CN103451307A - Shigella flora/serotype multiplex-PCR (polymerase chain reaction) detection primer set and kit - Google Patents
Shigella flora/serotype multiplex-PCR (polymerase chain reaction) detection primer set and kit Download PDFInfo
- Publication number
- CN103451307A CN103451307A CN2013104255679A CN201310425567A CN103451307A CN 103451307 A CN103451307 A CN 103451307A CN 2013104255679 A CN2013104255679 A CN 2013104255679A CN 201310425567 A CN201310425567 A CN 201310425567A CN 103451307 A CN103451307 A CN 103451307A
- Authority
- CN
- China
- Prior art keywords
- primer
- shigella
- shigellae
- seq
- pcr
- 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.)
- Pending
Links
Images
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
The invention discloses a Shigella flora/serotype multiplex-PCR (polymerase chain reaction) detection primer set and kit. The detection primer set is composed of primer pairs for detecting Shigella sonnei, type-1-5 Shigella flexneri, type-I Shigella dysenteriae, type-6 Shigella flexneri, and Shigella. According to the invention, a multiplex-PCR detection method based on a common PCR platform is built; according to the method, multiplex-PCR reaction is carried out on total DNA of bacteria extracted from a sample to be detected in a same reaction system by using the primer pairs obtained through analysis and design, and through electrophoretic analysis on a reaction product, whether the sample is Shigella can be determined and Shigella flora/serotype can be judged.
Description
Technical field
The invention belongs to the microorganism detection field, the multiple fast PCR that relates to Shigellae group/serotype detects primer sets and test kit.
Background technology
Shigellae is that a class has hyperinfection, endangers serious Grain-negative pathogen enterobacteria, the Shigellae property dysentery (shigellosis) caused by its clinical infection is one of important transmissible disease of developing country, is also the main pathogen of developed country's dysentery.Nearly 1.65 hundred million examples of Shigellae property dysentery case that the whole world is annual, wherein 1.63 hundred million occur in developing country, and cause 1,100,000 deaths, more than 60%, is wherein children below 5 years old.The vast Rural areas of China is because sanitary condition is poor, often has and is subject to Shigellae to pollute the report that causes the disease popularities such as dysentery, diarrhoea because of water source or food.
Shigellae is Shigella, is divided into four populations: shigella dysenteriae (A group), shigella flexneri (B group), Shigella bogdii (C group) and Shigella sonnei (D group).Shigellae antigen is comprised of O antigen (O antigen) and kantigen (K antigen), and the specificity of O antigen is good, can carry out serological typing to Shigellae accordingly.Shigella dysenteriae has 13 serotypes, and shigella flexneri has 6 serotypes, and Shigella bogdii has 18 serotypes, and Shigella sonnei only has a serotype.Wherein, shigella flexneri 6 types and 1-5 type O antigenic difference are larger.Four groups' Shigellae all can cause dysentery.According to the investigation that distributes of the bacterial type of Shigellae, China take Fu Shi and Shigella sonnei and infects as main; Shigella dysenteriae 1 type is the main bacterial strain that Shigellae breaks out.In recent years, the bacillary dysentery that shigella dysenteriae I type causes has developed into worldwide fashion trend, China at least in 10 provinces, that different scales has occurred in district is popular.The shigella dysenteriae of its alloytype and Shigella bogdii are more rare.
It is to using standard GB/T/T4789.5-2012 as foundation that the main method adopted at present detects to Shigellae in China, adopts bacterium separation and Culture, biochemical reaction and serological method to identify group and serotype.Whole detection obtains net result needs about 5 days, not only time-consuming but also require great effort, and it is limited to be limited to database information.Simultaneously, choosing colony is incomplete, bacterium colony is impure, bacterial concentration is too high or too low all can affect presenting of net result.
Molecular biology for detection provides good testing tool for Shigellae detects, and has all set up both at home and abroad and has adopted round pcr Shigellae to be carried out to the technology of rapid detection at present.Such as the Chinese Patent Application No. patent of invention " Shigellae detects by primer, detection method, detection kit " that is 200710030435.0, provide a kind of based on the loop-mediated isothermal amplification technique detection kit, the test kit that test kit detects primer sets and comprises this primer sets by one group of Shigellae detects the specific gene fragment of Shigellae in sample, to determine the situation that exists of Shigellae in sample.The patent of invention that Chinese Patent Application No. is 200710026604.3 " a kind of method and test kit thereof that detects Shigellae and ipah pathogenicity island thereof ", the method and the reagent that detect Shigella and ipah pathogenicity island in clinical sample are provided, have particularly related to method and test kit with real time fluorescent quantitative poly chain reaction technology for detection Shigella bacterium.The patent of invention that Chinese Patent Application No. is 200510132404.7 " gene chip and detection method and the detection test kit that detect for Shigellae serotype ", a kind of gene chip and detection method and detection test kit detected for Shigellae serotype is provided, by primer amplification 16S rRNA, O antigen encoding gene, PCR product and solid phase chip are hybridized to detect to the different serotypes of Shigellae.
Adopt substance PCR or real-time fluorescence PCR can identify fast the Shigellae kind, but group and serotype are still needed to identify by separation and Culture, biochemical reaction and serological method.Due to food and clinical samples complicated component, the nucleic acid after extraction still likely suppresses composition with PCR, false-negative result occurs, causes the undetected of Shigellae.
The cost of the technical barriers such as biochip can carry out the evaluation of system to group and the serotype of Shigellae, but carry out biochip test, need to be equipped with expensive instrument, and poor stability, repetition rate are low and costliness still restricts it and further develops and apply.
Summary of the invention
In order to solve problems of the prior art, the purpose of this invention is to provide primer sets and test kit that a kind of multiplex PCR detects Shigellae group/serotype, can judge quick, special and sensitively Shigella and differentiate Shigellae group and serotype, setting up a kind of multi-PCR detection method based on the regular-PCR platform.
In order to realize the object of the invention, at first the present invention provides the technical scheme of design and screening Auele Specific Primer and the primer sets that a kind of multiplex PCR detects Shigellae group/serotype:
1, design positive internal reference (IAC) amplimer and template:
Take the pET28a plasmid as the stencil design pair of primers, and the fragment of amplification 1176bp size wherein, as the IAC detected.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.13.
2, select the detection target spot of Shigella
Aggressive plasmid antigen H(ipaH) being present in the Shigellae of all groups and serotype, being present on karyomit(e) and the large plasmid of aggressive with multiple copied, not with the disappearance of going down to posterity, is the target spot of more satisfactory detection Shigella.Can judge Shigella by the amplification that detects the ipaH gene.
3, select group and the Serotype Identification primer of O antigen-specific
By read documents, selected key gene wzx in Shigellae O antigen route of synthesis and wzy group and the Serotype Identification target spot as the O antigen-specific.Design corresponding primer after obtaining the gene conservative region by sequence alignment.
4, primer candidate scheme design
The present invention uses primer premier6 software for all amplification target candidate genes or fragment design 1-3 cover alterant.
5, the primer candidate scheme of amplification target carried out to the Blast analysis
In Genbank, all primer candidate schemes are carried out to the Blast analysis, obtain primer scheme that specificity is higher as the experimental verification scheme, each pathogenic bacterium may be selected a plurality of amplification targets, and each target that increases has 1-3 experimental verification scheme.
6, each available experimental verification scheme is carried out to substance PCR checking, determine the operability of primer, comprise specificity assessment and sensitivity assessment.
7, the primer of all amplification targets is mixed and carries out multiplex PCR, the operability when checking primer increases jointly, need to be replaced 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 1) that obtains.
Table 1. Shigella, group and Serotype Identification multiple PCR primer summary sheet
Annotate: 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, does not affect the judgement of result.
Based on technique scheme, the invention provides the primer sets that a kind of multiplex PCR detects Shigellae group/serotype, it is comprised of the primer pair that detects Shigella sonnei, Fu Shi 1-5 type Shigellae, dysentery I type Shigellae, Fu Shi 6 type Shigellaes and Shigella;
The nucleotide sequence of the upstream and downstream primer of described detection Shigella sonnei primer pair is respectively by shown in SEQ ID NO.1, SEQ ID NO.2;
The nucleotide sequence of the upstream and downstream primer of described detection Fu Shi 1-5 type Shigellae primer pair is respectively by shown in SEQ ID NO.3, SEQ ID NO.4;
The nucleotide sequence of the upstream and downstream primer of described detection dysentery I type Shigellae primer pair is respectively by shown in SEQ ID NO.5, SEQ ID NO.6;
The nucleotide sequence of the upstream and downstream primer of described detection Shigella primer pair is respectively by shown in SEQ ID NO.7, SEQ ID NO.8;
The nucleotide sequence of the upstream and downstream primer of described detection Fu Shi 6 type Shigellae primer pairs is respectively by shown in SEQ ID NO.9, SEQ ID NO.10.
The present invention also provides a kind of method of Shigellae group/serotype being carried out to the multiplex PCR detection, total DNA that described primer sets and testing sample are extracted carries out multi-PRC reaction, after reaction finishes, pcr amplification product is carried out to electrophoretic analysis and take and determine that sample is whether as Shigella and judge Shigellae group/serotype.
Further, when carrying out multi-PRC reaction, also add positive internal reference primer pair in reaction system.
The nucleotide sequence of the upstream and downstream primer of described positive internal reference primer pair is respectively by shown in SEQ ID NO.11 and SEQ ID NO.12.
The method, by detecting the conserved sequence of Shigella, in the time of energy Rapid Screening Shigellae, can and be broken out strain and carry out the system detection domestic topmost Shigella spp strain.The IAC simultaneously added, differentiate false negative result as Quality Control.
Its technical scheme is specific as follows:
1, primer is synthetic
According to the primer sequence in table 1, in the synthetic all oligonucleotide sequences of gene Synesis Company.
2, DNA profiling extracts
Use boiling method or commercial kit to extract each population and the total DNA of Serotypes Shigellae as detecting sample.
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 0.5-3U, 5xPCR buffer(TrisHCl 100mM(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 0.4 μ M-4 μ M that comprises the target to be measured of IAC), pET28a0.0003-0.01ng, DNA profiling 2-5 μ L, deionized water is mended to 25 μ L.
The reaction conditions of multiple system is as follows:
a：95℃?4min；
b：95℃?30s，
c：55-68℃?30-60s，
D:72 ℃ of 30-120s, 30-35 reaction of b-d circulation;
h:72℃?5-10min。
4, multiple PCR products analysis and result are judged
By 5 μ L multiple PCR products loading to 2% agarose gel electrophoresis or use full automatic gel electrophoretic analysis PCR product clip size, determine its group and Serotypes when according to clip size synopsis 1, judging whether bacterial strain is Shigellae.
5, the checking of detection system
Carry out specificity checking and susceptibility checking for the Shigellae group who has set up and Serotype Identification multi-PCR detection method.
Specificity checking: select colon bacillus ATCC25922, Enterobacter sakazakii, streptococcus aureus, Salmonellas, Vibrio parahemolyticus, Listeria monocytogenes, vibrio cholerae, bacillus cereus, campylobacter jejuni, Campylobacter Coli, Enterobacter sakazakii, yersinia entero-colitica, have a liking for the aqueous vapor unit cell as specificity assessment bacterial strain, carry out nucleic acid extraction, adopt the reaction conditions of setting up early stage and optimizing to be detected.Result shows the positive internal reference of responded IAC() all positive.Except IAC, negative control and the specific band amplification nothing but of specificity checking bacterial strain, the duplicate detection result is all consistent.Show that this detection method has specificity preferably, non-object bacteria effectively can be distinguished.
Susceptibility checking: be 10 to starting point concentration
8the bacteria suspension of the Fu Shi 2a type Shigellae of CFU/mL, Fu Shi 6 type Shigellaes, dysentery I type Shigellae, Shigella sonnei extracts total DNA, further is diluted to 10
7cFU/mL, 10
6cFU/mL, 10
5cFU/mL, 10
4cFU/mL, 10
3cFU/mL.Adopt the reaction conditions of setting up early stage and optimizing to be detected, the detectability of all targets to be checked is all 10
5cFU/mL, the duplicate detection result is all consistent, shows that this detection method has susceptibility and the repeatability of height.
The present invention also provides a kind of quick detection kit, and described test kit comprises aforesaid primer sets, positive internal reference primer pair, positive internal reference template, archaeal dna polymerase, reaction system damping fluid, Mg
2+, dNTP and deionized water.
Beneficial effect of the present invention is:
Shigellae group and Serotype Identification multi-PCR detection method that the present invention sets up, can by detection time from traditional method within 3-5 days, shorten to 2 hours, reach following detection effect:
(1) Multiple detection
When the detection method that the present invention sets up can be identified Shigella in PCR reaction, in examination Fu Shi 1-5 type, Fu Shi 6 types, Song and dysentery I type Shigellae.Detection method has contained the topmost Shigella spp strain of China comprehensively and has broken out strain.Obtain fast detected result in 2 hours, save time, man power and material's cost.
(2) specificity is high
The specificity of detection method that the present invention sets up is mainly reflected in the specificity of a whole set of Auele Specific Primer, and all primers all pass through the Blast compare of analysis, has conservative property and the specificity of height; Specificity experiment simultaneously shows that primer of the present invention can be good at distinguishing the bacterium close with the Shigellae kind, that living environment is identical, comprises colon bacillus ATCC25922, Enterobacter sakazakii, streptococcus aureus, Salmonellas, Vibrio parahemolyticus, Listeria monocytogenes, vibrio cholerae, bacillus cereus, campylobacter jejuni, Campylobacter Coli, Enterobacter sakazakii, yersinia entero-colitica, has a liking for aqueous vapor unit cell etc.
(3) highly sensitive
When the detection method that the present invention sets up can realize 5 genes, detect, in reaction system, the detection sensitivity of each target gene all can reach 10
5cFU/ml is suitable with substance PCR detection sensitivity; Simultaneously heterogeneic detection sensitivity in the same horizontal line, has been avoided undetected to different Shigellae groups and serotype polyinfection.
(4) cost is lower
The multi-PCR detection method that the present invention sets up has reduced human cost and time cost on operability, and originally substance detects needs artificial and 5 times of times 5 times, uses now this method only to need 1 time manually and the time of 1 reaction; This multiple detection method has been saved the reagent consumption of the same sample of duplicate detection simultaneously, and maximum can be saved the reagent cost more than 50%.
(5) prevention false negative result
The IAC added in system, can effectively point out the false negative detected result.
The present invention provides complete solution for the Shigellae rapid screening, can realize that food poisoning occurs and the Shigellae group of epidemic after breaking out and the Rapid identification of Serotypes, for reasonable appropriate disposal epidemic situation provides reliable foundation.
The present invention overcomes the prior art complex operation, take time and effort, be difficult to carry out the shortcoming and defect such as homogeneous quality control, solves Shigella and group not and the difficult problem of Serotypes Rapid identification.O antigen encoding gene sequence based on the general and corresponding group of Shigella, serotype, do not design a set of Shigella, group not and the primer sequence identified of Serotypes, the multi-PCR detection method of foundation based on this primer sequence, add positive internal reference in system, in a reaction system, complete internal quality control simultaneously.
The accompanying drawing explanation
The pcr amplification product agarose gel electrophoresis result that Fig. 1 is specificity analyses in the embodiment of the present invention 2;
Wherein: M:AL5000DNA marker; 1.FS6-1; 2.FS6-1; 3.FS6-3; P. positive control.
The pcr amplification product agarose gel electrophoresis result that Fig. 2 is sensitivity assessment in the embodiment of the present invention 2;
Wherein: M:AL5000DNA marker; 1.FS6-1; 2.FS6-1; 3.FS6-3.
Fig. 3 is Shigellae group in the embodiment of the present invention 4/serotype multiplex PCR amplified production agarose gel electrophoresis result;
Wherein: M:AL5000DNA marker; 1. Fu Shi 2a type Shigellae; 2. Fu Shi 6 type Shigellaes; 3. Shigella sonnei; 4. dysentery I type Shigellae; 5. Shigella bogdii; 6. blank; 7. positive control.
Embodiment
Following examples are used for the present invention is described, but are not used for limiting the scope of the invention.
The design of primers of embodiment 1 Fu Shi 6 type O antigen encoding gene wzx is with synthetic
Search 10 of the full length sequences of Fu Shi 6 type O antigen encoding gene wzx (Fu Shi 6 types) in Genbank, 10 wzx (Fu Shi 6 types) full length gene sequence is imported in Mega4 software, use the conserved sequence of alignment function compare of analysis wzx (Fu Shi 6 types) gene, obtain the conserved sequence section, base sequence is as shown in sequence table SEQ ID No.14.
In wzx (Fu Shi 6 types) gene order input primer premier6 software, automatic analysis obtains multiple design of primers scheme.Manual regulation primer location and sequence length, be set as 55 ± 5 ℃ by the Tm value on this basis, and GC content 35%～65% 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 the Blast analysis on the NCBI website, if wherein there are cross reaction in any primer pair and non-object bacteria, need to readjust position and the sequence length of primer, until obtain wzx (Fu Shi 6 types) the gene primer sequence of high specific.Final acquisition three kinds of wzx (Fu Shi 6 types) gene primer alternatives as shown in table 2.
Table 2 wzx (Fu Shi 6 types) gene primer alternatives
| The primer numbering | Upstream sequence (5 '-3 ') | Downstream sequence (5 '-3 ') | Amplification length (bp) |
| FS6-1 | ACTTGTTAAACCAAGTCTGG | AGCACTTAGTAATCCTCCAT | 143 |
| FS6-2 | AGCTACCATACCCTTATACTTG | GCACTTAGTAATCCTCCATGA | 159 |
| FS6-3 | AGCTACCATACCCTTATACTTG | ATAGCACTTAGTAATCCTC | 162 |
The primer design method of other genes is identical with wzx (Fu Shi 6 types) gene.The primer alternatives obtained is all synthetic in invitrogen company.
Wzx in his-and-hers watches 2 (Fu Shi 6 types) gene primer alternatives is screened, and mainly assesses specificity and the susceptibility of primer.
Specificity assessment: extract colon bacillus ATCC25922, Enterobacter sakazakii, streptococcus aureus, Salmonellas, Vibrio parahemolyticus, Listeria monocytogenes, vibrio cholerae, bacillus cereus, campylobacter jejuni, Campylobacter Coli, Enterobacter sakazakii, yersinia entero-colitica, have a liking for total DNA of the bacterium such as aqueous vapor unit cell, every kind of bacterium is got 10 μ L extracting solutions, be mixed into a comprehensive DNA profiling, as specificity checking template, use Nanodrop to measure its DNA content for 183.37ng/ μ L.
Use TE(pH8.0) after solution is diluted to respectively the stock solution of 100 μ M by primer, then be mixed with corresponding use liquid.According to following operative configuration 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), primer 1 μ L(10 μ M to be assessed); Specificity validating DNA template 5 μ L, deionized 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 ℃ of 45s, b-d 30 reactions that circulate.
As shown in Figure 1, under the prerequisite of setting up at positive control, three groups of Shigellae primer pair FS6-1 of design, FS6-2, FS6-3 and colon bacillus ATCC25922, Enterobacter sakazakii, streptococcus aureus, Salmonellas, Vibrio parahemolyticus, Listeria monocytogenes, vibrio cholerae, bacillus cereus, campylobacter jejuni, Campylobacter Coli, Enterobacter sakazakii, yersinia entero-colitica, have a liking for that the habitat such as aqueous vapor unit cell is similar, high frequency the equal no cross reaction of bacterium deposited.Therefore can think, three couples of primers F S6-1, FS6-2, FS6-3 all have specificity preferably.
Sensitivity assessment: select Fu Shi 6 type Shigellaes to be cultivated, adjust bacterial concentration to 10
8cFU/mL, gradient dilution to 10
5cFU/mL, as the template of FS6 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%) 5 μ L, dNTP1.25 μ L(10mM), MgCl
24 μ L(25mM), primer 1 μ L(10 μ M to be assessed); Template 2 μ L, deionized water is mended to 25 μ L.
The experiment of PCR reaction conditions homospecificity.
Overall sensitivity and specificity assessment result, select FS6-3 as wzx(Fu Shi 6 types) the final primer of gene amplification.
The appraisal procedure that other Shigella, group and serotype detect primer is identical with Fu Shi 6 type primers.
The establishment of embodiment 3 Shigellae groups and Serotype Identification multiple PCR detection kit
This test kit consists of 2 * reaction system damping fluid, archaeal dna polymerase, 10 * primer mixed solution, positive control, deionized 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 every kind of primer concentration that comprises the IAC primer is 2um), positive control (hybrid template of Fu Shi 2a type, Fu Shi 6 types, dysentery I type and Shigella sonnei, every kind 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, deionized water 7 μ L.
The operation of embodiment 4 test kits and result judgement
1, the extraction of nucleic acid
Get enrichment liquid or streak culture bacterium colony soluble in water, extract the test kit specification sheets according to bacterial genomes and extract template DNA.Measure the concentration of extracting total DNA with NanoDrop.
2, the preparation of reaction system
Get the reaction system of the PCR pipe configuration 25 μ L of 200 μ L, its configuration is as follows: 2 * PCR Buffer12.5 μ L; 25xDNA polysaccharase 1 μ L; 10 * primer mixed solution, 2.5 μ L, template 2 μ L, deionized water 7 μ L.
3, PCR reaction
The PCR pipe is put into to Bio-Rad C1000 type PCR instrument, after opening the heat lid, carry out the PCR reaction according to following program: 95 ℃ of 4min; 95 ℃ of 30s, 62 ℃ of 30s, 72 ℃ of 90s, 30 circulations; 72 ℃ of 5min.
4, agarose gel electrophoresis is analyzed the PCR product
Get the PCR product of 5 μ L and the 6xloading buffer of 1 μ L and mix, join in the well of sepharose.Adopt 2% sepharose, 5V/cm electrophoresis 60min, used the gel imaging instrument to show the size of amplification target stripe.
5, result judgement
The pcr amplification result as shown in Figure 3, as shown in Figure 3:
A. all swimming lanes that comprise blank all have a band amplification at least, and blank has the amplification of IAC, other detections have target stripe and (or) amplification of IAC, show that all PCR reactions all set up, get rid of false-negative result.Otherwise invalid depending on experiment, need repetition.
B. all Shigellaes have the ipaH amplified band of a 210bp; On this basis, Shigella sonnei has the amplified band of a 505bp, and the shigella flexneri of 1-5 type has the amplified band of 372bp, and dysentery I type Shigellae has the amplified band of 279bp, and Fu Shi 6 types have the amplified band of a 162bp.Positive control is in Fu Shi 2a type, Fu Shi 6 types, Song, the hybrid template of dysentery I type Shigellae, and all bands all have amplification, show that this test kit has the ability that detects polyinfection.
The preservation period test of embodiment 5 test kits
With 10
5in the Fu Shi 2a type of CFU/mL, Fu Shi 6 types, Song, dysentery I type Shigellae hybrid template for assessment with detecting sample.Be positioned over-20 ℃ 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 the preservation period test.The preservation period detected result is as shown in table 3:
Table 3 preservation period test-results
| Preservation period | The positive number (comprising IAC) of target gene |
| The |
6 |
| The |
6 |
| The |
6 |
| The |
6 |
| The |
6 |
| The |
6 |
| The |
6 |
| The |
6 |
| The |
6 |
Annotate: the each check of positive internal reference is all positive.
The specific test of embodiment 6 test kits
Select colon bacillus ATCC25922, Enterobacter sakazakii, streptococcus aureus, Salmonellas, Vibrio parahemolyticus, Listeria monocytogenes, vibrio cholerae, bacillus cereus, campylobacter jejuni, Campylobacter Coli, Enterobacter sakazakii, yersinia entero-colitica, to have a liking for aqueous vapor unit cell etc. close with the Shigellae kind, exists like environmental facies bacterium as bacterium to be checked.Apply test kit of the present invention and detect these bacteriums to be checked, positive internal reference is all positive, proves the detection system establishment; Nonspecific assorted band does not all appear in bacterium to be checked.And, when object bacteria is detected, except specific band, without assorted band, produce.Show that test kit of the present invention can effectively distinguish non-target bacteria, there is specificity preferably.
The sensitivity test of embodiment 7 test kits
Assessment is with detecting sample: the Shigellae bacterial strain of selecting 4 particular detection: Fu Shi 2a type, Fu Shi 6 types, Song are interior, dysentery I type Shigellae, 1 non-detection aimed strain: Boydii I type.The concentration of 5 templates is adjusted to respectively to 10
9cFU/mL, equal proportion is mixed into integrated template.The integrated template gradient dilution is become to 10
8cFU/mL, 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 hybrid templates.According to example 4, operate and the result judgement, the sensitivity test result of 5 target genes of test kit detection (containing IAC) is as shown in table 4:
Table 4 test kit detects Shigellae sensitivity test result
As seen from Table 4, the susceptibility of test kit detection Fu Shi 1-5 type, Fu Shi 6 types, dysentery I type, Shigella sonnei and other type Shigellaes all can reach 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, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (6)
1. the primer sets of a multiplex PCR detection Shigellae group/serotype, is characterized in that, it is comprised of the primer pair that detects Shigella sonnei, Fu Shi 1-5 type Shigellae, dysentery I type Shigellae, Fu Shi 6 type Shigellaes and Shigella;
The nucleotide sequence of the upstream and downstream primer of described detection Shigella sonnei primer pair is respectively by shown in SEQ ID NO.1, SEQ ID NO.2;
The nucleotide sequence of the upstream and downstream primer of described detection Fu Shi 1-5 type Shigellae primer pair is respectively by shown in SEQ ID NO.3, SEQ ID NO.4;
The nucleotide sequence of the upstream and downstream primer of described detection dysentery I type Shigellae primer pair is respectively by shown in SEQ ID NO.5, SEQ ID NO.6;
The nucleotide sequence of the upstream and downstream primer of described detection Shigella primer pair is respectively by shown in SEQ ID NO.7, SEQ ID NO.8;
The nucleotide sequence of the upstream and downstream primer of described detection Fu Shi 6 type Shigellae primer pairs is respectively by shown in SEQ ID NO.9, SEQ ID NO.10.
2. a method of Shigellae group/serotype being carried out to the multiplex PCR detection, it is characterized in that, total DNA that the described primer sets of claim 1 and testing sample are extracted carries out multi-PRC reaction, after reaction finishes, pcr amplification product is carried out to electrophoretic analysis and take and determine that sample is whether as Shigella and judge Shigellae group/serotype.
3. method according to claim 2, is characterized in that, when carrying out multi-PRC reaction, also adds positive internal reference primer pair in reaction system.
4. method according to claim 3, is characterized in that, the nucleotide sequence of the upstream and downstream primer of described positive internal reference primer pair is respectively by shown in SEQ ID NO.11 and SEQ ID NO.12.
5. according to the described method of claim 2-4 any one, it is characterized in that, described multi-PRC reaction carries out according to the following steps:
a：95℃?4min；
b：95℃?30s，
c：55-68℃?30-60s，
D:72 ℃ of 30-120s, 30-35 reaction of b-d circulation;
e：72℃?5-10min。
6. a quick detection kit, is characterized in that, described test kit comprises primer sets claimed in claim 1, positive internal reference primer pair, positive internal reference template, archaeal dna polymerase, reaction system damping fluid, Mg
2+, dNTP and deionized water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013104255679A CN103451307A (en) | 2013-09-17 | 2013-09-17 | Shigella flora/serotype multiplex-PCR (polymerase chain reaction) detection primer set and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013104255679A CN103451307A (en) | 2013-09-17 | 2013-09-17 | Shigella flora/serotype multiplex-PCR (polymerase chain reaction) detection primer set and kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103451307A true CN103451307A (en) | 2013-12-18 |
Family
ID=49734140
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013104255679A Pending CN103451307A (en) | 2013-09-17 | 2013-09-17 | Shigella flora/serotype multiplex-PCR (polymerase chain reaction) detection primer set and kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103451307A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962652A (en) * | 2015-07-27 | 2015-10-07 | 内蒙古出入境检验检疫局检验检疫技术中心 | wbgZ-gene-based real-time fluorescent PCR (polymerase chain reaction) method and kit for identifying Shigella sonnei |
| CN107058514A (en) * | 2017-02-23 | 2017-08-18 | 浙江工商大学 | For Shigella and the primer of the RPA quick detections of tetracycline resistance gene |
| CN108384866A (en) * | 2018-04-16 | 2018-08-10 | 南开大学 | A kind of shigella sonnei specific nucleotide PCR detection kit |
| CN111662994A (en) * | 2020-07-20 | 2020-09-15 | 嘉兴市疾病预防控制中心 | Primer group and kit for shigella detection and typing based on fluorescence PCR technology and application of primer group and kit |
| CN112646910A (en) * | 2021-01-22 | 2021-04-13 | 中国科学院北京基因组研究所(国家生物信息中心) | Primer combination, kit and detection method for identifying related strains of Shigella |
| CN113215290A (en) * | 2020-02-05 | 2021-08-06 | 厦门大学 | Method and kit for detecting serotype of shigella |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102329861A (en) * | 2011-08-29 | 2012-01-25 | 中国疾病预防控制中心传染病预防控制所 | Primer for detecting serotype of shigella flexneri and multiplex amplification using same |
-
2013
- 2013-09-17 CN CN2013104255679A patent/CN103451307A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102329861A (en) * | 2011-08-29 | 2012-01-25 | 中国疾病预防控制中心传染病预防控制所 | Primer for detecting serotype of shigella flexneri and multiplex amplification using same |
Non-Patent Citations (3)
| Title |
|---|
| YAYUE LI ET AL.: "Molecular detection of all 34 distinct O-antigen forms of Shigella", 《JOURNAL OF MEDICAL MICROBIOLOGY》, 31 January 2009 (2009-01-31), pages 69 - 77 * |
| 张晓云，邓文松，姜利群: "多重PCR法在志贺菌基因分型鉴定中的应用价值", 《实用临床医药杂志》, 31 August 2013 (2013-08-31), pages 54 - 55 * |
| 李智琼，邓尚廉，孙承谋: "用检测ipaH基因的PCR检测志贺菌属", 《临床检验杂志》, 30 June 2009 (2009-06-30), pages 185 - 186 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962652A (en) * | 2015-07-27 | 2015-10-07 | 内蒙古出入境检验检疫局检验检疫技术中心 | wbgZ-gene-based real-time fluorescent PCR (polymerase chain reaction) method and kit for identifying Shigella sonnei |
| CN107058514A (en) * | 2017-02-23 | 2017-08-18 | 浙江工商大学 | For Shigella and the primer of the RPA quick detections of tetracycline resistance gene |
| CN107058514B (en) * | 2017-02-23 | 2022-11-11 | 浙江工商大学 | Primer for rapid detection of RPA of Shigella and tetracycline drug-resistant gene |
| CN108384866A (en) * | 2018-04-16 | 2018-08-10 | 南开大学 | A kind of shigella sonnei specific nucleotide PCR detection kit |
| CN113215290A (en) * | 2020-02-05 | 2021-08-06 | 厦门大学 | Method and kit for detecting serotype of shigella |
| CN113215290B (en) * | 2020-02-05 | 2022-11-11 | 厦门大学 | Method and kit for detecting serotype of shigella |
| CN111662994A (en) * | 2020-07-20 | 2020-09-15 | 嘉兴市疾病预防控制中心 | Primer group and kit for shigella detection and typing based on fluorescence PCR technology and application of primer group and kit |
| CN111662994B (en) * | 2020-07-20 | 2023-06-02 | 嘉兴市疾病预防控制中心 | Primer group and kit for shigella detection and typing based on fluorescent PCR technology and application of primer group and kit |
| CN112646910A (en) * | 2021-01-22 | 2021-04-13 | 中国科学院北京基因组研究所(国家生物信息中心) | Primer combination, kit and detection method for identifying related strains of Shigella |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103484546B (en) | Fourteen-food-borne pathogenic bacterium multiplex PCR detection primer set and kit | |
| CN103468811B (en) | Yersinia enterocolitica virulence gene multiplex-PCR (Polymerase Chain Reaction) detection primer group and kit | |
| CN102605055B (en) | Multiplex quantitative PCR (polymerase chain reaction) detection kit for vibrio parahaemolyticus and detection method | |
| CN103451307A (en) | Shigella flora/serotype multiplex-PCR (polymerase chain reaction) detection primer set and kit | |
| CN105420373A (en) | Multiple PCR primer set and probes and detecting method for simultaneously detecting three kinds of aeromonas | |
| CN104059975B (en) | To Providence O3, the Nucleotide that O4, O8, O12, O13 and O20 are special and application thereof | |
| CN103898108A (en) | Nucleotide specific to Vibrio fluvialis O2, O4, O13, O15 and O18 and application thereof | |
| CN111154900B (en) | Pseudomonas aeruginosa specific new molecular target and rapid detection method thereof | |
| CN101348831B (en) | Fluorescence quantitative PCR method for fast detecting Campylobacter jejuni macrolide drug resistant mutational site | |
| CN103451305B (en) | Detect the primer of disperse adhesivity colon bacillus, probe and method and test kit | |
| CN101768636A (en) | Composition and kit for detecting vibrio cholerae and detection method | |
| CN101748197A (en) | Target sequence for detection of yersinia enterocolitica and kit | |
| CN103993090A (en) | Specific nucleotides for providencia O31, O41, O42, O43 and O50 and application of specific nucleotides | |
| CN102154487A (en) | Reagent for detecting francisella tularensis and complex probe and fluorescent quantitative polymerase chain reaction (PCR) method for detecting francisella tularensis | |
| Zhai et al. | A duplex real-time NASBA assay targeting a serotype-specific gene for rapid detection of viable Salmonella Paratyphi C in retail foods of animal origin | |
| CN105154559A (en) | Specific nucleotide for vibrio parahaemolyticus K36, K37 and K68 and application thereof | |
| CN103014164B (en) | Duplex fluorescent quantitation RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit for shellfish Bonamia and Perkinsus | |
| CN102533981B (en) | Primer and detection method as well as kit for rapidly detecting bacillus subtilis | |
| CN102533967B (en) | Reagent kit and method for detecting multiple real-time fluorescent quantitative polymerase chain reaction (PCR) of vibrio cholerae toxin genes | |
| CN105200122A (en) | Quantitative detection kit for wheat stripe rust and application thereof | |
| CN105331677A (en) | Primer group used for multiple PCR detection by aiming at salmonella and Shigella in food, kit and detection method thereof | |
| CN101768633A (en) | Composition for detecting O139 group vibrio cholerae, kit and detection method | |
| CN107385057A (en) | Detect the RPA IAC primers and method of comma bacillus | |
| CN106222293B (en) | Fluorescence quantification PCR primer probe and kit and the method for detecting three kinds of bacillus | |
| CN105256041A (en) | Specific nucleotide for aeromonas hydrophila O44, O24, O25 and O28 and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20131218 |