SNP (Single Nucleotide Polymorphism) molecular marker, primer pair, kit, method and application for identifying oncomelania hirsuta of Yunnan province

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker, a primer pair, a kit, a method and application for identifying oncomelania snails in Sichuan and Yunnan regions, and belongs to the technical field of molecular biology. The nucleotide sequence of the SNP molecular marker is as shown in SEQ ID NO.1, and the single nucleotide polymorphism of a basic group at the 75th site is 75A / G or 75T; and the single nucleotide polymorphism of the basic group at the 420th site is 420C / T or 420A. According to the invention, a method capable of distinguishing oncomelania snail populations in Sichuan and Yunnan is established for the first time, whether oncomelania snails are from Sichuan or Yunnan can be rapidly distinguished by utilizing the provided SNP molecular marker or through simple PCR amplification and electrophoresis detection, and scientific support is provided for frequently-communicated input (positive) oncomelania snails in two adjacent places.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and specifically relates to a SNP molecular marker, a primer pair, a kit, a method and uses for identifying Oncomelania hupensis in Sichuan and Yunnan. Background Art

[0002] Schistosomiasis japonicum is a zoonotic parasitic disease caused by infection with Schistosoma japonicum, which seriously endangers the health of the people and hinders the development of social economy. As the only intermediate host of Schistosoma japonicum, the distribution and spread of Oncomelania hupensis (Oncomelania hupensis) directly determine the prevalence and spread of schistosomiasis. In recent years, the large-scale construction of ecological protection and people-friendly projects such as wetland construction, landscaping, water diversion and water regulation, and the frequent occurrence of natural disasters such as mountain torrents, rainstorms, and earthquakes have caused the continuous occurrence of Oncomelania snail import and spread events, greatly increasing the risk of schistosomiasis transmission and the difficulty of prevention and control. Therefore, the accurate identification of the source of Oncomelania snails is of great significance for determining the occurrence and transmission risk of schistosomiasis and achieving precise prevention and control of schistosomiasis.

[0003] At present, the identification and differentiation of Oncomelania snails mainly adopt morphological observation. The difference in shell morphology can be used to simply distinguish the nominate subspecies (large individuals with ribbed shells) originating from lakes such as Hunan, Hubei, and Anhui, and the Yunnan-Sichuan subspecies (smaller individuals with smooth shells) distributed in Sichuan and Yunnan. However, the degree of morphological variation of Oncomelania snails in the neighboring Sichuan and Yunnan regions is extremely small, and it is impossible to identify them by morphological characteristics. In recent years, molecular biology techniques have been increasingly used in the study of population regional genetics of Oncomelania snails, but existing studies have mostly focused on the comparison of the nominate and Yunnan-Sichuan subspecies, and there are no reports on the differences in the genetic structure of Oncomelania snails in Sichuan and Yunnan. At present, there is no method for distinguishing Oncomelania snails from different regions based on molecular detection. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a SNP molecular marker, a primer pair, a kit, a method and uses for identifying Oncomelania snails in Sichuan and Yunnan. The provided SNP site combination or simple PCR amplification and electrophoresis detection can be used to quickly distinguish Oncomelania snails from Sichuan and Yunnan, providing scientific support for determining the source of imported (positive) Oncomelania snails between the two neighboring areas with frequent exchanges.

[0005] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0006] The purpose of the present invention is to provide a SNP molecular marker for identifying snails in Sichuan and Yunnan, the nucleotide sequence of which is shown in SEQ ID NO.1, the single nucleotide polymorphism of the base at the 75th position is 75A / G or 75T; the single nucleotide polymorphism of the base at the 420th position is 420C / T or 420A. The specific sequence is as follows:

[0007]

[0008] Another object of the present invention is to provide a method for identifying Oncomelania hupensis in Sichuan and Yunnan, by using primers to amplify the Oncomelania hupensis target gene cox1, and sequencing the amplified fragment. If the base at the 75th position is A or G, the Oncomelania hupensis individual is from Sichuan or Yunnan; if the base at the 75th position is T, the Oncomelania hupensis individual is from other regions outside Sichuan and Yunnan.

[0009] When the snail is identified as originating from Sichuan or Yunnan by the base at the 75th base, if the 420th base is C or T, the snail is originating from Sichuan; if the 420th base is A, the snail is originating from Yunnan.

[0010] Another object of the present invention is to provide a reagent for detecting the above-mentioned SNP molecular markers for use in identifying Oncomelania snails in Sichuan and Yunnan or in preparing products for tracing the origin of Oncomelania snails in Sichuan and Yunnan.

[0011] Furthermore, the reagent is a primer, a probe or a chip.

[0012] Another object of the present invention is to provide a primer pair developed based on the above-mentioned SNP molecular marker, the primer pair comprising primers for a first round of PCR reaction and a second round of PCR reaction; the primer sequence F0 / R0 for the first round of PCR reaction is shown in SEQ ID NOs. 2 and 3; the primer sequence F1 / R1 for the second round of PCR reaction is shown in SEQ ID NOs. 4 and 5, and the specific sequences are as follows:

[0013] F0:5'-TTGGTATATGGGCAGGACTT-3'; (SEQ ID NO.2)

[0014] R0:5'-ACAGGAAGAGATAGCAAGAGTA-3'; (SEQ ID NO.3)

[0015] F1:5'-TCACGCTGGTGGTTCTGTA-3', (SEQ ID NO.4)

[0016] R1:5'-CGGTCGGTTAGGAGTATTGT-3'. (SEQ ID NO.5)

[0017] Another object of the present invention is to provide the use of the above primer pair in identifying Oncomelania snails in Sichuan and Yunnan or in preparing products for tracing the origin of Oncomelania snails in Sichuan and Yunnan.

[0018] Another object of the present invention is to provide a kit for identifying Oncomelania hupensis in Sichuan and Yunnan, comprising the above-mentioned primer pair.

[0019] Furthermore, the kit also includes 2×Taq-PCR amplification reagents, amplification primers, positive controls and negative controls.

[0020] Furthermore, the 2×Taq-PCR amplification reagents include Taq DNA polymerase (1.25 U / 25 μL), dNTPs (including dGTP, dCTP, dATP, dTTP, each with a concentration of 400 μM), and a reaction buffer (including 20 mM Tris-HCl, 100 mM KCl, 4 mM MgCl 2 ).

[0021] Furthermore, the negative control for both rounds of reactions was the genomic DNA of Oncomelania hupensis from Sichuan, the positive control for the first round of reactions was the genomic DNA of the nominate subspecies of Oncomelania hupensis (other Oncomelania hupensis except those from Sichuan and Yunnan), and the positive control for the second round of reactions was the genomic DNA of Oncomelania hupensis from Yunnan.

[0022] Furthermore, the two rounds of reactions used the same amplification system, which was a 25 μL reaction system, including 12.5 μL of 2× Taq-PCR amplification reagent, 9.5 μL of nuclease-free water, 1 μL each of the upstream and downstream primers of a 10 μM primer pair, and 1 μL of sample template DNA.

[0023] Furthermore, the conditions for the first round of PCR amplification were: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 40 s, 30 cycles; and extended extension at 72°C for 5 min.

[0024] Furthermore, the conditions for the second round of PCR amplification were: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 20 s, 30 cycles; and extended extension at 72°C for 5 min.

[0025] Another object of the present invention is to provide a method for identifying Oncomelania snails in Sichuan and Yunnan, using the above primer pair or kit to amplify the Oncomelania snail cox1 gene, and determining the region of origin of the Oncomelania snail based on the amplification result.

[0026] Furthermore, the first round of amplified fragments were subjected to electrophoresis detection. If a specific band appeared at 538 bp, the snail was a snail other than Sichuan and Yunnan; if no band was found, the snail was from Sichuan or Yunnan, and then the next round of PCR detection was performed;

[0027] The second round amplified fragments were subjected to electrophoresis detection. If a specific band appeared at 228 bp, it was an Oncomelania hupensis from Yunnan; if no band was found, it was an Oncomelania hupensis from Sichuan.

[0028] Beneficial effects of the present invention:

[0029] The present invention has established for the first time a method for distinguishing snail populations in Sichuan and Yunnan. By using the provided SNP molecular markers or through simple PCR amplification and electrophoresis detection, it can be quickly distinguished whether the snail is from Sichuan or Yunnan, providing scientific support for determining the source of imported (positive) snails between the two neighboring areas with frequent exchanges. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the sequence comparison result of the single nucleotide polymorphism position at the 75th base provided by the present invention of the mitochondrial sequence of Oncomelania snails in different regions of the country; wherein: SC represents Sichuan, YN represents Yunnan, HB represents Hubei, AN represents Anhui, ZJ represents Zhejiang, HN represents Hunan, JS represents Jiangsu, FJ represents Fujian, GX represents Guangxi, GD represents Guangdong, and JX represents Jiangxi;

[0031] Figure 2 It is the sequence comparison result of the single nucleotide polymorphism position at the 420th base provided by the present invention of the mitochondrial sequences of Oncomelania snails in Sichuan and Yunnan; wherein: SC represents Sichuan, and YN represents Yunnan;

[0032] Figure 3 The first round of PCR product electrophoresis detection result diagram of the present invention; wherein: wells 1 and 2 are amplified products of Oncomelania hupensis from Sichuan; wells 3 and 4 are amplified products of Oncomelania hupensis from Yunnan; wells 6 and 11 are amplified products of Oncomelania hupensis from Hunan; wells 7, 12 and 18 are amplified products of Oncomelania hupensis from Anhui; wells 8 and 13 are amplified products of Oncomelania hupensis from Jiangsu; wells 9, 14 and 19 are amplified products of Oncomelania hupensis from Fujian; wells 10, 15 and 20 are amplified products of Oncomelania hupensis from Hubei; wells 5 and 16 are DNA molecular weight standards, each consisting of 6 bands, the band sizes of well 5 are 600, 500, 400, 300, 200 and 100 bp respectively, and the band sizes of well 16 are 1200, 900, 700, 500, 300 and 100 bp respectively; well 17 is a no-template control;

[0033] Figure 4 The figure is a graph of the electrophoresis detection results of the second round of PCR products performed in the present invention; wherein: wells 1-10 are amplified products from Oncomelania hupensis in Sichuan; wells 11-18 are amplified products from Oncomelania hupensis in Yunnan; well 19 is a no-template control; well 20 is a DNA molecular weight standard, consisting of 6 bands, with sizes of 600, 500, 400, 300, 200, and 100 bp, respectively. DETAILED DESCRIPTION

[0034] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0035] Example 1 Site screening and primer design for identifying Oncomelania snails in Sichuan and Yunnan

[0036] The reference sequences of the mitochondrial genes of Oncomelania hupensis published in GenBank from different epidemic areas across the country were retrieved and downloaded. At the same time, the mitochondrial genes of Oncomelania hupensis collected from Sichuan, Yunnan, Hubei, Hunan, Fujian, Jiangsu, Anhui and other regions were sequenced and analyzed. Through sequence comparison, multiple single nucleotide polymorphism sites with potential regional correlation were found for the first time. On this basis, two single nucleotide polymorphism sites from the cox1 gene were obtained through multiple rounds of screening, which can be used as effective genetic markers for identifying Oncomelania hupensis populations in Sichuan and Yunnan. The sequence of the SNP molecular marker is shown in SEQ ID NO.1. The single nucleotide polymorphism site is located at the 75th and 420th bases; if the base at the 75th base is A or G, the Oncomelania hupensis individual comes from Sichuan or Yunnan; if the base at the 75th base is T, the Oncomelania hupensis individual comes from other regions outside Sichuan and Yunnan. If the base at the 420th base is C or T, the Oncomelania hupensis individual comes from Sichuan; if the base at the 420th base is A, the Oncomelania hupensis individual comes from Yunnan.

[0037] According to the SNP molecular marker, the following specific primer pair was designed and synthesized, and the specific sequence of the primer pair is:

[0038] F0:5'-TTGGTATATGGGCAGGACTT-3'; (SEQ ID NO.2)

[0039] R0:5'-ACAGGAAGAGATAGCAAGAGTA-3'; (SEQ ID NO.3)

[0040] F1:5'-TCACGCTGGTGGTTCTGTA-3', (SEQ ID NO.4)

[0041] R1:5'-CGGTCGGTTAGGAGTATTGT-3'. (SEQ ID NO.5)

[0042] Example 2 Identification of Oncomelania snails in Sichuan and Yunnan

[0043] 1. Sample collection

[0044] Oncomelania snail samples were collected from Dayi, Pidu, Pujiang, Dujiangyan, Xinjin, Eastern New Area, Tianfu New Area, Mianzhu, Zhongjiang, Luojiang, Xichang, Dechang, Puge, Xide, Huili, Mianning, Zhaojue, Yanbian, Renshou, Dongpo, Pengshan, Lushan, Tianquan, Yucheng, Jiajiang, Shawan, Wutongqiao, Jiangyou, Lezhi in Sichuan Province, Chuxiong, Yongsheng, Dali, Eryuan, Heqing, Xiangyun in Yunnan Province, Changle, Fuqing in Fujian Province, Gong'an, Songzi, Zhongxiang in Hubei Province, Guichi in Anhui Province, Yueyang in Hunan Province and Jiangsu Province, placed in 75% ethanol and stored at room temperature for later use.

[0045] 2. DNA Extraction

[0046] The DNeasy Blood & Tissue Kit produced by QIAGEN (Germany) was used to extract genomic DNA from Oncomelania snail tissue. The specific method is as follows:

[0047] After crushing and shelling a single snail, put its soft tissue into a 1.5mL centrifuge tube, grind it, add 180μL of ATL buffer and 20μL of proteinase K provided in the kit, shake and mix, and incubate in a 56℃ water bath until the tissue is completely lysed. After taking the centrifuge tube out of the water bath, shake and mix for 15s, add 200μL of buffer AL, and shake and mix. Then add 200μL of anhydrous ethanol and shake and mix. Transfer all the liquid in the tube into a DNeasy filter column, centrifuge at ≥8000rpm for 1min, and discard the filtrate. Add 500μL of buffer AW1 to the filter column, centrifuge at ≥8000rpm for 1min, and discard the filtrate. Add 500μL of buffer AW2 to the extraction column, centrifuge at 14000rpm for 3min, and discard the filtrate. Move the filter column into a new centrifuge tube, add 200 μL buffer AE, let stand at room temperature for 1 min, centrifuge at ≥8000 rpm for 1 min, and the filtrate obtained is the Oncomelania snail genomic DNA, which should be stored at -20°C for future use.

[0048] 3. Single Nucleotide Polymorphism Analysis

[0049] The snail cox1 gene was used as the detection target for sequencing analysis. After proofreading and splicing, the sequences were aligned using MEGA software to display polymorphic sites. The results are shown in Figure 1 and Figure 2 .

[0050] like Figure 1 and Figure 2 As shown in the figure, there are obvious differences between the snail samples from Sichuan and Yunnan and those from other regions, as well as between the snail samples from Sichuan and Yunnan. Specifically, at the 75th position, the base of the snails from Sichuan or Yunnan is A or G, while that from other regions is T ( Figure 1 ), at the 420th position, the base of Sichuan snail is C / T, and that of Yunnan is A ( Figure 2 ).

[0051] 4. PCR Identification

[0052] Both rounds of PCR reactions used a 25 μL PCR reaction system, adding 12.5 μL of 2×Taq PCR amplification reagent, 1 μL of 10 μM upstream and downstream primers, 9.5 μL of nuclease-free water, and 1 μL of sample template DNA. At the same time, negative and positive controls were set, and nuclease-free water was used as a template and a blank control, and the system was prepared according to the above method.

[0053] Place the prepared PCR reaction tube into the PCR instrument and set the reaction program according to the following conditions:

[0054] The first round of reaction: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 40 s, 30 cycles; extended extension at 72°C for 5 min.

[0055] The second round of reaction: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 20 s, 30 cycles; extended extension at 72°C for 5 min.

[0056] Take 10 μL of PCR amplification product and run it in 2% (w / v) agarose gel at 3-5 V / cm constant voltage electrophoresis for 45 min. Observe the results in gel imaging analysis system, with DNA molecular weight standard as reference. Figure 3-4 As shown in the figure, the snails with a single band at the target product size (538 bp) in the first round of PCR amplification were from Hubei, Hunan, Anhui, Fujian and Jiangsu, and the snails with negative amplification results were from Sichuan and Yunnan. Figure 3 ); The snails with a single band at the target product size (228 bp) in the second round of PCR amplification were all from Yunnan, and the snails with negative amplification results were all collected from Sichuan ( Figure 4 ).

[0057] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A SNP molecular marker for identifying Oncomelania hupensis in Sichuan and Yunnan, characterized in that: The nucleotide sequence is shown in SEQ ID NO. 1, and the single nucleotide polymorphism of the base at the 75th position is 75A / G or 75T; the single nucleotide polymorphism of the base at the 420th position is 420C / T or 420A.

2. Use of a reagent for detecting the SNP molecular marker described in claim 1 in identifying Oncomelania snails in Sichuan and Yunnan or in preparing products for tracing the origin of Oncomelania snails in Sichuan and Yunnan.

3. The use according to claim 2, characterized in that: The reagents are primers, probes or chips.

4. A primer pair for identifying Oncomelania hupensis from Sichuan and Yunnan, characterized in that: The primer pair includes primers for the first round of PCR reaction and the second round of PCR reaction; the primer sequences for the first round of PCR reaction are shown in SEQ ID NOs. 2 and 3; the primer sequences for the second round of PCR reaction are shown in SEQ ID NOs. 4 and 5.

5. Use of the primer pair according to claim 4 in identifying Oncomelania snails in Sichuan and Yunnan or in preparing products for tracing the origin of Oncomelania snails in Sichuan and Yunnan.

6. A kit for identifying Oncomelania hupensis from Sichuan and Yunnan, characterized in that: Comprising the primer pair described in claim 4.

7. The kit according to claim 6, characterized in that The kit also includes 2×TaqPCR amplification reagents, amplification primers, positive controls and negative controls.

8. A method for identifying snails in Sichuan and Yunnan, characterized in that: The primer pair described in claim 4 or the kit described in claim 6 is used to amplify the snail cox1 gene. The amplification is divided into two rounds. The first round of amplification results are used to determine whether the snail is from Yunnan and Sichuan, and the second round of amplification results are used to identify whether the snail belongs to Yunnan or Sichuan.

9. The method according to claim 8, characterized in that Perform electrophoresis on the first round of amplified fragments. If a specific band appears at 538 bp, the snail is from a species other than Sichuan or Yunnan. If no band is found, the snail is from Sichuan or Yunnan, and then perform the next round of PCR testing. The second round of amplified fragments were tested by electrophoresis. If a specific band appeared at 228 bp, it was a snail from Yunnan. If there is no band, it is a snail from Sichuan.

Images