CN103320501B - Primer, probe and kit for detecting bradyzoite of toxoplasma gondii - Google Patents
Primer, probe and kit for detecting bradyzoite of toxoplasma gondii Download PDFInfo
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- CN103320501B CN103320501B CN201210072527.6A CN201210072527A CN103320501B CN 103320501 B CN103320501 B CN 103320501B CN 201210072527 A CN201210072527 A CN 201210072527A CN 103320501 B CN103320501 B CN 103320501B
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Abstract
The invention discloses primers and a probe for detecting bradyzoite of toxoplasma gondii, wherein the primers are a pair of primers for amplifying a conserved region of a specific antigen BAG1 gene of T. gondii of bradyzoite, and the probe is hybridized with the conserved region of the BAG1 gene of toxoplasma gondii of bradyzoite, amplified by the primers. The invention further discloses a kit for detecting the bradyzoite of toxoplasma gondii. The primers, the probe and the kit are used for detecting the bradyzoite of toxoplasma gondii by a real-time fluorogenic quantitative PCR method, can accurately and rapidly detect infection of the bradyzoite of toxoplasma gondii, have advantages of high sensitivity and strong singularity, are suitable for clinic detection, or are used for researching formation and activation mechanism of the bradyzoite of toxoplasma gondii.
Description
Technical field
The present invention relates to technical field of bioengineering, relate in particular to a kind of primer for detection of toxoplasma gondii bradyzoite and probe and test kit.
Background technology
Toxoplasma gondii, also referred to as bow slurry worm, the traditional Chinese medical science, three corpse worms, belongs to top mixture subphylum, sporozoa, Eucoccida, isospora section, toxoplasma, be cytozoon, arrive each position of whole body with blood flow, destroy brain, heart, eyeground, cause people's immunity degradation, thereby suffer from various diseases.Toxoplasma gondii is a kind of parasitosis of propagating by He Shi source, water source, and humans and animals health is had to very big potentially dangerous, and they parasitize in host cell, can infect all temperature vertebrates including people.In some cases, consequence is seriously even in peril of one's life arch insect infection people very much, the fetus of for example pregnancy duration and the crowd of immune deficiency.Zoogenetic infection toxoplasma gondii can cause dam miscarriage, mummy tire, stillborn foetus, and this disease is broken out on pig farm, and mortality ratio is especially up to more than 60%.But, detecting at present the method for toxoplasma gondii, ubiquity susceptibility is low, the shortcoming of poor specificity.
Toxoplasma gondii bradyzoite, is the trophont in packing, and diameter 5-10 μ m constantly breeds in packing, reaches several extremely hundreds of, and packing can break under certain condition, and bradyzoite reenters new cell and forms new packing, can existence in tissue for a long time.
BAG1, toxoplasma gondii bradyzoite stage antigens 1, is one of main stage-specific antigen of toxoplasma gondii bradyzoite, can't detect this gene in the tachyzoite phase.The early stage quilt that BAG1 changes to bradyzoite at induction toxoplasma tachyzoite shows, and is the major protein of mature tissue's packing.BAG1 Chang Zuowei toxoplasma tachyzoite to bradyzoite transform indicator protein and studied, be a kind of endochylema antigen.
Real-time PCR, real-time quantitative PCR, refers to during the amplification of PCR index and immediately measures the amount of specificity product by the variation of continuous monitoring fluorescent signal power, and infer accordingly the original bulk of goal gene, does not need to take out PCR product and separates.Real-time quantitative PCR, as an extremely effective experimental technique, has been widely used in the every field of molecular biology research at present.
Summary of the invention
Detection method ubiquity susceptibility that the present invention will solve current toxoplasma gondii is low, the technical problem of poor specificity, a kind of primer for detection of toxoplasma gondii bradyzoite and probe are provided, utilizing this primer and probe to carry out real-time fluorescence quantitative PCR (Real-time PCR) detects, the infection of toxoplasma gondii bradyzoite can be detected quickly and accurately, its susceptibility is high, high specificity.
In addition, also need to provide a kind of test kit that comprises above-mentioned primer and probe.
In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:
In one aspect of the invention, provide a kind of primer for detection of toxoplasma gondii bradyzoite, comprised forward primer and reverse primer, this forward primer and reverse primer are the pair of primers of amplification toxoplasma gondii bradyzoite stage-specific antigen BAG1 gene conservative region.
Preferably, the nucleotide sequence of described forward primer is as shown in SEQ ID NO.3, and the nucleotide sequence of described reverse primer is as shown in SEQ ID NO.4.
In another aspect of this invention, also provide a kind of probe for detection of toxoplasma gondii bradyzoite, the toxoplasma gondii BAG1 gene conservative region of primer amplification hybridization described in this probe and claim 1 or 2.
Preferably, the nucleotide sequence of described probe is as shown in SEQ ID NO.5.
In another aspect of this invention, also provide a kind of test kit for detection of toxoplasma gondii bradyzoite, comprised above-mentioned primer.
Described test kit also comprises above-mentioned probe.
Described test kit also comprises standard substance, and these standard substance are the recombinant plasmid that contains toxoplasma gondii bradyzoite stage-specific antigen BAG1 gene.
In another aspect of this invention, also provide a kind of method that detects toxoplasma gondii bradyzoite, comprised the following steps:
Extract sample to be tested RNA, and taking it as template, carry out real-time fluorescence quantitative PCR detection with test kit claimed in claim 5.
In another aspect of this invention, also provide the application of a kind of above-mentioned primer in the product of preparation detection toxoplasma gondii bradyzoite.
In another aspect of this invention, also provide the application of a kind of above-mentioned probe in the product of preparation detection toxoplasma gondii bradyzoite.
The present invention is for detection of primer and probe and the test kit of toxoplasma gondii bradyzoite, detect toxoplasma gondii bradyzoite by real time fluorescence quantifying PCR method, the infection of toxoplasma gondii bradyzoite can be detected quickly and accurately, susceptibility is high, high specificity, be applicable to clinical detection, or study for formation and the activation mechanism of toxoplasma gondii bradyzoite.
Brief description of the drawings
Below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation.
Fig. 1 is the RT-PCR result figure of the toxoplasma gondii bradyzoite RNA of the embodiment of the present invention 1;
Fig. 2 is the Real-time PCR canonical plotting of the embodiment of the present invention 1;
Fig. 3 is Real-time PCR result figure in the embodiment of the present invention 2 susceptibility comparison tests.
Embodiment
In the following example, the experimental technique of unreceipted actual conditions, conventionally condition routinely, as " fine works molecular biology experiment guide " (F.M. Ao Sibai, R.E. James Kingston, the chief editors such as J.G. Sai Deman, Ma Xuejun, Su Yuelong translates. Beijing: Science Press, 2004) described in method carry out.
The invention provides primer and probe for detection of toxoplasma gondii bradyzoite, and utilize this primer and probe to set up toxoplasma gondii bradyzoite Real-time PCR detection method.The present invention is by amplification and the order-checking of gene, clone has obtained toxoplasma gondii bradyzoite stage-specific antigen BAG1 gene, its encoding gene reading frame is 690bp, and whole gene is subcloned into PMD-18T cloning vector, has built recombinant clone plasmid as Real-time PCR standard substance.Using BAG1 gene as the target gene detecting, special primer and probe are designed, the Real-time PCR that has set up a kind of new TaqMan probe method detects the technology of toxoplasma gondii bradyzoite, the RNA that the toxoplasma gondii bradyzoite that application cell in vitro is cultivated extracts, carry out the condition of detection method and grope, and compare analysis with conventional PCR method.The method that application is set up simultaneously detects the sample of laboratory animal.Result has shown successfully to construct the Real-time PCR detection technique of the toxoplasma gondii bradyzoite of susceptibility height and high specificity, the method can be used for clinical detection, also can be used for detecting the formation of toxoplasma gondii bradyzoite, for the formation of toxoplasma gondii bradyzoite and the research of activation mechanism provide basis.
The foundation of embodiment 1 toxoplasma gondii bradyzoite Real-time PCR detection method
1. method
1.1 Toxoplasma gondii Strains and the preparation of RNA sample: test is the PLK strain by this laboratory conservation recovery with toxoplasma gondii, going down to posterity to cultivate through human foreskin fibroblast (HFF) obtains.For the cultivation of toxoplasma tachyzoite, polypide is taken out from liquid nitrogen to the HFF cell that is inoculated into vitro culture after recovering, 37 DEG C, pH7.2-7.4DMEM substratum, contains 8% heat inactivation foetal calf serum, 5%CO
2cultivate.For the cultivation of toxoplasma gondii bradyzoite, changing the DMEM substratum of tachyzoite above into condition is that RPMI1640, pH are adjusted to 8.1, within every two days, changes a subculture, cultured continuously 6 days.Microscopic examination polypide pseudocyst.For the purifying of polypide, by rinsing, suspend with the PBS of precooling with worm cell, cross 27-G syringe needle three times, 5 μ m filter membrane (Millipore, USA) filter, then centrifugal 10 minutes of 1500rpm, collects polypide, PBS suspends, blood counting chamber counting, then uses Rneasy Tissue Kit (Qiagen, USA) to extract test kit and extracts total RNA, measure concentration and purity, set up and detect template for method.1.2 target gene clones and restructuring positive plasmid build: according to the toxoplasma gondii BAG1 gene order (X82213) having logged on GenBank, design primer, as upstream and downstream primer, amplify object fragment taking P1:5 '-ATGGCGCCGTCAGCATCG-3 ' (SEQ ID NO.1) and P2:5 '-CTACTTC ACGCTGATTTG-3 ' (SEQ ID NO.2).PCR product glue is reclaimed, and fragment is connected on PMD18-T carrier, and plasmid transforms and enters DH5 α, through the screening of resistance culture base, picking positive colony.Positive colony turns and is inoculated on liquid LB substratum, extracts plasmid.Plasmid sequence verification.
1.3 primers and probe design: utilize Oligo6.0 software design Real-time PCR primer and application Primer express software (Applied Biosystems, USA) designing probe.Primer: P3:5 '-CAATGGACAACGGCGTTCTA-3 ' (SEQ ID NO.3); P4:5 '-ACGCTGATTTGTTGCTTTGC-3 ' (SEQ ID NO.4), probe (Probe): 5 ' FAM-TGTCACGATCAAGGTC-MGB 3 ' (SEQ ID NO.5), amplified fragments size is 73bp.
1.4 reaction systems and condition: use Superscript II ThermoScript II (Takara, China) to carry out the reverse transcription of RNA, in the system that is 25 μ l at a final volume, react.Real-time PCR reaction system: Master Mix 12.5 μ l, 1 μ L uracil-DNA-glycosylase, the each 1 μ L of upstream and downstream primer (P3, P4), probe 0.8 μ L, template 1 μ L, adds appropriate amount of deionized water, makes system reach 25 μ L; Reaction conditions: 50 DEG C of 2min, 95 DEG C of 10min denaturations, 95 DEG C of 15s, 52 DEG C of 10s, 72 DEG C of 15s, 40 circulations.Primer and probe are all diluted to the concentration of 10pmol/ μ L with distilled water.Real-time PCR reaction is carried out on Applied Biosystems 7500 fluorescence real-time quantitative PCR instrument, and result carries software analysis by fluorescence real-time quantitative PCR.Conventional PCR reaction system: cumulative volume 25 μ L, comprise 2.5 μ L 10 × PCR damping fluids, 2.5 μ L2mM dNTP, 0.2 μ L 0.5U AmpliTaq Gold Taq archaeal dna polymerase (Applied Biosystems, USA), 1 μ L P1 primer, 1 μ L P2 primer, 1 μ L template DNA, and 16.8 μ L deionized waters.Reaction conditions is 94 DEG C of denaturation 3min, then 94 DEG C of 30s, and 55 DEG C of 30s, 72 DEG C of 1min, totally 35 circulations, after loop ends, 72 DEG C are extended 7min.1.5 standard curve making: by the plasmid standard building,, operate according to above-mentioned system and reaction conditions as template by 8 gradients of 10 times of doubling dilutions of deionized water.Repeatedly grope, the template gradient of selecting optimum plasmid concentration to set up as typical curve, and as subsequent experimental standard substance dilution gradient.
2. result
2.1 plasmid standards build
The toxoplasma gondii bradyzoite of vitro culture extracts RNA and does template, application BAG1 gene fragment Auele Specific Primer carries out RT-PCR, specific amplification goal gene, and contrast does not amplify fragment (Fig. 1) from the RNA of toxoplasma tachyzoite, goal gene is connected to T carrier, construct the recombinant plasmid containing 690bp gene fragment, sequencing result shows to successfully construct.In Fig. 1,1: bradyzoite, 2: tachyzoite.
The foundation of 2.2 typical curves
Different concns dilution using the plasmid that builds draws Real-time PCR typical curve (Fig. 2) as template.Relevant parameter analysis discovery, the relevance parameter R2=0.999 of typical curve and amplification efficiency E=1.02 are all better.
The application of embodiment 2 toxoplasma gondii bradyzoite Real-time PCR detection methods
1. materials and methods
1.1Real-time PCR specific test: Trichinella spiralis, Trypanosoma evansi, eimeria tenella, Cryptosporidium and Schistosoma japonicum provide by zooparasite key lab of the Ben Suo Ministry of Agriculture.Extracting all polypide RNA is template, by Real-time pcr amplification BAG1 target gene, observes its specificity with above-mentioned P3, P4 primer and probe.
1.2Real-time PCR susceptibility comparison test: the RNA that purifying toxoplasma gondii bradyzoite extracts, 8 gradients of doubling dilution, take respectively two kinds of methods of conventional PCR and Real-time PCR in the situation that template amount is consistent, to carry out comparison and detection with above-mentioned primer, observe it and detect lower limit.
The clinical detection of 1.3 zoogenetic infection toxoplasma gondii bradyzoites: application small white mouse is cooked model, culturing in vivo toxoplasma gondii bradyzoite, gets afterwards cerebral tissue and extracts RNA, adopts respectively conventional PCR and two kinds of methods of Real-time PCR to detect sample with above-mentioned primer.
2. result
The specificity analyses of 2.1Real-time PCR
The RNA of various parasite polypides is carried out to the specificity analyses of Real-time PCR, result shows only toxoplasma gondii bradyzoite RNA is had to very strong specificity, to other parasite without specific amplification.
The sensitivity analysis of 2.2Real-time PCR
Real-time PCR detected result as shown in Figure 3, initial concentration is 0.1ng, 10 times of dilutions 8 gradients (0.1ng, 10pg, 1pg, 100fg, 10fg, 1fg, 0.1fg and 0.01fg), under detection, be limited to 1fg (Fig. 3), and conventional PCR is limited to 100fg under detecting, Real-time PCR sensitivity is 100 times of conventional PCR as can be seen here.
The clinical detection of 2.3 zoogenetic infection toxoplasma gondii bradyzoites: contrast conventional PCR and Real-time PCR detection method, Real-time PCR method can detect the infection of toxoplasma gondii bradyzoite from lower concentration template.
The above embodiment has only expressed embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Sequence table
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Claims (7)
1. the primer for detection of toxoplasma gondii bradyzoite, comprise forward primer and reverse primer, it is characterized in that, this forward primer and reverse primer are the pair of primers of amplification toxoplasma gondii bradyzoite stage-specific antigen BAG1 gene conservative region, the nucleotide sequence of described forward primer is as shown in SEQ ID NO.3, and the nucleotide sequence of described reverse primer is as shown in SEQ ID NO.4.
2. for detection of a probe for toxoplasma gondii bradyzoite, it is characterized in that, the toxoplasma gondii BAG1 gene conservative region of primer amplification hybridization described in this probe and claim 1, the nucleotide sequence of described probe is as shown in SEQ ID NO.5.
3. for detection of a test kit for toxoplasma gondii bradyzoite, it is characterized in that, comprise primer claimed in claim 1.
4. test kit according to claim 3, is characterized in that, also comprises probe claimed in claim 2.
5. test kit according to claim 4, is characterized in that, also comprises standard substance, and these standard substance are the recombinant plasmid that contains toxoplasma gondii bradyzoite stage-specific antigen BAG1 gene.
6. the application of primer in the product of preparation detection toxoplasma gondii bradyzoite described in claim 1.
7. the application of probe in the product of preparation detection toxoplasma gondii bradyzoite described in claim 2.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101343638A (en) * | 2008-08-20 | 2009-01-14 | 中国人民解放军军事医学科学院军事兽医研究所 | Toxoplasma transgenosis carrier based on piggyback transposon and construction method |
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| CN101343638A (en) * | 2008-08-20 | 2009-01-14 | 中国人民解放军军事医学科学院军事兽医研究所 | Toxoplasma transgenosis carrier based on piggyback transposon and construction method |
Non-Patent Citations (4)
| Title |
|---|
| 弓形虫Real-timePCR检测方法的建立和初步应用;蔺智兵;《畜牧与兽医》;20111231;第43卷(第12期);988页 * |
| 张彦雷.表达期特异性双荧光蛋白的转基因弓形虫的构建.《中国人兽共患病学报》.2011,第27卷(第11期), |
| 蔺智兵.弓形虫Real-timePCR检测方法的建立和初步应用.《畜牧与兽医》.2011,第43卷(第12期), |
| 表达期特异性双荧光蛋白的转基因弓形虫的构建;张彦雷;《中国人兽共患病学报》;20111231;第27卷(第11期);21页 * |
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