A method for simultaneous quantitative detection of Schistosoma japonicum and its host, Oncomelania hupehensis.

Abstract

This invention discloses a method for the simultaneous quantitative detection of *Schistosoma japonicum* and its host, *Oncomelania hupehensis*, belonging to the field of molecular biology. The primer sequences involved are as follows: *Oncomelania hupehensis* pre-primer: 5'-GCCAGTGTGAAAAGCGAAAAAT-3'; *Oncomelania hupehensis* post-primer: 5'-AAAAGGACTTTCACCTCTACCTTACG-3'; fluorescent probe: 5'-CATGCGGTTTCGGC-3'; *Schistosoma japonicum* pre-primer: 5'-GCGTTCGAACCTATGCCAAA-3'; *Schistosoma japonicum* post-primer: 5'-GAGCGGTAATCTAGAGTCACAGCTAA-3'; fluorescent probe: 5'-AGGACAACAAGCCTC-3'. This method can rapidly diagnose *Oncomelania hupehensis* and *Schistosoma japonicum* infection.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and more specifically to a method for the simultaneous quantitative detection of Schistosoma japonicum and its host, Oncomelania hubeiensis. Background Technology

[0002] Schistosomiasis japonicus is a serious zoonotic disease. Acute schistosomiasis patients mainly present with fever, diarrhea, and abdominal pain. They may also experience tenderness in the right upper quadrant due to hepatomegaly. Red rashes may appear on the skin and mucous membranes where the parasites invade. Chronic schistosomiasis patients may experience chronic diarrhea, hepatosplenomegaly, and other symptoms. Late-stage schistosomiasis patients may experience increased fibrous connective tissue around the portal venous system, leading to symptoms such as hematemesis, abdominal distension, lower extremity edema, and dyspnea, which can ultimately be life-threatening.

[0003] With the implementation of prevention and control policies, schistosomiasis japonicus has entered the next elimination phase, and improved monitoring capabilities are crucial for further consolidating elimination achievements. However, the intermediate host of Schistosoma japonicum—Oncomelania hubeiensis—is easily confused with similar snails (such as those in the genera *Oncomelania* and *Syngonium*) due to its small size. This leads to investigators easily collecting snails other than Oncomelania hubeiensis during snail surveys, potentially causing errors in snail data and increasing the difficulty of later detection of Schistosoma japonicum in snails. Currently, the main methods used in Oncomelania japonicum laboratory testing are crushing microscopy or exocercariae assays. These methods often require waiting for the parasite to develop into microscopically observable sporocysts or cercariae within the snail, making it difficult to quickly assess the risk of schistosomiasis. Furthermore, the large number of snail samples often obtained on-site can easily lead to fatigue among laboratory personnel, resulting in missed detections. Whether it's on-site investigation or laboratory testing for snails and their infection status, experienced staff are required. If staff in areas where schistosomiasis is prevalent or at risk of prevalence are not trained regularly, the risk of disease transmission may not be effectively identified due to a decline in schistosomiasis detection capabilities.

[0004] Therefore, providing a method to improve the detection capability of Schistosoma japonicum is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a method for the simultaneous quantitative detection of Schistosoma japonicum and its host, Oncomelania hubeiensis, utilizing multiplex real-time fluorescence PCR technology to achieve rapid diagnosis of Oncomelania hubeiensis infection status and Schistosoma japonicum infection. This invention can help schistosomiasis control personnel quickly determine whether the obtained snails are Oncomelania hubeiensis and whether they are infected with Schistosoma japonicum. Based on different samples (snail samples, environmental samples, etc.), the risk of schistosomiasis transmission from Oncomelania hubeiensis or the environment can be directly assessed, thus providing a reference for schistosomiasis control work.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for simultaneous quantitative detection of Schistosoma japonicum and its host, Oncomelania hubeiensis, employs multiplex PCR and involves the following primer sequences:

[0008] Hubei Oncomelania preparata pre-primer: 5'-GCCAGTGTGAAAAGCGAAAAAT-3';

[0009] Hubei Oncomelania hupensis post-primer: 5'-AAAAGGACTTTCACCTCTACCTTACG-3';

[0010] Fluorescent probe: 5'-CATGCGGTTTCGGC-3';

[0011] Preprimer for Schistosoma japonicum: 5'-GCGTTCGAACCTATGCCAAA-3';

[0012] Japanese Schistosoma posterior primer: 5'-GAGCGGTAATCTAGAGTCACAGCTAA-3';

[0013] Fluorescent probe: 5'-AGGACAACAAGCCTC-3'.

[0014] Preferably, the multiplex PCR reaction system includes 10 μL of 2*AceQ U+Probe Master Mix, 1 μL of cDNATemplate, 0.4 μL of upstream primer from *Oncomelania hupehensis*, 0.4 μL of downstream primer from *Oncomelania hupehensis*, 0.2 μL of primer and probe from *Oncomelania hupehensis*, 0.4 μL of upstream primer from *Schistosoma japonicum*, 0.4 μL of downstream primer from *Schistosoma japonicum*, 0.2 μL of primer and probe from *Schistosoma japonicum*, and 7 μL of ddH2O.

[0015] Preferably, the multiplex PCR reaction program includes 37°C for 2 min; 95°C for 5 min; 95°C for 10 s; 60°C for 34 s; 45 cycles.

[0016] Another object of the present invention is to provide the application of the above method in the determination of potential risk of detecting Schistosoma japonicum.

[0017] Beneficial effects:

[0018] (1). This invention utilizes multiplex qPCR technology to simultaneously identify Oncomelania hupehensis and Schistosoma japonicum at the gene level. Compared with traditional methods that rely on experience, this method can serve as an effective means of identifying these two species.

[0019] (2). Multiplex qPCR can detect a large number of samples at once. Compared with traditional snail identification methods that rely on a lot of manpower, this method not only provides more accurate results, but also saves time and effort, making it suitable for on-site snail inspection requirements.

[0020] (3) This invention uses an absolute quantitative method to evaluate the density of Oncomelania snails, which can serve as a supplement to traditional snail detection methods to some extent. In addition, since this technology can quantitatively evaluate the schistosomiasis load of infected (positive) Oncomelania hupensis snails, it can more accurately evaluate the snail infection status compared to traditional microscopic estimation techniques.

[0021] (4). As a gene diagnostic technology, this invention can be used to detect nucleic acid fragments in water and soil environmental samples in addition to tissue samples in the future, so as to directly evaluate the risk of schistosomiasis transmission in the relevant environment. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0023] Figure 1 The image shows the melting curve of primer 1, obtained using an Applied Biosystems 7500 PCR instrument.

[0024] Figure 2 The image shows the melting curve of primer 2, obtained using an Applied Biosystems 7500 PCR instrument.

[0025] Figure 3 The image shows the melting curve of primer 3, obtained using an Applied Biosystems 7500 PCR instrument.

[0026] Figure 4 The image shows the melting curve of primer 4, obtained using an Applied Biosystems 7500 PCR instrument.

[0027] Figure 5 The image shows the melting curve of primer 5, obtained using an Applied Biosystems 7500 PCR instrument.

[0028] Figure 6 The image shows the melting curve of primer 6, obtained using an Applied Biosystems 7500 PCR instrument.

[0029] Figure 7The image shows the melting curve of primer 7, obtained using an Applied Biosystems 7500 PCR instrument.

[0030] Figure 8 The image shows the melting curve of primer 8, obtained using an Applied Biosystems 7500 PCR instrument.

[0031] Figure 9 The results show the experimental results of two primers and their probe combinations in detecting positive Oncomelania hupehensis (the primer for Oncomelania hupehensis is the blue FAM channel, and the primer for Schistosoma japonicum is the green JOE channel; the CT values ​​of both are between 10 and 30).

[0032] Figure 10 The results show the experimental results of two primers and their probe combinations in detecting negative Oncomelania hupehensis (the Oncomelania hupehensis primer is represented by the blue FAM channel, and the Schistosoma japonicum primer is represented by the green JOE channel; the CT value of the Oncomelania hupehensis primer is between 10 and 30, and the CT value of the Schistosoma japonicum primer is greater than 30).

[0033] Figure 11 The results show the experimental results of two primers and their probe combinations in detecting Schistosoma japonicum (the blue FAM channel is for the Hubei snail primer, the green JOE channel is for the Schistosoma japonicum primer, the CT value of Schistosoma japonicum is between 10 and 30, and the CT value of the Hubei snail primer is greater than 30).

[0034] Figure 12 The FAM channel results showed that the multiplex qPCR reaction system could specifically identify Oncomelania hupensis. Except for Oncomelania hupensis, whose Ct value was between 15 and 30, the Ct values ​​of other snails and shellfish were not in this range.

[0035] Figure 13 The HEX channel results showed that this multiplex qPCR reaction system could specifically recognize Schistosoma japonicum. Except for Schistosoma japonicum, whose Ct value was between 15 and 30, the Ct values ​​of other trematodes were not in this range.

[0036] Figure 14 This is a schematic diagram of a plasmid containing the target gene from the snail Oncomelania hupehensis.

[0037] Figure 15 This is a schematic diagram of a plasmid containing the target gene of Schistosoma japonicum.

[0038] Figure 16 The results of qPCR amplification of plasmids containing the target gene of Oncomelania hupehensis and plasmids containing the target gene of Schistosoma japonicum are shown.

[0039] Figure 17 The standard curve (for Oncomelania hupensis) is the result of multiplex qPCR for Oncomelania hupensis and Schistosoma japonicum.

[0040] Figure 18 The standard curve for multiplex qPCR of *Oncomelania hupensis* and *Schistosoma japonicum* (*Schistosoma japonicum*) is shown. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] Example 1

[0043] 1. Using the Nucleotide database on the NCBI website, the mitochondrial sequences of *Oncomelania hupehensis* and the gene sequences of *Schistosoma japonicum* were searched. BLAST was used to obtain similar sequences from the same species. To further improve the representativeness and specificity of the primers, similar fragment regions from different uploaded sequences of the same species were selected using BioEdit (v7.2) software, excluding similar fragment regions between similar species and the target species. Several target fragments from *Schistosoma japonicum* and *Oncomelania hupehensis* were selected. Primers and probes for real-time quantitative PCR were designed using Primer Express software on the selected base sequences. Four pairs of primers and probes were designed for each species (see Table 1). The probes for *Oncomelania hupehensis* were labeled with the FAM reporter group, and the probes for *Schistosoma japonicum* were labeled with the HEX reporter group.

[0044] Table 1

[0045]

[0046]

[0047]

[0048] 2. Genomic DNA was extracted from the gastropod tissue of *Oncomelania hupensis* (negative-negative snails - uninfected) and the adult worms of *Schistosoma japonicum* using a tissue genomic extraction kit, serving as templates for subsequent primer efficacy evaluation. Dye-based qPCR (e.g., ChamQ Universal SYBR qPCR Master Mix, Vazyme) was performed using a real-time fluorescent directional PCR instrument (e.g., Applied Biosystems 7500, Roche Applied Science LightCycler 480). The CT value of the primers was checked to ensure they were within the effective amplification range, and the melting curve was single-peaked and stable (see Appendix). Figure 1 -Appendix Figure 8Primer sets were screened, with two primer pairs selected for each species. Primers 3, 4, 7, and 8 were ultimately chosen for subsequent experiments. The qPCR reaction system is shown in Table 2, and the reaction procedure is shown in Table 3.

[0049] Table 2

[0050] reagents Reference volume (20 μL) 2*ChamQ Universal SYBR qPCR Master Mix 10 cDNA Template 1 upstream primer 0.8 Downstream primer 0.8 <![CDATA[ddH2O]]> 7.4

[0051] Table 3

[0052] Insulation stage Cyclic Phase (40 cycles) Melt curve stage 95℃ for 1 minute 95℃ for 10 seconds 95℃ for 15 seconds 60℃ 30sec 60℃ for 1 minute 95℃ for 15 seconds

[0053] Example 2

[0054] Using *Oncomelania hupensis* (uninfected) and *Schistosoma japonicum* as templates, primer and probe combinations were screened using primers 3, 4, 7, and 8 in Table 1 (3 and 7, 3 and 8, 4 and 7, 4 and 8). Multiplex qPCR amplification of the templates was performed using probe-based qPCR reagents (such as AceQ U+Probe Master Mix, AceQ qPCR Probe Master Mix, etc.). (Since the qPCR instrument used was an Applied Biosystems 7500, the probe for the HEX reporter group used the JOE channel, which has a similar spectrum to that of this machine). Primer combinations that could identify negative *Oncomelania hupensis* (Ct value of *Oncomelania hupensis* primer <30, Ct value of *Schistosoma japonicum* primer >30), positive *Oncomelania hupensis* (Ct values ​​of both *Oncomelania hupensis* and *Schistosoma japonicum* primers <30), and *Schistosoma japonicum* (Ct value of *Oncomelania hupensis* primer >30, Ct value of *Schistosoma japonicum* primer <30) were screened (Table 6). Compared to other primer combinations, primers 4 and 8 provided the most stable detection results and were less prone to false positives. See the appendix for the detection performance. Figure 9-11 .

[0055] The multiplex qPCR reaction system for this embodiment is shown in Table 4, and the reaction procedure is shown in Table 5.

[0056] Table 4

[0057] reagents Reference volume (20 μL) <![CDATA[2*AceQ U + Probe Master Mix]]> 10 cDNA Template 1 upstream primers of Oncomelania hupensis in Hubei 0.4 downstream primers of Oncomelania hupensis in Hubei 0.4 Hubei Oncomelania primer probe 0.2 upstream primers for Schistosoma japonicum 0.4 Downstream primers for Schistosoma japonicum 0.4 Schistosoma japonicum primer probe 0.2 <![CDATA[ddH2O]]> 7

[0058] Table 5

[0059] Insulation stage Pre-deformation stage Cyclic Phase (45 cycles) 37℃ for 2 minutes 95℃ for 5 minutes 95℃ for 10 seconds 60℃ 34sec

[0060] Table 6

[0061]

[0062]

[0063] Example 3

[0064] 1. Identification of primer specificity

[0065] Genome extraction kits were used to extract genomes from snails and mollusks (such as *Oncomelania hupehensis*, *Pomacea canaliculata*, *Spatholobus suberectus*, *Clams maculatus*, *Clams stenoptera*, *Clams stenoptera*, *Clams stenoptera*, *Clams stenoptera*, and *Clams stenoptera*), as well as closely related snails (*Oncomelania hupehensis*) and trematodes closely related to *Schistosoma japonicum* (*Schistosoma hemlock* and *Schistosoma mansoni*). The specificity of primers 4 and 8 obtained in Example 2 was verified using a probe-based qPCR reagent (AceQ qPCRProbe Master Mix, Vazyme) (Roche Applied Science LightCycler 480). The reaction system was the same as in Example 2, and the reaction program was 95°C pre-denaturation for 5 min, 95°C for 10 sec, and 60°C for 30 sec (40 cycles). The results showed that this multiplex qPCR reaction system could specifically identify *Oncomelania hupehensis* and *Schistosoma japonicum* (Table 7, Appendix). Figure 12 and attached Figure 13 ).

[0066] Table 7

[0067]

[0068]

[0069] 2. Primer sensitivity determination

[0070] Synthesize plasmids containing target genes from *Oncomelania hupehensis* and *Schistosoma japonicum* (see appendix). Figure 14 and 15 The initial concentrations of the two were measured to be 122.5 ng / μL and 138.6 ng / μL, respectively. According to the relevant formula [6.02*10]... 23 *Plasmid concentration (ng / μL)*10 -9 After converting the units by [ / (plasmid base length * 660)], the calculated concentrations are 3.85 * 10⁻⁶. 10 Copies / μL and 4.35*10 10 Copies / μL. Perform serial dilutions of the plasmid at a ratio of 1:10 (10 μL). 9 -10 -2 (Scale), reaction conditions and instruments used were the same as above, and multiplex qPCR amplification was performed. Results are shown in the appendix. Figure 16 For the Hubei snail gene plasmid, the mean Ct value of the blank control was 36.18, and at a concentration of 10... 1When the concentration was on the order of 100 copies, one sample well showed an undetectable Ct value while the Ct values ​​of the remaining wells were all greater than 31, indicating that this method can detect *Oncomelania hupehensis* templates at a concentration of 100 copies. For *Schistosoma japonicum* plasmids, the mean Ct value of the blank control was 36.07, and at a concentration of 10... -1 When the sample Ct value is greater than 31 at a concentration of 1 copy, it indicates that this method can detect Schistosoma japonicum template at a concentration of 1 copy.

[0071] Currently, molecular detection techniques for *Oncomelania hupensis* in Hubei Province are mainly used for taxonomic research, with limited sensitivity detection methods available. Referring to relevant molecular detection techniques for *Schistosoma japonicum*, the detection limit of conventional PCR is 10 pg / μL (Establishment of a PCR method for detecting *Schistosoma japonicum*-infected *Oncomelania hupensis* by Chen Junhu et al.), the detection limit of recombinase-mediated isothermal amplification of nucleic acids is 10 copies (Rapid detection of *Schistosoma japonicum* gene fragments by Zhao Song et al. combining recombinase-mediated isothermal amplification of nucleic acids with fluorescent probes), and the detection limit of qPCR is 100 copies (Establishment and evaluation of a method for extracting exogenous *Schistosoma japonicum* small molecule DNA fragments from urine by Zhang Qiaoqiao et al.). Therefore, the method of this invention exhibits superior sensitivity compared to most molecular techniques, reaching the highest sensitivity level.

[0072] 3. Quantitative determination

[0073] Based on the above sensitivity detection data, a standard curve was constructed using the Ct values ​​corresponding to more than six different plasmid concentrations (see Appendix). Figure 17 and Figure 18 This enables quantitative detection of the concentration of the sample to be tested (Table 8).

[0074] Table 8

[0075]

[0076] In summary, (1) this invention utilizes multiplex qPCR technology to simultaneously identify Oncomelania hupehensis and Schistosoma japonicum at the gene level. Compared with traditional methods that rely on experience, this method can serve as an effective means of identifying these two species.

[0077] (2). Multiplex qPCR can detect a large number of samples at once. Compared with traditional snail identification methods that rely on a lot of manpower, this method not only provides more accurate results, but also saves time and effort, making it suitable for on-site snail inspection requirements.

[0078] (3) This invention uses an absolute quantitative method to evaluate the density of Oncomelania snails, which can serve as a supplement to traditional snail detection methods to some extent. In addition, since this technology can quantitatively evaluate the schistosomiasis load of infected (positive) Oncomelania hupensis snails, it can more accurately evaluate the snail infection status compared to traditional microscopic estimation techniques.

[0079] (4). As a gene diagnostic technology, this invention can be used to detect nucleic acid fragments in water and soil environmental samples in addition to tissue samples in the future, so as to directly evaluate the risk of schistosomiasis transmission in the relevant environment.

[0080] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0081] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for simultaneous quantitative detection of Schistosoma japonicum and its host Oncomelania hupensis for non-diagnostic and therapeutic purposes, characterized in that, The multiple PCR method is used for determination, and the primer sequences involved are as follows: Schistosoma japonicum forward primer: 5'-GCGTTCGAACCTATGCCAAA-3'; Schistosoma japonicum reverse primer: 5'-GAGCGGTAATCTAGAGTCACAGCTAA-3'; fluorescent probe: 5'-AGGACAACAAGCCTC-3'. The reaction system of the multiple PCR comprises 2* AceQ U+ Probe Master Mix 10 μL, cDNA Template 1 μL, Schistosoma japonicum upstream primer 0.4 μL, Schistosoma japonicum downstream primer 0.4 μL, Schistosoma japonicum primer probe 0.2 μL, Schistosoma japonicum upstream primer 0.4 μL, Schistosoma japonicum downstream primer 0.4 μL, Schistosoma japonicum primer probe 0.2 μL, and ddH2O 7 μL. The reaction program of the multiple PCR comprises 37℃ 2min; 95℃ 5min; 95℃ 10s, 60℃ 34s, 45 cycles. The nucleotide sequences of the primer combinations are shown in SEQ ID NO. 10-SEQ ID NO. 12 and SEQ ID NO. 22-SEQ ID NO.

24.

2. The method for simultaneous quantitative detection of Schistosoma japonicum and its host Oncomelania hupensis for non-diagnostic and therapeutic purposes according to claim 1, characterized in that, 5. Use of the primer combination in claim 4 in the preparation of a Schistosoma japonicum and its host Oncomelania hupensis detection product.

3. The method for simultaneous quantitative detection of Schistosoma japonicum and its host Oncomelania hupensis for non-diagnostic and therapeutic purposes according to claim 1, characterized in that, ​ 4. A primer combination for simultaneously and quantitatively detecting Schistosoma japonicum and its host Oncomelania hupensis, characterized in that, ​ ​

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