Primer pair, kit and application of kit

Abstract

The invention discloses a primer pair, a kit and application of the kit, specifically, Cas12a2 nuclease is combined with PCR amplification to detect entamoeba histolytica nucleic acid, and compared with ELISA, IFA and other methods, entamoeba histolytica can be detected more quickly and conveniently, the detection reliability and accuracy are improved, and the kit has the advantages that the detection time is shortened, and the detection cost is reduced. The lower detection limit reaches 1 nucleic acid copy per microliter, and the method has a wide application prospect in the field of nucleic acid detection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a primer pair, a reagent kit, and the application of the reagent kit. Background Technology

[0002] Entamoeba histolytica is a parasitic protozoan that causes amoebiasis. The main symptoms after infection include amoebic colitis or extraintestinal abscesses, causing diarrhea, fever, and other discomfort. The life cycle of Entamoeba histolytica includes two stages: trophozoite and cyst. Human infection is primarily through ingestion, via consuming food or water contaminated with feces containing mature cysts, or using contaminated utensils. Amoebic colitis is a leading cause of severe diarrhea in children under 5 years old in low-income countries. Amoebiasis is a global health problem; according to the World Health Organization, approximately 500 million people are infected with this parasite. About 90% are asymptomatic carriers, and the remaining 10% exhibit clinical symptoms such as colitis, amoebic dysentery, and extraintestinal amoebiasis. It causes 55,000 deaths annually, making it the second leading cause of parasitic death after malaria. The most common clinical manifestation of extraintestinal infection is amoebic liver abscess (ALA), which can be fatal if not diagnosed and treated promptly. Although amoebiasis is prevalent worldwide, especially in developing countries, there is still no vaccine to prevent the disease. Therefore, it is particularly important to detect Entamoeba histolytica in drinking water, food, etc., in order to block the source of infection.

[0003] In recent years, with the development of disciplines such as parasitology, immunology, and molecular biology, many methods have been applied to the detection of amoebae. The diagnosis of amoebae mainly relies on clinical symptoms and laboratory test results. Laboratory diagnosis can be performed through microscopic examination, in vitro culture, antigen-antibody reactions, and molecular detection. Microscopic observation of amoebae in fecal, bodily fluid, or tissue samples is considered the "gold standard" for diagnosis. Indirect hemagglutination assays (IHA), enzyme-linked immunosorbent assays (ELISA), and indirect immunofluorescence assays (IFA), developed based on the principle of antigen-antibody reactions, detect specific antibodies against *Entamoeba histolytica* in serum. Specific primers are used to amplify amoebae DNA via polymerase chain reaction (PCR). These methods are effective for the detection of *Entamoeba histolytica*, but they still have certain limitations in practical applications. For example, microscopic observation is only applicable to intestinal amoeba infection and cannot diagnose patients with amoebic liver abscess. IHA test has low sensitivity and specificity and is prone to cross-reaction with other parasites, resulting in false positive results. ELISA and IFA are expensive, require professional operation, are time-consuming, and can only be performed after antibodies appear, which is a deficiency in the early diagnosis of infection. At the same time, it is difficult to distinguish between past and current infection. PCR has the problem of non-specific amplification.

[0004] Chinese patent CN116626293A discloses a microfluidic chip for detecting *Entamoeba histolytica* infection. The microfluidic chip's channels are modified to allow the surface of the channels to bind recombinant *Entamoeba histolytica* antigens. The surface of the channels is then further bound to these recombinant antigens, resulting in a microfluidic chip for detecting *Entamoeba histolytica* infection. Each recombinant *Entamoeba histolytica* antigen bound to the channel surface can bind up to one *Entamoeba histolytica*-specific antibody, and each *Entamoeba histolytica*-specific antibody can be bound to up to one gold nanosphere bound to anti-human IgG antibody. After adding a biological sample, the number of gold nanospheres determines whether the sample is infected with *Entamoeba histolytica*. This microfluidic chip's detection relies on the presence of *Entamoeba histolytica*-specific antibodies and cannot be used for early infection detection. Furthermore, the judgment result depends on the relative number of gold nanospheres, making it susceptible to misjudgment due to human error.

[0005] With the rapid development of the CRISPR-Cas system, different Cas effector proteins are being used to identify pathogens based on their unique properties. The principle involves a ternary complex formed by the nucleic acid fragment of the pathogen to be detected, crRNA, and Cas protein. After the nucleic acid fragment is cleaved, the trans-cleavage activity of the Cas protein is activated, cleaving a fluorescent probe with fluorescent and fluorescence quenching groups at both ends. This cleavage causes the fluorescent and fluorescence quenching groups to separate, resulting in fluorescence. This method can improve detection sensitivity and lower the limit of detection. However, there is currently no Cas protein-based detection technology for Entamoeba histolytica. Summary of the Invention

[0006] In order to overcome the above-mentioned shortcomings of the existing technology, the purpose of this invention is to provide a SuCas12a2 reaction system for detecting Entamoeba histolytica and its application.

[0007] The objective of this invention is achieved by at least one of the following technical solutions.

[0008] A first aspect of the present invention is to provide a primer pair, comprising an upstream primer and a downstream primer; the upstream primer sequence is shown in SEQ ID NO.1; and the downstream primer sequence is shown in SEQ ID NO.2.

[0009] SEQ ID NO.1: 5'-TTGTGAAGTTATTGGAGTGAGTGT-3'

[0010] SEQ ID NO.2: 5'-AACTATTCTGATTGTTTCTTCCGT-3'

[0011] Furthermore, an RNA polymerase promoter sequence is added to the 5' end of the upstream primer.

[0012] Furthermore, the RNA polymerase promoter sequence is the T7 promoter sequence, and the upstream primer sequence for adding the T7 promoter sequence is shown in SEQ ID NO.3.

[0013] SEQ ID NO.3: 5'-GAAATTAATACGACTCACTATAGGGTTGTGAAGTTATTGGAGTG AGTGT-3'

[0014] Furthermore, the primer pair specifically recognizes the target gene of Entamoeba histolytica prx gene.

[0015] A second aspect of the present invention is to provide a kit comprising the primer pair, Cas12a2 nuclease, and crRNA as described in any of the preceding claims.

[0016] Furthermore, the sequence of the crRNA is shown in SEQ ID NO.4.

[0017] SEQ ID NO.4:5'-GUCUAAACCGACCCAAUAAUUUCUACUGUUGUAGAUCUACUCCUCCUUUACUUUUAUCUGC-3'

[0018] Furthermore, the Cas12a2 nuclease includes the SuCas12a2 nuclease, which is prepared by the following method:

[0019] The pET52b-SuCas12a2 plasmid was transformed into competent cells for prokaryotic expression to obtain expressed bacterial precipitate. The SuCas12a2 nuclease was obtained by lysing the bacteria, Ni-NTA affinity chromatography, and molecular exclusion chromatography.

[0020] SuCas12a2 nuclease is an RNA-guided nuclease capable of cleaving ssDNA, dsDNA, and ssRNA. Currently, there is no nucleic acid detection technology based on SuCas12a2 nuclease. In this invention, the SuCas12a2 nuclease is a protein purified by affinity chromatography and size exclusion chromatography, containing a His tag at its C-terminus, and expressed in prokaryotes using expression bacteria. The encoding nucleic acid sequence of the SuCas12a2 nuclease is shown in SEQ ID NO. 5.

[0021]

[0022] Furthermore, the kit also includes a fluorescent probe selected from ssRNA fluorescent probes, ssDNA fluorescent probes, or dsDNA fluorescent probes.

[0023] Furthermore, the fluorescent probe is an ssRNA fluorescent probe, and the sequence of the ssRNA fluorescent probe is 5'-UUUUU-3', with a fluorescent reporter group attached to the 5' end and a non-fluorescent quencher group attached to the 3' end.

[0024] Preferably, the fluorescent reporter group is 6-FAM and the non-fluorescent quencher group is BHQ1.

[0025] Furthermore, the kit also includes RNA polymerase.

[0026] Furthermore, the RNA polymerase is T7 RNA polymerase.

[0027] Furthermore, the kit also includes buffer, dNTP mixture, and RNA inhibitor.

[0028] A third aspect of the present invention is to provide the application of the kit described in any of the above claims for the detection of Entamoeba histolytica for non-diagnostic purposes.

[0029] Mature cysts of Entamoeba histolytica can survive in water, soil, and contaminated food. Detecting the presence of Entamoeba histolytica in environmental or food samples can help control the source of infection and thus prevent amoebiasis.

[0030] Furthermore, the application of the kit in detecting Entamoeba histolytica specifically includes the following steps:

[0031] (1) Extract DNA from the sample to be tested;

[0032] (2) Using the DNA of the sample to be tested as a template, PCR amplification reaction was performed to obtain the amplification product. PCR amplification reaction was performed with RNase-free water as a negative control.

[0033] (3) Take the above amplification product, add fluorescent probe, SuCas12a2 nuclease and crRNA, and perform SuCas12a2 reaction to obtain Cas12a2 fluorescent cleavage reaction product.

[0034] (4) Measure the fluorescence signal of the Cas12a2 fluorescence cleavage reaction product and determine whether it contains Entamoeba histolytica based on the fluorescence signal.

[0035] Furthermore, the reaction system of the SuCas12a2 reaction described in step (3) includes 10-50 nM of SuCas12a2 nuclease.

[0036] The SuCas12a2 nuclease exhibits strong cleavage activity at concentrations ranging from 10 nM to 50 nM.

[0037] Preferably, in step (3), the concentration of SuCas12a2 is 50 nM.

[0038] Furthermore, the reaction system of the SuCas12a2 reaction in step (3) also includes 200 nM crRNA, 1 mM NTP mixture, 250 nM fluorescent probe, 2 U / μl RNA inhibitor and 5 U / μl T7 RNA polymerase.

[0039] Furthermore, in step (3), the temperature of the SuCas12a2 reaction is 37°C, and the reaction time is 15-45 min.

[0040] Preferably, in step (3), the reaction time of SuCas12a2 is 30 min.

[0041] If the fluorescence signal value of the test sample is statistically different from that of the negative control, then the test sample is infected with Entamoeba histolytica; if the fluorescence signal value of the test sample is not statistically different from that of the negative control, then the test sample is not infected with Entamoeba histolytica.

[0042] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0043] (1) The kit of the present invention combines the characteristics of PCR and Cas12a2 enzyme to achieve qualitative detection of Entamoeba histolytica nucleic acid.

[0044] (2) The kit of the present invention is based on a SuCas12a2 reaction system developed using the SuCas12a2 enzyme for detecting Entamoeba histolytica. This system has extremely high sensitivity, with a detection limit of 1 copy per microliter. Compared with methods such as ELISA and IFA, the present invention can detect Entamoeba histolytica more quickly and conveniently, while reducing costs and improving the reliability and accuracy of detection. It ensures the repeatability and accuracy of the detection results and can accurately detect Entamoeba histolytica in the early stages of infection.

[0045] (3) The kit of the present invention is based on nucleic acid for the detection of Entamoeba histolytica, and the detection limit is 1 copy per microliter, which can be used to detect whether Entamoeba histolytica is present in environmental / food samples.

[0046] (4) The kit of the present invention is specific for the recognition of Entamoeba histolytica nucleic acid and will not be misdetected due to the influence of other types of amoeba or parasites. Attached Figure Description

[0047] Figure 1 This is a schematic diagram illustrating the experimental principle for detecting Entamoeba histolytica in this invention.

[0048] Figure 2 This is a bar chart showing the fluorescence values ​​at the endpoint (45 min) of the five suspected infected samples in Example 2.

[0049] Figure 3 The image shows a line graph of fluorescence values ​​at different time points for five suspected infected samples from Example 2.

[0050] Figure 4 This is a bar chart showing the fluorescence values ​​at the detection endpoint (45 min) of different concentrations of amplified products in Example 3.

[0051] Figure 5 This is a bar chart showing the fluorescence values ​​at the endpoint (45 min) for detecting different types of amoebas or parasites in Example 4.

[0052] Figure 6 The image shows the fluorescence results of serum samples from 5 suspected infected individuals tested using the IFA method in Comparative Example 1. Detailed Implementation

[0053] The specific implementation of the present invention will be further described below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0054] The experimental principle of this invention for detecting Entamoeba histolytica is as follows: Figure 1 As shown, the target fragment of the Entamoeba histolytica prx gene was first amplified by PCR (the amplicon contained the SuCas12a2 recognition sequence). Then, the PCR product was purified, and finally, 1 μl of the purified PCR product was subjected to the SuCas12a2 reaction (37℃, reaction time 30-45 minutes).

[0055] The specific process of the SuCas12a2 reaction is as follows: the amplification product containing the T7 promoter sequence is transcribed into RNA by T7 RNA polymerase and NTP mixture. RNA, crRNA and SuCas12a2 nuclease form a ternary complex. At this time, the trans-cleavage activity of SuCas12a2 nuclease is activated, which non-specifically cleaves the additional fluorescent probe added in the test tube. The presence of Entamoeba histolytica infection is identified by the fluorescence signal.

[0056] The SuCas12a2 reaction depends on the concentration of SuCas12a2 nuclease. In the reaction system, a concentration of 10 nM to 50 nM of SuCas12a2 nuclease has strong cleavage activity.

[0057] Example 1

[0058] Preparation of SuCas12a2 nuclease

[0059] The SuCas12a2 gene was synthesized from its entirety, digested with two enzymes, and ligated into the pET52b multiple cloning site to construct the pET52b-SuCas12a2 plasmid. The pET52b-SuCas12a2 plasmid was transformed into *E. coli* Nico21(DE3) competent cells and cultured overnight at 37°C on LB Broth agar plates containing 100 μg / mL ampicillin. Clones containing the SuCas12a2 plasmid were isolated from the plates and incubated in LB Broth medium with shaking at 220 rpm at 37°C until the optical density (OD600) at 600 nm reached 0.6–0.8. Isopropyl β-thiogalactoside (IPTG) was added to the LB medium to a final concentration of 1 mM, and protein expression was induced at 18°C ​​for 18 h. Cells were placed on ice for 30 min, then collected by centrifugation at 10,000 rpm for 10 min at 4°C and stored at -80°C until purification.

[0060] Proteins were purified by affinity chromatography and size exclusion chromatography. All procedures were performed on ice. Cell particles were resuspended in 20 mL of lysis buffer (25 mM Tris-HCl pH 7.2, 150 mM NaCl, 20 mM imidazole, 20% glycerol) and sonicated at 200 W for 30 min, while adding PMSF protease inhibitor to a final concentration of 2.5 mM and Nonidet P-40 to a final mass fraction of 0.15%. Lysozyme (GPC biotechnology; CAS No.: 9001-63-2) to a final concentration of 200 μg / mL was then added, and lysis was performed at 30 °C and 100 rpm for 45 min. The lysate was sonicated again for 30 min in the same manner, followed by centrifugation at 11,000 rpm for 30 min, and large insoluble molecules were removed using a 0.45 μm filter. Add the filtered supernatant to 2 mL of Ni-NTA resin (Merck, 69670) and rotate at 4°C for 1 h, then allow it to pass through the resin twice naturally. Wash with 50 mL of wash buffer I (25 mM Tris-HCl pH 7.2, 300 mM NaCl, 50 mM imidazole, 10% glycerol), then wash with 5 mL of wash buffer II (25 mM Tris-HCl pH 7.2, 300 mM NaCl, 100 mM imidazole, 10% glycerol) containing 100 mM imidazole. Elute the protein with 10 mL of Ni-NTA elution buffer (25 mM Tris-HCl pH 7.2, 500 mM NaCl, 250 mM imidazole, 10% glycerol, 1 mM DTT), then concentrate the eluent containing SuCas12a2 nuclease to 5 mL using a 100 kDa MWKO Vivaspin 500 concentrator. Further purification was performed using size exclusion chromatography on a HiLoad™ 16 / 600 Superdex™ 200 pg column. The elution buffer was converted to 2 mL of storage buffer (100 mM HEPES pH 7.2, 150 mM KCl, 2 mM MgCl2, 10% glycerol, 1 mM DTT), rapidly frozen in liquid nitrogen, and stored at -80 °C for later use. Protein purity was identified by SDS-PAGE and Coomassie Brilliant Blue staining, and protein quantification was performed using a Qubit protein assay kit. The purified SuCas12a2 nuclease was obtained.

[0061] Example 2

[0062] Two tissue samples and three fecal samples from suspected Entamoeba histolytica infections were selected as substrate templates. The Entamoeba histolytica prx gene fragment was amplified using Ex Taq polymerase with primers containing the T7 promoter sequence. The amplification product contained a crRNA-binding sequence.

[0063] The 20 μl PCR reaction system includes:

[0064]

[0065]

[0066] Simultaneously, the DNA sample to be tested was replaced with RNase-free water as a negative control. The PCR program was set as follows: 94℃ for 3 min, 94℃ for 15 s, 58℃ for 30 s, 72℃ for 30 s, and 72℃ for 7 min, for a total of 40 cycles. The amplified products were then purified using the AxyPrep™ PCR purification kit. The purified products were then subjected to a SuCas12a2 reaction.

[0067] The 20 μl SuCas12a2 reaction mixture contained 2 μl 10×HOLMES buffer 1 (TOLOBIO; 32005), 1 μl PCR product, and purified SuCas12a2 nuclease to a final concentration of 50 nM, 200 nM crRNA, 1 mM NTP mixture (ATP, CTP, GTP, and UTP all to a final concentration of 1 mM), 2 U / μl RNase inhibitor, 5 U / μl T7 RNA polymerase, and 250 nM ssRNA fluorescent probe (5' / 6-FAM-UUUUU-BHQ1 / 3'). Nuclease-free water was added if necessary. The mixture was transferred to a qPCR plate and incubated at 37°C in an ABI 7500 instrument for 45 min, with fluorescence intensity measured every 30 s. The endpoint fluorescence values ​​of each group were statistically analyzed using Graphpad, and then divided by 500,000 to obtain the relative fluorescence values. These values ​​were compared with the endpoint fluorescence values ​​of the negative control group to analyze the statistical differences and determine whether the serum samples were from individuals infected with amoeba.

[0068] like Figure 2 As shown, all five samples suspected of being infected with Entamoeba histolytica produced significant fluorescence signals at the endpoint, indicating that all five samples were positive for Entamoeba histolytica. Furthermore, the fluorescence intensity of samples 4 and 5 was higher than that of samples 1-3, suggesting that samples 4 and 5 had higher levels of Entamoeba histolytica nucleic acid, indicating a deeper degree of infection.

[0069] like Figure 3 As shown, the results of dynamic fluorescence values ​​indicate that samples 4 and 5 can quickly reach the fluorescence plateau phase in about 10 minutes, while samples 1-3 only produce fluorescence values ​​that are statistically different from the negative control in about 25 minutes. This suggests that the detection time and sensitivity are related to the degree of infection in clinical samples. The deeper the infection, the faster a positive result can be detected.

[0070] Example 3

[0071] Determination of the lowest detection limit of Entamoeba histolytica using SuCas12a2 combined with PCR method

[0072] Genomic DNA was extracted from *Entamoeba histolytica*. PCR amplification was performed using 100 ng of *Entamoeba histolytica* genomic DNA as a template, with prx-dsDNA upstream primer (SEQ ID NO.3) and prx-dsDNA downstream primer (SEQ ID NO.2). RNase-free water was used as a negative control. The amplification program was: 94℃ for 3 min, 94℃ for 15 s, 58℃ for 30 s, 72℃ for 30 s, and 72℃ for 7 min, for a total of 25 cycles. The PCR products were purified, quantified using a Qubit assay kit, and converted to copy number. The PCR products were serially diluted with RNase-free water to achieve concentrations of 10-1 ppm. 4 10 3 10 2 10 1 10 0 10 -1 10 -2 Copy the sample, then add 1 μL of each to the SuCas12a2 reaction system. The 20 μL SuCas12a2 reaction system contains 2 μL of 10×HOLMES buffer, 1 μL of PCR amplification product, purified SuCas12a2 nuclease (final concentration 50 nM), 200 nM crRNA, 1 mM NTP mixture, 2 U / μL RNase inhibitor, 5 U / μL T7 RNA polymerase, and 250 nM ssRNA fluorescent probe. Add nuclease-free water if necessary. Incubate at 37°C for 45 minutes in an ABI 7500 real-time quantitative PCR instrument, detecting fluorescence every 30 seconds. Divide the endpoint fluorescence value by 500,000 and analyze in Graphpad to determine the limit of detection for Entamoeba histolytica using this method.

[0073] The results are as follows Figure 4 As shown, 10 per microliter 4 10 3 10 2 10 1 10 0 The copy numbers were significantly different from those of the negative control, indicating that the detection limit of the present invention for Entamoeba histolytica is 1 copy per microliter, which has extremely high sensitivity. When environmental / food samples are contaminated and contain trace amounts of Entamoeba histolytica cysts, they can be accurately detected.

[0074] Example 4

[0075] Specificity of SuCas12a2 combined with PCR method for the detection of Entamoeba histolytica

[0076] The whole genome DNA of various amoebae (Desparnea, Entamoeba histolytica, Entamoeba nori, and Giardia lamblia) was extracted. Using the primers described above, PCR amplification was performed with 100 ng of DNA as a template. RNase-free water was used as a negative control. The amplification program was: 94℃ for 3 minutes, 94℃ for 15 seconds, 58℃ for 30 seconds, 72℃ for 30 seconds, and 72℃ for 7 minutes, with 35 cycles. After purification, 1 μL of the PCR product was added to the SuCas12a2 reaction system. The 20 μL SuCas12a2 reaction system contained 2 μL of 10×HOLMES buffer 1, a final concentration of 50 nM purified SuCas12a2 nuclease, a final concentration of 200 nM crRNA, a final concentration of 1 mM NTP mixture, 40 U RNase inhibitor, 100 U T7 RNA polymerase, 1 μL of PCR amplification product, and a final concentration of 250 nM ssRNA fluorescent probe. Nuclease-free water was added if necessary. The reaction was incubated at 37°C for 45 minutes in an ABI 7500 real-time quantitative PCR instrument, with fluorescence signals detected every 30 seconds. The endpoint fluorescence value was divided by 500,000 and analyzed in GraphPad.

[0077] The results are as follows Figure 5 As shown, when the test samples were *Entamoeba dispar*, *Entamoeba moss*, *Entamoeba nori*, and *Giardia lamblia*, their fluorescence intensity showed no significant statistical difference compared with the control group. This indicates that the method of the present invention is specific for the recognition of *Entamoeba histolytica* nucleic acid and is not affected by other types of amoebas or other parasites.

[0078] Comparative Example 1

[0079] Indirect immunofluorescence (IFA) detection of specific antibodies against Entamoeba histolytica in serum

[0080] 1. Collect the cultured Entamoeba histolytica trophozoites, wash the worms twice with 2 mL PBS, and remove the culture medium.

[0081] 2. Resuspend the worms in 2 mL of PBS, take 10 μl and fill the cell to count (1 × 10⁻⁶). 5 500 μl (5 × 10⁶ worms / mL) was taken. 4 (One worm body) was placed in an Eppendorf tube, centrifuged at 500 rcf for 5 minutes, and the supernatant was discarded.

[0082] 3. Add 1 mL of 4% paraformaldehyde and fix for 15 minutes.

[0083] 4. Centrifuge at 500 rcf for 5 minutes using a horizontal rotor.

[0084] 5. Resuspend the precipitated worms in 900 μl of PBS.

[0085] 6. Centrifuge the resuspended fluid to transfer the parasites onto glass slides using a cell smear machine. Add 100 μl of the resuspended fluid to each slide and centrifuge at 800 rpm for 10 minutes.

[0086] 7. Add 200 μl of 4% paraformaldehyde to the slide and fix for 15 minutes.

[0087] 8. Remove the paraformaldehyde, add 2 mL of PBS and wash gently.

[0088] 9. Add 100 μl of 5% BSA to each slide and seal for 15 minutes.

[0089] 10. Serum samples from the five suspected Entamoeba histolytica infections described in Example 2 were diluted 1:64 with 5% BSA as the primary antibody, and serum from healthy individuals was diluted 1:64 with 5% BSA as the negative control. 200 μl of each diluted serum sample was added to each slide, and the slides were incubated in a humidified chamber at room temperature for 30 minutes.

[0090] 11. Wash three times with 2 mL PBS, 5 minutes each time, for a total of 15 minutes.

[0091] 12. Add Alexa Fluor anti-human IgG secondary antibody (Thermo; Alex 488goat antihuman IgG) diluted 1:100, and incubate in a humidified chamber for 30 minutes.

[0092] 13. Wash three times with 2 mL PBS, 5 minutes each time, for a total of 15 minutes.

[0093] 14. Cover with a coverslip, seal the slide, and observe and photograph it under a fluorescence microscope.

[0094] The results are as follows Figure 6 As shown, samples 1-5 were all positive, with samples 4 and 5 exhibiting stronger fluorescence signals. This is consistent with the results of Example 2. Compared to indirect immunofluorescence (IFA) detection of Entamoeba histolytica-specific antibodies in serum, the method of this invention is lower in cost, easier to operate, and applicable to early infection detection.

[0095] The above embodiments are merely preferred embodiments of the present invention and are only used to explain the present invention, not to limit the present invention. Any changes, substitutions, modifications, etc., made by those skilled in the art without departing from the spirit and essence of the present invention should be within the protection scope of the present invention.

[0096]

[0097]

[0098]

Claims

1. A pair of primers, characterized in that, comprising an upstream primer and a downstream primer; the sequence of the upstream primer is shown as SEQ ID NO. 1; the sequence of the downstream primer is shown as SEQ ID NO.

2.

2. The primer pair according to claim 1, characterized in that, the upstream primer is added with a RNA polymerase promoter sequence at the 5' end; the RNA polymerase promoter sequence is a T7 promoter sequence, and the sequence of the upstream primer added with the T7 promoter sequence is shown as SEQ ID NO.

3.

3. A kit characterized in that, comprising the primer pair of any one of claims 1-2, a Cas12a2 nuclease, and a crRNA.

4. The kit of claim 3, wherein the sequence of the crRNA is shown as SEQ ID NO. 4; and / or, the Cas12a2 nuclease comprises a SuCas12a2 nuclease, which is prepared by the following method: the pET52b-SuCas12a2 plasmid is transformed into a competent cell for prokaryotic expression to obtain an expression bacterial precipitate, and the SuCas12a2 nuclease is obtained by lysing the bacteria, Ni-NTA affinity chromatography, and molecular exclusion chromatography.

5. The kit of claim 3, wherein the kit further comprises a fluorescent probe selected from an ssRNA fluorescent probe, an ssDNA fluorescent probe, or a dsDNA fluorescent probe; the fluorescent probe is an ssRNA fluorescent probe, the sequence of the ssRNA fluorescent probe is 5'-UUUUU-3', the 5' end of the ssRNA fluorescent probe is connected with a fluorescent reporter group, and the 3' end is connected with a non-fluorescent quenching group.

6. The kit of claim 3, wherein the kit further comprises a RNA polymerase; the RNA polymerase is a T7 RNA polymerase.

7. Use of the kit of any one of claims 3-6 in detecting Entamoeba histolytica for non-diagnostic purposes.

8. Use according to claim 7, characterized in that, comprising the following steps: (1) extracting DNA of a sample to be tested; (2) performing a PCR amplification reaction with the DNA of the sample to be tested as a template to obtain an amplification product, and performing a PCR amplification reaction with RNase-free water as a negative control; (3) taking the amplification product, adding a fluorescent probe, a SuCas12a2 nuclease, and a crRNA, and performing a SuCas12a2 reaction to obtain a Cas12a2 fluorescent cleavage reaction product; (4) measuring the fluorescence signal of the Cas12a2 fluorescent cleavage reaction product, and determining whether the sample to be tested contains Entamoeba histolytica according to the fluorescence signal.

9. Use according to claim 8, characterized in that, The reaction system of the SuCas12a2 reaction of step (3) comprises 10-50 nM of the SuCas12a2 nuclease.

10. Use according to claim 8, characterized in that, The reaction system of the SuCas12a2 reaction of step (3) further comprises 200 nM of the crRNA, 1 mM of an NTP mixture, 250 nM of the fluorescent probe, 2 U / μl of an RNA inhibitor, and 5 U / μl of the T7 RNA polymerase; and / or, The temperature of the SuCas12a2 reaction is 37℃, and the time of the SuCas12a2 reaction is 15-45 min.

Images