Use of sjtr1 microsatellite sequence for the preparation of reagents for detecting schistosomiasis

Abstract

The present application belongs to the field of molecular biology detection, and particularly relates to detection of blood flukes, and more particularly to the use of SjTR1 microsatellite sequence for preparing reagents for detecting blood fluke disease. The present application provides the use of SjTR1 microsatellite sequence of Schistosoma japonicum for preparing reagents for detecting blood fluke disease. The PCR detection reagent prepared by using the new target point discovered by the present application can obtain higher sensitivity and specificity compared with the existing target point of Schistosoma japonicum, such as SJR2. In particular, in the design process of multiplex PCR, the new target point can make the whole system obtain better sensitivity and better specificity.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection, specifically, it relates to the detection of schistosomiasis, and more specifically, it relates to the use of the SjTR1 microsatellite sequence in the preparation of reagents for the detection of schistosomiasis. Background Technology

[0002] Schistosomiasis, commonly known as "the belly disease," is an acute or chronic illness caused by schistosomes. There are six types of schistosomes that infect humans: *Schistosoma japonicum*, *Schistosoma mansoni*, *Schistosoma haematobium*, *Schistosoma mekongiensis*, *Schistosoma guinea*, and *Schistosoma intercalatum*. Three of these cause the most significant harm to humans: *Schistosoma japonicum*, prevalent in Asia; *Schistosoma mansoni*, prevalent in Latin America and Central Africa; and *Schistosoma haematobium*, prevalent in North Africa. The main symptoms of schistosomiasis include dermatitis at the site of infection, accompanied by fever, abdominal pain, diarrhea, and tenderness in the liver area. Because the source of infection is difficult to completely eliminate and the transmission routes cannot be completely severed, the risk of transmission persists.

[0003] Diagnosis plays a crucial role in controlling and eliminating the source of infection in schistosomiasis prevention and control. Common diagnostic methods include examination of stool and urine specimens, and sometimes intestinal or bladder tissue specimens; blood tests are also sometimes used for diagnosis. However, these tests cannot indicate the severity of the infection and have limitations.

[0004] Nucleic acid detection boasts high sensitivity and specificity. Previously established nucleic acid diagnostic technologies are limited to primers and probes developed for existing targets, and their sensitivity and specificity need improvement. For example, Li, Juan, Zhao, Guang-Hui et al. reported a real-time PCR assay combined with high-resolution melting (HRM) targeting a portion of nuclear 18S rDNA to detect, identify, and differentiate four major schistosome species. However, its detection of genomic DNA from schistosomes is limited to a minimum amount of genomic DNA (10T) when the Ct value is <30. -5 The sensitivity needs improvement, and the melting curves of *Schistosoma japonicum* and *Schistosoma mekongiensis* have the same main peak value of 83.65℃. The difference lies in the fact that the melting curve of *Schistosoma mekongiensis* has a secondary peak to the left of the main peak. Using this to distinguish it from *Schistosoma japonicum* is not easy and may lead to misdiagnosis.

[0005] Therefore, there is a need in the field for a product that can diagnose schistosomiasis more quickly and effectively, with higher sensitivity and better specificity. Summary of the Invention

[0006] The subject matter of this article was developed under a project funded by the Hunan Provincial Science and Technology Innovation Plan (Project No.: 2022WZ1025).

[0007] 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 for detecting Schistosoma japonicum, the SjTR1 microsatellite sequence (SiTR1 region), and the gene sequence is: MW631938.1.

[0008] In a first aspect, the present invention provides the use of the SjTR1 microsatellite sequence of Schistosoma japonicum for preparing reagents for detecting schistosomiasis.

[0009] Furthermore, the reagent is a PCR reagent; even further, the reagent is a multiplex PCR reagent.

[0010] In some specific implementations, the reagent is a primer and / or a probe.

[0011] PCR detection reagents prepared using the novel target discovered in this invention can achieve higher sensitivity and specificity compared to existing targets of Schistosoma japonicum, such as SJR2. In particular, the novel target can enable the entire system to achieve better sensitivity and specificity in the design of multiplex PCR.

[0012] Secondly, the present invention provides a composition for detecting schistosomiasis, comprising:

[0013] Upstream and downstream primers and probes for detecting the SjTR1 microsatellite sequence of Schistosoma japonicum;

[0014] Primers and probes for detecting the SM1-7 sequence of Schistosoma mansoni; and

[0015] Upstream and downstream primers and probes used to detect the Dra1 sequence of Schistosoma haematobium.

[0016] Furthermore, the composition also includes an internal control gene. In some specific embodiments, the internal control is a human housekeeping gene.

[0017] Furthermore, the fluorescent groups of the probes in the above-described compositions of the present invention are different from each other and do not interfere with each other.

[0018] In this article, "dissimilar and non-interfering" means that each probe in the composition uses a different fluorophore and will not affect the detection of each other, i.e., different channels can be used for detection. For example, ATTO425, Quasar705, FAM, HEX, ROX, and CY5 can be used. These groups have different absorbance values ​​and can be selected in different channels, thus avoiding mutual interference.

[0019] In some specific implementations, the fluorescent reporter group of the probe for the SjTR1 microsatellite sequence is FAM; the fluorescent reporter group of the probe for the internal standard is HEX (or VIC); the fluorescent reporter group of the probe for the SM1-7 sequence is ROX; and the fluorescent reporter group of the probe for the Dra1 sequence is CY5.

[0020] Furthermore, the composition also includes upstream and downstream primers and probes for detecting the SJR2 sequence of Schistosoma japonicum.

[0021] By adding the aforementioned primer and probe sequences, the composition of the present invention exhibits higher sensitivity.

[0022] In some specific implementations, the probe for detecting the SJR2 sequence of Schistosoma japonicum can have the same or different fluorescent groups as the probe for the SjTR1 microsatellite sequence.

[0023] In some specific implementation schemes, the upstream and downstream primers and probes for detecting the SJR2 sequence of Schistosoma japonicum are shown in SEOID NO.1-3.

[0024] In some specific implementations, the upstream and downstream primers and probes for detecting the SjTR1 microsatellite sequence of Schistosoma japonicum are at least one pair as shown in SEO ID NO.4–6, or SEO ID NO.7–9, or SEO ID NO.10–12.

[0025] In some specific implementations, the upstream and downstream primers and probes for detecting the SjTR1 microsatellite sequence of Schistosoma japonicum are at least two pairs as shown in SEO ID NO.4–6, or SEO ID NO.7–9, or SEO ID NO.10–12.

[0026] In some specific implementation schemes, the upstream and downstream primers and probes for detecting the SjTR1 microsatellite sequence of Schistosoma japonicum are as shown in SEO ID NO.4–6, SEO ID NO.7–9, and SEO ID NO.10–12.

[0027] In some specific implementations, the upstream and downstream primers and probes for detecting the SM1-7 sequence are shown in SEO ID NO.13-15.

[0028] In some specific implementations, the upstream and downstream primers and probes used to detect the Dra1 sequence are shown in SEO IDs 16-18.

[0029] In some specific implementation schemes, the upstream and downstream primers and probes for detecting the internal standard are shown in SEO ID NO.19–21.

[0030] Furthermore, in some embodiments, the composition of the present invention may simultaneously include one or more pairs of the primer and probe pairs described above. In the present invention, a "pair" refers to a mutually matched upstream and downstream primer and probe for detecting a target.

[0031] The compositions of this invention can be arbitrarily combined to detect any combination of five corresponding targets. Those skilled in the art can combine them as needed, determining which targets to detect by combining the primer and probe pairs corresponding to those targets. All such combinations are included in this invention.

[0032] For example, it may include any 6 pairs of the above 7 pairs of primers and probes, any 5 pairs of the above 7 pairs of primers and probes, any 4 pairs of the above 7 pairs of primers and probes, any 3 pairs of the above 7 pairs of primers and probes, any 2 pairs of the above 7 pairs of primers and probes, or any 1 pair of the above 7 pairs of primers and probes.

[0033] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0034] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0035] Furthermore, the 3' end of the probe also has a quenching group, such as BHQ1 or BHQ2.

[0036] In one specific implementation, the 3' end of the probe is BHQ1.

[0037] In one specific embodiment, each component of the composition of the present invention is contained in a separate package.

[0038] In one specific embodiment, the components of the composition of the present invention are contained in the same package.

[0039] Furthermore, the components of the composition of the present invention exist in a mixed form.

[0040] Thirdly, the present invention provides the use of the above-described composition in the preparation of a schistosomiasis pathogen detection kit.

[0041] Furthermore, the present invention provides the use of the above-described composition of the present invention in the preparation of a kit for the joint detection and differentiation of schistosomiasis pathogens, wherein the pathogens are Schistosoma japonicum, Schistosoma mansoni, and Schistosoma haematobium.

[0042] Fourthly, the present invention provides a kit for detecting schistosomiasis pathogens, the kit comprising the composition of the present invention as described above.

[0043] Furthermore, the kit also includes negative and positive controls.

[0044] In one specific implementation, the negative control is at least one of DEPC H2O, physiological saline, and an internal standard gene. The positive control is at least one fragment of DNA from Schistosoma japonicum, Schistosoma mansoni, and Schistosoma haematobium.

[0045] Furthermore, the kit also includes dNTPs (U)s, PCR buffer, and Mg... 2+ At least one of them.

[0046] Furthermore, the kit also includes at least one of the following: a nucleic acid release reagent, a nucleic acid extraction reagent, and a DNA polymerase.

[0047] Furthermore, the kit also includes nucleic acid release reagents, nucleic acid extraction reagents, dNTPs, dUTPs, uracil glycosylase (UNG), DNA polymerase, PCR buffer, and Mg2+. 2+ At least one of them.

[0048] Furthermore, the concentration of the DNA polymerase is 3 U / reaction to 15 U / reaction, for example, the DNA polymerase can be Taq polymerase.

[0049] In one specific embodiment, the kit of the present invention includes Taq enzyme, UNG enzyme, and Mg. 2+ dNTPs (U), primers, probes, and PCR buffer.

[0050] Common PCR buffers consist of buffer systems such as Tris-HCl, MgCl2, KCl, and Triton X-100. The total volume of a single PCR reaction tube is generally 20 μl to 200 μl.

[0051] Fifthly, the invention provides the use of a composition for preparing a reagent for detecting and differentiating schistosomiasis pathogens, the detection comprising the following steps:

[0052] 1) Extract or release nucleic acid from the sample to be tested;

[0053] 2) Perform quantitative real-time PCR on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;

[0054] 3) Obtain and analyze the results.

[0055] In this invention, the sample used for detection can be blood, tissue fluid, etc., but is not limited to these.

[0056] Furthermore, the reaction conditions for the real-time PCR are as follows:

[0057] UNG enzyme reaction: temperature 40–60℃, time 30–150 seconds, 1 cycle; Taq enzyme activation: temperature 94℃, time 3–6 min, 1 cycle; denaturation: temperature 94℃, time 5–20 seconds; annealing: temperature 55℃–60℃, time 10–60 seconds, 30–50 cycles; fluorescence collection.

[0058] In one specific implementation, a method is provided for the combined detection and differentiation of schistosomiasis pathogens for non-diagnostic purposes, the method comprising the following steps:

[0059] 1) Extract or release nucleic acid from the sample to be tested;

[0060] 2) Perform quantitative real-time PCR on the nucleic acid obtained in step 1) using the composition or kit of the present invention as described above;

[0061] 3) Obtain and analyze the results.

[0062] Furthermore, the reaction conditions for the real-time PCR are as follows:

[0063] UNG enzyme reaction: temperature 40–60℃, time 30–150 seconds, 1 cycle; Taq enzyme activation: temperature 94℃, time 3–6 min, 1 cycle; denaturation: temperature 94℃, time 5–20 seconds; annealing: temperature 55–60℃, time 10–60 seconds, 30–50 cycles; fluorescence collection.

[0064] In this article, the term "non-diagnostic purpose" refers to a method not intended to obtain information about whether an individual is infected with the aforementioned pathogens and has schistosomiasis. For example, the method may be used to detect the presence of the aforementioned pathogens in a test culture (e.g., blood). Attached Figure Description

[0065] Figure 1 This is a graph showing the detection results of composition 1 of the present invention;

[0066] Figure 2 This is a graph showing the detection results of composition 2 of the present invention;

[0067] Figure 3 This is a graph showing the detection results of composition 3 of the present invention;

[0068] Figure 4 This is a graph showing the detection results of composition 4 of the present invention. Detailed Implementation

[0069] The present invention will be described in detail below with reference to specific implementation schemes and embodiments, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific implementation schemes and embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0070] Example 1: Primers and probes used in this invention

[0071] The primers and probes used in this invention are shown in Table 1 below.

[0072] Table 1

[0073]

[0074]

[0075] Example 2: Method for detecting schistosomiasis pathogen

[0076] 1. Reagent preparation:

[0077] Based on the number of negative controls, test samples, and positive controls, take the corresponding amounts of PCR reaction solution and enzyme mixture in proportion (38 μL / person PCR reaction solution + 2 μL / person enzyme mixture), mix thoroughly to form PCR Mix, centrifuge at 2000 rpm for 10 seconds, and set aside for later use.

[0078] PCR reaction system:

[0079]

[0080]

[0081] Preparation of mixed enzymes:

[0082] The enzyme mixture consists of H-Taq enzyme and UNG enzyme. H-Taq enzyme (15 U / μL) and UNG enzyme (2 U / μL) are mixed in a certain ratio (each person's dose is 1.7 μL of H-Taq enzyme and 0.3 μL of UNG enzyme).

[0083] 2. Sample processing and loading

[0084] 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) of Sansure Biotech Co., Ltd. to perform nucleic acid extraction according to its instructions.

[0085] Add 10 μL each of the negative control, the extracted nucleic acid sample, and the positive control to their respective 0.2 mL PCR reaction tubes. Add 40 μL of PCR Mix to each tube and cap the tubes.

[0086] 3. PCR amplification

[0087] Perform PCR amplification using the SLAN-96P fully automated medical PCR analysis system according to the procedure in the table below:

[0088]

[0089] 4. Interpretation of test results

[0090] If the sample shows a clear S-shaped amplification curve in the FAM, HEX(VIC), ROX, and CY5 channels, and the Ct value is ≤40, it is considered positive. If the sample shows no amplification curve (No Ct) or a Ct value >40 in the FAM, ROX, and CY5 channels, and the HEX(VIC) internal standard channel is positive (Ct value ≤40), it is considered negative. Details are as follows:

[0091]

[0092] Example 3: Detection results of test samples of the composition of the present invention

[0093] Using the primers and probes shown in Example 1 (composition 1: SEQ ID NO. 4-6, SEQ ID NO. 13-21), the *Schistosoma japonicum*, *Schistosoma mansoni*, and *Schistosoma haematobium* in the sample were simultaneously detected according to the method of Example 2. The results are as follows: Figure 1 As shown, from Figure 1 As can be seen, composition 1 can detect and distinguish various schistosomiasis species in the sample.

[0094] Using the primers and probes shown in Example 1 (composition 2: SEQ ID NO. 7-9, SEQ ID NO. 13-21), the *Schistosoma japonicum*, *Schistosoma mansoni*, and *Schistosoma haematobium* in the sample were simultaneously detected according to the method of Example 2. The results are as follows: Figure 2 As shown, from Figure 2 As can be seen, composition 2 is able to detect and distinguish various schistosomiasis species in the sample.

[0095] 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 in Example 2. The results are as follows: Figure 3 As shown, from Figure 3 As can be seen, composition 3 is able to detect and distinguish various schistosomiasis species in the sample.

[0096] 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 in Example 2. The results are as follows: Figure 4 As shown, from Figure 4 As can be seen, composition 4 is able to detect and distinguish various schistosomiasis species in the sample.

[0097] Example 4: Sensitivity of the composition of the present invention

[0098] Synthetic plasmids of Schistosoma japonicum, Schistosoma mansoni, and Schistosoma haematobium were mixed and serially diluted with negative serum samples to 1.00E+03 copies / mL, 4.00E+02 copies / mL, 2.00E+02 copies / mL, and 1.00E+02 copies / mL as test samples. The limit of detection of this reagent was set at 95%. The results of the sensitivity test showed that the limit of detection of the present invention composition 1 against the pathogens of Schistosoma japonicum / Schistosoma mansoni / Schistosoma haematobium was 4.00E+02 copies / mL (see Table 2).

[0099] Table 2

[0100]

[0101] Furthermore, the detection limits for compositions 2 and 3 of the present invention against Schistosoma japonicum / Schistosoma mansoni / Schistosoma haematobium pathogens are both 4.00E+02 copies / mL (see Tables 3 and 4).

[0102] Table 3

[0103]

[0104]

[0105] Table 4

[0106]

[0107] The detection limits of the present invention composition 4 for Schistosoma mansoni / Schistosoma heptaphyllum pathogens are 4.00E+02 copies / mL, and the detection limit for Schistosoma japonicum is as low as 2.00E+02 copies / mL (see Table 5).

[0108] Table 5

[0109]

[0110]

[0111] Example 5: Specificity of the composition of the present invention

[0112] Specificity experiments showed that the method of the present invention has no cross-reactivity with 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) etc. (sample information is shown in Table 6).

[0113] Table 6. Statistical Table of Information on Different Similar Pathogens

[0114]

[0115] The above samples were used for specificity analysis, with a mixed plasmid of Schistosoma japonicum, Schistosoma mansoni, and Schistosoma haematobium diluted to 1.00E+06 copies / mL used as a control sample.

[0116] Three batches of reagents (composition 4) were tested using the SLAN-96P fully automated medical PCR analysis system. The specificity of the kit was evaluated by detecting the positive and negative concordance rates. The test results are shown in Table 7 below:

[0117] Table 7. Statistics of Cross-Reactivity Results of Three Batches of Reagents

[0118]

[0119]

[0120] Conclusion: Using three batches of reagents, the cross-samples all showed negative results for Schistosoma, indicating that this kit has excellent specificity. Furthermore, verification showed that other high concentrations of pathogens listed in the table above did not affect the nucleic acid detection of Schistosoma japonicum, Schistosoma mansoni, or Schistosoma haematobium.

[0121] Example 6: Anti-interference and stability of the composition of the present invention

[0122] To investigate the impact of potential endogenous / exogenous substances in the samples on the detection results, the nucleic acid of the pre-determined *Schistosoma japonicum* sample was mixed with plasmids of *Schistosoma haematobium* and *Schistosoma mansoni*, diluted to the lowest detection limit, and divided into several aliquots. Each aliquot was supplemented with a specific concentration of endogenous interfering substances: plasma proteins, leukocytes, total bilirubin, triglycerides, total IgG, and interferon α; and exogenous interfering substance: praziquantel. The anti-interference ability of the kit was examined by comparing the results with a control sample. The experiment was conducted using composition 1 of this invention, and the results are shown in Table 8.

[0123] Table 8. Interference Test Verification Results

[0124] Interfering substances FAM HEX ROX CY5 control sample 36.00 34.08 34.43 37.05 plasma proteins 35.87 34.00 35.55 36.23 Total bilirubin 37.17 33.77 34.35 35.52 Triglycerides 35.24 34.28 36.48 36.79 leukocyte 37.37 34.21 35.81 35.18 Total IgG 36.55 34.98 35.10 38.10 Interferon alpha 36.21 34.98 34.93 37.45 Praziquantel 35.36 35.84 34.49 39.37

[0125] Comparative Example 1: The Influence of the New Target and Known Targets of this Application on Sensitivity

[0126] Synthetic plasmids of *Schistosoma japonicum*, *Schistosoma mansoni*, and *Schistosoma haematobium* were mixed and serially diluted with negative serum samples to 1.00E+03 copies / mL, 4.00E+02 copies / mL, 2.00E+02 copies / mL, and 1.00E+02 copies / mL as test samples. The limit of detection (LOD) of this reagent was set at 95%. Analytical sensitivity tests showed that the LOD of composition 5 of this invention (composition 5: SEQ ID NO. 1–3, SEQ ID NO. 13–21) against *Schistosoma japonicum*, *Schistosoma mansoni*, and *Schistosoma haematobium* pathogens was 1.00E+03.

[0127] copies / mL (see Table 9).

[0128] Table 9

[0129]

[0130] A comparison of Tables 9 and 2-4 shows that compositions 1, 2, and 3 using primers and probes designed with the new target all have a sensitivity of 4.00 ± 0.2 copies / mL, while composition 5 using primers and probes designed with the known target has a sensitivity of only 1.00 ± 0.3 copies / mL. The primers and probes designed with the new target in this application have higher sensitivity than those with the known target.

Claims

1. A composition for detecting schistosomiasis, characterized in that, Primers and probes for detecting the SjTR1 microsatellite sequence of Schistosoma japonicum, primers and probes for detecting the SM1-7 sequence of Schistosoma mansoni, primers and probes for detecting the Dra1 sequence of Schistosoma haematobium, primers and probes for detecting the SJR2 sequence of Schistosoma japonicum, and primers and probes for detecting the internal standard sequence, among which, The upstream and downstream primers and probes for detecting the SjTR1 microsatellite sequence of Schistosoma japonicum are at least one pair as shown in SEO ID NO.4~6, or SEO ID NO.7~9, or SEO ID NO.10~12; The upstream and downstream primers and probes for detecting the SM1-7 sequence of Schistosoma mansoni are shown in SEO ID NO. 13-15; the upstream and downstream primers and probes for detecting the Dra1 sequence of Schistosoma haematobium are shown in SEO ID NO. 16-18; the upstream and downstream primers and probes for detecting the SJR2 sequence of Schistosoma japonicum are shown in SEO ID NO. 1-3; and the upstream and downstream primers and probes for detecting the internal standard are shown in SEO ID NO. 19-21.

2. The composition according to claim 1, characterized in that, The components of the composition are contained in the same package.

3. Use of the composition according to claim 1 or 2 in the preparation of a schistosomiasis pathogen detection kit.

4. A kit for detecting schistosomiasis pathogens, said kit comprising the composition as described in claim 1 or 2.

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