Visualization method for rapidly detecting bovine babesiosis and application thereof

Abstract

The invention belongs to the technical field of biological detection, and provides a visualization method for rapidly detecting bovine babesia disease and application of the method. Specific SBP2 multi-copy conservative genes of bovine babesia are targeted, high-specificity ERA primers are designed, an ERA-CRISPR / Cas12a two-stage signal amplification system is established, specific recognition is conducted on amplification products through a CRISPR / Cas12a system, and the specific Babesia disease is detected. And activating a fluorescence signal. Results show that the method has no cross reaction on five common blood pathogens such as babesia vole and the like, the detection sensitivity for the babesia bovis reaches 10 copies / mu L, and on-site visual diagnosis can be completed within 40 minutes.

Description

Technical Field

[0001] This invention belongs to the field of parasitic disease detection technology, and relates to bovine babesiosis, specifically to a rapid visualization method for detecting bovine babesiosis and its application. Background Technology

[0002] Bovine babesiosis is a global animal blood disease transmitted by hard ticks (ixodidae). It is caused by various Babesia parasites. Infected cattle often exhibit symptoms such as fever, hemolytic anemia, hemoglobinuria, and jaundice; acute cases can lead to death. Commonly used drugs for treatment include imidazolidinedione, triazine, and atorvatroquinone. However, the toxicity and side effects of these chemical drugs can damage the animal's body and lead to drug resistance. Bovine babesiosis is widespread in Africa, Australia, Central America, and South America. In China, it affects 21 regions, including Gansu, Qinghai, and Xinjiang, hindering the development of the global cattle industry. Accurate early diagnosis can effectively control the spread of bovine babesiosis. Currently, the commonly used laboratory diagnostic methods are PCR and ELISA. When Babesia develops into a merozoite, the spheroids within it secrete 4 proteins, from SBP1 to SBP4. SBP2 shows high conservation among different geographical isolates and is encoded by a multi-copy gene (13 copies). Chung et al. established an ELISA detection method using spheroid proteins from Bovine Babesia (B. bovis) as molecular targets. Its sensitivity (98.7%) was higher than that of the ELISA detection of rod-shaped proteins from Bovine Babesia (60%), indicating that spheroid proteins have serological diagnostic value in the diagnosis and detection of Babesia.

[0003] Diagnostic methods for bovine babesiosis can be broadly categorized into pathogen microscopic detection, serological diagnostic methods, and molecular biological techniques. At the grassroots level, disease surveillance departments still rely on traditional microscopic examination for diagnosis, which is susceptible to errors due to factors such as slide preparation and staining techniques, particularly in differentiating between morphologically similar parasites. Conventional serological diagnostic methods suffer from insufficient antigen sources and poor sensitivity and specificity. In our region, a large number of cattle die from babesiosis annually from March to July. While methods such as PCR, nested PCR, multiplex PCR, and reverse linear blot hybridization (RLB) are becoming increasingly sophisticated due to their high specificity and sensitivity, they require expensive equipment, demanding high standards for operating environments and personnel, and have long reaction times (approximately 2 hours). Therefore, the only feasible approach is to develop a simple, convenient, and practical method for the differential diagnosis of bovine babesiosis, which holds significant importance in clinical diagnosis, treatment, and research.

[0004] Therefore, this invention aims to provide a rapid visualization method for detecting bovine babesiosis and its application, employing enzymatic recombinase amplification (ERA) combined with CRISPR / Cas12a technology. Enzymatic recombinase amplification (ERA) is a highly efficient on-site detection method due to its simple primer design, low temperature requirements, and ease of storage. ERA-CRISPR Cas12a can amplify the target gene fragment and amplify the fluorescence signal at 37°C. This method offers advantages such as ease of operation, rapid amplification, and real-time monitoring of results.

[0005] While the SBP2 gene of bovine babesiosa has been proven to have diagnostic value, the issue of fluctuating detection sensitivity due to its 13-copy characteristic remains unresolved. This invention significantly improves the detection rate of low-load samples by designing ERA primers targeting the conserved region and combining them with a dual validation mechanism of CRISPR / Cas12a. Summary of the Invention

[0006] The purpose of this invention is to provide a rapid visualization method for detecting bovine babesiosis and its application. This invention has no cross-reactivity with a variety of common bovine pathogens such as Theileria annulata, Babesia villiformis, Trypanosoma eeris, Rickettsia, and Aplasticia, exhibiting good specificity and high sensitivity, and can detect samples with low parasite loads.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] This invention provides an ERA-CRISPR / Cas12a detection method for bovine babesiosis, comprising an ERA primer pair in an ERA amplification system, a crRNA primer in a CRISPR / Cas12a detection system, and an ssDNA probe used in conjunction with the crRNA primer. The ERA primer pair includes an upstream primer SBP2-F and a downstream primer SBP2-R.

[0009] The upstream primer SBP2-F: 5'-GATTGATGTATCCAGGTTTGATAATAGTTC-3', and the downstream primer SBP2-R: 5'-ACAAGCACGCCACTAGATTCATAAATAA-3';

[0010] The crRNA primers:

[0011] 5'-UAAUUUCUACUAAGUGUAGAUAGUUGGUAACUCUGACGACUUGU-3'; The ssDNA probe: 6-FAM-TTATT-BHQ1.

[0012] This invention provides a rapid visualization method for detecting bovine babesiosis, characterized in that the specific steps of the detection method include:

[0013] (1) Extraction of DNA from the sample;

[0014] (2) Using sample DNA as the template to be tested, the target DNA is amplified isothermally by the ERA primer pair described in claim 1, and the amplification product is coupled to the CRISPR / Cas12a detection system.

[0015] (3) Result judgment: The fluorescent reporter molecule was activated by the trans-cleavage activity of Cas12a. The infection status of bovine Babesia was determined by the fluorescence signal under blue light / ultraviolet light excitation. The positive product was green under blue light and grayish-white under ultraviolet light.

[0016] Preferably, the ERA isothermal amplification in step (2) specifically involves mixing 20 μL of reconstitution buffer, 2.5 μL each of the upstream and downstream primers of the ERA described in claim 1, 21 μL of deionized water, and 2 μL of sample DNA. Finally, 2 μL of activator is added, followed by brief centrifugation and incubation at 37℃~42℃ for 10~30 minutes to complete the amplification.

[0017] Preferably, the ERA isothermal amplification reaction conditions in step (2) are: incubation at 37°C for 20 minutes.

[0018] Preferably, the CRISPR / Cas12a detection system and ERA amplification product in step (2) are coupled as follows: 2 μL ERA amplification product, 2 μL NEBuffer r2.1, 1 μL single-stranded DNA, 1 μL crRNA, 1 μL Cas12a and 15 μL nuclease-free water are mixed thoroughly and then incubated at 37°C for 20-30 minutes.

[0019] Preferably, the concentration of the ssDNA probe is 500–1250 nM.

[0020] Preferably, the concentration of the ssDNA probe is 1250 nM.

[0021] This invention also provides the application of an ERA primer pair, a crRNA primer, and an ssDNA probe used in conjunction with the crRNA primer in the detection of bovine babesiosis.

[0022] The present invention also provides a visualization method for rapid detection of bovine babesiosis, and its application in the detection of bovine babesiosis.

[0023] The present invention also provides a kit for rapid detection of bovine babesiosis, comprising the above-mentioned ERA primer pair, crRNA primer and ssDNA probe used in conjunction with the crRNA primer or the above-mentioned detection method.

[0024] The present invention also provides an application of the above-mentioned kit in the detection of bovine babesiosis.

[0025] The beneficial effects of this invention are:

[0026] This invention designs specific ERA primers targeting the conserved region of the SBP2 gene, utilizing their multi-copy characteristic to enhance signal intensity for initial signal amplification. The ERA amplification product activates the trans-cleavage activity of Cas12a, achieving secondary fluorescence signal amplification, thus establishing a visual and rapid detection method for bovine babesiosis. Results show that the detection method of this invention has high specificity, and through fluorescence signal interpretation under blue / ultraviolet light, it can achieve visual and rapid detection of bovine babesiosis. Attached Figure Description

[0027] Figure 1 This is a schematic diagram showing the results of using the optimal primers in the embodiments of the present invention (NC is the blank control);

[0028] Figure 2 This is a schematic diagram showing the results of the optimal reaction temperature in an embodiment of the present invention;

[0029] Figure 3 This is a schematic diagram showing the optimal reaction time in an embodiment of the present invention;

[0030] Figure 4 This is a schematic diagram of the crRNA screening results in an embodiment of the present invention (A shows the results under blue light and ultraviolet light; B shows the gray value analysis of fluorescence intensity; NC is the blank control, crDNA1+plasmid:crRNA1+10). 6 copies / μL plasmid, crDNA2+plasmid:crRNA2+10 6 copies / μL plasmid, crDNA1+NC1:crRNA1+enzyme-free water, crDNA1+NC2:crRNA2+enzyme-free water; ***P<0.001, intergroup comparison);

[0031] Figure 5 This is a schematic diagram of ssDNA concentration optimization in an embodiment of the present invention (A is the result diagram under blue light and ultraviolet light; B is the gray value analysis of fluorescence intensity; NC is the blank control, and PC is the gray value of ssDNA at different concentrations);

[0032] Figure 6This is a schematic diagram of the specific detection results in the embodiments of the present invention (A is the result diagram under blue light and ultraviolet light; B is the gray value analysis of fluorescence intensity; where Bbov is bovine Babesia, Bm is vole Babesia, Tann is bovine Theileria ringosa, Anap is anaplasm, Rick is Rickettsia, Teva is Trypanosoma eiri; NC is blank control; ****P<0.0001, inter-group comparison);

[0033] Figure 7 This is a schematic diagram of the sensitivity detection results in an embodiment of the present invention (NC is the blank control);

[0034] ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05);

[0035] Figure 8 This is a schematic diagram of the ERA-CRISPR / Cas12a clinical sample validation results in an embodiment of the present invention;

[0036] Figure 9 This is a schematic diagram of the results of PCR testing of clinical samples in an embodiment of the present invention (NC is a blank control). Detailed Implementation

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0040] Example

[0041] 1 Experimental Methods

[0042] 1.1 Construction of positive standard plasmids

[0043] DNA extraction was performed according to the DNA extraction kit instructions. Based on the GenBank database and the published SBP2 gene sequence (GenBank JN974305.1), the SBP2 gene was amplified using existing primers in the laboratory. After analysis of the PCR product by 1% agarose gel electrophoresis, the SBP2 gene was cloned into the PMD 19-T vector and then transformed into E. coli DH5α competent cells. After amplification culture in Amp+ / LB medium, the plasmid was sequenced for verification. The concentration was determined using a micro spectrophotometer and calculated using the formula: copy number = [6.02 × 10⁻⁶]. 23Calculate the copy number by dividing the plasmid concentration (g / mL) by the relative molecular mass of the plasmid (g / mol). Dilute the plasmid sample to a final concentration of 10 with 1×TE buffer. 6 -10 0 The sample was collected at 1 copy / μL and stored at -20℃ as a positive standard plasmid for later use.

[0044] 1.2 ERA Primer Design

[0045] Based on conserved gene sequences already registered in GenBank (accession number: JN974305.1), using

[0046] Primer Premier 5.0 software was used to design primers according to the ERA primer requirements. Three pairs of primers were designed for screening, and primers suitable for detection were selected. The primer sequences are shown in Table 1. The primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0047] Table 1 ERA primer sequences

[0048]

[0049] 1.3crRNA and ssDNA design

[0050] Based on the principles of crRNA primer design, two crRNAs were designed using the Liang Cpf1 online CRISPR design tool. The fluorescent reporter probe (ssDNA Reporter) is a short single-stranded DNA sequence with a 5' label of 6-carboxyfluorescein (6-FAM) and a 3' modification of black pore quencher-1 (BHQ1). The probe sequence is 6-FAM-TTATT-BHQI. Both crRNA and ssDNA were synthesized and purified by Shanghai Sangon Biotech Co., Ltd.

[0051] Table 2 shows the crRNA and ssDNA sequences.

[0052]

[0053] 1.4 Two-step method for constructing ERA-CRISPR / Cas12a

[0054] First, the SBP2 gene fragment of *Babesia bovis* was isothermally amplified using a basic ERA kit. A 48 μL reaction mixture was prepared by mixing 20 μL of reconstitution buffer, 2.5 μL each of forward and reverse primers (10 μM), 21 μL of deionized water, and 2 μL of template. This premixed solution was transferred to a reaction tube containing a lyophilized enzyme precipitate, and 2 μL of activator was added. After brief centrifugation, the mixture was incubated at 37°C for 20 minutes. The CRISPR / Cas12a detection system consisted of 2 μL of amplification product, 2 μL of NEBuffer r2.1, 1 μL of ssDNA (10 μM), 1 μL of crRNA (1 μM), 1 μL of Cas12a (1 μM), and nuclease-free water to a final volume of 20 μL. After thorough mixing, the system was incubated at 37°C for 20–30 minutes. Fluorescence signals were observed by irradiating the reaction tubes with blue light. To optimize experimental conditions, parameters such as ERA primer selection, reaction time, temperature, crRNA selection, and ssDNA concentration were systematically evaluated. The entire experiment was conducted at 10 6 Experiments were conducted using template concentrations of copies / μL to obtain optimal conditions.

[0055] 1.4.1 ERA primer screening:

[0056] ERA basic amplification was performed using the three primer pairs in Table 1, with a template of 10. 6 Copies / μL, incubated at 37℃ for 20 min, and then analyzed by electrophoresis after the reaction to select the best primers.

[0057] 1.4.2 Optimal reaction temperature for ERA amplification:

[0058] Since the reaction temperature range of the ERA system is between 37℃ and 42℃, in order to determine the optimal reaction temperature for amplification, 10 6 Plasmid standards of copies / μL were amplified at temperatures of 35℃, 37℃, 39℃, and 41℃ for 30 min. After the reaction, the products were analyzed by electrophoresis to select the optimal reaction temperature.

[0059] 1.4.3 Optimal reaction time for ERA amplification:

[0060] Copy number 10 6 Plasmid standards of copies / μL were incubated at 37℃ for 10 min, 15 min, 20 min, and 30 min, respectively. The products were then analyzed by electrophoresis to select the optimal reaction time.

[0061] 1.4.4 crRNA screening

[0062] According to the two sets of crRNA sequences designed in Table 2, different crRNA reaction systems were configured. After the reaction was completed, the results were observed under blue light and UV light. Under blue light, the positive product was green, and under ultraviolet light, the positive product was grayish-white. The optimal crRNA sequence was selected, and the gray value was analyzed.

[0063] 1.4.5 ssDNA concentration screening

[0064] The CRISPR system was optimized by preparing reaction systems using ssDNA at concentrations of 500 nM, 750 nM, 1000 nM, and 1250 nM.

[0065] 1.5B. Bovis-ERA-CRISPR / Cas12a Specificity Test

[0066] The established method was used to detect positive DNA samples of several pathogens, including bovine babesi (Bbov), vole babesi (Bm), bovine annular Theileria (Tann), anaplasmosis (Anaplasma), Rickettsia (Rick), and Teva (Teva), with ddH2O as a negative control. ERA-CRISPR / Cas12a specificity detection was performed at 37°C.

[0067] 1.6B. Bovis-ERA-CRISPR / Cas12a Sensitivity Test

[0068] 10 respectively 6 -10 0 Seven copies / μL of plasmid were used as templates for ERA amplification using primers SBP2-F1 and SBP2-R1. 2 μL of the ERA product was then used as dsDNA, and ddH2O was set up as a negative control. The detection system was prepared using fluorescent reporter molecules according to the determined optimal reaction system, and the reaction was repeated three times. After the reaction, the fluorescence status was observed under a blue light gel imaging system.

[0069] 1.7 Data Analysis

[0070] All results were analyzed for grayscale values ​​using ImageJ, and all data were analyzed using GraphPad Prism 8.0. Differences between groups were assessed using one-way ANOVA in SPSS 27.0. A p-value less than 0.05 was considered statistically significant.

[0071] 2 Experimental Results

[0072] 2.1 ERA Primer Screening

[0073] The results are as follows Figure 1As shown, the F1R1 band was the brightest and no nonspecific bands were produced, so F1R1 was used as the optimal primer pair in later experiments.

[0074] 2.2 Screening for the optimal reaction temperature for ERA amplification

[0075] The results are as follows Figure 2 As shown, the bands of B. bovis-ERA gradually brighten with increasing temperature; the amplified bands gradually lighten at 37℃. Therefore, the optimal reaction temperature for B. bovis is 37℃.

[0076] 2.3 Screening for optimal reaction time for ERA amplification

[0077] The results are as follows Figure 3 As shown, a specific band was obtained after 10 minutes of reaction, and the band became clearer after 15 minutes. Gray-scale analysis of the electrophoresis results showed that the product yield was highest at 20 minutes of B. bovis-ERA reaction, significantly different from the product yields at 15 and 30 minutes. Figure 3 Therefore, its optimal reaction time was determined to be 20 minutes.

[0078] 2.4CrRNA Primer Screening

[0079] The results showed that crRNA1 showed no fluorescent signal, while crRNA2 appeared green under blue light and grayish-white under ultraviolet light, exhibiting strong fluorescence intensity and showing a significant difference from the negative result. Figure 4 A) indicates that the designed crRNA2 is effective. The experimental results were analyzed using ImageJ software to determine the fluorescence intensity (grayscale analysis). Figure 4 B), the crRNA+plasmid group showed extremely significant differences from other groups (P<0.001).

[0080] 2.5ssDNA concentration screening

[0081] The results are as follows Figure 5 As shown, the fluorescence intensity increases with increasing concentration. Figure 5 A) When the concentration is 1250 nM, the fluorescence intensity is the strongest, and the grayscale analysis also increases with increasing concentration. Figure 5 B). Therefore, 1250 nM was chosen as the optimal concentration of ssDNA for B. bovis.

[0082] The final CRISPR / Cas12a assay system consisted of: 2 μL amplification product, 2 μL NEBuffer r2.1, 1 μL ssDNA (1250 nM), 1 μL crRNA (1 μM), 1 μL Cas12a (1 μM), and nuclease-free water to a final volume of 20 μL.

[0083] 2.6B. Bovis-ERA-CRISPR / Cas12a Specificity Test

[0084] like Figure 6 As shown, the ERA-CRISPR / Cas12a detection results and grayscale results (P<0.0001) revealed that only the bovine babesi sample triggered specific cleavage of CRISPR / Cas12a to produce a positive result. Other pathogens did not produce a fluorescent signal due to the lack of conserved SBP2 target sites, indicating that this method has good specificity for B. bovis.

[0085] 2.7B. Bovis-ERA-CRISPR / Cas12a Sensitivity Test

[0086] Based on the ERA-CRISPR / Cas12a detection results and grayscale results, it was found that ( Figure 7 The detection limit of 10 copies / μL (AB) validates the synergistic effect of multi-copy targets and dual-system amplification, which can effectively detect early low-load infections.

[0087] Application Examples

[0088] 1. Experimental Procedure

[0089] Total DNA was extracted from 16 bovine blood samples and stored at -20°C. Bovine babesiosis was detected in the 16 bovine blood samples using an optimized final version of the ERA-CRISPR / Cas12a detection method and conventional PCR.

[0090] 2 Experimental Results

[0091] The results are shown in Table 3. Figure 8 and Figure 9 As shown, PCR detected 4 positive samples of B. bovis, with a positive rate of 25%; the ERA method also detected 4 positive samples, showing 100% consistency with the PCR method. Comparing the two methods for detecting B. bovis, the ERA method showed 100% specificity and sensitivity, but significantly reduced detection time, further demonstrating that the ERA method is faster and more time-saving than the PCR method.

[0092] Table 3. Detection results of clinical samples

[0093]

[0094] In summary, the visual detection method for bovine babesioma established in this invention uses primers designed based on the conserved multi-copy SBP2 gene. After ERA isothermal amplification, it is combined with the CRISPR system to ensure zero cross-reactivity with several pathogens with similar clinical symptoms, including *Theileria annulata*, *Babesia voles*, *Rickettsia*, *Trypanosoma eeris*, and *Aphalomyeloidea*. Utilizing the 13-copy characteristic of the SBP2 gene to enhance signal intensity, it can stably detect early-stage infection samples with parasite loads as low as 10 copies / μL. The ERA-CRISPR / Cas12a dual-system detection process is optimized to be completed in 40 minutes, meeting the needs of rapid screening in epidemic areas.

[0095] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. An ERA-CRISPR / Cas12a detection method for bovine babesiosis, characterized in that, This includes the ERA primer pair in the ERA amplification system, the crRNA primer in the CRISPR / Cas12a detection system, and the ssDNA probe used in conjunction with the crRNA primer. The ERA primer pair contains the upstream primer SBP2-F and the downstream primer SBP2-R. The upstream primer SBP2-F: 5'-GATTGATGTATCCAGGTTTGATAATAGTTC-3', the downstream primer SBP2-R: 5'-ACAAGCACGCCACTAGATTCATAAATAA-3'; the crRNA primer: 5'-UAAUUUCUACUAAGUGUAGAUAGUUGGUAACUCUGACGACUUGU-3'; The ssDNA probe: 6-FAM-TTATT-BHQ1.

2. A rapid visualization method for detecting bovine babesiosis, characterized in that, The specific steps of the detection method include: (1) Extraction of DNA from the sample; (2) Using sample DNA as the template to be tested, the target DNA is amplified isothermally by the ERA primer pair described in claim 1, and the amplification product is coupled to the CRISPR / Cas12a detection system. (3) Result judgment: The infection status of bovine Babesia was determined by fluorescence signal under blue light / ultraviolet light excitation. The positive product was green under blue light and grayish-white under ultraviolet light.

3. The visualization method for rapid detection of bovine babesiosis according to claim 2, characterized in that, The ERA isothermal amplification in step (2) is specifically as follows: 20 μL of reconstitution buffer, 2.5 μL each of the 10 μM ERA upstream and downstream primers described in claim 1, 21 μL of deionized water and 2 μL of sample DNA are mixed, 2 μL of activator is added and the mixture is briefly centrifuged, and then incubated at 37℃~42℃ for 10~30 minutes to complete the amplification.

4. The visualization method for rapid detection of bovine babesiosis according to claim 3, characterized in that, The reaction conditions for the ERA isothermal amplification in step (2) are: incubation at 37°C for 20 minutes.

5. The visualization method for rapid detection of bovine babesiosis according to claim 2, characterized in that, The CRISPR / Cas12a detection system and ERA amplification product coupling in step (2) are specifically as follows: 2 μL of ERA amplification product, 2 μL of NEBuffer r2.1, 1 μL of ssDNA, 1 μL of crRNA, 1 μL of Cas12a, and nuclease-free water are added to a final volume of 20 μL. After thorough mixing, the mixture is incubated at 37°C for 20–30 minutes.

6. The visualization method for rapid detection of bovine babesiosis according to claim 5, characterized in that, The concentration of the ssDNA probe is 500–1250 nM.

7. The visualization method for rapid detection of bovine babesiosis according to claim 6, characterized in that, The concentration of the ssDNA probe was 1250 nM.

8. The application of the ERA primer pair, crRNA primer, and ssDNA probe used in conjunction with the crRNA primer as described in claim 1, or the detection method as described in any one of claims 2-7, in the detection of bovine babesiosis.

9. A kit for rapid detection of bovine babesiosis, characterized in that, Includes the ERA primer pair, crRNA primer and ssDNA probe used in conjunction with the crRNA primer as described in claim 1, or the detection method as described in any one of claims 2-7.

10. The application of the kit as described in claim 9 in the detection of bovine babesiosis.

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