Primer pair, kit and method for detecting echinococcosis based on circulating free DNA

Abstract

The invention discloses a primer pair, a kit and a method for detecting echinococcosis based on circulating free DNA, and relates to the field of molecular biological detection. According to the method, a sample to be detected is added into an RPA-CRISPR amplification detection system for synchronous isothermal amplification and CRISPR / Cas reaction, the sample to be detected is cfDNA extracted from blood of a patient, and the RPA-CRISPR amplification detection system comprises an RPA primer pair, crRNA, a Cas12a reporter probe, Cas12a protein, a buffer solution and nuclease-free water. Circulating free DNA of a patient is taken as a detection sample, a tissue sample of the patient does not need to be obtained, the patient cannot be wounded, implementation is easy, and the patient acceptability is high; the RPA-CRISPR amplification detection system constructed by the invention can simultaneously perform nucleic acid amplification and detection, so that the detection period is shortened, the cost is reduced, the operation process is simplified, a nucleic acid amplification product does not need to be uncovered and transferred, and the risk that a sample to be detected is polluted can be reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology detection, and particularly relates to a primer pair, a kit and a method for detecting echinococcosis based on circulating free DNA. BACKGROUND

[0002] Echinococcosis is one of the neglected tropical diseases listed by the World Health Organization (WHO), which is an important zoonosis caused by the larval stage of Echinococcus spp. Echinococcosis is mainly divided into cystic echinococcosis (CE) and alveolar echinococcosis (AE), which are caused by Echinococcus granulosus (Eg) and Echinococcus multilocularis (Em), respectively. Both cystic and alveolar echinococcosis are serious diseases, with a higher incidence of CE, while AE has a much higher disability rate and mortality than CE due to its cancer-like characteristics.

[0003] Both CE and AE show a long asymptomatic incubation period after infection. Most human AE mainly involves the liver and can be transferred to other organs through invasive behavior, damaging the function of local organs. The treatment of echinococcosis is complex, including extensive surgery, percutaneous sterilization and long-term antiparasitic treatment. However, if there is a lack of timely diagnosis and careful management, especially in the late stage of AE, the mortality rate is extremely high. Therefore, accurate and early diagnosis is crucial for timely intervention and improved prognosis.

[0004] Generally, the diagnosis of echinococcosis is based on epidemiological data, imaging tools, nucleic acid detection and serological tests. Imaging technology has advantages in lesion positioning, size and invasiveness determination, but has limitations in early infection detection. Serological antigens and antibodies can be detected in the early stage of infection, but due to non-specific cross-reactions and the influence of immunosuppressive therapy, the specificity and sensitivity of serological methods are poor. Nucleic acid detection techniques, including polymerase chain reaction (PCR) and isothermal amplification techniques, have high sensitivity and specificity, and are suitable for early differential diagnosis, but traditional molecular detection methods usually rely on infected tissue samples.

[0005] Therefore, it is of great significance to provide a high-performance molecular diagnostic method based on non-invasive samples for large-scale screening and early diagnosis of echinococcosis. SUMMARY

[0006] The present application discloses a primer, a kit and a method for detecting echinococcosis based on circulating free DNA, to solve the defects of the echinococcosis detection method in the related art.

[0007] In order to solve the above problems, the technical scheme adopted by the present application is as follows: The application is based on a primer pair for detecting echinococcosis by circulating free DNA, comprising a primer pair used for amplifying circulating free DNA of a patient by RPA, For Echinococcus granulosus, the primer pair comprises one or more of F1-R1, F1-R2, F2-R1, F2-R2, F2-R3, F3-R1, F3-R2, F3-R3, wherein, F1: AACGATCACTCCTATTCTAGCATGTGTGTGAA; R1: AGTGAGATGAGTGAGAAGGAGTGCTCGCACCG; F1: AACGATCACTCCTATTCTAGCATGTGTGTGAA; R2: AGATATTTCACATTCAGTGAGATGAGTGAGAA; F2: AATGCAAGCAGCAGATGCCTACCCATCCGGAA; R1: AGTGAGATGAGTGAGAAGGAGTGCTCGCACCGA; F2: AATGCAAGCAGCAGATGCCTACCCATCCGGAA; R2: AGATATTTCACATTCAGTGAGATGAGTGAGAA; F2: AATGCAAGCAGCAGATGCCTACCCATCCGGAA; R3: AAGTTGCCTTCGTTAGGTGGAGATATTTCACA; F3: AGCAGATGCCTACCCATCCGGAAAAGCATTTAA; R1: AGTGAGATGAGTGAGAAGGAGTGCTCGCACCGA; F3: AGCAGATGCCTACCCATCCGGAAAAGCATTTAA; R2: AGATATTTCACATTCAGTGAGATGAGTGAGAA; F3: AGCAGATGCCTACCCATCCGGAAAAGCATTTAA; R3: AAGTTGCCTTCGTTAGGTGGAGATATTTCACA; For E. granulosus, the primer pair comprises one or more of F1-R1, F1-R3, F1-R4, F2-R1, F2-R3, F2-R4, F3-R2, F3-R4, wherein, F1: TAGGACACAGTGCCAGCATCTGCGGTTAGTC; R1: AGAGTAAATTAATAGGTGGACCATCCTTTAC; F1: TAGGACACAGTGCCAGCATCTGCGGTTAGTC; R3: GTGTACCTGAGCTAAACTCAATTCACATCAACA; F1: TAGGACACAGTGCCAGCATCTGCGGTTAGTC; R4: GTCCTAAACAATACCATATTACAACAATATTCC; F2: TGTTAAGATATATGTGGTGACAGGGATTAGATAC; R1: AGAGTAAATTAATAGGTGGACCATCCTTTAC; F2: TGTTAAGATATATGTGGTGACAGGGATTAGATAC; R3: GTGTACCTGAGCTAAACTCAATTCACATCAACA; F2: TGTTAAGATATATGTGGTGACAGGGATTAGATAC; R4: GTCCTAAACAATACCATATTACAACAATATTCC; F3: TAAAGGATGGTCCACCTATTAATTTACTCTT; R2: CTCTTATAAGCAGCACATAGACTTAGCTTAACTA; F3: TAAAGGATGGTCCACCTATTAATTTACTCTT; R4: GTCCTAAACAATACCATATTACAACAATATTCC.

[0008] According to one optional embodiment, for E. granulosus, the primer pair is F1-R1 and / or F2-R3; for E. multilocularis, the primer pair is F1-R1 and / or F2-R4.

[0009] The application is based on a kit for detecting echinococcosis by circulating free DNA, comprising an RPA primer pair, crRNA, a Cas12a reporter probe, Cas12a protein, buffer and nuclease-free water, and the RPA primer pair is mixed with the crRNA, Cas12a reporter probe, Cas12a protein, buffer and nuclease-free water to construct an RPA-CRISPR amplification detection system.

[0010] According to an optional embodiment, the crRNA sequence is a combination of a crRNA target sequence and an LbCas12a scaffold sequence.

[0011] According to an optional embodiment, for Echinococcus granulosus, the crRNA sequence is one of crRNA1, crRNA2, crRNA1: uaauuucuacuaaguguagauGGCAUCUAGUGAAGGAAAAAUAA; crRNA2: uaauuucuacuaaguguagauCUUCACUAGAUGCCGAAACUACC; For Echinococcus multilocularis, the crRNA sequence is one of crRNA1, crRNA2 and crRNA3, crRNA1: uaauuucuacuaaguguagauACCUAACAUUGCACAUAAAUA; crRNA2: uaauuucuacuaaguguagauCUAUGCAUCUUCCCCUAACAA; crRNA3: uaauuucuacuaaguguagauGCAGUGAGUGAUUCUUGUUAG.

[0012] According to an optional embodiment, in the RPA-CRISPR amplification detection system, 1 muL of the RPA primer pair, 1 muL of the crRNA, 0.6 muL of the Cas12a reporter probe, 1 muL of the Cas12a protein, 2 muL of the buffer and 14.4 muL of the nuclease-free water are included.

[0013] The application is based on a method for detecting echinococcosis by circulating free DNA, comprising the following steps: Step 100: Obtain cfDNA of a sample as a sample to be tested; Step 200: adding the sample to be tested into the kit, and synchronously performing constant temperature amplification and CRISPR / Cas reaction on the sample to be tested in the RPA-CRISPR amplification detection system to collect the fluorescence signal, The kit is a kit for detecting echinococcosis based on circulating free DNA according to any one of the technical solutions of the present application.

[0014] According to an optional embodiment, the cfDNA is extracted from peripheral blood of a patient as the sample to be tested.

[0015] According to an optional embodiment, the RPA-CRISPR amplification detection system is incubated at 35-45 DEG C for 40-70 min to synchronously perform constant temperature amplification and CRISPR / Cas reaction.

[0016] According to an optional embodiment, the RPA-CRISPR amplification detection system is incubated at 40 DEG C for 60 min to synchronously perform constant temperature amplification and CRISPR / Cas reaction.

[0017] The technical solutions adopted by the present application can achieve the following beneficial effects: In the first aspect, the present application provides primers, kits and methods for detecting echinococcosis based on circulating free DNA, which uses circulating free DNA of a patient as a detection sample. The circulating free DNA can exist in the blood of the patient. The circulating free DNA is separated, and then constant temperature amplification is performed on the circulating free DNA by using the primers to meet the detection requirements. Compared with the existing nucleic acid detection method relying on tissue samples, the detection method of the present application does not need to obtain a tissue sample of the patient, does not cause trauma to the patient, is easy to implement, and has high patient acceptance.

[0018] In the second aspect, the present application provides a method for detecting echinococcosis based on circulating free DNA. The RPA primer pair, crRNA, Cas12a reporter probe, Cas12a protein, buffer and nuclease-free water are mixed to construct an RPA-CRISPR amplification detection system. The sample to be tested is added to the RPA-CRISPR amplification detection system. The RPA-CRISPR amplification detection system can simultaneously perform constant temperature nucleic acid amplification and detection. Compared with the prior art, which performs nucleic acid amplification on the sample to be tested and then performs detection again, the present application shortens the detection period, reduces the cost, simplifies the operation process, and does not need to open the cover to transfer the nucleic acid amplification product, thereby reducing the risk of contamination of the sample to be tested. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description only constitute some embodiments of the present application, and other accompanying drawings can be obtained by those skilled in the art without any creative effort.

[0020] Figure 1 The end-point fluorescence intensity map of each RPA primer pair of Echinococcus granulosus in Example 1 is shown; Figure 2 The electrophoresis map of each RPA primer pair of Echinococcus granulosus in Example 1 is shown; Figure 3 The negative control electrophoresis map of each RPA primer pair of Echinococcus granulosus in Example 1 is shown; Figure 4 The real-time fluorescence curve map of each RPA primer pair of Echinococcus granulosus in Example 1 is shown; Figure 5 The end-point fluorescence intensity map of each RPA primer pair of Echinococcus multilocularis in Example 1 is shown; Figure 6 The electrophoresis map of each RPA primer pair of Echinococcus multilocularis in Example 1 is shown; Figure 7 The negative control electrophoresis map of each RPA primer pair of Echinococcus multilocularis in Example 1 is shown; Figure 8 The real-time fluorescence curve map of each RPA primer pair of Echinococcus multilocularis in Example 1 is shown; Figure 9 The detection map of crRNA activity evaluation based on selected RPA primers in Example 2 is shown, wherein A is the activity detection result map of crRNA for Echinococcus granulosus target in two-step RPA-CRISPR reaction, B is the activity detection result map of crRNA for Echinococcus multilocularis target in two-step RPA-CRISPR reaction, C is the effect evaluation result map of crRNA for Echinococcus granulosus target in one-pot RPA-CRISPR reaction, and D is the effect evaluation result map of crRNA for Echinococcus multilocularis target in one-pot RPA-CRISPR reaction; Figure 10 The performance analysis result map of RPA-CRISPR amplification detection system in Example 3 is shown, wherein A is the detection sensitivity result map for Echinococcus granulosus target, B is the linear analysis map of detection sensitivity for Echinococcus granulosus target, C is the detection sensitivity result map for Echinococcus multilocularis target, D is the linear analysis map of detection sensitivity for Echinococcus multilocularis target, and E is the specificity analysis map of RPA-CRISPR amplification detection system; Figure 11 FIG. 3 shows a diagram of RPA primer pairs and crRNA design for Echinococcus granulosus in Example 3, wherein a is RPA primer pairs and crRNA for E. granulosus repetitive sequence, and b is RPA primer pairs and crRNA for E. multilocularis mitochondrial sequence; Figure 12 FIG. 4 shows a diagram of clinical sample detection results in Example 4. DETAILED DESCRIPTION

[0021] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0022] The terms "first", "second", and the like in the specification and claims of the present application are used to distinguish similar objects, and are not used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged under appropriate circumstances, so that the embodiments of the present application can be implemented in an order other than those illustrated or described herein, and the objects distinguished by "first", "second", etc. are generally a category and do not limit the number of objects, for example, the first object can be one or more. In addition, "and / or" in the specification and claims means at least one of the connected objects, and the character " / ", generally represents a "or" relationship between the front and rear associated objects.

[0023] Several sequencing-based methods have confirmed the presence of Echinococcus circulating free DNA (cfDNA) fragments in patient plasma, which exhibit even better sensitivity than PCR detection of lesion samples, indicating that Echinococcus cfDNA has great potential as a non-invasive biomarker for liquid biopsy. Although the content of cfDNA in plasma is insufficient, the reported diagnostic sensitivity is low, but the recombinase polymerase amplification (RPA) technology can exponentially amplify nucleic acid fragments in a few minutes at room temperature, which is very suitable for deployment in resource-limited areas. In addition, given the binding of specific repeat sequence short palindromic repeat (CRISPR) RNA (crRNA) to CRISPR-associated (Cas) protein, the complementary target sequence can activate the transcleavage activity of the Cas protein, thereby achieving cascade signal amplification. Therefore, the integration of RPA and CRISPR system can further improve the detection sensitivity of low-concentration sequences.

[0024] Based on this, the application provides a primer pair, a kit and a method for detecting hydatidosis based on circulating free DNA, which separates circulating free DNA, and then the primer can be used for isothermal amplification, so as to meet the detection requirement. Compared with the existing nucleic acid detection method which depends on tissue samples, the detection method of the application does not need to obtain patient tissue samples, does not cause trauma to the patient, is easy to implement, and has high patient acceptance.

[0025] The primer pair, the kit and the method for detecting hydatidosis based on circulating free DNA provided by the application will be described in detail below through specific examples and application scenarios.

[0026] Embodiment 1 This embodiment specifically describes the screening of the primer pair.

[0027] This embodiment specifically describes the screening of the primer pair, which includes the following steps. Step 110: The repeat sequence of Eg (GenBank number: DQ157697.1) and the mitochondrial sequence of Em (GenBank number: AB018440.2) were downloaded from the National Center for Biotechnology Information database (NCBI). The target sequences of Eg and Em were synthesized and cloned into a pUC57 vector to construct standard plasmids.

[0028] The sequence of DQ157697.1 is as follows: ACACCACGCATGAGGATTACTGACTTGAACGATCACTCCTATTCTAGCATGTGTGTGAATGCAAGCAGCAGATGCCTACCCATCCGGAAAAGCATTTAACTTACCAGTGGGCCGTGCTGTGGAGGTAGTTTCGGCATCTAGTGAAGGAAAAATAAGACATCGGTGCGAGCACTCCTTCTCACTCATCTCACTGAATGTGAAATATCTCCACCTAACGAAGGCAACTTCACTGCGATTGCCCCCAAGATGCAACATTTCAAATGCTCGGT.

[0029]

[0030] Step 120: Based on the specific conserved regions of DQ157697.1 sequence and AB018440.2 sequence, RPA primers were designed by Primer & probe design for RPA / RAA online tool, and the primer sequences are shown in Table 1.

[0031] Table 1. RPA primer pairs involved in the present embodiment Step 130: The optimal primer combination was determined by online verification and experimental screening. Specifically, the RPA / RAA isothermal DNA amplification basic kit (lyophilized powder) of Ezassay Biotechnology was used for the reaction. The specific operation was as follows: in a 20 μL reaction system, 10 μL 2x resuspension buffer, 0.5 μL forward and reverse primers (20 μM), 2 μL DNA template, 2 μL 10x start-up liquid and 5 μL nuclease-free water were added. The PCR thermal cycler was used for amplification at 40℃ for 20 minutes. When real-time fluorescence detection was performed, 0.5x Evagreen dye was added. To obtain pure amplification products, 2 μL proteinase K (20 mg / ml) was used for treatment at 60℃ for 20 minutes, and then the products were identified by polyacrylamide gel electrophoresis. Finally, the primer combination that could produce amplification products with appropriate abundance and size was selected.

[0032] Figures 1-4 The end-point fluorescence intensity diagram, electropherogram, negative control electropherogram and real-time fluorescence curve diagram of each RPA primer pair of E. granulosus are shown respectively. From Figure 1 and Figure 4 it can be seen that for E. granulosus, the F1-R1 primer pair and the F2-R3 primer pair showed the best performance in terms of positive fluorescence intensity and false positive-false negative discrimination. The RPA positive products were further verified by electrophoresis, and showed high-density specific bands of the expected size, as shown in Figure 2 and Figure 3 .

[0033] Similarly, Figures 5-8 The end-point fluorescence intensity diagram, electropherogram, negative control electropherogram and real-time fluorescence curve diagram of each RPA primer pair of E. multilocularis are shown respectively. From Figures 5-8 it can be seen that for E. multilocularis, the F1-R1 primer pair and the F2-R4 primer pair showed the best efficiency and specificity.

[0034] Therefore, for E. granulosus, the F1-R1 primer pair and the F2-R3 primer pair were selected for subsequent experiments; for E. multilocularis, the F1-R1 primer pair and the F2-R4 primer pair were selected for subsequent experiments.

[0035] Example 2 This embodiment details the method for detecting hydatidosis based on circulating free DNA.

[0036] The method for detecting hydatidosis based on circulating free DNA of the present application comprises the following steps: Step 210: Obtain cfDNA of the sample as the sample to be tested.

[0037] The cfDNA can exist in the blood of the patient, and efficient separation of cfDNA is crucial for the detection of low-concentration target sequences. Specifically, the Quick-cfDNA Serum & Plasma kit (No. D4076) purchased from Zymo Research Company is used to extract plasma cfDNA, and this method exhibits the best target cfDNA separation efficiency and stable CRISPR signal. All operation steps are carried out at room temperature, and unless otherwise specified, the operation guidelines of the manufacturer are strictly followed. The specific steps are as follows: mix 1 mL of separated plasma with 250 µL S&P 5× digestion buffer and 100 µL proteinase K (20 mg / mL), incubate at 55°C for 30 minutes, then add 2.7 L S&P DNA binding buffer, and use Zymo-Spin III-S column to capture target DNA (centrifuge at 1000 g for 2 minutes). After two washes, 400 µL S&P DNA pretreatment buffer and 700 µL S&P DNA wash buffer are used respectively, and finally 50 µL DNA elution buffer is used to elute cfDNA at 60°C, and store at -80°C for use.

[0038] Step 220: Mix RPA primer pair, crRNA, Cas12a reporter probe, Cas12a protein, buffer and nuclease-free water to construct RPA-CRISPR amplification detection system. Add the sample to be tested to the RPA-CRISPR amplification detection system, and simultaneously perform constant temperature amplification and CRISPR / Cas reaction on the RPA-CRISPR amplification detection system to collect the fluorescence signal.

[0039] Preferably, the RPA primer pair is the one described in Example 1 for detecting echinococcosis based on circulating free DNA. More preferably, for E. granulosus, the primer pair comprises one or more of F1-R1, F1-R2, F2-R1, F2-R2, F2-R3, F3-R1, F3-R2, F3-R3; for E. multilocularis, the primer pair comprises one or more of F1-R1, F1-R3, F1-R4, F2-R1, F2-R3, F2-R4, F3-R2, F3-R4. Particularly preferably, for E. granulosus, the primer pair is F1-R1 and / or F2-R3; for E. multilocularis, the primer pair is F1-R1 and / or F2-R4.

[0040] Specifically, the Cas12a reporter probe, Cas12a protein, buffer and nuclease-free water are conventional substances. By using the transcleavage activity of the Cas12a protein, detection of pre-amplified double-stranded target DNA is achieved.

[0041] Preferably, the target sequence of the crRNA is designed within the selected RPA primer region and combined with the scaffold sequence (UAAUUUCUACUAAGUGUAGAU) of LbCas12a to activate Cas12a.

[0042] More specifically, to improve the detection sensitivity of low-abundance E. granulosus cfDNA in peripheral blood, the RPA-CRISPR amplification detection system needs to use high-activity crRNA. Based on the screened RPA primer product sequences, 2 and 3 crRNA candidate sequences were designed for E. granulosus and E. multilocularis, as shown in Table 2.

[0043] Table 2. crRNA sequence table based on RPA product The performance of each crRNA was evaluated by a two-step RPA-CRISPR reaction (i.e., the conventional first constant temperature amplification step, and then the detection step), and the results showed that except for crRNA2 combined with the F1-R1 primer of E. multilocularis, the rest of the crRNAs showed high activity (A-B). Figure 9 A-B).

[0044] To further verify the compatibility of RPA and CRISPR technology, a single-tube RPA-CRISPR reaction was performed in this embodiment. Specifically, the CRISPR / Cas12a transcleavage experiment was performed in a 20 μL system, containing 2 μL cleavage buffer (10x), 0.6 μL reporter probe (4 μM), 1 μL Cas12a protein (1 μM), 1 μL crRNA (1 μM), 1 μL RPA primer pair, and 14.4 μL nuclease-free water. The reaction system was placed in a SLAN-96P real-time fluorescence quantitative PCR instrument, and incubated at 40°C for 60 minutes to synchronize the isothermal amplification and CRISPR / Cas reaction. To ensure the activation of the CRISPR / Cas12a complex, an isothermal recombinase polymerase amplification technology (RPA) was introduced to generate sufficient target sequences.

[0045] The results showed that the F2 / R3 primer pair targeting E. granulosus combined with crRNA1, and the F1-R1 primer pair targeting E. multilocularis combined with crRNA3, showed the best amplification kinetics and the highest end-point fluorescence intensity (Fmax) Figure 9 C-D).

[0046] Example 3 In this embodiment, the characteristics of the RPA-CRISPR amplification detection system were analyzed.

[0047] The limit of detection (LOD) of the RPA-CRISPR amplification detection system was evaluated using plasmid templates at a range of concentrations from 1 to 100 copies / test.

[0048] Specifically, under isothermal conditions, a significant increase in end-point fluorescence signal was observed when target DNA was present. The results showed that the detection limit of the RPA-CRISPR amplification detection system for E. granulosus and E. multilocularis was both single copy / test Figure 10 A and C). Notably, the fluorescence intensity was positively correlated with the target concentration in the range of 1 to 100 copies / test Figure 10 B and D). Although previous studies have shown that Cas12a can recognize suboptimal PAM sites, thereby expanding the detectable target range and enabling rapid and specific detection of echinococcosis, the RPA-CRISPR amplification detection system in this embodiment using a classic PAM site Figure 11 ) showed higher sensitivity, which is particularly important for the detection of low-abundance circulating free DNA in liquid biopsies.

[0049] In addition, in order to verify the specificity of the RPA-CRISPR amplification detection system, 6 common pathogens causing liver abscesses were tested, including Eg, Em, KP, EC, SA and MTB isolated from clinical samples. As shown in Fig. 2, the RPA-CRISPR amplification detection system for specific target pathogens showed positive results, while other non-target pathogens showed negative results. These results confirmed that the RPA-CRISPR amplification detection system has high specificity in detecting echinococcosis. Figure 10

[0050] Example 4 This example illustrates the application of the RPA-CRISPR amplification detection system in liquid biopsy of echinococcosis. This example included and analyzed 25 suspected patients with echinococcosis, and the cfDNA isolated from their blood was detected by the RPA-CRISPR amplification detection system. The detection results are shown in Fig. 3. Figure 12 As can be seen from Fig. 3, the RPA-CRISPR amplification detection system can detect positive and negative patients. Figure 12

[0051] As can be seen from Fig. 3, the RPA-CRISPR amplification detection system can detect positive and negative patients.

[0052] It should be noted that in this document, the terms "comprising", "including" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or further includes elements inherent to such a process, method, article or apparatus. Without more limitations, the element defined by the statement "comprising a" does not exclude the presence of other identical elements in the process, method, article or apparatus including the element.

[0053] ​​Furthermore, it is to be understood that the scope of the methods and apparatus of the present application are not limited to the details of the foregoing description, but can be practiced with modifications within the scope of the appended claims. It is also to be understood that the foregoing description and the following examples are illustrative of the present application and are not in any sense limiting.

[0054] The above descriptions are only specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, which should be covered in the protection scope of the present application.

Claims

1. A primer pair for detecting echinococcosis based on circulating free DNA, characterized in that: Including primer pairs used to amplify patients' circulating free DNA using RPA, For Echinococcus granulosus, the primer pair includes one or more of F1-R1, F1-R2, F2-R1, F2-R2, F2-R3, F3-R1, F3-R2, and F3-R3, wherein: F1:AACGATCACTCCTATTCTAGCATGTGTGTGAA; R1: AGTGAGATGAGTGAGAAGGAGTGCTCGCACCG; F1:AACGATCACTCCTATTCTAGCATGTGTGTGAA; R2: AGATATTTCACATTCAGTGAGATGAGTGAGAA; F2:AATGCAAGCAGCAGATGCCTACCCATCCGGAA; R1: AGTGAGATGAGTGAGAAGGAGTGCTCGCACCGA; F2:AATGCAAGCAGCAGATGCCTACCCATCCGGAA; R2: AGATATTTCACATTCAGTGAGATGAGTGAGAA; F2:AATGCAAGCAGCAGATGCCTACCCATCCGGAA; R3: AAGTTGCCTTCGTTAGGTGGAGATATTTCACA; F3: AGCAGATGCCTACCCATCCGGAAAAGCATTTAA; R1: AGTGAGATGAGTGAGAAGGAGTGCTCGCACCGA; F3: AGCAGATGCCTACCCATCCGGAAAAGCATTTAA; R2: AGATATTTCACATTCAGTGAGATGAGTGAGAA; F3: AGCAGATGCCTACCCATCCGGAAAAGCATTTAA; R3: AAGTTGCCTTCGTTAGGTGGAGATATTTCACA; For Echinococcus multilocularis, the primer pair includes one or more of F1-R1, F1-R3, F1-R4, F2-R1, F2-R3, F2-R4, F3-R2, and F3-R4, wherein: F1: TAGGACACAGTGCCAGCATCTGCGGTTAGTC; R1:AGAGTAAATTAATAGGTGGACCATCCTTTAC; F1: TAGGACACAGTGCCAGCATCTGCGGTTAGTC; R3: GTGTACCTGAGCTAAACTCAATTCACATCAACA; F1: TAGGACACAGTGCCAGCATCTGCGGTTAGTC; R4: GTCCTAAACAATACCATATTACAACAATATTCC; F2: TGTTAAGATATATGTGGTGACAGGGATTAGATAC; R1:AGAGTAAATTAATAGGTGGACCATCCTTTAC; F2: TGTTAAGATATATGTGGTGACAGGGATTAGATAC; R3: GTGTACCTGAGCTAAACTCAATTCACATCAACA; F2: TGTTAAGATATATGTGGTGACAGGGATTAGATAC; R4: GTCCTAAACAATACCATATTACAACAATATTCC; F3: TAAAGGATGGTCCACCTATTAATTTACTCTT; R2: CTCTTATAAGCAGCACATAGACTTAGCTTAACTA; F3: TAAAGGATGGTCCACCTATTAATTTACTCTT; R4: GTCCTAAACAATACCATATTACAACAATATTCC.

2. The primer pair for detecting echinococcosis based on circulating free DNA according to claim 1, characterized in that: For Echinococcus granulosus, the primer pair is F1-R1 and / or F2-R3; For Echinococcus multilocularis, the primer pairs are F1-R1 and / or F2-R4.

3. A kit for detecting echinococcosis based on circulating free DNA, characterized in that: The kit includes an RPA primer pair, crRNA, a Cas12a reporter probe, a Cas12a protein, a buffer, and nuclease-free water, and the RPA primer pair is mixed with the crRNA, the Cas12a reporter probe, the Cas12a protein, a buffer, and nuclease-free water to construct an RPA-CRISPR amplification detection system; Among them, the RPA primer pair is the primer pair for detecting echinococcosis based on circulating free DNA as described in claim 1 or 2.

4. The kit for detecting echinococcosis based on circulating free DNA according to claim 3, characterized in that The crRNA sequence is a combination of the crRNA target sequence and the LbCas12a scaffold sequence.

5. The kit for detecting echinococcosis based on circulating free DNA according to claim 4, characterized in that For Echinococcus granulosus, the crRNA sequence is one of crRNA1 and crRNA2. crRNA1: uaauuucuacuaaguguagauGGCAUCUAGUGAAGGAAAAAUAA; crRNA2: uaauuucuacuaaguguagauCUUCACUAGAUGCCGAAACUACC; For Echinococcus multilocularis, the crRNA sequence is one of crRNA1, crRNA2, and crRNA3. crRNA1: uaauuucuacuaaguguagauACCUAACAUUGCACAUAAAUA; crRNA2: uaauuucuacuaaguguagauCUAUGCAUCUUCCCCUACAA; crRNA3:uaauuucuacuaaguguagauGCAGUGAGUGAUUCUUGUUAG.

6. The kit for detecting echinococcosis based on circulating free DNA according to claim 3, characterized in that The RPA-CRISPR amplification detection system includes 1 μL of RPA primer pair, 1 μL of crRNA, 0.6 μL of Cas12a reporter probe, 1 μL of Cas12a protein, 2 μL of buffer and 14.4 μL of nuclease-free water.

7. A method for detecting echinococcosis based on circulating free DNA, characterized in that: The steps include: Step 100: Obtain cfDNA from a sample as a sample to be tested; Step 200: Add the sample to be tested into the kit, and conduct isothermal amplification and CRISPR / Cas reaction simultaneously in the RPA-CRISPR amplification detection system to collect fluorescence signals. Wherein, the kit is a kit for detecting echinococcosis based on circulating free DNA according to any one of claims 3 to 6.

8. The method for detecting echinococcosis based on circulating free DNA according to claim 7, characterized in that: cfDNA was extracted from the patient's peripheral blood as the sample to be tested.

9. The method for detecting echinococcosis based on circulating free DNA according to claim 7, characterized in that: The RPA-CRISPR amplification detection system was incubated at 35-45°C for 40-70 minutes to simultaneously perform isothermal amplification and CRISPR / Cas reactions.

10. The method for detecting echinococcosis based on circulating free DNA according to claim 7, characterized in that: The RPA-CRISPR amplification detection system was incubated at 40°C for 60 min to simultaneously perform isothermal amplification and CRISPR / Cas reactions.

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