Detection kit for identifying oncomelania hirsuta of Yunnan province

Abstract

The invention discloses a detection kit for identifying oncomelania hirsuta of Yunnan province and belongs to the technical field of molecular biology. The nucleotide sequence of the SNP molecular marker is shown as 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 application belongs to the technical field of molecular biology, and particularly relates to a detection kit for identifying Oncomelania hupensis from Sichuan and Yunnan. BACKGROUND

[0002] Japanese blood fluke disease is a parasitic disease of man and animals caused by infection of Japanese blood fluke, which seriously endangers people's health and hinders social and economic development. Oncomelania hupensis (hereinafter referred to as oncomelania) as the only intermediate host of Japanese blood fluke, its distribution and spread directly determines the prevalence and transmission of blood fluke disease. In recent years, a large number of ecological protection and people-benefiting engineering constructions such as wetland construction, landscaping, water diversion, etc. and frequent natural disasters such as mountain torrents, rainstorms, earthquakes, etc. have caused oncomelania input and diffusion events to occur continuously, greatly increasing the risk of blood fluke disease transmission and the difficulty of prevention and control. Therefore, accurate identification of oncomelania source is of great significance for determining the risk of blood fluke disease occurrence and transmission and realizing precise prevention and control of blood fluke disease.

[0003] At present, oncomelania identification and differentiation mainly adopts morphological observation, and oncomelania from Hunan, Hubei, Anhui and other lake areas (individual large, shell surface with ribs) can be simply distinguished from Sichuan and Yunnan (individual small, shell surface smooth) through shell morphology difference, while oncomelania from Sichuan and Yunnan has extremely small morphological variation and cannot be identified through morphological characteristics. In recent years, molecular biology technology has been increasingly applied to oncomelania population geographical genetic research, but existing researches are mostly focused on comparison of the nominal and Sichuan-Yunnan subspecies, and there is no related report on genetic structure difference of oncomelania from Sichuan and Yunnan, and currently there is no oncomelania identification method based on molecular detection from different regions. SUMMARY

[0004] In view of the problem that oncomelania identification and differentiation currently adopts morphological observation and is distinguished through shell morphology difference, while oncomelania from Sichuan and Yunnan has extremely small morphological variation and cannot be identified through morphological characteristics, the application provides a detection kit for identifying oncomelania from Sichuan and Yunnan, which identifies oncomelania from Sichuan and Yunnan through specific primers.

[0005] In one aspect, the application provides a detection kit for identifying oncomelania from Sichuan and Yunnan, which comprises two-round PCR amplification primers. The forward primer sequence of the first-round PCR reaction is shown in SEQ ID NO. 2: F0: 5'-TTGGTATATGGGCAGGACTT-3' (SEQ ID NO. 2); The reverse primer sequence of the first-round PCR reaction is shown in SEQ ID NO. 3: R0: 5'-ACAGGAAGAGATAGCAAGAGTA-3' (SEQ ID NO. 3); The sequence of the forward primer of the second round of PCR reaction is shown in SEQ ID NO. 4: F1: 5'-TCACGCTGGTGGTTCTGTA-3' (SEQ ID NO. 4); The sequence of the reverse primer of the second round of PCR reaction is shown in SEQ ID NO. 5: R1: 5'-CGGTCGGTTAGGAGTATTGT-3' (SEQ ID NO. 5). Preferably, the kit further comprises 2x Taq PCR amplification reagent, positive control and negative control, wherein the 2x Taq PCR amplification reagent comprises: Taq DNA polymerase (1.25 U / 25 μL), dNTPs (dGTP, dCTP, dATP, dTTP, each at a concentration of 400 μM), and reaction buffer (20 mM Tris-HCl, 100 mM KCl, 4 mM MgCl2).

[0006] Another aspect of the present application provides a detection method for identifying Oncomelania hunchunensis and Oncomelania hengdongensis, which comprises the following steps: (1) Sample lysis: crush and peel the Oncomelania, and then put the soft tissues into a 1.5 mL centrifuge tube, grind and add ATL buffer and proteinase K, mix by oscillation, incubate at 56°C for lysis, and centrifuge to obtain the supernatant as the sample lysis solution; (2) Sample extraction: purify and elute the sample lysis solution by column method to obtain the Oncomelania genomic DNA; (3) PCR amplification: prepare the PCR system for the Oncomelania genomic DNA, set the reaction program for amplification, and analyze the electrophoresis result to identify Oncomelania hunchunensis and Oncomelania hengdongensis.

[0007] Preferably, the reaction program in step (3) is as follows: First round of reaction: 94°C pre-denaturation for 2 min; 94°C denaturation for 30 s, 50°C annealing for 30 s, 72°C elongation for 40 s, 30 cycles; 72°C elongation for 5 min; Second round of reaction: 94°C pre-denaturation for 2 min; 94°C denaturation for 30 s, 50°C annealing for 30 s, 72°C elongation for 20 s, 30 cycles; 72°C elongation for 5 min.

[0008] Preferably, the electrophoresis in step (3) is 2% agarose gel electrophoresis, constant voltage electrophoresis at 3-5 V / cm for 45 min, and the result is observed in a gel imaging analysis system.

[0009] Preferably, the method for identifying Oncomelania hupensis from Sichuan and Yunnan in step (3) is: (1) the first round of amplified fragments are detected by electrophoresis, if specific bands appear at 538bp, the Oncomelania hupensis is from Sichuan and Yunnan, if no bands appear, the Oncomelania hupensis is from Sichuan and Yunnan; (2) the second round of amplified fragments are detected by electrophoresis, if specific bands appear at 228bp, the Oncomelania hupensis is from Yunnan, if no bands appear, the Oncomelania hupensis is from Sichuan.

[0010] Preferably, the detection method further comprises a SNP molecular marker for identifying Oncomelania hupensis from Sichuan and Yunnan, the nucleotide sequence 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.

[0011] Preferably, the identification is that: at the 75th position, the base of the Oncomelania hupensis from Sichuan or Yunnan is A or G, and the base of the Oncomelania hupensis from other regions is T; at the 420th position, the base of the Oncomelania hupensis from Sichuan is C / T, and the base of the Oncomelania hupensis from Yunnan is A.

[0012] Preferably, the reagent of the SNP molecular marker can be a primer, a probe or a chip.

[0013] Preferably, the detection method is used for distinguishing Oncomelania hupensis from Sichuan and Yunnan.

[0014] The embodiment of the present application has the following beneficial effects: the present application first establishes a method for distinguishing Oncomelania hupensis from Sichuan and Yunnan, and by using the provided SNP molecular marker or through simple PCR amplification and electrophoresis detection, the Oncomelania hupensis from Sichuan or Yunnan can be quickly distinguished, which provides scientific support for determining the source of input (positive) Oncomelania hupensis from the two adjacent regions with frequent exchanges. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The sequence alignment result sequence diagram of the second single nucleotide polymorphism site provided in the present application for the mitochondrial sequences of Oncomelania hupensis from different regions in China in embodiment 2; at the 75th position, the base of the Oncomelania hupensis from Sichuan and Yunnan is A, 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; Figure 2Sequence alignment result sequence diagram of the second single nucleotide polymorphism site of the mitochondrial sequences of snails in different regions of the country in Example 2 of the present application; at the 75th site, the base of Sichuan-Yunnan is G, 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; Figure 3 Sequence alignment result sequence diagram of the second single nucleotide polymorphism site of the mitochondrial sequences of snails in Sichuan and Yunnan in Example 2 of the present application; wherein: SC represents Sichuan, and YN represents Yunnan; Figure 4 Electrophoresis detection result diagram of the first round of PCR products using the kit provided by the present application in Example 2 of the present application; wherein: 1, 2 wells are the amplification products of snails from Sichuan; 3, 4 wells are the amplification products of snails from Yunnan; 6, 11 wells are the amplification products of snails from Hunan; 7, 12, 18 wells are the amplification products of snails from Anhui; 8, 13 wells are the amplification products of snails from Jiangsu; 9, 14, 19 wells are the amplification products of snails from Fujian; 10, 15, 20 wells are the amplification products of snails from Hubei; 5, 16 wells are DNA molecular weight standards, each consisting of 6 bands, and the sizes of the bands in 5 wells are 600, 500, 400, 300, 200, and 100 bp, respectively, and the sizes of the bands in 16 wells are 1200, 900, 700, 500, 300, and 100 bp, respectively; 17 well is a no-template control; Figure 5 Electrophoresis detection result diagram of the second round of PCR products using the kit provided by the present application in Example 2 of the present application; wherein: 1-10 wells are the amplification products of snails from Sichuan; 11-18 wells are the amplification products of snails from Yunnan; 19 well is a no-template control; 20 well is a DNA molecular weight standard, consisting of 6 bands, and the sizes are 600, 500, 400, 300, 200, and 100 bp, respectively. DETAILED DESCRIPTION

[0016] The technical solutions of the present application will be described below in conjunction with examples, obviously, the described examples are only a part of the examples of the present application, rather than all the examples. Based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0017] Example 1 Site screening and primer design for identifying snails in Sichuan and Yunnan (1) By searching and downloading the reference sequences of Oncomelania hupensis mitochondrial genes from different epidemic areas in China, and sequencing the mitochondrial genes collected from Sichuan and Yunnan provinces and other areas such as Hubei, Hunan, Fujian, Jiangsu, and Anhui, a plurality of single nucleotide polymorphism sites with potential regional correlation were first discovered through sequence alignment. On this basis, two single nucleotide polymorphism sites derived from cox1 gene were obtained through multiple rounds of screening, and the sequences are shown in SEQ ID NO. 1: SEQ ID NO. 1: atgcgttgat tattttcaac aaatcataaa gatattggga ctttatatat tttatttggt 60 atatgagcag ggttagtcgg tactgctctt agattactaa ttcgtgcaga gttagggcag 120 cctggggctt tacttggaga tgatcaactt tataatgtaa ttgttacagc acatgcattt 180 gtaataattt ttttcttggt gataccaata ataattggcg gctttggtaa ctgactagtt 240 ccacttatac ttggggctcc tgatatagca tttcctcgac tgaataacat aagattttga 300 ctccttcctc cagcactatt attgcttcta tcctcagcgg ctgttgagag gggagctggt 360 actgggtgaa cagtatatcc gccgcttgcc ggaaatttag cccatgccgg tggctctgtt 420 gatttagcta ttttttcttt acacttagct ggtgtttcat ctattttagg tgctgtaaac 480 tttattacca ctattattaa tatacgatga cgaggaatgc aatttgaacg acttccttta 540 tttgtttggt cggtaataat cactgctgtg ctactcttat tatctcttcc tgtcttagct 600 ggggccatta caatactttt aactgaccga aattttaata cggctttttt tgatccggct 660 ggggggggcg atccaattct ttatcaacac cttttttgat tttttggcca ccccgaagta 720 tatattttaa ttttgcctgg gtttggaatg atttctcata ttgtaagaca ttattcagct 780 aagaaagaaa cgtttgggac tttaggaatg atttatgcaa tgttggccat tggtgttcta 840 ggttttattg tttgggctca ccacatattc acggttggta tagatgtaga tactcgggca 900 tattttacag cagctacaat aattattgct gtacccactg gtattaaggt atttagttga 960 cttgctacta ttcatggggc caaaatcaaa tatgaaacac caatattgtg ggctctaggg 1020 tttatttttc tatttactgt aggtgggttg acaggaattg tactatcgaa ttcttcgttg 1080 gacatcatgt tgcacgatac ctactatgtg gtagcacatt ttcattatgt actttcaatg 1140 ggggcggttt ttgctttatt tggagctttt aactattgat ttccgctatt aactggagta 1200 acacttcatt ctcgatcaac taaagctcat ttttttatta tatttattgg tgttaatgtt 1260 accttttttc ctcagcattt tttagggcta gctggaatac cacggcgata ctctgattac 1320 ccagattgtt atactaaatg aaatgtaatc tcatcaattg gatctataat ttcttttgtt 1380 gcggtattat attttatagt aattgtgtga gaagccttag tttctcagcg aaaagttgtt 1440 tgaagaacac acctgagaac cgcactagag tgggataatt tattaccagc agatttccac 1500 aatgcgccag aaactggagc attggtttct gtatag 1560 (2) The single nucleotide polymorphism sites are located at the 75th and 420th bases. If the base at the 75th position is A or G, the Oncomelania hupensis individual is derived from Sichuan or Yunnan. If the base at the 75th position is T, the Oncomelania hupensis individual is derived from other areas outside Sichuan and Yunnan. If the base at the 420th position is C or T, the Oncomelania hupensis is derived from Sichuan. If the base at the 420th position is A, the Oncomelania hupensis is derived from Yunnan. According to the SNP site, specific primers are designed. The first round of primers is used to distinguish Oncomelania hupensis from Sichuan and Yunnan and other areas, and the forward primer is shown in SEQ ID NO. 2 and the reverse primer is shown in SEQ ID NO. 3. The second round of primers is used to distinguish Oncomelania hupensis from Yunnan and Sichuan, and the forward primer is shown in SEQ ID NO. 4 and the reverse primer is shown in SEQ ID NO. 5.

[0018] Example 2 Identification of Oncomelania hupensis in Sichuan and Yunnan (1) Sample collection: Oncomelania hupensis samples from Dayi, Pidu, Pujiang, Dujiangyan, Xintian, East New District, Tianfu New District, 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, as well as Oncomelania hupensis samples from Changle, Fuqing in Fujian Province, Gong'an, Songzi, Zhongxiang in Hubei Province, Guichi in Anhui Province, and Hunan and Jiangsu Province were collected and stored in 75% ethanol at room temperature for standby use.

[0019] (2) DNA extraction: The DNeasy Blood & Tissue Kit produced by QIAGEN (Germany) was used to extract the genomic DNA of Oncomelania hupensis. The specific method is as follows: 1) Lysis: After crushing and peeling the single Oncomelania hupensis, the soft tissue inside was placed in a 1.5 mL centrifuge tube, ground, and then 180 μL of ATL buffer and 20 μl of proteinase K provided by the kit were added. Shake well, incubate in a 56°C water bath until the tissue is completely lysed to obtain the lysate. 2) Centrifugal purification: After taking the lysis solution from the water bath, shake and mix for 15 s, add 200 μL buffer AL, shake and mix, then add 200 μL anhydrous ethanol, shake and mix; move all the liquid in the tube into the DNeasy filter column, centrifuge at 8000 rpm for 1 min, discard the filtrate; add 500 μL buffer AW1 to the filter column, centrifuge at 8000 rpm for 1 min, discard the filtrate; add 500 μL buffer AW2 to the extraction column, centrifuge at 14000 rpm for 3 min, discard the filtrate; 3) Elution: Move the filter column into a new centrifuge tube, add 200 μL buffer AE, stand at room temperature for 1 min, centrifuge at 8000 rpm for 1 min, the obtained filtrate is the Oncomelania genome sample template DNA, test the concentration and A260 / 280 OD value with Nano drop, dilute to 10 ng / μL, and store at -20°C for standby.

[0020] (3) Single nucleotide polymorphism detection: 1) Take the Oncomelania cox1 gene as the detection target, perform PCR amplification, sequence the amplification product, and after sequence correction and splicing, simultaneously combine the sequence of Oncomelania from Genbank, use MEGA software for multiple sequence alignment, and display the polymorphic sites.

[0021] 2) The results show that at the 75th site, as shown in the formula (I): Figure 1 the base of the Oncomelania in Sichuan and Yunnan is A, as shown in the formula (II): Figure 2 the base of the Oncomelania in Sichuan and Yunnan is G, with accession number OR661799.1; which can be used to distinguish the Oncomelania in Sichuan and Yunnan from the Oncomelania in other regions; at the 420th site, as shown in the formula (III): Figure 3 the base of the Oncomelania in Sichuan is C / T, and in Yunnan is A.

[0022] (4) PCR identification: 1) First round of PCR a. Prepare 25 μL of reaction system, as shown in Table 1, set up negative and positive controls, and use the target gene sequence plasmid as the positive control (synthesized by Shanghai Shengong), and use nuclease-free water as the negative control: Table 1 Preparation of first round of PCR system Reagent Volume (μL) 2X Taq PCR Amplification Reagent 12.5 Forward primer F0 1 Reverse primer R0 1 Sample Template DNA 1 Nuclease-Free Water 9.5 b. PCR reaction conditions: as shown in Table 2: Table 2 First round of PCR reaction conditions c. Agarose gel electrophoresis: 10 μL PCR amplification product was electrophoresed in 2% (w / v) agarose gel at 3-5 V / cm constant voltage for 45 min, and the results were observed in the gel imaging analysis system, with DNA molecular weight standard as the reference, and the results are shown in Fig. 2: the oneshell snail individuals with 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 amplification results of the shell snail individuals with negative results were from Sichuan and Yunnan. Thus, the shell snails with negative amplification results were selected for the second round of PCR experiment. Figure 4

[0023] 2) Second round of PCR: a. 25 μL of reaction system was prepared, as shown in Table 3, and positive and negative controls were set, and the target gene sequence plasmid was used as the positive control (synthesized by Shanghai Shengong), and nuclease-free water was used as the negative control: Table 3 Preparation of first round of PCR system Reagent Volume (μL) 2X Taq PCR Amplification Reagent 12.5 Forward primer F0 1 Reverse primer R0 1 Sample Template DNA 1 Nuclease-Free Water 9.5 b. PCR reaction conditions: as shown in Table 4: Table 4 First round of PCR reaction conditions c. Agarose gel electrophoresis: 10 μL PCR amplification product was electrophoresed in 2% (w / v) agarose gel at 3-5 V / cm constant voltage for 45 min, and the results were observed in the gel imaging analysis system, with DNA molecular weight standard as the reference, and the results are shown in Fig. 2: the oneshell snail individuals with 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 amplification results of the shell snail individuals with negative results were from Sichuan and Yunnan. Thus, the shell snails with negative amplification results were selected for the second round of PCR experiment. Figure 5

[0024] Example 3 Verification experiment of Sichuan snails Through the verification of Example 2, the negative was Sichuan snails. In order to distinguish Sichuan snails from negative controls, the samples with negative results in Example 2 and the negative controls were subjected to PCR amplification experiment for verification again: (1) The cytochrome oxidase (CO1) gene sequence was used, and the site was T5-13, the forward primer was as shown in SEQ ID NO. 5, and the reverse primer was as shown in SEQ ID NO. 6. The primer sequence was derived from the application of microsatellite and mitochondrial biotechnology in genetic differentiation research of Hubei snails: T5-13F: TAGTGGGACTTATTTGCTG (SEQ ID NO. 5); ​​T5-13R: R: AAGGCTGAGTGGTAGTTA (SEQ ID NO. 6).

[0025] (2) The PCR system is prepared as shown in Table 5: the volume of the negative control is the same as that of the DNA template, and the negative control is a no-template control using nuclease-free water: Table 5 Preparation of PCR system Reagent Volume (μL) 2X Taq PCR Amplification Reagent 12.5 Forward Primer T5-13F 1 Reverse Primer T5-13R 1 Sample Template DNA 1 Nuclease-Free Water 9.5 (3) The PCR reaction conditions are shown in Table 6: Table 6 PCR reaction conditions (4) Agarose gel electrophoresis: 10 μL of the PCR amplification product is electrophoresed in a 2% (w / v) agarose gel at a constant voltage of 3-5 V / cm for 45 min, and the results are observed in a gel imaging analysis system. The DNA template is imaged at about 245 bp, and the negative control shows no band.

[0026] In summary, the present application can quickly and accurately identify the geographical origin of snails through two rounds of PCR amplification and electrophoresis analysis. The method has the following advantages: high specificity: using specific SNP molecular markers (such as cox1 gene 75th and 420th base) to design primers ensures accurate differentiation of Sichuan and Yunnan snails: the first round of PCR (538 bp band) can exclude non-Chuan-Dian snails, and the second round of PCR (228 bp band) can distinguish Yunnan and Sichuan snails. Simple operation: only conventional PCR and agarose gel electrophoresis are required, without complex sequencing or high-end instruments, suitable for application in basic laboratories.

[0027] Although embodiments of the present application have been shown and described, it is to be understood that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the present application. Any equivalent changes and improvements made to the present application should still fall within the scope of the present patent.

Claims

1. A detection kit for identifying Oncomelania hunchatenis and Oncomelania hunchai, characterized in that, The kit comprises two rounds of PCR amplification primers; The sequence of the forward primer of the first round of PCR reaction is shown in SEQ ID NO. 2; The sequence of the reverse primer of the first round of PCR reaction is shown in SEQ ID NO. 3; The sequence of the forward primer of the second round of PCR reaction is shown in SEQ ID NO. 4; The sequence of the reverse primer of the second round of PCR reaction is shown in SEQ ID NO.

5.

2. The kit for discriminating between Oncomelania hunchuensis and Oncomelania qdliensis according to claim 1, wherein, The kit further comprises 2x Taq PCR amplification reagent, positive control and negative control.

3. A detection method for identifying Oncomelania hunchatenis and Oncomelania hunchatenis quadrasi, characterized in that, The detection method comprises the following steps: (1) Sample lysis: After crushing and shelling the oncomelania, put the soft tissue inside into a 1.5 mL centrifuge tube, grind and add ATL buffer and proteinase K for oscillation mixing, incubate at 56℃ for lysis, centrifuge to obtain the sample lysis solution; (2) Sample extraction: Column purification and elution of the sample lysis solution to obtain oncomelania genomic DNA; (3) PCR amplification: PCR system preparation of the oncomelania genomic DNA, setting of reaction program for amplification, analysis of electrophoresis results to identify oncomelania in Sichuan and Yunnan.

4. The method of claim 3, wherein the oncomelania hunchunensis and oncomelania qdoui are identified by detecting the expression of the gene. The reaction program in step (3) is: First round of reaction: 94℃ pre-denaturation for 2min; 94℃ denaturation for 30s, 50℃ annealing for 30s, 72℃ elongation for 40s, 30 cycles; 72℃ elongation for 5min; Second round of reaction: 94℃ pre-denaturation for 2min; 94℃ denaturation for 30s, 50℃ annealing for 30s, 72℃ elongation for 20s, 30 cycles; 72℃ elongation for 5min.

5. The method of claim 3, wherein the oncomelania hunchunensis and oncomelania qdaiensis are identified by detecting the expression of the gene. The electrophoresis in step (3) is 2% agarose gel electrophoresis.

6. The method of claim 3, wherein the oncomelania hunchunensis and oncomelania quadrasi are identified by detecting the expression of the gene. The method for analyzing the electrophoresis results to identify oncomelania in Sichuan and Yunnan in step (3) is: Electrophoresis detection of the first round of amplified fragments, if specific bands appear at 538bp, the oncomelania is from Sichuan and Yunnan, if no bands appear, the oncomelania is from Sichuan and Yunnan; Electrophoresis detection of the second round of amplified fragments, if specific bands appear at 228bp, the oncomelania is from Yunnan, if no bands appear, the oncomelania is from Sichuan.

7. The method of claim 3, wherein the oncomelania hunchunensis and oncomelania qdaiensis are identified by detecting the expression of the gene. The detection method further comprises SNP molecular marker identification of oncomelania in Sichuan and Yunnan, the nucleotide sequence 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. ​ 8. The method according to claim 7, wherein the method is used for identifying the oncomelania hunchunensis and oncomelania hesteri. The identification is that at the 75th position, the base of oncomelania in Sichuan or Yunnan is A or G, and that in other regions is T; at the 420th position, the base of oncomelania in Sichuan is C / T, and that in Yunnan is A. ​ 9. The detection method for identifying Oncomelania hupensis in Sichuan and Yunnan as described in claim 7, characterized in that, The reagent of the SNP molecular marker can be primer, probe or chip.

10. The method according to any one of claims 3-9, wherein the oncomelania hunchunensis and oncomelania haliangensis are discriminated by the method. The detection method is used for distinguishing oncomelania in Sichuan and Yunnan.

Images