Detection method of clonorchis sinensis for non-disease diagnosis purpose

Abstract

The invention belongs to the technical field of parasite detection, and particularly relates to a detection method of clonorchis sinensis for non-disease diagnosis purpose, which comprises the following steps: taking a primer group A for targeted amplification of clonorchis sinensis genes as a target primer, and taking a primer group B for targeted amplification of escherichia coli genes as an internal reference primer. Two sets of primer groups are combined, the primer group B for amplifying escherichia coli genes is used as an internal reference primer, whether nucleic acid of a sample and a quality control product is successfully extracted or not and whether a detection result is credible or not in the detection process can be monitored, and the technical problem that a conventional clonorchis sinensis detection technology cannot monitor whether an amplification experiment is abnormal or not is solved.

Description

Technical Field

[0001] The invention belongs to the technical field of parasite detection, and in particular relates to a detection method of Clonorchis sinensis for non-disease diagnosis purposes. Background Art

[0002] Clonorchiasis is an important foodborne parasitic disease caused by Clonorchiasis, a parasite that inhabits the biliary system. The exact clinical symptoms of clonorchiasis are unclear, except for late sequelae, as most infected individuals have low parasite loads and mild symptoms. Moderate to severe infections may be accompanied by dull pain in the right upper abdomen; in contrast, chronic or recurrent infections are associated with manifestations such as cholangitis, cholehepatitis, and biliary obstruction. Therefore, clonorchiasis is a serious public health problem in endemic areas.

[0003] Existing methods for foodborne parasitic diseases include etiological detection methods, immunological detection methods, and imaging detection methods. Etiological detection methods include direct sample smear, quantitative transparent thick smear method, egg collection method, and duodenal drainage bile egg examination; immunological detection methods include ELISA, IHA, and IFA; imaging detection methods include B-ultrasound and CT. In the "Diagnostic Criteria for Clonorchiasis Sinensis" (WS309-2009), ELISA and three etiological tests are recommended: modified Kato thick smear method, aldehyde ether centrifugation precipitation method, and capsule pulling method. Among them, sample egg examinations such as modified Kato thick smear method and formalin ether concentration method will have false negatives in low infection conditions, and it is difficult to distinguish fish-borne trematodes such as testicular flukes, opisthorchis flukes, and heteromorpha due to the similarity of eggs. Immunological detection methods for Clonorchiasis Sinensis have the advantages of objectivity and simple operation, and have a tendency to replace traditional detection methods, but on the other hand, these methods also have shortcomings such as false negatives, false positives, and cross reactions.

[0004] To overcome the detection limitations associated with traditional parasitological methods, a number of PCR-based tests have been developed that can rapidly amplify and identify trematode species directly from eggs in samples.

[0005] Chinese patent (publication number CN105132539A) discloses a PCR amplification kit and amplification primers for detecting the metacercariae of Clonorchis sinensis based on ribosomal DNA ITS1 gene, including a PCR amplification reaction solution, wherein the PCR amplification reaction solution includes: Tris-HCl 10mmol / L, KCl 50mmol / L, MgCl2 1.5mmol / L, dNTPs 0.8mmol / L, forward primer 0.4μmol / L, reverse primer 0.4μmol / L, DNA template 20ng / μL, Taq enzyme 0.075U / μL. The invention has high sensitivity, strong specificity, rapid and accurate detection, and provides an effective technical means for the diagnosis, treatment and prevention of Clonorchis sinensis infection.

[0006] Chinese patent (publication number CN114959082 A) discloses primer probe sets, kits and applications for detecting multiple animal-derived food pathogens. The primer probe set includes specific primer pairs and probes for multiple animal-derived food pathogens, including Salmonella, Escherichia coli 0157:H7, Shigella, Staphylococcus aureus, Listeria monocytogenes, Streptococcus suis type 2, Brucella, Mycobacterium tuberculosis, Trichinella spiralis, Toxoplasma gondii, Cysticercus, Echinococcus, Angiostrongylus cantonensis, Clonorchis sinensis and Hepatitis E virus. The primer probe set can quickly, efficiently and specifically detect pathogens in animal-derived foods, which is of great significance for ensuring food safety and human health.

[0007] The journal article "Real-time fluorescence quantitative PCR detection of nucleotide residues in asparaginase preparations for injection" discloses the design of amplification primers by selecting the Escherichia coli 23S ribosomal RNA gene as the target gene.

[0008] In the above-mentioned prior art, the primers involved in CN105132539A only include primers designed based on the gene sequence of the transcribed spacer region 1 of Clonorchis sinensis; although Escherichia coli primers and Clonorchis sinensis primers are included in CN114959082 A, Escherichia coli is not used as an internal reference; although the above-mentioned journal document discloses the selection of Escherichia coli 23S ribosomal RNA gene as the target gene to design amplification primers, Escherichia coli is not used as an internal reference. It can be seen that the prior art does not design internal reference primers, and it is impossible to monitor whether the amplification experiment is abnormal, such as whether there is a false positive or false negative, and thus the authenticity of the experimental results cannot be guaranteed. Summary of the invention

[0009] In view of the above problems, the present invention provides a detection method for Clonorchis sinensis for non-disease diagnosis purposes, which is combined with two sets of primer groups, wherein primer group B for amplifying Escherichia coli genes is used as an internal reference primer, which can monitor whether the nucleic acids of samples and quality control products are successfully extracted during the detection process and whether the test results are reliable, thereby solving the technical problem that the previous Clonorchis sinensis detection technology cannot monitor whether the amplification experiment is abnormal.

[0010] To achieve the above object, the present invention adopts the following technical solution: A method for detecting Clonorchis sinensis for non-disease diagnosis purposes: Primer set A for targeted amplification of Clonorchis sinensis genes is used as the target primer, and primer set B for targeted amplification of Escherichia coli genes is used as the internal reference primer. By independently designing a new primer-probe combination, the specific gene sequences of Clonorchis sinensis and Escherichia coli are targeted for amplification, respectively, with high specificity, which can highly match multiple copy fragments of Clonorchis sinensis genes and Escherichia coli genes, and has high amplification efficiency.

[0011] Preferably, the Clonorchis sinensis gene is a conserved sequence in the transcriptional spacer region 1 of Clonorchis sinensis (sequence number KX378008.1), and the Escherichia coli gene is a conserved sequence in the 23S RNA of Escherichia coli (sequence number CP139887.1). When the internal reference gene is a conserved sequence in the 23S RNA of Escherichia coli, the matching degree reaches 100%, which greatly improves the amplification efficiency.

[0012] Preferably, the primer set A comprises primer F1, primer R1 and probe P1, the nucleotide sequence of primer F1 is shown in SEQ ID No.1; the nucleotide sequence of primer R1 is shown in SEQ ID No.2; the nucleotide sequence of probe P1 is shown in SEQ ID No.3.

[0013] Preferably, the probe P1 has a 6-FAM fluorescent group at its 3' end and a BHQ1 fluorescent quenching group at its 5' end.

[0014] Preferably, the primer set B comprises primer F2, primer R2 and probe P2, the nucleotide sequence of the primer F2 is shown in SEQ ID No.4; the nucleotide sequence of the primer R2 is shown in SEQ ID No.5; the nucleotide sequence of the probe P2 is shown in SEQ ID No.6.

[0015] Preferably, the probe P2 has a CY5 fluorescent group at its 3' end and a BHQ2 fluorescent quenching group at its 5' end.

[0016] Preferably, the detection method of Clonorchis sinensis for non-disease diagnosis purposes includes extraction of sample nucleic acid, extraction of positive control nucleic acid, and extraction of negative control nucleic acid, and anti-inhibitors are added to the extraction of sample nucleic acid, extraction of positive control nucleic acid, and extraction of negative control nucleic acid.

[0017] Preferably, the anti-inhibitor is InhibitEX Buffer. Adding the anti-inhibitor InhibitEX Buffer during the nucleic acid extraction process can significantly inhibit the activity of PCR inhibitors in the sample, greatly improve the PCR amplification efficiency, detection efficiency and detection throughput, and improve the sensitivity of the detection.

[0018] Preferably, the anti-inhibitor is added in an amount of 1 mL / 250 mg dry stool or 1 mL / 1 mL loose stool.

[0019] Preferably, the extraction of sample nucleic acid, the extraction of positive control nucleic acid, and the extraction of negative control nucleic acid are all performed by magnetic bead method. The nucleic acid extraction or purification reagent of Guangzhou Daan Gene Co., Ltd., item number HYK0139, is used as the extraction reagent.

[0020] Preferably, in the extraction process of the positive control nucleic acid, a plasmid containing Clonorchis sinensis DNA and a negative sample are used as positive quality control products.

[0021] Preferably, the plasmid is obtained as follows: a 300 bp sequence in the transcriptional spacer region 1 of Clonorchis sinensis is inserted into the pUC57 (2710 bp) vector to synthesize and construct a plasmid. After synthesis, the plasmid is diluted to 1×10 6 copies / uL, 1×10 5 copies / uL, 1×10 4 copies / uL, 1×10 3 Four concentration gradients of 1000 copies / uL are used as the detection limit reference. Multiple different concentrations of plasmid + negative samples constitute the positive reference, and one of the concentrations is selected as the positive quality control product.

[0022] Preferably, the 300 bp sequence is shown as SEQ ID No.7.

[0023] Preferably, nuclease-free water is used as a negative control product during the extraction of the negative control nucleic acid.

[0024] A quality control system is formed by combining positive quality control plasmid, negative quality control product and internal reference primer. This quality control system is involved in sample pretreatment, DNA extraction and PCR detection process. Among them, nuclease-free water is used as negative quality control product to monitor whether the background of the entire batch of detection is contaminated; a certain concentration of plasmid + negative sample is used as positive quality control product to monitor the efficiency of the batch of detection; internal reference primers directly amplify the Escherichia coli nucleic acid extracted from the sample to monitor whether the sample and quality control product nucleic acid are extracted successfully and whether the test results are reliable.

[0025] The present invention has the following beneficial effects: 1. The present invention optimizes and innovates the existing primer probes for detecting Clonorchis sinensis, and adopts two sets of independently designed new primer groups for combination, respectively performing targeted amplification on the target gene (Clonorchis sinensis gene) and the internal reference gene (Escherichia coli gene), which can quickly and accurately match and detect the target pathogen gene. Among them, the primer group B for amplifying the Escherichia coli gene is used as the internal reference primer, which can monitor whether the nucleic acid of the sample and the quality control product is successfully extracted during the detection process and whether the test results are credible. It has the characteristics of few false positives or false negatives, high detection specificity and sensitivity, and solves the technical problem that the previous Clonorchis sinensis detection technology cannot monitor whether the amplification experiment is abnormal.

[0026] 2. The detection method of the present invention comprises the above-mentioned two sets of primer groups for Clonorchis sinensis and internal reference genes, which can quickly detect the target pathogen and the internal reference gene, and can detect multiple samples at the same time, greatly shortening the detection time and helping the clinic to quickly identify the pathogen; at the same time, because the internal reference primers are included, the quality control of the experiment can be carried out; the unique primer design makes the detection method have high specificity and obvious timeliness.

[0027] 3. The detection method of the present invention is simple and easy, can detect pathogens and internal reference nucleic acids at the same time, can detect multiple samples at the same time, has high specificity and obvious timeliness, greatly shortens the detection time, has ideal sensitivity, specificity, accuracy and repeatability, and provides technical support for basic research on the distribution of Clonorchis sinensis and the genetic variation rules of Clonorchis sinensis in epidemiological surveys. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the amplification of the stool sample in Example 1 of the present invention; Figure 2 is the amplification of a stool sample processed in Example 2 of the present invention; Figure 3 is the amplification of 2 pairs of stool samples processed by Example 2 of the present invention; Figure 4 is the amplification of 3 pairs of stool samples processed by Example 2 of the present invention; Figure 5 This is the comparison result of monitoring the amplification efficiency of stool samples by using the internal reference gene of Escherichia coli and the human internal reference gene in Example 6 of the present invention. DETAILED DESCRIPTION

[0029] In order to better present the present invention, it is illustrated by specific implementation cases, which belong to the protection scope of the present invention but do not limit the protection scope of the present invention.

[0030] Example 1 1. Acquisition of primers and plasmids 1.1 Two sets of targeted primers are provided: The first set is a primer set A for targeted amplification of a conserved sequence in the transcribed spacer region 1 of Clonorchis sinensis, comprising primer F1, primer R1 and probe P1, wherein the 3' end of P1 carries a 6-FAM fluorescent group and the 5' end carries a BHQ1 fluorescent quenching group. The second set is a primer set B for targeted amplification of a conserved sequence in the 23S RNA of Escherichia coli, comprising primers F2 and R2, and probe P2, wherein the 3' end of P2 carries a CY5 fluorescent group and the 5' end carries a BHQ2 fluorescent quenching group.

[0031] 1.2 Obtaining positive quality control plasmid A 300 bp sequence from the transcribed spacer region 1 of Clonorchis sinensis was inserted into the pUC57 (2710 bp) vector to construct a positive quality control plasmid. After synthesis, the plasmid was diluted to 1×10 6 copies / uL, 1×10 5 copies / uL, 1×10 4 copies / uL, 1×10 3 Four concentration gradients of copies / uL were used as detection limit references.

[0032] Table 1: Sequences of primers, probes, and plasmids

[0033] Note: The sequences in the table default to the 5' end on the left and the 3' end on the right.

[0034] 2. Sample pretreatment and nucleic acid extraction The sample type of this embodiment is a stool sample, which is collected according to the microbiological test sample collection standard. For dry stool samples, it is recommended to collect more than 2.5g, and for loose stool samples, it is recommended to collect more than 2mL.

[0035] 2.1 Sample pretreatment: 1) Solid samples: Take about 250 mg of sample with a sterile cotton swab or pipette tip and place it in a sterile 2 mL centrifuge tube (soybean size). Add 400 µL of nuclease-free water and 1 mL of InhibitEX Buffer and glass beads from a broken tube in the extraction reagent. Vortex for 1 minute or until the sample is completely homogenized. Heat the suspension at 90°C for 5 min and vortex for 15 seconds. Centrifuge the sample at 6000 rpm for 1 minute to precipitate insoluble matter. Aspirate 800 µL of supernatant and set aside.

[0036] 2) Liquid samples: Take 1 mL of sample and place it in a sterile 2 mL centrifuge tube. Centrifuge at 12000g for 2 min to keep the precipitate and 400 µL of supernatant. Add 1 mL of InhibitEX Buffer and glass beads in a broken tube in the extraction reagent. Vortex for 1 minute or until the sample is completely homogenized. Heat the suspension at 90°C for 5 min and vortex for 15 seconds. Centrifuge the sample at 6000 rpm for 1 minute to precipitate insoluble matter. Pipette 800 µL of supernatant for later use.

[0037] 3) Pretreatment of positive quality control: Take about 250 mg of negative sample and place it in a sterile 2 mL centrifuge tube (soybean size), add 400 µL of nuclease-free water and 1 mL of InhibitEX Buffer and glass beads in a broken tube in the extraction reagent, and then add 1.4 uL of plasmid of corresponding concentration as required (1×10 6 copies / uL as a strong positive quality control, 1×10 4 The suspension was heated at 90℃ for 5 min and vortexed for 15 seconds. The sample was centrifuged at 6000rpm for 1 minute to precipitate insoluble matter, and 800μL of supernatant was aspirated for later use.

[0038] 4) Pretreatment of negative quality control products: Take 1 mL of nuclease-free water and place it in a sterile 2 mL centrifuge tube. Centrifuge at 12000g for 2 minutes to keep the precipitate and 400 µL of supernatant. Add 1 mL of InhibitEX Buffer and glass beads in a broken tube in the extraction reagent. Vortex for 1 minute or until the sample is completely homogenized. Heat the suspension at 90℃ for 5 minutes and vortex for 15 seconds. Centrifuge the sample at 6000rpm for 1 minute to precipitate insoluble matter. Aspirate 800 µL of supernatant and set aside.

[0039] 2.2 Nucleic acid extraction: The nucleic acid extraction or purification reagent (magnetic bead method) of Guangzhou Daan Gene Co., Ltd., catalog number HYK0139, was used to extract nucleic acid from the above samples.

[0040] 1) Place the supernatant in a 2 mL centrifuge tube, add 30 µL of streptavidin magnetic beads and 40 µL of proteinase K, and vortex thoroughly to mix.

[0041] 2) Place the centrifuge tube in a constant temperature shaking metal bath, incubate at 70°C for 12 min (speed is 1000 rpm / min), and centrifuge briefly.

[0042] 3) Place the centrifuge tube on the magnetic rack. After the magnetic beads are completely adsorbed, carefully remove the liquid with a pipette, being careful not to absorb the magnetic beads.

[0043] 4) Add 800 µL of washing solution I to the centrifuge tube, vortex to mix for 1 min, and centrifuge briefly.

[0044] 5) Place the centrifuge tube on the magnetic rack. After the magnetic beads are completely adsorbed, carefully remove the liquid with a pipette, being careful not to absorb the magnetic beads.

[0045] 6) Add 800 µL of Washing Buffer II to the centrifuge tube, vortex to mix for 1 min, and centrifuge briefly.

[0046] 7) Place the centrifuge tube on the magnetic rack. After the magnetic beads are completely adsorbed, carefully remove the liquid with a pipette, being careful not to absorb the magnetic beads.

[0047] 8) Repeat the above steps 6) and 7).

[0048] 9) Centrifuge the tube for 10 seconds, place the tube on the magnetic rack, and after the magnetic beads are completely adsorbed, carefully remove the liquid with a pipette, being careful not to absorb the magnetic beads. Open the lid and let it dry for 3 to 5 minutes.

[0049] 10) Add 100 µL of elution buffer to the centrifuge tube, vortex to mix, incubate at room temperature for 5 minutes, place on a magnetic rack, and transfer the nucleic acid solution to a new sterile centrifuge tube after the magnetic beads are completely adsorbed. Use Qubit4.0 Nucleic Acid Quantifier for DNA quantification.

[0050] 3. Reaction system preparation and operation 3.1 Preparation of PCR Reagents The main components and specifications of PCR reaction reagents are shown in Table 2: Table 2: Main components and specifications of PCR reaction reagents

[0051] 3.2 Preparation 1) Take out the PCR MIX reaction solution, primer set A, and primer set B, melt them on ice, shake and mix them, and centrifuge them at 8000 rpm for a few seconds before use.

[0052] 2) Take 2 1.5 mL EP tubes and prepare the PCR reaction solutions for two channels. Tube 1: 12 μL of PCR MIX reaction solution, 0.5 μL of P1, 1 μL of F1, and 1 μL of R1. Tube 2: 12 μL of PCR MIX reaction solution, 0.5 μL of P2, 1 μL of F2, and 1 μL of R2.

[0053] 3.3 Sample Loading Add 6.5 μL of the extracted nucleic acid of the sample to be tested, negative control nucleic acid, and positive control nucleic acid to the corresponding reaction wells above, tightly cap the tube lids, centrifuge at 8000 rpm for a few seconds, and then transfer to the amplification area.

[0054] 3.4 PCR Amplification: 1) Place the reaction tubes into the sample slots of the fluorescence quantitative PCR instrument, set the positive control, negative control, and the sample to be tested in the corresponding order, and set the sample names.

[0055] 2) Fluorescence detection channel selection: Select the FAM channel to detect Clonorchis sinensis nucleic acid. Select the CY5 channel to detect the internal reference.

[0056] 3) After setting, save the file and run the reaction program.

[0057] Table 3: PCR Reaction Program

[0058] 4. Result Analysis 1) Negative control: No Ct value in the FAM channel, no Ct value in the internal reference CY5 channel.

[0059] 2) Positive control: Ct values in both the FAM channel and the internal reference CY5 channel are ≤ 35.

[0060] 3) The above requirements need to be met simultaneously in the same experiment. Otherwise, this experiment is invalid and needs to be repeated.

[0061] 4) For samples with a typical S-shaped amplification curve detected in the FAM channel and a Ct value ≤ 37 in this channel, it is judged as positive for Clonorchis sinensis.

[0062] 5) For samples where no typical S-shaped amplification curve (No Ct) is detected in the FAM channel, there is an amplification curve in the CY5 channel, and the Ct value ≤ 37, it is judged as negative for Clonorchis sinensis.

[0063] 6) For samples where no typical S-shaped amplification curve is detected in the FAM channel, 37 < Ct value ≤ 40, there is an amplification curve in the CY5 channel, and Ct ≤ 37, it is determined to be in the gray area. Repeat the amplification experiment. If there is still a Ct value in the FAM channel, it is judged as positive for Clonorchis sinensis. If No Ct, it is judged as negative for Clonorchis sinensis.

[0064] 7) If no typical S-shaped amplification curve (No Ct) is detected in the FAM channel and the CY5 channel, or the Ct values ​​are both >37, the test results of the sample are invalid and need to be repeated.

[0065] 5. Five stool specimens that were positive under microscopic examination were selected and amplified for Clonorchis sinensis according to the above method. The results were as follows: Figure 1 As shown, in the five samples, all the extracted Clonorchis sinensis genes were detected to be amplified, and the internal reference gene of Escherichia coli was also amplified.

[0066] Example 2 Referring to the detection method of Example 1, different treatment groups were set up with sample pretreatment and nucleic acid extraction methods as variables to investigate the effects of different sample pretreatment and nucleic acid extraction methods on the detection results.

[0067] 1. Treatment 1: Use conventional column extraction method instead of magnetic bead method, and use PBS buffer instead of InhibitEX Buffer.

[0068] The specific operations are as follows: 1.1 Sample pretreatment: 1) Solid feces: Use a sterile cotton swab or pipette tip to take about 250 mg of sample and place it in a sterile 2 mL centrifuge tube (soybean size). Add 1 mL of PBS buffer and vortex for 1 minute or until the fecal sample is completely homogenized. Heat the suspension at 90°C for 5 min and vortex for 15 seconds. Centrifuge the sample at 6000 rpm for 1 minute to precipitate insoluble matter. Aspirate 800 µL of supernatant and extract using column extraction reagent.

[0069] 2) Liquid feces: Take 1 mL of sample and place it in a sterile 2 mL centrifuge tube. Centrifuge at 12000g for 2 min to keep the precipitate and 400 µL of supernatant. Add 1 mL of PBS buffer and vortex for 1 minute or until the fecal sample is completely homogenized. Heat the suspension at 90°C for 5 min and vortex for 15 seconds. Centrifuge the sample at 6000rpm for 1 minute to precipitate insoluble matter. Pipet 800 µL for later use.

[0070] Note: The main components of PBS buffer are Na2HPO4, KH2PO4, NaCl, and KCl, which act as solvents to dissolve protective reagents.

[0071] 1.2 When using column extraction reagents for hand extraction, if the nucleic acid concentration exceeds 5ng / ul, the nucleic acid needs to be diluted to below 5ng / ul before use.

[0072] 2. Treatment 2: Replace the PBS buffer in Treatment 1 with InhibitEX Buffer, and keep other operations unchanged.

[0073] 3. Treatment 3: Based on Example 1, PBS buffer was used instead of InhibitEX Buffer, and other operations remained unchanged.

[0074] 4. Five stool specimens that were positive under microscopic examination were selected and processed according to treatments 1 to 3 in Example 2 for Clonorchis sinensis amplification.

[0075] Results: In treatment 1, only 2 samples were detected with amplification of the Clonorchis sinensis gene, and only 3 samples were detected with amplification of the Escherichia coli internal reference gene; in treatment 2, only 3 samples were detected with amplification of the Clonorchis sinensis gene, and no amplification of the Escherichia coli internal reference gene was detected; in treatment 3, only 1 sample was detected with amplification of the Clonorchis sinensis gene, and only one sample was detected with amplification of the Escherichia coli internal reference gene. Figures 2~4 shown.

[0076] By comparing the detection results of treatments 1 to 3 in Example 2 with those of Example 1, it can be seen that the detection effect can be greatly improved by using the magnetic bead method and InhibitEX Buffer in the present invention.

[0077] Example 3 Confirmation of accuracy by microscopic examination of positive and negative samples 1. A total of 70 clinical samples (50 positive stool samples and 20 negative stool samples) with known results (traditional microscopy) and meeting the sample quality control requirements were selected for PCR testing of Clonorchis sinensis and compared with the clinical results for accuracy verification.

[0078] 2. The detection results are as follows: Table 4: Detection of Clonorchis sinensis PCR test in negative and positive samples

[0079] 3. Based on the accuracy test of clinical samples, the detection kit of the present invention has a detection sensitivity of 98.11% (52 / 53), a specificity of 100% (17 / 17), and an overall accuracy of 98.57% (69 / 70).

[0080] Example 4 Repeatability and detection limit confirmation 1. Repeatability 1.1 1×10 4 Copies / uL concentration of positive plasmid was added to 20 negative stool samples to make simulated positive references for nucleic acid extraction and PCR experiments. After 20 repeated tests, the results of the 20 tests were analyzed.

[0081] 1.2 All 20 repeatability verifications were able to detect correctly, with a repeatability of 100% (20 / 20), and the Ct values ​​of the 20 repetitions were basically consistent (coefficient of variation CV=3.48%). The results showed that the repeatability of the nucleic acid detection of Clonorchis sinensis by the present invention was good, and the positive simulation reference product could be used as a positive quality control product.

[0082] 2. Detection Limit 2.1 Add 4 concentrations of plasmid to negative samples for extraction and then perform PCR detection of Clonorchis sinensis (each concentration is repeated 3 times). Record the lowest concentration that can be detected in three repetitions (as the sample concentration of the repeatability simulation reference) as Cy, dilute Cy gradient into three concentrations of 0.5Cy, 0.25Cy, and 0.125Cy, and perform nucleic acid extraction and PCR verification 5 times for each concentration. Record the lowest concentration that can be detected in 5 repetitions as the minimum detection limit.

[0083] 2.2 The results show that the detection kit of the present invention has a reference concentration of 2.5×10 3 Copies / mL, it can be stably detected (5 / 5), and at a concentration of 1.25×10 3 Copies / mL, the detection rate was 80% (4 / 5), so the detection limit of the detection kit involved in the present invention was 2.5×10 3 Copies / mL.

[0084] Example 5: Determination of the amount of inhibitor InhibitEX added to investigate the interfering substance inhibitor InhibitEX in fecal sample PCR amplification The dosage of InhibitEX was set to 300 μL, 500 uL, 1000 μL and 2000 μL, using 1×10 4 The simulated positive reference made of plasmid with a concentration of 100 copies / uL was tested 5 times, and 9 positive samples were verified at different gradients. The experimental results are as follows: Table 5: Positive detection rate at different InhibitEX addition amounts

[0085] The experimental results show that a decrease in the concentration of InhibitEX buffer may lead to PCR inhibition and thus reduce the sensitivity of the detection. While ensuring sufficient detection sensitivity, the optimal addition amount is determined to be 1000 μL.

[0086] Example 6 Comparison of Escherichia coli endogenous genes and human endogenous reference genes in monitoring amplification efficiency of stool samples An internal reference primer (with a Cy5 fluorescent group at 3') was designed based on the human β-actin encoding gene (ACTB) and added to the Clonorchis sinensis amplification system. Fecal samples, pus, bronchoalveolar lavage fluid, and blood samples were tested, and the specific results are shown in Table 6.

[0087] Table 6: Comparison of E. coli endogenous genes and human endogenous reference genes in monitoring amplification efficiency of stool samples

[0088] From Table 6 and Figure 5 It can be seen that ACTB can correctly amplify the ACTB gene in non-fecal samples, indicating that the amplification of human internal reference primers in non-fecal samples is feasible, but difficult to amplify in fecal samples. Escherichia coli internal reference primers can be correctly amplified in fecal samples, but because the internal reference of Escherichia coli is for feces, there is no Escherichia coli in non-fecal samples such as blood, so it will not be amplified in non-fecal samples such as blood. In contrast, Escherichia coli internal reference primers are more suitable as internal reference primers for the detection of Chinese flukes in fecal samples, and can effectively monitor the amplification efficiency in samples.

[0089] The above contents are further detailed descriptions of the present invention in combination with specific / preferred implementations, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, they can also make several substitutions or modifications to these described embodiments without departing from the concept of the present invention, and these substitutions or modifications should be regarded as belonging to the protection scope of the present invention.

Claims

1. A method for detecting Clonorchis sinensis for non-disease diagnosis purposes, characterized in that: Primer set A for targeted amplification of Clonorchis sinensis genes was used as target primers, and primer set B for targeted amplification of Escherichia coli genes was used as internal reference primers.

2. The method for detecting Clonorchis sinensis for non-disease diagnosis purposes as claimed in claim 1, characterized in that: The Clonorchis sinensis gene is a conserved sequence in the transcriptional spacer region 1 of Clonorchis sinensis, and the Escherichia coli gene is a conserved sequence in the Escherichia coli 23S RNA.

3. The method for detecting Clonorchis sinensis for non-disease diagnosis purposes as claimed in claim 1, characterized in that: The primer set A comprises primer F1, primer R1 and probe P1, the nucleotide sequence of primer F1 is shown as SEQ ID No.1; the nucleotide sequence of primer R1 is shown as SEQ ID No.2; the nucleotide sequence of probe P1 is shown as SEQ ID No.

3.

4. The method for detecting Clonorchis sinensis for non-disease diagnosis purposes as claimed in claim 1, characterized in that: The primer set B comprises primer F2, primer R2 and probe P2, the nucleotide sequence of the primer F2 is shown in SEQ ID No.4; the nucleotide sequence of the primer R2 is shown in SEQ ID No.5; the nucleotide sequence of the probe P2 is shown in SEQ ID No.

6.

5. The method for detecting Clonorchis sinensis for non-disease diagnosis purposes as claimed in claim 1, characterized in that: The process includes extraction of sample nucleic acid, extraction of positive control nucleic acid and extraction of negative control nucleic acid, and anti-inhibitors are added to the extraction of sample nucleic acid, extraction of positive control nucleic acid and extraction of negative control nucleic acid.

6. The method for detecting Clonorchis sinensis for non-disease diagnosis purposes as claimed in claim 5, characterized in that: The anti-inhibitor is InhibitEX Buffer.

7. The method for detecting Clonorchis sinensis for non-disease diagnosis purposes as claimed in claim 5, characterized in that: The amount of the anti-inhibitor added is 1 mL / 250 mg dry stool or 1 mL / 1 mL loose stool.

8. The method for detecting Clonorchis sinensis for non-disease diagnosis purposes as claimed in claim 5, characterized in that: The extraction of sample nucleic acid, the extraction of positive control nucleic acid and the extraction of negative control nucleic acid all adopt the magnetic bead method for nucleic acid extraction.

9. The method for detecting Clonorchis sinensis for non-disease diagnosis purposes as claimed in claim 5, characterized in that: The extraction of the positive control nucleic acid uses a plasmid containing Clonorchis sinensis DNA and a negative sample as a positive quality control product.

10. The method for detecting Clonorchis sinensis for non-disease diagnosis purposes according to claim 5, characterized in that: The extraction of the negative control nucleic acid uses nuclease-free water as a negative control product.

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