CN117106932A - Application of SjTR1 microsatellite sequence in preparation of reagent for detecting schistosomiasis - Google Patents

Abstract

The present invention belongs to the field of molecular biology detection, specifically, relates to the detection of schistosomiasis, and more specifically, relates to the use of the SjTR1 microsatellite sequence for preparing reagents for detecting schistosomiasis. The present invention provides a use of the Schistosoma japonicum SjTR1 microsatellite sequence for preparing reagents for detecting schistosomiasis. The PCR detection reagent prepared using the new target discovered by the present invention is better than the existing targets of Schistosoma japonicum, such as SJR2. It can achieve higher sensitivity and specificity. In particular, during the design process of multiplex PCR, new targets can make the entire system achieve better sensitivity and better specificity.

Description

Use of SjTR1 microsatellite sequence for preparing reagents for detecting schistosomiasis

Technical field

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

Background technique

Schistosomiasis, commonly known as "potbelly disease", is an acute and chronic disease caused by schistosomiasis. There are six types of schistosomiasis that infect humans: Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, Schistosoma mekongi, Schistosoma guinea and Schistosoma intersperse. There are three types of schistosomiasis that cause major harm to humans: Schistosoma japonicum, which is prevalent in Asia, and Schistosoma japonicum, which is prevalent in Latin America and Schistosoma mansoni in Central Africa and Schistosoma haematobium , which is endemic in North Africa. Schistosomiasis mainly manifests as dermatitis at the invasion site, accompanied by symptoms such as fever, abdominal pain, diarrhea, and tenderness in the liver area. The source of schistosomiasis infection is difficult to completely eliminate, the transmission route cannot be completely cut off, and the risk of transmission continues to exist.

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

Nucleic acid detection has high sensitivity and specificity. Previously established nucleic acid diagnostic technologies are limited to primers and 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 The melting (HRM) assay targets a portion of the nuclear 18S rDNA to detect, identify and differentiate the four major Schistosoma species, but it detects genomic DNA from Schistosoma and the assay is limited to the minimum amount of genomic DNA at a Ct value <30 , is 10 ^-5 ng, the sensitivity needs to be improved, and the main peaks of the melting curves of Schistosoma japonicum and Schistosoma mekongi are the same, both at 83.65°C. 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 mekongi. It is difficult to distinguish it from Schistosoma japonicum based on this, which is not easy to interpret and may cause misjudgment.

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

Contents of the invention

The subject disclosed in this article was made under a project funded by the Hunan Provincial Science and Technology Innovation Plan (Project No.: 2022WZ1025).

In view of this, the applicant used the Schistosoma japonicum gene sequence with the 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 .

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

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

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

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

In a second aspect, the present invention provides a composition for detecting schistosomiasis, comprising

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

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 sequence.

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

Furthermore, the fluorescent groups of the probes of the composition of the present invention are different from each other and do not interfere with each other.

In this article, "different from each other and not interfering with each other" means that the fluorescent 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, ATTO425, Quasar705, FAM, HEX, ROX and CY5 can be used. The absorption 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 is HEX (or VIC); the fluorescent reporter group of the probe of the SM1-7 sequence The group is ROX; the fluorescent reporter group of the Dra1 sequence probe is CY5.

Further, 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 composition of the present invention has higher sensitivity.

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

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

In some specific embodiments, the upstream and downstream primers and probes for detecting the Schistosoma japonicum SjTR1 microsatellite sequence are as shown in SEO ID NO. 4 to 6, or as shown in SEO ID NO. 7 to 9, or as shown in SEO ID NO. At least one pair shown in 10 to 12.

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

In some specific embodiments, the upstream and downstream primers and probes for detecting the Schistosoma japonicum SjTR1 microsatellite sequence are as shown in SEO ID NO.4-6, SEO ID NO.7-9, and SEO ID NO.10 ~12 shown.

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

In some specific embodiments, the upstream and downstream primers and probes used to detect the Dra1 sequence are shown in SEO IDNOs. 16-18.

In some specific embodiments, the upstream and downstream primers and probes for detecting the internal standard are as shown in SEO ID NO. 19-21.

Further, in some embodiments, the composition of the present invention may simultaneously include one or more pairs of the above-mentioned primer and probe pairs. In the present invention, "pair" refers to matching upstream and downstream primers and probes for detecting a target point.

The composition of the present invention can be arbitrarily combined into any combination for detecting 5 target points. Those skilled in the art can combine as needed to detect which target points, that is, just combine primers and probe pairs corresponding to the target points. These combinations are included in the present invention.

For example, it may include any 6 pairs of the above 7 pairs of primers and probes, may include any 5 pairs of the above 7 pairs of primers and probes, may include any 4 pairs of the above 7 pairs of primers and probes, may It includes any 3 pairs of the above 7 pairs of primers and probes, can include any 2 pairs of the above 7 pairs of primers and probes, and can also include any 1 pair of the above 7 pairs of primers and probes.

In some specific embodiments, compositions of the invention are used in fluorescent PCR.

Furthermore, the 3’ end of the probe also has a non-fluorescent quencher.

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

In a specific embodiment, the 3&apos; end of the probe is BHQ1.

In a specific embodiment, each component of the composition of the invention is presented in a separate package.

In a specific embodiment, the ingredients of the composition of the invention are present in the same package.

Further, each component of the composition of the present invention is present in a mixed form.

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

Further, the present invention provides the use of the above-mentioned composition of the present invention in preparing a kit for joint detection and differentiation of schistosomiasis pathogens, wherein the pathogens are Schistosoma japonicum, Schistosoma mansoni, and Schistosoma haematobium.

In a fourth aspect, the present invention provides a kit for detecting schistosomiasis pathogens, which kit includes the composition of the present invention as described above.

Further, the kit also includes negative quality control products and positive quality control products.

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

Further, 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 release reagent, a nucleic acid extraction reagent, and a DNA polymerase.

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

Further, the concentration of the DNA polymerase ranges from 3 U/reaction to 15 U/reaction. For example, the DNA polymerase can be Taq enzyme.

In a specific embodiment, the kit of the invention 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 and other buffer systems. Generally, the total volume in a single PCR reaction tube is 20 μl to 200 μl.

In a fifth aspect, there is provided a use of a composition for preparing a reagent for detecting and distinguishing schistosomiasis pathogens, and the detection includes the following steps:

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

2) Use the composition of the present invention as described above or the kit of the present invention as described above to perform fluorescence quantitative PCR on the nucleic acid obtained in step 1);

3) Obtain and analyze the results.

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

Further, the reaction conditions of the fluorescent quantitative PCR are:

UNG enzyme reaction, the temperature is 40~60℃, the time is 30~150 seconds, one cycle; Taq enzyme activation, the temperature is 94℃, the time is 3~6min, one cycle; denaturation, the temperature is 94℃, the time is 5-20 seconds, annealing, temperature 55°C-60°C, time 10-60 seconds, 30-50 cycles, and fluorescence collection.

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 the nucleic acid of the sample to be tested;

2) Use the above-mentioned composition of the present invention or the above-mentioned kit of the present invention to perform fluorescence quantitative PCR on the nucleic acid obtained in step 1);

3) Obtain and analyze the results.

Further, the reaction conditions of the fluorescent quantitative PCR are:

UNG enzyme reaction, the temperature is 40~60℃, the time is 30~150 seconds, one cycle; Taq enzyme activation, the temperature is 94℃, the time is 3~6min, one cycle; denaturation, the temperature is 94℃, the time is 5-20 seconds, annealing, temperature 55-60°C, time 10-60 seconds, 30-50 cycles, fluorescence collection.

In this context, the term "non-diagnostic purposes" means that it is not intended to obtain information on whether an individual is infected with the pathogens listed above and suffers from schistosomiasis. For example, the method may be required to detect the presence of the above-mentioned pathogens in a test culture (e.g., blood).

Description of drawings

Figure 1 is a graph showing the test results of composition 1 of the present invention;

Figure 2 is a graph showing the test results of composition 2 of the present invention;

Figure 3 is a graph showing the test results of composition 3 of the present invention;

Figure 4 is a graph showing the test results of composition 4 of the present invention.

Detailed ways

The present invention will be described in detail below in conjunction with specific embodiments and examples, from which the advantages and various effects of the present invention will be more clearly presented. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present invention but not to limit the present invention.

Example 1. Primers and probes used in the present invention

The primers and probes used in the present invention 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, samples to be tested, and positive controls, take corresponding amounts of PCR reaction solution and enzyme mixture in proportion (PCR reaction solution 38 μL/person + enzyme mixture 2 μL/person), and mix thoroughly to form PCR Mix , centrifuge at 2000rpm for 10s and set aside.

PCR reaction system:

Preparation of mixed enzyme:

The enzyme mixture consists of H-Taq enzyme and UNG enzyme. Mix H-Taq enzyme (15U/μL) and UNG enzyme (2U/μL) according to a certain ratio (each serving is 1.7μL H-Taq enzyme and 0.3μL UNG enzyme).

2. Sample processing and adding

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 Shengxiang Biotechnology Co., Ltd. to perform nucleic acid extraction according to its instructions.

Take 10 μL each of the negative control, the extracted sample nucleic acid, and the positive control and add them to the corresponding 0.2 mL PCR reaction tubes. Add 40 μL PCR Mix to each tube and cap the tube.

3. PCR amplification

On the SLAN-96P fully automatic medical PCR analysis system, perform PCR amplification according to the procedure in the table below:

4. Interpretation of test results

If the sample has an obvious S-type amplification curve in 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 in FAM, ROX, and CY5 channels (No Ct) Or if the Ct value is >40 and the HEX (VIC) internal standard channel is positive (Ct value ≤ 40), it will be judged as negative. details as follows:

Example 3. Test results of test samples of the composition of the present invention

The primers and probes shown in Example 1 (Composition 1: SEQ ID NO. 4-6, SEQ ID NO. 13-21) were used according to the method of Example 2 to detect Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium was detected simultaneously, and the results are shown in Figure 1. It can be seen from Figure 1 that composition 1 can detect and distinguish various schistosomiasis 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 according to the method of Example 2 to detect Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium was detected simultaneously, and the results are shown in Figure 2. It can be seen from Figure 2 that composition 2 can detect and distinguish various schistosomiasis 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 as follows As shown in Figure 3, it can be seen from Figure 3 that composition 3 can detect and differentiate various schistosomiasis in the sample.

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

Example 4. Sensitivity of the composition of the present invention

Mix the synthetic plasmids of Schistosoma japonicum, Schistosoma mansoni, and Schistosoma haematobium and use negative serum samples to gradient dilute them to 1.00E+03copies/mL, 4.00E+02copies/mL, 2.00E+02copies/mL, and 1.00E+02copies/mL. As a sample to be tested, the detection level of 95% is used as the minimum detection limit of this reagent. The analytical sensitivity test results show that the detection limit of the composition 1 of the present invention for the Schistosoma japonicus/Mansoni/Haematoma pathogen is: 4.00E+02 copies/mL (See Table 2).

Table 2

Furthermore, the detection limits of Compositions 2 and 3 of the present invention for Schistosoma japonicus/Mansoni/Schistosoma haematobium pathogens are both: 4.00E+02 copies/mL (see Tables 3 and 4)

table 3

Table 4

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

table 5

Example 5. Specificity of the composition of the present invention

Specificity experiments show that the method of the present invention is effective against 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) have no cross-reactivity (see Table 6 for sample information).

Table 6. Statistics table of information on different similar pathogens

Specificity analysis uses the above samples, and uses Schistosoma japonicum, Schistosoma mansoni, and Schistosoma haematobium mixed plasmids diluted to 1.00E+06 copies/mL as control samples for detection.

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

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

Conclusion: Using three batches of reagents, the cross-sample schistosomiasis detection results were all negative, 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 composition of the present invention

In order to examine the impact of endogenous/exogenous substances that may be present in the sample on the test results, the nucleic acid of the determined Schistosoma japonicum sample was mixed with the plasmids of Schistosoma haematobium and Schistosoma mansoni, diluted to the lowest detection limit, and divided into several parts. A certain concentration of endogenous interfering substances: plasma protein, leukocytes, total bilirubin, triglycerides, total IgG, interferon α was added to each portion; exogenous interfering substances: praziquantel, and compared with the control sample , test the anti-interference ability of the kit. The composition 1 of the present invention was used for testing, and the test results are shown in Table 8:

Table 8. Interference test verification test results

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

Comparative Example 1. Effect of new targets and known targets of this application on sensitivity

Mix the synthetic plasmids of Schistosoma japonicum, Schistosoma mansoni, and Schistosoma haematobium and use negative serum samples to gradient dilute them to 1.00E+03copies/mL, 4.00E+02copies/mL, 2.00E+02copies/mL, and 1.00E+02copies/mL. As a sample to be tested, the detection level of 95% is used as the minimum detection limit of this reagent. The analytical sensitivity test results show that composition 5 of the present invention (composition 5: SEQ ID NO. 1 to 3, SEQ ID NO. 13 to 21) The detection limits for Japanese/Mansoni/Schistosoma haematobium pathogens are: 1.00E+03

copies/mL (see Table 9).

Table 9

From the comparison between Table 9 and Table 2-4, it can be seen that the sensitivity of compositions 1, 2 and 3 of primers and probes designed using new targets are all 4.00+02copies/mL, while those designed using known targets The sensitivity of primer and probe combination 5 is 1.00+03 copies/mL. The sensitivity of the primers and probes designed for the new target in this application is higher than that of the known target.

Claims (10)

1. The application of the schistosoma japonicum SjTR1 microsatellite sequence in preparing reagent for detecting schistosomiasis.

2. The use according to claim 1, wherein the reagent is a PCR reagent.

3. A composition for detecting schistosomiasis, comprising an upstream primer and a downstream primer for detecting schistosoma japonicum SjTR1 microsatellite sequences and a probe.

4. A composition according to claim 3, wherein the upstream and downstream primers and probes for detecting the sequence of the microsatellite of SjTR1 of schistosoma japonicum are at least one pair of those shown as SEO ID nos. 4 to 6, or SEO ID nos. 7 to 9, or SEO ID nos. 10 to 12.

5. The composition according to claim 3 or 4, wherein the composition further comprises at least one pair of an upstream primer and a downstream primer for detecting the schistosoma mansoni SM1-7 sequence, an upstream primer and a downstream probe for detecting the schistosoma japonicum Dra1 sequence, an upstream primer and a downstream primer and a probe for detecting the schistosoma japonicum SJR2 sequence, and an upstream primer and a downstream primer and a probe for detecting an internal standard sequence.

6. The composition according to claim 5, wherein the upstream and downstream primers and probes for detecting the sequence of S.mansoni SM1-7 are as shown in SEO ID No. 13-15; the upstream and downstream primers and probes for detecting the Dra1 sequence of the schistosoma japonicum are shown as SEO ID NO. 16-18; the upstream and downstream primers and probes for detecting the Schistosoma japonicum SJR2 sequence are shown in SEO ID No. 1-3; the upstream and downstream primers and probes for detecting the internal standard are shown as SEO ID No. 19-21.

7. The composition of claim 6, wherein the composition comprises an upstream and a downstream primer and a probe for detecting the SjTR1 microsatellite sequence of schistosoma japonicum, an upstream and a downstream primer and a probe for detecting the SM1-7 sequence of schistosoma mansoni, an upstream and a downstream primer and a probe for detecting the Dra1 sequence of schistosoma japonicum, an upstream and a downstream primer and a probe for detecting the SJR2 sequence of schistosoma japonicum, and an upstream and a downstream primer and a probe for detecting the internal standard sequence.

8. The composition of claim 5, wherein the components of the composition are in the same package.

9. Use of a composition according to any one of claims 3 to 8 in the preparation of a schistosomiasis pathogen detection kit.

10. A kit for detecting a schistosomiasis pathogen, the kit comprising the composition of any one of claims 3-8.