CN107012237A - A kind of fluorescence PCR method and kit of specific detection gondii nucleic acid - Google Patents
A kind of fluorescence PCR method and kit of specific detection gondii nucleic acid Download PDFInfo
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- CN107012237A CN107012237A CN201710308761.7A CN201710308761A CN107012237A CN 107012237 A CN107012237 A CN 107012237A CN 201710308761 A CN201710308761 A CN 201710308761A CN 107012237 A CN107012237 A CN 107012237A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The present invention relates to a kind of fluorescent PCR kit of specific detection gondii nucleic acid, including specific primer pair and probe, described specific primer to for：Forward primer：5’‑CCGGGTGAAACAATAGAGAGTACTG‑3’；Reverse primer：5’‑GGTCTACGTCGATGGCATGA‑3’；Described probe is：5’‑AACGTCGCCGCTACTGCCCAGTT‑3’.The kit of the present invention contains the specific primer pair and probe, the characteristics of with sensitivity height, high specificity, high reaction efficiency, it is possible to achieve to the qualitative detection of Infection of Toxoplasma Gondii, can turn into and effectively aid in detection instrument.
Description
Technical field
The invention belongs to microorganism beyond body nucleic acid detection field, and in particular to a kind of specific detection gondii nucleic acid it is glimmering
Light PCR method and kit, can carry out qualitative detection to the DNA of Infection of Toxoplasma Gondii, can be used as effective detection instrument.
Background technology
Toxoplasmosis is a kind of in worldwide distribution and serious as caused by toxoplasma gondii (Toxoplasma gondii)
Endanger the parasitic zoonoses of human health.Infection of Toxoplasma Gondii is a kind of special sexual cell endoparasitism protozoon, can infect the mankind and
Most of mammal.According to incompletely statistics, the whole world there are about 1/3rd Adult infections' Infection of Toxoplasma Gondii.It is most by
The adult of arch insect infection can't cause serious symptom, but for immune deficiencies such as organ transplant, malignant tumour, AIDS
Person, and pregnant woman etc., arch insect infection can cause a series of serious conditions.The all one's life of Infection of Toxoplasma Gondii needs definitive host and centre simultaneously
Host completes its zoogamy and asexual reproduction.Cat and other cats are the definitive hosts of Infection of Toxoplasma Gondii.Infection of Toxoplasma Gondii
Main infection approach is：One, oral cavity intake infection；Two, blood borne infection；Three, congenital transmission infection.Congenital transmission master
If mother-to-baby transmission.It is worth noting that, pregnant woman early pregnancy toxoplasma gondii infection, can cause miscarriage, stillborn foetus situations such as, and
Be pregnant middle and later periods toxoplasma gondii infection, then development of fetus can be caused slow, blinds, encephalitis, and the symptom such as nervous system disorders, is to need weight
The problem of point is solved.
For the detection of Infection of Toxoplasma Gondii, serological test, such as ELISA, RIA and IFA being used clinical labororatory, detection is special more
Heterogenetic antibody.Serum Infection of Toxoplasma Gondii-IgM the positives prove recent infection, and oneself continues 2~4 months.Serum Infection of Toxoplasma Gondii-IgG the positives are then carried
Show previous infection.Because toxoplasma antigen source is difficult, the still difficult popularization of serological test, and also it is anti-after some population infections virus
Precursor reactant is very weak or does not produce antibody, therefore only can not accurately judge arch insect infection situation with antibody test result, need to be aided with
Antigen or detection of nucleic acids result are used as diagnosis and the foundation treated.The nucleic acid detection method of current comparative maturity is to use fluorescent PCR
Method, the method overcome Standard PCR method poor specificity, easily pollution, the shortcomings of interpretation of result operating procedure is more, using TaqMan
Method or double mark sonde methods (fluorescent quenching probe or FQ probes), i.e., hold one fluorescence report of mark respectively at its 5 ' end and 3 '
Accuse group and a fluorescent quenching group.The specific probe of target sequence and specific PCR primer are used simultaneously, design should
Probe is annealed in the range of upstream and downstream PCR primer, is incorporated into target gene fragment jointly by three oligonucleotides, is made its special
Property is greatly enhanced.In the PCR extension stage, the exonuclease activities of Taq archaeal dna polymerases 5 ' -3 ' by fluorescent reporter group from
Cut down on probe.With the increase of PCR cycle number, the quantity of free reporter group constantly increases, and detects in real time from trip
From the fluorescence signal discharged on fluorophor, so as to carry out qualitative analysis to target sequence.PCR reactions are entered in closed system
OK, analysis result is without Kaifeng, therefore, it is to avoid the generation of PCR primer pollution.
At present, also there is fluorescence PCR method to be used to detecting the report of Infection of Toxoplasma Gondii, but develop it is more effectively, it is quick, accurate,
Sensitivity is high, the primer and probe of the fluorescence PCR method of high specificity, and this primer and probe is used for into detection kit
It is the target that this area is pursued.
The content of the invention
The technical problems to be solved by the invention are to overcome the deficiencies in the prior art there is provided a kind of specific detection Infection of Toxoplasma Gondii
The fluorescence PCR method and kit of nucleic acid, the kit have the characteristics of sensitive, special, reaction efficiency is high, can realize qualitative
Detect the purpose of gondii nucleic acid.
To solve above technical problem, the present invention is adopted the following technical scheme that：
It is an object of the present invention to provide a kind of fluorescent PCR kit of specific detection gondii nucleic acid, including spy
Specific primer pair and probe, described specific primer to for：
Forward primer：5’-CCGGGTGAAACAATAGAGAGTACTG-3’；
Reverse primer：5’-GGTCTACGTCGATGGCATGA-3’；
Described probe is：5’-AACGTCGCCGCTACTGCCCAGTT-3’.
In the present invention, 5 ' ends of described probe mark reporter fluorescences element or fluorescent dye, 3 ' end mark quenching groups.
Specifically, described reporter fluorescence element or fluorescent dye be FAM or other it is any can be marked as probe it is glimmering
Light element or fluorescent dye；Quenching group be TAMRA or other it is any can as quenching group chemical substance.
Preferably, other any fluoresceins that can be marked as probe or fluorescent dye such as HEX, TET, JOE,
Yakima Yellow、Cy3、Cy3.5、Cy5、NED、VIC、PET、ROX、LIZ、RED、Texas Red；It is other any to make
For chemical substance such as Dabcyl, BHQ1, BHQ2 of quenching group.
Preferably, described kit also includes positive quality control product, and the positive quality control product contains artificial synthesized sequence, described
Artificial synthesized sequence for it is artificial synthesized containing the Infection of Toxoplasma Gondii specific primer to the sequence of amplified production fragment.This is artificial synthesized
The content of sequence is：
CAAACTGCAACAACTGCTCTAGCGTGTTCGTCTCCATTCCGTACAGTCTTCAAAAATACA
AAAGAGAACATTCCAGCAACTTCTGCCTTTGTTCTTTTAGCCTCAATAGCAGGATGACGC
CTCCCTCCTATCTTTCAGCCAACCCAGCAAACACCGACGAACTCTCTGTAGAGTAACAAA
GAGAAGGCAAAACGCGCCATCACGAACACTCGCAGAGATGATACAGAGACGTGTCATCAG
GACAAGGTTGGTCGCTTAATTTTCTGTATATAGCATTTTTAGAATGCACCTTTCGGACCT
CAACAACCGTGCAAAAGGATCGCCACCTGGTGTCTCTTCAAGCGTCAAAACGAACTATCT
GTATATCTCTCAAGGAGGACTGGCAACCTGGTGTCGACAACAGAACAGCTGCAGTCCGGA
AATAGAAAGCCATGAGGCACTCCAACGGGCGAGTAGCACCTGAGGAGATACAAACTGCTA
AACGGTCCGGGTGAAACAATAGAGAGTACTGGAACGTCGCCGCTACTGCCCAGTTGTCAT
GCCATCGACGTAGACCCAGAAATGAGGCGAGAAATTAATATTGTTAGTAAAGCATTCAAA
AAGTTCCGGTCGAGAGGCTAAACCACAAAAGTGCAAACCATGCGCAGCCATCAGCTTAAC
AAAAGCAGTTGGTGATGGTTGCCTCGAGTTCCTTCTGAAAATGGATTACTTCATCAACGA
GCCCACCACGCAGAATCATGCTTTCCCAGTG。
According to an embodiment, described positive quality control product is described artificial by being inserted in pUC57 plasmid vectors
Synthesis is recombinated to the sequence of amplified production fragment containing the Infection of Toxoplasma Gondii specific primer and is made.
Further, the concentration of described pUC57 vector plasmids is 1ng/ μ L.
Preferably, described kit also includes negative quality-control product, and the negative quality-control product contains the artificial synthesized mankind
The specific sequence of house-keeping gene GAPDH Partial Fragments.
According to an embodiment, for expanding drawing for described artificial synthesized mankind's house-keeping gene GAPDH Partial Fragments
Thing is to as follows：
Forward primer：5’-CGGGAAGGAAATGAATGG-3’；
Reverse primer：5’-CAAAGGGCAGGAGTAAAG-3’.
According to an embodiment, described negative quality-control product is the part inserted with mankind's house-keeping gene GAPDH
The pTG19-T vector plasmids of fragment sequence.
Further, the concentration of described pTG19-T vector plasmids is 1ng/ μ L.
According to an embodiment, the specific sequence contained by the negative quality-control product is：
CGGGAAGGAAATGAATGGGCAGCCGTTAGGAAAGCCTGCCGGTGACTAAC
CCTGCGCTCCTGCCTCGATGGGTGGAGTCGCGTGTGGCGGGGAAGTCAGG
TGGAGCGAGGCTAGCTGGCCCGATTTCTCCTCCGGGTGATGCTTTTCCTA
GATTATTCTCTGGTAAATCAAAGAAGTGGGTTTATGGAGGTCCTCTTGTG
TCCCCTCCCCGCAGAGGTGTGGTGGCTGTGGCATGGTGCCAAGCCGGGAG
AAGCTGAGTCATGGGTAGTTGGAAAAGGACATTTCCACCGCAAAATGGCC
CCTCTGGTGGTGGCCCCTTCCTGCAGCGCCGGCTCACCTCACGGCCCCGC
CCTTCCCCTGCCAGCCTAGCGTTGACCCGACCCCAAAGGCCAGGCTGTAA
ATGTCACCGGGAGGATTGGGTGTCTGGGCGCCTCGGGGAACCTGCCCTTC
TCCCCATTCCGTCTTCCGGAAACCAGATCTCCCACCGCACCCTGGTCTGA
GGTTAAATATAGCTGCTGACCTTTCTGTAGCTGGGGGCCTGGGCTGGGGC
TCTCTCCCATCCCTTCTCCCCACACACATGCACTTACCTGTGCTCCCACT
CCTGATTTCTGGAAAAGAGCTAGGAAGGACAGGCAACTTGGCAAATCAAA
GCCCTGGGACTAGGGGGTTAAAATACAGCTTCCCCTCTTCCCACCCGCCC
CAGTCTCTGTCCCTTTTGTAGGAGGGACTTAGAGAAGGGGTGGGCTTGCC
CTGTCCAGTTAATTTCTGACCTTTACTCCTGCCCTTTG。
In the present invention, kit also includes PCR reaction solutions, and described PCR reaction solutions include Taq enzyme, dNTP, buffer solution etc.
Composition.
It is described it is a further object to provide a kind of fluorescence PCR method of specific detection gondii nucleic acid
Fluorescence PCR method uses described fluorescent PCR kit.
The present invention devises specific primer pair and probe according to the DNA sequence dna of Infection of Toxoplasma Gondii B1 genes, and probe uses fluorescence
Element mark, carries out the amplification of nucleic acid for sample to be measured with fluorescent PCR instrument, analyses whether the amplification of the virus nucleic acid segment
Product, so as to reach the detection viral purpose.
In the present invention, the reaction system of described kit is 20 μ L-50 μ L, if 20 μ L systems, then include：PCR
The μ L of reaction solution (2x) 10, the μ L of forward primer 0.5 (10 μM of ol/mL), the μ L of reverse primer 0.5 (10 μM of ol/mL), the μ of fluorescence probe 0.5
L (10 μM of ol/mL), detected material cell, tissue, the μ L of DNA extracts 4 of body fluid (secretion, sputum, urine etc.), sterilizing
Ultra-pure water adds to 20 μ L；If 50 μ L systems, then include：The μ L of PCR reaction solutions (2x) 25, the μ L of forward primer 1.25 (10 μM of ol/
ML), the μ L of reverse primer 1.25 (10 μM of ol/mL), the μ L of fluorescence probe 1.25 (10 μM of ol/mL), detected material cell, tissue, body
The DNA extract 4-10 μ L of liquid (secretion, sputum, urine etc.), sterilizing ultra-pure water adds to 50 μ L.
Use described kit carry out fluorescent PCR amplification reaction time and temperature for：50 DEG C, 1 circulation of 2min
(being omitted if PCR reaction solutions are free of UNG enzymes)；95 DEG C, 2-10min (depending on the PCR reaction solutions of separate sources) 1 is followed
Ring；95 DEG C, 15s, 60 DEG C, 1min, 40 circulate and collect fluorescence signal.
Due to the implementation of above-mentioned technical proposal, the present invention has the following advantages that compared with prior art：
Kit of the present invention by being detected to a large amount of clinical samples, can effective detection go out Infection of Toxoplasma Gondii, it is a large amount of clinical
Validity, the large-scale use of the detection of sample also to kit, which are provided, to be ensured.
Kit of the present invention, the reaction efficiency of Infection of Toxoplasma Gondii fluorescent PCR detection is up to 88.5%, is imitated with higher reaction
Rate.
Brief description of the drawings
Fig. 1 is the response diagram of Infection of Toxoplasma Gondii fluorescent PCR, and the curve in figure from left to right corresponds to the concentration of positive quality control product respectively
From high to low；
Fig. 2 is the corresponding canonical plotting of Infection of Toxoplasma Gondii Fluorescence PCR in Fig. 1；
Fig. 3 is the quality inspection electrophoretogram of Infection of Toxoplasma Gondii fluorescence PCR primer pair, wherein, 1 is that Infection of Toxoplasma Gondii Fluorescence PCR forward direction is drawn
Thing；2 be Infection of Toxoplasma Gondii Fluorescence PCR reverse primer；M is electrophoresis Marker, and it is (single that length is followed successively by 20,24,36 from top to bottom
Position：Base)；
Fig. 4 schemes for the quality inspection HPLC of Infection of Toxoplasma Gondii fluorescent PCR probe；
Fig. 5 is that pUC57 recombinant plasmids constitute schematic diagram；
Fig. 6 is that pTG19-T plasmid vectors constitute schematic diagram.
Embodiment
The kit of the present invention is using the Fluorescence PCR specific primer pair and fluorescence probe of autonomous Design, manually conjunction
Into positive quality control product, artificial synthesized negative quality-control product, with sensitivity is high, high specificity, the characteristics of reaction efficiency is high can
To realize the qualitative detection to Infection of Toxoplasma Gondii, effective auxiliary detection instrument can be turned into.
With reference to specific embodiment, the present invention will be further described in detail, should illustrate that some embodiment is only used
In illustrating, of the invention rather than limitation is of the invention.
Embodiment 1：The preparation of kit
1st, the design and synthesis of specific primer pair and probe
Using the softwares of Primer Express 3.0, with the DNA sequence dna of Infection of Toxoplasma Gondii B1 genes design specific primer pair and
Its specific probe, primer and probe is synthesized by the biological Co., Ltd of Shanghai JaRa, has been carried out on the probe of synthesis glimmering
Light reporter group and fluorescent quenching group mark.Wherein, primer purifies for PAGE, and probe purifies for HPLC.
The specific primer pair and probe designed are as follows：
Forward primer：5’-CCGGGTGAAACAATAGAGAGTACTG-3’(SEQ ID NO.1)；
Reverse primer：5’-GGTCTACGTCGATGGCATGA-3’(SEQ ID NO.2)；
Probe is：5’-AACGTCGCCGCTACTGCCCAGTT-3’(SEQ ID NO.3).
Lyophilized powder is prepared into after primer and probe synthesis, 100 μM of concentration is then diluted to 1 × TE buffer
It is used as mother liquor, -20 DEG C of preservations.Working solution is that mother liquor is made for 10 times by sterilized water dilution, is used for conventional.
Quality inspection analysis is carried out to specific primer pair and fluorescence probe, as a result as shown in Figure 3 and Figure 4.
2nd, the preparation of positive quality control product and negative quality-control product
The preparation of positive quality control product：Specific primer containing the present invention is to the specific sequence fragment of amplified production by Nanjing
The synthesis of Jin Sirui biologies Co., Ltd, is then manually fitted into pUC57 plasmid vectors, then lead to recombinant dna gene engineering method
Cross the steps such as conversion Escherichia coli, plasmid amplification and plasmid extraction and obtain recombinant plasmid dna, this DNA is exactly examination of the present invention
Positive quality control product used in agent box, the specific sequence contained by the positive quality control product is as shown in SEQ ID NO.4.
The preparation of negative quality-control product：It is, using mankind's house-keeping gene GAPDH genes as template, to design Standard PCR primer, primer
Sequence is entered performing PCR and expanded as shown in SEQ ID NO.5 and SEQ ID NO.6 in the genomic DNA extracted from human tissue cells
Increase, obtain amplified production, then carried amplified production manual assembly to pTG19-T plasmids with the method for recombinant dna gene engineering
In body, then by converting the steps such as Escherichia coli, plasmid amplification and plasmid extraction acquisition recombinant plasmid dna, this DNA is exactly
Negative quality-control product used in kit of the present invention, the specific sequence such as SEQ ID NO.7 institutes contained by the negative quality-control product
Show.
3rd, PCR reaction solutions
PCR reaction solutions include the compositions such as Taq enzyme, dNTP, buffer solution, can use the Fluorescence PCR purchased from ABI companies
Mixed liquor, title isUniversal Master Mix II,with UNG。
Embodiment 2：The extraction of sample nucleic acid
Use the blood/tissue/cellular genome extracts kit of TIANGEN Biotech (Beijing) Co., Ltd.
(DP304), according to kit specification the step of, sample nucleic acid is extracted.After the completion of extraction, product puts -20 DEG C of preservations.
Embodiment 3：The making of standard curve
Positive quality control product is chosen as reference material, diluted concentration is then proceeded to 10 times of gradient dilutions five to 1 μ g/mL
Concentration, comes to 6 concentration gradients, i.e. 1 μ g/mL, 10-1μg/mL、10-2μg/mL、10-3μg/mL、10-4μg/mL、10-5μg/
mL.Then it regard the sample of this six concentration gradients as reaction system and reaction condition of the sample to be tested according to following embodiments 4
Fluorescent PCR amplified reaction is carried out, with the Software Create standard curve supporting with ABI7500 instruments.As a result as depicted in figs. 1 and 2,
And the system of instrument is shown, reaction efficiency is up to 88.5%.
Embodiment 4：Fluorescent PCR is expanded and interpretation of result
1) reaction system that fluorescent PCR amplified reaction is used is as shown in table 1.Configured in special eight union of fluorescent PCR
After each system, be put into ABI7500 fluorescent PCR instruments (or other types of open quantitative fluorescent PCR instrument), prepare into
Row reaction.Experiment is required for configuring the reaction tube of 1 positive quality control product as the negative quality-control product of positive control and one every time
Reaction tube is used as negative control.
Table 1
2) shown in the reaction condition table 2 that fluorescent PCR amplified reaction is used.
Table 2
3) interpretation of result：
(1) after, reaction terminates, first, the selection of baseline is set to " automatic ".The principle of threshold value (threshold) setting
It is the peak for the amplification curve that threshold line is just above negative quality-control product, makes its Ct value=40 or " Undetermined ".
(2), set after good threshold, whether the amplification curve of observation positive quality control product is normal S types Increasing Curve of Logarithm,
And Ct value≤37.Meet above-mentioned condition and then illustrate that this experiment reaction is normal, otherwise, it is necessary to reform.
(3), result judgement.Ct value≤37, it is the positive, Ct values to represent the sample results>37 or " Undetermined ", table
It is feminine gender to show the sample results.
Application Example
124 tissue samples being collected into are detected using kit of the present invention.124 samples are collected to be clinical
Fetal tissue, be divided to two groups：Normal induced labor group (control group) 42, spontaneous abortion group (ill group) 82.According to above-mentioned reality
Apply example 2：The extraction of sample nucleic acid.Then according to above-described embodiment 4：Fluorescent PCR is expanded and interpretation of result, uses present invention examination
Agent box Infection of Toxoplasma Gondii fluorescent PCR testing result such as table 3 below in ABI7500 fluorescent PCRs instrument carries out fluorescent PCR detection, fetal tissue
It is shown.
Table 3
Can be drawn from the result in table 3, kit of the present invention can successfully detect Infection of Toxoplasma Gondii, and from normal group and
From the point of view of the positive rate result of illness group, kit of the present invention can distinguish Normal group and ill group well, and both are positive
The ratio of rate is 1 to 8.20, and the infection rate that is Infection of Toxoplasma Gondii is organized in illness is 8 times of Normal group, as a result shows this hair
Bright kit has good sensitivity and specificity, can be used as the effective tool for detecting Infection of Toxoplasma Gondii.A large amount of clinical samples
Detect that the validity to kit, large-scale use are provided to ensure.
Kit of the present invention, the reaction efficiency of Infection of Toxoplasma Gondii detection is up to 88.5%, with higher reaction efficiency.
The present invention is described in detail above, its object is to allow the personage for being familiar with this art to understand this
The content of invention is simultaneously carried out, and it is not intended to limit the scope of the present invention, and the invention is not restricted to above-mentioned implementation
Example, the equivalent change or modification that all Spirit Essences according to the present invention are made, should all be included within the scope of the present invention.
SEQUENCE LISTING
<110>Zhangjiagang indigo plant is revived thing Engineering Co., Ltd
<120>A kind of fluorescence PCR method and kit of specific detection gondii nucleic acid
<130> 2017
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213>Artificial sequence
<400> 1
ccgggtgaaa caatagagag tactg 25
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
ggtctacgtc gatggcatga 20
<210> 3
<211> 23
<212> DNA
<213>Artificial sequence
<400> 3
aacgtcgccg ctactgccca gtt 23
<210> 4
<211> 751
<212> DNA
<213>Artificial sequence
<400> 4
caaactgcaa caactgctct agcgtgttcg tctccattcc gtacagtctt caaaaataca 60
aaagagaaca ttccagcaac ttctgccttt gttcttttag cctcaatagc aggatgacgc 120
ctccctccta tctttcagcc aacccagcaa acaccgacga actctctgta gagtaacaaa 180
gagaaggcaa aacgcgccat cacgaacact cgcagagatg atacagagac gtgtcatcag 240
gacaaggttg gtcgcttaat tttctgtata tagcattttt agaatgcacc tttcggacct 300
caacaaccgt gcaaaaggat cgccacctgg tgtctcttca agcgtcaaaa cgaactatct 360
gtatatctct caaggaggac tggcaacctg gtgtcgacaa cagaacagct gcagtccgga 420
aatagaaagc catgaggcac tccaacgggc gagtagcacc tgaggagata caaactgcta 480
aacggtccgg gtgaaacaat agagagtact ggaacgtcgc cgctactgcc cagttgtcat 540
gccatcgacg tagacccaga aatgaggcga gaaattaata ttgttagtaa agcattcaaa 600
aagttccggt cgagaggcta aaccacaaaa gtgcaaacca tgcgcagcca tcagcttaac 660
aaaagcagtt ggtgatggtt gcctcgagtt ccttctgaaa atggattact tcatcaacga 720
gcccaccacg cagaatcatg ctttcccagt g 751
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence
<400> 5
cgggaaggaa atgaatgg 18
<210> 6
<211> 18
<212> DNA
<213>Artificial sequence
<400> 6
caaagggcag gagtaaag 18
<210> 7
<211> 788
<212> DNA
<213>Artificial sequence
<400> 7
cgggaaggaa atgaatgggc agccgttagg aaagcctgcc ggtgactaac cctgcgctcc 60
tgcctcgatg ggtggagtcg cgtgtggcgg ggaagtcagg tggagcgagg ctagctggcc 120
cgatttctcc tccgggtgat gcttttccta gattattctc tggtaaatca aagaagtggg 180
tttatggagg tcctcttgtg tcccctcccc gcagaggtgt ggtggctgtg gcatggtgcc 240
aagccgggag aagctgagtc atgggtagtt ggaaaaggac atttccaccg caaaatggcc 300
cctctggtgg tggccccttc ctgcagcgcc ggctcacctc acggccccgc ccttcccctg 360
ccagcctagc gttgacccga ccccaaaggc caggctgtaa atgtcaccgg gaggattggg 420
tgtctgggcg cctcggggaa cctgcccttc tccccattcc gtcttccgga aaccagatct 480
cccaccgcac cctggtctga ggttaaatat agctgctgac ctttctgtag ctgggggcct 540
gggctggggc tctctcccat cccttctccc cacacacatg cacttacctg tgctcccact 600
cctgatttct ggaaaagagc taggaaggac aggcaacttg gcaaatcaaa gccctgggac 660
tagggggtta aaatacagct tcccctcttc ccacccgccc cagtctctgt cccttttgta 720
ggagggactt agagaagggg tgggcttgcc ctgtccagtt aatttctgac ctttactcct 780
gccctttg 788
Claims (10)
1. a kind of fluorescent PCR kit of specific detection gondii nucleic acid, including specific primer pair and probe, its feature exist
In：
Described specific primer to for：
Forward primer：5’-CCGGGTGAAACAATAGAGAGTACTG-3’；
Reverse primer：5’-GGTCTACGTCGATGGCATGA-3’；
Described probe is：5’-AACGTCGCCGCTACTGCCCAGTT-3’.
2. the fluorescent PCR kit of specific detection gondii nucleic acid according to claim 1, it is characterised in that：It is described
Probe 5 ' end mark reporter fluorescences element or fluorescent dye, 3 ' end mark quenching groups.
3. the fluorescent PCR kit of specific detection gondii nucleic acid according to claim 2, it is characterised in that：It is described
Reporter fluorescence element or fluorescent dye be any fluoresceins or fluorescent dye that can be marked as probe of FAM or other；It is described
Quenching group for TAMRA or other it is any can as quenching group chemical substance.
4. the fluorescent PCR kit of specific detection gondii nucleic acid according to claim 1, it is characterised in that：It is described
Kit also include PCR reaction solutions, positive quality control product and negative quality-control product.
5. the fluorescent PCR kit of specific detection gondii nucleic acid according to claim 4, it is characterised in that：It is described
Positive quality control product contain artificial synthesized sequence, described artificial synthesized sequence contains the Infection of Toxoplasma Gondii specific primer to be artificial synthesized
To the sequence of amplified production fragment.
6. the fluorescent PCR kit of specific detection gondii nucleic acid according to claim 5, it is characterised in that：It is described
Positive quality control product contained by artificial synthesized sequence be：
CAAACTGCAACAACTGCTCTAGCGTGTTCGTCTCCATTCCGTACAGTCTTCAAAAATACA
AAAGAGAACATTCCAGCAACTTCTGCCTTTGTTCTTTTAGCCTCAATAGCAGGATGACGC
CTCCCTCCTATCTTTCAGCCAACCCAGCAAACACCGACGAACTCTCTGTAGAGTAACAAA
GAGAAGGCAAAACGCGCCATCACGAACACTCGCAGAGATGATACAGAGACGTGTCATCAG
GACAAGGTTGGTCGCTTAATTTTCTGTATATAGCATTTTTAGAATGCACCTTTCGGACCT
CAACAACCGTGCAAAAGGATCGCCACCTGGTGTCTCTTCAAGCGTCAAAACGAACTATCT
GTATATCTCTCAAGGAGGACTGGCAACCTGGTGTCGACAACAGAACAGCTGCAGTCCGGA
AATAGAAAGCCATGAGGCACTCCAACGGGCGAGTAGCACCTGAGGAGATACAAACTGCTA
AACGGTCCGGGTGAAACAATAGAGAGTACTGGAACGTCGCCGCTACTGCCCAGTTGTCAT
GCCATCGACGTAGACCCAGAAATGAGGCGAGAAATTAATATTGTTAGTAAAGCATTCAAA
AAGTTCCGGTCGAGAGGCTAAACCACAAAAGTGCAAACCATGCGCAGCCATCAGCTTAAC
AAAAGCAGTTGGTGATGGTTGCCTCGAGTTCCTTCTGAAAATGGATTACTTCATCAACGA
GCCCACCACGCAGAATCATGCTTTCCCAGTG。
7. the fluorescent PCR kit of specific detection gondii nucleic acid according to claim 4, it is characterised in that：It is described
Negative quality-control product contain the specific sequences of artificial synthesized mankind's house-keeping gene GAPDH Partial Fragments.
8. the fluorescent PCR kit of specific detection gondii nucleic acid according to claim 7, it is characterised in that：For
The primer pair of the described artificial synthesized mankind's house-keeping gene GAPDH Partial Fragments of amplification is as follows：
Forward primer：5’-CGGGAAGGAAATGAATGG-3’；
Reverse primer：5’-CAAAGGGCAGGAGTAAAG-3’.
9. the fluorescent PCR kit of specific detection gondii nucleic acid according to claim 7, it is characterised in that：It is described
Negative quality-control product contained by specific sequence be：
CGGGAAGGAAATGAATGGGCAGCCGTTAGGAAAGCCTGCCGGTGACTAAC
CCTGCGCTCCTGCCTCGATGGGTGGAGTCGCGTGTGGCGGGGAAGTCAGG
TGGAGCGAGGCTAGCTGGCCCGATTTCTCCTCCGGGTGATGCTTTTCCTA
GATTATTCTCTGGTAAATCAAAGAAGTGGGTTTATGGAGGTCCTCTTGTG
TCCCCTCCCCGCAGAGGTGTGGTGGCTGTGGCATGGTGCCAAGCCGGGAG
AAGCTGAGTCATGGGTAGTTGGAAAAGGACATTTCCACCGCAAAATGGCC
CCTCTGGTGGTGGCCCCTTCCTGCAGCGCCGGCTCACCTCACGGCCCCGC
CCTTCCCCTGCCAGCCTAGCGTTGACCCGACCCCAAAGGCCAGGCTGTAA
ATGTCACCGGGAGGATTGGGTGTCTGGGCGCCTCGGGGAACCTGCCCTTC
TCCCCATTCCGTCTTCCGGAAACCAGATCTCCCACCGCACCCTGGTCTGA
GGTTAAATATAGCTGCTGACCTTTCTGTAGCTGGGGGCCTGGGCTGGGGC
TCTCTCCCATCCCTTCTCCCCACACACATGCACTTACCTGTGCTCCCACT
CCTGATTTCTGGAAAAGAGCTAGGAAGGACAGGCAACTTGGCAAATCAAA
GCCCTGGGACTAGGGGGTTAAAATACAGCTTCCCCTCTTCCCACCCGCCC
CAGTCTCTGTCCCTTTTGTAGGAGGGACTTAGAGAAGGGGTGGGCTTGCC
CTGTCCAGTTAATTTCTGACCTTTACTCCTGCCCTTTG。
10. a kind of fluorescence PCR method of specific detection gondii nucleic acid, it is characterised in that：Described fluorescence PCR method is used
Fluorescent PCR kit any one of claim 1 to 9.
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