophor is selected from FAM, HEX, ROX and Cy5, and described quenching group is selected from DABCYL and BHQ.
8. a diarrheagenic E. coli detects and classifying method, comprises the steps:
Obtain and the processing sample, be added in the diarrheagenic E. coli detection kit,
Carry out the quantitative fluorescent PCR reaction,
Obtain detected result,
It is characterized in that described quantitative fluorescent PCR reaction utilizes to be carried out as each described diarrheagenic E. coli detection kit among the claim 1-7.
9. diarrheagenic E. coli as claimed in claim 8 detects and classifying method, it is characterized in that, the response procedures of described quantitative fluorescent PCR reaction is:
95 ℃ of pre-sex change 3min;
95 ℃ of sex change 10s, 58 ℃ of annealing 20s, 72 ℃ are extended 15s, totally 5 circulations;
95 ℃ of sex change 10s, 55 ℃ of annealing 20s, 72 ℃ are extended 15s, totally 40 circulations.
10. diarrheagenic E. coli as claimed in claim 8 detects and classifying method, it is characterized in that the mode of result of determination is as follows in the described acquisition detected result step:
0＜Ct≤35 are positive,
Ct 〉=38 or be to be judged as feminine gender at 0 o'clock,
The Ct value is 35 between 38 the time, in 35＜Ct＜38 and there is obvious index amplification to be judged as the positive, otherwise negative.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561326A (en) * | 2015-01-16 | 2015-04-29 | 天津医科大学 | Typing method based on pilus diversity for uropathogenic escherichia coli |
| CN105567820A (en) * | 2016-01-13 | 2016-05-11 | 江苏和创生物科技有限公司 | Kit for fluorescent PCR detection of escherichia coli |
| CN106978506A (en) * | 2017-05-05 | 2017-07-25 | 武汉爱基百客生物科技有限公司 | A kind of fluorescence quantifying PCR method for detecting Escherichia coli |
| CN107760766A (en) * | 2017-12-05 | 2018-03-06 | 苏州国科闻普生物科技有限公司 | Method, kit and its application of multiple five kinds of Diarrheogenil Escherichia colis of qPCR quick detections |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101760514A (en) * | 2008-12-25 | 2010-06-30 | 天津生物芯片技术有限责任公司 | Gene chip and kit for detecting diarrheagenic escherichia coli in food and clinical samples |
| CN102140507A (en) * | 2010-12-20 | 2011-08-03 | 南开大学 | Detection genetic chip and detection kit for infectious diarrhea |
-
2013
- 2013-04-10 CN CN201310123546.1A patent/CN103215358B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101760514A (en) * | 2008-12-25 | 2010-06-30 | 天津生物芯片技术有限责任公司 | Gene chip and kit for detecting diarrheagenic escherichia coli in food and clinical samples |
| CN102140507A (en) * | 2010-12-20 | 2011-08-03 | 南开大学 | Detection genetic chip and detection kit for infectious diarrhea |
Non-Patent Citations (3)
| Title |
|---|
| OSCAR G. GÓMEZ-DUARTE: "Detection of Escherichia coli, Salmonella spp., Shigella spp., Yersinia enterocolitica, Vibrio cholerae, and Campylobacter spp. enteropathogens by 3-reaction multiplex polymerase chain reaction", 《DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE》, vol. 63, no. 1, 31 January 2009 (2009-01-31), pages 1 - 9, XP025796359, DOI: doi:10.1016/j.diagmicrobio.2008.09.006 * |
| 朱敏等: "多重PCR快速检测致泻大肠埃希菌", 《中华预防医学杂志》, vol. 44, no. 4, 31 December 2010 (2010-12-31), pages 345 - 347 * |
| 赵爱兰: "鉴定五类致泻性大肠埃希菌和志贺菌的多重PCR方法", 《疾病监测》, vol. 26, no. 1, 30 January 2011 (2011-01-30), pages 65 - 67 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561326A (en) * | 2015-01-16 | 2015-04-29 | 天津医科大学 | Typing method based on pilus diversity for uropathogenic escherichia coli |
| CN105567820A (en) * | 2016-01-13 | 2016-05-11 | 江苏和创生物科技有限公司 | Kit for fluorescent PCR detection of escherichia coli |
| CN106978506A (en) * | 2017-05-05 | 2017-07-25 | 武汉爱基百客生物科技有限公司 | A kind of fluorescence quantifying PCR method for detecting Escherichia coli |
| CN107760766A (en) * | 2017-12-05 | 2018-03-06 | 苏州国科闻普生物科技有限公司 | Method, kit and its application of multiple five kinds of Diarrheogenil Escherichia colis of qPCR quick detections |
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