Molecular markers for discriminating schistosoma japonicum and paragonimus westermani and application thereof

Abstract

The application provides a molecular marker which can be used for distinguishing and identifying Schistosoma japonicum and Trichobilharzia orientalis, and constructs a qPCR detection method (SYBR Green dye method and TaqMan probe method) which has high sensitivity, strong specificity and can distinguish and identify Schistosoma japonicum and Trichobilharzia orientalis, and can accurately and quickly distinguish Schistosoma japonicum and Trichobilharzia orientalis.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology, specifically to molecular markers for identifying Schistosoma japonicum and Paragonimus westermani, and their applications. More specifically, it relates to the establishment and application of a Real-time PCR detection method based on Paragonimus westermani specific genes that can distinguish between Schistosoma japonicum and Paragonimus westermani. Background Technology

[0002] Orientobilharzia turkestanicum (Ot) belongs to the family Orientobilharzia and the genus Orientobilharzia. Orientobilharzia disease is caused by the parasite Orientobilharzia turkestanicum, which infects the portal and mesenteric venous systems of mammals. Adult worms, eggs, cercariae, and larvae can all cause a series of damages to the host, leading to mass mortality in cattle and sheep, seriously threatening the healthy development of animal husbandry. The most common species is Orientobilharzia turkestanicum. This disease is reportedly widespread in my country, often endemic. Host animals include sheep, goats, cattle, buffalo, camels, equines, and some wild mammals, primarily affecting cattle and sheep. Furthermore, Orientobilharzia cercariae can cause cercarial dermatitis in humans, posing a serious threat to the health of people living around endemic areas, making it a serious zoonotic parasitic disease.

[0003] Schistosoma japonicum (Sj) belongs to the class Trematoda, subclass Digenea, order Digenea, family Schistosomatidae, and genus Schistosoma. The definitive hosts of Schistosoma japonicum include more than 40 species of mammals, such as humans, buffalo, cattle, goats, sheep, pigs, horses, mules, and dogs. Among these, infected livestock such as buffalo, cattle, goats, and sheep are the main sources of infection. Currently, approximately 200 million people in 74 countries and regions worldwide are infected with schistosomiasis, of whom 120 million exhibit clinical symptoms, 20 million are severely ill, and 600 million are at risk of infection. Schistosomiasis remains a serious public health issue and is listed as one of the six major tropical diseases requiring priority research, detection, and control by the World Bank, the World Health Organization (WHO), and the United Nations Special Programme for Research and Development in Tropical Diseases (TDR).

[0004] Schistosomiasis japonicus is prevalent in provinces along the Yangtze River in my country, including Anhui, Jiangsu, Hunan, Hubei, Jiangxi, Sichuan, and Yunnan. Fluke disease is prevalent in 20 provinces, municipalities, and autonomous regions in my country, including Gansu, Inner Mongolia, Sichuan, Hunan, Xinjiang, Jiangsu, Shaanxi, Guizhou, Qinghai, Ningxia, Liaoning, Jilin, Heilongjiang, Guangxi, Beijing, Shanxi, Hubei, Fujian, and Yunnan. The provinces where fluke disease and schistosomiasis japonicus overlap are Anhui, Jiangsu, Hunan, Hubei, Sichuan, and Yunnan.

[0005] With the widespread use of drug treatment in countries endemic to schistosomiasis, the infection rate and intensity of schistosomiasis japonicus in humans and livestock have significantly decreased, and the elimination of this disease has been placed on the agenda of the Healthy China 2030 national strategic plan. Currently, commonly used diagnostic methods for schistosomiasis japonicus include etiological diagnosis and immunological diagnosis, with etiological diagnosis being the gold standard. However, research on the diagnosis of schistosomiasis japonicus is relatively limited, and current research mainly focuses on immunological diagnosis (Ji et al., 2020).

[0006] Nucleic acid diagnostics are characterized by high sensitivity and specificity, and have the potential for early diagnosis, differential diagnosis, and efficacy evaluation of schistosomiasis. Currently, nucleic acid diagnostic methods are widely used in disease diagnosis, tumor diagnosis, etc., and their sensitivity and specificity have been recognized. However, due to the lack of genomic data for *Schistosoma japonicum*, molecular biological diagnostic methods for *Schistosoma japonicum* have been almost entirely unreported. In recent years, no cross-reactivity experiments have been conducted between Schistosomiasis japonicus and other schistosomiasis-related diseases in molecular biological diagnostic methods. For example, in the establishment and preliminary evaluation of the recombinase polymerase amplification (RPA) method for detecting Schistosomiasis japonicus nucleic acid, cross-reactivity experiments were conducted between Schistosomiasis japonicus and Schistosoma mansoni, Schistosoma haematobium, Clonorchis sinensis, Corynebacterium fasciatus, Paragonimus westermani, and Cynodon dactylum (Wang et al., 2020). In the research on the establishment and preliminary application of nucleic acid detection methods for Schistosomiasis japonicus, only cross-reactivity experiments were conducted between Schistosomiasis japonicus and other schistosomiasis-related diseases such as Pallasia flavomarginata, Corynebacterium fasciatus, Toxoplasma gondii, Trichinella spiralis, Spirocera, Sarcocystis, Cryptosporidium, and Haemaphysema contortus (Guo et al., 2021).

[0007] Our research revealed that both *Schistosoma japonicum* and *Clonorchis sinensis* hatch into miracidia from their eggs, and the size and movement trajectory of these miracidia are remarkably similar. Traditional etiological diagnosis (miracidia hatching method, MH) struggles to distinguish between the two species. Furthermore, current molecular biological diagnostic research lacks effective evidence to differentiate between *Schistosoma japonicum* and *Clonorchis sinensis*, and our experiments showed a degree of cross-reactivity between the two species in molecular biological diagnosis. This could easily lead to misdiagnosis of schistosomiasis, thereby impacting national plans to further control and eliminate schistosomiasis. Therefore, there is an urgent need to develop more sensitive and accurate detection methods for the precise diagnosis and differentiation of these two diseases. Summary of the Invention

[0008] This invention provides molecular markers that can be used to distinguish and identify Schistosoma japonicum and Paragonimus westermani, and constructs a highly sensitive and specific qPCR detection method (SYBR Green dye method and TaqMan probe method) that can distinguish and identify Schistosoma japonicum and Paragonimus westermani, enabling accurate and rapid differentiation between the two species.

[0009] First, this invention provides a molecular marker that can accurately and rapidly distinguish between Schistosoma japonicum and Paragonimus westermani, wherein the molecular marker is:

[0010] Ot-F:5'-CTCAGATAACCGCCTATTCTTA-3'

[0011] Ot-R:5'-CAGTCTTCTATCGAACCTCTC-3'

[0012] This invention provides a qPCR (SYBR Green dye method) detection method for differentiating *Schistosoma japonicum* from *O. japonicum*. The method utilizes a set of highly sensitive and specific primers for *O. japonicum* detection, with the primer nucleotide sequence being Ot-F: 5'-CTCAGATAACCGCCTATTCTTA-3'.

[0013] Ot-R:5'-CAGTCTTCTATCGAACCTCTC-3'

[0014] The amplified fragment is 130bp.

[0015] The reaction system and amplification conditions for real-time quantitative PCR (SYBR Green dye method) used in this invention are as follows:

[0016] qPCR (SYBR Green method) reaction system: 10 μl SYBR Master Mix, 0.4 μl each of forward and reverse primers, 1 μl template, and 8.2 μl deionized water to bring the total volume to 20 μl. Dilute the primers with double-distilled water to a concentration of 10 pmol / μL; the recommended template concentration range is 5 pg / μL-0.5 ng / μL.

[0017] qPCR (SYBR Green method) amplification conditions: 95℃ for 30s pre-denaturation, 95℃ for 15s, 60℃ for 30s, 72℃ for 15s, 35 cycles. Validated using experimental samples, this method can be used to detect *Schistosoma japonicum* in differentiation from *Schistosoma japonicum*.

[0018] To analyze qPCR (SYBR Green method) amplification products, any of the following methods can be selected:

[0019] 1. The qPCR (SYBR Green method) products were analyzed by agarose gel electrophoresis.

[0020] 2. The amplification curve of qPCR (SYBR Green method) is combined with the melting curve for judgment.

[0021] The above detection method uses a nucleic acid extraction kit to extract the genomic DNA of the insect from the sample to be tested.

[0022] The above detection method indicates a positive result if the amplification curve of the product forms a characteristic peak when the CT value is less than 33 and the melting curve of the product forms a characteristic peak at 84℃; if the amplification curve forms a characteristic peak when the CT value is between 33 and 34 and the melting curve forms a characteristic peak at 84℃, the sample is suspected; if the amplification curve forms a characteristic peak when the CT value is greater than 34 and the melting curve forms a characteristic peak at 84℃, the sample is negative.

[0023] The present invention can also be amplified using a conventional PCR instrument, and the products can be analyzed and judged by agarose gel electrophoresis.

[0024] Secondly, this invention provides a molecular marker that can accurately and rapidly identify Schistosoma japonicum and Paragonimus westermani, wherein the molecular marker is:

[0025] Sj-F1:5'-TCAGACAATCGTTTATTCTTAGC-3'

[0026] Sj-R1:5'-TGATGGCAATATGGTCTATTTG-3'

[0027] Sj-P1:5'-CATAAGGAGAGACACCGCCTAACG-3'

[0028] Ot-F2:5'-CTCAGATAACCGCCTATTCTTA-3'

[0029] Ot-R2:5'-CAGTCTTCTATCGAACCTCTC-3'

[0030] Ot-P2:5'-CGAACACCAACTTTAGGCATAGGGAA-3'

[0031] Furthermore, this invention provides a nucleic acid detection method for identifying Schistosoma japonicum and Clonorchis sinensis. The method uses two sets of highly sensitive and specific primers and probes for Clonorchis sinensis detection, the nucleotide sequences of which are as follows:

[0032] Sj-F1:5'-TCAGACAATCGTTTATTCTTAGC-3'

[0033] Sj-R1:5'-TGATGGCAATATGGTCTATTTG-3'

[0034] Sj-P1:5'-CATAAGGAGAGACACCGCCTAACG-3'

[0035] Ot-F2:5'-CTCAGATAACCGCCTATTCTTA-3'

[0036] Ot-R2:5'-CAGTCTTCTATCGAACCTCTC-3'

[0037] Ot-P2:5'-CGAACACCAACTTTAGGCATAGGGAA-3'

[0038] Another objective of this application is to provide qPCR (TaqMan probe method) reaction systems and amplification conditions for identification detection, allowing users to select the appropriate reaction system and amplification conditions according to their actual needs.

[0039] The reaction system is as follows: 10 μl of TaqMan Master Mix, 0.6 μl each of forward and reverse primers F1, R1, F2, and R2, 0.6 μl of probe P1, 0.4 μl of probe P2, 1 μl of template, and 5.2 μl of deionized water to bring the total volume to 20 μl. Dilute the primers with double-distilled water to a concentration of 10 pmol / μL; the recommended template concentration range is 5 pg / μL–0.5 ng / μL.

[0040] The qPCR (TaqMan probe method) amplification conditions are as follows: 95℃ for 30s pre-denaturation, 95℃ for 15s, 60℃ for 30s, 72℃ for 15s, for 40 cycles. Experimental samples have verified that this method can be used for the differential detection of Schistosoma japonicum and Clonorchis sinensis.

[0041] To analyze qPCR (TaqMan probe method) amplification products, the amplification curve of Real-time PCR can be used for judgment.

[0042] The above detection method uses a nucleic acid extraction kit to extract DNA from the sample to be tested.

[0043] The above detection method indicates that the amplification curve of the product shows specific amplification when the CT value is less than 35, which is a positive result; when the amplification curve shows specific amplification between 35 and 37, the sample is suspected; and when the amplification curve shows specific amplification at a CT value greater than 37, the sample is negative.

[0044] This application uses a molecular marker and detection method to distinguish between Schistosoma japonicum and Paragonimus westermani; it can be used to monitor and differentiate whether water bodies contain miracidia and cercariae of Schistosoma japonicum and Paragonimus westermani, and can also be used to identify the species of Schistosoma japonicum and Paragonimus westermani. Attached Figure Description

[0045] Figure 1 Comparison of target gene sequences of Schistosoma japonicum and Paragonimus westermani;

[0046] Figure 2. qPCR (SYBR Green assay) results: Figure 2-1 Amplification curve; Figure 2-2 Melting curve;

[0047] Figure 3 Agarose gel electrophoresis results: 1, 2: genome of *Schistosoma japonicum*; 3: blank control; 4, 5: genome of *Schistosoma japonicum*.

[0048] Figure 4 Recommended template concentration test results: The initial concentration is 0.5 ng / μL, and 10-fold dilutions are performed in 5 gradients (0.5 ng / μL, 50 pg / μL, 5 pg / μL, 500 fg / μL, 50 fg / μL, 5 fg / μL).

[0049] Figure 5: qPCR (TaqMan probe method) results: Figure 5-1 Detection of amplification curves of *Clerodendrum thoracis*; Figure 5-2 Detection of Schistosoma japonicum amplification curve;

[0050] Figure 6: Results of specificity test: Figure 6-1 : Detection of specific amplification curves for *Cladosporium fasciatus*; Figure 6-2 : Detection of specific amplification curves for Schistosoma japonicum;

[0051] Figure 7: Sensitivity test results: Figure 7-1 Sensitivity for detecting East Asian trematode; Figure 7-2 Sensitivity for detecting Schistosoma japonicum;

[0052] Figure 8: Standard curve results: Figure 8-1 Standard curve of *Strombus haematomarginatus*; Figure 8-2 Standard curve of Schistosoma japonicum. Detailed Implementation

[0053] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0054] Given the limited current research on *Schistosoma japonicum*, the difficulty in obtaining *Schistosoma japonicum* cells, and the lack of *Schistosoma japonicum* genomic data, we designed degenerate primers based on high-copy sequences (SjCHGCS15, SjCHGCS16, SjCHGCS17, SjCHGCS18, SjCHGCS19, SjCHGCS20, SjCHGCS21, etc.) reported in the *Schistosoma japonicum* genome. Using the *Schistosoma japonicum* genome as a template, we amplified these sequences, sequenced and aligned them, and then screened for candidate target sequences. Through alignment and screening, we found that the *SjCHGCS20* sequence of *Schistosoma japonicum* differs from the corresponding sequence in the *Schistosoma japonicum* genome, making it suitable as a molecular target for differentiating the two parasites. Therefore, we used this sequence as a molecular marker for diagnostic research. Target sequence sites are as follows: Figure 1 As shown.

[0055] Example 1:

[0056] (1) Preparation of Schistosoma japonicum and Epistylis esculenta strains and genomic nucleic acid samples: The Schistosoma japonicum and Epistylis esculenta strains used in the experiment were preserved in our laboratory. The parasites were ground for 2 min, then centrifuged at 12000 rpm for 2 min, and the supernatant was discarded. Then, the genomic DNA of the parasites was extracted using a nucleic acid extraction kit, and the concentration and purity were determined, which was used as the detection template for the established method.

[0057] (2) Primer design: Primers were designed using software based on the target sequences screened in the laboratory in the early stage. Primer: Ot-F:

[0058] 5'-CTCAGATAACCGCCTATTCTTA-3', Ot-R:5'-CAGTCTTCTATCGAACCTCTC-3', the amplified fragment size is 130bp.

[0059] (3) Reaction system:

[0060]

[0061] Amplification conditions: 95℃ for 30s pre-denaturation, 95℃ for 15s, 60℃ for 30s, 72℃ for 15s, 35 cycles.

[0062] (4) Result determination:

[0063] Melting curve method detection: The qPCR (SYBR Green method) amplification curve formed a characteristic peak at 84℃ when the CT value was less than 33 (Figure 2), and the sample was East Asian fluke.

[0064] Agarose gel electrophoresis detection: The amplified product size was 130 bp. Figure 3 If the sample is *Echinochloa crus-galli*, then the target fragment has been amplified.

[0065] (5) Recommended template concentration: The initial concentration of the insect genome at 0.5 ng / μL was serially diluted 10-fold, for a total of 5 dilutions, and then detected by qPCR (SYBR Green method).

[0066] The test results showed that the optimal template concentration range was 5 pg / μL-0.5 ng / μL. Figure 4 ).

[0067] The above studies show that this method has good sensitivity and specificity and can distinguish between schistosomiasis japonicus and trematode fluke.

[0068] Example 2:

[0069] (1) Preparation of Schistosoma japonicum and Epistylis esculenta strains and genomic nucleic acid samples: The Schistosoma japonicum and Epistylis esculenta strains used in the experiment were preserved in our laboratory. The parasites were ground for 2 min, then centrifuged at 12000 rpm for 2 min, and the supernatant was discarded. Then, the genomic DNA of the parasites was extracted using a nucleic acid extraction kit, and the concentration and purity were determined, which was used as the detection template for the established method.

[0070] (2) Primer and probe design: Based on previous nucleic acid diagnostic research in the laboratory, a gene with diagnostic potential was identified. Primers were designed using Beacon Designer software based on this gene. Primers Sj-F1: 5'-CTCAGATAACCGCCTATTCTTA-3', Sj-R1: ​​5'-CAGTCTTCTATCGAACCTCTC-3', probe Sj-P1: 5'-VIC-CATAAGGAGAGACACCGCCTAACG-BHQ1-3', the amplified fragment is as shown in SEQ ID NO.1, 118bp; primers Ot-F2: 5'-CTCAGATAACCGCCTATTCTTA-3', Ot-R2: 5'-CAGTCTTCTATCGAACCTCTC-3', probe...

[0071] Ot-P2:5'-FAM-CGAACACCAACTTTAGGCATAGGGAA-BHQ1-3', the amplified fragment is as shown in SEQ ID NO.4, and the size is 130bp.

[0072] (3) Reaction system:

[0073]

[0074] Amplification conditions: 95℃ for 30s pre-denaturation, 95℃ for 15s, 60℃ for 30s, 72℃ for 15s, 40 cycles.

[0075] (4) Result determination:

[0076] Amplification curve method detection: a) FAM channel result interpretation: The amplification curve of the product shows specific amplification when the CT value is less than 35, indicating a positive result; when the amplification curve shows specific amplification between 35 and 37, the sample is suspected; when the amplification curve shows specific amplification at a CT value greater than 37, the sample is negative. b) VIC channel result interpretation: The amplification curve of the product shows specific amplification when the CT value is less than 35, indicating a positive result; when the amplification curve shows specific amplification between 35 and 37, the sample is suspected; when the amplification curve shows specific amplification at a CT value greater than 37, the sample is negative (Figure 5).

[0077] (5) Cross-reactivity test: The genomes of Toxoplasma gondii, Fasciola heliotropium, Paleopeliae fasciculata, Sarcocystis jirovecii, Trichinella spiralis, and Echinococcus granulosus were all provided by the Key Laboratory of Animal Parasitology of the Ministry of Agriculture of our institute. Using the genomic DNA of these six parasites as templates, qPCR (TaqMan probe method) was performed under the above reaction system and conditions, and the amplification results were observed.

[0078] Results: No amplification curves were observed after qPCR (TaqMan probe method) amplification of the genomic DNA of the above 6 parasites, proving that there was no cross-reaction with the above 6 parasites under the reaction system and conditions (Figure 6).

[0079] (6) Sensitivity test: plasmids were constructed based on the amplified target sequences of Schistosoma japonicum and Schistosoma esculentum. The constructed recombinant plasmids were serially diluted in 9 gradients, with an initial concentration of 1 ng / μL diluted to 1 ag / μL. The plasmids of different concentrations were used as templates for qPCR (TaqMan probe method) reaction to detect the detection limit of the method.

[0080] Results: Under the above qPCR (TaqMan probe method) reaction system and conditions, the detection limit for plasmids containing the target sequence of Schistosoma japonicum was 0.1 fg, and the detection limit for plasmids containing the target sequence of Plasmodium styracifolium was also 0.1 fg (Figure 7).

[0081] (7) Standard curve preparation: Real-time PCR standard curves were obtained by using different concentration dilutions of the constructed plasmid as templates. The reaction system and conditions were the same as those for the sensitivity test.

[0082] Results; Correlation analysis revealed that the correlation parameter R of the FAM channel standard curve for detecting *Cladosporium fasciatus* was... 2 =1.00 and amplification efficiency E=1.89 are good; the correlation parameter R of the standard curve for detecting the VIC channel of Schistosoma japonicum is good. 2 =1.00 and amplification efficiency E=1.91 are better (Figure 8).

[0083] The embodiments of the invention have been disclosed above. Although the embodiments have been optimized, they are not intended to limit the invention. Anyone skilled in the art can make modifications and alterations without departing from the spirit and scope of the invention. Therefore, the protection of the invention should be determined by the claims.

Claims

1. A molecular marker for distinguishing between Schistosoma japonicum and Coriolis tangutica, the sequence of which is shown in SEQ ID NO.1 and SEQ ID NO.

4.

2. A composition for identifying the molecular markers used in claim 1 to distinguish between Schistosoma japonicum and Coriolis tangutica, characterized in that... The composition comprises two sets of primers and corresponding probes, the nucleotide sequences of which are as follows: Sj-F1:5'-TCAGACAATCGTTTATTCTTAGC-3' Sj-R1: ​​5'- TGATGGCAATATGGTCTATTTG-3' Sj-P1:5'- CATAAGGAGAGACACCGCCTAACG-3' Ot-F2:5'- CTCAGATAACCGCCTATTCTTA-3' Ot-R2:5'- CAGTCTTCTATCGAACCTCTC-3' Ot-P2:5'-CGAACACCAACTTTAGGCATAGGGAA-3'.

3. The application of the composition according to claim 2 in the nucleic acid detection for non-diagnostic purposes to differentiate Schistosoma japonicum and Paragonimus westermani, wherein the application is qPCR TaqMan probe method: The reaction system is as follows: 10 μl of TaqMan Master Mix, 0.6 μl each of forward and reverse primers F1, R1, F2, and R2, 0.6 μl of probe P1, 0.4 μl of probe P2, 1 μl of template, and 5.6 μl of deionized water to bring the total system volume to 20 μl; the primers were diluted with double-distilled water to a concentration of 10 pmol / μL. The qPCR reaction conditions are as follows: Step 1: 95℃ for 30 seconds; Step 2: 95℃ for 15s, 60℃ for 30s, 72℃ for 15s, 40 cycles.

4. The application according to claim 3, characterized in that, The specific application involves labeling probes with fluorescent groups: probe Sj-P1: 5'-VIC-CATAAGGAGAGACACCGCCTAACG-BHQ1-3' and probe Ot-P2: 5'-FAM-CGAACACCAACTTTAGGCATAGGGAA-BHQ1-3'. Using two pairs of specific primers and probes, DNA extracted from the sample is amplified by PCR. The PCR amplification products are analyzed by observing amplification curves under different fluorescence signals. If specific amplification is observed under VIC fluorescence signal and the CT value of the amplification curve is less than 35, it is considered that a fragment of *Schistosoma japonicum* has been amplified, and the sample is identified as positive for *Schistosoma japonicum*. If specific amplification is observed under FAM fluorescence signal and the CT value of the amplification curve is less than 35, it is considered that a fragment of *Clonorchis sinensis* has been amplified, and the sample is identified as positive for *Clonorchis sinensis*.

Images