WO2025055559A1 - Use of sjtr1 microsatellite sequence for preparation of reagent for detecting schistosomiasis - Google Patents

Abstract

The present disclosure belongs to the field of molecular biological detection, and specifically relates to the detection of schistosomiasis, more specifically to the use of an SjTR1 microsatellite sequence for the preparation of a reagent for detecting schistosomiasis. Provided in the present disclosure is the use of an Schistosoma japonicum Katsurada SjR1 microsatellite sequence for the preparation of a reagent for detecting schistosomiasis. The PCR detection reagent prepared by using a new target discovered by the present disclosure can realize higher sensitivity and specificity as compared to existing targets for Schistosoma japonicum Katsurada, such as SJR2. In particular, in the design process of multiplex PCR, the new target can enable the entire system to obtain better sensitivity and better specificity.

Description

Use of SjTR1 microsatellite sequence for preparing reagents for detecting schistosomiasis Technical Field

The present disclosure belongs to the field of molecular biological detection, and specifically, relates to the detection of schistosomiasis, and more specifically, to the use of SjTR1 microsatellite sequence for preparing reagents for detecting schistosomiasis.

Background Art

Schistosomiasis, commonly known as "big belly disease", is an acute and chronic disease caused by schistosomes. There are 6 types of schistosomes that infect humans: Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, Schistosoma mekongi, Schistosoma guineensis and Schistosoma intercalatum. There are 3 types of schistosomes that cause major harm to humans: Schistosoma japonicum, which is prevalent in Asia, Schistosoma mansoni, which is prevalent in Latin America and Central Africa, and Schistosoma haematobium, which is prevalent in North Africa. The main manifestation of schistosomiasis is dermatitis at the site of invasion, accompanied by fever, abdominal pain, diarrhea, tenderness in the liver area and other symptoms. The source of infection of schistosomiasis is difficult to completely eliminate, and the transmission route cannot be completely cut off, so the risk of transmission continues to exist.

Diagnosis plays a key role in the prevention and control of schistosomiasis, controlling and eliminating the source of infection. Common tests include stool and urine specimens, and sometimes intestinal or bladder tissue specimens are required for examination; blood tests are sometimes used for diagnosis. However, these tests cannot indicate the severity of the infection and have limitations.

Nucleic acid detection has high sensitivity and specificity. Previously established nucleic acid diagnostic techniques are limited to primer probes developed for existing targets, and the sensitivity and specificity need to be improved. For example, Li, Juan, Zhao, Guang-Hui et al. reported a real-time PCR assay combined with high-resolution melting (HRM) assay, targeting a portion of nuclear 18S rDNA to detect, identify and distinguish four major schistosome species. However, the detection of genomic DNA from schistosomes was limited to the minimum amount of genomic DNA when the Ct value was <30, which was ^10-5 ng. The sensitivity needs to be improved, and the main peak of the melting curve of Schistosoma japonicum and Schistosoma mekongense is the same, both at 83.65℃. The difference between the two is that there is a secondary peak on the left side of the main peak of the melting curve of Schistosoma mekongense, which is not easy to distinguish from Schistosoma japonicum, and may cause misjudgment.

Therefore, there is a need in the art for a product that can diagnose schistosomiasis more quickly and effectively, with higher sensitivity and better specificity.

Summary of the invention

In view of this, the applicant used the gene sequence of Schistosoma japonicum with accession number NC_002544.1 as the detection target and discovered a new target SjTR1 microsatellite sequence (SiTR1 region) for detecting Schistosoma japonicum. The gene sequence is: MW631938.1. When the SjTR1 microsatellite sequence of Schistosoma japonicum is used to prepare a test kit for schistosomiasis, it can diagnose schistosomiasis more quickly and effectively with high sensitivity and good specificity.

In a first aspect, the present disclosure provides a use of a Schistosoma japonicum SjTR1 microsatellite sequence for preparing a reagent for detecting schistosomiasis.

Furthermore, the reagent is a PCR reagent; further, the reagent is a multiplex PCR reagent.

In a second aspect, the present disclosure provides a composition for detecting schistosomiasis, the composition comprising upstream and downstream primers and probes for detecting the SjTR1 microsatellite sequence of Schistosoma japonicum.

In some specific embodiments, a composition for detecting schistosomiasis comprises

Upstream and downstream primers and probes for detecting Schistosoma japonicum SjTR1 microsatellite sequences;

Upstream and downstream primers and probes for detecting Schistosoma mansoni SM1-7 sequences; and

Upstream and downstream primers and probes for detecting Schistosoma haematobium Dra1 sequences.

Furthermore, the composition also includes: upstream and downstream primers and probes for detecting the SJR2 sequence of Schistosoma japonicum.

In some specific embodiments, the upstream and downstream primers and probes for detecting the Schistosoma japonicum SJR2 sequence are shown in SEQ ID NO.1~3.

In some specific embodiments, the upstream and downstream primers and probes for detecting the SjTR1 microsatellite sequence of Schistosoma japonicum are at least one group shown in SEQ ID NO.4-6, or SEQ ID NO.7-9, or SEQ ID NO.10-12.

In some specific embodiments, the upstream and downstream primers and probes for detecting the SjTR1 microsatellite sequence of Schistosoma japonicum are as shown in SEQ ID NOs. 4 to 6, or SEQ ID NOs. 7 to 9, or SEQ ID At least two groups among those shown in NO. 10 to 12.

In some specific embodiments, the upstream and downstream primers and probes for detecting the SjTR1 microsatellite sequence of Schistosoma japonicum are as shown in SEQ ID NOs. 4 to 6, SEQ ID NOs. 7 to 9, and SEQ ID NOs. 10 to 12.

In some specific embodiments, the upstream and downstream primers and probes for detecting SM1-7 sequences are shown as SEQ ID NO.13 to 15.

In some specific embodiments, the upstream and downstream primers and probes used to detect Dra1 sequences are shown as SEQ ID NO.16~18.

Furthermore, the composition also includes an internal standard gene.

In some specific embodiments, the internal standard gene is a human housekeeping gene.

Furthermore, the composition also includes an upstream primer, a downstream primer and a probe for detecting an internal standard gene.

In some specific implementation schemes, the upstream and downstream primers and probes for detecting the internal standard gene are shown as SEQ ID NO.19~21.

In a third aspect, the present disclosure provides use of the above composition in preparing a schistosomiasis pathogen detection kit.

In a fourth aspect, the present disclosure provides a kit for detecting schistosomiasis pathogens, wherein the kit comprises the composition of the present disclosure as described above.

In a fifth aspect, a method for detecting and distinguishing schistosomiasis pathogens is provided, wherein the detection comprises the following steps:

1) Extract or release nucleic acid from the sample to be tested;

2) performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition disclosed above or the kit disclosed above;

3) Obtain and analyze the results.

In a specific embodiment, a method for joint detection and differentiation of schistosomiasis pathogens for non-diagnostic purposes is provided, the method comprising the following steps:

1) Extract or release nucleic acid from the sample to be tested;

2) performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition disclosed above or the kit disclosed above;

3) Obtain and analyze the results.

The PCR detection reagent prepared for the new target SjTR1 microsatellite sequence discovered in the present invention can achieve higher sensitivity and specificity than the existing targets of Schistosoma japonicum, such as SJR2. In particular, in the design process of multiplex PCR, the new target can enable the entire system to achieve better sensitivity and better specificity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the test results of composition 1 of the present disclosure.

FIG. 2 is a graph showing the test results of composition 2 of the present disclosure.

FIG. 3 is a diagram showing the test results of composition 3 of the present disclosure.

FIG. 4 is a graph showing the test results of composition 4 of the present disclosure.

DETAILED DESCRIPTION

The present disclosure will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present disclosure and should not be construed as limiting the scope of protection of the present disclosure. All technologies implemented based on the above content of the present disclosure are included in the scope of protection intended by the present disclosure.

Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the reagents, materials, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

In the description of the present disclosure, it should be noted that the terms "first", "second", etc. are only used for descriptive purposes and do not indicate or imply relative importance.

In view of this, the applicant used the gene sequence of Schistosoma japonicum with accession number NC_002544.1 in the GenBank database as the detection target and discovered a new target SjTR1 microsatellite sequence (SjTR1 region) for detecting Schistosoma japonicum. The gene sequence is: MW631938.1 (GenBank database number). The sequence of MW631938.1 is shown in SEQ ID NO.22. SEQ ID NO.22 is TAGGGGGGAATGTCGTGCACAACCTTTCTTCCCCATATAAAGATGATGGCGAGATGTTGTGGGTGTAAGTTGATTGGCGGTGATGAGTTGTGCATGAAAAAA.

In a first aspect, the present disclosure provides a use of a Schistosoma japonicum SjTR1 microsatellite sequence for preparing a reagent for detecting schistosomiasis.

In one embodiment, the reagent is a PCR reagent.

In one embodiment, the reagent is a multiplex PCR reagent.

In some specific embodiments, the reagents comprise primers and/or probes.

The PCR detection reagent prepared using the new target discovered in the present disclosure can achieve higher sensitivity and specificity than the existing Schistosoma japonicum targets, such as SJR2. In particular, in the design process of multiplex PCR, the new target can enable the entire system to achieve better sensitivity and better specificity.

In a second aspect, the present disclosure provides a composition for detecting schistosomiasis, the composition comprising upstream and downstream primers and probes for detecting the SjTR1 microsatellite sequence of Schistosoma japonicum.

In some specific embodiments, a composition for detecting schistosomiasis comprises

Upstream and downstream primers and probes for detecting Schistosoma japonicum SjTR1 microsatellite sequences;

Upstream and downstream primers and probes for detecting Schistosoma mansoni SM1-7 sequences; and

Upstream and downstream primers and probes for detecting Schistosoma haematobium Dra1 sequences.

Furthermore, the composition also includes an internal standard gene. In some specific embodiments, the internal standard gene is a human housekeeping gene.

In some embodiments, the composition further comprises an upstream primer, a downstream primer, and a probe for detecting an internal standard gene.

In some specific embodiments, the composition also includes upstream and downstream primers and probes for detecting Schistosoma mansoni SM1-7 sequences, upstream and downstream primers and probes for detecting Schistosoma haematobium Dra1 sequences, upstream and downstream primers and probes for detecting Schistosoma japonicum SJR2 sequences, and at least one set of upstream and downstream primers and probes for detecting internal standard gene sequences.

In some embodiments, the probe of the disclosed compositions comprises a fluorescent reporter group. In an embodiment, the probe in the composition is labeled with a fluorescent reporter group at the 5' end.

Furthermore, the fluorescent reporter groups of different probes in the above-mentioned composition of the present disclosure are different from each other and do not interfere with each other.

In this article, "different and non-interfering" means that the fluorescent reporter groups used in each probe in the composition are different and will not affect each other's detection, that is, different channels can be used for detection. For example, ATTO 425, Quasar 705, FAM, HEX, ROX and CY5 can be used. The absorbance values of these groups are not close, and different channels can be selected, so they will not interfere with each other.

In some specific embodiments, the fluorescent reporter group of the probe of the SjTR1 microsatellite sequence is FAM; the fluorescent reporter group of the probe of the internal standard gene is HEX (or VIC); the fluorescent reporter group of the probe of the SM1-7 sequence is ROX; and the fluorescent reporter group of the probe of the Dra1 sequence is CY5.

Furthermore, the composition also includes: upstream and downstream primers and probes for detecting the SJR2 sequence of Schistosoma japonicum.

After adding the above primer and probe sequences, the sensitivity of the composition of the present disclosure is higher.

In some specific embodiments, the fluorescent reporter group of the probe for detecting the SJR2 sequence of Schistosoma japonicum may be the same as or different from that of the probe for the SjTR1 microsatellite sequence.

In some specific implementation schemes, the upstream and downstream primers and probes for detecting the SJR2 sequence of Schistosoma japonicum are shown in SEQ ID NO.1 to 3. Among them, SEQ ID NO.1 is the upstream primer, SEQ ID NO.2 is the downstream primer, and SEQ ID NO.3 is the probe.

In some specific embodiments, the sequences of the upstream and downstream primers and probes for detecting the SjTR1 microsatellite sequence of Schistosoma japonicum are at least one of the sequences shown in SEQ ID NO.4 to 6, the sequences shown in SEQ ID NO.7 to 9, or the sequences shown in SEQ ID NO.10 to 12. Among them, SEQ ID NO.4 is an upstream primer, SEQ ID NO.5 is a downstream primer, and SEQ ID NO.6 is a probe; SEQ ID NO.7 is an upstream primer, SEQ ID NO.8 is a downstream primer, and SEQ ID NO.9 is a probe; SEQ ID NO.10 is an upstream primer, SEQ ID NO.11 is a downstream primer, and SEQ ID NO.12 is a probe.

In some specific embodiments, the upstream and downstream primers and probes for detecting the SjTR1 microsatellite sequence of Schistosoma japonicum are the sequences shown in SEQ ID NOs. 4 to 6, the sequences shown in SEQ ID NOs. 7 to 9, and the sequences shown in SEQ ID NOs. At least two of the sequences shown in ID NOs. 10 to 12.

In some specific embodiments, the upstream and downstream primers and probes for detecting the SjTR1 microsatellite sequence of Schistosoma japonicum include sequences shown in SEQ ID NOs. 4 to 6, sequences shown in SEQ ID NOs. 7 to 9, and sequences shown in SEQ ID NOs. 10 to 12.

In some specific embodiments, the upstream and downstream primers and probes for detecting the SM1-7 sequence are shown in SEQ ID NO. 13 to 15. Among them, SEQ ID NO. 13 is an upstream primer, SEQ ID NO. 14 is a downstream primer, and SEQ ID NO. 15 is a probe.

In some specific embodiments, the upstream and downstream primers and probes for detecting the Dra1 sequence are shown in SEQ ID NO. 16 to 18. Among them, SEQ ID NO. 16 is an upstream primer, SEQ ID NO. 17 is a downstream primer, and SEQ ID NO. 18 is a probe.

In some specific implementation schemes, the upstream and downstream primers and probes for detecting the internal standard gene are shown in SEQ ID NO. 19 to 21. Among them, SEQ ID NO. 19 is the upstream primer, SEQ ID NO. 20 is the downstream primer, and SEQ ID NO. 21 is the probe.

Further on the basis of the aforementioned combination, the composition includes upstream and downstream primers and probes for detecting the SjTR1 microsatellite sequence of Schistosoma japonicum, upstream and downstream primers and probes for detecting the SM1-7 sequence of Schistosoma mansoni, upstream and downstream primers and probes for detecting the Dra1 sequence of Schistosoma haematobium, upstream and downstream primers and probes for detecting the SJR2 sequence of Schistosoma japonicum, and upstream and downstream primers and probes for detecting the internal standard gene sequence.

Further, in some embodiments, the composition of the present disclosure may include one or more of the above primer and probe sets. In the present disclosure, a "set" refers to mutually matching upstream and downstream primers and probes for detecting a target.

The compositions disclosed herein can be combined into any combination of the corresponding five targets. Those skilled in the art can combine them as needed to detect which targets, that is, to combine the primers and probe pairs corresponding to the targets. These combinations are all included in the present disclosure.

For example, any 6 of the above 7 primer sets and probes may be included, any 5 of the above 7 primer pairs and probes may be included, or any 4 of the above 7 primer pairs and probes may be included. It may include any three of the seven sets of primer pairs and probes, any two of the seven sets of primer pairs and probes, or any one of the seven sets of primer pairs and probes.

In some specific embodiments, the disclosed compositions are used for fluorescent PCR.

Furthermore, the 3' end of the probe also has a non-fluorescent quencher, such as NFQ and QSY.

Furthermore, the 3' end of the probe also has a quencher group, such as BHQ1 or BHQ2.

In a specific embodiment, the 3' end of the probe is BHQ1.

In a specific embodiment, each component of the composition of the present disclosure is present in a separate package.

In a specific embodiment, the components of the composition of the present disclosure are present in the same package.

Furthermore, the components of the composition of the present disclosure are present in a mixed form.

In a third aspect, the present disclosure provides use of the above composition in preparing a schistosomiasis pathogen detection kit.

Furthermore, the present disclosure provides use of the above-mentioned composition of the present disclosure in preparing a kit for joint detection and differentiation of schistosomiasis pathogens, wherein the pathogen is at least one of Schistosoma japonicum, Schistosoma mansoni and Schistosoma haematobium.

In a fourth aspect, the present disclosure provides a kit for detecting schistosomiasis pathogens, wherein the kit comprises the composition of the present disclosure as described above.

Furthermore, the kit also includes a negative quality control product and a positive quality control product.

In a specific embodiment, the negative control is at least one of DEPC H ₂ O, physiological saline, and an internal standard gene. The positive control is at least one of the fragmented DNA of Schistosoma japonicum, the fragmented DNA of Schistosoma mansoni, and the fragmented DNA of Schistosoma haematobium.

Furthermore, the kit also includes at least one of dNTP(U)s, PCR buffer and Mg ²⁺ .

Furthermore, the kit also includes: at least one of a nucleic acid releasing reagent, a nucleic acid extracting reagent, and a DNA polymerase.

Furthermore, the kit also includes a nucleic acid releasing reagent, a nucleic acid extracting reagent, dNTP, At least one of dUTP, uracil glycosylase (UNG), DNA polymerase, PCR buffer and Mg ²⁺ .

Furthermore, the concentration of the DNA polymerase is 3U/reaction to 15U/reaction, for example, the DNA polymerase may be Taq enzyme.

In a specific embodiment, the disclosed kit includes Taq enzyme, UNG enzyme, Mg ²⁺ , dNTP(U)s, primers, probes and PCR buffer.

Common PCR buffers are composed of Tris-HCl, MgCl ₂ , KCl, Triton X-100, etc. The total volume in a single PCR reaction tube is generally 20 μl to 200 μl.

In a fifth aspect, a method for detecting and distinguishing schistosomiasis pathogens is provided, wherein the detection comprises the following steps:

1) Extract or release nucleic acid from the sample to be tested;

2) performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition disclosed above or the kit disclosed above;

3) Obtain and analyze the results.

In the present disclosure, the sample used for detection may be blood, tissue fluid, etc., but is not limited thereto.

Furthermore, the reaction conditions of the fluorescent quantitative PCR are as follows: UNG enzyme reaction, temperature is 40-60°C, time is 30-150 seconds, 1 cycle; Taq enzyme activation, temperature is 94°C, time is 3-6 minutes, 1 cycle; denaturation, temperature is 94°C, time is 5-20 seconds, annealing, temperature is 55°C-60°C, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

The present disclosure also provides a use of a composition for preparing a reagent for detecting and distinguishing schistosomiasis pathogens, the method comprising the following steps:

1) Extracting or releasing nucleic acid from the sample to be tested;

2) performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition disclosed above or the kit disclosed above;

3) Obtain and analyze the results.

In one embodiment, the method can be used for diagnosis and treatment of diseases, and can be used for auxiliary treatment of diseases. It can be used to assist in diagnosis, but it may not be used for the diagnosis and treatment of diseases.

In one embodiment, the method is not used for diagnosis of the disease, that is, non-diagnostic purposes. In this article, the term "non-diagnostic purpose" means that it is not intended to obtain information on whether an individual is infected with the above-mentioned pathogen and suffers from schistosomiasis. For example, the method can be used to detect whether the above-mentioned pathogen is present in a test culture (such as blood).

In a specific embodiment, a method for joint detection and differentiation of schistosomiasis pathogens for non-diagnostic purposes is provided, the method comprising the following steps:

1) Extracting or releasing nucleic acid from the sample to be tested;

2) using the composition disclosed above or the kit disclosed above to perform fluorescent quantitative PCR on the nucleic acid obtained in step 1);

3) Obtain and analyze the results.

Furthermore, the reaction conditions of the fluorescent quantitative PCR are as follows: UNG enzyme reaction, temperature is 40-60°C, time is 30-150 seconds, 1 cycle; Taq enzyme activation, temperature is 94°C, time is 3-6 minutes, 1 cycle; denaturation, temperature is 94°C, time is 5-20 seconds, annealing, temperature is 55-60°C, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

Example 1. Primers and probes used in the present disclosure

The primers and probes used in the present disclosure are shown in Table 1 below.

Table 1

Example 2: Method for detecting schistosomiasis pathogens

1. Reagent preparation

According to the number of negative controls, test samples, and positive controls, take the corresponding amount of PCR reaction solution and enzyme mixture according to the proportion (PCR reaction solution 38μL/person + enzyme mixture 2μL/person), mix them thoroughly to form PCR Mix, and centrifuge at 2000rpm for 10s before use.

PCR reaction system:

Preparation of mixed enzyme:

The enzyme mixture consists of H-Taq enzyme and UNG enzyme. H-Taq enzyme (15U/μL) and UNG enzyme (2U/μL) are mixed in a certain ratio (each person is a mixture of 1.7μL H-Taq enzyme and 0.3μL UNG enzyme).

2. Sample processing and sample addition

Take 200 μL of the sample to be tested into a 1.5 mL centrifuge tube and use the Nucleic acid extraction or purification reagent (S10015) is used for nucleic acid extraction according to its instructions.

Pipette 10 μL each of the negative control, the sample nucleic acid extracted above, and the positive control and add them into the corresponding 0.2 mL PCR reaction tubes, add 40 μL PCR Mix to each tube, and cover the tubes.

3. PCR amplification

On the SLAN-96P fully automated medical PCR analysis system, PCR amplification was performed according to the procedure in the following table:

4. Interpretation of test results

If the sample has an obvious S-shaped amplification curve in the FAM, HEX (VIC), ROX, and CY5 channels, and the Ct value is ≤40, it is judged as positive; if the sample has no amplification curve (No Ct) or the Ct value is greater than 40 in the FAM, ROX, and CY5 channels, and the HEX (VIC) internal standard channel is positive (Ct value ≤40), it is judged as negative. The details are as follows:

Example 3. Test results of the test samples of the disclosed composition

The primers and probes shown in Example 1 (Composition 1: SEQ ID NOs. 4 to 6, SEQ ID NOs. 13 to 21) were used to simultaneously detect Schistosoma japonicum, Schistosoma mansoni, and Schistosoma haematobium in the sample according to the method of Example 2. The results are shown in FIG1 . As can be seen from FIG1 , Composition 1 is capable of detecting and distinguishing various schistosomes in the sample.

The primers and probes shown in Example 1 (Composition 2: SEQ ID NO.7-9, SEQ ID NO.13-21) were used to simultaneously detect Schistosoma japonicum, Schistosoma mansoni and Schistosoma haematobium in the sample according to the method of Example 2. The results are shown in Figure 2. It can be seen from Figure 2 that Composition 2 is able to detect and distinguish various schistosomes in the sample.

The primers and probes shown in Example 1 (Composition 3: SEQ ID NO. 10-21) were used to simultaneously detect Schistosoma japonicum, Schistosoma mansoni and Schistosoma haematobium in the sample according to the method of Example 2. The results are shown in Figure 3. It can be seen from Figure 3 that Composition 3 is able to detect and distinguish various schistosomes in the sample.

The primers and probes shown in Example 1 (Composition 4: SEQ ID NO.1-6, SEQ ID NO.13-21) were used to simultaneously detect Schistosoma japonicum, Schistosoma mansoni and Schistosoma haematobium in the sample according to the method of Example 2. The results are shown in Figure 4. It can be seen from Figure 4 that Composition 4 is able to detect and distinguish various schistosomes in the sample.

Example 4. Sensitivity of the disclosed composition

The artificially synthesized plasmids of Schistosoma japonicum, Schistosoma mansoni and Schistosoma haematobium were mixed and gradiently diluted to 1.00E+03copies/mL, 4.00E+02copies/mL, 2.00E+02copies/mL and 1.00E+02copies/mL using negative serum samples as test samples, and the 95% detection level was taken as the minimum detection limit of this reagent. The results of the analytical sensitivity test showed that the detection limit of the composition 1 disclosed herein for Schistosoma japonicum/Mansoni/Hematobium pathogens was 4.00E+02copies/mL (see Table 2).

The detection rate is expressed as "number of detections/number of tests". "Number of tests" refers to the total number of tested samples, for example, if 20 samples are tested, the number of tests is 20; "number of detections" refers to the number of samples that are positive, for example, if 10 samples show a qualified amplification curve, the number of detections is 10.

Table 2

Furthermore, the detection limits of the compositions 2 and 3 of the present disclosure for Schistosoma japonicum/Mansoniae/Heghamdium pathogens are all 4.00E+02 copies/mL (see Tables 3 and 4).

Table 3

Table 4

The detection limit of the disclosed composition 4 for the pathogens of Schistosoma mansoni/Schistosoma haematobium is 4.00E+02 copies/mL, and the detection limit for Schistosoma japonicum is as low as 2.00E+02 copies/mL (see Table 5).

Table 5

Example 5. Specificity of the disclosed composition

Specific experiments show that the method disclosed in the present invention has no cross-reaction to common parasitic pathogens and infectious viruses (Toxoplasma gondii (TOXO), Trichinella spiralis (Trichina), human parvovirus B19 (HPV B19), rubella virus (RV), cytomegalovirus (CMV) and herpes simplex virus (HSV1/2) (see Table 6 for sample information).

Table 6. Statistical table of information on different similar pathogens

The specificity analysis was performed using the above samples and a mixed plasmid of Schistosoma japonicum, Schistosoma mansoni, and Schistosoma haematobium diluted to 1.00E+06 copies/mL as a control sample.

Three batches of reagents (composition 4) were used for testing in the SLAN-96P fully automatic medical PCR analysis system, and the specificity of the kit was examined by testing the positive and negative coincidence rates. The test results are shown in Table 7 below:

Table 7. Statistics of cross-reaction results of three batches of reagents

Conclusion: The results of cross-sample schistosoma detection were all negative using three batches of reagents, indicating that this kit has good specificity. At the same time, it has been verified that other high-concentration pathogens in the above table will not affect the nucleic acid detection of Schistosoma japonicum, Schistosoma mansoni, and Schistosoma haematobium.

Example 6. Anti-interference and stability of the disclosed composition

In order to investigate the influence of endogenous/exogenous substances in the sample on the test results, the The nucleic acid of Schistosoma japonicum sample was mixed with the plasmids of Schistosoma haematobium and Schistosoma mansoni and diluted to the minimum detection limit, and divided into several portions, each of which was added with a certain concentration of endogenous interfering substances: plasma protein, leukocytes, total bilirubin, triglycerides, total IgG, interferon α; exogenous interfering substances: praziquantel, and the anti-interference ability of the kit was examined by comparison with the control sample. The composition 1 described in Example 3 of the present disclosure was used for the test, and the test results are shown in Table 8.

Table 8. Interference test verification test results

Comparative Example 1: Effects of the New Targets and Known Targets of the Present Disclosure on Sensitivity

The artificially synthesized plasmids of Schistosoma japonicum, Schistosoma mansoni and Schistosoma haematobium were mixed and gradiently diluted to 1.00E+03copies/mL, 4.00E+02copies/mL, 2.00E+02copies/mL and 1.00E+02copies/mL using negative serum samples as test samples, and 95% detection level was taken as the minimum detection limit of this reagent. The results of analytical sensitivity test showed that the detection limit of composition 5 described in Example 3 of the present disclosure (composition 5: SEQ ID NO.1-3, SEQ ID NO.13-21) for Schistosoma japonicum/S. mansoni/S. haematobium pathogens was 1.00E+03copies/mL (see Table 9).

Table 9

From the comparison between Table 9 and Tables 2-4, it can be seen that the sensitivity of compositions 1, 2 and 3 using primers and probes designed for new targets is 4.00E+02 copies/mL, while the sensitivity of composition 5 using primers and probes designed for known targets is 1.00E+03 copies/mL. The sensitivity of the primers and probes designed for new targets in the present disclosure is higher than that of known targets.

The above describes the implementation methods of the present disclosure. However, the present disclosure is not limited to the above implementation methods. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the protection scope of the present disclosure.

Claims (15)

A use of a Schistosoma japonicum SjTR1 microsatellite sequence for preparing a reagent for detecting schistosomiasis. The use according to claim 1, characterized in that the reagent is a PCR reagent. A composition for detecting schistosomiasis, characterized in that the composition comprises upstream and downstream primers and probes for detecting the SjTR1 microsatellite sequence of Schistosoma japonicum. The composition according to claim 3 is characterized in that the upstream and downstream primers and probes for detecting the SjTR1 microsatellite sequence of Schistosoma japonicum include at least one of the primer groups shown in SEQ ID NOs. 4 to 6, the primer groups shown in SEQ ID NOs. 7 to 9, or the primer groups shown in SEQ ID NOs. 10 to 12. The composition according to claim 3 or 4 is characterized in that the composition also includes at least one set of upstream and downstream primers and probes for detecting Schistosoma mansoni SM1-7 sequences, upstream and downstream primers and probes for detecting Schistosoma haematobium Dra1 sequences, upstream and downstream primers and probes for detecting Schistosoma japonicum SJR2 sequences, and upstream and downstream primers and probes for detecting internal standard gene sequences. The composition according to any one of claims 3 to 5, characterized in that the upstream and downstream primers and probes for detecting the Schistosoma mansoni SM1-7 sequence are as shown in SEQ ID NO.13 to 15; and/or The upstream and downstream primers and probes for detecting Schistosoma haematobium Dra1 sequences are shown in SEQ ID NOs. 16 to 18; and/or The upstream and downstream primers and probes for detecting the Schistosoma japonicum SJR2 sequence are shown in SEQ ID NO.1 to 3; and/or The upstream and downstream primers and probes for detecting the internal standard gene are shown in SEQ ID NO.19~21. The composition according to any one of claims 3 to 6, characterized in that the composition comprises upstream and downstream primers and probes for detecting the SjTR1 microsatellite sequence of Schistosoma japonicum, upstream and downstream primers and probes for detecting the SM1-7 sequence of Schistosoma mansoni, upstream and downstream primers and probes for detecting the Dra1 sequence of Schistosoma haematobium, upstream and downstream primers and probes for detecting the SJR2 sequence of Schistosoma japonicum, and upstream and downstream primers and probes for detecting the internal standard gene sequence. The composition according to claim 5, characterized in that the components of the composition are present In the same packaging. The composition according to any one of claims 5 to 7, characterized in that the internal standard gene is a human housekeeping gene. The composition according to any one of claims 3 to 9, characterized in that the probe comprises a fluorescent reporter group. The composition according to claim 10 is characterized in that the fluorescent reporter groups of different probes are different from each other and do not interfere with each other. The composition according to claim 10 or 11, characterized in that the fluorescent reporter group of the probe of the SjTR1 microsatellite sequence is FAM; and/or The fluorescent reporter group of the probe of the internal standard gene is HEX or VIC; and/or The fluorescent reporter group of the probe of SM1-7 sequence is ROX; and/or The fluorescent reporter group of the probe of Dra1 sequence is CY5. Use of the composition according to any one of claims 3 to 12 in the preparation of a schistosomiasis pathogen detection kit. A kit for detecting schistosomiasis pathogens, the kit comprising the composition according to any one of claims 3 to 12. A method for detecting and distinguishing schistosomiasis pathogens, characterized in that the detection comprises the following steps: 1) Extract or release nucleic acid from the sample to be tested; 2) performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition described in any one of claims 3 to 12 or the kit described in claim 14; 3) Obtain and analyze the results.