Schistosoma japonicum circulating DNA and application thereof

Abstract

The invention provides circulating DNA (deoxyribonucleic acid) of schistosoma japonicum katsurada and application thereof, and particularly provides a kit for detecting the infection condition of schistosoma japonicum katsurada in a patient, which comprises a component for detecting a circulating DNA sequence of schistosoma japonicum katsurada, the circulating DNA sequence of the schistosoma japonicum katsurada is selected from any one or a combination of more of the following groups: nucleotide sequences as shown in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13 and SEQ ID NO: 16. The circulating DNA provided by the invention can be used for curative effect examination of schistosoma japonicum katsurada chemotherapy, and also can be used for diagnosing schistosoma japonicum katsurada; according to the detection method provided by the invention, a sample can be obtained by collecting peripheral blood, the blood can be collected at any time in the treatment process, and the chemotherapy effect is dynamically monitored in real time; the method has the advantages of low cost and effectiveness, is a promising method for examining the curative effect of the schistosomiasis, and is also used for nucleic acid diagnosis of the schistosomiasis.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology and diagnosis. Specifically, it relates to a Schistosoma japonicum circulating DNA and its applications. Background Art

[0002] Schistosomiasis is a major infectious disease that seriously endangers human health and affects global economic development. It is prevalent in 78 countries and regions around the world and is one of the major public health problems in tropical and subtropical regions. As of 2022, 75% of the 452 endemic counties in China have reached the elimination standard and are in a low-prevalence state. However, there are still some difficulties and challenges in achieving the overall goal of completely eliminating schistosomiasis. The epidemic factors have not changed fundamentally; the role of animal reservoirs, especially wild animals, as the natural reservoir hosts of Schistosoma in nature is difficult to eliminate, and unexpected natural disasters and other emergencies exacerbate the risk of Oncomelania snail dispersion and transmission. Therefore, it is urgent to continuously strengthen the prevention and control of schistosomiasis and further establish and improve the effect evaluation mechanism.

[0003] Currently, the widely used schistosomiasis etiological methods cannot meet the needs in endemic areas due to their disadvantages such as low throughput, time-consuming, and high requirements for operator experience; immunological methods, due to their rapidity, simplicity, and relatively high sensitivity and specificity, have played an important role in the prevention and control of schistosomiasis. Currently commonly used methods include indirect hemagglutination assay (IHA), enzyme-linked immunosorbent assay (ELISA), and colloidal dye immunoassay (DDIA), etc. These methods have relatively high sensitivity and good specificity and have been widely applied in the field. However, after a host is infected with Schistosoma, the antibodies (mainly IgG) produced by the body in response to antigen stimulation will remain in the host's body fluid for a long time. Even if the infected person has received effective treatment (the worms in the body have died), the antibody level in their body still remains at a high level and will continue for several years without disappearing in the absence of reinfection. Therefore, it is impossible to determine whether further treatment is needed, often resulting in repeated medication for cured patients, which not only increases the mental burden of the medication users but may also reduce the sensitivity of Schistosoma to drugs.

[0004] Therefore, there is an urgent need in this field to develop a short-term marker that disappears relatively quickly after treatment to promote the effective development of effective efficacy assessment methods in schistosomiasis prevention and control research. Summary of the Invention

[0005] The object of the present invention is to provide a class of Schistosoma japonicum circulating DNA, which has strong specificity and high detection rate for schistosomiasis.

[0006] In the first aspect of the present invention, there is provided a kit for detecting Schistosoma japonicum infection in a patient, the kit comprising components for detecting Schistosoma japonicum circulating DNA sequences, and the Schistosoma japonicum circulating DNA sequences being selected from any one or more combinations of the following groups:

[0007] (1) The nucleotide sequence shown in SEQ ID NO: 1;

[0008] (2) The nucleotide sequence shown in SEQ ID NO: 4;

[0009] (3) The nucleotide sequence shown in SEQ ID NO: 7;

[0010] (4) The nucleotide sequence shown in SEQ ID NO: 10;

[0011] (5) The nucleotide sequence shown in SEQ ID NO: 13;

[0012] (6) The nucleotide sequence shown in SEQ ID NO: 16.

[0013] In another preferred example, the kit comprises one or more primer pairs selected from the following groups:

[0014] (1) The nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3;

[0015] (2) The nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6;

[0016] (3) The nucleotide sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9;

[0017] (4) The nucleotide sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12;

[0018] (5) The nucleotide sequences shown in SEQ ID NO: 14 and SEQ ID NO: 15;

[0019] (6) The nucleotide sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18.

[0020] In another preferred example, the kit further comprises any one or more sequences selected from the following groups:

[0021] (1) The nucleotide sequence shown in SEQ ID NO: 1;

[0022] (2) The nucleotide sequence shown in SEQ ID NO: 4;

[0023] (3) The nucleotide sequence shown in SEQ ID NO: 7;

[0024] (4) The nucleotide sequence shown in SEQ ID NO: 10;

[0025] (5) The nucleotide sequence shown in SEQ ID NO: 13;

[0026] (6) The nucleotide sequence shown in SEQ ID NO: 16.

[0027] In another preferred example, the kit further includes an instruction manual, and the following steps are recorded in the instruction manual:

[0028] (i) Detect the level of one or more circular DNA fragments selected from the following group in the sample using the components in the kit:

[0029] (1) The nucleotide sequence shown in SEQ ID NO: 1;

[0030] (2) The nucleotide sequence shown in SEQ ID NO: 4;

[0031] (3) The nucleotide sequence shown in SEQ ID NO: 7;

[0032] (4) The nucleotide sequence shown in SEQ ID NO: 10;

[0033] (5) The nucleotide sequence shown in SEQ ID NO: 13;

[0034] (6) The nucleotide sequence shown in SEQ ID NO: 16;

[0035] (ii) Judge the Schistosoma japonicum infection status of the patient according to the detected level of circular DNA fragments.

[0036] In another preferred example, the sample is a peripheral blood sample of the patient.

[0037] In another preferred example, the following steps are also recorded in the instruction manual: When one or more circular DNA fragments are detected as positive in the sample, judge the sample as a positive sample.

[0038] In another preferred example, the detection includes the following steps:

[0039] (a) Extract circular DNA: Extract circular DNA from the peripheral blood of the host;

[0040] (b) Prepare the PCR system: Prepare PCR tubes for the extracted circular DNA and the primer combination respectively;

[0041] (c) PCR amplification reaction: Place the prepared PCR tube in a PCR instrument for amplification reaction;

[0042] (d) Detection of PCR results.

[0043] In another preferred embodiment, the PCR result is detected by agarose gel electrophoresis to detect the result of the PCR amplification reaction.

[0044] In the second aspect of the present invention, a method for screening or identifying a potential therapeutic agent for treating or alleviating schistosomiasis japonica is provided, including the following steps:

[0045] (a) Administer a candidate substance to a non-human mammalian model of schistosomiasis japonica, and set up a blank control group;

[0046] (b) Detect the level of circulating DNA in the serum of the non-human mammalian model, and compare it with the control group.

[0047] If the positive detection rate of circulating DNA in the animal model administered with the candidate substance is reduced compared with the control group, it is determined that the candidate substance has a therapeutic effect on schistosomiasis japonica.

[0048] Wherein, the nucleotide sequence of the schistosoma japonicum circulating DNA is selected from any one or a combination of the following groups:

[0049] (1) The nucleotide sequence shown in SEQ ID NO: 1;

[0050] (2) The nucleotide sequence shown in SEQ ID NO: 4;

[0051] (3) The nucleotide sequence shown in SEQ ID NO: 7;

[0052] (4) The nucleotide sequence shown in SEQ ID NO: 10;

[0053] (5) The nucleotide sequence shown in SEQ ID NO: 13;

[0054] (6) The nucleotide sequence shown in SEQ ID NO: 16.

[0055] In another preferred embodiment, the screening includes the step of administering a candidate substance to the non-human mammalian model of schistosomiasis japonica, so as to evaluate the effect of the candidate substance on the positive detection rate of schistosoma japonicum circulating DNA in the serum of the model animal.

[0056] In another preferred embodiment, the screening further includes the step of sacrificing the non-human mammalian model.

[0057] In another preferred example, the reduction includes: a decrease in the positive detection rate of circulating DNA in an animal model administered with a candidate substance, and this decrease is statistically significantly different compared to the control group.

[0058] In another preferred example, the method further includes: detecting using one or more primer pairs selected from the group consisting of:

[0059] (1) The nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3;

[0060] (2) The nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6;

[0061] (3) The nucleotide sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9;

[0062] (4) The nucleotide sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12;

[0063] (5) The nucleotide sequences shown in SEQ ID NO: 14 and SEQ ID NO: 15;

[0064] (6) The nucleotide sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18.

[0065] In the third aspect of the present invention, there is provided a Schistosoma japonicum circulating DNA sequence composition, and the sequence composition is selected from any two or more sequence combinations in the following group:

[0066] (1) The nucleotide sequence shown in SEQ ID NO: 1;

[0067] (2) The nucleotide sequence shown in SEQ ID NO: 4;

[0068] (3) The nucleotide sequence shown in SEQ ID NO: 7;

[0069] (4) The nucleotide sequence shown in SEQ ID NO: 10;

[0070] (5) The nucleotide sequence shown in SEQ ID NO: 13;

[0071] (6) The nucleotide sequence shown in SEQ ID NO: 16.

[0072] In another preferred example, the nucleotide sequences of the composition are those shown in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 16.

[0073] In the fourth aspect of the present invention, there is provided a primer combination for amplifying Schistosoma japonicum circular DNA sequences, and the nucleotide sequences of the primer combination are selected from any one or more combinations of the following groups:

[0074] (1) The nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3;

[0075] (2) The nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6;

[0076] (3) The nucleotide sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9;

[0077] (4) The nucleotide sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12;

[0078] (5) The nucleotide sequences shown in SEQ ID NO: 14 and SEQ ID NO: 15;

[0079] (6) The nucleotide sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18.

[0080] In another preferred example, the nucleotide sequences of the primer combination are those shown in SEQ ID NO: 2 and SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 18.

[0081] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 It shows a schematic diagram of the screening process of cDNA fragments based on resequencing.

[0083] Figure 2 It shows the detection results of serum K81-55 of 3 mice in the normal group of Schistosoma japonicum before praziquantel treatment. Among them, M represents Marker, and N represents negative control (amplification with H2O as template).

[0084] Figure 3Shown are the test results of serum K81-55 of 10 mice in the Schistosoma japonicum infection group before praziquantel treatment. Among them, M represents Marker; N represents the negative control, that is, the serum of normal C57 mice not infected with Schistosoma japonicum. Extract its nucleic acid as the negative control. Only when there is no band in the negative control can the integrity of the experiment and the reliability of the results be ensured. The molecular weight of the target band of the detected K81-55 is 80 bp.

[0085] Figure 4 Shown are the test results of serum K81-55 of 5 mice in the Schistosoma japonicum infection group 3 weeks (3w) after praziquantel treatment. Among them, M represents Marker, and N represents the negative control, that is, the serum of normal C57 mice not infected with Schistosoma japonicum. Extract its nucleic acid as the negative control.

[0086] Figure 5 Shown are the test results of serum K81-55 of 5 mice in the Schistosoma japonicum infection group 6 weeks (6w) after praziquantel treatment. Among them, M represents Marker, and N represents the negative control, that is, the serum of normal C57 mice not infected with Schistosoma japonicum. Extract its nucleic acid as the negative control. Detailed implementation mode

[0087] Through extensive and in-depth research, the present inventors for the first time adopted the resequencing technology to analyze serum samples in a mouse challenge infection model and screened 6 circulating DNA (circulating DNA, cDNA) fragments (K81-47, K81-25, K81-41, K81-55, K81-68, K81-18) from Schistosoma japonicum. The specific sequence information is shown in Table 2 below. They have the characteristics of strong specificity and high detection rate for Schistosoma japonicum disease. On this basis, the present invention was completed.

[0088] The present invention discovers multiple circulating DNAs from Schistosoma japonicum based on resequencing technology. In addition, the present invention also provides a dynamic evaluation method for evaluating the curative effect after praziquantel gavage treatment. This method uses the circulating DNA in the serum of infected mice as a template, designs and screens primers with strong specificity and high sensitivity for PCR and agarose gel electrophoresis verification. The detection points are 3 weeks and 6 weeks after drug chemotherapy. According to the presence or absence and intensity of the result bands, it is further indicated whether the schistosomes in the mice are killed or not. Specifically, it includes the following steps: establishment of a mouse challenge model, preparation of serum samples before praziquantel treatment, 3 weeks after treatment, and 6 weeks after treatment; extraction and resequencing of circulating DNA; screening of circulating DNA fragments from Schistosoma japonicum; design of fragment-specific primers; PCR detection and agarose gel verification; determination of chemotherapy effect.

[0089] Terms

[0090] Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0091] As used herein, the terms "comprising" or "including" can be open-ended, semi-closed, and closed-ended. In other words, these terms also include "consisting essentially of" or "consisting of".

[0092] As used herein, the terms "circular DNA", "cDNA", "circulating DNA", (cDNA fragment) can be used interchangeably and all refer to circular DNA fragments.

[0093] Schistosoma japonicum

[0094] Schistosomes, also known as schistosomula or blood flukes, belong to the class Trematoda, order Digenea, family Schistosomatidae, and genus Schistosoma. There are mainly six species of schistosomes that parasitize humans, namely Schistosoma mansoni, Schistosoma japonicum, Schistosoma haematobium, Schistosoma intercalatum, Schistosoma mekongi, and Schistosoma malayensis. Among them, schistosomiasis caused by Schistosoma mansoni, Schistosoma japonicum, and Schistosoma haematobium has the widest prevalence and the greatest harm. Schistosomiasis is mainly distributed in Asia, Africa, and Latin America. In China, Schistosoma japonicum disease is prevalent, and the condition caused by Schistosoma japonicum infection is also the most serious and the most difficult to control, mainly due to the characteristics of a large number of animal hosts of Schistosoma japonicum, a long lifespan of adult worms, poor concomitant immunity after infection and immunity after cure, and difficulty in controlling the intermediate host, Oncomelania hupensis.

[0095] Adult Schistosoma japonicum are dioecious. Male worms are milky white, 10 - 20 mm in length, with a smooth body surface, and well-developed oral and ventral suckers at the front end. Below the ventral sucker, the body extends to both sides and curls slightly towards the ventral surface, forming a gynecophoral canal where the female worm stays and is in a copulatory embrace with the male worm. Female worms are 12 - 28 mm in length, dark black, with a small front end and a thick round rear end. Schistosoma japonicum disease is caused by cercariae infecting definitive hosts such as humans and animals that come into contact with contaminated water.

[0096] Circular DNA

[0097] Circulating DNA exists in the host body fluid as DNA fragments derived from the growth and development of parasites (here referring to Schistosoma japonicum). Therefore, this circulating DNA in the host body fluid is highly pathogen-specific. Samples can be obtained by collecting peripheral blood, and blood can be collected at any time during the treatment process to monitor the chemotherapy effect in real-time and dynamically. The PCR-based analysis has the advantages of low cost and effectiveness, and is a promising method for evaluating the efficacy of schistosomiasis treatment. It is also used for the nucleic acid diagnosis of schistosomiasis.

[0098] Detection kit

[0099] Based on the new discovery of the present inventors, a kit for detecting schistosomiasis is also provided. The kit contains: cDNA molecules of Schistosoma japonicum; or primers specifically detecting cDNA molecules of Schistosoma japonicum.

[0100] The main advantages of the present invention include:

[0101] (1) The circulating DNA provided by the present invention can be used for evaluating the efficacy of chemotherapy for Schistosoma japonicum, and can also diagnose Schistosoma japonicum.

[0102] (2) The detection method provided by the present invention can obtain samples by collecting peripheral blood, and blood can be collected at any time during the treatment process to monitor the chemotherapy effect in real-time and dynamically.

[0103] (3) The PCR-based analysis of the present invention has the advantages of low cost and effectiveness, and is a promising method for evaluating the efficacy of schistosomiasis treatment. It is also used for the nucleic acid diagnosis of schistosomiasis.

[0104] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0105] Example 1

[0106] 1.1 Establishment of C57BL / 6 mouse challenge model and acquisition of serum samples

[0107] After 42 days of raising 10 male C57BL / 6 mice at 8 weeks old and infected with 60 cercariae, all mice were given praziquantel by gavage for 4 consecutive days. Each mouse was administered the drug at a dosage of 12.5 mg per 25 g of body weight, using methylcellulose as the solvent for praziquantel. The mice were randomly divided into 2 groups, with 5 mice in each group, and mouse serum samples were obtained at the 3rd week and the 6th week respectively. All infected mice were provided by the Shanghai Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention.

[0108] 1.2 Extraction of genomic DNA from Schistosoma japonicum adult worms and DNA from serum samples

[0109] Extraction of peripheral blood samples: After the mice were anesthetized, blood was collected from the eye socket and collected in a sterile 1.5 ml centrifuge tube; the blood sample was transferred to a centrifuge, centrifuged at 3500 rpm at 4 °C for 15 min; the supernatant was aspirated into a new sterile 1.5 ml centrifuge tube; centrifuged at 3500 rpm at 4 °C for 15 min; the supernatant was aspirated into a new sterile 1.5 ml centrifuge tube; for the serum samples obtained by this method, the total circulating DNA was extracted using the cell-free DNA magnetic bead extraction kit (product number: A29319) produced by Thermo Fisher Scientific (China) Co., Ltd., and the specific steps were referred to the instruction manual.

[0110] Extraction of adult worm DNA: 42 days after cercariae infection, the abdominal cavity was opened, the hepatic portal vein was gently severed, and the adult worms at the hepatic portal were picked out. The adult worms could also be obtained by perfusion method. The genomic DNA of adult worms was extracted using the Dneasy Blood and Tissue Kit (product number: 69504) produced by Qiagen (Shanghai) Co., Ltd., and the specific steps were referred to the instruction manual. If the extracted nucleic acids were not used temporarily, they were stored at -80 °C.

[0111] 1.3 Resequencing of serum cDNA samples and screening of target fragments

[0112] For 2 mice infected with 60 cercariae for 42 days, serum cDNA (sample 1 and sample 2) was isolated for resequencing, which was completed by Shanghai OE Biotech Co., Ltd., and the resequencing results were obtained. According to Figure 1 the flow chart and Table 1 shown below, the following steps were analyzed:

[0113] Table 1

[0114]

[0115] (1) Filter out the background fragments that match the mouse genome;

[0116] (2) Select the fragments that match the Schistosoma japonicum genome in NCBI and are present in both samples;

[0117] (3) Filter out the fragments less than 140 bp, which is due to the need for designing specific primers;

[0118] (4) Perform de novo genome assembly of BLASTN on NCBI to obtain the final 63 cDNAs.

[0119] The present inventors conducted a large number of screening works and finally determined six specific cDNA fragments derived from Schistosoma japonicum: K81-47, K81-25, K81-41, K81-55, K81-68, and K81-18, which were used for the evaluation of therapeutic efficacy assessment.

[0120] 1.4 Primer design for the target cDNA

[0121] Based on the results obtained from resequencing, the nucleotide sequences of the above six cDNA fragments were derived, and primer design was performed using software dedicated to primer design (Primer Premier 5). The design principle was the general PCR principle, and the primer pairs were screened with a PrimerPremier 5 software score > 95; the amplified fragment size was between 60 and 250; the primers were all synthesized by Shanghai BGI Tech Co., Ltd. The above six cDNA fragments and their primer sequences are shown in Table 2 below:

[0122] Table 2

[0123]

[0124]

[0125] 1.5 Specificity verification of primers and cDNA fragments

[0126] The designed primers and cDNA fragments were subjected to BLAST specificity verification in NCBI and other databases. The results showed that these six cDNA fragments were specific to the species Schistosoma japonicum, and no other species or genera were found. Therefore, they could be used for the therapeutic efficacy assessment and diagnosis of schistosomiasis.

[0127] 1.6 PCR and agarose gel electrophoresis

[0128] Using the extracted circular DNA as a template, the circular DNA fragment was detected using the designed specific primer pair. The PCR reagent was purchased from Hunan Aikery Biotechnology Co., Ltd. (product number: AG11023), and the following operations were performed:

[0129] (1) The PCR reaction system was 50 μl:

[0130]

[0131] (2) The PCR reaction program:

[0132]

[0133] After PCR amplification, 5 μl of the amplified product was taken for detection by 2% agarose gel electrophoresis.

[0134] Detection of serum K81-55 in Schistosoma japonicum-infected mice and normal mice before praziquantel treatment in Example 2

[0135] (1) Based on the content of Example 1, sera from infected mice before praziquantel treatment (infected group, n = 10) and normal C57 mice without any treatment (normal group, n = 3) were used as samples to amplify the Schistosoma-derived K81-55 gene fragment in the sera. The length of the amplified product was approximately 80 bp. The primer sequences used were as shown in SEQ ID NO: 2 and SEQ ID NO: 3.

[0136] (2) After PCR amplification, 5 μl of the amplified product was taken for detection by 2% agarose gel electrophoresis. The presence of a band corresponding to the size of the target fragment in the gel electrophoresis was used as the criterion for determination. The results were as Figures 2-3 and Table 3 showed:

[0137] Table 3

[0138]

[0139] The results showed that in the serum samples of 3 mice in the normal group, no target band K81-55 was detected on the electrophoresis map ( Figure 2 ), and the positive detection rate was 0% (0 / 3). In the serum samples of 10 mice in the infected group before praziquantel treatment, the target band K81-55 was detected on the electrophoresis map ( Figure 3 ), and the positive detection rate was 100% (10 / 10).

[0140] Example 3 Detection of serum K81-55 in Schistosoma japonicum-infected mice 3 weeks and 6 weeks after praziquantel treatment Based on the content of Example 1, after praziquantel treatment, sera from 3 weeks (n = 5) and 6 weeks (n = 5) of infection were used as samples for detection. The specific implementation method was the same as that in Example 2. The detection results were as Figures 4-5 and Table 4 showed, where Table 4 showed the results of the efficacy assessment of cDNA in the sera of mice in the infected group 3 weeks and 6 weeks after praziquantel treatment:

[0141] Table 4

[0142]

[0143] The results showed that at 3 weeks ( Figure 4 ) and 6 weeks ( Figure 5For each of the 5 mice in the Schistosoma japonicum infection group, no target band K81-55 was detected on the electrophoresis pattern, and the negative conversion rate was 100% (5 / 5). Therefore, the K81-55 fragment in the present invention can quickly and accurately determine the curative effect of schistosomiasis.

[0144] Based on the above method, the inventor analyzed the results of 6 cDNAs, namely K81-47, K81-25, K81-41, K81-55, K81-68, and K81-18. The results are shown in Tables 5-7 below. Tables 5 and 6 are the detection results of cDNA in the serum of mice in the infection group before praziquantel treatment and normal group mice, respectively. Compared with the normal group mice, K81-47, K81-25, K81-41, K81-55, K81-68, and K81-18 had a high positive detection rate and strong specificity for mice in the Schistosoma japonicum infection group (Tables 5-6). Table 7 is the detection results of these 6 cDNA fragments in the serum of mice in the Schistosoma japonicum infection group 3 weeks and 6 weeks after praziquantel treatment, and these 6 cDNA fragments showed relatively good negative conversion rates (Table 7).

[0145] Table 5

[0146] Circular DNA fragment Positive number Negative number Positive detection rate K81-47 9 1 90％ K81-25 9 1 90％ K81-41 6 4 60％ K81-55 10 0 100％ K81-68 10 0 100％ K81-18 9 1 90％

[0147] Table 6

[0148]

[0149]

[0150] Table 7

[0151]

[0152] Therefore, when the number of target band indicators detected by PCR amplification and agarose gel electrophoresis of the 6 cDNAs provided in the present invention decreases, it indicates that the current treatment is effective; when all bands are detected and highlighted, it indicates that there are still adult worms in the body at this time, and treatment still needs to continue, but the presence or absence of circulating DNA needs to be continuously detected regularly; when all bands cannot be detected, it indicates that the adult worms have been killed and chemotherapy can be stopped.

[0153] Any one of the six cDNA sequences, namely K81-47, K81-25, K81-41, K81-55, K81-68, and K81-18, provided in the present invention can be used for efficacy evaluation. Among them, K81-55 and K81-68 are more stable and reliable. At the same time, if the number of target bands detected in two consecutive tests decreases, it also indicates that the treatment is effective, and the treatment should continue until all bands disappear. The detection method of the present invention is simple, rapid, highly sensitive, and has good repeatability. Moreover, the interpretation of its results is very clear and intuitive. Especially for repeated detections of a large number of different actual patients, it can conduct real-time dynamic monitoring of treatment efficacy assessment, and can be used for the diagnosis of Schistosoma japonicum. The (qualitative) results are all accurate, highly reliable, and have low economic costs, making it suitable for large-scale promotion.

[0154] Discussion

[0155] The developmental stage of Schistosoma japonicum from cercariae to adult worms laying eggs is completed in the host. This process not only enables the schistosome to complete its own growth and development but also causes a series of immune responses and pathological changes in the host. Schistosomiasis japonica remains a zoonotic parasitic disease prevalent worldwide. Although China has achieved great success in the prevention and control of schistosomiasis, there is still a lack of effective methods for evaluating the treatment efficacy of Schistosoma japonicum.

[0156] Circulating DNA exists in the host body fluid as DNA fragments derived from the growth and development of parasites (here referring to schistosomes). Therefore, this circulating DNA in the host body fluid has high pathogen specificity. Samples can be obtained by collecting peripheral blood, and blood can be collected at any time during the treatment process to monitor the chemotherapy effect in real-time and dynamically. PCR-based analysis has the advantages of low cost and effectiveness and is a promising method for evaluating the treatment efficacy of schistosomiasis, and is also used for the nucleic acid diagnosis of schistosomiasis. So far, there have been no research reports on the application of the six circulating DNA fragments (K81-47, K81-25, K81-41, K81-55, K81-68, K81-18) of the present invention in the evaluation of the treatment efficacy of schistosomiasis.

[0157] With the development and application of sequencing technologies, the present inventors screened specific small DNA fragments in the sera of mice infected with Schistosoma japonicum by re-sequencing; designed specific primers for PCR and agarose gel electrophoresis, and determined the therapeutic effect of drugs and the infection status based on the presence or absence of detected bands. Based on the cDNA fragments specific to the pathogen in the mouse body discovered by re-sequencing, the present invention detected the cDNA in the sera of mice before treatment with praziquantel, 3 weeks and 6 weeks after treatment, and normal mice. The results showed that the positive detection rates of K81-47, K81-25, K81-41, K81-55, K81-68, and K81-18 in the infected mice were high. The statistical results are shown in Tables 5-7, and the specificity was strong. After treatment with praziquantel, these 6 cDNA fragments in the sera of the infected mice at 3 weeks and 6 weeks showed relatively good negative conversion rates.

[0158] Therefore, when the number of target band indicators detected by PCR amplification and agarose gel electrophoresis of the 6 cDNAs provided in the present invention decreases, it indicates that the current treatment is effective; when all bands are detected and highlighted, it indicates that there are still adult worms in the body at this time, and continuous treatment is still required, but the presence or absence of circulating DNA needs to be continuously detected regularly; when all bands cannot be detected, it indicates that the adult worms have been killed and chemotherapy can be stopped. Any one of the 6 cDNAs of K81-47, K81-25, K81-41, K81-55, K81-68, and K81-18 provided in the present invention can be used for efficacy evaluation, and K81-55 is particularly more stable and reliable. At the same time, if the number of detected target bands decreases in two consecutive detections, it also indicates that the treatment is indeed effective, and treatment should be continued until all bands disappear. This detection method is simple, rapid, highly sensitive, and has good repeatability, and the interpretation of its results is very clear and intuitive. Especially for repeated detections of a large number of different actual patients, it can be used for real-time dynamic monitoring of efficacy assessment, and can be used for the diagnosis of Schistosoma japonicum. The (qualitative) results are all accurate, highly reliable, and have low economic costs, and can be widely promoted.

[0159] All documents mentioned in the present invention are incorporated herein by reference as if each individual document was specifically and individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present invention.

Claims

1. A kit for detecting Schistosoma japonicum infection in a patient, characterized in that, The kit includes components for detecting Schistosoma japonicum circulating DNA sequences, and the Schistosoma japonicum circulating DNA sequences are selected from any one or more combinations of the following groups: (1) The nucleotide sequence shown in SEQ ID NO: 1; (2) The nucleotide sequence shown in SEQ ID NO: 4; (3) The nucleotide sequence shown in SEQ ID NO: 7; (4) The nucleotide sequence shown in SEQ ID NO: 10; (5) The nucleotide sequence shown in SEQ ID NO: 13; (6) The nucleotide sequence shown in SEQ ID NO:

16.

2. The kit according to claim 1, wherein The kit includes one or more primer pairs selected from the following groups: (1) The nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3; (2) The nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6; (3) The nucleotide sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9; (4) The nucleotide sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12; (5) The nucleotide sequences shown in SEQ ID NO: 14 and SEQ ID NO: 15; (6) The nucleotide sequences shown in SEQ ID NO: 17 and SEQ ID NO:

18.

3. The kit according to claim 1, characterized in that, The kit further includes any one or more sequences selected from the following groups: (1) The nucleotide sequence shown in SEQ ID NO: 1; (2) The nucleotide sequence shown in SEQ ID NO: 4; (3) The nucleotide sequence shown in SEQ ID NO: 7; (4) The nucleotide sequence shown in SEQ ID NO: 10; (5) The nucleotide sequence shown in SEQ ID NO: 13; (6) The nucleotide sequence shown in SEQ ID NO:

16.

4. The kit according to claim 1, characterized in that, The kit further includes an instruction manual, and the following steps are recorded in the instruction manual: (i) Detect the level of one or more circulating DNA fragments selected from the following groups in the sample using the components in the kit: (1) The nucleotide sequence shown in SEQ ID NO: 1; (2) The nucleotide sequence shown in SEQ ID NO: 4; (3) The nucleotide sequence shown in SEQ ID NO: 7; (4) The nucleotide sequence shown in SEQ ID NO: 10; (5) The nucleotide sequence shown in SEQ ID NO: 13; (6) The nucleotide sequence shown in SEQ ID NO: 16; (ii) Judge the Schistosoma japonicum infection status of the patient according to the detected level of the circulating DNA fragment.

5. The kit according to claim 4, characterized in that, The sample is a peripheral blood sample of the patient.

6. The kit according to claim 4, wherein The following step is also recorded in the instruction manual: When one or more circulating DNA fragments are detected as positive in the sample, judge the sample as a positive sample.

7. A method for screening or identifying potential therapeutic agents for treating or alleviating Schistosoma japonicum disease, comprising the following steps: (a) Administer a candidate substance to a non-human mammalian model of Schistosoma japonicum disease, and set up a blank control group; (b) Detect the level of circulating DNA in the serum of the non-human mammalian model and compare it with the control group. If the positive detection rate of circulating DNA in the non-human mammalian model administered with the candidate substance is reduced compared with the control group, it is determined that the candidate substance has a therapeutic effect on schistosomiasis japonica. Among them, The nucleotide sequences of the schistosoma japonicum circulating DNA are selected from any one or more combinations of the following groups: (1) The nucleotide sequence shown in SEQ ID NO: 1; (2) The nucleotide sequence shown in SEQ ID NO: 4; (3) The nucleotide sequence shown in SEQ ID NO: 7; (4) The nucleotide sequence shown in SEQ ID NO: 10; (5) The nucleotide sequence shown in SEQ ID NO: 13; (6) The nucleotide sequence shown in SEQ ID NO:

16.

8. The method according to claim 7, wherein The method further includes: detecting with one or more sets of primer pairs selected from the following groups: (1) The nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3; (2) The nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6; (3) The nucleotide sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9; (4) The nucleotide sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12; (5) The nucleotide sequences shown in SEQ ID NO: 14 and SEQ ID NO: 15; (6) The nucleotide sequences shown in SEQ ID NO: 17 and SEQ ID NO:

18.

9. A Schistosoma japonicum circular DNA sequence composition, characterized in that, The sequence composition is selected from any two or more sequence combinations of the following groups: (1) The nucleotide sequence shown in SEQ ID NO: 1; (2) The nucleotide sequence shown in SEQ ID NO: 4; (3) The nucleotide sequence shown in SEQ ID NO: 7; (4) The nucleotide sequence shown in SEQ ID NO: 10; (5) The nucleotide sequence shown in SEQ ID NO: 13; (6) The nucleotide sequence shown in SEQ ID NO:

16.

10. A primer combination for amplifying Schistosoma japonicum circular DNA sequences, characterized in that, The nucleotide sequences of the primer combination are selected from any one or more combinations of the following groups: (1) The nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3; (2) The nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6; (3) The nucleotide sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9; (4) The nucleotide sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12; (5) The nucleotide sequences shown in SEQ ID NO: 14 and SEQ ID NO: 15; (6) The nucleotide sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18.

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