CN112695103A - Multiple PCR primer combination for determining oncomelania individual genotype, kit and application - Google Patents

Multiple PCR primer combination for determining oncomelania individual genotype, kit and application Download PDF

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CN112695103A
CN112695103A CN202110182197.5A CN202110182197A CN112695103A CN 112695103 A CN112695103 A CN 112695103A CN 202110182197 A CN202110182197 A CN 202110182197A CN 112695103 A CN112695103 A CN 112695103A
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oncomelania
kit
multiplex pcr
determining
primer combination
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吕大兵
顾漫漫
周支军
孙梦涛
张洁英
余秋夫
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Suzhou University
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract

The application relates to a multiplex PCR primer combination, a kit and application for determining the individual genotype of oncomelania, wherein the multiplex primer combination comprises 5 sites with high polymorphism selected from a plurality of microsatellite sites, and is formed by optimization on the basis of repeated experiments; meanwhile, an allele threshold is established based on amplification data of the oncomelania samples in thousands of different areas, and popularization, use and comparison are facilitated. The method is used for carrying out genetic research and traceability analysis on Oncomelania hupensis population by determining the genotype of an Oncomelania hupensis specimen as an intermediate host of schistosoma.

Description

Multiple PCR primer combination for determining oncomelania individual genotype, kit and application
Technical Field
The invention relates to a multiplex PCR primer combination, a kit and application for determining the individual genotype of oncomelania, belonging to the technical field of biological detection.
Background
Schistosomiasis is a major infectious disease that seriously harms human health, and is classified as one of 20 overlooked tropical diseases by the world health organization. At present, 78 countries and regions are epidemic schistosomiasis, and more than 2 hundred million infected people and nearly 8 hundred million people face infection threat. The Japanese schistosomiasis is popular in China, and the Oncomelania hupensis is the only intermediate host of the Japanese schistosomiasis. Establishing a multi-site genotyping system, genotyping oncomelania specimens, analyzing genetic diversity and population structure of the oncomelania specimens, and performing traceability analysis to explore the reasons and influence factors of the oncomelania in one environment, has important significance for monitoring and controlling oncomelania diffusion, and is particularly suitable for schistosomiasis propagation blocking areas mainly based on oncomelania monitoring.
Microsatellites (also called Short Tandem Repeat (STR) or simple repeat (SSR) are composed of 2-6 nucleotide fragments which are repeatedly connected in series, are widely distributed in the oncomelania genome, show higher polymorphism in the repeat sequences and the length, are easy to detect, have co-dominant inheritance, are neutral in selection, can measure heterozygosity, and have the advantages of high repeatability, good stability and the like. However, the commonly used single PCR reaction, i.e., one microsatellite locus is amplified at a time, has low efficiency and high cost, and is easy to increase experimental operation errors. Therefore, the multiple PCR reaction method is adopted for amplifying a plurality of microsatellite loci at one time, so that the efficiency is greatly improved, the cost is reduced, and particularly, the workload of a laboratory is greatly reduced.
Disclosure of Invention
The invention aims to provide a multiplex PCR primer combination, a kit and application for determining the oncomelania individual genotype, and aims to perform population genetics research and traceability analysis on oncomelania by determining the genotype of an oncomelania specimen which is a schistosome intermediate host.
In order to achieve the purpose, the invention provides the following technical scheme: a multiplex PCR primer combination for determining the genotype of an individual of a snail, said primer combination comprising: DH01-F, DH01-R, DH02-F, DH02-R, T6-17-F, T6-17-R, B14-F, B14-R, and C22-F and C22-R, wherein the sequence of each primer in the primer combination is SEQ ID NO.1-SEQ ID NO.10 in sequence.
The invention also provides a kit containing the multiple PCR primer combination and used for amplifying the schistosoma japonicum multi-site sequence.
Further, the kit is based on Qiagen MM and comprises dNTPs, Taq DNA polymerase and Mg2+And PCR reaction buffer.
Further, the kit further comprises any one or more of TAMAR, HEX, FAM or ROX fluorescent markers for marking the forward primer.
The invention also provides application of the kit in determining the individual genotype of the oncomelania.
The present invention also provides a multiplex PCR method for determining the genotype of an individual oncomelania using the kit, the method comprising the steps of:
s1, extracting target DNA;
s2, performing multiplex PCR amplification reaction by using the target DNA as a template and the primer combination;
s3, analyzing the amplification product.
Further, in the multiplex PCR method, the multiplex PCR reaction system is calculated as 15 μ L:
DH01-F:0.045μL；DH01-R:0.045μL；DH02-F:0.03μL；DH02-R:0.03μL；T6-17-F:0.0225μL；T6-17-R:0.0225μL；B14-F:0.03μL；B14-R:0.03μL；C22-F:0.03μL；C22-R:0.03μL；PCR MM：7.5μL；ddH2o: 5.685 μ L; DNA template: 1.5. mu.L.
Further, the multiplex PCR reaction conditions were: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 60s, and extension at 72 ℃ for 30s for 30 cycles, and finally extension at 65 ℃ for 30 min.
Compared with the prior art, the invention has the beneficial effects that: the multiplex PCR primer combination, the kit and the application for determining the individual genotype of the oncomelania comprise the multiplex primer combination which is formed by selecting 5 sites with high polymorphism from a plurality of microsatellite sites and optimizing the sites on the basis of repeated experiments; meanwhile, an allele threshold is established based on amplification data of the oncomelania samples in thousands of different areas, and popularization, use and comparison are facilitated. The method is used for carrying out genetic research and traceability analysis on Oncomelania hupensis population by determining the genotype of an Oncomelania hupensis specimen as an intermediate host of schistosoma.
The foregoing is a summary of the present invention, and in order to provide a clear understanding of the technical means of the present invention and to be implemented in accordance with the present specification, the following is a detailed description of the preferred embodiments of the present invention.
Detailed Description
The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The multiplex PCR reaction system can simultaneously amplify 5 oncomelania microsatellite loci, and the size of the allele can be determined according to the allele threshold value of each locus. The 5 microsatellite loci comprising: DH01(GenBank: GU204242), DH02(GenBank: GU204045), T6-17(GenBank: GU204108), B14(GenBank: GU204050), C22(GenBank: GU 204145).
Firstly, designing a primer combination
According to 5 locus gene sequences published by GenBank, 5 pairs of primers are designed and synthesized, wherein the 5 pairs of primer sequences are respectively as follows:
DH01-F:GATCAATACCAGCAACTACT(SEQ ID NO.1)；
DH01-R:ATGATGCAGACTTGTAACGC(SEQ ID NO.2)；
DH02-F:AGGCGATTTATGCAGTTCCC(SEQ ID NO.3)；
DH02-R:CATTTGTGGTGCCCCTACGA(SEQ ID NO.4)；
T6-17-F:GCTGTCCTTTTACCAACTGC(SEQ ID NO.5)；
T6-17-R:TATCAAAGGATTATGCCGAG(SEQ ID NO.6)。
B14-F:GGCAGTTTTGATTAGCTTGGT(SEQ ID NO.7)；
B14-R:ACCTGATGTCAAATACCAGTTAG(SEQ ID NO.8)；
C22-F:CGGTACATCTGGATAGTGG(SEQ ID NO.9)；
C22-R:TGCGAAACAGTTGCAGACAC(SEQ ID NO.10)。
the fluorescent markers of the 5 pairs of primers are respectively as follows: DH01, FAM blue; DH02, HEX green; t6-17, TAMAR yellow; (ii) a B14, FAM blue; c22, ROX red.
Two, multiplex PCR reactions
In the multiplex PCR reaction system of this example, the concentrations of the primers are: DH01, 0.3. mu.M; DH02, 0.2. mu.M; t6-17, 0.15. mu.M; b14, 0.2. mu.M; c22, 0.2. mu.M.
The specific method comprises the following steps:
1. DNA extraction
According to the instruction, the genomic DNA of the oncomelania is extracted by adopting a DNA extraction kit.
2. Multiplex PCR amplification
And (2) carrying out multiplex PCR amplification by using the DNA extracted in the step 1 as a template by using 5 pairs of primers including DH01-F, DH01-R, DH02-F, DH02-R, T6-17-F, T6-17-R, B14-F, B14-R, C22-F and C22-R.
One reaction system, 15. mu.l, contained each pair of primers, PCR MM, water and template DNA as follows:
F R
DH01 0.045 0.045
DH02 0.03 0.03
T6-17 0.0225 0.0225
B14 0.03 0.03
C22 0.03 0.03
Qiagen MM 7.5
ddH2O 5.685
sample DNA 1.5
In this example, the conditions of the multiple PCR thermal cycling were: pre-denaturation at 95 ℃ for 5 min; 30 cycles of denaturation at 95 ℃ for 30sec, annealing at 60 ℃ for 60sec, and elongation at 72 ℃ for 30 sec; finally, extension is carried out for 30min at 65 ℃.
After a large number of oncomelania samples are amplified, the inventor finds that the threshold value of the size of each allele of 5 loci is as follows:
Figure BDA0002942432570000041
Figure BDA0002942432570000051
Figure BDA0002942432570000061
Figure BDA0002942432570000071
Figure BDA0002942432570000081
Figure BDA0002942432570000091
Figure BDA0002942432570000101
Figure BDA0002942432570000111
Figure BDA0002942432570000121
Figure BDA0002942432570000131
Figure BDA0002942432570000141
Figure BDA0002942432570000151
Figure BDA0002942432570000161
Figure BDA0002942432570000171
Figure BDA0002942432570000181
Figure BDA0002942432570000191
the allele size threshold of the 5 sites is provided, and the kit and the method are combined, so that the genetic typing of the oncomelania individual is facilitated, and the promotion is carried out.
In summary, the following steps: the multiplex PCR primer combination, the kit and the application for determining the individual genotype of the oncomelania comprise 5 sites with high polymorphism selected from a plurality of microsatellite sites, and are optimized and formed on the basis of repeated experiments; meanwhile, an allele threshold is established based on amplification data of the oncomelania samples in thousands of different areas, and popularization, use and comparison are facilitated. The method is used for carrying out genetic research and traceability analysis on Oncomelania hupensis population by determining the genotype of an Oncomelania hupensis specimen as an intermediate host of schistosoma.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Figure BDA0002942432570000211
Figure BDA0002942432570000221
Figure BDA0002942432570000231
Sequence listing
<110> Suzhou university
<120> multiplex PCR primer combination for determining oncomelania individual genotype, kit and application
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
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<211> 20
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 2
atgatgcaga cttgtaacgc 20
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<211> 20
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<213> Artificial Synthesis (Artificial Sequence)
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aggcgattta tgcagttccc 20
<210> 4
<211> 20
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 4
catttgtggt gcccctacga 20
<210> 5
<211> 20
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 5
gctgtccttt taccaactgc 20
<210> 6
<211> 20
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 6
tatcaaagga ttatgccgag 20
<210> 7
<211> 21
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 7
ggcagttttg attagcttgg t 21
<210> 8
<211> 23
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 8
acctgatgtc aaataccagt tag 23
<210> 9
<211> 19
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 9
cggtacatct ggatagtgg 19
<210> 10
<211> 20
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 10
tgcgaaacag ttgcagacac 20

Claims (8)

1. A multiplex PCR primer combination for determining the genotype of an individual of a snail, said primer combination comprising: DH01-F, DH01-R, DH02-F, DH02-R, T6-17-F, T6-17-R, B14-F, B14-R, and C22-F and C22-R, wherein the sequence of each primer in the primer combination is SEQ ID NO.1-SEQ ID NO.10 in sequence.
2. A kit for amplifying a multi-site sequence of Schistosoma japonicum comprising the multiplex PCR primer set according to claim 1.
3. The kit of claim 2, wherein the kit is based on Qiagen MM, comprising dNTPs, Taq DNA polymerase, Mg2+And PCR reaction buffer.
4. The kit of claim 3, further comprising any one or more of a TAMAR, HEX, FAM or ROX fluorescent marker for marking the forward primer.
5. Use of a kit according to any one of claims 2 to 4 for determining the genotype of an individual of a oncomelania.
6. A multiplex PCR method for use in determining the genotype of an individual of a Oncomelania snail, using a kit according to any one of claims 2 to 4, the method comprising the steps of:
s1, extracting target DNA;
s2, performing multiplex PCR amplification reaction by using the target DNA as a template and the primer combination;
s3, analyzing the amplification product.
7. The multiplex PCR method according to claim 6, wherein in the multiplex PCR method, the multiplex PCR reaction system is, in 15 μ L:
DH01-F:0.045μL；DH01-R:0.045μL；DH02-F:0.03μL；DH02-R:0.03μL；T6-17-F:0.0225μL；T6-17-R:0.0225μL；B14-F:0.03μL；B14-R:0.03μL；C22-F:0.03μL；C22-R:0.03μL；PCR MM：7.5μL；ddH2o: 5.685 μ L; DNA template: 1.5. mu.L.
8. The multiplex PCR method according to claim 6, wherein the multiplex PCR reaction conditions are: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 60s, and extension at 72 ℃ for 30s for 30 cycles, and finally extension at 65 ℃ for 30 min.
CN202110182197.5A 2021-02-10 2021-02-10 Multiple PCR primer combination for determining oncomelania individual genotype, kit and application Pending CN112695103A (en)

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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
丁焕: "应用微卫星位点研究安徽、江苏地区湖北钉螺的群体遗传结构", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 *

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