CN101182571A - Animals toxoplasmosis PCR detection reagent kit - Google Patents
Animals toxoplasmosis PCR detection reagent kit Download PDFInfo
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- CN101182571A CN101182571A CNA2007100321591A CN200710032159A CN101182571A CN 101182571 A CN101182571 A CN 101182571A CN A2007100321591 A CNA2007100321591 A CN A2007100321591A CN 200710032159 A CN200710032159 A CN 200710032159A CN 101182571 A CN101182571 A CN 101182571A
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Abstract
The invention discloses a PCR detection reagent box of animal toxoplasmosis; the box contains DNA lysate, red blood cell lysate, white blood cell lysate, PCR reaction liquid and toxoplasmosis gene DNA positive control liquid; the 529bp repeated DNA segment of toxoplasmin is taken as genetic mark and special primer is taken to optimize the PCR reaction conditions such as the magnesium ion consistency, the anneal temperature and the regulation of the cycle number. The invention can detect the toxoplasmosis infection of pig, dog and cat in a fast special sensible way; the operation is convenient and the sensibility is high; the invention can be used for diagnosing the toxoplasmosis of pig, dog and cat and detecting and testing the drug effect appraisal of the selection of the toxoplasmosis medicines.
Description
Technical field
The present invention relates to the diagnosis and the detection technique field of animals toxoplasmosis, relate in particular to animals toxoplasmosis PCR detection reagent kit and using method thereof.
Background technology
Toxoplasmosis is to suffer from parasitosis altogether by arch insect infection people and the caused people beast of various animal, is distributed widely in China and reaches all over the world, and the average infection rate of western countries' human body is up to about 30% (Tenter et al, 2000; Xie Mingquan and Li Guoqing, 2003), the average infection rate of China's human body reaches 7.88%, and the whole nation has 100,000,000 people to infect (Lv Yuancong and Cui Junzhao, 1994 approximately; Chen Zhaoyi etc., 2005; Feng Yueju etc., 2005; Xu Longqi etc., 2005; Xu Xiangzhen etc., 2005; Open and wait 2006 quietly), zoogenetic infection rate higher (shed China etc., 2004).
Toxoplasmosis is not only one of human modal infectious diseases, and also is immunosuppression and immune deficiency crowd's main one of the cause of disease (the Luft et al.1984 that causes death; Zangerleet al, 1991; Luft﹠amp; Remington, 1992; Crigg etc., 2001; The pure and mild woods of Liu De is gloomy clearly, and 2001), it and prenatal and postnatal care also have substantial connection, may directly influence the growth of fetus after the pregnant woman infects, and can cause fetus deformity even death when serious.Therefore, toxoplasmosis is serious harm China people's life and health not only, and has a strong impact on the quality of the Chinese nation.
The animal species of China's energy natural infection toxoplasmosis has nearly 200 kinds (Yin Guorong etc., 1996 such as pig, ox, buffalo, horse, goat, sheep, deer, cat, dog, chicken, rabbit; Ye Yumei etc., 2002; Yu Yonglan etc., 2006, Zhang Han etc., 2007), wherein higher with the infection rate of pig, endanger the most seriously, be main contagium (Zhang Decai etc., 1994 of human body toxoplasmosis.Zhu Jibin, 2006).In China people's dietary structure, pig is topmost meat product source, and whether its health influences huge numbers of families.At present the region outbreak of epidemic at China's pig toxoplasmosis also takes place often, and is especially also more serious on the intensification pig farm, swinery sickness rate height, and mortality ratio is also high, and also ubiquity (Fu Bin etc., 1995 of the caused sow miscarriage of toxoplasmosis and stillborn foetus; Liu Jiusheng and Chen Sihui, 1996; By pavilion etc., 1998; Kuang Yongjuan, 2002; Xie Mingquan and Li Guoqing, 2003; Shed China etc., 2004), and the pig toxoplasmosis is also frequent and other transmissible disease polyinfection (Lin Xiaozhi, 2001; Cheng Haiqing etc., 2002; Ma Jun opens and Wu Xiufeng, and 2002).Along with the development of China's pig industry, boar and pork pig circulation unimpeded more quickened the propagation of pig toxoplasmosis and popular, causes serious financial loss to pig industry, and the serious threat HUMAN HEALTH.According to estimates, annual nearly 225,000 new cases of the U.S., 50% case infects (Mead et al, 1999) by food.Also become main source (Dubey et al, 1990) at U.S.'s pork for people's toxoplasma gondii infection disease.
Accurate diagnosis to the humans and animals toxoplasmosis is the prerequisite of effective prevention and control toxoplasmosis, and Chinese scholars has been done many-sided exploration for this reason, comprises etiology, immunology and diagnosis of molecular biology method (Xie Mingquan and Li Guoqing, 2003; Shed China etc., 2004; Liao Shenquan etc., 2006).Finding the existence of toxoplasma gondii from pathological material of disease, is reliable detection method, and pathological material of disease can be made contact or tissue slice carries out chromoscopy, or pathological material of disease is inoculated in susceptible animal separates polypide.Dyeing microscopic examination is usually with Giemsa dyeing or Wright ' s dyeing, but symptom is slight or be about to the sick pig of anti-mistake, and the sick pig of using sulfa drugs to treat, and often is difficult for detecting toxoplasma gondii.The pathological material of disease limitation of gathering in addition is also easily by omission (shed China etc., 2004; Liao Shenquan etc., 2006).
Release cycle antigen and induce and produce resisting toxoplasmosis antibody in vivo behind the arch insect infection humans and animals can adopt immunological method to detect intravital arch insect circulating antigen of humans and animals and resisting toxoplasmosis antibody (Li Hui etc., 2006, Li Jian etc., 2006).The method that can be used for the toxoplasmosis immunodiagnosis has a lot (multitudinous etc. in grace, 1992), comprises various agglutination tests (Dubey etal, 1995; Dubey, 1997; Shed China etc., 2004; Liao Shenquan etc., 2006) (indirect hemagglutination test for example, IHA), indirect fluorescent antibody test (IFAT) (Arko-Mensah et al, 2000), enzyme linked immunosorbent assay (ELISA) and improve one's methods (Chen Ziqing etc., 1995; Wu Xusu and meter are great, and 1995; Zhao Hengmei etc., 1997; Ceng Mingan etc., 2000; Zheng Lanyan and Wang Haipeng, 2000; Damriyasa et al, 2004), colloidal gold immunity chromatography (Zhang Yanggen, 1996 such as (ColloidalGold Immunochromatography); Guo Lanying and Wan volume virtue, 2000).But still there are a lot of problems in the immunological detection method of toxoplasmosis, as cross reaction, recall rate are not high, false positive is more, waste time and energy, again influences that are subjected to immune status mistaken diagnosis occurs and fail to pinpoint a disease in diagnosis easily, can not be as (Liao Shenquan etc., 2006) such as diagnosis basis of existing disease.
In recent years, based on the molecular biology method of polymerase chain reaction (PCR) in the widespread use of parasitology field (Zhu Xingquan and Gasser, 1999; He Fang etc., 2004; Zhuet al., 2007).Advantages such as PCR is easy, quick, special with it, sensitivity have been used (Dan Lianyu etc., 2003 in the diagnosis of people and animals toxoplasmosis; Remington et al, 2004; Switaj et al, 2005; Liao Shenquan etc., 2006; Zhang Han etc., 2007), toxoplasmosis specific PCR diagnostic method is exactly wherein the most frequently used a kind of (Burg et al, 1989; Savva et al., 1990; Ho-yen et al, 1992; Hurtado et al, 2001; Cultrera et al, 2002; Buchbinder et al, 2003; Filisetti et a1,2003; Contini et al, 2003,2005; Liao Shenquan etc., 2006; Zhang Han etc., 2007), the key of setting up toxoplasmosis specific PCR diagnostic method is to seek responsive, special genetic marker.Yet, at present B1 gene (Pelloux et al, 1998 of adopting more; Contini etal, 2002) and rDNA internal transcribed spacer district (ITS) sequence (Jauregui et a1,2001; Shed China etc., 2005) set up toxoplasmosis specific PCR diagnostic method as genetic marker.
In sum, conventional detection exists that cross reaction, recall rate are not high, false positive is more, wastes time and energy, occur mistaken diagnosis easily and fail to pinpoint a disease in diagnosis, problems such as susceptibility is limited, but also be not used in the detection of dog, cat toxoplasmosis, and dog, cat also are the animals that contacts closely with the mankind, through investigation, go to a doctor in 40 dogs of pet clinic because of other disease clinically, can amplify toxoplasma gondii 529bp dna sequence dna in 9 the blood, illustrate that the arch insect infection of dog is also very general.From commercially available 10 cats that are used for eating, can amplify toxoplasma gondii 529bp dna sequence dna in 1 the blood, illustrate that the arch insect infection of cat is also very general, should cause showing great attention to of relevant department, pet owners and household.Press for inquire into a kind of easy to use, highly sensitive and can detect the detection method of dog, cat animal toxoplasma gondii.
Homan etc. (2000) identify the 529bp repetition DNA fragment of one 200-300 copy from the toxoplasma cdna group, and think the desirable genetic marker of toxoplasmosis diagnosis.The 529bp sequence has higher susceptibility and accuracy (Reischi et al, 2003 than B1 gene; Edvinsson et al, 2006).But the toxoplasma gondii detection method and the test kit of this scientific payoffs design invention do not appear utilizing at present.
Summary of the invention
Purpose of the present invention aims to provide a kind of animals toxoplasmosis PCR detection reagent kit, is particularly useful for the test kit that pig, dog or cat toxoplasmosis detect.
Another object of the present invention provides the using method of described animals toxoplasmosis PCR detection reagent kit.
Purpose of the present invention is achieved through the following technical solutions:
A kind of animals toxoplasmosis PCR detection reagent kit is provided, and it contains:
(1) dna cleavage liquid;
(2) erythrocyte cracked liquid;
(3) write cell lysis buffer;
(4) PCR reaction solution;
(5) toxoplasma cdna DNA positive control solution.
25mM EDTA, the W/V of 10mM Tris-Cl, the pH8.0 of NaCl, pH8.0 that described dna cleavage liquid is 100mM is 1% the SDS and the mixing solutions of 4 μ g/ μ L Proteinase Ks; Described erythrocyte cracked liquid is the 10mM Tris-Cl of NaCl, pH8.0 of 10mM and the MgCl of 5mM
2Mixing solutions; Described write cell lysis buffer is that the 1mM EDTA of 10mMTris-C1, pH8.0 of NaCl, pH8.0 of 100mM and W/V are the mixing solutions of 0.5% SDS; Described PCR reaction solution is that final concentration respectively is dATP, dTTP, dGTP, the dCTP of 200 μ M; Final concentration respectively is primer TOX4, the TOX5 of 0.5pmol/ μ L and the MgCl of 2mM
2Mixing solutions.
The using method of described test kit may further comprise the steps:
(1) DNA of extraction blood sample or tissue sample;
(2) pcr amplification;
(3) get PCR product application of sample after the amplification on sepharose, electrophoresis is placed on observations and photographic analysis under the ultraviolet transilluminator, draws detected result.
The DNA of the described extraction blood sample of step (1) is for adopting whole blood sample and 0.5M/L EDTA with 1: 9 mixed anti-freezing, centrifugal removal blood plasma, add the erythrocyte cracked liquid splitting erythrocyte, the centrifugation white corpuscle, add write cell lysis buffer cracking white corpuscle, water-bath behind the adding dna cleavage liquid, phenol-chloroform method extracts DNA.
The DNA of the described extraction tissue sample of step (1) adds dna cleavage liquid for shredding tissue sample, places incubator digestion to spend the night, and phenol-chloroform method extracts DNA.
Test kit Biospin Blood Genomic DNA Extraction Kit that the also available BioFlux of blood sample and tissue sample company produces and Biospin TissueGenomic DNA Extraction Kit by specification extract.
The described pcr amplification of step (2) is to get centrifuge tube and mark according to the number that the amplification sample number adds after 2, adds sterilization deionization distilled water, 10 * buffer, MgCl in centrifuge tube successively
2, dNTPs, upstream and downstream primer TOX4 and TOX5, TaqDNA polysaccharase, vortex vibration mixing; Instantaneous centrifugal after, be sub-packed in the centrifuge tube of 0.2 μ L, add at last sample template DNA or positive and negative the contrast; Vortex vibration mixing is instantaneous centrifugal again, places the pcr amplification instrument to increase.
Described TaqDNA polysaccharase is pressed 1.25U by each PCR reaction and is added.
The condition of described pcr amplification is Mg
++Concentration gradient is that 1.5mM～2.5mM, annealing temperature are that 50 ℃～65 ℃ and cycle number are 30～45, and the condition of preferred amplification is that annealing temperature is 57 ℃; Mg
++Concentration be 2.0mM; Cycle number is 38.
The pcr amplification system sees Table 1:
Table 1 pcr amplification system
| Reagent | Volume (μ L) |
| ddH 2O 10×PCR buffer MgCl 2DNTPs (2.5mM) TOX4 (50pmol/ μ L) TOX5 (50pmol/ μ L) Taq enzyme (5U/ μ L) template DNA | 16.75 2.5 2 2 0.25 0.25 0.25 1 |
The pcr amplification condition:
94 ℃ of pre-sex change 5min
Extend 10min after 72 ℃
The optimum condition of described pcr amplification is Mg
++Concentration gradient is that 1.5mM～2.5mM, annealing temperature are that 50 ℃～65 ℃ and cycle number are 30～45.
The top condition of described pcr amplification is that annealing temperature is 57 ℃; MgCl
2Concentration be 2.0mM; Cycle number is 38.
The present invention uses that Homan etc. publishes identifies the special primer TOX4 and the TOX5 of the 529bp repetitive dna sequence that is used to increase from the toxoplasma cdna group, its nucleotide sequence is composed as follows:
Upstream primer TOX4 sequence is:
5’-CGCTGCAGGGAGGAAGACGAAAGTTG-3’，
Downstream primer TOX5 sequence is:
5’-CGCTGCAGACACAGTGCATCTGGATT-3’，
The about 530bp of expection amplified fragments size.
The invention has the beneficial effects as follows: the present invention identifies on the basis of the distinctive 529bp dna fragmentation of toxoplasma gondii in (2000) such as Homan, set up with the specific PCR detection method of 529bp dna fragmentation, and in pig, dog, cat toxoplasmosis detect, used as genetic marker; This test kit also can be used for the evaluating drug effect of experiment pig toxoplasmosis drug screening.
Whether now the specific PCR method that the present invention sets up can be used for detecting dog, cat disease toxoplasma gondii infection.Experiment shows, the specific PCR diagnostic method that the present invention is set up with toxoplasma gondii 529bp tumor-necrosis factor glycoproteins has sensitivity, characteristics such as special, quick, both can be used for one of pig, the detection of mouse experiment toxoplasmosis, detection means of drug screening, also can be used for pig, dog, the existing disease diagnosis of infection of cat toxoplasmosis.In sum, the present invention's sequencing simple to operate, the method high specificity, the susceptibility height, the result judges objective, is specially adapted to the detection and the investigation of the existing disease toxoplasma gondii infection of pig, dog or cat; Also can be used for the detection of toxoplasma gondii in the pork; One of means of evaluating drug effect when also can be used as the screening of laboratory animal toxoplasmosis medicine.
Embodiment
The following examples are intended to the present invention is described in more detail.
The composition of embodiment 1 test kit
Test kit includes dna cleavage liquid 30ml, wherein contains the SDS of EDTA, 1% (W/V) of 25mM of Tris-Cl, pH8.0 of 10mM of NaCl, pH8.0 of 100mM and the Proteinase K of 4 μ g/ μ L; Erythrocyte cracked liquid 100ml wherein contains Tris-Cl, the MgCl of 5mM of 10mM of NaCl, the pH8.0 of 10mM
2Write cell lysis buffer 100ml wherein contains the SDS of EDTA, 0.5% (W/V) of imM of Tris-Cl, pH8.0 of 10mM of NaCl, the pH8.0 of 100mM; 100 reactions of PCR reaction solution (25 μ L/ reaction) are primer TOX4 and the TOX5 of dATP, the dTTP of each 200 μ M of final concentration, dGTP, dCTP, each 0.5pmol/ μ L of final concentration, the MgCl of 2mM
2, the Taq enzyme is pressed 1.25U by each PCR reaction and is added 1 of toxoplasma cdna group DNA positive control.
The test of embodiment 2 test kit specificitys
With each 1 μ L of 6 control sample DNA such as the dog neospora that passes through the DNA validation verification, trichomonas, cryptosporidium parvum, plasmodium falciparum, Eimeria tenella, ascaris suum is template, reaction conditions according to test kit carries out the specific PCR amplification, establishes blank and test kit positive control simultaneously.
The pcr amplification condition is:
94 ℃ of pre-sex change 5min
Extend 10min after 72 ℃
The PCR product in 0.8% TBE sepharose behind the electrophoresis, observations under the ultraviolet transilluminator, gel imaging system shooting.
The result has only toxoplasma gondii test kit positive control dna sample amplification to go out the band of about 530bp, and above-mentioned other 6 kinds contrast parasites and negative control all do not have band and occur.
The sensitivity test of embodiment 3 test kits
At first will count the extractive toxoplasma tachyzoite DNA in back and dilute, vortex vibration mixing, the working specification of pressing Eppendorf Biophotometer nucleic acid-protein determinator detects its total dna content.Adopt coubling dilution by 10 *, 100 *, 1000 *, 2000 *, 4000 * dilution tachyzoite DNA.The pcr amplification condition is the same, establishes blank simultaneously.The PCR product detects through 0.8% agarose gel electrophoresis, to determine its susceptibility.The PCR reaction of the bow-shaped worm dna by 5 concentration gradients shows that the low energy of this PCR detection method detects the DNA of 0.5 toxoplasma tachyzoite.
The preservation period test of embodiment 4 test kits
To detect known sample with-20 ℃ of test kits of preserving 1 month, 3 months, 6 months, 9 months at 4 ℃, amplification condition is with aforementioned.The result shows that test kit can be in 4 ℃ (except Taq enzymes ,-20 ℃ of preservations of palpus) and-20 ℃ of preservation prolonged preservation, and in 9 months of test period, positive all can amplify the purpose bright band under suitable preservation condition, and negative control does not have the band appearance.
The application of embodiment 5 test kits in pig toxoplasmosis drug test
1. the foundation of pig toxoplasmosis animal model
Liquid nitrogen is protected the toxoplasma tachyzoite continuous recovery three generations in the mouse body who plants, and disconnected neck method is put to death mouse, an amount of normal saline flushing abdominal cavity, and the taking-up washings, count with the white blood cell count(WBC) plate suitable dilution back.The dosage of pig toxoplasma gondii infection is 1 * 10
7Individual tachyzoite/head, ascites is diluted to 2 * 10 with physiological saline
6Individual tachyzoite/mL, every pig infects 5mL.
2. the specific PCR of pig experiment toxoplasmosis drug screening effect detects and estimates
Finish the experiment that pig is tested toxoplasmosis drug screening by the Master degree candidate Liao Shenquan of veterinary parasitology research department of Agricultural University Of South China, the blood sample and the tissue sample of experiment pig is provided.After extracting genomic dna, increase with the 529bp specific PCR of this institute's foundation and to detect bow-shaped worm dna in each experimental group porcine tissue and the blood sample.
Test pig is divided into 4 groups at random, and wherein each 3 of groups, 2 of blank groups are not treated in 2 medication therapy groups, infection.The experimental observation phase is 20 days.Infective dose is a PYS strain tachyzoite 1 * 10
7Individual/head.Test grouping and medication see Table 2.
The medicine and the medication of table 2 pig toxoplasmosis
| Group | Medicine | Medication | Dosage | Administration time |
| Organize two positive controls blank groups | Sulfamonomethoxine+Pyrimethamine hcl+TMP Fu side Sulfamethoxazole+acetylspiramycin physiological saline physiological saline | Irritate stomach and irritate stomach filling stomach filling stomach | 50mg/Kg.d+ 0.2mL/ 0.2mL/ of 2.5mg/Kg.d+ 10mg/Kg.d 50mg/Kg.d+ 20mg/Kg.d only | Infect back 2h and begin administration, connect and give 5 days, every day 2 times is the same the same |
Every pig blood sample divides two parts of preservations (anti-freezing and non-anticoagulation) before and after the test, slaughters 1 pig at random in infecting 6,11,21 days each groups in back, and every test pig is carried out the precaval vein blood sampling before slaughtering, and blood divides two parts of preservations.It is frozen to get the ascites, liver, lungs, hilar lymph node, heart, spleen, the mesenteric lymph nodes that cut open pig extremely then.Each 0.01g of edge that gets frozen internal organs extracts DNA.
3. the extraction of pig whole blood DNA
The antithrombotics of blood and 0.5M/L EDTA was with anti-freezing in 1: 9.Phenol-chloroform method is adopted in the extraction of blood sample DNA, and concrete steps are as follows:
(1) get the 1mL anticoagulation and place the 2mL centrifuge tube, 4 ℃, 3000rpm, centrifugal 20min removes blood plasma.
(2) add erythrocyte cracked liquid about 3 times of volumes in the sedimentary hemocyte, 4 ℃, 3000rpm, centrifugal 10min abandons supernatant.Repeat once, undesirable if red corpuscle is removed, can repeat again once, cold erythrocyte cracked liquid is very important.
(3) will add the 1mL write cell lysis buffer in the sedimentary white corpuscle, mix the back and add 10 μ L Proteinase Ks (25mg/mL), mix being placed on more than 50 ℃ of water-bath 4h.
(4) should clarify brightly through the postdigestive liquid that spends the night, add the saturated phenol of equal-volume Tris: chloroform (3: 1), abundant mixing, 4 ℃, 3000rpm, centrifugal 10min.
(5) the limpid colourless liquid in careful sucking-off upper strata places new centrifuge tube, and it is rapid to repeat previous step.
(6) liquid after the sucking-off extracting for the second time places new centrifuge tube, adds the above dehydrated alcohol of 2 times of volumes, and thorough mixing places-20 ℃, and 30～60min allows DNA precipitate.
(7) the centrifugal 10min of 12000rpm.
(8) hand-held centrifuge tube is 45, the DNA precipitation towards outside, be close to tube wall sucking-off ethanol gently with micropipet from another side, do not shake the DNA precipitation.Before the DNA precipitation reclaims result's evaluation, preserve the ethanol of sucking-off with another centrifuge tube.
(9) add 70% ethanol, 2/3 volume to the pipe, rinsing is to remove remaining salt, and the centrifugal 2min of 12000rpm abandons supernatant.
(10) do not cover the pipe lid, place 10～15min in the room temperature, make the ethanol volatilization.
(11) suspend DNA again in 50 μ L sterile purified waters or TE, blow and beat the DNA precipitation repeatedly, it is fully dissolved with the micropipette rifle.
4. the extraction of porcine tissue DNA
Respectively get the hilar lymph node and the about 0.01g of submandibular lymph nodes tissue of infected pigs, shred and add dna cleavage liquid and digest, 37 ℃ of effect 12～24h shake centrifuge tube therebetween frequently, make pathological material of disease organize abundant cracking.
Phenol-chloroform method is adopted in the extraction of tissue sample DNA, and concrete grammar and step are as follows:
(1) the centrifugal 2min of sample 10000rpm that digestion is spent the night gets supernatant liquor in 2mL sterilization centrifuge tube, adds 500 μ L sterilization distilled water, adds the saturated phenol solution of isopyknic Tris again, slowly gently with its mixing.
(2) the centrifugal 1min of 12000rpm draws in the new centrifuge tube of upper strata water to.
(3) add equal-volume phenol/chloroform (1: 1) solution, the centrifugal 1min of 12000rpm draws in the new centrifuge tube of upper strata water to.
(4) add the equal-volume chloroformic solution, the centrifugal 1min of 12000rpm sucts in the new centrifuge tube of layer water to one.
(5) add the NaAc solution of 1/10 volume 3mol/L, to final concentration be 0.3mol/L.
(6) behind the mixing, add accurately 2 times of volume dehydrated alcohols of amount,, place-20 ℃ of 30～60min with the solution mixing, allow DNA precipitate.
(7) the centrifugal 10min of 12000rpm.
(8) hand-held centrifuge tube is 45, the DNA precipitation towards outside, be close to tube wall sucking-off ethanol gently with micropipet from another side, do not shake the DNA precipitation.Before the DNA precipitation reclaims result's evaluation, preserve the ethanol of sucking-off with another centrifuge tube.
(9) add 70% ethanol, 2/3 volume to the pipe, rinsing is to remove remaining salt, and the centrifugal 2min of 12000rpm abandons supernatant.
(10) do not cover the pipe lid, place 10～15min in the room temperature, make the ethanol volatilization.
(11) suspend DNA again in 50 μ L sterile purified waters or TE, blow and beat the DNA precipitation repeatedly, it is fully dissolved with the micropipette rifle.
5.PCR amplification
Use this test kit and carry out pcr amplification, set up positive control and negative control simultaneously.
6. agarose electrophoretic analysis
The PCR product in 0.8% TBE sepharose behind the electrophoresis, observations under the ultraviolet transilluminator, gel imaging system shooting.
7. result and analysis
The artificial challenge contrasts the pig sample and all can expand except that heart and toxoplasma gondii specificity 529bp dna fragmentation, sulfamonomethoxine+Pyrimethamine hcl+TMP treatment group and Fu side's Sulfamethoxazole+acetylspiramycin treatment group then only expands from blood, hilus pulumonis and toxoplasma gondii specificity 529bpDNA fragment, proves that these two kinds of methods of treatment have certain result of treatment.The result shows that test kit of the present invention can be used for testing the evaluating drug effect of arc medicine screening.
The application of embodiment 6 test kits in pig toxoplasmosis clinical case detects
1. pathological material of disease and DNA sample
The pathological material of disease of two parts of doubtful pig toxoplasmosiss is respectively from two pig farms in Fanyu, Guangzhou and Zengcheng, Guangzhou.
2. the extraction of pathological material of disease tissue DNA
With pathological material of disease tissues such as the lung of the pig that dies of illness of doubtful pig toxoplasmosis, hilar lymph nodes, respectively get about 0.01g, shred and digest, the extraction of pathological material of disease DNA is as described above.
3.PCR amplification
Use test kit above-mentioned extractive genomic dna is carried out pcr amplification, do negative control and positive control simultaneously.
4. agarose electrophoretic analysis
The PCR product in 0.8% TBE sepharose behind the electrophoresis, observations under the ultraviolet transilluminator, gel imaging system shooting.
5. the determined dna sequence of amplified production
Get a positive PCR product of representativeness and send Guangzhou Top Genomics Ltd. and Shanghai Bo Shang Bioisystech Co., Ltd respectively, carry out two-way order-checking with toxoplasma gondii TOX4 and TOX5 special primer.Sequencing result is analyzed with DNAstar software, carries out sequence alignment with BLAST.
6. result and analysis
All amplify the band of about 530bp from the DNA sample of the hilar lymph node of two cases, and the band of amplification is more clear.Proved that the toxoplasma gondii specific PCR method that we set up can be used for the detection of doubtful pig toxoplasmosis pathological material of disease, and can be used for the detection and the epidemiology survey of pig toxoplasmosis.
Embodiment 7 test kits are in the application in dog, cat toxoplasmosis detect
1. dog, cat whole blood sample
The dog blood system does not pick up from Shenzhen auspicious roc pet clinic and this school veterinary college pets hospital; Cat blood picks up from market, road, Milky Way Changxing, Guangzhou.
2. the extraction of dog, cat whole blood DNA
With dog, the cat whole blood of the different varieties randomly drawed, the antithrombotics of blood and 0.5M/L EDTA was with anti-freezing in 1: 9.Phenol-chloroform method is adopted in the extraction of blood sample DNA, and concrete steps as described above.
3.PCR amplification
Use test kit above-mentioned extractive genomic dna is carried out pcr amplification, do the positive and negative control simultaneously.
4. agarose electrophoretic analysis
The PCR product in 0.8% TBE sepharose behind the electrophoresis, observations under the ultraviolet transilluminator, gel imaging system shooting.
5. the determined dna sequence of amplified production
Get a positive PCR product of representativeness and send Guangzhou Top Genomics Ltd. and Shanghai Bo Shang Bioisystech Co., Ltd respectively, carry out two-way order-checking with toxoplasma gondii TOX4 and TOX5 special primer.Sequencing result is analyzed with DNAstar software, carries out sequence alignment with BLAST.
6. result and analysis
Verified effective 40 parts of dog DNA samples increase with this test kit, and the result amplifies toxoplasma gondii 529bp specific fragment from 9 samples, and the band of amplification is more clear, and positive rate reaches 22.5%.To 10 parts of cat blood DNA samples, increase with this test kit, the result amplifies toxoplasma gondii 529bp specific fragment from 1 sample, and the band of amplification is more clear, positive rate 10%.Get the fragment that expands in a positive dog and the positive cat and check order through two tame order-checking companies, the two companies row that check order are in full accord.BLAST analysis revealed, dna fragmentation really are toxoplasma gondii specificity 529bp tumor-necrosis factor glycoproteins.
Claims (10)
1. animals toxoplasmosis PCR detection reagent kit is characterized in that it contains:
(1) dna cleavage liquid;
(2) erythrocyte cracked liquid;
(3) write cell lysis buffer;
(4) PCR reaction solution;
(5) toxoplasma cdna DNA positive control solution.
2. test kit according to claim 1,25mM EDTA, the W/V that it is characterized in that 10mM Tris-Cl, pH8.0 that described dna cleavage liquid is the NaCl of 100mM, pH8.0 are 1% the SDS and the mixing solutions of 4 μ g/ μ L Proteinase Ks; Described erythrocyte cracked liquid is the 10mM Tris-C1 of NaCl, pH8.0 of 10mM and the MgCl of 5mM
2Mixing solutions; Described write cell lysis buffer is that the 1mM EDTA of 10mM Tris-C1, pH8.0 of NaCl, pH8.0 of 100mM and W/V are the mixing solutions of 0.5% SDS; Described PCR reaction solution is that final concentration respectively is dATP, dTTP, dGTP, the dCTP of 200 μ M; Final concentration respectively is primer TOX4, the TOX5 of 0.5pmol/ μ L and the MgCl of 2mM
2Mixing solutions.
3. test kit according to claim 2 is characterized in that described primer sequence is:
Upstream primer TOX4:5 '-CGCTGCAGGGAGGAAGACGAAAGTTG-3 ',
Downstream primer TOX5:5 '-CGCTGCAGACACAGTGCATCTGGATT-3 '.
4. the using method of the described test kit of claim 1 is characterized in that may further comprise the steps:
(1) DNA of extraction blood sample or tissue sample;
(2) pcr amplification;
(3) get PCR product application of sample after the amplification on sepharose, electrophoresis is placed on observations and photographic analysis under the ultraviolet transilluminator, draws detected result.
5. according to the using method of the described test kit of claim 4, the DNA that it is characterized in that the described extraction blood sample of step (1) is for adopting whole blood sample and 0.5M/L EDTA with 1: 9 mixed anti-freezing, centrifugal removal blood plasma, add the erythrocyte cracked liquid splitting erythrocyte, the centrifugation white corpuscle, add write cell lysis buffer cracking white corpuscle, water-bath behind the adding dna cleavage liquid, phenol-chloroform method extracts DNA.
6. according to the using method of the described test kit of claim 4, the DNA that it is characterized in that the described extraction tissue sample of step (1) adds dna cleavage liquid for shredding tissue sample, places incubator digestion to spend the night, and phenol-chloroform method extracts DNA.
7. according to the using method of the described test kit of claim 4, it is characterized in that the described pcr amplification of step (2) is to get centrifuge tube and mark according to the number that the amplification sample number adds after 2, adds sterilization deionization distilled water, 10 * buffer, MgCl successively in centrifuge tube
2, dNTPs, upstream and downstream primer TOX4 and TOX5, TaqDNA polysaccharase, vortex vibration mixing; Instantaneous centrifugal after, be sub-packed in the centrifuge tube of 0.2 μ L, add at last sample template DNA or positive and negative the contrast; Vortex vibration mixing is instantaneous centrifugal again, places the pcr amplification instrument to increase.
8. according to the using method of the described test kit of claim 7, it is characterized in that described TaqDNA polysaccharase adds by 1.25U by each PCR reaction.
9. according to the using method of the described test kit of claim 4, the condition that it is characterized in that described pcr amplification is Mg
++Concentration gradient is that 1.5mM～2.5mM, annealing temperature are that 50 ℃～65 ℃ and cycle number are 30～45.
10. according to the using method of the described test kit of claim 9, the condition that it is characterized in that described pcr amplification is that annealing temperature is 57 ℃; Mg
++Concentration be 2.0mM; Cycle number is 38.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100321591A CN101182571A (en) | 2007-12-06 | 2007-12-06 | Animals toxoplasmosis PCR detection reagent kit |
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2007100321591A CN101182571A (en) | 2007-12-06 | 2007-12-06 | Animals toxoplasmosis PCR detection reagent kit |
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| CNA2007100321591A Pending CN101182571A (en) | 2007-12-06 | 2007-12-06 | Animals toxoplasmosis PCR detection reagent kit |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102304578A (en) * | 2011-08-29 | 2012-01-04 | 浙江大学 | Kit for detecting cysticercus cellulosae, swine trichinella and swine toxoplasmosis and application |
| CN106834504A (en) * | 2017-03-09 | 2017-06-13 | 华南农业大学 | The specific detection primer and detection kit of a kind of toxplasmosis in pigs |
| CN108614117A (en) * | 2018-03-29 | 2018-10-02 | 杭州泰熙生物技术有限公司 | A kind of preparation of toxoplasma recombinant antigen, Toxophasma gondii detecting kit and preparation method thereof |
| CN110534202A (en) * | 2019-08-21 | 2019-12-03 | 江南大学附属医院（无锡市第四人民医院） | A kind of system that the expression for Sox10 in triple negative breast cancer is analyzed |
-
2007
- 2007-12-06 CN CNA2007100321591A patent/CN101182571A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102304578A (en) * | 2011-08-29 | 2012-01-04 | 浙江大学 | Kit for detecting cysticercus cellulosae, swine trichinella and swine toxoplasmosis and application |
| CN102304578B (en) * | 2011-08-29 | 2012-12-26 | 浙江大学 | Kit for detecting cysticercus cellulosae, swine trichinella and swine toxoplasmosis and application |
| CN106834504A (en) * | 2017-03-09 | 2017-06-13 | 华南农业大学 | The specific detection primer and detection kit of a kind of toxplasmosis in pigs |
| CN108614117A (en) * | 2018-03-29 | 2018-10-02 | 杭州泰熙生物技术有限公司 | A kind of preparation of toxoplasma recombinant antigen, Toxophasma gondii detecting kit and preparation method thereof |
| CN110534202A (en) * | 2019-08-21 | 2019-12-03 | 江南大学附属医院（无锡市第四人民医院） | A kind of system that the expression for Sox10 in triple negative breast cancer is analyzed |
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