CN103320501A - Primer, probe and kit for detecting bradyzoite of toxoplasma gondii - Google Patents

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

Primer and probe and test kit for detection of the toxoplasma gondii bradyzoite

Technical field

The present invention relates to technical field of bioengineering, relate in particular to a kind of primer for detection of the toxoplasma gondii bradyzoite and probe and test kit.

Background technology

Toxoplasma gondii, be also referred to as bow slurry worm, the traditional Chinese medical science belongs to top mixture subphylum three corpse worms, sporozoa, Eucoccida, isospora section, toxoplasma are cytozoons, 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 that can propagate by water source and food source, have very big potentially dangerous to humans and animals health, and they parasitize in the host cell, can infect all temperature vertebrates that comprise the people.The arch insect infection people in some cases, consequence is very serious even be in peril of one's life, for example the crowd of the fetus of pregnancy duration and immune deficiency.The zoogenetic infection toxoplasma gondii can cause dam miscarriage, mummy tire, stillborn foetus, and this disease is broken out on the pig farm, and mortality ratio is especially up to more than 60%.But, detecting the method for toxoplasma gondii at present, ubiquity susceptibility is low, the shortcoming of poor specificity.

The toxoplasma gondii bradyzoite is the trophont in the packing, and the about 5-10 μ of diameter m constantly breeds in packing, reaches several to 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 phase specific antigens of toxoplasma gondii bradyzoite, detects less than this gene in the tachyzoite phase.BAG1 is the major protein of mature tissue's packing inducing toxoplasma tachyzoite to show to the early stage quilt that bradyzoite changes.The indicator protein that BAG1 Chang Zuowei toxoplasma tachyzoite transforms to bradyzoite and being studied is a kind of endochylema antigen.

Real-time PCR, real-time quantitative PCR refers to measure the amount of specificity product immediately by the variation of continuous monitoring fluorescent signal power during the amplification of PCR index, and infers the original bulk of goal gene accordingly, does not need to take out the PCR product and separates.Real-time quantitative PCR has been widely used in the every field of molecular biology research as an extremely effective experimental technique at present.

Summary of the invention

The detection method ubiquity susceptibility that the present invention will solve present toxoplasma gondii is low, the technical problem of poor specificity, a kind of primer for detection of the toxoplasma gondii bradyzoite and probe are provided, utilizing this primer and probe to carry out real-time fluorescence quantitative PCR (Real-time PCR) detects, can detect the infection of toxoplasma gondii bradyzoite quickly and accurately, its susceptibility height, 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 the toxoplasma gondii bradyzoite, comprised forward primer and reverse primer, this forward primer and reverse primer are a pair of primers of amplification toxoplasma gondii bradyzoite phase specific antigens BAG1 gene conservative region.

Preferably, the nucleotide sequence of described forward primer is shown in SEQ ID NO.3, and the nucleotide sequence of described reverse primer is shown in SEQ ID NO.4.

In another aspect of this invention, also provide a kind of probe for detection of the toxoplasma gondii bradyzoite, the toxoplasma gondii BAG1 gene conservative region hybridization of this probe and claim 1 or 2 described primer amplifications.

Preferably, the nucleotide sequence of described probe is shown in SEQ ID NO.5.

In another aspect of this invention, also provide a kind of test kit for detection of the 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 phase specific antigens BAG1 gene.

In another aspect of this invention, also provide a kind of method that detects the toxoplasma gondii bradyzoite, may further comprise the steps:

Extract sample to be tested RNA, and be template with it, carry out the real-time fluorescence quantitative PCR detection with the described test kit of 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 the toxoplasma gondii bradyzoite by real time fluorescence quantifying PCR method, can detect the infection of toxoplasma gondii bradyzoite quickly and accurately, susceptibility height, high specificity, be applicable to clinical detection, or be used for formation and the activation mechanism research of toxoplasma gondii bradyzoite.

Description of drawings

The present invention is further detailed explanation below in conjunction with the drawings and specific embodiments.

Fig. 1 is the RT-PCR figure as a result of the toxoplasma gondii bradyzoite RNA of the embodiment of the invention 1;

Fig. 2 is the Real-time PCR canonical plotting of the embodiment of the invention 1;

Fig. 3 is Real-time PCR figure as a result in the embodiment of the invention 2 susceptibility comparison tests.

Embodiment

In the following example, the experimental technique of unreceipted actual conditions, usually condition routinely, as " fine works molecular biology experiment guide " (F.M. Ao Sibai, R.E. James Kingston, chief editors such as J.G. Sai Deman, Ma Xuejun, the Su Yuelong translates. Beijing: Science Press, 2004) described in method carry out.

The invention provides primer and probe for detection of the 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, the clone has obtained toxoplasma gondii bradyzoite phase specific antigens BAG1 gene, it is 690bp that its encoding gene is read frame, and whole gene subclone to the PMD-18T cloning vector, has been made up the recombinant clone plasmid as Real-time PCR standard substance.With the target gene of BAG1 gene as detection, special primer and probe have been 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 the 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 simultaneously set up detects the sample of laboratory animal.The result shows the Real-time PCR detection technique of the toxoplasma gondii bradyzoite that has successfully constructed susceptibility height and high specificity, this method can be used for clinical detection, also can be used for detecting the formation of toxoplasma gondii bradyzoite, for the research of the formation of toxoplasma gondii bradyzoite and activation mechanism provides the basis.

The foundation of embodiment 1 toxoplasma gondii bradyzoite Real-time PCR detection method

1. method

1.1 the strain of toxoplasma gondii worm and the preparation of RNA sample: test is protected kind of the PLK strain of recovery with toxoplasma gondii by this laboratory, going down to posterity to cultivate through human foreskin fibroblast (HFF) obtains.For the cultivation of toxoplasma tachyzoite, be inoculated into the HFF cell of vitro culture polypide taken out recovery from liquid nitrogen after, 37 ℃, the 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 top tachyzoite into condition is that RPMI1640, pH are adjusted to 8.1, changes a subculture, cultured continuously 6 days in per two days.Microscopic examination polypide pseudocyst.For the purifying of polypide, with PBS flushing, the suspension of band worm cell with precooling, cross 27-G syringe needle three times, 5 μ m filter membrane (Millipore, USA) filter, 1500rpm is centrifugal 10 minutes then, collects polypide, PBS suspends, the blood counting chamber counting, (Qiagen USA) extracts test kit and extracts total RNA to use Rneasy Tissue Kit then, measure concentration and purity, be used for method and set up the detection template.1.2 target gene clone and reorganization positive plasmid make up: according to the toxoplasma gondii BAG1 gene order of having landed on the GenBank (X82213), the design primer, be the upstream and downstream primer with P1:5 '-ATGGCGCCGTCAGCATCG-3 ' (SEQ ID NO.1) and P2:5 '-CTACTTC ACGCTGATTTG-3 ' (SEQ ID NO.2), amplify the purpose fragment.PCR product glue is reclaimed, and fragment is connected on the PMD18-T carrier, and plasmid transforms and enters DH5 α, through the screening of resistance substratum, picking positive colony.The positive colony commentaries on classics is inoculated on the liquid LB substratum, extracts plasmid.The plasmid sequence verification.

1.3 primer and probe design: utilize Oligo6.0 software design Real-time PCR primer and use 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), the amplified fragments size is 73bp.

1.4 reaction system and condition: (Takara China) carries out the reverse transcription of RNA, reacts in the system that a final volume is 25 μ l to use Superscript II ThermoScript II.Real-time PCR reaction system: Master Mix 12.5 μ l, 1 μ L uracil-DNA-glycosylase, 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 ℃ of 2min, 95 ℃ of pre-sex change of 10min, 95 ℃ of 15s, 52 ℃ of 10s, 72 ℃ of 15s, 40 circulations.Primer and probe all are diluted to the concentration of 10pmol/ μ L with distilled water.Real-time PCR is reflected on the Applied Biosystems 7500 fluorescence real-time quantitative PCR instrument and carries out, and the 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 fluid, 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 ℃ of pre-sex change 3min, 94 ℃ of 30s then, and 55 ℃ of 30s, 72 ℃ of 1min, totally 35 circulations, 72 ℃ are extended 7min after the loop ends.1.5 standard curve making: with the plasmid standard that builds,, operate according to above-mentioned system and reaction conditions as template with 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 standard makes up

The toxoplasma gondii bradyzoite of vitro culture extracts RNA and does template, use BAG1 gene fragment Auele Specific Primer and carry out RT-PCR, the specific amplification goal gene, and contrast does not amplify fragment (Fig. 1) from the RNA of toxoplasma tachyzoite, goal gene is connected to the T carrier, construct the recombinant plasmid that contains the 690bp gene fragment, sequencing result shows and successfully constructs.Among Fig. 1,1: bradyzoite, 2: tachyzoite.

2.2 the foundation of typical curve

Different concns dilution with the plasmid that makes up draws Real-time PCR typical curve (Fig. 2) as template.The relevant parameter analysis finds that 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 the test of PCR specificity: Trichinella spiralis, Trypanosoma evansi, eimeria tenella, Cryptosporidium and Schistosoma japonicum provide by zooparasite key lab of the Ministry of Agriculture of this institute.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 two kinds of methods of conventional PCR and Real-time PCR under the consistent situation of template amount, to compare detection respectively with above-mentioned primer, observe it and detect lower limit.

1.3 the clinical detection of zoogenetic infection toxoplasma gondii bradyzoite: use small white mouse and make model, culturing in vivo toxoplasma gondii bradyzoite is got cerebral tissue afterwards and is extracted RNA, adopts conventional PCR and two kinds of methods of Real-time PCR that sample is detected respectively with above-mentioned primer.

2. result

2.1Real-time the specificity analyses of PCR

The RNA of various parasite polypides is carried out the specificity analyses of Real-time PCR, and the result only shows that toxoplasma gondii bradyzoite RNA is had very strong specificity, and other parasite is not had specific amplification.

2.2Real-time the sensitivity analysis of 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), be limited to 1fg (Fig. 3) under detecting, and conventional PCR is limited to 100fg under detecting, and this shows that Real-time PCR sensitivity is 100 times of conventional PCR.

2.3 the clinical detection of zoogenetic infection toxoplasma gondii bradyzoite: contrast conventional PCR and Real-time PCR detection method, the Real-time PCR method can detect the infection of toxoplasma gondii bradyzoite from the 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 claim of the present invention.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

＜110〉China Agriculture Academe Shanghai Veterinary Institute

＜120〉for detection of primer and probe and the test kit of toxoplasma gondii bradyzoite

<160>5

<170>PatentIn version 3.3

<210>1

<211>18

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(18)

＜223〉primer

<400>1

atggcgccgt cagcatcg 18

<210>2

<211>18

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(18)

＜223〉primer

<400>2

ctacttcacg ctgatttg 18

<210>3

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(20)

＜223〉primer

<400>3

caatggacaa cggcgttcta 20

<210>4

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(20)

＜223〉primer

<400>4

acgctgattt gttgctttgc 20

<210>5

<211>16

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(16)

＜223〉probe

<400>5

tgtcacgatc aaggtc 16

Claims (10)

1. the primer for detection of the toxoplasma gondii bradyzoite comprises forward primer and reverse primer, it is characterized in that, this forward primer and reverse primer are a pair of primers of amplification toxoplasma gondii bradyzoite phase specific antigens BAG1 gene conservative region.

2. primer according to claim 1 is characterized in that, the nucleotide sequence of described forward primer is shown in SEQ ID NO.3, and the nucleotide sequence of described reverse primer is shown in SEQ ID NO.4.

3. the probe for detection of the toxoplasma gondii bradyzoite is characterized in that, the toxoplasma gondii BAG1 gene conservative region hybridization of this probe and claim 1 or 2 described primer amplifications.

4. probe according to claim 3 is characterized in that, the nucleotide sequence of described probe is shown in SEQ ID NO.5.

5. the test kit for detection of the toxoplasma gondii bradyzoite is characterized in that, comprises claim 1 or 2 described primers.

6. test kit according to claim 5 is characterized in that, also comprises claim 3 or 4 described probes.

7. test kit according to claim 6 is characterized in that, also comprises standard substance, and these standard substance are the recombinant plasmid that contains toxoplasma gondii bradyzoite phase specific antigens BAG1 gene.

8. a method that detects the toxoplasma gondii bradyzoite is characterized in that, may further comprise the steps:

Extract sample to be tested RNA, and be template with it, carry out the real-time fluorescence quantitative PCR detection with the described test kit of claim 5.

9. claim 1 or the 2 described primers application in the product of preparation detection toxoplasma gondii bradyzoite.

10. claim 3 or the 4 described probes application in the product of preparation detection toxoplasma gondii bradyzoite.

Images