A specific primer pair and kit for identifying clonorchis sinensis adult, cysticercus and worm egg and application thereof

Abstract

The application provides a specific primer pair and kit for identifying Clonorchis sinensis adult, cysticercus and worm eggs and application, and belongs to the technical field of biological molecule detection. In order to solve the problems of insufficient specificity and insufficient sensitivity of the primer in the basic diagnosis of Clonorchis sinensis, the application discloses a specific primer pair for identifying Clonorchis sinensis adult, cysticercus and worm eggs, wherein the upstream primer sequence of the primer pair is shown as SEQ ID NO. 1, and the downstream primer sequence is shown as SEQ ID NO. 2. The detection method is simple, efficient and objective, the specific primer is used, and the Clonorchis sinensis can be quickly identified through simple PCR technology; the prepared kit is low in cost, high in accuracy and simple in operation, and can meet the operation of non-professionals.

Description

Technical Field

[0001] This invention belongs to the field of biomolecular detection technology, specifically relating to a specific primer pair and kit for identifying adult Clonorchis sinensis, metacercariae and eggs, as well as their applications. Background Technology

[0002] Clonorchis sinensis (also known as liver fluke) is an important zoonotic pathogen. Humans and other mammals become infected by consuming raw or undercooked freshwater fish and shrimp containing Clonorchis sinensis metacercariae. Adult worms can survive in the definitive host for decades, making the infected person a long-term carrier. The adult worms lay eggs, which enter the water with the definitive host's feces. These eggs are ingested by freshwater snails (the first intermediate host), where they develop into cercariae. Once mature, the cercariae escape from the snail and encounter the second intermediate host (freshwater fish or shrimp), where they invade the muscle and other tissues and develop into metacercariae. These metacercariae are then ingested by the definitive host, where they develop into adult worms that parasitize the bile ducts and gallbladder. Early symptoms are often subtle, but the infection gradually damages the liver and gallbladder, leading to symptoms such as hepatomegaly, cholangitis, biliary obstruction, cirrhosis, and cholangiocarcinoma. In severe cases, it can even cause death. In 2009, the WHO classified Clonorchis sinensis as a Group 1 biological carcinogen. In recent years, with the diversification of dietary culture and changes in people's dietary structure, the consumption of pickled foods, raw fish, and raw shrimp has increased significantly. Therefore, adopting a prevention-oriented control approach is essential.

[0003] Currently, the gold standard for diagnosing Clonorchis sinensis is mainly the pathogenic biological diagnostic method, which involves detecting whether there are eggs in the feces of the definitive host, and whether there are metacercariae in the digestion of fish and shrimp. However, this method requires professional personnel to operate, is time-consuming and labor-intensive, and has disadvantages such as difficulty in identifying similar parasite morphological characteristics, low sensitivity, no excretion of eggs in the early stage of infection, easy to miss or misdiagnose, and difficulty in large-scale diagnosis. It has been greatly limited in the integrated prevention and control of Clonorchis sinensis.

[0004] Immunological diagnostic methods for Clonorchis sinensis currently include enzyme-linked immunosorbent assay (ELISA), indirect hemagglutination assay, immunofluorescence antibody technology, and immunogold immunoassay, which to some extent fill the gaps in pathogen detection methods. However, these methods suffer from drawbacks such as false negatives, false positives, and cross-reactivity, and their antigen sensitivity and specificity are still not very high, limiting their application in practical detection. In recent years, molecular biology detection technologies have made continuous progress and have been widely used in the diagnosis of parasitic diseases. Among them, conventional PCR plays an extremely important role, possessing advantages such as high sensitivity, good specificity, small diagnostic error, and large detection capacity. At present, with the continuous development of PCR technology, this method is also widely used in clinical practice, playing an irreplaceable role in epidemiological surveys and specific diagnosis. This invention provides a simple, highly sensitive, and highly specific primer, reagent kit, and detection method for detecting adult Clonorchis sinensis and metacercariae based on conventional PCR detection methods, providing an effective technical means for the detection of Clonorchis sinensis. Summary of the Invention

[0005] The purpose of this invention is to solve the problems of insufficient primer specificity and low sensitivity in the primary diagnosis of Clonorchis sinensis.

[0006] This invention, based on the mitochondrial genome sequence of Clonorchis sinensis, screens out new target sequences for the first time for nucleic acid detection of Clonorchis sinensis. The primers provided by this invention not only have extremely high specificity and sensitivity, but can also detect the entire life cycle of Clonorchis sinensis (adult, larva, and egg) and provide technical support for the identification of Clonorchis sinensis and precision medicine.

[0007] This invention provides a specific primer pair for identifying adult Clonorchis sinensis, metacercariae, and eggs. The upstream primer sequence is shown in SEQ ID NO.1, and the downstream primer sequence is shown in SEQ ID NO.2.

[0008] This invention provides a kit for identifying adult Clonorchis sinensis, metacercariae, and eggs. The kit includes the specific primer pair described in claim 1, ddH2O, a standard positive control, and a PCR reaction solution.

[0009] Further specified, the standard positive control was a Clonorchis sinensis DNA sample; the PCR reaction solution included dNTPs, Taq DNA polymerase, Tris-HCl, KCl and MgCl2.

[0010] This invention provides a method for using the above-described reagent kit for non-diagnostic purposes, the specific steps of which are as follows:

[0011] Step 1: Extract genomic DNA from the pathogen to be identified;

[0012] Step 2: Use specific primers to amplify the genomic DNA of the pathogen to be identified by PCR. The amplification products are subjected to agarose gel electrophoresis and observed under ultraviolet conditions for the presence of specific amplification bands to determine whether the sample is Clonorchis sinensis. If a single 200bp DNA band is observed, it is identified as Clonorchis sinensis.

[0013] Further specifying the reaction conditions for the PCR technology described in step 2, the reaction conditions are as follows: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 56℃ annealing for 30 seconds, 72℃ extension for 12 seconds, for 30 cycles; and 72℃ final extension for 5 minutes.

[0014] To further define the reaction system for PCR in step 2, the reaction system is as follows: 20 ng template DNA, 0.5 μL upstream primer, 0.5 μL downstream primer, 12.5 μL PCR reaction solution, and ddH2O added to bring the total volume to 25 μL.

[0015] Further specified, the concentrations of both the upstream and downstream primers were 10 μmol / L.

[0016] This invention provides the application of the above-mentioned specific primer pair in the preparation of a kit for identifying adult Clonorchis sinensis, metacercariae, and eggs.

[0017] This invention provides the application of the above-mentioned specific primer pair in the preparation of a kit for detecting adult Clonorchis sinensis, metacercariae, and eggs.

[0018] Beneficial effects: By comparing the mitochondrial genome sequences of Clonorchis sinensis with those of various other parasites, highly specific primers for detecting Clonorchis sinensis were screened, which can accurately identify adult Clonorchis sinensis, metacercariae, and eggs. The detection method provided is simple, efficient, and objective. Using the aforementioned specific primers and simple PCR technology, Clonorchis sinensis can be quickly identified. The prepared kit is low in cost, highly accurate, and easy to operate, and can meet the needs of non-professionals. Attached Figure Description

[0019] Figure 1 The results of the amplification reaction are shown below. M is the DNA Marker, 1 is the adult Clonorchis sinensis, 2 is the Clonorchis sinensis metacercariae, 3 is the Clonorchis sinensis egg, and 4 is the negative control.

[0020] Figure 2 To indicate the specificity of the amplification reaction, M is the DNA Marker, 1 is Clonorchis sinensis, 2 is Clonorchis orientalis, 3 is Fasciolopsis buski, 4 is Taenia asiatica, 5 is Taenia solium, 6 is Hookworm, 7 is Cryptosporidium, and 8 is the negative control.

[0021] Figure 3To show the sensitivity of the amplification reaction, M is the DNA Marker, 1-9 are serially diluted Clonorchis sinensis DNA, which are 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, and 1fg, respectively, and 10 is the negative control.

[0022] Figure 4 This image shows the amplification results of the target fragment from Clonorchis sinensis. M is the DNA Marker, 1-2 are adult Clonorchis sinensis, 3-4 are Clonorchis sinensis metacercariae, 5-6 are Clonorchis sinensis eggs, and 7 is the negative control.

[0023] Figure 5 Results of mitochondrial sequence homology analysis of Clonorchis sinensis. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to the embodiments and accompanying drawings, but it should not be construed as a limitation of the present invention.

[0025] Example 1. Primer Design

[0026] 1. A method for designing specific primers for identifying adult Clonorchis sinensis, larvae, and eggs, comprising the following steps:

[0027] 1) Obtain the whole mitochondrial genome sequence of Clonorchis sinensis from Genbank, and use online software (https: / / proksee.ca / ) for sequence alignment analysis to select Clonorchis sinensis-specific conserved sequences as candidate primer regions; 2) Use Primer 5 software to analyze the candidate region sequences, set selection conditions, and obtain candidate primers; 3) Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0028] 2. The obtained primer sequences are: upstream primer: 5'-GGGAGAGGAGATCCAAAGAAGG-3' (SEQ ID NO.1), and downstream primer sequence: 5'-AGTCCCCGGATACCACCATC-3' (SEQ ID NO.2).

[0029] Example 2. Extraction of genomic DNA

[0030] Genomic DNA was extracted from adult Clonorchis sinensis (adults, metacercariae, and eggs), Cibotium orientalis, Fasciolopsis buski, Taenia asiatica, Taenia solium, Hookworm, and Cryptosporidium. The specific steps are as follows: The worms were placed in sterile centrifuge tubes and washed three times repeatedly with ddH2O, discarding the ddH2O. 450 μL of lysis buffer and 20 μL of proteinase K were added, and the mixture was digested at 56°C for 2 hours, inverting the centrifuge tube during digestion to promote digestion. 450 μL of DNA extraction phenol was added to the completely digested centrifuge tube, and the mixture was inverted and centrifuged at 12,000 rpm for 10 minutes. The supernatant was transferred to a new 1.5 mL sterile centrifuge tube, and 225 μL of chloroform and 225 μL of DNA extraction phenol were added. After mixing, the mixture was incubated on ice for 15 minutes. Centrifuge at 12,000 rpm for 10 min; transfer the supernatant to a new 1.5 mL sterile centrifuge tube, add an equal volume of isopropanol, then add sodium acetate (to a final concentration of 0.1–0.25 M), invert and shake well, and let stand for 12 min; centrifuge at 12,000 rpm for 5 min, transfer the precipitate to a new 1.5 mL sterile centrifuge tube, wash twice with 75% alcohol, and after the alcohol evaporates, dissolve in ddH2O and store at -20°C.

[0031] Example 3. Detection kit for adult Clonorchis sinensis, larvae, and eggs and its application.

[0032] The test kit includes specific primers, ddH2O, a standard positive control, and PCR reaction solution.

[0033] The standard positive control was a Clonorchis sinensis DNA sample, and the PCR reaction solution included dNTPs, Taq DNA polymerase, Tris-HCl, KCl, and MgCl2.

[0034] The application of the test kit includes the following steps:

[0035] 1) The amplification reaction system is as follows: 20 ng template DNA, 0.5 μL upstream primer (10 μmol / L), 0.5 μL downstream primer (10 μmol / L), 12.5 μL PCR reaction solution, add ddH2O to make up to 25 μL, and mix well.

[0036] 2) The amplification reaction conditions were: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 56℃ annealing for 30 seconds, 72℃ extension for 12 seconds, for 30 cycles; and 72℃ extension for 5 minutes.

[0037] 3) Specificity analysis: Genomic DNA from Clonorchis sinensis, Clonorchis orientalis, Fasciolopsis buski, Taenia asiatica, Taenia solium, Hookworm, and Cryptosporidium were used as templates for PCR amplification using the specific primers described above. The PCR products were then analyzed by agarose gel electrophoresis. The electrophoresis results are shown below. Figure 2As shown, only Clonorchis sinensis genomic DNA could be effectively amplified, proving that the above primers have strong specificity for detecting Clonorchis sinensis.

[0038] 4) Sensitivity analysis: Clonorchis sinensis DNA was serially diluted, and 100 ng, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg of template DNA and a negative control (ddH2O) were added respectively. PCR was performed using the primers described above, and the PCR products were analyzed by agarose gel electrophoresis. The electrophoresis results are shown below. Figure 3 As shown, the minimum detection limit of the above primers for Clonorchis sinensis DNA is 1 pg, indicating high sensitivity.

[0039] Example 4. Amplification and sequencing analysis of fragments from adult Clonorchis sinensis, metacercariae, and eggs.

[0040] 1) Amplification of the target sequence: Following the steps described in Example 3, the target fragments from adult Clonorchis sinensis, metacercariae, and eggs were amplified. The amplified products were analyzed by agarose gel electrophoresis. The electrophoresis results are as follows: Figure 4 As shown. Bands 1-2 represent adult Clonorchis sinensis, 3-4 represent Clonorchis sinensis metacercariae, 5-6 represent Clonorchis sinensis eggs, and 7 represents the negative control.

[0041] 2) Sequencing of amplified fragments: The amplified bands are recovered from the gel, ligated, transformed, and then sequenced.

[0042] The results are as follows: the mitochondrial sequencing results of Clonorchis sinensis adult worm 1 are shown in SEQ ID NO.3; the mitochondrial sequencing results of Clonorchis sinensis adult worm 2 are shown in SEQ ID NO.4; the mitochondrial sequencing results of Clonorchis sinensis cyst 1 are shown in SEQ ID NO.5; the mitochondrial sequencing results of Clonorchis sinensis metacercariae 2 are shown in SEQ ID NO.6; the mitochondrial sequencing results of Clonorchis sinensis egg 1 are shown in SEQ ID NO.7; and the mitochondrial sequencing results of Clonorchis sinensis egg 2 are shown in SEQ ID NO.8.

[0043] 3) Homology analysis: Homology analysis of the sequencing results was performed using MegAlign. The intraspecific differences in the Clonorchis sinensis mitochondrial sequences (SEQ ID NO. 3-8) were 0%-1%, as shown in the following results. Figure 5 .

[0044] Example 5. Detection effect of three primer pairs designed based on target gene region.

[0045] This invention utilizes the comparison of the mitochondrial genome of Clonorchis sinensis with that of other trematodes to screen for specific target gene sequences with a length of 741 bp. Three pairs of amplification primers were designed, namely:

[0046] CSF1:5'-GGGAGAGGAGATCCAAAGAAGG-3',

[0047] CSR1:5'-AGTCCCCGGATAACCACCATC-3';

[0048] CSF2:5'-TGTGGAAGGTGACTTGGTGTT-3'(SEQ ID NO.9),

[0049] CSR2:5'-GGCTAATTGCCATCATACCCC-3'(SEQ ID NO.10);

[0050] CSF3:5'-ATGTGGAAGGTGACTTGGTGT-3'(SEQ ID NO.11),

[0051] CSR3: 5'-GCCATCATACCCCTATACACCG-3' (SEQ ID NO. 12).

[0052] Only the CSF1 / CSR1 primer pair showed high specificity and sensitivity, while CSF2 / CSR2 and CSF3 / CSR3 showed varying degrees of cross-reactivity with other parasites and poor specificity.

[0053] The primer design method disclosed in this invention is based on the mitochondrial sequence of Clonorchis sinensis, and the resulting primers are sensitive and efficient. Using these primers for the identification of Clonorchis sinensis demonstrates high accuracy, overcoming the shortcomings of traditional pathogenic examination methods, such as difficulty in identifying morphological characteristics, long processing times, and a high risk of missed or false detections. The detection kit disclosed in this invention can identify a large number of samples in a short time, with good specificity and small error, providing an effective technical means for the accurate identification of Clonorchis sinensis.

[0054]

[0055]

Claims

1. A specific primer pair for identifying adult Clonorchis sinensis, metacercariae, and eggs, characterized in that, The upstream primer sequence of the primer pair is shown in SEQ ID NO.1, and the downstream primer sequence is shown in SEQ ID NO.

2.

2. A kit for identifying adult Clonorchis sinensis worms, metacercariae, and eggs, characterized in that, The kit includes the specific primer pair as described in claim 1, ddH2O, a standard positive control, and a PCR reaction solution.

3. The reagent kit according to claim 2, characterized in that, The standard positive control was a Clonorchis sinensis DNA sample; the PCR reaction solution included dNTPs, Taq DNA polymerase, Tris-HCl, KCl, and MgCl2.

4. A method for using the kit according to claim 2 or 3 for a non-diagnostic purpose, characterized in that, The specific steps of the method are as follows: Step 1: Extract genomic DNA from the pathogen to be identified; Step 2: Use the specific primers described in claim 1 to amplify the genomic DNA of the pathogen to be identified by PCR. The amplification products are subjected to agarose gel electrophoresis and observed under ultraviolet conditions to determine whether there is a specific amplification band. If a single 200 bp DNA band is shown, it is identified as Clonorchis sinensis.

5. The method according to claim 4, characterized in that, The reaction conditions for the PCR technology described in step 2 are as follows: pre-denaturation at 94℃ for 5 minutes; denaturation at 94℃ for 30 seconds, annealing at 56℃ for 30 seconds, extension at 72℃ for 12 seconds, for 30 cycles; and final extension at 72℃ for 5 minutes.

6. The method according to claim 4, characterized in that, The reaction system for PCR in step 2 is as follows: 20 ng template DNA, 0.5 μL upstream primer, 0.5 μL downstream primer, 12.5 μL PCR reaction solution, and ddH2O added to bring the total to 25 μL.

7. The method according to claim 6, characterized in that, The concentrations of both the upstream and downstream primers were 10 μmol / L.

8. The use of the specific primer pair according to claim 1 in the preparation of a kit for identifying adult Clonorchis sinensis, metacercariae and eggs.

9. The use of the specific primer pair according to claim 1 in the preparation of a kit for detecting adult Clonorchis sinensis, metacercariae and eggs.

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