Primer combination, reagent and kit for detecting echinococcus granulosus

Abstract

The invention relates to the technical field of biological medicine detection, in particular to a primer combination, a reagent and a kit for detecting echinococcus granulosus, and the primer combination comprises an echinococcus co1 subunit gene primer pair and an echinococcus nd5 subunit gene primer pair. The invention establishes an FAM / HEX double-probe fluorescent RPA (recombinase polymerase amplification) method capable of rapidly detecting co1 and nd5 genes of echinococcus, and the method not only can detect echinococcus parasites, but also can further identify echinococcus granulosus.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical detection, and in particular to a primer combination, a reagent and a kit for detecting Echinococcus granulosus. Background Art

[0002] Echinococcosis is a serious zoonosis caused by echinococcosis parasitizing the liver, lungs and other tissues and organs of the host. It is widely distributed in areas around the world dominated by agricultural and animal husbandry production, seriously endangering human health and animal husbandry development. Echinococcosis has a long incubation period, and the early onset characteristics are not obvious. In addition, the education level of the population in the epidemic area is generally not high. Therefore, patients only choose to seek medical treatment when they have obvious abdominal swelling and discomfort. At this time, echinococcosis has reached the middle and late stages, and surgical treatment is not effective or even not suitable. In the transmission route of echinococcosis, canines, as the terminal hosts of echinococcosis, are in close contact with herders in the echinococcosis epidemic areas in northwest my country, and the risk of infection is high. Therefore, monitoring the infection of the terminal hosts and taking targeted and effective measures to control the source of infection are important means to block the spread of echinococcosis.

[0003] Adult Echinococcus parasites live in the upper small intestine of definitive hosts such as dogs, wolves, and foxes. Mature gravid segments or eggs are excreted with feces, contaminating animal fur and the surrounding environment. When the eggs or gravid segments are ingested by the intermediate host, they develop into echinococcosis in the intermediate host, leading to the occurrence of echinococcosis. Therefore, monitoring the infection status of the definitive host is of great significance for the prevention and control of echinococcosis. Canines are mainly infected with Echinococcus granulosus (Eg) and Echinococcus multilocularis (Em). Commonly used detection methods are ELISA and nucleic acid detection of their feces. Among them, ELISA detection often has false positive problems, while nucleic acid detection is more sensitive and accurate. Polymerase Chain Reaction (PCR) technology such as nested PCR and real-time quantitative PCR is commonly used for nucleic acid detection of dog fecal samples. However, traditional PCR detection technology has complex procedures, cumbersome operations, long time, and is prone to contamination. It also has high requirements for experimental equipment, experimental environment and conditions, as well as the technical level of detection personnel, and is difficult to use for the detection of a large number of samples on site. Multiplex PCR technology solves the problem of time-consuming and labor-intensive detection of a single worm species. It has been reported that this method can simultaneously detect Echinococcus granulosus and Echinococcus multilocularis, but it still cannot avoid the problems of cumbersome operation and long time in PCR technology itself. Therefore, it is necessary to further develop fast, convenient, and suitable technologies for on-site sample detection. Summary of the invention

[0004] The object of the present invention is to provide a primer combination, a reagent and a kit for detecting Echinococcus granulosus.

[0005] The technical problem of the present invention is solved by the following technical solutions:

[0006] In the first aspect of the present invention, a primer combination for detecting Echinococcus granulosus is provided, and the primer combination includes an Echinococcus co1 subunit gene primer pair and an Echinococcus nd5 subunit gene primer pair;

[0007] The nucleotide sequence of the upstream primer of the Echinococcus co1 subunit gene primer pair has more than 80% identity with SEQ ID NO: 1; the nucleotide sequence of the downstream primer of the Echinococcus co1 subunit gene primer pair has more than 80% identity with SEQ ID NO: 2;

[0008] The nucleotide sequence of the upstream primer of the Echinococcus nd5 subunit gene primer pair has more than 80% identity with SEQ ID NO: 3; the nucleotide sequence of the downstream primer of the Echinococcus nd5 subunit gene primer pair has more than 80% identity with SEQ ID NO: 4.

[0009] Cytochrome C oxidase (COX) gene and mitochondrial NADH dehydrogenase (ND) gene are widely present in various Echinococcus species and are the most commonly used target genes for the detection of Echinococcus. The present invention will establish a rapid and efficient FAM / HEX dual-probe fluorescence RPA detection technology for the Echinococcus co1 subunit gene and nd5 subunit gene, providing technical support for the monitoring of Echinococcus definitive host infection.

[0010] Based on the genomic DNA of Echinococcus granulosus, the present invention designs specific primers for dual-probe fluorescence RPA, so that the FAM probe can specifically recognize the co1 gene in the genomic DNA of Echinococcus granulosus and Echinococcus multilocularis, and the HEX probe can specifically recognize the nd5 gene in the genomic DNA of Echinococcus granulosus. On the basis of improving sensitivity and reducing detection time, the present invention can detect and identify Echinococcus species faster, more conveniently and accurately through multi-target simultaneous detection.

[0011] In some embodiments of the present invention, the nucleotide sequence of the upstream primer of the Echinococcus co1 subunit gene primer pair has more than 90% identity with SEQ ID NO: 1; the nucleotide sequence of the downstream primer of the Echinococcus co1 subunit gene primer pair has more than 90% identity with SEQ ID NO: 2;

[0012] The nucleotide sequence of the upstream primer of the Echinococcus granulosus nd5 subunit gene primer pair has more than 90% identity with SEQ ID NO: 3; the nucleotide sequence of the downstream primer of the Echinococcus granulosus nd5 subunit gene primer pair has more than 90% identity with SEQ ID NO: 4.

[0013] In some embodiments of the present invention, the upstream primer of the Echinococcus granulosus co1 subunit gene primer pair has the nucleotide sequence shown in SEQ ID NO: 1; the downstream primer of the Echinococcus granulosus co1 subunit gene primer pair has the nucleotide sequence shown in SEQ ID NO: 2;

[0014] The upstream primer of the Echinococcus granulosus nd5 subunit gene primer pair has the nucleotide sequence shown in SEQ ID NO: 3; the downstream primer of the Echinococcus granulosus nd5 subunit gene primer pair has the nucleotide sequence shown in SEQ ID NO: 4.

[0015] In some embodiments of the present invention, the primer combination further includes a co1 subunit gene probe and an Echinococcus granulosus nd5 subunit gene probe.

[0016] In some embodiments of the present invention, the nucleotide sequence of the co1 subunit gene probe has more than 80% identity with SEQ ID NO: 5;

[0017] The nucleotide sequence of the Echinococcus granulosus nd5 subunit gene probe has more than 80% identity with SEQ ID NO: 6.

[0018] In some embodiments of the present invention, the nucleotide sequence of the co1 subunit gene probe has more than 90% identity with SEQ ID NO: 5;

[0019] The nucleotide sequence of the Echinococcus granulosus nd5 subunit gene probe has more than 90% identity with SEQ ID NO: 6.

[0020] In some embodiments of the present invention, the co1 subunit gene probe has the nucleotide sequence shown in SEQ ID NO: 5;

[0021] The Echinococcus granulosus nd5 subunit gene probe has the nucleotide sequence shown in SEQ ID NO: 6.

[0022] In some embodiments of the present invention, the fluorophore of the co1 subunit gene probe is FAM, and the fluorophore of the nd5 subunit gene probe is HEX.

[0023] The second aspect of the present invention provides a reagent for detecting Echinococcus granulosus, and the reagent includes the above-mentioned primer combination.

[0024] The third aspect of the present invention provides a kit for detecting Echinococcus granulosus, and the reagent includes the above primer combination or reagent.

[0025] The present invention has the following beneficial effects: The present invention has established a FAM / HEX dual-probe fluorescence RPA method capable of rapidly detecting the co1 and nd5 genes of Echinococcus. This method can not only detect parasites of the genus Echinococcus, but also further identify Echinococcus granulosus. This method has low requirements for equipment, is simple and fast, and can perform rapid detection of a large number of samples. Compared with the traditional ELISA detection method, this method can effectively improve the detection accuracy; compared with ordinary PCR detection, it can greatly shorten the detection time and improve the detection efficiency. Subsequently, it is planned to further optimize and prepare a test kit for rapid detection of field samples. Description of the Drawings

[0026] Figure 1 Evaluation of the amplification effect of the dual-probe fluorescence RPA primer-probe combination on the genomic DNA of Echinococcus granulosus and Echinococcus multilocularis.

[0027] Figure 2 Amplification effect of the dual-probe fluorescence RPA method on the genomic DNA of Echinococcus granulosus at different temperatures.

[0028] Figure 3 Ratio of the fluorescence signal of the target gene amplification of Echinococcus granulosus to the fluorescence signal of the blank control at different temperatures.

[0029] Figure 4 Amplification signal intensity of FAM fluorescence for different concentrations of plasmids.

[0030] Figure 5 Amplification signal intensity of HEX fluorescence for different concentrations of plasmids.

[0031] Figure 6 Relative intensity of FAM fluorescence amplified by the dual-probe fluorescence RPA method for the genomic DNA of different parasites.

[0032] Figure 7 Relative intensity of HEX fluorescence amplified by the dual-probe fluorescence RPA method for the genomic DNA of different parasites.

[0033] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0034] As used in the present invention, the term "prevention" refers to preventing the occurrence of a disease and / or preventing the recurrence of a disease. Detailed Description of the Invention

[0035] The specification of the present invention describes the specific implementation embodiments in detail. Those skilled in the art should recognize that the following implementation embodiments are exemplary and should not be construed as limiting the present invention. For those skilled in the art, without departing from the principle of the present invention, by making several improvements and modifications to the present invention, the technical solutions obtained by these improvements and modifications also fall within the protection scope of the claims of the present invention. The beneficial effects of the present invention are specifically described below through examples.

[0036] Detection of Echinococcus granulosus by Dual - probe Fluorescent Recombinase Polymerase Amplification

[0037] 1 Materials and Methods

[0038] 1.1 Source of Materials

[0039] Genomic DNA of Echinococcus granulosus, Echinococcus multilocularis, Taenia solium, Schistosoma japonicum, Necator americanus, Nippostrongylus brasiliensis, Clonorchis sinensis, Giardia lamblia, Cryptosporidium parvum, Toxoplasma gondii and Babesia microti, as well as positive DNA of Echinococcus granulosus in dog feces were all provided by the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (National Center for Tropical Diseases Research).

[0040] After collection, dog fecal samples were stored at -80°C for 3 months, and then nucleic acids were extracted using the fecal genomic DNA extraction kit from Qiagen, Germany. After nested PCR detection, they were determined to be positive.

[0041] 1.2 Primer Design

[0042] Based on the sequences of the Echinococcus granulosus gene (accession number: KJ831062.1) and the Echinococcus multilocularis gene (accession number: NC_044548.1) in NCBI, the fluorescence RPA primers and probes were designed using the primer design software Primer Premier 6. The primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. After primer interference screening and amplification screening, the final determined optimal primer and probe sequences are shown in the following table (Table 1):

[0043] Table 1 Primer and Fluorescent Probe Sequences

[0044]

[0045]

[0046] 1.3 DNA Extraction

[0047] For the extraction of genomic DNA from the protoscoleces of Echinococcus, the operation was carried out according to the instructions of the tissue and blood kit from Qiagen, Germany. The extracted DNA was stored at -80°C for standby.

[0048] 1.4 Establishment of Fluorescent RPA and Dual-probe Fluorescent RPA Reaction Systems

[0049] Fluorescent RPA reaction system for the co1 subunit gene of Echinococcus granulosus: Transfer a reaction premix with a total volume of 47 μl containing 25 μl of buffer, 2.1 μl of co1-F (10 nmol / μl), 2.1 μl of co1-R (10 nmol / μl), 0.6 μl of FAM-P (10 nmol / μl), 2.0 μl of target gene DNA to be detected, and 15.2 μl of ddH 2 O to the reaction unit containing freeze-dried enzyme, mix well and transfer to a detection tube. Drop 3.0 μl of initiator (234 mM magnesium acetate aqueous solution) on the tube cap, carefully tighten the cap and centrifuge. Then place it on a metal bath and react with shaking at 39°C and 300 rpm for 4 min, and then transfer it to a fluorescence quantitative PCR instrument for continued reaction. The reaction detection conditions are 39°C and 12.5 min, and fluorescence signals are collected every 20 s. The total reaction duration is 16.5 min.

[0050] Fluorescent RPA reaction system for the nd5 subunit gene of Echinococcus granulosus: Transfer a reaction premix with a total volume of 47 μl containing 25 μl of buffer, 2.1 μl of nd5-F (10 nmol / μl), 2.1 μl of nd5-R (10 nmol / μl), 0.6 μl of HEX-P (10 nmol / μl), 2.0 μl of target gene DNA to be detected, and 15.2 μl of ddH 2 O to the reaction unit containing freeze-dried enzyme, mix well and transfer to a detection tube. Drop 3.0 μl of initiator (234 mM magnesium acetate aqueous solution) on the tube cap, carefully tighten the cap and centrifuge. Then place it on a metal bath and react with shaking at 39°C and 300 rpm for 4 min, and then transfer it to a fluorescence quantitative PCR instrument for continued reaction. The reaction detection conditions are 39°C and 12.5 min, and fluorescence signals are collected every 20 s. The total reaction duration is 16.5 min.

[0051] The dual-probe fluorescent RPA reaction system is as follows: 1.0 μl of co1-F (10 nmol / μl), 1.0 μl of nd5-F (10 nmol / μl), 1.0 μl of co1-R (10 nmol / μl), 1.0 μl of nd5-R (10 nmol / μl), 1.0 μl of FAM-P (10 nmol / μl), 1.0 μl of HEX-P (10 nmol / μl), 2.5 μl of target gene DNA to be detected, 5.0 μl of ddH 2Pre - mix 1.5 μl of the initiator (234 mM magnesium acetate aqueous solution) and transfer it to the detection tube; transfer 25.0 μl of the buffer to the reaction unit containing the freeze - dried enzyme, and mix well to prepare the dissolution solution; take 10.0 μl of the dissolution solution and drop it on the cap of the detection tube containing the premixed solution, carefully tighten the cap and centrifuge, then place it on a metal bath at 39 °C and shake at 300 rpm for 4 min, and then transfer it to a fluorescence quantitative PCR instrument for continuous reaction. The reaction detection conditions are 39 °C for 12.5 min, and fluorescence signals are collected every 20 s. The total reaction duration is 16.5 min.

[0052] Example 1

[0053] Amplification effect of dual - probe fluorescence RPA method on genomic DNA of E.g and E.m

[0054] Configure the fluorescence RPA reaction system, use sterile pure water as the template DNA for amplification, detect the FAM fluorescence probe and the HEX fluorescence probe respectively. After screening non - interfering primer pairs, amplify the genomic DNA of the protoscoleces of Echinococcus granulosus, screen out primer pairs sensitive to the FAM / HEX fluorescence probe, then amplify the genomic DNA of the protoscoleces of Echinococcus multilocularis, and screen out primer combinations that only detect one fluorescence signal (FAM or HEX). This primer combination uses the dual - probe fluorescence RPA reaction system to amplify the genomic DNA of the protoscoleces of Echinococcus granulosus and the genomic DNA of the protoscoleces of Echinococcus multilocularis, and determine the optimal primer combination by combining the fluorescence amplification intensity and the peak of the amplification curve.

[0055] Figure 1 It is for the evaluation of the amplification effect of the dual - probe fluorescence RPA primer - probe combination on the genomic DNA of Echinococcus granulosus and Echinococcus multilocularis.

[0056] After designing primer - probe combinations for the genomic DNA of Echinococcus, through primer interference screening and amplification screening, the primer - FAM probe combination in the dual - probe fluorescence RPA method can specifically recognize the co1 gene in the genomic DNA of Echinococcus granulosus and Echinococcus multilocularis, and the primer - HEX probe combination can specifically recognize only the nd5 gene in the genomic DNA of Echinococcus granulosus and can perform effective amplification, expressing obvious specific fluorescence signals ( Figure 1 ).

[0057] Example 2

[0058] Effect of temperature on the dual - probe fluorescence RPA reaction

[0059] Using the genomic DNA of Echinococcus granulosus and Echinococcus multilocularis as templates, dual-probe fluorescence RPA reactions were carried out at 37, 38, 39, and 40 °C respectively, and the fluorescence signal intensity was detected by qPCR to evaluate the amplification effect.

[0060] Figure 2 Amplification effect of the dual-probe fluorescence RPA method on the genomic DNA of Echinococcus granulosus at different temperatures. Figure 3 Ratio of the fluorescence signal of the target gene amplification of Echinococcus granulosus to the fluorescence signal of the blank control at different temperatures.

[0061] The results of qPCR detection of the genomic DNA of Echinococcus granulosus protoscoleces by the dual-probe fluorescence RPA at different temperatures showed that when the temperature was 39 °C and 40 °C, after 4 min of metal bath shaking reaction at 300 rpm, when the qPCR reaction time was 12.5 min (total reaction time 16.5 min), the two fluorescence signals were significantly distinguishable from the signal of the blank control group, and the reproducibility was better ( Figure 2 ), and the ratio of the two fluorescence signal intensities was close at 39 °C ( Figure 3 ). Finally, 39 °C, after 4 min of metal bath shaking reaction at 300 rpm, the qPCR reaction time of 12.5 min, and the total reaction duration of 16.5 min were selected as the final reaction program.

[0062] Example 3

[0063] Sensitivity evaluation of the dual-probe fluorescence RPA method

[0064] The amplification product with the co1 gene as the template was ligated to the pUC-SP cloning vector, and the amplification product with the nd5 gene as the template was ligated to the pUC57 cloning vector to construct recombinant plasmids respectively. The plasmids were synthesized by Sangon Biotech (Shanghai) Co., Ltd. 2.5 μl of 10 4 , 10 3 , 10 2 , 10, and 1 copy / μl of the recombinant plasmids were taken, and ddH 2 O was used as the blank control for the dual-probe fluorescence RPA reaction. The fluorescence signal intensity was detected by qPCR to evaluate the amplification effect. If the fluorescence signal intensity of the sample was significantly enhanced compared with the final absorbance of the blank control sample, it was considered a positive result. If there was no significant difference or it was lower than the final absorbance of the blank control sample, it was considered a negative result.

[0065] Figure 4 Amplification signal intensity of FAM fluorescence for plasmids with different concentrations. Figure 5 Amplification signal intensity of HEX fluorescence for plasmids with different concentrations.

[0066] The results of amplifying and detecting the pUC-SP-co1 recombinant plasmid and pUC57-nd5 recombinant plasmid of Echinococcus granulosus at different concentrations (copies / μl) by the dual-probe fluorescence RPA method showed that the lowest detection limit of this method for the pUC-SP-co1 recombinant plasmid was 10 copies / μl (FAM fluorescence, Figure 4 ), and the lowest detection limit for the pUC57-nd5 recombinant plasmid was 100 copies / μl (HEX fluorescence, Figure 5 ).

[0067] Example 4

[0068] Specificity evaluation of the dual-probe fluorescence RPA method

[0069] Using the genomic DNA of Echinococcus granulosus, Echinococcus multilocularis, Taenia solium, Babesia microtus, Clonorchis sinensis, Nippostrongylus braziliensis, Necator americanus, Toxoplasma gondii, Schistosoma japonicum, Giardia lamblia, and Cryptosporidium parvum as templates, and ddH 2 O as the template for the negative control group, a dual-probe fluorescence RPA reaction was carried out. The amplification effect was evaluated by detecting the fluorescence signal intensity through qPCR. If the fluorescence signal intensity of the sample was significantly enhanced compared with the final absorbance of the blank control sample, it was considered a positive result; if there was no significant difference or it was lower than the final absorbance of the blank control sample, it was considered a negative result.

[0070] Figure 6 It is the relative intensity of FAM fluorescence amplified from the genomic DNA of different parasites by the dual-probe fluorescence RPA method. Figure 7 It is the relative intensity of HEX fluorescence amplified from the genomic DNA of different parasites by the dual-probe fluorescence RPA method.

[0071] The detection results of the genomic DNA of Taenia solium, Babesia microtus, Clonorchis sinensis, Nippostrongylus braziliensis, Necator americanus, Toxoplasma gondii, Schistosoma japonicum, Giardia lamblia, and Cryptosporidium parvum using the dual-probe fluorescence RPA method for Echinococcus granulosus were all negative. Only FAM (co1 gene) signals were detected for the genomic DNA of Echinococcus multilocularis, and both FAM (co1 gene) and HEX (nd5 gene) signals were detected for the genomic DNA of Echinococcus granulosus ( Figure 6 and Figure 7 ).

[0072] Example 5

[0073] Evaluation of the Detection Effect of the Dual - Probe Fluorescent RPA Method for Simulated DNA Samples and Positive DNA Samples of Echinococcus granulosus in Field Dog Feces

[0074] Using 23 simulated DNA samples (8 mixed DNA samples of Echinococcus granulosus, 7 mixed DNA samples of Echinococcus multilocularis, 8 mixed DNA samples of Echinococcus granulosus and Echinococcus multilocularis. The mixed DNA samples are all mixed with DNA extracted from protoscoleces and negative dog feces, and the final DNA sample concentration is in the range of 2.0 - 5.0 ng / μl. All samples are determined to be positive by PCR amplification detection) and 5 DNA samples of field dog feces positive for Echinococcus granulosus infection (the nucleic acid extraction concentrations are 5.2, 4.9, 5.0, 2.2, 4.2 ng / μl, and all samples are determined to be positive by PCR amplification detection) to verify the feasibility of the dual - probe fluorescent RPA method. The result judgment basis is that samples containing the genomic DNA of Echinococcus granulosus should produce double - positive signals; samples containing only the genomic DNA of Echinococcus multilocularis without the genomic DNA of Echinococcus granulosus should produce single - positive signals; positive DNA samples of Echinococcus granulosus in field dog feces should produce single - positive signals; other samples without any positive signals are negative results.

[0075] The 23 simulated positive DNA samples of dog feces (8 mixed DNA samples of Echinococcus granulosus, 7 mixed DNA samples of Echinococcus multilocularis, 8 mixed DNA samples of Echinococcus granulosus and Echinococcus multilocularis) and 5 positive DNA samples of Echinococcus granulosus in field - collected dog feces used are all determined to be positive by PCR amplification detection. The detection results of the dual - probe fluorescent RPA method show that samples containing the genomic DNA of Echinococcus granulosus produce double - positive FAM / HEX signals; samples containing only the genomic DNA of Echinococcus multilocularis should produce single - positive FAM signals; positive DNA samples of Echinococcus granulosus in field - collected dog feces produce double - positive FAM / HEX signals; other DNA samples without any positive signals are negative results (Table 2).

[0076] Table 2 Comparison of Detection Results between the Dual - Probe Fluorescent RPA Method and the PCR Method

[0077]

[0078] In summary, based on the genomic DNA of Echinococcus granulosus, specific primers for dual-probe fluorescence RPA were designed, enabling the FAM probe to specifically recognize the co1 gene in the genomic DNA of Echinococcus granulosus and Echinococcus multilocularis, and the HEX probe to specifically recognize the nd5 gene in the genomic DNA of Echinococcus granulosus. Although there were significant differences in the fluorescence signals at 39°C and 40°C compared to the blank control group, the two fluorescence signal intensities were closer at 39°C. Therefore, 39°C was selected as the optimal temperature. The total detection time was only 16.5 minutes, slightly shorter than the reaction time of about 20 minutes for detecting other parasites by fluorescence RPA. Compared with the complex temperature-changing system and longer reaction time of PCR technology, the stable temperature system and shorter detection time are more suitable for the detection of a large number of field samples.

[0079] The minimum detection limits of the dual-probe fluorescence RPA method for pUC-SP-co1 and pUC57-nd5 recombinant plasmids were only 10 copies / μl and 100 copies / μl, respectively, showing high sensitivity, similar to that of the fluorescence RPA method for detecting other parasites. However, the recognition of the nd5 gene itself utilizes the mismatch tolerance of the probe, so the sensitivity increased slightly compared to that of other fluorescence RPA methods for detecting parasites. It can accurately detect the genomic DNA of Echinococcus and Echinococcus granulosus, and there was no signal for the genomic DNA of Taenia solium, Schistosoma japonicum, Necator americanus, Nippostrongylus brasiliensis, Clonorchis sinensis, Giardia lamblia, Cryptosporidium parvum, Toxoplasma gondii, and Babesia microti, showing high specificity. The detection of positive dog fecal DNA samples of Echinococcus granulosus collected by simulation and in the field showed that the detection results of the dual-probe fluorescence RPA method were reliable.

[0080] The present invention established a FAM / HEX dual-probe fluorescence RPA method for rapidly detecting the co1 and nd5 genes of Echinococcus. This method can not only detect parasites of the genus Echinococcus but also further identify Echinococcus granulosus. This method has low requirements for equipment, is simple and fast, and can perform rapid detection of a large number of samples. Compared with the traditional ELISA detection method, this method can effectively improve the detection accuracy; compared with ordinary PCR detection, it can greatly shorten the detection time and improve the detection efficiency. Subsequently, it is planned to further optimize and prepare a test kit for rapid detection of field samples.

[0081] The above embodiments merely illustrate the principles and effects of the present invention and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A primer combination for detecting Echinococcus granulosus, characterized in that: The primer combination includes a primer pair for the Echinococcus co1 subunit gene and a primer pair for the Echinococcus nd5 subunit gene; The upstream primer nucleotide sequence of the Echinococcus co1 subunit gene primer pair has greater than 80% identity with SEQ ID NO: 1; the downstream primer nucleotide sequence of the Echinococcus co1 subunit gene primer pair has greater than 80% identity with SEQ ID NO: 2; The upstream primer nucleotide sequence of the Echinococcus nd5 subunit gene primer pair has greater than 80% identity with SEQ ID NO:3; the downstream primer nucleotide sequence of the Echinococcus nd5 subunit gene primer pair has greater than 80% identity with SEQ ID NO:

4.

2. A primer combination for detecting Echinococcus granulosus according to claim 1, characterized in that: The upstream primer nucleotide sequence of the Echinococcus co1 subunit gene primer pair has greater than 90% identity with SEQ ID NO: 1; the downstream primer nucleotide sequence of the Echinococcus co1 subunit gene primer pair has greater than 90% identity with SEQ ID NO: 2; The upstream primer nucleotide sequence of the Echinococcus nd5 subunit gene primer pair has greater than 90% identity with SEQ ID NO:3; the downstream primer nucleotide sequence of the Echinococcus nd5 subunit gene primer pair has greater than 90% identity with SEQ ID NO:

4.

3. A primer combination for detecting Echinococcus granulosus according to claim 1, characterized in that: The upstream primer of the Echinococcus co1 subunit gene primer pair has a nucleotide sequence as shown in SEQ ID NO: 1; the downstream primer of the Echinococcus co1 subunit gene primer pair has a nucleotide sequence as shown in SEQ ID NO: 2; The upstream primer of the Echinococcus tapeworm nd5 subunit gene primer pair has a nucleotide sequence as shown in SEQ ID NO:3; the downstream primer of the Echinococcus tapeworm nd5 subunit gene primer pair has a nucleotide sequence as shown in SEQ ID NO:

4.

4. A primer combination for detecting Echinococcus granulosus according to claim 1 or 2, characterized in that: The primer combination also includes a co1 subunit gene probe and an Echinococcus tapeworm nd5 subunit gene probe.

5. A primer combination for detecting Echinococcus granulosus according to claim 3, characterized in that: The nucleotide sequence of the co1 subunit gene probe has greater than 80% identity with SEQ ID NO:5; The nucleotide sequence of the Echinococcus nd5 subunit gene probe has greater than 80% identity with SEQ ID NO:

6.

6. A primer combination for detecting Echinococcus granulosus according to claim 4, characterized in that: The nucleotide sequence of the co1 subunit gene probe has greater than 90% identity with SEQ ID NO:5; The nucleotide sequence of the Echinococcus tapeworm nd5 subunit gene probe has greater than 90% identity with SEQ ID NO:

6.

7. A primer combination for detecting Echinococcus granulosus according to claim 4, characterized in that: The co1 subunit gene probe has a nucleotide sequence as shown in SEQ ID NO:5; The Echinococcus tapeworm nd5 subunit gene probe has a nucleotide sequence as shown in SEQ ID NO:

6.

8. A primer combination for detecting Echinococcus granulosus according to claim 4, characterized in that: The fluorescent group of the co1 subunit gene probe is FAM, and the fluorescent group of the nd5 subunit gene probe is HEX.

9. A reagent for detecting Echinococcus granulosus, characterized in that: The reagent comprises the primer combination according to any one of claims 1 to 8.

10. A kit for detecting Echinococcus granulosus, characterized in that: The reagent comprises the primer combination according to any one of claims 1 to 8 or the reagent according to claim 9.

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