A primer-probe combination, kit and method for detecting Cryptosporidium tayzei

Abstract

The present invention is applicable to the field of biological detection technology, and provides a primer-probe composition, a kit and a method for detecting Cryptosporidium tyzeri. The primer-probe composition includes an upstream primer, a downstream primer and a probe, and the nucleotide sequences of the upstream primer, the downstream primer and the probe are shown in SEQ ID NO.1 to SEQ ID NO.3. The present invention has practical significance in the actual application of the detection of Cryptosporidium tyzeri infection in experimental mice, reduces the dependence on detection equipment, shortens the detection time, and ensures the accuracy of the detection results. The test results of clinical samples showed that after amplification at 39°C for 30 minutes, the amplification product was dripped onto a test strip, and the results could be obtained in a short time, and the minimum detection limit for Cryptosporidium tyzeri oocysts reached 10 ‑2 While having good specificity, this detection method also achieves rapid and efficient detection process and intuitive visualization of detection results.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a primer-probe combination, a kit and a method for detecting Cryptosporidium tyzeri. Background Art

[0002] Cryptosporidium tyzzeri is a natural susceptible pathogen that parasitizes the mouse intestine and can infect rodents such as mice, guinea pigs, and gerbils. On the one hand, the presence of Cryptosporidium tyzzeri is relatively common in experimental animals such as mice, which not only affects the quality of experimental animals, but also affects the experimental results to a certain extent. On the other hand, the Cryptosporidium tyzzeri mouse infection model can be used as an animal model for evaluating vaccines and drugs for the prevention and treatment of Cryptosporidium parvum. At present, the methods for diagnosing animal infection with Cryptosporidium tyzzeri mainly include microscopy and molecular detection technology. However, these methods have their own limitations: microscopy includes direct smear microscopy and staining microscopy. Although Cryptosporidium can be identified by the size, shape and internal structure of the oocysts, it faces many challenges in actual operation. The oocysts are small in diameter (only 4 to 6 µm) and have a fuzzy internal structure, making observation difficult. In addition, it is difficult to effectively distinguish oocysts from small particles such as mold and food debris in feces, resulting in a time-consuming identification process and requiring extensive professional knowledge. In contrast, molecular detection technology provides a new approach for the detection of Cryptosporidium tyzeri with its high sensitivity, stability and specificity. Among them, methods such as PCR and quantitative real-time polymerase chain reaction (qRT-PCR) have been developed and applied to the detection of Cryptosporidium tyzeri. However, these methods also have shortcomings, such as long time consumption and high dependence on large-scale detection equipment, which limits their extensive application in practical applications. Therefore, a rapid and simple detection method is of great practical significance for the detection and monitoring of Cryptosporidium tyzeri infection in experimental animals.

[0003] Recombinase-mediated amplification lateral flow chromatography (RAA-LFD) is a rapid on-site detection system established by combining recombinase-mediated amplification RAA (Recombinase-aid amplification RAA) technology with lateral flow chromatography test strips (Lateral flow dipsticks LFD). It is particularly suitable for in vitro diagnosis, veterinary medicine, food safety, biosafety and agriculture, and has the potential to be developed into a rapid detection kit. However, there is no report on the use of RAA-LFD for the detection of Cryptosporidium infection in Tyzer. To this end, the present invention proposes a primer probe composition, a kit and a method for detecting Cryptosporidium Tyzer. Summary of the invention

[0004] The object of the present invention is to provide a primer-probe combination, a kit and a method for detecting Cryptosporidium tyzeri, aiming to solve the problems raised in the above-mentioned background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A primer-probe combination for detecting Cryptosporidium tyzeri, comprising an upstream primer, a downstream primer and a probe, wherein the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1; the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2; and the nucleotide sequence of the probe is shown in SEQ ID NO.3;

[0007] The 5' end of the downstream primer is biotinylated; the 5' end of the probe is modified by 6-carboxyfluorescein, the relevant G base is replaced by a [THF] residue, and the 3' end is modified by a C3 Spacer.

[0008] A kit for detecting Cryptosporidium tyzeri, the kit comprising the primer-probe combination described above, wherein the primer-probe combination comprises 1.0 μL of a 10 μM upstream primer, 1.0 μL of a 10 μM downstream primer and 0.6 μL of a 10 μM probe.

[0009] Furthermore, the kit also includes RAA amplification reaction reagents, controls and lateral flow chromatography test strips.

[0010] Furthermore, the RAA amplification reaction reagent includes a reaction tube containing RAA freeze-dried particles, a reaction buffer, a starter and purified water; the components of the reaction tube containing RAA freeze-dried particles include recombinase, DNA polymerase, single-stranded DNA binding protein, dNTP and exonuclease.

[0011] Furthermore, the control includes a positive control and a negative control; the DNA template of the positive control is fecal genomic DNA infected with Cryptosporidium tyzeri; the DNA template of the negative control is fecal genomic DNA not infected with Cryptosporidium tyzeri.

[0012] A method for detecting Cryptosporidium tyzeri using the above-mentioned kit comprises the following steps:

[0013] Step S1, extracting genomic DNA of the sample to be tested;

[0014] Step S2, RAA amplification: mixing the primer-probe combination and the extracted genomic DNA of the sample to be tested in a reaction tube of a RAA amplification reaction reagent to form an amplification reaction system, and performing a RAA amplification reaction;

[0015] Step S3, LFD detection of RAA amplification products: using lateral flow chromatography test strips to detect RAA amplification products.

[0016] Furthermore, the reaction temperature of the RAA amplification reaction is 39° C., and the reaction time is 30 min.

[0017] Furthermore, the specific process of step S3 is as follows:

[0018] Dilute the RAA amplification product to 350 μL with PBS, take 10 μL of the amplification product and drop it on the sample pad, then drop 100 μL of the test strip buffer on the sample pad, react at room temperature for 5 minutes, and observe the results with the naked eye:

[0019] If both the control line and the test line of the lateral flow chromatography test strip are colored, the RAA amplification product is positive and the sample contains Cryptosporidium tyzeri DNA;

[0020] If only the control line of the lateral flow chromatography test strip shows a band, and no band appears on the test line, the RAA amplification product is negative, and there is no Cryptosporidium tyzeri DNA in the sample;

[0021] If the quality control line of the lateral flow chromatography test strip does not show color, the test result is invalid.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention has practical significance in the detection of experimental mice infected with Cryptosporidium tyzeri. It not only reduces the dependence on detection equipment, but also shortens the detection time, while ensuring the accuracy of the detection results. The test results of clinical samples showed that after amplification at 39°C for 30 minutes, the amplification product only needs to be dripped onto the test strip, and the results can be obtained in a short time, and the minimum detection limit of Cryptosporidium tyzeri oocysts reaches 10 -2 While having good specificity, this detection method also achieves rapid and efficient detection process and intuitive visualization of detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The specificity of RAA-LFD in detecting the genomic DNA of Cryptosporidium tyzeri; genomic DNAs 1-9 are: negative control group, Cryptosporidium tyzeri, Giardia duodenalis, Pentatrichomoniasis hominis, Toxoplasma gondii, Cryptosporidium parvum, Escherichia coli, Salmonella, and Tritrichomonas muris.

[0025] Figure 2 Optimization of reaction temperature for RAA-LFD detection of Cryptosporidium tyzeri genomic DNA; 1-6: the reaction temperatures were 33℃, 35℃, 37℃, 39℃, 41℃, and 43℃, respectively.

[0026] Figure 3Optimization of reaction time for RAA-LFD detection of Cryptosporidium tyzeri genomic DNA; 1-6: reaction times were 10min, 15min, 20min, 25min, 30min and 35min respectively.

[0027] Figure 4 This is the sensitivity test of RAA-LFD for detecting genomic DNA of Cryptosporidium typhimurium; 1-8: 1-7 means the number of oocysts is 10 3 10 2 10, 10 0 10 -1 pcs, -10 -2 10 -3 8 were negative control groups.

[0028] Figure 5 The results of conventional PCR and RAA-LFD detection of Cryptosporidium in 24 rat feces samples; (a) is the result of conventional PCR detection, and (b) is the result of RAA-LFD detection. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0031] Example 1. One embodiment of the present invention provides a primer-probe combination for detecting Cryptosporidium tyzeri, the primer-probe combination comprising an upstream primer, a downstream primer and a probe; the 5' end of the downstream primer is biotinylated; the 5' end of the probe is modified with 6-carboxyfluorescein (6-FAM), the relevant G base is replaced by a [THF] residue, and the 3' end is modified with C3Spacer.

[0032] The nucleotide sequence of the primer-probe combination is shown in Table 1:

[0033] Table 1 Nucleotide sequences of primer-probe combinations

[0034]

[0035] Example 2, specificity test of RAA-LFD for detecting genomic DNA of Cryptosporidium tayzeri;

[0036] In order to verify the specificity of the RAA-LFD detection method, we prepared a 50μL RAA amplification reaction system: 40.9μL reaction buffer, 1.0μL 10μM upstream primer, 1.0μL 10μM downstream primer, 0.6μL 10μM probe, 2.0μL purified water, 2.0μL genomic DNA template and 2.5μL initiator were added to the reaction tube containing RAA freeze-dried particles. After fully mixing, the mixture was reacted in a 39℃ water bath for 30min, and fecal genomic DNA not infected with Cryptosporidium tyzeri was used as a negative control. In this experiment, Cryptosporidium tyzeri, Giardia duodenalis, Trichomonas humanis, Toxoplasma gondii, Trichomonas muris, Cryptosporidium parvum, Escherichia coli and Salmonella were selected for detection to determine whether the RAA-LFD detection method established in this experiment is specific.

[0037] Use lateral flow chromatography test strips to detect RAA amplification products: dilute the RAA amplification product to 350 μL with PBS, take 10 μL and drop it on the sample pad of the test strip, then add 100 μL of test strip buffer on the sample pad, react at room temperature for 5 minutes, and observe the results with the naked eye.

[0038] The results are as follows Figure 1 As shown, only the genome of Cryptosporidium tyzeri could be amplified well, which proved that this method had strong specificity for detecting Cryptosporidium tyzeri.

[0039] Example 3, Optimization of reaction conditions for detecting Cryptosporidium tayzeri genomic DNA by RAA-LFD;

[0040] RAA-LFD detection was carried out according to the reaction system described in Example 2, and the reaction temperature and reaction time were optimized.

[0041] 1) Reaction temperature optimization:

[0042] The reaction temperature was set to 6 gradients: 33°C, 35°C, 37°C, 39°C, 41°C and 43°C. The reaction time was set to 30 min. The reaction temperature at which the detection line showed obvious color was determined as the optimal reaction temperature.

[0043] The results of reaction temperature optimization are as follows Figure 2 As shown, the optimal reaction temperature is 39°C.

[0044] 2) Response time optimization:

[0045] The reaction time was set to 7 gradients: 10 min, 15 min, 20 min, 25 min, 30 min and 35 min, and the reaction temperature was set to 39 °C. The reaction time when the detection line was obviously colored was determined as the optimal reaction time.

[0046] The reaction time optimization results are as follows Figure 3As shown, the optimal reaction time is 30 min.

[0047] Example 4, sensitivity test of RAA-LFD in detecting genomic DNA of Cryptosporidium tayzeri;

[0048] To evaluate the sensitivity of the RAA-LFD assay, genomic DNA was extracted from purified Cryptosporidium tyzeri oocysts and the samples were analyzed by 10 3 10 2 10, 10 0 10 -1 pcs, -10 -2 10 -3 The genomic DNA was diluted in a gradient manner according to the number of oocysts, and the diluted genomic DNA was used as the template for RAA amplification reaction. The amplification reaction was carried out in a 39°C water bath for 30 minutes. Figure 4 As shown in Figure 2, with the increase of template concentration, the color of the detection line gradually deepened. When the number of oocysts reached 10 -2 The test line begins to show color when the number of oocysts reaches 10 -1 When the number of oocysts reaches or exceeds 10, the test line will be clearly colored. -2 When the number of samples was 0.04, all samples could be detected by the RAA-LFD detection method, which proved that the method had high sensitivity.

[0049] Example 5: Detection of clinical samples;

[0050] In order to verify the accuracy of the RAA-LFD detection method in clinical applications, we used RAA-LFD and conventional PCR methods to detect 24 mouse fecal samples. Figure 5 As shown in (b), three of the mouse feces samples showed positive characteristics. At the same time, the results of conventional PCR detection (such as Figure 5 (a) also shows that 3 of the 24 samples also showed positive characteristics. The test results of the two methods are consistent, as shown in Table 2:

[0051] Table 2 Results of RAA-LFD and conventional PCR detection samples

[0052]

[0053] This indicates that the RAA-LFD detection method has high accuracy and reliability in clinical applications.

[0054] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A primer-probe combination for detecting Cryptosporidium tayze, characterized in that: It includes an upstream primer, a downstream primer and a probe, wherein the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1; the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2; and the nucleotide sequence of the probe is as follows: [6-FAM]-CGGAGAAGGTACTCCAGCTGCGACCTTGAAGT[THF]TGGTGGTTACACTATTG-[C3Spacer]; The 5' end of the downstream primer is biotinylated; the 5' end of the probe is modified by 6-carboxyfluorescein, the relevant G base is replaced by a [THF] residue, and the 3' end is modified by a C3 Spacer.

2. A kit for detecting Cryptosporidium tayze, characterized in that: The kit comprises the primer-probe combination of claim 1, wherein the primer-probe combination comprises 1.0 μL of a 10 μM upstream primer, 1.0 μL of a 10 μM downstream primer, and 0.6 μL of a 10 μM probe.

3. The kit according to claim 2, characterized in that The kit also includes a RAA amplification reaction reagent, a positive control, a negative control and a lateral flow chromatography test strip.

4. The kit according to claim 3, characterized in that The RAA amplification reaction reagent comprises a reaction tube containing RAA freeze-dried particles, a reaction buffer, a starter and purified water; the components of the reaction tube containing RAA freeze-dried particles include recombinase, DNA polymerase, single-stranded DNA binding protein, dNTP and exonuclease.

5. The kit according to claim 3, characterized in that The DNA template of the positive control is the genomic DNA of feces infected with Cryptosporidium tyzeri; and the DNA template of the negative control is the genomic DNA of feces not infected with Cryptosporidium tyzeri.

6. A method for detecting Cryptosporidium tyzeri using the kit according to any one of claims 2 to 5 for non-disease diagnosis purposes, characterized in that: The following steps are involved: Step S1, extracting genomic DNA of the sample to be tested; Step S2, RAA amplification: mixing the primer-probe combination and the extracted genomic DNA of the sample to be tested in a reaction tube of a RAA amplification reaction reagent to form an amplification reaction system, and performing a RAA amplification reaction; Step S3, LFD detection of RAA amplification products: using lateral flow chromatography test strips to detect RAA amplification products.

7. The method according to claim 6, characterized in that The reaction temperature of the RAA amplification reaction is 39° C. and the reaction time is 30 min.

8. The method according to claim 6, characterized in that The specific process of step S3 is as follows: Dilute the RAA amplification product to 350 μL with PBS, take 10 μL of the amplification product and drop it on the sample pad, then drop 100 μL of the test strip buffer on the sample pad, react at room temperature for 5 minutes, and observe the results with the naked eye: If both the control line and the test line of the lateral flow chromatography test strip are colored, the RAA amplification product is positive and the sample contains Cryptosporidium tyzeri DNA; If only the control line of the lateral flow chromatography test strip shows a band, and no band appears on the test line, the RAA amplification product is negative, and there is no Cryptosporidium tyzeri DNA in the sample; If the quality control line of the lateral flow chromatography test strip does not show color, the test result is invalid.

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