CN106434901A - Method for detecting six types of diarrheagenic escherichia coli and shigella through multiple PCRs (Polymerase Chain Reactions) - Google Patents
Method for detecting six types of diarrheagenic escherichia coli and shigella through multiple PCRs (Polymerase Chain Reactions) Download PDFInfo
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Abstract
The invention provides a primer group for detecting six types of diarrheagenic escherichia coli and shigella through multiple PCRs (Polymerase Chain Reactions), wherein the primer group comprises primers which are as shown by SEQ ID NO. 1 to SEQ ID NO. 29. The invention also provides a method and a kit for detecting the six types of diarrheagenic escherichia coli and the shigella. Through the technical scheme, by using the primer group, the method and the kit, a complete technical means is provided for the quick and accurate screening on the six types of diarrheagenic escherichia coli and the shigella; the quick sieving detection and identification on the six types of diarrheagenic escherichia coli and the shigella after a food poisoning event happens and an epidemic situation outbreaks can be realized; a reliable basis is provided for reasonably and appropriately disposing the epidemic situation.
Description
Technical field
The present invention relates to biological technical field, in particular it relates to multiplex PCR detects six kinds of Diarrheogenil Escherichia colis and will
Hayes bacterium primer sets and test kit and its detection method.
Background technology
Shigella is that a class has highly infectious, endangers serious Grain-negative pathogenic entero becteria, by its clinic sense
Contaminating led to shigella property dysentery (shigellosis) is one of important infectious disease of developing country, is also flourishing state
The main pathogen of family's diarrhoeal diseasess.
Escherichia coli (Escherichia coli) is important normal flora in intestinal.But decline in host immunity
Or after antibacterial invades parenterally histoorgan, you can become conditionality pathogenic bacterium, cause extra intestinal infection.Secondly, some are big
Intestinal Escherichia bacterial strain carries Disease-causing gene, can cause diarrhoea, referred to as Diarrheogenil Escherichia coli.Know that diarrhea inducing large intestine angstrom is wished
Salmonella has：Intestinal aggressive colon bacillus (enteroinvasive E.coli, EIEC), produce enterotoxin E
Bacterium (enterotoxigenic E.coli, ETEC), enteropathic colon bacillus (enteropathogenic
E.coli, EPEC), intestinal bleeding colon bacillus (enterohemorrhagic E.coli, EHEC), intestinal concentration
Colon bacillus (enteroaggregative E.coli, EAEC), and intestinal disperse adhesiveness colon bacillus
(diffusively adherent E.coli, DAEC).
The main method that China adopts at present to Diarrheogenil Escherichia coli and shigella detection is with country's mark respectively
As foundation, detection process is to obtain sample to quasi- GB/T4789.6-2003 and GB/T4789.5-2012, increasing bacterium, separation and Culture,
Gram stain microscopy/biochemistry and the experiment of bacterium colony observation/biochemical reaction, enterotoxin inspection/serological Identification.Whole detection process
Complex operation, the time is long, needs 4-7 days about, cumbersome and time consuming, and detecting that target is single, and be limited to database information has
The factors such as limit, the detection data of acquisition is unreliable.
Molecular biology for detection is Diarrheogenil Escherichia coli and shigella detection provides good detection work
Tool, all establishes skill Diarrheogenil Escherichia coli and shigella being used for quickly detecting using round pcr at present both at home and abroad
Art.But how to be detected using substance round pcr, the annealing temperature of multipair primer and concentration are not quite similar, need many wheel PCR anti-
Should detect one by one, this method complex operation, expend time and manpower, need to carry out respectively the quality control of each reaction system
System.
The scheme that existing use multiple PCR method is detected to colon bacillus and shigella all can not be fine
Differentiation diarrhea inducing and non-Diarrheogenil Escherichia coli, nor accurate judge its pathological form.A kind of document " multiple PCR method
7 kinds of common enteric pathogenic bacterias of Rapid identification " report the technical scheme simultaneously detecting 7 kinds of common pathogenic entero becterias.But the method
EstIa and estIb gene amplification fragment differ only by 15bp, be cannot be distinguished by using the sepharose electrophoresis less than 2.5% at all；Husky
Door bacterium invA gene amplification product and stx1A gene amplification fragment cannot be distinguished from, and need the spy according to bacterium colony on selectivity plate
Point is tentatively distinguished again；No amplification interior label is it is impossible to effectively differentiate false negative result；The method virulence gene diarrhea inducing type simultaneously
Other coverage is inadequate, and sensitivity is general.
Therefore, it is necessary to provide a kind of new can be simultaneously to six kinds of Diarrheogenil Escherichia coli pathological forms and will Hayes
Bacterium carries out the technical scheme of quick, special, sensitive detection.
Content of the invention
Present invention aim to address current to six kinds of Diarrheogenil Escherichia coli pathological forms, that is, EPEC, EHEC, ETEC,
EAEC, DAEC, EIEC, and shigella carries out problem present in detection technique simultaneously, provide a kind of new can be simultaneously
Six kinds of Diarrheogenil Escherichia coli pathological forms and shigella are carried out with the technical scheme of quick, special, sensitive detection.
For reaching object above, the invention provides a kind of multiplex PCR detects that six kinds of Diarrheogenil Escherichia colis and will are congratulated
Salmonella primer sets, wherein, described primer sets include the primer shown in SEQ ID NO.1-29；Described six kinds of diarrhea inducing large intestines angstrom are wished
Salmonella includes intestinal aggressive colon bacillus (enteroinvasive E.coli, EIEC), produces enterotoxin E
Bacterium (enterotoxigenic Escherichia coli, ETEC), enteropathic colon bacillus
(enteropathogenic E.coli, EPEC), intestinal bleeding colon bacillus (enterohemorrhagic
E.coli, EHEC), intestinal concentration colon bacillus (enteroaggregative E.coli, EAEC) and intestinal disperse
Adhesiveness colon bacillus (diffusively adherent E.coli, DAEC).
Present invention also offers the detection method of six kinds of Diarrheogenil Escherichia colis and shigella, the method includes as follows
Step：
(1) extract the STb gene of sample to be tested；
(2) with described STb gene as template, and use primer sets of the present invention, carry out multi-PRC reaction, obtain many
Material after weight PCR amplification；
(3) nucleic acid electrophoresis detection is carried out to the material after described multiplexed PCR amplification, obtain the result of nucleic acid electrophoresis detection.
Present invention also offers a kind of multiplex PCR detects the test kit of six kinds of Diarrheogenil Escherichia colis and shigella,
Described test kit includes primer sets of the present invention and archaeal dna polymerase.
On the other hand, present invention also offers primer sets as above are in preparation six kinds of Diarrheogenil Escherichia colis of detection
With the purposes in the test kit of shigella；Wherein, described six kinds of Diarrheogenil Escherichia colis include intestinal aggressive large intestine angstrom
Uncommon Salmonella, product enterotoxin colon bacillus, enteropathic colon bacillus, intestinal bleeding colon bacillus, intestinal
Road concentration colon bacillus and intestinal disperse adhesiveness colon bacillus.
Six kinds of Diarrheogenil Escherichia colis and shigella identification multi-PCR detection method that the present invention sets up, can be by
Detection time shortened to from 3-5 days of traditional method 3 hours, reached following Detection results：
(1) Multiple detection covers comprehensively
The detection method that the present invention is set up can check whether containing Diarrheogenil Escherichia coli in a PCR reaction
While with shigella, determine the pathological form of six kinds of Diarrheogenil Escherichia colis.Quick acquisition testing result in 3 hours,
Time-consuming, man power and material's cost.
(2) specificity is high
The specificity of the set up detection method of the present invention is mainly reflected in the specificity of a whole set of primer, all primers all warps
Cross blast and compare analysis, there is conservative and the specificity of height；Specificity experiments show that the primer of the present invention can be very simultaneously
Good differentiation is close with Diarrheogenil Escherichia coli and shigella kind, living environment identical antibacterial, including slope rugged intestinal bar
Bacterium, staphylococcus aureuses, Salmonella, vibrio parahaemolyticus, Listeria monocytogenes, vibrio cholera, wax
Sample bacillus cereuss, campylobacter jejuni, Campylobacter Coli, Enterobacter sakazakii, yersinia enterocolitica and thermophilic aqueous vapor unit cell
Bacterium etc..
(3) sensitivity is high
The detection method that the present invention is set up is capable of detection, each target base in reaction system while 14 genes
The detection sensitivity of cause all can reach 103CFU/ml is suitable with substance real-time PCR detection sensitivity；Different genes simultaneously
Detection sensitivity in the same horizontal line, it is to avoid leakage to six kinds of Diarrheogenil Escherichia colis and shigella mixed infection
Inspection.The main cause that sensitivity of the present invention improves is that the present invention is directed to the specific primer of all target genes to sequential design
Specific LHP sequence, the homology universal primer in LHP sequence can improve the amplification of multi-PRC reaction in second stage PCR
Efficiency, reduces the non-specific amplification of multi-PRC reaction and the generation of primer dimer, and 6 base catenation sequences in LHP
When ensure that homology universal primer amplification different virulence gene, there is identical amplification efficiency, it is to avoid multiplexed PCR amplification process
Middle different target gene yield unbalance, thus on the whole improve multiplex PCR detection sensitivity.
(4) cost is relatively low
The multi-PCR detection method that the present invention is set up reduces human cost and time cost in operability, originally
14 artificial and 14 times of times of substance detection needs, currently use the method and only need to 1 artificial and 1 time reacted；Should
Multiple detection method saves the reagent consumption of the same sample of duplicate detection simultaneously, and the reagent that maximum can save more than 50% becomes
This.
(5) prevent false negative result
The positive internal reference adding in system, can effectively point out false negative testing result.
The present invention is six kinds of Diarrheogenil Escherichia colis and shigella rapid screening provides complete solution, energy
Realize six kinds of Diarrheogenil Escherichia coli pathological forms after food poisoning occurs to break out and shigella with epidemic
Rapid identification, for rationally appropriately dispose epidemic situation provide reliable foundation.
Instant invention overcomes prior art operation is loaded down with trivial details, take time and effort, be difficult to the shortcomings of carry out homogeneous quality control and not
Foot, solves a difficult problem for six kinds of Diarrheogenil Escherichia colis and shigella Rapid identification simultaneously.
IpaH gene based on EIEC/ Shigella, the universal genetic uidA of E Pseudomonas/Shigella
With its virulence associated gene, design a set of six kinds of Diarrheogenil Escherichia colis and the primer sequence of shigella identification, set up base
In the multi-PCR detection method of this primer sequence, add amplification interior label in system simultaneously, in a reaction system, complete inside
Quality control.
Other feature and advantage of the disclosure will be described in detail in subsequent specific embodiment part.
Specific embodiment
Specific embodiment of this disclosure is described in detail below.It should be appreciated that it is described herein concrete
Embodiment is merely to illustrate and explains the disclosure, is not limited to the disclosure.
The invention provides a kind of multiplex PCR detects six kinds of Diarrheogenil Escherichia colis and shigella primer sets, its
In, described primer sets include the primer shown in SEQ ID NO.1-29；Described six kinds of Diarrheogenil Escherichia colis include intestinal and invade
Attacking property colon bacillus, product enterotoxin colon bacillus, enteropathic colon bacillus, intestinal bleeding large intestine angstrom
Uncommon Salmonella, intestinal concentration colon bacillus and intestinal disperse adhesiveness colon bacillus.
Wherein, detect the nucleotide sequence of six kinds of Diarrheogenil Escherichia colis and shigella homology universal primer such as
Shown in SEQ ID NO.29；
Present invention also offers LHP sequence is used in six kinds of Diarrheogenil Escherichia colis of detection and the detection of shigella long primer,
Its nucleotide sequence is as shown in SEQ ID NO.30；
Wherein, described primer sets also include the primer shown in SEQ ID NO.31-32.Shown in SEQ ID NO.31-32
Primer is the pair of primers with pET28a plasmid as stencil design, as positive internal reference primer pair.
Primer sets provided by the present invention are using 14 genes as designed by target amplification sequence, wherein, uidA base
Because judging E Pseudomonas, the molecular target of Shigella；Elt, estla and estlb identify target spot for ETEC；
BfpB and escV is the identification target spot of EPEC；EscV, stx1 and stx2 are the identification target spot of EHEC；IpaH is the identification target of EIEC
Point；AstA, aggR and pic are the identification target spot of EAEC；DaaC and afa is the identification target spot (as shown in table 1) of DAEC.
1 six kinds of Diarrheogenil Escherichia colis of table and shigella identification multiple PCR primer summary sheet
In one embodiment of the invention, there is provided the detection side of six kinds of Diarrheogenil Escherichia colis and shigella
Method, the method comprises the steps：(1) extract the STb gene of sample to be tested；(2) with described STb gene as template, and use this
Bright described primer sets, carry out multi-PRC reaction, obtain the material after multiplexed PCR amplification；(3) after to described multiplexed PCR amplification
Material carry out nucleic acid electrophoresis detection, obtain nucleic acid electrophoresis detection result.Result according to nucleic acid electrophoresis carries out sample judgement.
Sample judgment basis are as shown in table 2.
Table 2 sample judgment basis
Wherein it is preferred to, the final concentration of the primer shown in SEQ ID NO.1-28 is respectively 0.1-0.8 μM, SEQ
The final concentration of the primer shown in ID NO.29 is 0.4-0.8 μM.
Wherein it is preferred to, the step that the condition of multi-PRC reaction includes following a-h：
a：94-96 DEG C, 4-6min；
b：94-96 DEG C, 15-60s,
c：50-60 DEG C, 15-60s,
d：71-73 DEG C, 60-120s, carry out the circulation of 10-15 b-d；
e：94-96 DEG C, 15-60s,
f：55-65 DEG C, 15-60s,
g：71-73 DEG C, 30-120s, carry out the circulation of 30-35 e-g；
h：71-73 DEG C, 5-10min.
Wherein, described sample to be tested can include but is not limited in food, medicine, Excreta, vomitus and body fluid extremely
Few one kind.Preferably, the multiplex PCR of the present invention detect the detection method of six kinds of Diarrheogenil Escherichia colis and shigella without
In diagnosis.Whether the qualitative and quantitative result of six kinds of Diarrheogenil Escherichia colis and shigella and disease occur without in other words
One-to-one dependency, is not belonging to diagnostic result, but can be as average information, for reference for clinicians.
Wherein it is preferred to, described nucleic acid electrophoresis detection includes nucleic acid gel electrophoresis and/or nucleic acid capillary electrophoresis.
In one embodiment of the invention, divided as a result using QIaxcel full-automatic capillary electrophoresis analysis instrument
The platform of analysis.Full automatic working based on QIaxcel and the intelligent interpretation of result program setting, thus realize the automatic of result
Decision analysis.
One aspect of the present invention additionally provides a kind of multiplex PCR and detects six kinds of Diarrheogenil Escherichia colis and shigella
Test kit, wherein, described test kit includes primer sets of the present invention and archaeal dna polymerase；Described six kinds of diarrhea inducing large intestines angstrom
Uncommon Salmonella include intestinal aggressive colon bacillus, produce enterotoxin colon bacillus, enteropathic colon bacillus,
Intestinal bleeding colon bacillus, intestinal concentration colon bacillus and intestinal disperse adhesiveness colon bacillus.
Preferably, described test kit also includes the required PCR reaction reagent of PCR reaction, and described reaction reagent can be PCR
Reaction buffer and dNTP.
On the other hand, present invention also offers primer sets as above are in preparation six kinds of Diarrheogenil Escherichia colis of detection
With the purposes in the test kit of shigella；Wherein, described six kinds of Diarrheogenil Escherichia colis include intestinal aggressive large intestine angstrom
Uncommon Salmonella, product enterotoxin colon bacillus, enteropathic colon bacillus, intestinal bleeding colon bacillus, intestinal
Road concentration colon bacillus and intestinal disperse adhesiveness colon bacillus.
Following examples are used for the present invention is described, but are not limited to the scope of the present invention.
In following examples, intestinal aggressive colon bacillus and shigella are all from Chinese Medicine antibacterial preservation pipe
Reason center.
1 six kinds of Diarrheogenil Escherichia colis of embodiment and the establishment of shigella identification multiple PCR detection kit
This test kit is by 2 × reaction system buffer, archaeal dna polymerase, 10 × primer mixed liquor, positive control, deionization
Water is constituted, and its concrete component is as follows：2 × PCR Buffer (Tris HCl 40mM (pH8.3), KCl 100mM, tween-20
0.08%, 0.0006ng/ μ L pET28a, 1.5mM dNTP, 8mM MgCl2)；25 × archaeal dna polymerase (2U/ μ L)；10 × primer
(the every kind of long primer concentration including IAC primer is 2 μM to mixed liquor, and homology universal primer SEQ ID NO.29 concentration is 8
μM), positive control (hybrid template of EPEC, EHEC, ETEC, EAEC, DAEC, EIEC/ shigella, every kind of 106CFU/mL),
Positive internal reference is pET28a plasmid.
The reaction system of test kit detection is 25 μ L, and its configuration is as follows：2×PCR Buffer 12.5μL；25 × DNA gathers
Synthase 1 μ L；10 × primer mixed liquor 2.5 μ L；Template 2 μ L, deionized water 7 μ L.
The operation of embodiment 2 test kit and result judge
1st, the extraction of nucleic acid
Take enrichment liquid or streak culture bacterium colony soluble in water, extract mould according to bacterial genomes extracts kit description
Plate DNA.Measure the concentration extracting STb gene with NanoDrop.
2nd, the preparation of reaction system
Take the reaction system of the PCR pipe configuration 25 μ L of 200 μ L, its configuration is as follows：2×PCR Buffer 12.5μL；
25xDNA polymerase 1 μ L；10 × primer mixed liquor 2.5 μ L, template 2 μ L, deionized water 7 μ L.
3rd, PCR reaction
PCR pipe is put in Bio-Rad C1000 type PCR instrument, after opening heat lid, enters performing PCR reaction according to following program：
a：95℃4min；
b：95 DEG C of 30s,
c：55 DEG C of 30s,
d：72 DEG C of 90s, b-d circulate 10 reactions；
e：95 DEG C of 30s,
f：60 DEG C of 30s,
g：72 DEG C of 90s, e-g circulate 30 reactions；
h：72 DEG C, 5min.
4th, full-automatic capillary electrophoresis analysis PCR primer
PCR reaction tube is put into full-automatic capillary electrophoresis apparatus Qiaxcel Advanced (Kai Jie company), using high score
Resolution clip, selects suitable alignment marker and size marker, automatically analyzes the size of target stripe.
5th, result judges
A. all at least one band amplification of all swimming lanes including blank, blank has positive internal reference
Amplification, other detections have target stripe and (or) the amplification of positive internal reference, show that all PCR reactions are all set up, exclusion is false
Negative result.Otherwise regard experiment invalid, need to repeat.
B. all sample standard deviations have the amplified band of uidA (1673bp), that is, be colon bacillus or shigella.Its
His sample also has the amplification of virulence gene, is Lapactic colon bacillus.On this basis, concrete judgement is as follows：
While sample has uidA (1673bp), the amplification of escV (548bp) band, also there is the amplification of bfpB (172bp)
Band, and two kinds of shiga toxin stx1 (242bp), stx2 (305bp) are all negative, are judged to typical EPEC.
While sample has uidA (1673bp), the amplification of escV (548bp) band, two kinds of shiga toxin stx1 (242bp),
Stx2 (305bp) is all positive, and bfpB (172bp) no expands, and is judged to EHEC.
While sample has the amplification of uidA (1673bp) band, heat-labile toxin elt (339bp), heat-stable toxin
EstIb (221bp) is all positive, is judged to ETEC.
While sample has the amplification of uidA (1673bp) band, ipaH (433bp) is positive, is judged to EIEC/ shigella.
While sample has the amplification of uidA (1673bp) band, astA (153bp), aggR (492bp), pic (965bp) are all
The positive, is judged to EAEC.
Sample have uidA (1673bp), escV (548bp) band amplification while, stx1 (242bp), stx2 (305bp),
BfpB (172bp) is all negative, is judged to the EPEC that is not true to type.
While sample has the amplification of uidA (1673bp) band, heat-labile toxin elt (339bp), astA (153bp) sun
Property, in the case of simultaneous with other type virulence genes, astA, not as judgment basis, is judged to ETEC.
While sample has the amplification of uidA (1673bp) band, astA (153bp) is all positive, is judged to EAEC.
All purposes band contained by positive control all can be amplified out, shows that this test kit has detection mixed infection
Ability.
The specific test of embodiment 3 test kit
Select Enterobacter sakazakii (purchased from Chinese medicine DSMZ, numbering is 21665), staphylococcus aureuses
(purchased from Chinese medicine DSMZ, numbering is 26003), Salmonella (purchased from Chinese medicine DSMZ, are numbered
For 50001), vibrio parahaemolyticus (purchased from Chinese medicine DSMZ, numbering is 21617), the Hypertrophic Liszt of monokaryon
Salmonella (purchased from Chinese medicine DSMZ, numbering is 54002), vibrio cholera (purchased from Chinese medicine DSMZ,
Numbering is 54002), campylobacter jejuni (purchased from Chinese medicine DSMZ, numbering is 54102), Campylobacter Coli (be purchased from
Chinese medicine DSMZ, numbering is 47016), Aeromonas hydrophila (purchased from Chinese medicine DSMZ, numbers
For 44016) as antibacterial to be checked.Test kit of the present invention is applied to detect these antibacterials to be checked, amplification interior label is the positive it was demonstrated that examining
Examining system is set up；All nonspecific miscellaneous band in antibacterial to be checked.And when object bacteria is detected, in addition to specific band, no
Miscellaneous band produces.Show that test kit of the present invention can effectively distinguish non-targeted antibacterial, there is preferable specificity.
The minimum detectability test of embodiment 4 test kit
Assessment detection sample：Select the bacterial strain of particular detection：EPEC, EHEC, ETEC, EAEC, DAEC, EIEC/ will is congratulated
Salmonella.The concentration of 6 templates is separately adjusted to angularly 108CFU/mL, equal proportion is mixed into integrated template.Will be dilute for integrated template gradient
It is interpreted into 106CFU/mL, 105CFU/mL, 104CFU/mL, 103CFU/mL, 102The detection sample of CFU/mL.
Detect different dilution hybrid templates using test kit of the present invention respectively.Operated and result according to example 2
Judge, test kit detects that the minimum detectability result of the test of 14 target genes (without IAC) is as shown in table 3：
Table 3 test kit detects the minimum detectability result of the test of 14 target genes
| Numbering | Amplified band title | 106CFU/mL | 105CFU/mL | 104CFU/mL | 103CFU/mL | 102CFU/mL |
| 1 | ipaH | + | + | + | + | - |
| 2 | uidA | + | + | + | + | - |
| 3 | elt | + | + | + | + | - |
| 4 | estIa | + | + | + | + | - |
| 5 | estIb | + | + | + | + | - |
| 6 | bfpB | + | + | + | + | - |
| 7 | escV | + | + | + | + | - |
| 8 | stx1 | + | + | + | + | - |
| 9 | stx2 | + | + | + | + | - |
| 10 | astA | + | + | + | + | - |
| 11 | aggR | + | + | + | + | - |
| 12 | pic | + | + | + | + | - |
| 13 | afa | + | + | + | + | - |
| 14 | daaC | + | + | + | + | - |
As seen from Table 3, the minimum detectability of test kit six kinds of Diarrheogenil Escherichia colis of detection and shigella is all permissible
Reach 103CFU/mL.
The detection of embodiment 5 test kit and the comparison of literature method
Evaluate test kit of the present invention and document report (Claudia Toma, Yan Lu, 1 Naomi Higa, Noboru
Nakasone,Isabel Chinen,Ariela Baschkier,Marta Rivas,and Masaaki
Iwanaga.Multiplex PCR Assay for Identification of Human Diarrheagenic
Escherichia coli.American Society for Microbiology, Dec.2004, p.5849 5853.) effect
Relatively, mainly it is compared in terms of minimum detectability, specificity and coverage three.
Evaluation positive sample：EPEC, EHEC, ETEC, EAEC, DAEC, EIEC, and each 1 plant of shigella, totally 7 plants.
Evaluation negative sample：Enterobacter sakazakii (purchased from Chinese medicine DSMZ, numbering is 21665), golden yellow
Color staphylococcuses (purchased from Chinese medicine DSMZ, numbering is 26003), Salmonella (are protected purchased from Chinese medicine strain
Tibetan center, numbering is 50001), vibrio parahaemolyticus (purchased from Chinese medicine DSMZ, numbering is 21617), monokaryon increase
Natural disposition listeria spp (purchased from Chinese medicine DSMZ, numbering is 54002), vibrio cholera are (purchased from Chinese medicine bacterium
Kind of collection, numbering is 54002), campylobacter jejuni (purchased from Chinese medicine DSMZ, numbering is 54102), colon
Campylobacter spp (purchased from Chinese medicine DSMZ, numbering is 47016), Aeromonas hydrophila (protect purchased from Chinese medicine strain
Tibetan center, numbering is 44016).Above bacterial strain is extracted after nucleic acid, equal proportion is mixed into the negative assessment DNA profiling of.
Specificity is assessed：Carry out specificity evaluation operation according to the embodiment of the present invention 1 and 2；According to literature procedure inspection
Survey uidA, ipaH, elt, estIa, estIb, bfpB, escV, stx1, stx2, astA, aggR, pic, afa, daaC gene and
Positive internal reference.
Coverage is assessed：Above-mentioned 7 plants of evaluation positive sample are detected respectively using test kit of the present invention and literature method, often
Individual bacterial strain DNA profiling 5-10ng/uL.
Minimum detectability is assessed：All 7 plants of evaluation positive sample are diluted to 10 respectively6CFU/mL, 105CFU/mL,
104CFU/mL, 103CFU/mL, 102CFU/mL, extracts nucleic acid preparation positive DNA profiling, using test kit of the present invention and literary composition
Method of offering detects the gradient dilution DNA profiling of 7 plants of evaluation positive sample respectively.
Comparative result：Test kit of the present invention and literature procedure detection negative sample result are feminine gender, show all have
There is preferable specificity.
Target gene in test kit of the present invention and literature procedure 7 plants of evaluation positive sample of detection, all obtains sun
Property result, wherein literature procedure can only detect ipaH, eae, elt, est, aggR, stx gene；And test kit energy of the present invention
Detect the target gene of all kinds in positive sample, including uidA, ipaH, elt, estIa, estIb, bfpB, escV,
Stx1, stx2, astA, aggR, pic, afa, daaC gene and positive internal reference.
Test kit of the present invention and literature procedure detect the minimum detectability of target gene in 7 plants of positive sample to such as
Table 4 below：
Table 4
As seen from the above table, test kit of the present invention detects uidA, ipaH, elt, estIa, estIb, bfpB, escV, stx1,
The minimum detectability of stx2, astA, aggR, pic, afa, daaC target gene all can reach 103The level of CFU/mL, and document
Report method only ipaH can reach 103The level of CFU/mL, eae, elt, est, stx can only achieve 104The water of CFU/mL
Flat, aggR can only achieve 105The level of CFU/mL.The minimum detectability of test kit detection of the present invention is significantly better than document report side
Method.
The preferred implementation of the disclosure described in detail above, but, the disclosure is not limited in above-mentioned embodiment
Detail, in the range of the technology design of the disclosure, multiple simple variant can be carried out with technical scheme of this disclosure, this
A little simple variant belong to the protection domain of the disclosure.
It is further to note that each particular technique feature described in above-mentioned specific embodiment, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the disclosure to various can
The compound mode of energy no longer separately illustrates.
Additionally, combination in any can also be carried out between the various different embodiment of the disclosure, as long as it is without prejudice to this
Disclosed thought, it equally should be considered as disclosure disclosure of that.
Claims (9)
1. a kind of multiplex PCR detects six kinds of Diarrheogenil Escherichia colis and shigella primer sets, wherein, described primer sets bag
Include the primer shown in SEQ ID NO.1-29；Described six kinds of Diarrheogenil Escherichia colis include intestinal aggressive E
Bacterium, product enterotoxin colon bacillus, enteropathic colon bacillus, intestinal bleeding colon bacillus, intestinal collection
Poly- property colon bacillus and intestinal disperse adhesiveness colon bacillus.
2. primer sets according to claim 1, wherein, described primer sets also include drawing shown in SEQ ID NO.31-32
Thing.
3. the detection method of six kinds of Diarrheogenil Escherichia colis and shigella is it is characterised in that the method comprises the steps：
(1) extract the STb gene of sample to be tested；
(2) with described STb gene as template, and usage right requires the primer sets described in 1 or 2, carries out multi-PRC reaction, obtains
Material after multiplexed PCR amplification；
(3) nucleic acid electrophoresis detection is carried out to the material after described multiplexed PCR amplification, obtain the result of nucleic acid electrophoresis detection.
4. detection method according to claim 3, wherein, the final concentration of the primer shown in SEQ ID NO.1-28
It is respectively 0.1-0.8 μM, the final concentration of the primer shown in SEQ ID NO.29 is 0.4-0.8 μM.
5. the detection method according to claim 3 or 4, wherein, the step that the condition of multi-PRC reaction includes following a-h：
a：94-96 DEG C, 4-6min；
b：94-96 DEG C, 15-60s,
c：50-60 DEG C, 15-60s,
d：71-73 DEG C, 60-120s, carry out the circulation of 10-15 b-d；
e：94-96 DEG C, 15-60s,
f：55-65 DEG C, 15-60s,
g：71-73 DEG C, 30-120s, carry out the circulation of 30-35 e-g；
h：71-73 DEG C, 5-10min.
6. the detection method according to claim 3 or 4, wherein, the detection of described nucleic acid electrophoresis includes nucleic acid gel electrophoresis with/
Or nucleic acid capillary electrophoresis.
7. a kind of multiplex PCR detects the test kit of six kinds of Diarrheogenil Escherichia colis and shigella it is characterised in that described examination
Agent box includes primer sets and archaeal dna polymerase described in claim 1 or 2；Described six kinds of Diarrheogenil Escherichia colis include intestinal
Aggressive colon bacillus, product enterotoxin colon bacillus, enteropathic colon bacillus, intestinal bleeding large intestine
Escherichia, intestinal concentration colon bacillus and intestinal disperse adhesiveness colon bacillus.
8. test kit according to claim 7 it is characterised in that described test kit also include PCR reaction buffer and
dNTP.
9. the primer sets described in claim 1 or 2 are in the reagent of preparation six kinds of Diarrheogenil Escherichia colis of detection and shigella
Purposes in box；Wherein, described six kinds of Diarrheogenil Escherichia colis include intestinal aggressive colon bacillus, produce enterotoxin big
Intestinal Escherichia, enteropathic colon bacillus, intestinal bleeding colon bacillus, intestinal concentration E
Bacterium and intestinal disperse adhesiveness colon bacillus.
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