CN103451306B - Multi-PCR (Polymerase Chain Reaction) detecting primer group for Escherichia coli O104:H4 and reagent kit - Google Patents

Abstract

The invention discloses a multi-PCR (Polymerase Chain Reaction) detecting primer group for Escherichia coli O104:H4 and a reagent kit. The primer group for detecting serotype genes and pathogenic genes of the O104:H4 is composed of six primer pairs for detecting an orfO104 gene, a fliCH4 gene, an stx1 gene, an stx2 gene, an eaeA gene and an aggR gene. A multi-PCR detecting method is established based on a common PCR platform; the method comprises the steps: carrying out multi-PCR reaction on a DNA (Deoxyribonucleic Acid) by using the primer pairs, wherein the DNA is extracted from a sample to be detected, and the primer pairs are obtained through analysis and design; carrying out electrophoretic analysis on a PCR product after the reaction is ended. The method can be used for determining the kinds of virulence genes in the sample while determining whether the sample belongs to the serotype of the Escherichia coli O104:H4 and can also be used for effectively detecting a novel recombinant mode of potential EAEC (Enterohemorrhagic E. Coli) and EHEC (Enteroaggregative E. Coli).

Description

Colon bacillus O104:H4 multiple PCR detection primer group and test kit

Technical field

The invention belongs to microorganism detection field, relate to the multiple fast PCR of colon bacillus O104:H4 and detect primer sets and test kit.

Background technology

In May, 2011, there is the epidemic outbreaks that caused by colon bacillus O104:H4 in Germany, and involves the ground such as other 13 countries of European Union and the U.S., Canada.Owing to causing, the Serotypc comparison of colon bacillus of this epidemic situation is rare, the general susceptible of crowd, and epidemic situation spreads fast.Until July, epidemic situation stopped then, report altogether colon bacillus O104:H4 cases of infection more than 4400 examples.

The clinical manifestation of infecting colon bacillus O104:H4 mainly contains abdominal colic and diarrhoea, and some cases are bloody stool sample diarrhoea.Rehabilitation in most of patients 10 days,, can there is severe complication in small number of patients, as the Hemolytic―uraemic taking acute renal failure, hemolytic anemia and thrombopenia as feature (HUS).Follow up a case by regular visits to and find that the prognosis of infection O104:H4 is poor, the infected of 20% is converted into HUS.Once HUS occurs, and case fatality rate will significantly increase.

This time causing the colon bacillus O104:H4 bacterial strain of epidemic situation all different with the Lapactic colon bacillus of finding in the past, is a kind of newfound colon bacillus new variant with strong virus force.O104:H4 genome sequencing is found, O104:H4 is the new bacterial strain that EHEC (EHEC) and intestines concentration colon bacillus (EAEC) restructuring produce.O104:H4 had both contained EAEC intermediary leads the aggR that concentration sticks, and contains again the shiga-like toxin 2(stx2 that can cause HUS in EHEC).But to pathogenic other the relevant virulence genes of EHEC, as adhesion factor gene eaeA, shiga-like toxin 1(stx1) in O104:H4, all do not find.Wherein, eaeA mediation thalline adheres to, and the mechanism of causing a disease of stx1 and stx2 is similar.

Novel colon bacillus O104:H4, with the genetic characteristics of EAEC and EHEC, therefore has larger hazardness.The aggR containing in O104:H4, can make thalline produce concentration on intestinal cells surface and stick, and decrease uptake, produces diarrhoea.Its shiga-like toxin 2(stx2 carrying simultaneously), cause intestinal mucosa epithelial cell destruction, mucosa ulcer, hemorrhage, vascular endothelial cell damage, haemolysis, thrombopenia.More seriously, renal cells necrosis, obstruction and renal glomerulus destroy, and cause hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC).Severe patient can develop into multiple organ dysfunction syndrome, causes infected patient death.

O104:H4 type colon bacillus is mainly by contaminated food transmission, according to experience previously, in states such as America and Europes, the milk preparation of contaminated hamburger filling and non-sterile is an important contagium, and more contaminated agricultural-food also can become contagium.

Before research shows that O104:H4 breaks out in crowd, between animal, propagating for 10 years.Therefore carry out the active detecting of O104:H4, the Novel heavy group mode of potential EAEC and EHEC is explored, set up and effectively break out early warning mechanism, the prevention and control of O104:H4 and novel restructuring pathogenic infection are of great importance.

Adopt traditional method qualification O104:H4, as increased bacterium cultivation, biochemical identification and serum agglutination, need to after bacterium enrichment, to adopt biochemical method to determine be colon bacillus increasing, determine its O antigen and H antigen by serum agglutination method.But after determining that a strain colon bacillus is O104:H4 serotype, because not knowing its virulence gene Carriage, still can not judge whether this strain bacterium is pathogenic bacterium, cannot determine detect whether meaningful.And traditional detection method is wasted time and energy, result is affected greatly by serum quality and subjective judgement.

Except traditional method, some molecular biological detection methods are set up.The colon bacillus Reference Lab recommendation substance real-time fluorescence of European Union is to stx2, rfb _o104and fliC _h4gene examination one by one (Detection and identification of Verocytotoxin-producing Escherichia coli (VTEC) O104:H4in food by Real time PCR).Chinese Patent Application No. 201110376158.5 discloses one and has caused and rush down intestinal bacteria quadruple fluorescent quantificationally PCR detecting kit, by a reaction, can from sample, detect the existence of the colon bacillus of O157:H7 and O104:H4 serotype, but can only determine whether it is the colon bacillus of O104:H4 serotype, can not judge whether this bacterium is pathogenic strain.Chinese Patent Application No. 201210068180.8 discloses enterohemorrhagic Escherichia coli O104:H4 detection kit and using method thereof, pass through quantitative fluorescent PCR, detect and whether contain wzy(O104), fliC (H4) and aggR gene, thereby judge that tested bacteria is enterohemorrhagic Escherichia coli O104:H4.Chinese Patent Application No. 201110197208.3 discloses multiple fluorescence PCR detection reagent box and the detection method of a kind of enterohemorrhagic Escherichia coli O104:H4, detect shiga-like toxin 2 type genes (stx2), O antigen Flippases gene (wzx) and H4 flagellar antigen gene (fliC _h4), if Stx2, wzx and fliC _h4all be positive, judge that tested bacteria is enterohemorrhagic Escherichia coli O104:H4.

Adopt the detection of molecular biology method to O104:H4, be confined to common substance PCR and fluorescent PCR more, the former need to be to relevant O antigen, H antigen encoding gene and shiga-like toxin and adhesion genes involved detect one by one, waste time and energy, cannot meet Emergent detection to time sex-limited requirement, the latter's fluorescent quantitation detection platform drops into high, reagent costliness, and be limited by the restriction of fluorescence channel, can not be by O104:H4 serotype and the examination of pathogenic related gene system, and the Novel heavy group mode to potential EAEC and EHEC cannot detect, greatly limit its application in a line detects.

Summary of the invention

In order to solve problems of the prior art, the object of this invention is to provide a kind of colon bacillus O104:H4 multiple PCR detection primer group and test kit, can rapid screening colon bacillus O104:H4 serotype and pathogenic strain and recombinant strain, set up a kind of multi-PCR detection method based on regular-PCR platform.

In order to realize the object of the invention, first the present invention provides the technical scheme of design and screening Auele Specific Primer and the primer sets of a kind of multiplex PCR detection colon bacillus O104:H4:

1, design positive internal reference (IAC) amplimer and gene

Taking pET28a plasmid as IAC stencil design pair of primers, the fragment of amplification 1176bp size wherein, as the IAC detecting.IAC upstream primer sequence is 5 '-TGCAGGTCGACTCTAGAGGA-3 ', and the sequence of IAC downstream primer is 5 '-TTCGAGCTCGGTACCCGGGGA-3 '.The base sequence of pET28a is as shown in SEQ ID No.15.

2, the gene of screening detection target or DNA fragmentation are for design of primers

The present invention is by document analysis and detect demand analysis, has obtained the gene of each target to be checked or DNA fragmentation as amplification target candidate (in table 1).

Table 1 colon bacillus O104:H4 multiplex PCR detects candidate target gene

Target to be checked	Candidate's amplification gene
		O104 antigen	orf O104
H4 antigen	fliC H4
		Shiga-like toxin 1	stx1
Shiga-like toxin 2	stx2
		Adhesion factor gene	eaeA
Mediation concentration adheres to	aggR

3, primer candidate scheme design

The present invention uses primer premier6 software for all amplification target candidate genes or fragment design 1-2 cover alterant.

4, the primer candidate scheme of amplification target gene is carried out to Blast analysis

In Genbank, all primer candidate schemes are carried out to Blast analysis, obtain the higher primer scheme of specificity as experimental verification scheme, each amplification target has 1-3 experimental verification scheme.

5, each available experimental verification scheme is carried out to substance PCR checking, determine the operability of primer, comprise specificity assessment and sensitivity assessment.

6, the primer of all amplification targets is mixed and carries out multiplex PCR, operability when checking primer increases jointly, need to replace according to the new primer of step 2-4 design iterations inoperable primer.The final a set of special primer sequence provided by the invention (referring to table 2) that obtains.

Table 2 colon bacillus O104:H4 multiple PCR primer summary sheet

Note: although the gene of selected detection target or DNA fragmentation are conservative region, the possible type that still has indivedual bases to insert or lack, the size of PCR product also can increase thereupon or reduce several bp.

Based on technique scheme, the invention provides a kind of colon bacillus O104:H4 multiple PCR detection primer group, it is made up of the primer pair that detects serotype and pathogenic gene;

Wherein, the primer pair of detection serotype and pathogenic gene is by detecting orf _o104gene, fliC _h46 primer pair compositions of gene, stx1 gene, stx2 gene, eaeA gene and aggR gene;

Described detection orf _o104the nucleotide sequence of the right upstream and downstream primer of gene primer is respectively by shown in SEQ ID NO.1, SEQ ID NO.2;

Described detection fliC _h4the nucleotide sequence of the right upstream and downstream primer of gene primer is respectively by shown in SEQ ID NO.3, SEQ ID NO.4;

The described nucleotide sequence that detects the right upstream and downstream primer of stx1 gene primer is respectively by shown in SEQ ID NO.5, SEQ ID NO.6;

The described nucleotide sequence that detects the right upstream and downstream primer of stx2 gene primer is respectively by shown in SEQ ID NO.7, SEQ ID NO.8;

The described nucleotide sequence that detects the right upstream and downstream primer of eaeA gene primer is respectively by shown in SEQ ID NO.9, SEQ ID NO.10;

The described nucleotide sequence that detects the right upstream and downstream primer of aggR gene primer is respectively by shown in SEQ ID NO.11, SEQ ID NO.12;

The present invention also provides a kind of method of colon bacillus O104:H4 being carried out to multiplex PCR detection, the DNA of described primer sets and testing sample is carried out to multi-PRC reaction, reaction finishes rear PCR product to be carried out to electrophoretic analysis, to determine that whether sample is as colon bacillus O104:H4, determine its virulence gene kind containing simultaneously, judge whether it is pathogenic strain and (or) recombinant strain.

Further, in the time carrying out multi-PRC reaction, in reaction system, also add positive internal reference primer pair.

The nucleotide sequence of the upstream and downstream primer of described positive internal reference primer pair is respectively by shown in SEQ ID NO.13 and SEQ ID NO.14.

When the method can be determined colon bacillus O104:H4 serotype fast, determine whether it contains adhere to and cause a disease to concentration relevant stx1, stx2, eaeA and aggR, and then determine whether the Novel heavy group mode of EAEC and EHEC, the technical scheme of its realization is as follows:

1, primer is synthetic

According to the primer sequence in table 2, in the synthetic all oligonucleotide sequences of gene Synesis Company.

2, DNA profiling extracts

Select Salmonellas, Shigellae, Vibrio parahemolyticus, campylobacter jejuni, Campylobacter Coli, streptococcus aureus, bacillus cereus, Listeria monocytogenes, yersinia entero-colitica, Enterobacter sakazakii, vibrio cholerae, non-Diarrheogenil Escherichia coli, EHEC (EHEC, O157:H7 serotype), intestines concentration colon bacillus (EAEC, O9:K99 serotype), Aeromonas hydrophila and the outburst strain of colon bacillus O104:H4(Germany, aggR+, stx2+) as the target microorganism of setting up detection method, according to GB or industry standard method, corresponding microorganism being increased to bacterium respectively cultivates, adopt bacterial genomes to extract test kit and extract DNA profiling.

3, use regular-PCR platform construction multiplex PCR detection system

In definite multiple reaction system, after primer sequence, also need to be optimized respectively from aspects such as primer concentration, annealing temperature, reaction system configuration, reaction conditionss.The configuration of reaction system is as follows:

Taq archaeal dna polymerase 1-2U, 5 × PCR buffer(TrisHCl100mM(PH8.3), KCl250mM, tween-200.2%) and 5 μ L, the dNTP0.5-1.5 μ L of 10mm, the MgCl of 25mM ₂1-5 μ L, 10 × primer mixture, 2.5 μ L (the primer concentration 1 μ M-8 μ M of the each target to be measured including positive internal reference), pET28a0.0003-0.01ng, DNA profiling 2-5 μ L, ultrapure water is mended to 25 μ L.The reaction conditions of multiple system is as follows:

a：95℃4min；

b：95℃15-60S，

c：55-65℃15-60s，

D:72 DEG C of 30-120s, 30-35 reaction of b-d circulation;

e：72℃5-10min。

4, multiple PCR products analysis and result are judged

5 μ L multiple PCR products are carried out to electrophoretic analysis PCR product clip size, judge the situation of amplification target gene according to clip size synopsis 2.

5, the checking of detection system

Carry out specificity checking and susceptibility checking for the colon bacillus O104:H4 multi-PCR detection method of having set up.

Specificity checking: select Salmonellas, Shigellae, Vibrio parahemolyticus, campylobacter jejuni, Campylobacter Coli, streptococcus aureus, bacillus cereus, Listeria monocytogenes, yersinia entero-colitica, Enterobacter sakazakii, vibrio cholerae, non-Diarrheogenil Escherichia coli, Aeromonas hydrophila, Vibrio flurialis, Bacillus anthracis, Webster genus bacillus, sheep listeria spp, bordetella pertussis, legionella, suis etc. are as specificity assessment bacterial strain, carry out nucleic acid extraction, adopt the reaction conditions of setting up and optimizing early stage, applying detection reagent of the present invention detects.

Result shows the positive internal reference of IAC() all positive.Except IAC, the specific band amplification nothing but of specificity checking bacterial strain, duplicate detection result is all consistent, shows that this detection method has good specificity, non-object bacteria effectively can be distinguished.

Susceptibility checking: be 10 to starting point concentration ⁸the colon bacillus O104:H4 of CFU/mL and O157:H7 bacterium liquid extract template, and the former verifies orf _o104gene, fliC _h4the detection sensitivity of gene, stx2 gene and aggR gene, the latter verifies the detection sensitivity of stx1 gene and eaeA gene, is diluted to respectively 10 ⁷cFU/mL, 10 ⁶cFU/mL, 10 ⁵cFU/mL, 10 ⁴cFU/mL.Adopt the reaction conditions of setting up early stage and optimize to detect, the detectability of every kind of target to be checked all can reach 10 ⁵cFU/mL, duplicate detection result is all consistent, shows that this detection method has the susceptibility of height and good repeatability.

The present invention also provides a kind of test kit that contains aforementioned multiple PCR detection primer group.

Beneficial effect of the present invention is:

The colon bacillus O104:H4 multi-PCR detection method that the present invention sets up, can realize serology, cause of disease cultivate learn, qualification work that immunology cannot complete, the weak point that overcomes other Protocols in Molecular Biology means qualification virulence genes, reaches following detection effect:

(1) Multiple detection

The detection method that the present invention sets up can examination and EHEC and EAEC pathogenic relevant stx1, stx2, eaeA and tetra-kinds of virulence genes of aggR comprehensively in PCR reaction, and the encoding gene orf of O104 and H4 _o104and fliC _h4, determine that fast whether sample is colon bacillus O104:H4 serotype and the virulence gene kind containing, and judges whether it is pathogenic strain and (or) recombinant strain.Save time, man power and material's cost.

(2) specificity is high

The detection method specificity that the present invention sets up is mainly reflected in the specificity of a whole set of primer, and all primers all pass through Blast compare of analysis, has conservative property and the specificity of height, simultaneously through specificity checking, can be good at distinguishing with detection target species symbolic animal of the birth year near, the bacterium that living environment is identical, comprise Salmonellas, Shigellae, Vibrio parahemolyticus, campylobacter jejuni, Campylobacter Coli, streptococcus aureus, bacillus cereus, Listeria monocytogenes, yersinia entero-colitica, Enterobacter sakazakii, vibrio cholerae, non-Diarrheogenil Escherichia coli, Aeromonas hydrophila, Vibrio flurialis, Bacillus anthracis, Webster genus bacillus, sheep listeria spp, bordetella pertussis, legionella, suis etc., prove that detection method has the specificity of height, non-target bacteria accurately can be distinguished.

(3) highly sensitive

The detection method that the present invention sets up can realize to causing a disease relevant stx1, stx2, eaeA and tetra-kinds of virulence gene examinations of aggR to EHEC and EAEC when, determine fast O104:H4 serotype, in each reaction system, the detection sensitivity of each detection target can reach 10 ⁵cFU/mL.

(4) cost is lower

The multi-PCR detection method that the present invention sets up has reduced human cost and time cost in operability, and originally substance detects needs artificial and 6 times of times 6 times, uses now this method only to need 1 time manually and the time of 1 secondary response; This multiple detection method has been saved the reagent consumption of the same sample of duplicate detection simultaneously, and maximum can be saved more than 50% reagent cost.

(5) prevention false negative result

The IAC adding in system, can effectively point out false negative detected result.

Serotype Identification and virulence gene rapid detection that the present invention is colon bacillus O104:H4 provide complete solution, can realize O104:H4 and break out the quick Emergent detection after rear and novel recombinant bacterial strain occurs, save the valuable time for disposing as early as possible epidemic situation.

In sum, the invention provides a kind of multiplex PCR and detect the primer sets of colon bacillus O104:H4, and set up a kind of multi-PCR detection method based on regular-PCR platform.The present invention adopts highly sensitive and specific primer sequence, ensures the quality of detected result; This detection method is simple to operate, time saving and energy saving; Detection flux is high, and reagent consumables cost is low; Can detect the enrichment liquid of sample, be compared to traditional detection method, can shift to an earlier date 24-48 hour and lock suspicious pathogenic bacterium; To detection platform and testing staff require lowly, can extensively promote at inspection and quarantine First Line.

Brief description of the drawings

Fig. 1 is the pcr amplification product agarose gel electrophoresis result of specificity analyses in the embodiment of the present invention 2;

Wherein: M:100bp DNA marker; 1.aggR-1; 2.aggR-2; 3.aggR-3; 4. positive control.

Fig. 2 is the pcr amplification product agarose gel electrophoresis result of sensitivity assessment in the embodiment of the present invention 2;

Wherein: M:100bp DNA marker; 1.aggR-1; 2.aggR-2; 3.aggR-3.

Fig. 3 is the amplification of three bacterial strains in the embodiment of the present invention 4;

Wherein: M:100bp DNA marker; 1. colon bacillus O104:H4(Germany outburst strain, stx2+, aggR+); 2.EHEC(O157:H7); 3.EAEC (O9:K99); 4. blank; 5. positive control (1 and 2 hybrid templates).

Embodiment

Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.

The design of embodiment 1aggR gene primer is with synthetic

In Genbank, download 10 of the full length sequences of colon bacillus O104:H4aggR gene, 10 aggR full length gene sequences are imported in Mega4 software, use the conserved sequence of alignment function compare of analysis aggR gene, obtain conserved sequence section, base sequence is as shown in SEQ ID No.16.

In the conserved sequence of above-mentioned aggR input primer premier6 software, automatic analysis obtains multiple design of primers scheme.Manual regulation primer location and sequence length on this basis, is set as 55 ± 5 DEG C by Tm value, and GC content 35%～60% does not produce hairpin structure and primer dimer as far as possible, and inerrancy causes generation.The design of primers scheme that above-mentioned manual regulation is obtained is carried out Blast analysis on NCBI website, if there is cross reaction in any primer pair and non-detection target wherein, need to readjust position and the sequence length of primer, until obtain the aggR gene primer sequence of high specific.Finally obtain as three of table 3 kinds of aggR gene primer alternativess.

Table 3aggR gene primer alternatives

The primer design method of other genes is identical with aggR gene.The primer alternatives obtaining is all synthetic in invitrogen company.

The test of embodiment 2aggR primer screening

The bacterium group of specificity assessment: comprise Shigellae, Vibrio parahemolyticus, campylobacter jejuni, Campylobacter Coli, streptococcus aureus, bacillus cereus, Listeria monocytogenes, yersinia entero-colitica, Enterobacter sakazakii, non-Diarrheogenil Escherichia coli, vibrio cholerae, Aeromonas hydrophila, Vibrio flurialis, Bacillus anthracis, Webster genus bacillus, sheep listeria spp, bordetella pertussis, legionella, the common food-borne pathogens such as suis.

Adopt bacterial genomes to extract test kit to above-mentioned bacterium and extract DNA profiling.The template of extraction is respectively got to 5 μ L and mix, as the template of specificity checking.

The specificity analyses test of colon bacillus O104:H4: use the primer pair having designed in embodiment 1, according to following project configuration PCR reaction system: Taq archaeal dna polymerase 0.4 μ L(5U/ μ L), 5 × PCR buffer(TrisHCl100mM(PH8.3), KCl250mM, tween-200.2%) 5 μ L, dNTP1.25 μ L(10mM), MgCl ₂4 μ L(25mM), Auele Specific Primer 1 μ L(10 μ M); Specificity validating DNA template 5 μ L, ultrapure water is mended to 25 μ L.Carry out pcr amplification according to following reaction conditions:

a：95℃4min；

b：95℃30S，

c：62℃30s，

D:72 DEG C of 90s, b-d 30 reactions that circulate;

e：72℃5min。

Pcr amplification product 5 μ L use 2% agarose gel electrophoresis analytical results as shown in Figure 1.As shown in Figure 1, under the prerequisite of setting up at positive control, three couples of aggR primer aggR-1 of design, aggR-2 and aggR-3 and Shigellae, Vibrio parahemolyticus, campylobacter jejuni, Campylobacter Coli, streptococcus aureus, bacillus cereus, Listeria monocytogenes, yersinia entero-colitica, Enterobacter sakazakii, non-Diarrheogenil Escherichia coli, vibrio cholerae, Aeromonas hydrophila, Vibrio flurialis, Bacillus anthracis, Webster genus bacillus, sheep listeria spp, bordetella pertussis, legionella, the habitats such as suis are similar, high frequency the equal no cross reaction of the bacterium of depositing.Therefore can think, three couples of primer aggR-1, aggR-2 and aggR-3 all have good specificity.

Sensitivity assessment: select the template of O104:H4, its starting point concentration is 10 ⁸cFU/mL, gradient dilution to 10 ⁵cFU/mL, as the template of aggR primer sensitivity assessment.System configurations is as follows: Taq archaeal dna polymerase 0.4 μ L(5U/ μ L), 5 × PCR buffer(TrisHCl100mM(PH8.3), KCl250mM, tween-200.2%) and 5 μ L, dNTP1.25 μ L(10mM), MgCl ₂4 μ L(25mM), Auele Specific Primer 1 μ L(10 μ M); O104:H4 template 2 μ L, ultrapure water is mended to 25 μ L.The experiment of PCR reaction conditions homospecificity.

Pcr amplification product 5 μ L use 2% agarose gel electrophoresis analytical results as shown in Figure 2.As seen from Figure 2, three primer pairs all can reach 10 ⁵cFU/mL, wherein the band brightness of primer pair aggR-2 is the highest, is secondly aggR-1, aggR-3.

Overall sensitivity and specific assessment result, select the primer of aggR-2 as aggR gene amplification.

The appraisal procedure of other genes is identical with aggR gene.

The establishment of embodiment 3 colon bacillus O104:H4 multiple PCR detection kits

This test kit is made up of 2 × reaction system damping fluid, archaeal dna polymerase, 10 × primer mixed solution, positive control, ultrapure water.

Its concrete component is as follows: 2 × PCR Buffer(TrisHCl40mM (PH8.3), KCl100mM, tween-200.08%, 0.0006ng/ μ L pET28a, 1mm dNTP, 8mm MgCl ₂); 25 × archaeal dna polymerase (2U/ μ L); 10 × primer mixed solution (the each 2 μ M of Auele Specific Primer including IAC); The hybrid template of positive control (colon bacillus O104:H4, EHEC(O157:H7), every kind is 10 ⁶cFU/ml).

The reaction system that test kit detects is 25 μ L, and its configuration is as follows: 2 × PCR Buffer12.5 μ L; 25 × archaeal dna polymerase, 1 μ L; 10 × primer mixed solution, 2.5 μ L; Template 2 μ L, ultrapure water 7 μ L.

The operation of embodiment 4 test kits and result judgement

1, genomic extraction

Get colon bacillus O104:H4, EHEC(O157:H7), EAEC(O9:K99) enrichment liquid or streak culture bacterium colony soluble in water, adjust bacterial suspension concentration to 10 with turbidometer ⁸cFU/ml, gets 1ml bacterial suspension, ice bath after boiling water bath 10min, and the centrifugal 10min of 13000rpm, gets supernatant as test kit detection template.

2, the preparation of reaction system

The reaction system of getting the PCR pipe configuration 25 μ L of 200 μ L, its configuration is as follows: 2 × PCR Buffer12.5 μ L; 25 × archaeal dna polymerase, 1 μ L; 10 × primer mixed solution, 2.5 μ L, template 2 μ L, ultrapure water 7 μ L.

3, PCR reaction

PCR pipe is put into Bio-Rad C1000 type PCR instrument, opens after heat lid, carry out PCR reaction according to following program:

a：95℃4min；

b：95℃30S，

c：62℃30s，

D:72 DEG C of 90s, b-d 30 reactions that circulate;

e：72℃5min。

4, agarose gel electrophoresis Platform Analysis PCR product

Get the PCR product of 5 μ L and the 6xloading buffer of 1 μ L mixes, join in the well of sepharose.Adopt 2% sepharose, 5V/cm electrophoresis 60min, uses gel imaging instrument to show the size of amplification target stripe.

5, result judgement

The result of three bacterial strain amplifications as shown in Figure 3, as shown in Figure 3, all swimming lanes including blank all have a band amplification at least, blank (4 swimming lane) has IAC(1176bp) amplification, other detections have the amplification of target stripe and (or) IAC, show all establishments of all PCR reactions, get rid of false-negative result.Otherwise invalid depending on experiment, need repetition.

On the basis of IAC amplification, also there are 4 articles of PCR products in O104:H4 (the 1st swimming lane), and that 192bp is fliC _h4the amplified band of gene, 253bp's is the amplified band of aggR gene, that 385bp is orf _o104the amplified band of gene, 640bp's is the amplified band of stx2 gene.EHEC(the 2nd swimming lane) what also occur 150bp is the amplified band of stx1 gene, and 501bp's is the amplified band of eaeA gene, and 640bp's is the amplified band of stx2 gene; EAEC(the 3rd swimming lane) also there is the band of 253bp, be the amplified production of aggR gene; Positive control (the 5th swimming lane) all increases 6 bar segment out, illustrates that the coamplification ability of system is strong.

The preservation period test of embodiment 5 test kits

With 10 ⁵colon bacillus O104:H4, the EHEC (O157:H7) of CFU/ml, EAEC(O9:K99) as assessment with detect sample, in the time of the 0th day, be distributed into 9 parts frozen in-70 DEG C of refrigerators.Be positioned over-20 DEG C of preservations by setting up complete test kit, the test kit of getting respectively 0,10,20,30,60,90,120,150 and 180 day carries out preservation period test.Preservation period detected result is as shown in table 4:

Table 4 preservation period test-results

As shown in Table 4, test kit is kept at-20 DEG C of refrigerators, all positive in the amplification of different preservation perives target to be checked, shows that the preservation period of this test kit is at least 6 months.

The specific test of embodiment 6 test kits

Selection comprises Shigellae, Vibrio parahemolyticus, campylobacter jejuni, Campylobacter Coli, streptococcus aureus, bacillus cereus, Listeria monocytogenes, yersinia entero-colitica, Enterobacter sakazakii, non-Diarrheogenil Escherichia coli, vibrio cholerae, Aeromonas hydrophila, Vibrio flurialis, Bacillus anthracis, Webster genus bacillus, sheep listeria spp, bordetella pertussis, legionella, suis etc. are close with detection object bacteria kind, exist like environmental facies bacterium as bacterium to be checked, all only has after testing an IAC band amplification, produce without assorted band.And when the object bacteria such as O104:H4, EHEC, EAEC are detected, except specific band, produce without assorted band.Show that test kit of the present invention can effectively distinguish non-target bacteria, there is good specificity.

The sensitivity test of embodiment 7 test kits

Assessment is with detecting sample: adjust colon bacillus O104:H4, EHEC (O157:H7), EAEC(O9:K99) bacterial concentration to 10 ⁸on the CFU/mL order of magnitude, extract DNA profiling.Each template gradient dilution becomes 10 ⁷cFU/mL, 10 ⁶cFU/mL, 10 ⁵cFU/mL, 10 ⁴the detection sample of CFU/mL.

Use test kit of the present invention to detect respectively different dilution To Templates to be measured.According to example 4 result determination methods, the sensitivity test result of test kit detection target gene is as shown in table 5:

Table 5 test kit detects testing gene sensitivity test result

As seen from Table 5, the susceptibility of target to be checked is 10 ⁵cFU/mL.

Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.

Claims (1)

1. a colon bacillus O104:H4 multiple PCR detection primer group, is characterized in that, it is made up of the primer pair that detects serotype and pathogenic gene;

Wherein, the primer pair of detection serotype and pathogenic gene is by detecting orf _o104gene, fliC _h46 primer pair compositions of gene, stx1 gene, stx2 gene, eaeA gene and aggR gene;

Described detection orf _o104the nucleotide sequence of the right upstream and downstream primer of gene primer is respectively by shown in SEQ ID NO.1, SEQ ID NO.2;

Described detection fliC _h4the nucleotide sequence of the right upstream and downstream primer of gene primer is respectively by shown in SEQ ID NO.3, SEQ ID NO.4;

The described nucleotide sequence that detects the right upstream and downstream primer of stx1 gene primer is respectively by shown in SEQ ID NO.5, SEQ ID NO.6;

The described nucleotide sequence that detects the right upstream and downstream primer of stx2 gene primer is respectively by shown in SEQ ID NO.7, SEQ ID NO.8;

The described nucleotide sequence that detects the right upstream and downstream primer of eaeA gene primer is respectively by shown in SEQ ID NO.9, SEQ ID NO.10;

The described nucleotide sequence that detects the right upstream and downstream primer of aggR gene primer is respectively by shown in SEQ ID NO.11, SEQ ID NO.12.