Toxoplasma gondii closed tube visual detection kit and detection method thereof

Abstract

The application provides a Toxoplasma gondii closed tube visual detection kit and a detection method thereof. + The application combines the SPA constant temperature amplification and the pH indicator BTB, and uses the RGB analysis technology of a smart phone, so that the closed tube detection of the Toxoplasma gondii is realized, and the detection kit has the characteristics of rapidness, strong specificity, high sensitivity, strong anti-interference, visualization and convenient operation.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, and in particular to a visual detection kit for Toxoplasma gondii occlusion and its detection method. Background Technology

[0002] Toxoplasma gondii is a widely distributed parasite that infects most warm-blooded animals. It can also be transmitted through food and contaminated water. Currently, over 30% of the world's population is believed to have chronic Toxoplasmosis. Therefore, toxoplasmosis is listed as a foodborne parasitic infection of global concern by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO). In recent years, with the rapid development of molecular biology, methods for identifying Toxoplasma gondii have expanded to include novel molecular biological approaches. Among the most common molecular biological methods are PCR and LAMP amplification, which utilize primers designed specifically for the Toxoplasma gondii genome to achieve rapid detection. With the continuous improvement of people's living standards, the demands of animal husbandry and the increasing rate of pet ownership are also rising, thus placing higher demands on rapid parasite detection, requiring features such as on-site detection, speed, high sensitivity, strong specificity, and low cost.

[0003] LAMP amplification is a mature isothermal amplification technique that utilizes inner and outer primers combined with polymerase to perform isothermal amplification under isothermal conditions. However, this method is complex, primer design is challenging, and it is prone to false positives. Currently, with the continuous advancement of nucleic acid amplification theory, more and more modified variants of LAMP amplification have been proposed. Among these methods, stem-loop primer-assisted isothermal amplification (SPA), based on a pair of ordinary primers and hairpin primers, undoubtedly has great potential. This method can improve response speed and anti-interference ability while ensuring design simplicity. However, this method lacks practical application and validation.

[0004] With the widespread application of isothermal amplification, signal output methods are constantly being enriched. When conditions permit, complex sensors, represented by electrochemical and surface plasmon resonance methods, are the best choice for obtaining high sensitivity. Customized devices, such as microfluidics, microarrays, and multichannel paper-based devices, are ideal for high-throughput detection. However, their high cost and process requirements often limit their application in laboratories. Due to the high sensitivity of isothermal amplification, traditional fluorescence and colorimetric methods have long been able to meet various detection requirements. Compared with fluorescence sensors, magnesium ion-based (Mg... 2+ ) and hydrogen ions (H + Colorimetric sensors using indicators are increasingly used in laboratory and commercial settings due to the advantage of visually perceptible results. However, currently, colorimetric methods are limited to Mg... 2+Indicators such as hydroxynaphthol blue (HNB) and acid chrome blue K (ACBK), as well as neutral red (NR), phenol red (PR), cresol red (CR), fuchsin, and xylenol orange (XO), etc. + Indicators. Although some new indicators have recently been applied to isothermal amplified signal outputs, their color changes are not obvious or they need to be added after the reaction.

[0005] Based on this, this application provides a rapid and visual detection kit for Toxoplasma gondii using a closed-tube method. The closed-tube colorimetric detection achieves rapid, contamination-free detection of Toxoplasma gondii through a one-step closed-tube reaction; the optimized SPA amplification system reduces non-specific SPA amplification; and visualization is achieved using the colorimetric indicator bromothymol blue (BTB). Summary of the Invention

[0006] In view of this, the present invention provides a rapid and visual detection kit for Toxoplasma gondii closed tubes and a detection method thereof. This invention combines SPA isothermal amplification with the pH indicator BTB and utilizes RGB analysis technology from a smartphone to achieve closed tube detection of Toxoplasma gondii. It features rapid detection, high specificity, high sensitivity, strong anti-interference capabilities, visualization, and convenient operation.

[0007] The purpose of this invention is to provide a rapid SPA closed-tube visualization detection method for detecting Toxoplasma gondii-specific genes (328-372 of AF146527.1) as target genes.

[0008] The technical solution adopted in this invention is to combine the SPA amplification system with the BTB colorimetric indicator to detect Toxoplasma gondii-specific genes.

[0009] The scope of application of this invention: rapid detection of Toxoplasma gondii in various fields.

[0010] The operating method of this invention is as follows: After the reaction is completed according to the required system and procedure, the results can be observed with the naked eye, or RGB auxiliary analysis can be performed using a smartphone.

[0011] The results of this invention are determined by observing the color change of the solution with the naked eye after the reaction is complete. A blue solution indicates the absence of Toxoplasma gondii; a yellow solution indicates the presence of a large number of Toxoplasma gondii; and a yellowish-green solution indicates the presence of a small number of Toxoplasma gondii.

[0012] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0013] On the one hand, this invention provides a visual detection method for Toxoplasma gondii closure tubes, which designs SPA primers using Toxoplasma gondii gene fragments as targets, performs SPA isothermal amplification, and combines H + Responding to relevant reagents enables closed-tube visual detection;

[0014] The SPA primers include:

[0015] Toxo-HF primers: sequence as shown in SEQ ID NO: 1;

[0016] Toxo-HR primers: sequence as shown in SEQ ID NO: 2;

[0017] Toxo-F primers: sequence as shown in SEQ ID NO: 3;

[0018] Toxo-R primers: sequence as shown in SEQ ID NO: 4;

[0019] The SPA isothermal amplification system is as follows:

[0020]

[0021] The preferred SPA isothermal amplification system is as follows:

[0022]

[0023] The H + The response-related reagent was a 0.1% BTB solution;

[0024] There are two methods for evaluating the detection method:

[0025] I. Direct visual observation: SPA isothermal amplification will cause pH changes in the reaction system. The concentration of the target gene can be determined based on the degree of color change of BTB.

[0026] II. Analysis using smartphones: Take a picture of the BTB colorimetric results, and then perform RGB analysis using smartphone software to determine the target gene concentration;

[0027] The amplification reaction program is 58-70℃ for 85-100 min, then 80℃ for 10 min;

[0028] Preferably, the amplification reaction procedure is to react at 67°C for 85 minutes.

[0029] On the other hand, the present invention provides a Toxoplasma gondii detection kit, comprising the following components: commercially available PCR tubes, SPA amplification system, and H... + Response-related reagents;

[0030] The commercially available PCR tubes are designed with an open cap.

[0031] The SPA isothermal amplification system includes SPA amplification primers, dNTPs, betaine, MgSO4, buffer, Bst DNA polymerase, SYTO-9 dye, and water.

[0032] The SPA amplification primers include:

[0033] Toxo-HF primers: sequence as shown in SEQ ID NO: 1;

[0034] Toxo-HR primers: sequence as shown in SEQ ID NO: 2;

[0035] Toxo-F primers: sequence as shown in SEQ ID NO: 3;

[0036] Toxo-R primers: sequence as shown in SEQ ID NO: 4.

[0037] The H + The response-related reagent was a 0.1% BTB solution.

[0038] Specifically, the SPA isothermal amplification system includes:

[0039]

[0040]

[0041] The amplification reaction program is 58-70℃ for 85-100 min, then 80℃ for 10 min;

[0042] Preferably, the amplification reaction procedure is to react at 67°C for 85 minutes.

[0043] After the reaction is complete, the presence or absence of the genome is determined by whether the solution changes from blue to yellow.

[0044] This invention uses Toxoplasma gondii as the research object and combines the SPA amplification system with the pH indicator BTB to achieve closed-tube detection of Toxoplasma gondii. It has the characteristics of being rapid, highly specific, highly sensitive, highly resistant to interference, visual, and easy to operate.

[0045] The technical effects of this invention are as follows:

[0046] (1) This invention is the first to use the commercial colorimetric indicator BTB to achieve endpoint indication of isothermal amplification.

[0047] (2) By utilizing the SPA isothermal amplification reaction and BTB colorimetric indicator visualization detection technology, rapid, closed-tube, visualized, and contamination-free detection of Toxoplasma gondii was achieved.

[0048] (3) This invention provides specific primers for amplifying and detecting the 529bp repeat sequence-specific gene (AF146527.1) of Toxoplasma gondii using SPA. The primers have good specificity and excellent anti-interference ability.

[0049] (4) This invention combines SPA amplification and BTB colorimetric indicator visualization for analysis, which can accurately determine whether the DNA extract of the sample contains Toxoplasma gondii. This method is fast and simple, and the test results can be read within 85 minutes. The results are easy to observe and are applicable to Toxoplasma gondii testing in various fields.

[0050] (5) In the rapid detection method of the present invention, SPA amplification can be completed at a constant temperature, so the requirements for temperature control instruments and equipment are not high, and water baths or plate heaters can meet the requirements.

[0051] (6) The rapid detection method of the present invention improves the accuracy of detection by combining smartphone-assisted RGB analysis technology. Attached Figure Description

[0052] Figure 1 The naked-eye indication effect of BTB. A, the colorimetric effect of BTB and Neutral Red (NR) in solutions of different pH values, 1-11 respectively, showing the indication effect of BTB and NR in different pH buffer solutions; B, the qualitative effect of BTB and NR as shown on the color wheel.

[0053] Figure 2 BTB RGB and luminance quantization results at different pH values.

[0054] Figure 3 BTB colorimetric performance test at different volumes.

[0055] Figure 4 BTB's colorimetric performance at 12 hours.

[0056] Figure 5 Colorimetric performance of BTB in 96-well plates.

[0057] Figure 6 Reaction temperature optimization. NC (Negative contrast) is the negative control, PC (Positive contrast) is the positive control, and TTP (Time to the positive) is the difference in Ct values ​​between the negative and positive controls.

[0058] Figure 7 Primer ratio optimization.

[0059] Figure 8 BTB addition amount optimized.

[0060] Figure 9 Optimization of reaction buffer.

[0061] Figure 10 Schematic diagram.

[0062] Figure 11Specificity experiments. "Other" refers to a mixed genome of Echinococcus granulosus and swine tapeworm.

[0063] Figure 12 The results of the linear relationship test were examined.

[0064] Figure 13 Detection limit test results.

[0065] Figure 14 RGB analysis results assisted by smartphone.

[0066] Figure 15 Results of resistance to interference from complex real fecal samples' genomes.

[0067] Figure 16 Actual sample test results. Detailed Implementation

[0068] This invention discloses a rapid visual detection kit for Toxoplasma gondii occulta and its detection method. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the same result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0069] The reagents or instruments used in the Toxoplasma gondii detection kit and detection method provided by this invention are all commercially available. The invention is further illustrated below with reference to specific examples.

[0070] Example 1: Extraction of Toxoplasma gondii genome.

[0071] Template DNA was extracted from Toxoplasma gondii using the CTAB method, and the quality of the extracted genome was determined by nanodrop (Table 1).

[0072] Table 1. Genome content of Toxoplasma gondii

[0073]

[0074] Example 2: Measurement of the effect of BTB indication.

[0075] The color change of BTB was tested in buffer solutions containing different pH values, and the colorimetric effect was observed with the naked eye. Optical images were acquired using a smartphone (auto camera mode, flash off) at an exposure of 6500K. Subsequently, the RGB (Red, Green, and Blue) values ​​were extracted from the images using ImageJ software.

[0076] result Figure 1 The results show that BTB appears blue at pH 8.8. As the pH of the solution decreases, the color of BTB changes sequentially from blue to blue-green, green, and yellow-green, finally turning yellow at pH 6.8. This phenomenon is more pronounced and exhibits a more gradual color change than Neutral Red (NR), the most commonly used pH indicator in academic research and commercial reagent kits. Further analysis on the color wheel reveals that BTB can achieve a color change from blue to its complementary color—yellow—while NR shows a much smaller change. Figure 2 The results show that as the pH of the solution decreases from 8.8 to 6.8, all RGB values ​​change significantly, especially the R value, which changes by -155.76. Based on the maximum value of RGB in decimal (255), the normalized R value change of BTB can be calculated to be 61.08%. These results demonstrate that BTB not only possesses good potential for naked-eye colorimetric analysis but also excellent RGB quantitative results.

[0077] Example 3: Optimization of the SPA colorimetric reaction system.

[0078] The SPA amplification system includes Toxo-HF, Toxo-HR, Toxo-F, Toxo-F, dNTPs, Bst DNA polymerase, 10× weakly buffered buffer, betaine, MgSO4, SYTO-9, 0.1% BTB, and H2O. The primer sequences for the SPA amplification system are shown in Table 2, and the SPA amplification system itself is shown in Table 3.

[0079] Table 2 Primer sequences

[0080]

[0081] Table 3 SPA reaction system

[0082]

[0083] 1. Indication effect of BTB at different reaction volumes and fluxes

[0084] First, the indication effect of BTB was tested at different reaction volumes, with 20 μL, 15 μL, 10 μL, and 5 μL as reaction volumes. The results are as follows: Figure 3 As shown, BTB can stably indicate reaction systems of 5-20 μL. The smaller volume (0.5-5 μL) also demonstrates BTB's excellent indicating ability. Figure 4 Test results on a 96-well plate ( Figure 5 This indicates that BTB also performs excellently in high-throughput testing.

[0085] 2. Temperature optimization of the SPA amplification system

[0086] Secondly, the optimal temperature for the SPA amplification system was optimized, and 58℃, 59.4℃, 61.3℃, 63.3℃, ​​65.5℃, 67℃, 68.0℃, and 69.7℃ were selected as the reaction temperatures. The results are as follows... Figure 6 As shown, the optimal reaction temperature is 67℃.

[0087] 3. Optimization of F / R and HF / R primer ratios in the SPA amplification system

[0088] Next, the F / R and HF / R primer ratios in the SPA amplification system were optimized, with ratios of 0:20, 1:19, 1:4, 1:1, 3:4, 19:1, 97:3, and 99:1 selected for optimization. The results are as follows... Figure 7 As shown, the optimal F / R and HF / R primer ratio is 99:1. However, in actual sample loading, achieving a 99:1 F / R and HF / R primer ratio requires adding 1.98 μL of F / R and 0.02 μL of HF / R, respectively. Such extremely small volume loading is difficult to achieve and results in significant operational errors. Even with a primer ratio of 97:1, the HF / R loading volume remains as low as 0.06 μL. Considering both the TTP in real-time quantification results and practical operability, a ratio of 19:1 was chosen.

[0089] 4. Optimization of BTB addition in the SPA amplification system

[0090] The amount of 0.1% BTB added to the SPA amplification system was optimized by selecting concentrations of 0.6 μL, 0.8 μL, 1.0 μL, 1.2 μL, and 1.4 μL, respectively. The results are as follows: Figure 8 As shown, considering both real-time quantitative Ct and RFU results, as well as naked-eye visualization results, the optimal amount of BTB added is 1.0 μL.

[0091] 5. Optimization of Tris concentration in the buffer solution of the SPA amplification system

[0092] The Tris concentration in the buffer solution of the SPA amplification system was optimized by selecting concentrations of 0 mM, 30 mM, 60 mM, and 200 mM, respectively. The results are as follows: Figure 9 As shown, real-time quantitative results indicate that the optimal Tris concentration in the buffer solution is 30 mM, but naked-eye colorimetric results indicate that the optimal Tris concentration in the buffer solution is 0 mM. Based primarily on the colorimetric results, the optimal Tris concentration in the buffer solution was selected as 0 mM.

[0093] Example 4: Reaction principle.

[0094] The SPA amplification system is combined with the BTB colorimetric indicator to detect Toxoplasma gondii-specific genes, as shown in the figure. Figure 10 The nucleic acid signal from *Toxoplasma gondii* is amplified using SPA, which consists of a pair of standard primers and a hairpin primer, targeting only two gene regions. Compared to LAMP, which requires 4-6 primers targeting 6-8 regions, SPA significantly reduces primer design difficulty and system complexity. At 67°C, the standard primers and hairpin primers are sequentially annealed and extended with the double-stranded genome target sites by Bst polymerase. Because the tun-back sequence is designed on the hairpin primer, the amplicon can be gradually extended and accelerated. The *Toxoplasma gondii* nucleic acid signal is then converted into a colorimetric signal using BTB, marking the first use of BTB in isothermal amplification. Specifically, each dNTP introduced into the amplicon during nucleic acid amplification generates an equal amount of H2O. + and P2O7 4- A large number of amplicones means a large number of H + The reaction occurs when the pH of the reaction system decreases significantly under weak pH buffer conditions, causing BTB to gradually change from its initial blue color to blue-green, then to green, and finally to yellow. This phenomenon is obvious and can be clearly observed with the naked eye. Combined with a smartphone, more accurate and stable RGB analysis can be achieved than visual observation. For a large number of test samples, high-throughput experiments can be conducted using commercial consumables such as 8-well and 96-well plates.

[0095] Example 5: Specificity experiment.

[0096] The genomes of two common parasites, *Echinococcus granulosus* and *Taenia solium*, were selected and mixed 1:1 as templates for the SPA reaction. An additional mixed genome was added to the positive control as the experimental group. The reaction was carried out according to the optimal reaction system and conditions obtained in the experiment, and the colorimetric indicators and real-time fluorescence curves were observed.

[0097] The results are as follows Figure 11 The results showed that real-time amplification curves only appeared in the presence of Toxoplasma gondii, and the reaction solution turned yellow after amplification, a phenomenon unaffected by the addition of other parasite genomes. These results indicate that the constructed biosensor has good specificity for Toxoplasma gondii and can be stably tested even with interference from other parasite genomes.

[0098] Example 6 Sensitivity.

[0099] The genomes of Toxoplasma gondii were serially diluted and used as templates for the SPA reaction. The reaction was carried out according to the optimal reaction system and reaction conditions obtained in the experiment, and the colorimetric indication results and real-time fluorescence curves were observed.

[0100] The results are as follows Figure 12 and 13 As shown in the figure, the constructed biosensor can detect 5 fg / μL of Toxoplasma gondii genome, or 0.071 Toxoplasma gondii per μL (calculated based on one Toxoplasma gondii containing 250 copies of the target gene). Naked-eye colorimetric results also show that after the reaction, the solution is blue when Toxoplasma gondii is absent; green when a small amount of Toxoplasma gondii is present; and predominantly yellow when a large amount of Toxoplasma gondii is present. The sensitivity of this method is comparable to that of PCR and LAMP detection methods.

[0101] Example 7: RGB analysis based on smartphone assistance.

[0102] After the reaction, the PCR tubes were photographed. The corresponding color areas were selected using color software, and RGB values ​​were analyzed. The R values ​​were used to help determine the reaction results.

[0103] The results are as follows Figure 14 As shown, after the 85-minute reaction, the R value of the negative control was very small, approaching 0, while the R value increased when Toxoplasma gondii was present. Based on this, it can be used for further determination of the presence or absence of Toxoplasma gondii, reducing subjective errors from naked-eye observation.

[0104] Example 8 Anti-interference analysis.

[0105] Genome samples from confirmed animal feces were selected and added at 1 μL to the positive control system of the SPA reaction as the experimental group. The reaction was carried out according to the optimal reaction system and conditions obtained in the experiment, and the real-time quantitative curve and the color change of the solution after the reaction were observed.

[0106] The results are as follows Figure 15 The results showed that the Ct values ​​of the experimental groups with added animal fecal genome were close to those of the positive control, and all experimental groups with added animal fecal genome and the positive control showed a yellow colorimetric result under naked eye. This indicates that the constructed biosensor has excellent anti-interference ability for extremely high concentrations of complex genomes, and even for genomes that may undergo non-specific amplification during PCR amplification.

[0107] Example 9: Actual sample detection.

[0108] The actual fecal genomes of 11 confirmed cases of dogs or cats were selected and used as templates for the SPA reaction. The reaction was carried out according to the optimal reaction system and conditions obtained from the experiment, and the real-time quantitative curves and color changes of the reaction tubes were observed.

[0109] The results are as follows Figure 16As shown, except for sample H, the Ct values ​​and naked-eye colorimetric results of all samples were close to those of the negative control. This result is consistent with the diagnostic results of real samples. For the questionable sample H, eight replicate experiments were performed. All eight tests turned yellow after the reaction, indicating the presence of Toxoplasma gondii in sample H. The above results show that the constructed biosensor has good detection performance in real samples and can detect cases missed by PCR.

[0110] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0111]

[0112]

Claims

1. A method for visually detecting Toxoplasma gondii genome closure for non-diagnostic purposes, characterized in that, SPA primers were designed using Toxoplasma gondii gene fragments as targets to perform isothermal amplification of SPA, combined with H + Responding to relevant reagents enables closed-tube visual detection; The Toxoplasma gondii gene fragment is 328-372 of GenBank: AF146527.1; The SPA primers are: Toxo-HF primers: sequence as shown in SEQ ID NO: 1; Toxo-HR primers: sequence as shown in SEQ ID NO: 2; Toxo-F primers: sequence as shown in SEQ ID NO: 3; and Toxo-R primers: sequence as shown in SEQ ID NO: 4; The system for SPA isothermal amplification is as follows: The H + The relevant reagent was a 0.1% BTB solution; There are two methods for evaluating the detection method: I. Direct visual observation: SPA isothermal amplification will cause pH changes in the reaction system. The concentration of the target gene can be preliminarily determined based on the degree of color change of BTB. II. Combined with smartphone analysis: Take a picture of the BTB colorimetric results, and then perform RGB analysis using mobile phone software to roughly determine the target gene concentration.

2. The detection method according to claim 1, characterized in that, The amplification reaction program is 58-70℃ for 85-100 min, then 80℃ for 10 min.

3. A non-diagnostic Toxoplasma gondii genome closed-tube visualization detection kit, characterized in that, Includes commercially available PCR tubes, SPA isothermal amplification system, and H + Response-related reagents; The commercially available PCR tubes are designed with an open cap. The SPA isothermal amplification system includes SPA amplification primers, dNTPs, betaine, MgSO4, buffer, Bst DNA polymerase, SYTO-9 dye, and water. The SPA amplification primers are: Toxo-HF primers: sequence as shown in SEQ ID NO: 1; Toxo-HR primers: sequence as shown in SEQ ID NO: 2; Toxo-F primers: sequence as shown in SEQ ID NO: 3; and Toxo-R primers: sequence as shown in SEQ ID NO: 4; The H + The response-related reagent was a 0.1% BTB solution.

4. The detection kit according to claim 3, characterized in that, The SPA isothermal amplification system is as follows: 。 5. The detection kit according to claim 3, characterized in that, The SPA isothermal amplification system is as follows: 。 6. The detection kit according to claim 3, characterized in that, The amplification reaction program is 58-70℃ for 80-100 min, then 80℃ for 10 min.

7. The detection kit according to claim 6, characterized in that, The amplification reaction program was 67℃ for 85 min, followed by 80℃ for 10 min.

8. The detection kit according to any one of claims 3-7, characterized in that, After the reaction is complete, observe the color change of the solution with the naked eye: if the solution is blue, there is no Toxoplasma gondii; if the solution is yellow, it contains a large number of Toxoplasma gondii; if the solution is yellowish-green, it contains a small number of Toxoplasma gondii.

9. The use of the test kit according to any one of claims 3-7 in the detection of Toxoplasma gondii for non-disease diagnosis purposes.

10. The application of the detection method according to any one of claims 1-2 in the detection of Toxoplasma gondii for non-disease diagnosis purposes.

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