Primer group and method for detecting toxoplasma gondii

Abstract

The invention relates to the technical field of biology, in particular to a primer group and a method for detecting toxoplasma gondii. The primer group for detecting the toxoplasma gondii, disclosed by the invention, comprises the nucleotide sequences of primers as shown in SEQ ID NO.1-SEQ ID NO.6; according to the present invention, the excrement sample can be directly collected and added into the LAMP reaction reagent integrated in the microfluidic chip, the detection process is automatically completed by the full-automatic detection instrument, the spectrum is analyzed through the spectrum sensor, the collected spectrum signal is comprehensively judged through the AI technology, the full-automatic operation is achieved, the detection time can be controlled within one hour, and the detection accuracy is high. The kit realizes instant diagnosis, does not need professionals and laboratory environments, is portable and light, can be applied to instant detection in scenes such as pet hospitals on site, adopts an innovative LAMP test method, does not need nucleic acid extraction, realizes sample result output, and is low in price.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a primer set and method for detecting Toxoplasma gondii. Background Art

[0002] Toxoplasma gondii is a genus of Toxoplasma in the family Sarcocystis of the order Eucocidia, also known as Toxoplasma gondii (TOX). Toxoplasma gondii is a crescent-shaped worm that can invade the human central nervous system.

[0003] The life cycle of Toxoplasma includes asexual and sexual reproduction stages, and requires two hosts. It reproduces sexually and asexually in cats and felines, and asexually in humans, mammals, birds, fish, and reptiles. Cats are both the final host and the intermediate host of Toxoplasma, while humans or other animals are the intermediate hosts. Toxoplasma parasitizes in the epithelial cells of the small intestine of cats, reproduces sexually, and produces oocysts. The oocysts are excreted with feces, and it takes more than a day to complete sporulation and acquire the ability to infect. After being ingested by other mammals or birds, the mature oocysts develop into tachyzoites and multiply rapidly. Toxoplasma can cause toxoplasmosis in humans. Congenital toxoplasmosis mainly damages the central nervous system of the fetus, and can cause miscarriage, stillbirth, brain necrosis, hydrocephalus, and mental disorders. Children who survive the infection often have intellectual disabilities and epilepsy due to congenital brain damage. Acquired toxoplasmosis generally has no obvious clinical symptoms in patients with complete immune function.

[0004] Toxoplasma develops in two stages, the extramucosal stage and the intramucosal stage. The former develops in various intermediate hosts and the final host's infectious tissue cells. The latter only develops in the final host's small intestinal mucosal epithelial cells.

[0005] 1. Extramucosal stage

[0006] After the oocysts, cysts or pseudocysts of Toxoplasma gondii are ingested by the intermediate host or the definitive host, sporozoites, bradyzoites or tachyzoites are released in the intestinal cavity respectively. The parasites can invade various nucleated cells of extraintestinal tissues and organs directly or through lymph and blood, or enter cells through phagocytes and phagocytosis. The parasites are mainly in the cytoplasm, and can also divide and reproduce in the nucleus. In the acute phase, tachyzoites rapidly divide and multiply, causing the invaded cells to rupture, and the tachyzoites invade new cells to proliferate. With the formation of the body's specific immunity, the proliferation of Toxoplasma tachyzoites in cells slows down and eventually develops into cysts. The parasite enters the bradyzoite stage. Cysts can exist in the host for a long time. When the host's immune function is low, the cysts rupture and release a large number of bradyzoites, forming parasitemia, and can invade new host cells and proliferate rapidly.

[0007] (II) Intestinal mucosal stage

[0008] After being ingested by the definitive host, the oocysts, cysts or pseudocysts enter the small intestine. Sporozoites, bradyzoites or tachyzoites can directly invade the epithelial cells of the small intestinal mucosa to reproduce asexually and form merozoites. After the cells are destroyed, the merozoites are released and then invade new epithelial cells. After several generations of proliferation, some merozoites develop into female and male gametes in the epithelial cells, which combine and fertilize to become zygotes, and finally develop into oocysts. After the oocysts mature, they fall out of the epithelial cells into the large intestine and are excreted from the body with feces. The excreted oocysts mature after 2-3 days of development in the outside world and are infectious.

[0009] There are obvious differences in the resistance of Toxoplasma gondii at different developmental stages. Trophozoites are sensitive to temperature and general disinfectants. They can survive for 10 minutes when heated to 54°C; they die in 1 minute in cresolsulfonic acid solution or 1% hydrochloric acid solution. Cysts have strong resistance and can survive for 68 days at 4°C and can tolerate 3 hours in gastric juice, but they are not resistant to dryness and high temperature and die in 10 to 15 minutes at 56°C. Oocysts are very resistant to common disinfectants such as acids and alkalis, but are weak to heat and die in 1 minute at 80°C.

[0010] To date, the most common and convenient method for detecting the presence of TOX is the fecal ELISA (Enzyme-linked immunosorbent assay), which is easy to operate and produces results quickly, but occasionally produces false positive or false negative results, so further testing may be required to confirm the diagnosis. TOX antibodies can also be detected in healthy cat populations, so this method has certain limitations. The enzyme-linked immunosorbent assay can also be judged by detecting antigen-antibody complexes in the body, but the positive rate of this test method is only 67%, which undoubtedly greatly limits the detection of the virus.

[0011] Veterinarians can also use molecular diagnostic technology - polymerase chain reaction (PCR) technology to diagnose TOX in fecal samples. This method is currently a relatively effective method with a relatively high detection rate of TOX. Primers targeting the conservative region of the viral genome are used for detection or primers are designed using the viral 529 repeat sequence region or B1 gene, and then PCR is used to detect Toxoplasma infection. The results are specific and sensitive. PCR technology is widely used due to its high detection sensitivity, wide range of applications, and simple operation. However, the detection technology is complex, requires sophisticated instruments, and takes a long time to detect, which is not conducive to on-site detection in non-laboratory environments and the promotion and application of grassroots laboratories. Summary of the invention

[0012] In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide a primer set and method for detecting Toxoplasma gondii.

[0013] In one aspect, the present invention provides a primer set for detecting Toxoplasma gondii, characterized in that the nucleotide sequence of the primers included in the primer set is as follows:

[0014] SEQ ID NO.1: 5'-TCTAGGGCACCCTTACTGC-3';

[0015] SEQ ID NO.2: 5'-TCTGGCATTTGCCATGAGAG-3';

[0016] SEQ ID NO.3:

[0017] 5'-CTGGATCTCTTCCCTTGAATGCGATTTTAAGTCGACCACCTGTTTCCT-3';

[0018] SEQ ID NO.4:

[0019] 5'-ATTCGAGACAAGAGAGGTCCGCCTTTTGAGGCAGCACAAGAATGTTG-3';

[0020] SEQ ID NO.5: 5'-TGTCGTACGTGACAGTGAAGA-3';

[0021] SEQ ID NO. 6: 5'-CCCACAAGACGGCTGAAGA-3'.

[0022] Among them, the forward outer primer F3, its nucleotide sequence is shown in SEQ ID NO.1; the reverse outer primer B3, its nucleotide sequence is shown in SEQ ID NO.2; the forward inner primer FIP, its nucleotide sequence is shown in SEQ ID NO.3; the reverse inner primer BIP, its nucleotide sequence is shown in SEQ ID NO.4, the loop primer LF, its nucleotide sequence is shown in SEQ ID NO.5, and the loop primer LB, its nucleotide sequence is shown in SEQ ID NO.6.

[0023] Specifically, the present invention also provides the application of the above-mentioned primer set, which can be used in the preparation of a Toxoplasma gondii kit, or the application of the primer set in the detection of Toxoplasma gondii, the application of the above-mentioned primer set in a microfluidic chip for detecting Toxoplasma gondii, and especially the detection of Toxoplasma gondii using a loop-mediated isothermal amplification-spectral sensor-artificial intelligence technology platform.

[0024] In another aspect, the present invention further provides a method for detecting Toxoplasma gondii, comprising: performing loop-mediated isothermal amplification on a sample to be tested using LAMP detection technology in combination with the primer set described above; and analyzing the amplified product.

[0025] Furthermore, the reaction temperature of the loop-mediated isothermal amplification is 65° C., and the reaction time is 35 minutes.

[0026] Furthermore, the loop-mediated isothermal amplification reaction is carried out in a microfluidic chip.

[0027] Furthermore, the method for analyzing the amplification product includes performing spectral analysis and / or colorimetric analysis on the LAMP reaction result.

[0028] The beneficial effect of the present invention is that the present invention innovatively develops a set of specific primer formulas for the TOX-B1 sequence of Toxoplasma gondii that do not require nucleic acid extraction and can directly use fecal samples for LAMP reaction, collects data through spectral sensors, and performs a method for rapid nucleic acid detection of Toxoplasma gondii. The method can directly use fecal samples of mammals such as cats and dogs, add them to the LAMP reaction reagent integrated in the microfluidic chip, and the detection process is automatically completed by a fully automatic detection instrument. The spectrum is analyzed by a spectral sensor, and the collected spectral signals are comprehensively judged by AI technology to achieve fully automatic operation, control the detection time within one hour, and achieve instant diagnosis. It does not require professionals and laboratory environments, is portable and lightweight, and can be used on-site for immediate detection in scenes such as pet hospitals. It adopts an innovative LAMP test method, does not require nucleic acid extraction, and achieves sample-in and result-out, and is low-priced.

[0029] Other features and advantages of the present invention will be described in the following description, and partly become apparent from the description, or understood by practicing the present invention. The purpose and other advantages of the present invention are realized and obtained by the structures particularly pointed out in the description and the drawings.

[0030] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, preferred embodiments are given below and described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 It is the screening result of LAMP reaction of 6 primer sets designed for the TOX-B1 sequence of Toxoplasma gondii;

[0033] Figure 2 It is the test result of the present invention for TOX positive samples and negative samples;

[0034] Figure 3 is the test result of the specific experiment of the present invention;

[0035] Figure 4 It is the detection result of the Toxoplasma gondii detection method of the present invention for fecal samples. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] In the embodiments of the present invention, the TOX standard positive sample is a synthetic TOX-B1 nucleic acid conservative sequence positive plasmid, Salmonella (Sal), feline calicivirus (FCV) and canine parvovirus (CPV) are all synthetic plasmid samples, and the primer sequences involved are all ordered from Sangon Biotech (Shanghai) Co., Ltd.

[0038] 1. Primer design and screening

[0039] By using a higher concentration of TOX standard positive samples, the LAMP primer set designed with TOX-B1 fragment as template was screened. The goal was to screen out a primer set with the best sensitivity and specificity.

[0040] The primer design method is:

[0041] (1) (1) Download the TOX-B1 sequence from NCBI (GenBank accession number: AF179871) as a template for primer design. The TOX-B1 sequence (SEQ ID NO. 7) is:

[0042]

[0043]

[0044] (2) Using PrimerExplorer V5

[0045] (http: / / primerexplorer.jp / lampv5e / index.html), and design specific primers using the TOX-B1 sequence as a template. LAMP reaction primers were designed based on six different regions on the TOX-B1 sequence, labeled F3, F2, F1, B1, B2, and B3, starting from the 5' end. F1, F2, F3, B1, B2, and B3 are about 20 bp long sequence fragments on the target gene, and F1c and B1c are complementary regions of F1 and B1, respectively. The four primers include: upstream external primer F3 (Forward Outer Primer), downstream external primer B3 (Backward Outer Primer), upstream internal primer FIP (Forward Inner Primer), and downstream internal primer BIP (Backward Inner Primer). At the same time, use the free software PrimerExplorer V5 to design 1-2 loop primers LF (Loop Primer Forward) and LB (Loop Primer Backward) between F1c and F2c, or between B1c and B2c to speed up the LAMP reaction. The primers are required to meet the Tm value (DNA chain melting temperature) of 64℃~66℃ for F1c and B1c, 59℃~61℃ for F2, F3, B2, B3, and 64℃~66℃ for loop primers. The free energy of the 3' end of F2 / B2, F3 / B3 and LF / LB and the 5' end of F1c and B1c should not be greater than -4kcal / mol. The free energy change (ΔG) is equal to the free energy of the product after the primer binds to the template minus the free energy of the initial reactant. Δ The smaller G is (the larger the absolute value of the negative number is), the easier it is for the primer to bind to the template. And the GC content in the primer sequence should be controlled at 30% to 65%. At the same time, ensure that each primer, especially the inner primer, is not easy to form a secondary structure to prevent the formation of primer dimers. There should be a certain appropriate spacing between the sequence fragments recognized by each primer. The spacing from the F2 end to the B2 end (LAMP amplification region) is 120 to 160 bases, the spacing from the 5' end of F2 to the 5' end of F1 (the region forming a loop structure) is 40 to 60 bases, and the spacing between F2 and F3 is 0 to 60 bases. All of the above conditions can be automatically calculated by the free software PrimerExplorer V5, and a list of designed primers is given;

[0046] (3) Multiple primer sets designed by PrimerExplorer V5 with different positions on the TOX-B1 sequence as templates were selected, and the target product sequences of different primer sets were input into NCBI Nucleotide BLAST

[0047] (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=Bl astSearch&LINK_L℃=blasthome) online tool to analyze the specificity of the primer set corresponding to the LAMP product to ensure that there is no non-specific amplification on the genome of cats and felines and other pathogens.

[0048] Through the above method, 6 sets of LAMP primer sets were preliminarily designed for selection. The nucleotide sequences of each set of primers are shown in Table 1 below:

[0049] Table 1 Primer design

[0050]

[0051]

[0052] The above six primer sets were used to perform LAMP amplification experiments with TOX standard positive samples as positive samples and sterile deionized water (ddH2O) as negative samples, and the color changes of the LAMP reaction system of each primer set were observed. Figure 1 As shown in the figure, the primer set of group 5 changed the color of the positive sample (+) in a short time (purple to bright yellow) and the negative sample (-) did not change color after 35 minutes, indicating that this primer set has higher sensitivity and specificity, while the other primer sets showed confusing positive and negative reactions and were not selected; in summary, group 5 is the primer set with the best sensitivity and specificity, therefore, the primer set of group 5 was selected as the primer set for detecting Toxoplasma gondii.

[0053] 2. Reaction conditions

[0054] After several rounds of testing of different reaction conditions, the following conditions were finally selected as the final reagent ratio and reaction conditions:

[0055] (1) Preparation of 10× primer mixture:

[0056] Table 2 Primer set mixing concentration ratio

[0057] Working concentration (μM) 10× Primer Mix Concentration (μM) FIP 1.6 16 BIP 1.6 16 F3 0.2 2 B3 0.2 2 LB 0.4 4 LF 0.4 4

[0058] Table 3

[0059] Primer concentration (μM) Primer name Prepare 40 reactions (μL) 100 FIP 8 100 BIP 8 10 F3 10 10 B3 10 100 LB 2 100 LF 2 Sterile deionized water 10 Total volume (μL) 50

[0060] (2) Primers, 2×LAMP premix and positive / negative sample ratios in the LAMP reaction system:

[0061] Table 4

[0062] 1 reaction (μL) 2×LAMP premix 6.25 10× Primer Mix 1.25 Positive / Negative samples 5.0

[0063] The 2×LAMP premix solution is a well-known formula, which includes MgSO4 (MgSO4 concentration is 6mM to 12mM), buffer, dNTPs, KCl, (NH4)2SO4, Bst DNA polymerase, phenol red, sterile enzyme-free water, etc.

[0064] The positive sample may be a TOX standard positive sample, that is, a synthesized TOX-B1 sequence positive plasmid.

[0065] The negative sample may be sterile deionized water (ddH2O).

[0066] (4) Reaction conditions

[0067] Under the condition of 65℃ temperature and constant temperature amplification for 35 minutes, better detection results were obtained.

[0068] Heating can be performed using a constant temperature heating device (water bath, metal bath, constant temperature oven, or PCR instrument, etc.).

[0069] 3. LAMP detection method for TOX

[0070] The present invention uses a TOX standard positive sample (synthesized TOX-B1 sequence positive plasmid) and performs detection according to the following steps:

[0071] Step 1: TOX positive / negative sample preparation

[0072] The synthesized TOX-B1 sequence positive plasmid was diluted with sterile deionized water, and the concentration of the TOX standard positive sample was diluted to 1000 copies / μL as a positive sample; sterile deionized water (ddH2O) was used as a negative sample.

[0073] Step 2: Prepare 10× primer mixture according to the method in Table 3 above.

[0074] Step 3: Take the Toxoplasma positive / negative samples prepared in the first step and prepare the LAMP reaction system for each reaction according to the method in Table 4 above.

[0075] Step 4: Heat at 65°C and react for 35 minutes.

[0076] The reaction results are as follows Figure 2 As shown, the LAMP reaction systems of 20 negative samples (-) were all purple-red, and the LAMP reaction systems of 20 positive samples (+) turned yellow. The positive accuracy rate and the negative accuracy rate in the reaction results were 100%, which showed that Toxoplasma gondii could be clearly detected.

[0077] 4. Specificity experiments

[0078] In order to test the specificity of the primer set for Toxoplasma gondii detection, Salmonella (Sal), feline calicivirus (FCV) and canine parvovirus (CPV) plasmid solutions at a concentration of 1000 copies / μL were used as cross-detection samples.

[0079] Step 1: Preparation of TOX positive / negative samples for cross-testing

[0080] Positive sample: The synthetic TOX-B1 sequence positive plasmid diluted to a concentration of 1000 copies / μL was used as the positive sample;

[0081] Negative samples: Salmonella (Sal), feline calicivirus (FCV) and canine parvovirus (CPV) plasmid solutions diluted to a concentration of 1000 copies / μL were used as negative samples.

[0082] Step 2: Prepare 10× primer mixture according to the method in Table 3 above.

[0083] Step 3: Take the positive samples and negative samples prepared in the first step respectively, and prepare the LAMP reaction system for each reaction according to the method in Table 4 above.

[0084] Step 4: Heat at 65°C and react for 35 minutes.

[0085] The reaction results are as follows Figure 3 As shown, the LAMP reaction systems of 5 positive samples containing synthetic TOX-B1 sequence positive plasmid solutions all turned yellow, and 5 negative samples containing Salmonella (Sal) plasmid solutions, 5 negative samples containing feline calicivirus (FCV) plasmid solutions, and 5 negative samples containing canine parvovirus (CPV) plasmid solutions did not change color, indicating that no false positive results occurred, demonstrating that the primer set for detecting Toxoplasma gondii of the present invention has good specificity.

[0086] according to Figure 2 and Figure 3 The sensitivity and specificity of LAMP for detecting Toxoplasma gondii were preliminarily calculated based on the experimental results. Figure 2 and Figure 3 According to the experimental results, 25 out of 25 positive samples showed clear positive results, so the sensitivity was calculated as: As well as 32 of the negative samples of 15 other bacterial species and 20 sterile deionized water (ddH2O), all showed clear negative results, so the specificity was calculated as:

[0087]

[0088] 5. LAMP detection method for Toxoplasma gondii in fecal samples

[0089] Step 1: Preparation of positive / negative stool samples

[0090] Use a cotton swab to insert it into different positions of the stool sample 5 to 8 times. When inserting, the head of the cotton swab should be completely immersed in the stool sample and try to cover all parts of the stool sample. If there is a lot of solid stool on the cotton swab, gently scrape off the excess stool solids; completely immerse the cotton swab with the stool sample into 1 mL of sterile deionized water prepared in advance, shake the cotton swab dozens of times to dissolve the stool sample in the water, and obtain a negative stool sample;

[0091] A positive stool sample can be obtained by adding a TOX standard positive sample (synthesized TOX-B1 sequence positive plasmid) at a certain dilution ratio to a portion of the negative stool sample prepared above.

[0092] Step 2: Prepare 10× primer mixture according to the method in Table 3 above.

[0093] Step 3: dilute the positive / negative stool samples prepared in the first step by 1 / 50 with sterile deionized water, and make the concentration of the synthetic TOX-B1 sequence positive plasmid in the positive stool sample 1000 copies / μL; prepare the LAMP reaction system for each reaction according to the method in Table 4 above.

[0094] Step 4: Heat at 65°C and react for 35 minutes.

[0095] The reaction results are as follows Figure 4 As shown, all 10 negative stool samples (-) showed clear negative results, and all 10 positive stool samples (+) turned yellow, indicating that Toxoplasma gondii in the stool samples can be clearly detected.

[0096] The LAMP detection method for Toxoplasma gondii in fecal samples is simple to collect and process, does not require nucleic acid extraction, simplifies the detection steps, and shortens the detection time. It only takes about 1 hour from sample collection to test results.

[0097] This detection method can also detect Toxoplasma gondii through the loop-mediated isothermal amplification-spectral sensor-artificial intelligence (LAMP-SpectralSensor-AI) technology platform. The diluted sample to be tested can be directly injected into the LAMP reaction reagent integrated in the microfluidic chip reaction chamber for amplification. The amplification product is analyzed by the spectral sensor, and the collected spectral signal is comprehensively judged by AI technology to quickly determine whether Toxoplasma gondii is present in the sample. The spectral detection method is combined with the AI ​​algorithm to achieve the detection accuracy of molecular detection. By limiting the sample to be tested to the microfluidic environment of the device, the risk of sample contamination is reduced, and the sample volume and reagents required for detection are minimized, thereby further reducing the overall cost of screening and detection. Moreover, this technology does not require any complex operations, only simple training, and can be completed on-site by medical staff in pet hospitals. It can realize fully automatic detection of samples in and results out, which is suitable for rapid and instant detection in scenarios such as pet hospitals.

[0098] Based on the above ideal embodiments of the present invention, the relevant staff can make various changes and modifications without departing from the technical concept of the present invention through the above description. The technical scope of the present invention is not limited to the contents of the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A primer set for detecting Toxoplasma gondii, characterized in that: The nucleotide sequences of the primers included in the primer set are as follows: SEQ ID NO.1: 5'-TCTAGGGCACCCTTACTGC-3'; SEQ ID NO.2: 5'-TCTGGCATTTGCCATGAGAG-3'; SEQ ID NO.3: 5'-CTGGATCTCTTCCCTTGAATGCGATTTTAAGTCGACCACCTGTTTCCT-3'; SEQ ID NO.4: 5'-ATTCGAGACAAGAGAGGTCCGCCTTTTGAGGCAGCACAAGAATGTTG-3'; SEQ ID NO.5: 5'-TGTCGTACGTGACAGTGAAGA-3'; SEQ ID NO. 6: 5'-CCCACAAGACGGCTGAAGA-3'.

2. Use of the primer set according to claim 1 in preparing a Toxoplasma gondii detection kit.

3. Use of the primer set according to claim 1 in detecting Toxoplasma gondii.

4. Use of the primer set according to claim 1 in a microfluidic chip for detecting Toxoplasma gondii.

5. A method for detecting Toxoplasma gondii, characterized in that: include: Using LAMP detection technology combined with the primer set as claimed in claim 1 to perform loop-mediated isothermal amplification on the sample to be tested; Analyze the results of the amplification products.

6. The method for detecting Toxoplasma gondii according to claim 5, characterized in that: The reaction temperature of the loop-mediated isothermal amplification was 65° C., and the reaction time was 35 minutes.

7. The method for detecting Toxoplasma gondii according to claim 5, characterized in that: The loop-mediated isothermal amplification reaction is carried out in a microfluidic chip.

8. The method for detecting Toxoplasma gondii according to claim 5, characterized in that: The method for analyzing the results of the amplification products includes performing spectral analysis and / or colorimetric analysis on the LAMP reaction results.

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