CN108570512A - Staphylococcus aureus, Escherichia coli, the primer of salmonella triple fluorescent quantitative PCR detections, probe and method foundation - Google Patents
Staphylococcus aureus, Escherichia coli, the primer of salmonella triple fluorescent quantitative PCR detections, probe and method foundation Download PDFInfo
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Abstract
It is established the present invention relates to primer, probe and method for staphylococcus aureus, Escherichia coli, the PCR detections of salmonella triple fluorescent quantitative, belongs to technical field of biological.The present invention provides the primer and probes that staphylococcus aureus, Escherichia coli, salmonella triple fluorescent quantitative PCR are detected, including respectively with staphylococcus aureus nuc genes, Escherichia coli O 157：H7 wzx genes, salmonella invA genes are three groups of primer and probes of object, and establish staphylococcus aureus, colon bacillus 0157：H7, salmonella triple fluorescent quantitative PCR method.The present invention PCR detection method high sensitivity and with well repeatability.
Description
Technical field
The invention belongs to technical field of biological, and in particular to staphylococcus aureus, Escherichia coli, salmonella three
Primer, probe and the method for weight fluorescence quantitative PCR detection are established.
Background technology
This 3 kinds of bacteriums of staphylococcus aureus, Escherichia coli, salmonella are the important diseases for causing mankind's food origin disease
Opportunistic pathogen.
Staphylococcus aureus is staphylococcus member, is one kind in gram-positive cocci, most grapes
Coccus is not pathogenic, and minority can cause mankind or animal to be caused a disease, and belong to infecting both domestic animals and human pathogenic bacteria.And staphylococcus aureus causes
Characteristic of disease is most strong, other than the change arm inflammation for often causing skin, tissue and organ, the enterotoxin generated can also pollute pork and
Cause to poison by food.
Escherichia coli O 157：H7 is the typical strain in enterohemorrhagic escherichia coli, with stronger acid resistance, 37 DEG C
Under, in pH2.5-3.0, it can tolerate 5h.Escherichia coli O 157：H7 is important food-borne pathogens, can be for a long time in animal
Exist in vivo, and constantly ambient enviroment and animal carcasses is polluted, it is easy to lead to food pollution from internal discharge.
Salmonella is the brevibacterium of both ends blunt circle, Gram-negative bacteria, no pod membrane and brood cell, removes avian infectious bronchitis nephritis virus
There is whole body flagellum outside with S. pullonum, can move, it is most of that there is pili.It is divided into kind of an antigenic structure, and thalline is anti-
Former (O), flagellar antigen (H) and surface antigen (Vi).The bacterium is not high to nutritional requirement, is grown on plain agar culture medium good
It is good, aerobic and facultative anaerobic bacteria.Growth temperature range is 5-46 DEG C, and the temperature of the most suitable growth is 35-37 DEG C, the pH of the most suitable growth
It is in mix only length in liquid medium for 6.8-7.8.
In recent years, the detection of pathogenic bacteria is had been a hot spot of research both at home and abroad, detection method includes mainly conventional detection
Method, immunological detection method and molecular biology for detection etc.
It is many that conventional PCR method has that quick, accurate, easy to operate, sensitivity and specificity are high, testing cost is low etc.
Advantage, however in food production and circulation, business and government portion mouth is both needed to carry out large quantities of food in the detection of pathogenic bacteria, sample
Amount is big, and the time is anxious, meanwhile, often existing pathogenic bacteria type is more in food samples, and once experiment can only be examined conventional PCR
Survey a kind of pathogenic bacteria.
Individual staphylococcus aureus, Escherichia coli, salmonella fluorescent quantitative PCR detection method technology are ripe
And there is special detection kit product, but the associated detecting method of three kinds of bacteriums not yet.If needing to examine with portion sample
Above-mentioned three kinds of pathogenic bacteria are surveyed, then need with different system and sample size, that is, to detect above-mentioned three kinds of pathogenic bacteria and need to do to try three times
It tests, be unfavorable for high-volume while detecting above-mentioned three kinds of pathogenic bacteria.Therefore there is an urgent need for a set of systems and method to be capable of detecting when above-mentioned three
The pollution condition of kind bacterium.
Invention content
A kind of staphylococcus aureus, Escherichia coli, sand are provided the purpose of the present invention is overcome the deficiencies in the prior art
Primer, probe and the method for door Salmonella triple fluorescent quantitative PCR detections are established.
The present invention adopts the following technical scheme that：
The primer and probe that staphylococcus aureus, Escherichia coli, salmonella triple fluorescent quantitative PCR are detected, including
Respectively with staphylococcus aureus nuc genes, Escherichia coli O 157：H7wzx genes, three that salmonella invA genes are object
Group primer and probe, three groups of primer and probes are respectively provided with following base-pair：
Using staphylococcus aureus nuc genes as object：
Sense primer (5 ' -3 ') TTGTAGTTTCAAGTCTAAGTAGCTCAGC
Downstream primer (5 ' -3 ') TTGCACTATATACTGTTGGATCTTCAG
Probe (5 ' -3 ') FAM-TGCATCACAAACAGATAACGGCGTAAATAG-BHQ1；
With colon bacillus 0157：H7wzx genes are object：
Sense primer (5 ' -3 ') TCATGCACGCAATGATACTCAA
Downstream primer (5 ' -3 ') CGAACACTACTAATATGAAGGCCA
Probe (5 ' -3 ') CY5-AGACGCTCAGAACATCATTGAAAATAGTGGG-BHQ2；
Using salmonella invA genes as object：
Sense primer (5 ' -3 ') CCTGATCGCACTGAATATCGTACT
Downstream primer (5 ' -3 ') GTGGTAATTAACAGTACCGCAGGA
Probe (5 ' -3 ') Vic-ATATTGGTGTTTATGGGGTCGTTCTACATTGAC-BHQ1.
The staphylococcus aureus, Escherichia coli, the PCR detections of salmonella triple fluorescent quantitative primer and probe
In staphylococcus aureus, colon bacillus 0157：H7, salmonella triple fluorescent quantitative PCR method foundation in application, packet
Include following steps：
Step 1：Design three groups of primer and probes；
Step 2：Primer specificity detects；
Step 3：Staphylococcus aureus nuc genes, Escherichia coli O 157：H7wzx genes, salmonella invA genes
Clone and plasmid extraction identification；
Step 4：Staphylococcus aureus, salmonella, Escherichia coli are carried out with probe substance specific assay respectively；
Step 5：Sonde method multiple specific detects；
Step 6：Multiple condition is explored；
Step 7：Standard curve is established.
Further, when primer specificity described in step 2 detects,
PCR system is as follows：
PCR reaction conditions are as follows：
Colon bacillus 0157：The PCR reaction conditions of H7 and salmonella：
The PCR reaction conditions of staphylococcus aureus：
Further, when substance specific assay described in step 4, qPCR systems are as follows：
QPCR conditions are as follows：50 DEG C, 2min；95 DEG C, 5min；(95 DEG C, 40s；60 DEG C, 40s；) × 40 recycle.
Further, when sonde method multiple specific described in step 5 detects, by staphylococcus aureus, Salmonella
Bacterium, Escherichia coli are placed on the same system and are expanded, and qPCR systems are as follows：
Further, when multiple condition described in step 6 is explored, Mix fixes 30 μ L, and probe sets 3 concentration (0.4 μ
L, 0.3 μ L, 0.2 μ L), finally selected concentration and probe concentration is 0.4 μ L；Probe is fixed as 0.4 μ L, mix and sets 30 μ L, 40 μ L, 35 mix
μ L finally select a concentration of 30 μ L of mix.
Further, the standard curve established in step 7 is：
Staphylococcus aureus：Y=-4.929X+58.824R2=0.999Eff%=86.721
Salmonella：Y=-4.74X+57.046R2=0.996Eff%=90.775
Escherichia coli O 157 H7：Y=-4.277X+52.119R2=0.997Eff%=84.861.
Compared with prior art, the present invention having the advantages that：
First, PCR detection method high sensitivity of the invention：Staphylococcus aureus and salmonella sensitivity are 105
Copies/ μ L, and Escherichia coli sensitivity 104copies/μL。
Second, the triple fluorescent RT-PCR that the present invention establishes have repeatability well, at 5 gradient variation within batch coefficients
Between 0.09%-1.71%, it is less than 10%, there is high repeatability, 5 gradient interassay coefficient of variation to be in 0.05%-
Between 6.62%, it is less than 15%-20%, there is high repeatability.
Description of the drawings
Fig. 1 is bacterial primers specificity identification electrophoretogram, wherein M:500DNAMarker 1、3、5：Staphylococcus aureus
Bacterium, Escherichia coli, salmonella negative control, 2, staphylococcus aureus (96bp), 4, Escherichia coli (104bp), 6, sramana
Salmonella (108bp)；
Fig. 2 is bacterium target gene plasmid identification electrophoretogram, wherein M:2000DNA Marker 1, staphylococcus aureus
(96bp), 2, Escherichia coli (104bp), 3, salmonella (108bp), 4, negative control；
Fig. 3-1 is staphylococcus aureus probe substance specific outcome figure；
Fig. 3-2 is salmonella probe substance specific outcome figure；
Fig. 3-3 is Escherichia coli probe substance specific outcome figure；
Fig. 4 is probe multiple specific outcome figure；
Fig. 5-1 is that probe multiple specificity mix fixation probes grope result figure (total figure)；
Fig. 5-2 is that mix is fixed as 30 μ L, and 0.2 μ L of probe grope result figure (component)；
Fig. 5-3 is that mix is fixed as 30 μ L, and 0.3 μ L of probe grope result figure (component)；
Fig. 5-4 is that mix is fixed as 30 μ L, and 0.4 μ L of probe grope result figure (component)；
Fig. 6-1 is that probe multiple specific probe fixation mix gropes result figure；
It is that 30 μ L grope result figure that Fig. 6-2 is fixed as 0.4 μ L, mix for probe；
It is that 35 μ L grope result figure that Fig. 6-3 is fixed as 0.4 μ L, mix for probe；
It is that 40 μ L grope result figure that Fig. 6-4 is fixed as 0.4 μ L, mix for probe；
Fig. 7-1 is three kinds of bacterium canonical plottings (total figure), and wherein abscissa Quantity is copy number gradient concentration, indulges and sits
It is designated as CT values；
Fig. 7-2 is three kinds of bacterium standard items amplification curve diagrams (total figure)；
Fig. 8-1 is staphylococcus aureus canonical plotting (component), Y=-4.929X+58.824R2=0.999Eff%
=86.721, wherein abscissa Quantity are copy number gradient concentration, and ordinate is CT values；
Fig. 8-2 is staphylococcus aureus standard items amplification curve diagram (component)；
Fig. 9-1 is salmonella gene standard curve result figure (component), Y=-4.74X+57.046R2=
0.996Eff%=90.775, wherein abscissa Quantity are copy number gradient concentration, and ordinate is CT values；
Fig. 9-2 is salmonella standard items amplification curve diagram (component)；
Figure 10-1 is bacillus coli gene standard curve result figure (component), Y=-4.277X+52.119R2=
0.997Eff%=84.861, wherein abscissa Quantity are copy number gradient concentration, and ordinate is CT values；
Figure 10-2 is Escherichia coli standard items amplification curve diagram (component)；
Figure 11-1 is triple qPCR sensitivitys amplification curves (total figure)；
Figure 11-2 is triple middle salmonella sensitivity amplification curves (component)；
Figure 11-3 is triple middle Escherichia coli sensitivity amplification curves (component)；
Figure 11-4 is triple middle staphylococcus aureus sensitivity amplification curves (component).
Specific implementation mode
Below by specific implementation mode, invention is further described in detail, but those skilled in the art will manage
Solution, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.
Embodiment
S1 primers are designed with probe
Respectively with staphylococcus aureus nuc genes, Escherichia coli O 157：H7wzx genes, salmonella invA genes are
Object, through ncbi database sequence alignment design primer：
S1.1 staphylococcus aureus nuc genes, 96bp
Sense primer (5 ' -3 ') TTGTAGTTTCAAGTCTAAGTAGCTCAGC (SEQ ID NO.1)
Downstream primer (5 ' -3 ') TTGCACTATATACTGTTGGATCTTCAG (SEQ ID NO.2)
Probe (5 ' -3 ') FAM-TGCATCACAAACAGATAACGGCGTAAATAG-BHQ1 (SEQ ID NO.3)
S1.2 colon bacillus 0157s：H7wzx genes, 104bp
Sense primer (5 ' -3 ') TCATGCACGCAATGATACTCAA (SEQ ID NO.4)
Downstream primer (5 ' -3 ') CGAACACTACTAATATGAAGGCCA (SEQ ID NO.5)
Probe (5 ' -3 ') CY5-AGACGCTCAGAACATCATTGAAAATAGTGGG-BHQ2 (SEQ ID NO.6)
S1.3 salmonella invA genes, 108bp
Sense primer (5 ' -3 ') CCTGATCGCACTGAATATCGTACT (SEQ ID NO.7)
Downstream primer (5 ' -3 ') GTGGTAATTAACAGTACCGCAGGA (SEQ ID NO.8)
Probe (5 ' -3 ') Vic-ATATTGGTGTTTATGGGGTCGTTCTACATTGAC-BHQ1 (SEQ ID NO.9)
S2 primer specificities detect
S2.1 PCR systems：
S2.2 PCR reaction conditions
Colon bacillus 0157：The PCR reaction conditions of H7 and salmonella：
The PCR reaction conditions of staphylococcus aureus：
S2.3 specificity identification electrophoretograms：
Primer specificity qualification result is shown in Fig. 1, and as seen from the figure, each target gene amplifies respective strap.
The clone of tri- kinds of bacterial genes of S3 and plasmid extraction identification
Plasmid PCR is carried out with Escherichia coli, salmonella, the sense primer of staphylococcus aureus and downstream primer respectively
Identification, gained PCR product carry out 6g/L agarose gel electrophoresis identifications.The correct recombinant plasmid of PCR Preliminary Identifications is sent
Company carries out sequencing.
S3.1 target gene is cloned and plasmid identification
PCR product is purified with small measuring cylinder DNA QIAquick Gel Extraction Kits after agarose gel electrophoresis is identified and recycles DNA.
Respectively by after purification target fragment and 16 DEG C of carrier T (size 3928bp) connection 2h, by connection product convert to
DH5 α competent cells, 37 DEG C of constant incubators, are incubated overnight.
Picking white monoclonal colonies are inoculated in the LB liquid medium containing Amp, 200r/min, 37 DEG C of culture 12h.Simultaneously
Bacterium colony PCR is carried out to same bacterium colony, plasmid extraction is carried out to the bacterium solution of the bacterium colony PCR positives.Three kinds of bacterium target gene plasmid mirror
Determine result and sees 2.
S3.2 plasmid order-checking results
By sequencing, three kinds of bacterium specificity target gene all obtain correct verification.
(SEQ ID NO.10) is sequenced in S3.2.1 staphylococcus aureuses
GCTACTAGATACGGCGATTGGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATG
GATATCTGCAGAATTGCCCTTTGTAGTTTCAAGTCTAAGTAGCTCAGCAAATGCATCACA
AACAGATAATGGCGTAAATAGAAGTGGTTCTGAAGATCCAACAGTATATAGTGCAAAAG
GGCAATTCCAGCACACTGGCGGCCGTTACTAGTGGATCCGAGCTCGGTACCAAGCTTGG
CGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAA
CATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCA
CATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGC
ATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCT
TCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCA
CTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGT
GAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTT
CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGC
GAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGC
TCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGC
GTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCC
AAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAA
CTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAG
(SEQ ID NO.11) is sequenced in S3.2.2 Escherichia coli
GGGCTACTCTATAGGGGCGATTGGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGT
GATGGATATCTGCAGAATTGCCCTTCGAACACTACTAATATGAAGGCCAATAAGAATGAT
GAAATACCCACTATTTTCAATGATGTTCTGAGCGTCTTTTTTATAAATTGAGTATCATTGCG
TGCATGAAAGGGCAATTCCAGCACACTGGCGGCCGTTACTAGTGGATCCGAGCTCGGTA
CCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAA
TTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTG
AGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCG
TGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCG
CTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGG
TATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGA
AAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTA
AAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAA
AATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGT
TTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACC
TGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATC
TCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCCGTTCA
GCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACG ACTTA
(SEQ ID NO.12) is sequenced in S3.2.3 salmonellas
GCAGACTCTATAGGGCGATTGGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGA
TGGATATCTGCAGAATTGCCC
TTGTGGTAATTAACAGTACCGCAGGAAACGTTGAAAAACTGAGGATTCTGTCAATGTAG
AACGACCCCATAAACACCAATATCGCCAGTACGATATTCAGTGCGATCAGGAAGGGCAA
TTCCAGCACACTGGCGGCCGTTACTAGTGGATCCGAGCTCGGTACCAAGCTTGGCGTAA
TCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATAC
GAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTA
ATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAA
TGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTC
GCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAA
AGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGC
AAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCAT
AGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAA
ACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCT
CCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTG
GCGCTTTCTCATAGCTCACGCTGTAGTATCTCAGTTCGGTGTAGTCGTTCGCTCCAAGCT
GGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCCTTATCCGGTAACTAT
CGTCTTGAGTCCAACCCGGTAAGACACGACTTA
S4 quantitative fluorescent PCR substance specific detections
Staphylococcus aureus, salmonella, Escherichia coli are carried out with probe substance specific assay respectively, as a result substance
PCR amplifies corresponding curve, sees Fig. 3-1 to Fig. 3-3.
QPCR systems are as shown in table 1 below：
1 qPCR systems of table
QPCR conditions (following all same) are as shown in table 2 below：
2 qPCR conditions of table
S5 sonde method multiple specifics detect
Staphylococcus aureus, salmonella, Escherichia coli are placed on the same system to expand, as a result 3 kinds of bacterium are all
Special linearity curve is amplified, sees Fig. 4.
QPCR systems are as shown in table 3 below：
3 qPCR systems of table
S6 multiple conditions are groped
S6.1 Mix fix 30 μ L, and probe sets 3 concentration (0.4 μ L, 0.3 μ L, 0.2 μ L), and finally selected concentration and probe concentration is
0.4 μ L, are as a result shown in Fig. 5-1 to Fig. 5-4.
QPCR systems are as shown in table 4 below：
4 qPCR systems of table
S6.2 probes are fixed as 0.4 μ L, mix and set 30 μ L, 40 μ L, 35 mix μ L, finally a concentration of 30 μ L of selected mix, knot
Fruit sees Fig. 6-1 to Fig. 6-4.
QPCR systems are as shown in table 5 below：
5 qPCR systems of table
S7 standard curves are established
QPCR systems are as shown in table 6 below：
6 qPCR systems of table
Three kinds of plasmids measure a concentration of through microplate reader：Staphylococcus aureus：36ng/ μ L, Escherichia coli：45ng/ μ L, sand
Door Salmonella：46.5ng/μL.
Copy number calculates：
The mass concentration of DNA copy number=DNA/DNA average molecular weight * 6.02 × 1023(unit：copies/μL)；
The base logarithm * 660Da of the average molecular weight of DNA=total；
The mass concentration of L-form staphylococcus aureus copy number=DNA/DNA average molecular weight * 6.02 × 1023
=(36 × 10-9/96*660)*6.02×1023
≈3.4×1011=3.4E+11
The mass concentration of e. coli dna copy number=DNA/DNA average molecular weight * 6.02 × 1023
=(45 × 10-9/104*660)*6.02×1023
≈3.94×1011=3.94E+11
The mass concentration of salmonella DNA copy number=DNA/DNA average molecular weight * 6.02 × 1023
=(46.5 × 10-9/104*660)*6.02×1023
≈3.92×1011=3.92E+11
Each bacterium starting template is pressed 10×Standard items are established by being diluted, then use 105、106、107、108、109、
1010Copy number gradient concentration template establishes standard curve, such as Fig. 7-1 and Fig. 7-2：
Standard curve is from top to bottom：
Staphylococcus aureus (Fig. 8-1 and 8-2)：Y=-4.929X+58.824R2=0.999Eff%=86.721
Salmonella (Fig. 9-1 and 9-2)：Y=-4.74X+57.046R2=0.996Eff%=90.775
Escherichia coli O 157 H7 (Figure 10-1 and 10-2)：Y=-4.277X+52.119R2=0.997Eff%=84.861
Amplification efficiency meets the requirements between 80%-110%；
R2It is all higher than 0.99, is met the requirements.
S8 sensitivity Detections
Due to the foundation of standard curve, multiple sensitivity examines us from 106It is diluted to 102The gradient concentration of copy number
Sensitivity inspection is carried out, positive findings judgement is generally limited with 35 cycles, the result is shown in Figure 1 1-1 to Figure 11-4：Golden yellow grape
Coccus and salmonella sensitivity are 105Copies/ μ L, and Escherichia coli sensitivity 104copies/μL。
S9 repeatability
Take 105-1095 gradients template, each gradient carries out 10 repetitions, calculate separately log concentration value batch in
The coefficient of variation and interassay coefficient of variation, the results are shown in Table 7.
The coefficient of variation (CV)=standard deviation SD/ means X 100%
Repeatability in batch：Reproducibility of the multiple multiple pipes of same sample in primary measure, CV are less than 10%.
Repeatability between batch：Same sample measured at more batches between reproducibility, CV be less than 15%-20%.
Show that the triple fluorescent RT-PCR established has repeatability well.
Table 7-1 triple fluorescent PCR system Repeatability checkings (first)
Table 7-2 triple fluorescent PCR system Repeatability checkings (second batch)
5 gradient variation within batch coefficients are between 0.09%-1.71%, are less than 10%, have high repeatability.
Repeatability checking between table 7-3 triple fluorescent PCR systems batch
5 gradient interassay coefficient of variation are between 0.05%-6.62%, are less than 15%-20%, have high repetition
Property.
Sequence involved in the application：
Staphylococcus aureus nuc genes, 96bp
Sense primer (5 ' -3 ') TTGTAGTTTCAAGTCTAAGTAGCTCAGC (SEQ ID NO.1)
Downstream primer (5 ' -3 ') TTGCACTATATACTGTTGGATCTTCAG (SEQ ID NO.2)
Probe (5 ' -3 ') FAM-TGCATCACAAACAGATAACGGCGTAAATAG-BHQ1 (SEQ ID NO.3)
Colon bacillus 0157：H7wzx genes, 104bp
Sense primer (5 ' -3 ') TCATGCACGCAATGATACTCAA (SEQ ID NO.4)
Downstream primer (5 ' -3 ') CGAACACTACTAATATGAAGGCCA (SEQ ID NO.5)
Probe (5 ' -3 ') CY5-AGACGCTCAGAACATCATTGAAAATAGTGGG-BHQ2 (SEQ ID NO.6)
Salmonella invA genes, 108bp
Sense primer (5 ' -3 ') CCTGATCGCACTGAATATCGTACT (SEQ ID NO.7)
Downstream primer (5 ' -3 ') GTGGTAATTAACAGTACCGCAGGA (SEQ ID NO.8)
Probe (5 ' -3 ') Vic-ATATTGGTGTTTATGGGGTCGTTCTACATTGAC-BHQ1 (SEQ ID NO.9)
(SEQ ID NO.10) is sequenced in staphylococcus aureus
GCTACTAGATACGGCGATTGGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATG
GATATCTGCAGAATTGCCCTTTGTAGTTTCAAGTCTAAGTAGCTCAGCAAATGCATCACA
AACAGATAATGGCGTAAATAGAAGTGGTTCTGAAGATCCAACAGTATATAGTGCAAAAG
GGCAATTCCAGCACACTGGCGGCCGTTACTAGTGGATCCGAGCTCGGTACCAAGCTTGG
CGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAA
CATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCA
CATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGC
ATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCT
TCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCA
CTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGT
GAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTT
CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGC
GAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGC
TCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGC
GTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCC
AAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAA
CTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAG
(SEQ ID NO.11) is sequenced in Escherichia coli
GGGCTACTCTATAGGGGCGATTGGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGT
GATGGATATCTGCAGAATTGCCCTTCGAACACTACTAATATGAAGGCCAATAAGAATGAT
GAAATACCCACTATTTTCAATGATGTTCTGAGCGTCTTTTTTATAAATTGAGTATCATTGCG
TGCATGAAAGGGCAATTCCAGCACACTGGCGGCCGTTACTAGTGGATCCGAGCTCGGTA
CCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAA
TTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTG
AGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCG
TGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCG
CTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGG
TATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGA
AAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTA
AAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAA
AATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGT
TTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACC
TGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATC
TCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCCGTTCA
GCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACG ACTTA
(SEQ ID NO.12) is sequenced in salmonella
GCAGACTCTATAGGGCGATTGGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGA
TGGATATCTGCAGAATTGCCC
TTGTGGTAATTAACAGTACCGCAGGAAACGTTGAAAAACTGAGGATTCTGTCAATG
TAGAACGACCCCATAAACACCAATATCGCCAGTACGATATTCAGTGCGATCAGGAAGGG
CAATTCCAGCACACTGGCGGCCGTTACTAGTGGATCCGAGCTCGGTACCAAGCTTGGCG
TAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACA
TACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACA
TTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCAT
TAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTC
CTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACT
CAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTG
AGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTC
CATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGC
GAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGC
TCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGC
GTGGCGCTTTCTCATAGCTCACGCTGTAGTATCTCAGTTCGGTGTAGTCGTTCGCTCCAA
GCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCCTTATCCGGTAAC
TATCGTCTTGAGTCCAACCCGGTAAGACACGACTTA
Embodiment described above is only to absolutely prove that preferred embodiment that is of the invention and being lifted, protection domain are unlimited
In this.Those skilled in the art on the basis of the present invention made by equivalent substitute or transformation, the present invention protection
Within the scope of, protection scope of the present invention is subject to claims.
Sequence table
<110>Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute
<120>Primer, probe and the side that staphylococcus aureus, Escherichia coli, salmonella triple fluorescent quantitative PCR are detected
Method is established
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 28
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ttgtagtttc aagtctaagt agctcagc 28
<210> 2
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ttgcactata tactgttgga tcttcag 27
<210> 3
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
tgcatcacaa acagataacg gcgtaaatag 30
<210> 4
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
tcatgcacgc aatgatactc aa 22
<210> 5
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
cgaacactac taatatgaag gcca 24
<210> 6
<211> 31
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
agacgctcag aacatcattg aaaatagtgg g 31
<210> 7
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
cctgatcgca ctgaatatcg tact 24
<210> 8
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
gtggtaatta acagtaccgc agga 24
<210> 9
<211> 33
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
atattggtgt ttatggggtc gttctacatt gac 33
<210> 10
<211> 1000
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
gctactagat acggcgattg ggccctctag atgcatgctc gagcggccgc cagtgtgatg 60
gatatctgca gaattgccct ttgtagtttc aagtctaagt agctcagcaa atgcatcaca 120
aacagataat ggcgtaaata gaagtggttc tgaagatcca acagtatata gtgcaaaagg 180
gcaattccag cacactggcg gccgttacta gtggatccga gctcggtacc aagcttggcg 240
taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 300
atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 360
ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 420
taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 480
tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 540
aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 600
aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 660
ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 720
acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 780
ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 840
tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 900
tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 960
gagtccaacc cggtaagaca cgacttatcg ccactggcag 1000
<210> 11
<211> 1000
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
gggctactct ataggggcga ttgggccctc tagatgcatg ctcgagcggc cgccagtgtg 60
atggatatct gcagaattgc ccttcgaaca ctactaatat gaaggccaat aagaatgatg 120
aaatacccac tattttcaat gatgttctga gcgtcttttt tataaattga gtatcattgc 180
gtgcatgaaa gggcaattcc agcacactgg cggccgttac tagtggatcc gagctcggta 240
ccaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca 300
attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg 360
agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 420
tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 480
tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 540
tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 600
aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 660
tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 720
tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 780
cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 840
agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 900
tccaagctgg gctgtgtgca cgaacccccc cgttcagccc gaccgctgcg ccttatccgg 960
taactatcgt cttgagtcca acccggtaag acacgactta 1000
<210> 12
<211> 1000
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
gcagactcta tagggcgatt gggccctcta gatgcatgct cgagcggccg ccagtgtgat 60
ggatatctgc agaattgccc ttgtggtaat taacagtacc gcaggaaacg ttgaaaaact 120
gaggattctg tcaatgtaga acgaccccat aaacaccaat atcgccagta cgatattcag 180
tgcgatcagg aagggcaatt ccagcacact ggcggccgtt actagtggat ccgagctcgg 240
taccaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 300
caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 360
tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 420
cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 480
gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 540
tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 600
agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 660
cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 720
ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 780
tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 840
gaagcgtggc gctttctcat agctcacgct gtagtatctc agttcggtgt agtcgttcgc 900
tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc ccttatccgg 960
taactatcgt cttgagtcca acccggtaag acacgactta 1000
Claims (7)
1. the primer and probe that staphylococcus aureus, Escherichia coli, salmonella triple fluorescent quantitative PCR are detected, feature
It is, including respectively with staphylococcus aureus nuc genes, Escherichia coli O 157：H7wzx genes, salmonella invA genes
For three groups of primer and probes of object, three groups of primer and probes are respectively provided with following base-pair：
Using staphylococcus aureus nuc genes as object：
Sense primer (5 ' -3 ') TTGTAGTTTCAAGTCTAAGTAGCTCAGC
Downstream primer (5 ' -3 ') TTGCACTATATACTGTTGGATCTTCAG
Probe (5 ' -3 ') FAM-TGCATCACAAACAGATAACGGCGTAAATAG-BHQ1；
With colon bacillus 0157：H7wzx genes are object：
Sense primer (5 ' -3 ') TCATGCACGCAATGATACTCAA
Downstream primer (5 ' -3 ') CGAACACTACTAATATGAAGGCCA
Probe (5 ' -3 ') CY5-AGACGCTCAGAACATCATTGAAAATAGTGGG-BHQ2；
Using salmonella invA genes as object：
Sense primer (5 ' -3 ') CCTGATCGCACTGAATATCGTACT
Downstream primer (5 ' -3 ') GTGGTAATTAACAGTACCGCAGGA
Probe (5 ' -3 ') Vic-ATATTGGTGTTTATGGGGTCGTTCTACATTGAC-BHQ1.
2. what staphylococcus aureus described in claim 1, Escherichia coli, salmonella triple fluorescent quantitative PCR were detected draws
Object and probe are in staphylococcus aureus, colon bacillus 0157：H7, salmonella triple fluorescent quantitative PCR method foundation in
Application, which is characterized in that include the following steps：
Step 1：Design three groups of primer and probes；
Step 2：Primer specificity detects；
Step 3：Staphylococcus aureus nuc genes, Escherichia coli O 157：Gram of H7wzx genes, salmonella invA genes
The identification of grand and plasmid extraction；
Step 4：Staphylococcus aureus, salmonella, Escherichia coli are carried out with probe substance specific assay respectively；
Step 5：Sonde method multiple specific detects；
Step 6：Multiple condition is explored；
Step 7：Standard curve is established.
3. staphylococcus aureus according to claim 2, Escherichia coli, the PCR detections of salmonella triple fluorescent quantitative
Primer and probe in staphylococcus aureus, colon bacillus 0157：H7, salmonella triple fluorescent quantitative PCR method are built
Application in vertical, which is characterized in that when primer specificity described in step 2 detects,
PCR system is as follows：
PCR reaction conditions are as follows：
Colon bacillus 0157：The PCR reaction conditions of H7 and salmonella：
The PCR reaction conditions of staphylococcus aureus：
4. staphylococcus aureus according to claim 2, Escherichia coli, the PCR detections of salmonella triple fluorescent quantitative
Primer and probe in staphylococcus aureus, colon bacillus 0157：H7, salmonella triple fluorescent quantitative PCR method are built
Application in vertical, which is characterized in that when substance specific assay described in step 4,
QPCR systems are as follows：
QPCR conditions are as follows：50 DEG C, 2min；95 DEG C, 5min；(95 DEG C, 40s；60 DEG C, 40s；) × 40 recycle.
5. staphylococcus aureus according to claim 2, Escherichia coli, the PCR detections of salmonella triple fluorescent quantitative
Primer and probe in staphylococcus aureus, colon bacillus 0157：H7, salmonella triple fluorescent quantitative PCR method are built
Application in vertical, which is characterized in that when sonde method multiple specific described in step 5 detects, by staphylococcus aureus, sand
Door Salmonella, Escherichia coli are placed on the same system and are expanded, and qPCR systems are as follows：
6. staphylococcus aureus according to claim 2, Escherichia coli, the PCR detections of salmonella triple fluorescent quantitative
Primer and probe in staphylococcus aureus, colon bacillus 0157：H7, salmonella triple fluorescent quantitative PCR method are built
Application in vertical, which is characterized in that when multiple condition described in step 6 is explored, Mix fixes 30 μ L, and probe sets 3 concentration
(0.4 μ L, 0.3 μ L, 0.2 μ L), finally selected concentration and probe concentration is 0.4 μ L；Probe is fixed as 0.4 μ L, mix set 30 μ L, 40 μ L,
35 μ L of mix finally select a concentration of 30 μ L of mix.
7. staphylococcus aureus according to claim 2, Escherichia coli, the PCR detections of salmonella triple fluorescent quantitative
Primer and probe in staphylococcus aureus, colon bacillus 0157：H7, salmonella triple fluorescent quantitative PCR method are built
Application in vertical, which is characterized in that the standard curve established in step 7 is：
Staphylococcus aureus：Y=-4.929X+58.824 R2=0.999 Eff%=86.721
Salmonella：Y=-4.74X+57.046 R2=0.996 Eff%=90.775
Escherichia coli O 157 H7：Y=-4.277X+52.119 R2=0.997 Eff%=84.861.
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