CN113504366A - ELISA detection method capable of distinguishing acute toxoplasma infection from chronic toxoplasma infection and application thereof - Google Patents
ELISA detection method capable of distinguishing acute toxoplasma infection from chronic toxoplasma infection and application thereof Download PDFInfo
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Abstract
The invention provides a detection method capable of simultaneously detecting acute and chronic toxoplasma infection and application thereof, and also provides an antigen for detecting toxoplasma infection and establishes an ELISA detection technology by utilizing the antigen. The invention further establishes an ELISA detection technology for detecting the toxoplasma infection, applies toxoplasma infection serum prepared in vivo to carry out condition exploration of a detection method, and carries out comparative analysis with a single protein coating method. And simultaneously, the established method is applied to detect the serum sample of the clinical animal. The result shows that the toxoplasma ELISA detection technology with high sensitivity and strong specificity is successfully constructed, and the method can be used for clinical detection.
Description
Technical Field
The invention relates to the technical field of biological engineering, in particular to an ELISA detection method capable of distinguishing acute toxoplasma infection from chronic toxoplasma infection and application thereof.
Background
Toxoplasma belongs to the phylum Toxoplasma, class Sporozoa, order Pediobolus, family Isosporaceae, genus Toxoplasma, is an obligate intracellular protozoan parasite capable of infecting a wide range of hosts, including humans and most warm-blooded animals. Approximately one third of the world's population is infected with toxoplasma, the parasites are controlled by host immunity, and infection in most people is asymptomatic; however, infection can lead to compromised immunity, can cause complications and even death, and toxoplasma infection in pregnant women can lead to fetal abortion, fetal death, and fatal encephalitis in aids patients. The toxoplasma transmitted in livestock and poultry can cause the abortion, mummy fetus and dead fetus of female animals, the outbreak of the disease in pig farms, the death rate is more than 60 percent, and great economic loss is caused to the animal husbandry. The existing toxoplasma detection method has the defects of low sensitivity and poor specificity.
Toxoplasma tachyzoites, also known as trophozoites, are parasitic within the nucleated cells of the intermediate host. Is in an asymmetric banana shape or crescent shape, one end is sharper, and the other end is blunt and round; the length is 4-7 μm, the width is 2-4 μm, the cytoplasm of the whole host is divided and proliferated in two parts in nucleated cells, the number of cytoplasm varies from dozens to dozens, the cytoplasm is surrounded by host cell membranes and is shaped like an encystment, and the encystment is called pseudo-encystment.
The slow reproduction of toxoplasma in the wall of the worm body to form capsule is the trophozoite with diameter of 5-10 microns, and the slow reproduction of toxoplasma in the capsule can reach several to several hundred. The capsule is spherical, has a diameter of 8-150 μm, but mostly 20-40 μm, and has a flexible wall, a cyst membrane and a granular layer, and has a thickness of about 0.5 μm.
Toxoplasma gondii is mainly in two different development stages of tachyzoite and bradyzoite, the tachyzoite is the main cause of toxoplasmosis, and its specificity parasitizes in nucleated cells, and the tachyzoite can make rapid replication and proliferation, and after penetrating through the cells, it is released into blood, and quickly spread to every tissue organ of whole body along with blood distribution. Tachyzoites can infect almost all nucleated cells, proliferate and spill rapidly in Toxoplasma modified Naphtheilus blebs, and continuously infect neighboring cells, causing the spread of Toxoplasma and causing acute Toxoplasma infection. The toxoplasma proliferated rapidly can activate the immune system of the host, and promote the body to eliminate most of the toxoplasma. Due to the existence of host immune pressure, a part of tachyzoites can escape from the pursuit of immune cells and then are converted into bradyzoites, so that tissue encapsulation is formed, and long-term chronic infection is formed in a host body. In contrast to tachyzoites, bradyzoites grow slowly and form mainly tissue encapsulation and colonize in vivo. The two stages of polypide have their own characteristics, mainly expressed in terms of morphology and molecular level. From the morphology, the bradyzoite is similar to the tachyzoite, is slightly larger, has a round and blunt end and a tapered end, is more close to a crescent or banana shape, and has a cell nucleus positioned at the rear end of the polypide. On the molecular level, some proteins expressed by the worms in the two phases are different and belong to phase-specific proteins.
In the prior art, the method for distinguishing the two stages of the insect bodies is mainly a molecular biological method, such as a Real-time PCR method, which can distinguish tachyzoites and bradyzoites, but only a blood sample can be collected for a living body, but the toxoplasma parasitism in the blood is only temporary and is difficult to detect, and most of infections are mixed infections of the tachyzoites and the bradyzoites, so that only serological methods such as ELISA and the like can be adopted to detect the toxoplasma infection of the living body. Toxoplasmosis is generally recessive infection, if the infection does not occur, the toxoplasmosis is basically chronic infection in a bradyzoite stage, and the toxoplasmosis only develops acute infection in a tachyzoite stage when the immunity of organisms is low, so that most of infected people are basically mixed infection, and no report shows that the stage of the worm can be distinguished by a serological method (ELISA).
SAG2, the membrane surface antigen 2 (or P22) of Toxoplasma gondii tachyzoite, is one of the specific antigens of Toxoplasma gondii tachyzoite main phase, and the surface protein which is not expressed in the sporozoite phase and the bradyzoite phase has stronger immune induction effect, the reaction intensity of the recombinant antigen to acute infection serum is obviously higher than that of chronic infection serum in serum detection, and the recombinant antigen can be applied to the diagnosis of acute Toxoplasma gondii.
SAG4, membrane surface antigen 4 (or P18) of toxoplasma bradyzoite, is a toxoplasma bradyzoite phase specific antigen. The host can be induced to produce specific antibody in early stage of toxoplasma infection, and can be recognized by toxoplasma infected mouse serum. Because the toxoplasma gondii is mostly recessive infection, the slow breeding period specific antigen has more significance in the detection, prevention and treatment of the toxoplasma gondii.
The combination of tachyzoite and bradyzoite antigens for toxoplasma diagnosis may more truly and effectively reflect toxoplasma infection in both humans and animals. In addition, the sensitivity of the detection technology is improved, so that the method has important significance for various aspects such as disease prevention, control, treatment, epidemiological investigation and the like.
Disclosure of Invention
The invention aims to solve the technical problems of low sensitivity and poor specificity of the existing serological detection method of Toxoplasma gondii, provides two antigens for detecting acute and chronic Toxoplasma gondii infection, and can accurately and rapidly detect the acute and chronic Toxoplasma gondii infection by using the antigens for ELISA detection, and the detection method has high sensitivity and strong specificity.
In order to solve the technical problems, the invention is realized by the following technical scheme:
in one aspect of the invention, an antigen mixture for detecting toxoplasma gondii is provided, which comprises two antigens, namely toxoplasma gondii tachyzoite stage specific antigen SAG2 and bradyzoite stage specific antigen SAG4 which are purified after expression.
Preferably, the primer nucleotide sequence of the antigen SAG2 is shown as SEQ ID NO.1 and SEQ ID NO.2, and the primer nucleotide sequence of the antigen SAG4 is shown as SEQ ID NO.3 and SEQ ID NO. 4.
In another aspect of the invention, an ELISA detection technique for detecting toxoplasma is also provided, comprising the above antigen.
The detection technology also comprises a positive control, wherein the control is positive serum for the toxoplasma infection.
In another aspect of the present invention, there is also provided a method of detecting Toxoplasma gondii, comprising the steps of:
and collecting serum to be detected, taking the serum as a sample, and performing ELISA detection.
The invention is used for detecting toxoplasma antigen and detection technology, detects toxoplasma infection by ELISA method, can accurately and rapidly detect acute and chronic toxoplasma infection, has high sensitivity and strong specificity, and is suitable for clinical detection.
After the SAG2 and the SAG4 proteins are mixed, samples infected by toxoplasma in an acute stage or a chronic stage can be detected, so that the detection sensitivity is improved, and the defects of omission and the like are avoided. The slow breeding period specificity SAG4 protein is used as an antigen for ELISA detection, and the fast breeding period specificity SAG2 protein is mixed, so that the defect that only the fast breeding period specificity protein is used and only acute infection can be detected is overcome, the detection sensitivity is improved, and the omission of detection is avoided.
Drawings
FIG. 1 is an amplification chart of the Toxoplasma gondii SAG2 gene of example 1 of the present invention; wherein, M: DNA molecular mass standard; 1: PCR amplification product of SAG2 gene; 2: blank control
FIG. 2 is an amplification chart of the Toxoplasma gondii SAG4 gene of example 1 of the present invention; wherein, M: DNA molecular mass standard; 1: RT-PCR amplification product of SAG4 gene; 2: blank control
FIG. 3 is an electrophoretogram of purified Toxoplasma gondii SAG2 and SAG4 proteins of example 1 of the present invention; wherein, 1: SAG 2; 2: SAG 4; m: molecular mass standard of protein
Fig. 4 is a graph of the test results of example 2 of the present invention, with the abscissa: 15 parts of pig serum; ordinate: and (4) OD value.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
In the following examples, the experimental procedures without specifying the specific conditions were generally carried out by the methods described in "molecular biology laboratory Manual of Fine text" (F.M. Osber, R.E. Kingston, J.G. Sedman, et al, Mashimi, Shujiong, et al, Beijing: scientific Press, 2004).
The invention provides an antigen for detecting toxoplasma infection, and establishes an ELISA detection technology for toxoplasma by using the antigen. According to the invention, the gene amplification and sequencing are carried out to clone a toxoplasma tachyzoite stage specific antigen SAG2 and a bradyzoite stage specific antigen SAG4 gene, and the two genes are respectively cloned to pET-28a and pET-30a expression vectors to construct a recombinant protein expression vector. And establishing an ELISA detection technology for detecting Toxoplasma gondii infection by using the mixed protein of SAG2 and SAG4 which is purified after expression as an envelope antigen, applying Toxoplasma gondii infection serum prepared in vivo, performing condition exploration of a detection method, and performing comparative analysis with a single protein envelope method. And simultaneously, the established method is applied to detect the serum sample of the clinical animal. The result shows that the toxoplasma ELISA detection technology with high sensitivity and strong specificity is successfully constructed, and the method can be used for clinical detection.
Example 1 establishment of Toxoplasma gondii ELISA detection method
Culturing toxoplasma gondii strains and extracting nucleic acid: the toxoplasma gondii RH strain used in the test is an international standard I type insect strain, is recovered by seed conservation in the laboratory and is obtained by subculturing Human Foreskin Fibroblast (HFF). For the culture of Toxoplasma gondii tachyzoite, the polypide is taken out from liquid nitrogen, recovered and inoculated to HFF cells cultured in vitro, at 37 ℃, pH 7.2-7.4 DMEM culture medium containing 8% heat inactivated fetal calf serum and 5% CO2Culturing in an incubator, and observing the polypide with a microscope. For purification of the worm bodies, the worm-carrying cells were washed with pre-cooled PBS, suspended, filtered three times through a 27-G needle, filtered through a 5 μm filter (Millipore, USA), centrifuged at 1500rpm for 10 minutes, the worm bodies were collected, suspended in PBS, and counted on a hemocytometer. Extracting the genome DNA of the RH strain by using a resin type genome DNA extraction kit, and storing at-20 ℃ for later use. RNA was extracted by TRIzol method and stored at-80 ℃.
Gene cloning and recombinant plasmid construction: primers were designed based on the sequence of the Toxoplasma gondii SAG2 protein-encoding gene (XM-018781602.1) that has been logged in on GenBank as P1: 5' -TTCATATGTCCACCACCGAGACGCCAGCG-3 '(SEQ ID NO. 1) and P2: 5' -TTGCGGCCGCCACAAACGTGATCAACAAACC-3' (SEQ ID NO. 2) as an upstream primer and a downstream primer, and the SAG2 gene was amplified using Toxoplasma gondii genomic DNA as a template. Primers were designed based on the sequence of the toxoplasma SAG4 protein-encoding gene (AF 340224) that had been logged in on GenBank, and the sequence was P3: 5' -TAGGATCCTTGGCGAGTGCGGGCAAAGAGC-3 '(SEQ ID NO. 3) and P4: 5' -TGCTCGAGCATTGATATCAACACAAAGGC’(SEQ ID NO. 4) as upstream and downstream primers, and SAG4 gene was amplified using cDNA prepared by reverse transcription of the extracted RNA using a reverse transcription kit as a PCR template. Amplification system (25 μ L): upstream, downstream primers and template were 1. mu.L each, 2 XTAQA Mix 12.5. mu.L, ddH2O9.5. mu.L. And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 1min for 30 cycles; extension at 72 ℃ for 10 min. The PCR product was detected by 1% agarose gel electrophoresis, and the desired fragment was recovered using an agarose gel recovery kit, and then ligated to pET28a or pET30a vector, and the ligation product was transformed into Escherichia coli (E.) (E.coli)E. coli) And (4) screening a resistance culture medium from DH5 alpha competent cells, and selecting positive clones. And (4) inoculating the positive clone to a liquid LB culture medium, extracting a plasmid, and verifying the plasmid sequencing. DNA extracted from Toxoplasma gondii RH strain (type I standard insect strain) is used as a template, and the SAG2 gene is amplified by a PCR method. The extracted RNA is reversely transcribed into cDNA as a template, the SAG4 gene is amplified by a PCR method, and the amplified product is analyzed by 1 percent agarose gel electrophoresis to have single bands of SAG2 and SAG4 at 499bp and 469bp respectively (figure 1 and figure 2).
Expression and identification of recombinant proteins: after the sequencing is correct, the recombinant plasmid is transformed into Escherichia coli (E.) (E. coli) BL21 (DE 3) competent cells were plated overnight, positive BL21 monoclonal colonies were picked, inoculated into LB liquid medium containing 100. mu.g/mL kanamycin, shaken overnight at 37 ℃ and 200rpm, enlarged cultured the next day at a ratio of 1:100 to A600=0.3-0.5, IPTG was added to a final concentration of 0.1mmol/L, induced expression was carried out at 37 ℃ and 180rpm for 6 hours, centrifuged at 12000rpm for 10min, the pellet was collected, washed 3 times with sterile 1XPBS, the pellet was resuspended with PBS of the original bacterial liquid volume 1/10, and the pellet was sonicated on ice, centrifuged at 12000rpm for 10min, the supernatant and the pellet were collected separately and analyzed by SDS-PAGE electrophoresis. And (3) purifying the recombinant protein: the bacteria liquid collected after expression is centrifuged for 10min at 12000rpm/min at 4 ℃, the supernatant is discarded, and the solution is washed with Tris-HCl for 2 to 3 times. Purification was performed using His bind Resin purification kit, and 10 ml Binding buffer was added first, followed by sonication on ice-water mixture for 5 min, followed by centrifugation at 12000rpm/min for 10min, and the supernatant was collected. Preparing Ni-MTA His resin, collecting 500 μ L of the resin, and mixingThe column was placed on a purification column and the equilibration column was washed 3 times with PBS. And combining the supernatant of the centrifuged bacterial liquid with Ni-MTA His resin, and rotating at 4 ℃ for 1-2 h or overnight at 4 ℃. Washing with Wash buffer for 1-3 times, eluting with Elution buffer of 200 μ L, dialyzing the eluate with PBS to obtain purified target protein (FIG. 3), and determining protein concentration by BCA method. ELISA reaction system and conditions: the antigens (SAG 2 and SAG 4) were diluted to 5. mu.g/mL with 0.05M CBS (pH 9.6) coating solution, and the diluted antigens were added to 96-well plates, 100. mu.L/well, and the freezer was kept overnight at 4 ℃. 1xPBST and 3% skim milk were prepared. The plate was washed once with a plate washer, 3% skim milk was added to block 100. mu.L/well, incubated at 37 ℃ in an incubator for 1 h, and the positive control antibody (1: 4000 dilution) or the serum to be tested (1: 100 dilution) was diluted with 3% skim milk during incubation. And taking out the 96-well plate from the incubator, throwing off the skim milk, washing the plate once, adding 100 mu L/hole of the serum to be detected, incubating at 37 ℃ for 1 h, and then washing the plate 6 times by using PBST. Corresponding animal secondary antibodies were prepared, diluted to 1:4000 using 3% skim milk, added to 100. mu.L/well and then placed at 37 ℃ for 1 h, after which the plates were washed 6 times. Preparing a substrate developing solution, wherein the substrate developing solution: ABTS: 30% H2O2=10 mL: 3 mg: 3 μ L. After the addition, the mixture is placed in an incubator at 37 ℃ for 15 min, and the result is checked by reading 415 nm of a microplate reader.
Example 2 application of Toxoplasma gondii ELISA detection method
ELISA specificity test: coccidium, cryptosporidium and schistosoma japonicum positive sera are all provided by the animal parasitology key open laboratory of the department of agriculture. Diluting all the positive serums of the polypide to be tested to be samples, detecting by an ELISA method by using the SAG2 and SAG4 as antigens, and observing the specificity of the samples. The result shows that the ELISA specificity analysis is carried out on the positive serum of various parasite bodies, and the result shows that the ELISA specificity analysis only has strong specificity reaction on the positive serum of the toxoplasma gondii and has no specificity reaction on other parasite sera. Comparative ELISA sensitivity test: diluting purified SAG2 or SAG2 and SAG4 mixed protein (SAG 2+ SAG 4) to 5 mu g/mL, adding 100 mu L of the mixed protein into each well, diluting positive toxoplasma infected mouse serum and healthy mouse serum in a concentration ratio of 1:200, 1:400, 1:800 and 1:1600 by a multiple ratio, 100 mu L/well, repeating the dilution by two times, performing ELISA test according to the above-described ELISA reaction system and conditions, and measuring OD value by using an ELISA reader at 415 nm. According to the measurement result, the purified protein can react with positive serum of a Toxoplasma gondii-infected mouse, even if the protein is diluted to 1:1600, the sensitivity of the SAG2+ SAG4 mixed antigen is still higher than that of the SAG2 antigen group, but the protein does not react with the negative serum, which indicates that the SAG2+ SAG4 mixed antigen has higher sensitivity and can be clinically used as an antigen to distinguish the negative serum from the positive serum (Table 1).
TABLE 1 ELISA sensitivity assay
Clinical detection of animal toxoplasma infection: 15 clinical serum samples with positive toxoplasma gondii strength are used, SAG2 alone or SAG2 and SAG4 mixed protein (SAG 2+ SAG 4) is used as a coating antigen to carry out ELISA method detection, contrast detection is carried out under the condition that the amount of samples to be detected is consistent, and the detection sensitivity is observed. Comparing the ELISA detection method of the two coated antigens of SAG2 and SAG2+ SAG4, the method of SAG2+ SAG4 is more sensitive, and weak positive toxoplasma infection can be detected (Table 2).
TABLE 2 clinical examination of animals infected with Toxoplasma gondii
The above-mentioned embodiments only express the embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> Shanghai animal doctor institute of Chinese academy of agricultural sciences (Shanghai center of Chinese centers of animal health and epidemiology)
< 120 > ELISA detection method capable of distinguishing acute toxoplasma infection from chronic toxoplasma infection and application thereof
〈160〉 4
〈170〉 PatentIn version 3.3
〈210〉 1
〈211〉 28
〈212〉 nt
〈213〉P1
〈400〉TTCATATGTCCACCACCGAGACGCCAGCG
〈210〉 2
〈211〉 28
〈212〉 nt
〈213〉P2
〈400〉TTGCGGCCGCCACAAACGTGATCAACAAACC
〈210〉 3
〈211〉 28
〈212〉 nt
〈213〉P3
〈400〉TAGGATCCTTGGCGAGTGCGGGCAAAGAGC
〈210〉 4
〈211〉 28
〈212〉 nt
〈213〉P4
〈400〉TGCTCGAGCATTGATATCAACACAAAGGC
Claims (4)
1. A kit for detecting Toxoplasma gondii infection is characterized by comprising two pairs of forward primers and reverse primers for amplifying two genes, wherein the forward primers and the reverse primers are two pairs of primers for amplifying a Toxoplasma gondii tachyzoite-stage specific antigen SAG2 and a bradyzoite-stage specific antigen SAG4 genes respectively.
2. The kit according to claim 1, wherein one pair of forward and reverse primers has the nucleotide sequences shown as SEQ ID No.1 and SEQ ID No.2, and the other pair of forward and reverse primers has the nucleotide sequences shown as SEQ ID No.3 and SEQ ID No. 4.
3. Use of a kit according to claim 1 for the detection of Toxoplasma gondii infections, further, for the detection of Toxoplasma gondii infections, in vitro environmental tests.
4. A kit capable of simultaneously detecting acute and chronic toxoplasma infection, which is characterized in that the antigens are toxoplasma tachyzoite stage specific antigen SAG2 and bradyzoite stage specific antigen SAG 4.
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| CN103320501A (en) * | 2012-03-19 | 2013-09-25 | 中国农业科学院上海兽医研究所 | Primer, probe and kit for detecting bradyzoite of toxoplasma gondii |
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| CN103320501A (en) * | 2012-03-19 | 2013-09-25 | 中国农业科学院上海兽医研究所 | Primer, probe and kit for detecting bradyzoite of toxoplasma gondii |
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