CN101315381A - Early detection method for arch insect infection - Google Patents
Early detection method for arch insect infection Download PDFInfo
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- CN101315381A CN101315381A CNA2008100628369A CN200810062836A CN101315381A CN 101315381 A CN101315381 A CN 101315381A CN A2008100628369 A CNA2008100628369 A CN A2008100628369A CN 200810062836 A CN200810062836 A CN 200810062836A CN 101315381 A CN101315381 A CN 101315381A
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Abstract
The invention relates to a method for detecting toxoplasma infection early. The nucleoside triphosphate hydrolase-II (NTPase-II) of toxoplasma is an indispensable key enzyme in the process that purine is remedied and synthesized by the toxoplasma by utilizing host cells, play an important part in parasitism and propagation of the toxoplasma, and is a major antigen for causing a body to generate immune reaction in the acute infection stage of the toxoplasma. The gene of the toxoplasma nucleoside triphosphate hydrolase-II is expanded by utilizing a polymerase chain reaction through the invention, expression plasmids are constructed and recombined in a expression vector, the recombined expression plasmids are transferred to procaryotic cells for expression, recombined fusion protein NTPase-II is obtained, the recombined fusion protein NTPase-II is used as an indirect ELISA method to establish a coating antigen, and is used as the method for detecting the toxoplasma infection early.
Description
Technical field
The present invention relates to the detection method of the entozoic opportunistic pathogenesis protozoon of a kind of special sexual cell, specifically relate to the method for arch insect infection early detection.
Background technology
Arc worm (Toxoplasma gondii) is the entozoic opportunistic pathogenesis protozoon of a kind of special sexual cell, and it causes the toxoplasmosis of infecting both domestic animals and human in the karyocyte endoparasitism and the breeding of humans and animals.Toxoplasmosis is worldwide distribution, and the average infection rate of human arc worm reaches 33%.Health adult generally tangible clinical symptoms can not occur after infecting, but can continue a lot of years in vivo.When immunity of organisms reduction or severe infections, this subclinical infection then can show significant toxoplasmosis symptom, as arc small holes caused by worms disease, arc worm hepatitis, arc worm lymphnoditis etc.The body of immunologic hypofunction or serious defect (as AIDS, congenital agammaglobulinemia's disease, Huppert's disease and other malignant tumor patient), arch insect infection usually is the lethal pathology.Arc worm is ranked first of the five big pathogen (TORCH) that pregnant woman's intrauterine infection causes embryo's deformity, the pregnant woman who infects is except that self is ill, average about 40% can infect arc worm to fetus by placenta, causes miscarriage, stillborn foetus, deformity or baby's congenital toxoplasmosis.The fetus extent of damage and gestational age are negative correlation, infect to take place more early, the fetus extent of damage can be serious more, the present effective ways that the toxoplasmosis early diagnosis is not also arranged as yet.
Summary of the invention
The method of early diagnosis that the objective of the invention is to overcome the deficiencies in the prior art and a kind of arc worm is provided.
For achieving the above object, the invention discloses a kind of method of arch insect infection early detection, the method for described arch insect infection early detection is made up of following steps:
1) make the coating buffer of 5 μ g/ml with the NTPase-II behind the carbonic acid buffer dilution purifying, wrap by 96 hole ELISA Plate in 100 μ l/ holes, and 4 ℃ of bags are spent the night;
2) PBST washes plate 3-5 time, 2-3min/ time, pats dry;
3) add 5% skimmed milk power, 37 ℃ of sealing 2h;
4) PBST washes plate 5-7 time, 2-3min/ time, pats dry;
5) the adding dilutability is 1: 100 a tested serum, and 1.5h is hatched for 37 ℃ in 100 μ l/ holes;
6) PBST washes plate 5-7 time, 2-3min/ time, pats dry;
7) the adding dilutability is 1: 4000 sheep anti mouse ELIAS secondary antibody IgM, and 1h is hatched for 37 ℃ in 100 μ l/ holes, and PBST washes plate 5-7 time, 2-3min/ time, pats dry;
8) add substrate colour developing liquid A liquid and B liquid, 37 ℃ of lucifuge colour developing 10-15min add stop buffer, 50 μ l/ holes;
9) microplate reader reading, the OD value is surveyed in 450nm place air zeroing back, with the OD of negative serum
450nmMean value and 3 times of standard deviation sums are as the upper limit of negative serum, as the OD of serum
450nm〉=X+3S person promptly is judged as positive serum, otherwise then negative.
The method of arch insect infection early detection of the present invention can also be made up of following steps:
1) make the coating buffer of 5 μ g/ml with the NTPase-II behind the carbonic acid buffer dilution purifying, wrap by 96 hole ELISA Plate in 100 μ l/ holes, and 4 ℃ of bags are spent the night;
2) PBST washes plate 3 times, 2-3min/ time, pats dry;
3) add 5% skimmed milk power, 37 ℃ of sealing 2h;
4) PBST washes plate 5 times, 2-3min/ time, pats dry;
5) the adding dilutability is 1: 200 a tested serum, and 1.5h is hatched for 37 ℃ in 100 μ l/ holes;
6) PBST washes plate 5-7 time, 2-3min/ time, pats dry;
7) the adding dilutability is 1: 4 000 sheep anti mouse ELIAS secondary antibody IgG, and 1h is hatched for 37 ℃ in 100 μ l/ holes, and PBST washes plate 7 times, 2-3min/ time, pats dry;
8) add substrate colour developing liquid A liquid and B liquid, 37 ℃ of lucifuge colour developing 10-15min add stop buffer, 50 μ l/ holes;
9) microplate reader reading, the OD value is surveyed in 450nm place air zeroing back, with the OD of negative serum
450nmMean value and 3 times of standard deviation sums are as the upper limit of negative serum, as the OD of serum
450nm〉=X+3S person promptly is judged as positive serum, otherwise then negative.
Because NTPase-II (research of nucleoside triphosphate hydrolase-II nucleosidetriphosphate hydrolase) is that arc worm can be at the entozoic key enzyme of host, also be to cause that body produces immunoreactive major antigen the arc worm acute infection stage, therefore utilize arc worm fusion NTPase-II that the early diagnosis of toxoplasmosis is had higher accuracy.
The invention will be further described below in conjunction with specific embodiment.
Embodiment
Be NTPase-II (nucleoside triphosphate hydrolase, the application specific embodiment in the toxoplasmosis early diagnosis of promptly arc worm fusion research of nucleoside triphosphate hydrolase-II) below.
Embodiment 1
The preparation method of the fusion NTPase-II of arc worm may further comprise the steps in this detection method:
(1) preparation of toxoplasma cdna group DNA;
(2) amplification of arc worm NTPase-II gene;
(3) arc worm NTPase-II gene inserts cloned plasmids;
(4) arc worm NTPase-II gene inserts expression plasmid;
(5) recon with (4) gained changes host cell over to;
(6) abduction delivering fusion NTPase-II;
The product of (7) separation and purification (5) obtains the NTPase-II expressing protein.
Concrete preparation process is as follows:
(1) preparation of toxoplasma cdna group DNA:
The RH strain of Toxoplasma gondii tachyzoite is continuously at the mouse interior generation, ascites is extracted in sterile working after infecting 3d, polypide is washed 3 times with physiological saline, add lymphocyte separation medium with polypide suspension equivalent, the centrifugal 8min after-acceleration of 800rpm is to the centrifugal 10min of 2000rpm, collect the middle level polypide, by DNA extraction kit instructions extracting toxoplasma cdna group DNA, with concentration and the purity of spectrophotometry DNA.
(2) amplification of arc worm NTPase-II gene:
According to arc worm NTPase-II reference nucleotide sequence and expression vector cloning site incision enzyme map analysis result; adopt Primer Designer primer-design software to design primer voluntarily; synthetic by last sea base health biotech company, primer sequence is as follows: upstream primer: 5 '-GA
AGATCTACAGACTCATCGTCACT CCG-3 ' (underscore is a Bgl II restriction enzyme site), downstream primer: 5 '-GC
AAGCTTTCACAGATTGTGAGAATATCC-3 ' (underscore is the HindIII restriction enzyme site).Adopt 2 * PCR Master Mix kit amplification NTPase-II gene, PCR reaction cumulative volume is 50 μ l: wherein contain template DNA 5 μ l (200ng), each 1 μ l (final concentration is 0.4 μ mol/L) of upstream and downstream primer.Set up blank simultaneously.PCR response parameter: 95 ℃ of 5min * 1; 95 ℃ of 45s, 58 ℃ of 45s, 72 ℃ of 60s * 30; 72 ℃ of 10min * 1.1.5% agarose gel electrophoresis is identified and is cut glue and reclaims.
(3) T-A clone and order-checking
The PCR product that reclaims spends the night for 16 ℃ with pGEM-T Easy carrier and is connected.Linked system is 10 μ l: the PCR product 3 μ l of purifying, 2 * buffer, 5 μ l, pGEM-T Easy carrier 1 μ l, T4 dna ligase 1 μ l.Connect product transformed competence colibacillus E.coli DH5 α, the competence bacterium that draws after 200 μ l transform is coated on the LB flat board that contains ampicillin screening positive clone.Positive recombinant carries out Bgl II and HindIII double digestion and PCR respectively and identifies after cultivation and extracting plasmid, and serves the order-checking of sea base health biotech company.The arc worm NTPase-II gene order that sequencing result adopts DNAMAN software and Genbank to announce compares.
(4) structure of recombinant expression plasmid:
With recombinant clone plasmid and pBAD-HisB Bgl II and HindIII double digestion, the enzyme system of cutting is 40 μ l: recombinant clone plasmid (pGEM-T Easy-NTPase-II) or pBAD-HisB20 μ l, 10 * buffer, 4 μ l, Bgl II 2 μ l, HindIII 2 μ l, nuclease-free water12 μ l; Press glue and reclaim kit explanation recovery purpose fragment; 16 ℃ of connections are spent the night, and linked system is 10 μ l:pBAD-HisB, 1 μ l, recovery back purpose fragment 3 μ l, T4 dna ligase 1 μ l, 2 * buffer, 5 μ l.
(5) recombinant expression plasmid changes host cell over to.
The recombinant expression plasmid that builds is added in the competent escherichia coli cell ice bath 30min; Put into 42 ℃ of water-bath thermal shock 90s rapidly; Rapid again ice bath 5min; The LB that adds antibiotic-free, 37 ℃ of 200rpm shaken cultivation 45-60min behind the mixing; Get and a certain amount ofly be coated with containing on the solid LB nutrient culture media of ampicillin, dry under the room temperature, be inverted then and be put in overnight incubation in 37 ℃ of incubators.
(6) abduction delivering.
Picking white colony from the flat board, in 37 ℃, the 200rpm overnight shaking is cultivated in 3ml LB fluid nutrient medium (containing 50 μ g/ml Amp).The bacterium liquid of getting 50 μ l incubated overnight adds 5mlLB-Amp fluid nutrient medium, 37 ℃, 200rpm shaken cultivation 3h; Get 1ml bacterium liquid as inducing preceding contrast, 12000rpm collected thalline, 4 ℃ of preservations in centrifugal 30 seconds.It is 0.2% abduction delivering that residue bacterium liquid adds 20% L-arabinose to final concentration, 37 ℃, and 200rpm shaken cultivation 4h.12,000rpm collected thalline ,-20 ℃ of preservations in centrifugal 30 seconds.Add 30 μ l distilled water and isopyknic 2 * albumen sample-loading buffer in the thalline of collecting, 100 ℃ are boiled 3min behind the mixing, and 12, the centrifugal 10min of 000rpm gets supernatant and does the SDS-PAGE electrophoretic analysis in right amount.
(7) purifying of recombinant protein
Purifying: the GuNTA-0 Buffer (20mmol/LTris-HCl pH7.9,0.5mol/L NaCl, 10%Glycerol, 6mol/L GuanidiumHCl) and the PMSF that add 1/20 cell growth volume.Resuspended back ultrasonication.Get supernatant after the ultrasonication bacterium is centrifugal, place-20 ℃ of preservations.
Chromatography: with the 5ml NTA resin suitable chromatographic column of packing into, chromatography is with the GuNTA-0 Buffer balance of 10 times of NTA volumes;
Sample is added in the NTA chromatographic column, and flow speed control is collected penetrating component about 15ml/h, be used for the SDS-PAGE analysing protein in conjunction with situation; Chromatography is washed with the GuNTA-0 Buffer of 5 times of NTA volumes, and flow speed control is about 30ml/h;
Use the GuNTA-20 of 5 times of NTA volumes respectively, GuNTA-40, GuNTA-60, GuNTA-100, the GuNTA-500 wash-out, flow speed control is collected eluent about 15ml/h;
Determine the distribution situation of target protein in eluent.With the distribution of SDS-PAGE analysing protein, get cleansing solution and each 20 μ l of eluent of each section collection, with 5 * albumen sample-loading buffer mixing of 5 μ l, after boiling water boils 5min, 5000rpm, centrifugal 5min, 10%SDS-PAGE identifies.
The dialysis renaturation of purification of recombinant proteins albumen
The processing of bag filter: at 300ml～500ml bag filter treating fluid (2%NaHCO
3, 1.0mmol/L EDTA, pH 8.0) in bag filter is boiled behind the 10min with the thorough rinsing of distilled water, place the EDTA (pH8.0) of 1.0mmol/L to boil 10min then, for several times with the about cleaning down bag filter of sterilization pure water;
The eluent of purifying gained is diluted with dislysate A, and the molecular cut off of packing into is in the bag filter of 14kDa, with magnetic stirrer, dialyses in the 1L dislysate B that the guanidine hydrochloride final concentration progressively reduces successively for 4 ℃, and every 10h carries out dislysate and changes liquid once;
Take out bag filter and place dislysate C, 4 ℃, 15h is with magnetic stirrer;
Take out bag filter and place dislysate D, 4 ℃, 10h is with magnetic stirrer.
The preparation of dislysate
Dislysate A:20mmol/L Tris-HCl, pH8.0;
Dislysate B: in dislysate A, add the guanidine hydrochloride of different amounts, be mixed with the dislysate B that the guanidine hydrochloride final concentration is respectively 6mol/L, 4mol/L, 2mol/L and 1mol/L;
Dislysate C:20mmol/L Tris-HCl, 0.6mmol/L L-arginine, 10% sucrose, 2mmol/L EDTA, 0.5mol/L guanidine hydrochloride, pH8.0;
Dislysate D:20mmol/L Tris-HCl, 25mmol/L NaCl, 0.2mol/L guanidine hydrochloride, pH8.0.
Among the above-mentioned preparation method, various test parameterss are operation selection routinely all, is decided by concrete experiment condition, and used cloning host cell can be bacillus coli DH 5 alpha, JM109 etc., and expression host cell can be e. coli bl21, DE3 etc.Abduction delivering fusion NTPase-II can adopt several different methods, for example uses IPTG, L-arabinose.
Separation and purification fusion NTPase-II also can adopt several different methods among the present invention, for example uses affinity chromatography, ion-exchange chromatography or the like.
The arch insect infection early detection
(1) arch insect infection BALB/c mouse.
The RH strain of Toxoplasma gondii tachyzoite is continuously at the mouse interior generation, and ascites is extracted in sterile working behind the infection 3d, and polypide adds physiological saline suspension polypide with physiological saline washing 3 times, counts under mirror with cell counting count board, and making concentration is the tachyzoite suspension of 2000/ml.Use disposable syringe to draw suspension, 0.5ml/ toxoplasma tachyzoite infects BALB/c mouse.10 BALB/c mouse are numbered respectively, and the tail vein is got blood before infecting, and infects the back and gets blood since second day tail vein, until mouse invasion death, infect back mouse survival 8 days, after death mouse is cut off belly, with the physiological saline washing, mirror is observed the tachyzoite infection conditions down.The serum of all samples is separated out, place-20 ℃ of preservations.
(2) indirect elisa method detects the titre of antibody in the mice serum.
With the NTPase-II behind the carbonic acid buffer dilution purifying (nucleosidetriphosphate hydrolase, promptly research of nucleoside triphosphate hydrolase-II) is made the coating buffer of 5 μ g/ml, wrap by 96 hole ELISA Plate in 100 μ l/ holes, 4 ℃ of bags are spent the night; PBST washes plate 3-5 time, gets final product, 2-3min/ time, pats dry for common 3 times; Add 5% skimmed milk power, 37 ℃ of sealing 2h; PBST washes plate 5-7 time, gets final product, 2-3min/ time, pats dry for common 5 times; The adding dilutability is 1: 100 an above-mentioned tested mice serum serum, and 1.5h is hatched for 37 ℃ in 100 μ l/ holes; PBST washes plate 5-7 time, is good with 7 times, 2-3min/ time, pats dry; The adding dilutability is 1: 4000 sheep anti mouse ELIAS secondary antibody IgM, and 1h is hatched for 37 ℃ in 100 μ l/ holes, and PBST washes plate 5-7 time, is good with 7 times, 2-3min/ time, pats dry; Add substrate colour developing liquid A liquid and B liquid, 37 ℃ of lucifuges colour developing 10-15min add the dense H of stop buffer 2mol/L
2SO
4Cessation reaction, 50 μ l/ holes; Use the absorbance of microplate reader detection reaction liquid, survey the OD value in wavelength 450nm place air zeroing back.OD with negative serum
450nmMean value and 3 times of standard deviation sums are as the upper limit of negative serum, as the OD of serum
450nm〉=X+3S person promptly is judged as positive serum, otherwise then negative.
IgM antibody ELISA result of the present invention is as shown in the table, and the IgM antibody titer in the visible mice serum positive reaction can occur on the 3rd day in infecting the back.
IgM antibody ELISA result such as following table
Embodiment 2
Make the coating buffer of 5 μ g/ml with the NTPase-II behind the carbonic acid buffer dilution purifying, wrap by 96 hole ELISA Plate in 100 μ l/ holes, and 4 ℃ of bags are spent the night; PBST washes plate 3-5 time, gets final product, 2-3min/ time, pats dry for common 3 times; Add 5% skimmed milk power, 37 ℃ of sealing 2h; PBST washes plate 3-5 time, gets final product, 2-3min/ time, pats dry for common 5 times; The adding dilutability is 1: 200 an above-mentioned tested mice serum, and 1.5h is hatched for 37 ℃ in 100 μ l/ holes; PBST washes plate 5-7 time, is good with 7 times, 2-3min/ time, pats dry; The adding dilutability is 1: 4 000 sheep anti mouse ELIAS secondary antibody IgG, and 1h is hatched for 37 ℃ in 100 μ l/ holes, and PBST washes plate 5-7 time, is good with 7 times, 2-3min/ time, pats dry; Add substrate colour developing liquid A liquid and B liquid, 37 ℃ of lucifuges colour developing 10-15min add the dense H of stop buffer 2mol/L
2SO
4Cessation reaction, 50 μ l/ holes; Use the absorbance of microplate reader detection reaction liquid, survey the OD value in wavelength 450nm place air zeroing back.OD with negative serum
450nmMean value and 3 times of standard deviation sums are as the upper limit of negative serum, as the OD of serum
450nm〉=X+3S person promptly is judged as positive serum, otherwise then negative.
IgG antibody ELISA result of the present invention is as shown in the table, and the IgG antibody titer in the visible mice serum began to occur positive reaction on the 7th day in infecting the back.
The IgG antibody ELISA is table as a result
Tested serum and the dilutability of sheep anti mouse ELIAS secondary antibody must be adjusted accordingly along with the sheep anti mouse ELIAS secondary antibody of different manufacturers is different in the above specific embodiment.
The detection specificity check:
NTPase-II behind the use purifying is as envelope antigen, and indirect ELISA detects the IgG antibody in the collected plasmodium infection in this laboratory, cerebral cysticercosis, snail fever and the paragonimiasis serum respectively, and calculates positive rate respectively.Reaction conditions and operation steps as previously mentioned, testing result is as shown in the table, visible detection specificity of the present invention is higher.
Indirect ELISA detection specificity testing result table
Claims (2)
1, a kind of method of arch insect infection early detection is characterized in that being made up of following steps:
1) make the coating buffer of 5 μ g/ml with the NTPase-II behind the carbonic acid buffer dilution purifying, wrap by 96 hole ELISA Plate in 100 μ l/ holes, and 4 ℃ of bags are spent the night;
2) PBST washes plate 3-5 time, 2-3min/ time, pats dry;
3) add 5% skimmed milk power, 37 ℃ of sealing 2h;
4) PBST washes plate 5-7 time, 2-3min/ time, pats dry;
5) the adding dilutability is 1: 100 a tested serum, and 1.5h is hatched for 37 ℃ in 100 μ l/ holes;
6) PBST washes plate 5-7 time, 2-3min/ time, pats dry;
7) the adding dilutability is 1: 4000 sheep anti mouse ELIAS secondary antibody IgM, and 1h is hatched for 37 ℃ in 100 μ l/ holes, and PBST washes plate 5-7 time, 2-3min/ time, pats dry;
8) add substrate colour developing liquid A liquid and B liquid, 37 ℃ of lucifuge colour developing 10-15min add stop buffer, 50 μ l/ holes;
9) microplate reader reading, the OD value is surveyed in 450nm place air zeroing back, with the OD of negative serum
450nmMean value and 3 times of standard deviation sums are as the upper limit of negative serum, as the OD of serum
450nm〉=X+3S person promptly is judged as positive serum, otherwise then negative.
2, a kind of method of arch insect infection early detection is characterized in that being made up of following steps:
1) make the coating buffer of 5 μ g/ml with the NTPase-II behind the carbonic acid buffer dilution purifying, wrap by 96 hole ELISA Plate in 100 μ l/ holes, and 4 ℃ of bags are spent the night;
2) PBST washes plate 3-5 time, 2-3min/ time, pats dry;
3) add 5% skimmed milk power, 37 ℃ of sealing 2h;
4) PBST washes plate 5-7 time, 2-3min/ time, pats dry;
5) the adding dilutability is 1: 200 a tested serum, and 1.5h is hatched for 37 ℃ in 100 μ l/ holes;
6) PBST washes plate 5-7 time, 2-3min/ time, pats dry;
7) the adding dilutability is 1: 4000 sheep anti mouse ELIAS secondary antibody IgG, and 1h is hatched for 37 ℃ in 100 μ l/ holes, and PBST washes plate 7 times, 2-3min/ time, pats dry;
8) add substrate colour developing liquid A liquid and B liquid, 37 ℃ of lucifuge colour developing 10-15min add stop buffer, 50 μ l/ holes;
9) microplate reader reading, the OD value is surveyed in 450nm place air zeroing back, with the OD of negative serum
450nmMean value and 3 times of standard deviation sums are as the upper limit of negative serum, as the OD of serum
450nm〉=X+3S person promptly is judged as positive serum, otherwise then negative.
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| CNA2008100628369A CN101315381A (en) | 2008-06-30 | 2008-06-30 | Early detection method for arch insect infection |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101865920A (en) * | 2010-06-21 | 2010-10-20 | 常州二十一世纪生物技术研究所有限公司 | Method for detecting toxoplasma antibody |
| CN104897834A (en) * | 2015-05-25 | 2015-09-09 | 中国农业科学院上海兽医研究所 | Method for detecting toxoplasma acute infection and target protein thereof |
| CN108265070A (en) * | 2016-12-30 | 2018-07-10 | 中国农业科学院上海兽医研究所 | The method of one species specific detection Infection of Toxoplasma Gondii |
| CN108614115A (en) * | 2016-12-13 | 2018-10-02 | 河南后羿实业集团有限公司 | One breeder egg drop syndrome virus (PRRSV) double-antibody sandwich ELISA detection method |
| CN109030836A (en) * | 2018-10-25 | 2018-12-18 | 苏州大学附属儿童医院 | Food proteins Specific IgA antibody detects ELISA kit and its application |
-
2008
- 2008-06-30 CN CNA2008100628369A patent/CN101315381A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101865920A (en) * | 2010-06-21 | 2010-10-20 | 常州二十一世纪生物技术研究所有限公司 | Method for detecting toxoplasma antibody |
| CN104897834A (en) * | 2015-05-25 | 2015-09-09 | 中国农业科学院上海兽医研究所 | Method for detecting toxoplasma acute infection and target protein thereof |
| CN104897834B (en) * | 2015-05-25 | 2017-02-08 | 中国农业科学院上海兽医研究所 | Method for detecting toxoplasma acute infection and target protein thereof |
| CN108614115A (en) * | 2016-12-13 | 2018-10-02 | 河南后羿实业集团有限公司 | One breeder egg drop syndrome virus (PRRSV) double-antibody sandwich ELISA detection method |
| CN108265070A (en) * | 2016-12-30 | 2018-07-10 | 中国农业科学院上海兽医研究所 | The method of one species specific detection Infection of Toxoplasma Gondii |
| CN108265070B (en) * | 2016-12-30 | 2022-07-26 | 中国农业科学院上海兽医研究所 | Specific toxoplasma gondii detection method |
| CN109030836A (en) * | 2018-10-25 | 2018-12-18 | 苏州大学附属儿童医院 | Food proteins Specific IgA antibody detects ELISA kit and its application |
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Application publication date: 20081203 |