CN109536606B - SNP site related to susceptibility of heavy metal poisoning and application thereof - Google Patents

Abstract

The application discloses an SNP locus related to susceptibility to heavy metal poisoning and application thereof. The SNP locus is the 103 th nucleotide C > T of the TRPV5 gene, and the sequence of the TRPV5 gene is shown as SEQ ID No. 10. The difference between C and T of the SNP locus between the high blood lead group and the low blood lead group is very obvious, wherein C is a lead poisoning susceptibility allele or a lead poisoning high risk allele, and T is a non-lead poisoning susceptibility allele or a lead poisoning low risk allele. Therefore, the base type of the site is detected, and the genotype of the site is judged, so that the method can be used for detecting the susceptibility to heavy metal (such as lead) poisoning or the risk of heavy metal poisoning, screening heavy metal poisoning patients, identifying or assisting in identifying the susceptibility genes of heavy metal poisoning, and detecting the single nucleotide polymorphism related to heavy metal poisoning or metabolism. The application and the product provided by the invention have important values in the aspect of reasonably preventing heavy metal poisoning by identifying the population susceptible to heavy metal poisoning.

Description

SNP site related to susceptibility of heavy metal poisoning and application thereof

Technical Field

The invention belongs to the technical field of biomedical molecular detection, and particularly relates to an SNP (single nucleotide polymorphism) site related to susceptibility of heavy metal poisoning and application thereof.

Background

The heavy metal is metal with specific gravity of more than 5 (generally, metal with density of more than 4.5 g per cubic centimeter), including gold, silver, copper, iron, mercury, lead, cadmium and the like, and the heavy metal is accumulated in a human body to a certain degree to cause chronic poisoning. Heavy metals in terms of environmental pollution are mainly heavy elements with significant biological toxicity, such as mercury (mercury), cadmium, lead, chromium, metalloid arsenic and the like. Heavy metals are very difficult to biodegrade, but instead can be concentrated hundreds of times under the action of biological amplification of the food chain and finally enter the human body. Heavy metals can interact strongly with proteins and enzymes in the human body, so that the heavy metals lose activity and can also accumulate in certain organs of the human body to cause chronic poisoning. Different populations exhibit different biological effects at the same level of heavy metal exposure, which depends in large part on the genetic susceptibility of the population. The method is beneficial to early identifying susceptible people and making corresponding preventive measures to protect the susceptible people by searching genetic susceptibility factors of heavy metal poisoning.

TRPV5 is a member of the TRPV subfamily channel among members of the Transient Receptor Potential (TRP) channel superfamily, which channel protein consists of 729 amino acids and has a molecular weight of approximately 83 kDa. The corresponding gene consists of 15 exons arranged on chromosome 7q34, and is mainly distributed in kidney, osteoclast, placenta, etc. Among them, TRPV5 is an important channel protein mediating osteoclastic bone resorption, although it is not expressed at a high level in kidney, but is also expressed in osteoclasts. The research proves that the calcium-calcium complex has the effect of reducing Ca in the kidney²⁺Active reabsorption is relevant. First, calcium ions enter cells through TRPV5, and diffuse to basolateral membranes by binding to calbindin D28K. Then, the calcium ions pass through a sodium-calcium exchanger (Na)⁺/Ca²⁺exchanger1, NCX1) and ATP-dependent plasma membrane Ca²⁺ATPase 1b (PMCA1b) is extracellularly excreted, and studies have shown that Ca is present²⁺Expression of transporter depends on TRPV 5-regulated Ca²⁺Influx and expression of TRPV 5.

At present, no research indicates that the polymorphism of the TRPV5 gene is related to susceptibility to heavy metal (such as lead) poisoning.

Disclosure of Invention

The invention aims to provide application of detecting a novel SNP in detecting susceptibility to heavy metal poisoning.

The inventor of the application finds in research that: two new SNP sites exist on the human TRPV5 gene (the DNA sequence is shown as SEQ ID No. 10): g.103(C > T) (mutation of C at position 103 of the sequence shown in SEQ ID No.10 to T) and g.654(G > A) (mutation of G at position 654 of the sequence shown in SEQ ID No.10 to A). The polymorphism or genotype of the two SNP sites is subsequently proved to be related to susceptibility to heavy metal (lead) poisoning by case-control analysis.

15 exons of the TRPV5 gene shown in SEQ ID No.10 are respectively: the DNA shown in SEQ ID No.10 has the positions 265-392, 3278-3375, 3544-3666, 4159-4296, 4604-4702, 4858-5033, 5490-565656565638, 7983-8195, 18156-18242, 18341-18417, 18678-18843, 19018-19084, 20974-21242, 24128-24234 and 24916-25210.

Therefore, the invention protects a SNP site related to the susceptibility to heavy metal poisoning, wherein the SNP site is the 103 th nucleotide C > T of the TRPV5 gene.

Further, the sequence of the TRPV5 gene is shown as SEQ ID No. 10.

In addition, in one aspect, the invention provides the application of the reagent for detecting the SNP locus of the subject in preparing a product for detecting the susceptibility of the subject to heavy metal poisoning; preferably, the heavy metal is gold, silver, copper, iron, mercury, lead and/or cadmium, more preferably, lead.

The product can also be a product of 1) to 3) as follows:

1) products for detecting single nucleotide polymorphisms associated with heavy metal poisoning or metabolism;

2) screening products of heavy metal poisoning patients;

3) identifying or assisting in identifying the products of the heavy metal poisoning susceptibility genes;

the heavy metal is gold, silver, copper, iron, mercury, lead and/or cadmium, preferably lead.

In another aspect, the present invention also provides a product containing a reagent for the SNP site of a subject as described above, wherein the reagent comprises:

a product for detecting single nucleotide polymorphism related to heavy metal poisoning, which contains a reagent for detecting the SNP locus of a subject;

a product for detecting susceptibility to heavy metal poisoning or risk of heavy metal poisoning, which contains a reagent for detecting the SNP site of a subject;

a product for screening heavy metal poisoning patients, wherein the product contains a reagent for detecting the SNP sites of a subject;

an article of manufacture for identifying or assisting in identifying a heavy metal poisoning susceptibility gene, the article of manufacture comprising a reagent for detecting the SNP site in a subject.

In the above application or product, the reagent for detecting the SNP site of the subject may be a reagent and/or an apparatus for determining the polymorphism or genotype of the SNP site by at least one of the following methods: DNA sequencing, restriction enzyme fragment length polymorphism, single-strand conformation polymorphism, denaturing high performance liquid chromatography, SNP chip, mass spectrum and fluorescent quantitative PCR; the product may be a reagent or kit, and may also be a combination of a reagent or kit and an instrument, such as a combination consisting of a PCR primer, a single base extension primer, and a mass spectrometer and related reagents.

In a specific embodiment, the reagent for detecting the SNP site of the subject comprises a PCR primer pair for amplifying the SNP site, so as to increase the copy number of the SNP site and perform further sequencing, hybridization, mass spectrometry and the like;

optionally, the reagent for detecting the SNP site of the subject further comprises a single base extension primer for amplifying the SNP site, so as to amplify the SNP site and analyze the amplified base.

In a specific embodiment, the upstream primer of the PCR primer pair is shown as SEQ ID No.1, and the downstream primer is shown as SEQ ID No. 2;

the single base extension primer is shown as SEQ ID No.7 or a complementary sequence thereof;

in one embodiment, the subject's reagent for the SNP site further comprises PCR reaction reagent, and/or alkaline phosphatase and its reaction buffer, and/or single base extension reaction reagent, and/or desalting resin;

the PCR reaction reagent comprises: dNTP, Mg²⁺PCR reaction preliminaryMixing, and DNA polymerase;

the single base extension reaction reagent comprises: ddNTP, single-base extension reaction buffer solution, single-base extension reaction stop solution and single-base extension enzyme.

In one embodiment, the reagent for detecting the SNP site can be used in combination with other products for detecting susceptibility of heavy metal poisoning of a subject;

for example, the above PCR primer pair may be used in multiplex PCR in combination with a PCR primer pair for specifically amplifying a PCR primer pair containing other SNP sites, so as to simultaneously analyze a plurality of SNP sites; can also be used independently, so that only 1 SNP locus is needed to be analyzed;

the single-base extension primer can also be mixed with a single-base extension primer for specifically amplifying other SNP sites so as to analyze a plurality of SNP sites simultaneously; it can also be used alone, so that it is necessary to analyze only 1 SNP site.

On the other hand, the invention also provides a PCR primer for detecting the susceptibility to heavy metal poisoning, wherein an upstream primer of the PCR primer is shown as SEQ ID No.1, and a downstream primer of the PCR primer is shown as SEQ ID No. 2.

Further, the PCR primer also comprises a single-base extension primer; preferably, the sequence of the single base extension primer is shown as SEQ ID No.7 or the complementary sequence thereof.

In another aspect, the present invention also provides a method for detecting susceptibility to heavy metal poisoning in a subject for non-therapeutic purposes, comprising the step of detecting polymorphism at nucleotide 103, C > T, of TRPV5 gene in the subject, wherein the TRPV5 gene is represented by SEQ ID No.10, and the determination conditions of the susceptibility are as follows:

the 103 th nucleotide C of the TRPV5 gene of the subject or the nucleotide G complementary with the base thereof is a high-risk subject with heavy metal poisoning, and the 103 th nucleotide T of the TRPV5 gene of the subject or the nucleotide A complementary with the base thereof is a low-risk subject with heavy metal poisoning;

preferably, the method comprises the step of amplifying using the PCR primers of claim 8 or 9.

In addition, the invention also provides a PCR primer combination for preparing any product, which comprises the following primer pairs:

the primer pair 1 is used for specifically amplifying a human genome DNA fragment with SNP sites TRPV5 gene 103 th nucleotide C > T, the upstream primer of the primer pair 1 is shown as SEQ ID No.1, and the downstream primer is shown as SEQ ID No. 2;

a primer pair 2 for specifically amplifying a human genome DNA fragment with the SNP site TRPV5 gene 654 th nucleotide G > A, wherein an upstream primer of the primer pair 2 is shown as SEQ ID No.3, and a downstream primer is shown as SEQ ID No. 4;

optionally, the method further includes: a primer pair 3 (used as a contrast) for specifically amplifying a human genome DNA fragment containing a SNP site TRPV5 gene 7240 th nucleotide G & gtA (rs1568760), wherein an upstream primer of the primer pair 3 is shown as SEQ ID No.5, and a downstream primer is shown as SEQ ID No. 6;

optionally, the method further includes: a single base extension primer for specifically amplifying the SNP locus, or a probe for detecting the SNP locus;

detecting the amplification product of the single base extension primer to obtain the genotype of the corresponding SNP site;

detecting the marker signal of the probe can also obtain the genotype of the corresponding SNP locus;

preferably, the single-base extension primers for specifically amplifying the SNP sites are as follows:

a single base extension primer A for specifically amplifying 103 th nucleotide C & gtT of a SNP locus TRPV5 gene, wherein the sequence is SEQ ID No.7 or a complementary sequence thereof;

a single base extension primer B for specifically amplifying 654 th nucleotide G & gtA of a SNP site TRPV5 gene, the sequence of which is SEQ ID No.8 or the complementary sequence thereof;

a single-base extension primer C for specifically amplifying the 7240 th nucleotide G > A (rs1568760) of the SNP site TRPV5 gene, and the sequence of the primer C is SEQ ID No.9 or a complementary sequence thereof.

The invention protects the application of the PCR primer combination in preparing any product.

The PCR primer combination provided by the invention can complete the amplification of a plurality of SNP locus products in one tube while keeping the high efficiency and sensitivity of PCR amplification, and the detection accuracy of a gene sequencing method is considered; after the sample DNA is extracted, the concentration does not need to be measured and diluted, and the sample is directly added for PCR amplification and subsequent SAP and iPLEX reaction, so that the influence factors such as probe hybridization are avoided, the time is saved, and the manual operation is reduced; the detection cost is better controlled, and the economic and social benefits are improved; the invention is beneficial to applying the MassARRAY mass spectrum technology to the inspection or correlation analysis of the susceptibility of heavy metal poisoning, and provides basis on the premise of making corresponding preventive measures for specific population.

Experiments prove that the difference between alleles C and T at the 103 th nucleotide (g.103) C > T of the TRPV5 gene at the SNP locus is extremely obvious (P < 0.01) between a case (high blood lead group) and a control (low blood lead group), which indicates that the alleles C and T at the g.103(C > T) SNP locus are related to lead poisoning susceptibility (OR is 1.29), the number of people with C at the alleles is obviously higher than that of a low blood lead group (control group) in the population of the high blood lead group (lead poisoning susceptibility group, case group), namely, the C at the g.103(C > T) SNP locus is judged to be a lead poisoning susceptibility allele OR a lead poisoning high-risk allele, and the T is a non-lead poisoning susceptibility allele OR a lead poisoning low-risk allele. Therefore, the base type of the site is detected, and the genotype of the site is judged, so that the method can be used for detecting the susceptibility to heavy metal (such as lead) poisoning or the risk of heavy metal poisoning, screening heavy metal poisoning patients, identifying or assisting in identifying the susceptibility genes of heavy metal poisoning, and detecting the single nucleotide polymorphism related to heavy metal poisoning or metabolism.

The application and the product provided by the invention have important values in the aspect of reasonably preventing heavy metal poisoning by identifying the population susceptible to heavy metal poisoning.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The inventor of the application discovers that two new SNP sites exist on a human TRPV5 gene (the DNA sequence is shown as SEQ ID No. 10) in research: g.103(C > T) (mutation of C at position 103 of the sequence shown in SEQ ID No.10 to T) and g.654(G > A) (mutation of G at position 654 of the sequence shown in SEQ ID No.10 to A). The polymorphism or genotype of the two SNP sites is subsequently proved to be related to susceptibility to heavy metal (lead) poisoning by case-control analysis.

15 exons of the TRPV5 gene shown in SEQ ID No.10 are respectively: the DNA shown in SEQ ID No.10 has the positions 265-392, 3278-3375, 3544-3666, 4159-4296, 4604-4702, 4858-5033, 5490-565656565638, 7983-8195, 18156-18242, 18341-18417, 18678-18843, 19018-19084, 20974-21242, 24128-24234 and 24916-25210.

Example 1 two newly discovered SNP sites on TRPV5 Gene are associated with susceptibility to heavy metal (lead) poisoning (case-control analysis)

1. With the people of Chinese Han worker 1301 who are exposed to the lead-polluted working environment for a long time (more than 2 years), according to the GBZ 37-2002 occupational chronic lead poisoning diagnosis standard, people with blood lead of more than or equal to 400 mu g/L are used as a high blood lead group (namely a lead poisoning susceptible group and a case group), and people with blood lead of less than 400 mu g/L are used as a low blood lead group (a control group). The age, sex, working age and other conditions of two groups of people are not statistically different (P is more than 0.05) through statistical analysis.

The blood lead content was measured by anodic stripping voltammetry using a model 3010B blood lead analyzer from ESA corporation, usa.

2. The genotypes of the SNP loci g.103(C > T) and g.654(G > A) of the population with the high blood lead group (lead poisoning susceptibility group) and the population with the low blood lead group are detected, and the SNP locus g.7240(G > A) (the 7240 th G of the DNA shown in SEQ ID No.10 is mutated into A, and the number in NCBI is rs1568760) which is known to be irrelevant to lead poisoning is used as a contrast.

3. The results of steps 1 and 2 were counted and the California was used to test whether the allele/genotype at each SNP site correlates with the case-control group, and the results are shown in Table 1.

TABLE 1 genotyping assay results

Note: through analysis, the genotype frequencies of the three SNPs are consistent with the genetic Hardy-Weinberg balance (P is more than 0.05) in the total population, and the research object is considered to have good representativeness.

N in table 1 represents a number; OR (95% CI) indicates the odds ratio (95% confidence interval), the OR value for different genotypes is an estimate of the relative risk of the gene causing disease for disease incidence, when the OR value is 1, the factor is not effective for disease occurrence, when the OR value is greater than 1, the factor is a risk factor, and when the OR value is less than 1, the factor is a protective factor; the P value represents the statistical significance, and when the P is less than 0.05, the comparative samples have significant difference; when P < 0.01, the comparative samples have very significant differences.

The results in table 1 show that the difference between the allele C at the SNP site g.103(C > T) and the allele T between the case and the control group is very significant (P < 0.01), which indicates that the alleles C and T at the SNP site g.103(C > T) are related to lead poisoning susceptibility (OR is 1.29), the number of people with C at the allele in the population of the high blood lead group (i.e. lead poisoning susceptibility group, case group) is significantly higher than that in the population of the low blood lead group (control group), i.e. C at the SNP site g.103(C > T) is determined to be lead poisoning susceptibility allele OR lead poisoning high risk allele, and T is non-lead poisoning susceptibility allele OR lead poisoning low risk allele;

similarly, the difference between the allele G at the SNP locus g.654(G > A) and the allele A between the case group and the control group is very significant (P < 0.01), which indicates that the allele G and the allele A at the SNP locus g.654(G > A) are related to lead poisoning susceptibility (OR is 1.37), the number of people who are G at the allele in the population of the high-blood lead group (i.e. the lead poisoning susceptibility group and the case group) is obviously higher than that of the low-blood lead group (the control group), i.e. the G at the SNP locus g.654(G > A) is judged to be the lead poisoning susceptibility allele OR the lead poisoning high-risk allele, and the A is the lead poisoning non-susceptibility allele OR the lead poisoning low-risk allele;

the difference between alleles G and A at the control SNP site g.7240(G > A) was not significantly different between case and control groups (P was 0.75-0.50), indicating that the site allele was not associated with susceptibility to lead poisoning.

The detection in the step 2 is specifically as follows:

a detection instrument: MassARRAY Genetic Analysis System DNA Mass Spectrometry Gene Analysis System (SEQUENOM Inc. SiGeno, USA); an AB-Vii A7 fluorescent quantitative PCR instrument (applied biosystems, USA, ABI); centrifuge (TL-5.0W, Shanghai); rotary shaker (shanghai).

The detection method comprises the following steps:

1) primer design

The sequence information of the genome in which SNP sites g.103(C > T) and g.654(G > A) and SNP site g.7240(G > A) (control, known to be irrelevant to susceptibility to lead poisoning) are located is retrieved from NCBI, after bioinformatics comparison analysis, a highly conserved region containing one of the three SNP sites is found, a primer and a single base extension primer corresponding to the SNP site are designed, and a primer combination with the best effect is found through analysis software and actual amplification search (multiplex PCR amplification sensitivity is highest, specificity of the primer in single base extension reaction is good, and molecular weight difference between single base extension products is more than 20 Da), and the sequences are shown in Table 2 and Table 3.

TABLE 2 multiplex PCR primer design

TABLE 3 design of single base extension primers

2) The genomic DNA in the blood samples was purified using a blood genome extraction kit from the blood anticoagulated with the above 1031 human EDTA stored in vitro at-20 ℃.

3) Multiplex PCR amplification: simultaneously using 3 primer pairs shown in Table 2, respectively taking the genomic DNA obtained in the step 2) as a template and setting water as a negative control of the template to carry out multiplex PCR amplification, wherein the reaction system is as follows:

reagent composition	Dosage of
		MgCl2(25mM)	1.5μL
PCR buffer (10X)	2.5μL
		dNTP mix (2.5mM)	2.0μL
1 μ M primer mixture (same molar concentration for each primer)	5.0μL
		Taq HS DNA polymerase	2U
Template (genome DNA solution)	2.0μL
		H2O	Make up to 25. mu.L

The reaction conditions for multiplex PCR amplification are as follows: pre-denaturation at 95 ℃ for 3 min; the following cycle was then performed 45 times: denaturation at 95 ℃ for 20s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 20 s; finally, extension is carried out for 3min at 72 ℃.

4) SAP reaction

Respectively taking 5 mu L of the liquid subjected to PCR amplification in the step 3), and adding 2 mu L of SAP reaction liquid to perform SAP reaction, wherein the formula of the SAP reaction liquid is as follows:

reagent composition	Dosage of
		Deionization of H2O	1.53μL
SAP buffer	0.17μL
		SAP enzyme (1.7U/. mu.L)	0.30μL
Total up to	2.00μL

The SAP reaction conditions were as follows: the mixture was stored at 37 ℃ for 40 minutes, 85 ℃ for 5 minutes and 4 ℃.

5) Single base extension reaction

Adding 2 mu L of extension reaction solution into the liquid reacted in the step 4) respectively to carry out single base extension reaction, wherein the extension reaction system is as follows:

components	Dosage of
		Deionization of H2O	0.619μL
iPLEX buffer solution	0.2μL
		iPLEX termination mix	0.2μL
An equal concentration of each single base extension primer (0.5. mu.M)	0.94μL
		iPLEX enzyme	0.041μL
Total up to	2μL

The conditions for the extension reaction were as follows:

6) resin desalination

After adding 41. mu.L of water to each well of the extension reaction solution containing the specimen, the mixture was centrifuged. Add 15mg of clean Resin (Resin): the sample plate is gently inverted and placed on the sample plate with the resin placed, and then the sample plate is inverted together with the sample plate (the two plates cannot move horizontally in the process), so that the resin falls into the holes. The plate was sealed with a membrane and placed on a rotator and shaken for 15 minutes. Plates were centrifuged for 5 minutes at 3200g (4000 rpm of standard plate centrifuge).

7) SpectroCHIP chip loading

Using MassARRAY^TMThe RS 1000 autosampler performs a volume test to find the appropriate speed of spotting. Suitable volumes for spotting are 8-12 nL. + -. 25%. Volume testing was performed with samples on the plate, followed by MassARRAY^TMThe RS 1000 autosampler spotted the reacted sample onto a 96-spot SpectroCHIP.

8) Mass spectrometric detection

And (3) carrying out mass spectrum detection and Analysis by using a MassARRAY Genetic Analysis System DNA mass spectrum gene Analysis System, and determining the base type of the corresponding SNP site in the multiplex PCR product according to the difference between the molecular weight of a mass spectrum detection peak and the molecular weight of a corresponding single-base amplification primer.

Those not described in detail in this specification are within the skill of the art. The above description is only an example of the present application and is not intended to limit the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Sequence listing

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gcagggacga atcctgtggg ctgcttcttc ctccaagtgt tttggggctt gcctcccacc 1140

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caggcatggt ggtgggcacc tgtagtccca gctactcggg aggctgaggc aggagaatca 1920

cttgaaccca ggaggcggaa gttgcagtga gccgagatcg caccactgca ctccagtctg 1980

ggcaatagag tgagacttcg tctcaaaaaa aataaataaa taaaataaaa acactttttc 2040

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tacacatatg tctagttgga gaggctccag ctttatctct ttctgggttc aaatctgggt 2220

atggtagaag caaagattaa cagaggtcct gtagaatgag tatctcaacc tgttttgcct 2280

tgagacaccc acataaacaa gctgtttaga cctttgcatc taagggtata atgcagaaga 2340

aagaggcacc ctgtaactca ccccacaact ctccaccccc tgcagggacc tgtatctaaa 2400

taacctgagg atgctgggcg caatgtaatc ccagcacttt gggaggccaa ggcaggcgga 2460

ttgcctgagc tcaggagttc gagatcagcc tggccaacat ggcaaaaccc catctctact 2520

aaaaatacaa aaaattagct gggcgtggtg gtgcgtgcct gtaatcccag ctactcggga 2580

gactgaggca caagaattgc ttgaacctgg gaggcagagg ctgcggtaag ctgagattgt 2640

accactgcac tccagcctag gcaacagagc aagactctgt ctcaaaaaaa aaaaaaaaaa 2700

aaaaaatagc ctgagggagt agtcagaact ttagtgcctc acaaatttca caaatttcaa 2760

aatgcctagg aaagatctag cagtcgatcc attcctcctc cctctgtgtt ttggtctccc 2820

aaactcccct tggttaaaga cagtagattc agcaggccca ggagcagggg gaccgcagat 2880

ggaagtgccc tctgtcccgc tctccaacta tccaggatct gagacagtta ctcaggttag 2940

atgaattcag ccaagcctag ggtcctcttc ttagagactc tcagaccact ctttccagac 3000

tacaggttct cttcctatta actcttcccc tccagcagag cttcaagggc catcctcaca 3060

ggagaatcac catgatgtga attgcaagga aggccctggg gctgaccctt gcccaagtcc 3120

accctcggtc attcgagtcc agtaggcact tctttccaat ttgtcactgc acgctgtgtg 3180

agtcacgttg gtcttaaaga atgtcttaga tcccctttgc tcttggcctc attctgctat 3240

gactgtctct tccaaactca cgttcacctt tccccaggat tctagagtct ccactgcttc 3300

gagcatccaa ggaaaatgac ctgtctgttc ttaggcaact tctactggac tgcacctgtg 3360

acgttcgaca aagaggtggg atctggattt aatgggatgg aggtgggggc agaagaacct 3420

actcctcctc cccaagactt cgagtttgtg aaatagggcc ttggagggtg tctctggagc 3480

ctgaggcccc ttcctcaagg gaatctgaaa agtgtctaac atctctgtct tactccaatc 3540

caggagccct gggggagacg gcgctgcaca tagcagccct ctatgacaac ttggaggcgg 3600

ccttggtgct gatggaggct gccccagagc tggtctttga gcccaccaca tgtgaggctt 3660

ttgcaggtaa ggagcctaca tggtaattca aggtggaaac tttgagatgg agggtgtgct 3720

gttgtctctt aaaattggcc tctgaaggat gggttggggt ggggtgatgg agggtcagga 3780

ggatgggctc cgttgagttg ggagaaatgc aaaggtcctg gtcttggctt ttaccaatct 3840

gtggcttcct tcagcatttc ctttcaaacc tcccatagtc cctgtaggac ctatctcaat 3900

ttctttctct catagcaaca tgagaatgat tatattcata gcaatcccct gctggctccc 3960

aataactgaa gggtaggtca atgagcaggg cagcctgagg gatgaattgc tcttagtccc 4020

aggccaccct gccaagccca gggcagaagt ctagcaaaga tctatcagga acctcctgag 4080

agggatggtg ggggatgctg tgtccctgag agaactgggg acagagggag accctgatga 4140

tggctgtctc tgggccaggt cagactgcac tgcacatcgc tgttgtgaac cagaatgtga 4200

acctggtgcg tgccctgctc acccgcaggg ccagtgtctc tgccagagcc acaggcactg 4260

ccttccgcca tagtccccgc aacctcatct actttggtga gagcagggtt gggcttgaag 4320

gatggttggc tggagaggcg ggcaaggcag agggaggcca gggcatgagg gtcctgcctt 4380

cagctccatc tgttgggcag ttgagagggg cagcccttct agggcttaat taagccagag 4440

gcccattccc ctgatggttc cttcctctgg ttctgctgga cgagtgtccc tcagcctaga 4500

actgagatat gggggatgag gaggaggccc ttatttgggg gagactgccc accccatcct 4560

gccatcctgc gttctcctgc catggatctc tctgtgtcca caggggagca ccctttgtcc 4620

tttgctgcct gtgtgaacag cgaggagatc gtgcggctgc tcattgagca tggagctgac 4680

atcagggccc aggactccct gggtaagagc tgggatgggg aggggagctg ggatgctgtg 4740

gggagcgagg gacagggtaa ggagtggagc tgggaccttt aggaagaccc cagagtgggg 4800

atggggacat cagggacttt aggccagtgg ccacatgata ccctgcgtcc tccccaggaa 4860

acacagtatt acacatcctc atcctccagc ccaacaaaac ctttgcctgc cagatgtaca 4920

acctgctgct gtcctatgat ggacatgggg accacctgca gcccctggac cttgtgccca 4980

atcaccaggg tctcaccccc ttcaagctgg ctggagtgga gggtaacact gtggtaagag 5040

acgctcccca tccctgaggt gtctttctaa tgaccctcaa cctatcctgc tggccctggg 5100

agaaatagag gtgaagggcc cacctttctc taaaatccct tttccccaaa ctccccaaag 5160

ggatggtggt cccttgcttc ctattccatc actgaaatct tcagcttcat ggtccttgac 5220

tcaagaggat ttgagaacct taccttaatg catgtgctgt gataacccaa agctctagct 5280

tcctttgcct attttcttct ttctgcactt tctggggttt tggagcagga aagatgacaa 5340

aaaagggaaa gcttgtagat gcagaggttt gctggttccc actgtctgtc tgtctcaagg 5400

ggtgggcaac tggctccatc caatggagtt gttctcctcg accaccttct cacacgttgg 5460

ccccctgact catcctggtg gtctctcaga tgttccagca cctgatgcag aagcggaggc 5520

acatccagtg gacgtatgga cccctgacct ccattctcta cgacctcacg gagatcgact 5580

cctggggaga ggagctgtcc ttcctggagc ttgtggtctc ctctgataaa cgagaggtat 5640

ggatgccaag gaggatcagg cctgtcatgt gagggaattt agtgtttgtg cagagtctgt 5700

ccacttacta agatcctggg cctgagacac ttcccagcct tataataggt ggagatgaga 5760

atagtctcct gggacatggg ggaaagtcca atgcacactc ctctcctgaa tcccctgtct 5820

ctctgcttag ctccagtccc ttctgcctac ctttcgcctt ctatcatttc agatgagaaa 5880

ctccaaataa gagctgtttc ctcatattca ttcattggtt cagcattgat atggtttgca 5940

tttgtgtcca aatgcaaaaa tttcatgtca aattgtaatc cacagtgttg gagaaggggc 6000

ctggtgggag gtgattggat catggggaca gacttccctc ttgatgttct cgtgatagtg 6060

agttttcaca agatccggtt gtttaaaagt gtgtggcacc tccccctgct ctctcttcct 6120

cctgctctct cttcctcctg ctccggccat gtaggatgtg cctgctttct tcttggcctt 6180

ttgccatgat tgtacgtttc ctcaggcctc tccagaacct gtacagcctg cagaaccatg 6240

agccaattaa acctttttat ttataaatta ccctgtgcca ggtagttctt tatagtaata 6300

tgagaatgga ctaatacaag catattgaac acccgctgta tgtcaggcac tgcacaagaa 6360

acaaacagta cagaaataaa tacaacacag ttcctgttca tcaacttcct caattctcag 6420

tccctgtcct gaataattgc aacagagcat gacatatgcc gtaatagttg tgtgtaccat 6480

gcatgcagac agccacattg aggtagagtt aacttggctt ggatagacca gagaagatgt 6540

cacagaggaa atgctggggc tggatcttca aggaagaatt gggagtcaca ggaagatgag 6600

agaaaaaaaa gagcttttta gagagggagc aacgtataga ccattcaaca atgctgggtt 6660

gagtgatgtg agggaaacga aaatttgttg aagactgcag tagagaggag aaaagacttc 6720

caggtttcag aacgttccat gagagtgcca agatgtagtt tctgttttgt atctgccaca 6780

gtgacaattt cccaggaatc cttatagatc cagaaaacat gaatgattat taatactaac 6840

gaatagaaga aaagttgagt ggctagtttg tgagatcttc ctcagagaat atccctggaa 6900

acagatacac tttgagcagg atttcttagg tattaaaagc acaatcccac tgtgtggatt 6960

tcgccaccac atatccttgg tctttgattt aagctattct caatattctc cagcaggttt 7020

cataagtgtc ttgggtcagt tgcttttttc ctccataaat cttcctttct agcccccttt 7080

cacatccttc actaccttca gctgcagttc agaagaccta gactcctgat ttaggacaaa 7140

aatagaggct ttctggctgg ggatccttca gcttcctgcc ctacactgcc gagcatgtct 7200

ggaaactgcc cactgtctct ctcctcccct ccagcttcag tagaaaagat attcatgctg 7260

ttaatggcta atcactctac tgtgctttga atctgtctct ttctaatttc tcagatgcct 7320

cctcccgtcc atcatcccct ctcctgtgta tctttaacgt ctcactggct gcttatttca 7380

gagcaccatc ccaccctgcc acatcctact gcctgtaacc tccattgctt ctttccagcc 7440

caataatact tctagagcaa atctgaccac ttccctgctt gaactcccca gttgctttct 7500

gttatattta gaaaaaacaa aaaatgcaaa cttcctacct tggcatttat gatgcatgat 7560

gacctggccc tgttcttcct cagtcattat catttctgac taccactccc aatagattct 7620

cccctctccc caaccacatc tcaactgcaa atgggcaatg gcttctcaga ccttcctgac 7680

ctggcaactg ctgttagctt atggtatgat ctttcctaac tctcccttta ctcatctaac 7740

tactactcat cctttaaatc ctctttggtg ctatggtgcc ttgtgtttac tgctatagtc 7800

atttacctgt atcactagca caaggcccag ccatatgcag gaaatatact atttgtccgt 7860

caaataagtg actgatgagc tgagtgagtg atggttgctg ccctgcctct tctggccccc 7920

agtgggataa acaacatcct tctgtgttgg tcaccccaag tttcactcta cttccatgcc 7980

aggctcgcca aattctggaa cagaccccag tgaaggagct ggtgagcttc aagtggaaca 8040

agtatggccg gccgtacttc tgcatcctgg ctgccttgta cctgctctac atgatctgct 8100

ttactacgtg ctgcgtctac cgccccctta agtttcgtgg tggcaaccgc actcattctc 8160

gagacatcac catcctccag caaaaactac tacaggtgat tctcctcaga aggggatgag 8220

agagaattgc atcaaggggt gatggtgctg gaaacgaagc catgctactc agggacgatg 8280

ccacgctctg ggttccagca ggtgtaggct cctaaacacc caggtagaaa ttagacatga 8340

gaccaggagg ccacagatgg ggtttaatga gcacaagagc atttgtccag ggagctgtgg 8400

attttctcac acaaactttg aaagagtgct tggcatgagt aagcctgtgc ttttgtgaaa 8460

ttttttggta atttacaaaa agtatcaggg ttttttagtg ggatgttcaa ggtaaactct 8520

attgctcaaa aggaggaaaa ttgtgctcca agtccatttc tgagtccatt tttgttgtat 8580

ttgggcaaca cagacagccc tgctggcctg caccctcttc tgtgtgctgt ctatatgggc 8640

ccatgggaat aatgtgtcag ctgtctgact gcctgtcaga cactctgcag aaggatgtct 8700

ggattgggta tcaggctgaa gtaaatactc tctgaggctc catttagtgt tgtgggcctg 8760

ccattctgtg ttgtggtggc caagggaaaa ctagggttag gaagaggact gattcgaggt 8820

acacagtaaa tatagaaatc tgctgttgaa gaaagtaaac ctcctttttg tgcaacagaa 8880

agggtagact ctaatgagca cttctgaata tatatattta acaactgtag ctatatttga 8940

ggggttgact ctttgtggat tactaaaatt tttgtcctgg tgagctgaaa atacacagaa 9000

ggtgatgtag gtatgtagca gaaagcaaaa gaccaggaaa aagaattttg caaagcaagt 9060

gagaactgag tgattggaga tttatctcaa tgcaggagag agcagccata ggtatagctc 9120

agcctggact aggaagcagg gtagtttcag tcggaaagag aatataaact ttctatagcc 9180

aagaagcaaa aatcctgaat ttgtttagac caaagacagc gatatgcaca gagaaagaac 9240

agggcaaaga gcccaaatga accctatgct aggtagagac actccatctc tcctgttttt 9300

agaattgcct ttatatatat acatgtatat gtatatatat atatatatgt atatatatac 9360

atgtatatgt ctatatatat atatatgtat atatatacat gtatatgtgt gtatatatat 9420

atgtatatat atacatgtat atgtatatat atatatatgt atatatatac atgtatatgt 9480

atatatatat atatgtatat atatacagtg tgtcatgtgt ccagatacac tgcctctcac 9540

caagtaggtc ttgctgcctg gggaggacaa gcctgaatgt gttagagaca ctggaagcag 9600

ttgtgctgca tgcatctcag tctggccgca aaagttgtcc ctggcatgga gacatgagga 9660

aaataaggac agttaggaga tggcaagtgg agggaggagt gattagaaat tgagaaaaca 9720

caaacaaggt ggggatgact ggacttttcc aatgctcctg gccctatttc ctcagatatg 9780

tcttctccaa ccttcctttg tctcaaattt acttggagat tttctgcatt ttgaaggtag 9840

acaggactct gggttaaaag gtgggcaatg ctgagccact gataaccaaa actctccttc 9900

ctgtgtgtct ctttctcttt gggacagggc tagttatgta caatcacatt tctttgatcc 9960

agatagtctg tatgtaaaag accgcctcct ttgcttcagg actcagaagc ataattatag 10020

ctgaacacct gacccatcct tctttctttc tctcttctgc cttccttctt gttccttggc 10080

atttttaccc tttctgcctt ctgcttcagt gaaagccact tcagctcaaa gagaagactt 10140

tggactttca gatattcttt tcttggtgga gaagtgggtg ggtaagagga ttgagaatgc 10200

tgctggaagg ggttagataa caaagaacgc taatgaaaag tccctatgct ttgagccctg 10260

gccctgattt atgggatgac tatgttcttc ttattcagta gtaagcccat ttggattaga 10320

tcaggggctc ctaaacttat catcatgatt tgaaagattc tgttttcaca gtgcaccaga 10380

ttttttaaat aataggaaat tccttttttt tcctactgtt ttcttacttg aaagtcttag 10440

aaaaaatgaa aacgcacaaa tacagtgtct tgatttgact catgcaattg ttgacatatt 10500

agttagctgg tggagctttt tcttagatga atgaatactt tactgtccaa ttatattaat 10560

aactctggga tcacagggat ctcagtcact tcaggacttc ctctgcagat ctcttgggat 10620

ctgtggaaat cactgggctc catcacccct aagatccttt ctagtttaat atctggcatt 10680

ttataagagt agtgcaacag caccatctca gaacagatga gagaaaacaa attcctctgt 10740

ataaggtagc aaaaaactac acaaaagctg atgtttgtaa agctggagtt agtttatttc 10800

tatccaattt gggggctatc ttaatgactc taagacacct cattgctcag ccataaaagt 10860

tctgctttac tagagatttc ccagaactct attaatttga gttaacttct tatctccata 10920

tatttctact tggtttaggc agagcagcat gcactatagt actagttctc ttaggtttcc 10980

aggtgagtaa aaagctcaaa gtcctggagt ccagcagctc gggttttagc cctaactctg 11040

aaatttaata cttgtttgac cttaggaaaa tcctacttcc tgccagatct gtgattttta 11100

acccagactg tacactagaa acctaggaag ctagtaaaat gactagctcc taggctacac 11160

tttagtagac ctaataagtt ggaatgtctg aagctgaggc tctggccttg gtatttttaa 11220

gaaactcttt gagtgagcca aatgttctgc caaggttgag aacccgtggc ttccatcttt 11280

gaggtaagtg cagctctgcc tttccagtct taggcccacc actctggtca tttagattcc 11340

cggattactg ttcagcaggg agttcaaatt tctcgtcaca tctccttttt gacttttgaa 11400

aaagattctc ttgagccctc ctttgatttt ctctttccca gaaatatttt gctaatccat 11460

tcattaaata aattattatt gagagcctca tataggctaa acagctgttc caggtgttgg 11520

aaattcaaca gccatggtct gagcaaaaca cacatgatgt ctgctttctt ggagtctaca 11580

gactggtttg tattaatcaa acgatcatac aaatagatac ttcattataa tctgtgactg 11640

ttggcatcaa tattattttt ttcttatgat gagaaaagca cagagtgggg aaaaaactta 11700

taacaggggc ctggtccctc tgggaggcca gagcctgctt ctgagaaaga tgactgagct 11760

gagatctagg tatgaattca acgtgaaaag agagttctag acagaaagaa tgcatattca 11820

aacgcctcaa ggcaggaata acctcaccaa agtctaggaa ctgaaagaag attcactaga 11880

gcacagagag ttgggttggg tacctccagg tgaggttgtc cagacagatc cttctcaaat 11940

tatatagtct taaagggttt tacgctttat ccccaggggg atgggaagtc agaaggattt 12000

taaacagggg gatgtacaga gtaatttttg ttttagaaaa attactctgc ttgcatggtg 12060

agtaacagat gtggggggtg gtaggaagga cattgggaga ccagtcagga ggctattaca 12120

gtttagcaag caaaagataa tgttggctgg gcacggtggc tcaggcttgt aatcccagca 12180

ctttgggagg ctgaggcggc ggatcacgag gtcaggagtt tgagaccagc ctggccaaca 12240

tagtgaaacc ccatctctcc taaaaataca aaaaattagc caggcatggt ggtgagcacc 12300

tgtaatccca gctattcggg aggctgagag aggagaatcg cttgaacgtg ggagtcagag 12360

gttgcagtga gccgagatca cgccattgca ctccaggcca ggcgacagta tgagactccg 12420

tctcaaaata aataaattta aaaaaatggt ggattggcct aggaatgtag tggggagata 12480

gagggaaggg attggcatca ggagacttat tgaaggcaaa atcatcccta ctgtgtttcc 12540

atcttttaaa aatcatcttc tacctatcac agtccgtggg atctatttgt ccacacatca 12600

tcatccctcg taagttctga acaatccaaa gcaccactgc tatttgaaaa gcaactgatt 12660

gccaactcta tacaactttg ttgaggggtc ctaaccaact catggtggaa caagcacagc 12720

acattataga cagggaaaga gatgaggaga accagggagc ccggtaggta aaaataacat 12780

ccactgagca ctgatcatga acacatgcat tcatcagtgt ttttgaggcc ttattaaact 12840

gtggattctg acagtaaggc tgggtgggac ctgagacttg gcatttccag caagctgcca 12900

ggtgaggcca atgttgtgat tctgtgcacc gcgttttgag tgacaaagca ttattctttc 12960

atttaatctt tgtagaaatc ctaacaaata aatgcaatca tcaccacttt aaagatggtg 13020

aatctgatgt caagagaggt tgaataattt ggcaaagaca ctgagctagc aaggacagca 13080

ctaggattgg gctgcaagtc tgcctccctt cagaatcctc acctttaaac accagacaag 13140

gccatttctc cctccagagt gatgtgctag gtcccagtct tctctattgg ctagccaggg 13200

cttgccacac tcacgtgtat ctacacatct catctttccc tgtcaagagg agaggcgctt 13260

gagaagtgct gtgggagagg atcagatata gaaagattca tacccccaga tactgattca 13320

tacccccaga tactcctctc cagttttgat aattggcact gttttttgat ggggttaacc 13380

tactggcata tagggcaagt cataattctg tgggaatggg tggagactct tcagaagaaa 13440

ggggtaatct cccaagccaa tacccctgag aaaatgcaga gactgggaca tgaaattaac 13500

ctttggaagg cagcttgact tacatgggaa ccagatctct ctctctctct ttatggccca 13560

gcggttctca acttgggata cacgtccaaa acgcctacag aacttttcaa gtaaatgcca 13620

ggttcttgat ccaaattaac ccagggttgg cctggacatg tgtgttgtga caaagactta 13680

acacttgatt ctgatgggca gtcttggtta ggaactcatg cccagttagg gaatcggaga 13740

agctctttgt aagagctagc agagctttct ctgaaaactt cattcctttg caagtctggg 13800

atcatggggg ggaaaaaggt ctagagctgg gctggacaac tggttctggg gcctacccac 13860

ttcccactgt ggtggcctat gttccccatc ctttccacat agtacatccc tggcccttac 13920

gacccagtag tgccccaggc actgctttgc ccactccttc tgggcatttc cttccaggca 13980

atgaactcag ctggctggga agtgagagca aatctcatta ggccaattct cagtgacgta 14040

taaatgccag cctgcatagg ggatttggtt gttgaaagct cattaaattg caagccatgt 14100

tgtggagtaa aaaattccag tcattgttag atgatattta tttttaaagg gatgtattgg 14160

agtaccttct ctgtgcgttg ttagccaggc ctgcttaggt gaccttgatc taggaatctg 14220

aaaatttcag aaaagcacga ttagttgtca ttcttttcct cttaggcctc cagataggtc 14280

tctccatccc cgcttcctgt cctccttacc acccttctgc gtctcttagg caatgcttaa 14340

cttgcacctg ctgtctcctc tctccttttt ctccttcctt ctttcctttc ttaccaactg 14400

agtgcctacc acattgtgtg catttgtgga taaaaagtag gagggaggca tccctgccct 14460

caggaagctc taagactact ggtgagacat ggggtaccca gacactttca gtgtgctggg 14520

agagggctgt caatacacag cagccccaca tgtcccagga gagacacatg acctgggcat 14580

ggaggggcag gaaaggaggc ccaggaggag tgttggcctg acctgagtct tgaggcatcg 14640

gtgagggtga gccaggtcaa catggtggag gaaggcattc tgacaaagtg gcatagcctg 14700

tgcagagacc caacttagca acccagaacc ccaatagctc aatgtggcca gagctcacac 14760

agggtatgag gacacaaggc aggggccata gcagggaggg ttttgtacat cccgtttggt 14820

actctggttt tattttttaa attcttatct cacagtgagc tttcctaatt aaaagaaaat 14880

gacgtacatc gtgctcatga cttacaaatc aagtggcaga tctgatagag agcaactgcg 14940

tcctggacag cccctctttc tcacccatgt cttactcaca ggggacaacc agtttttctt 15000

tcttttagct cttccttcct gtcatcacct ccacatccct ggaatcaaat ccttctatct 15060

ctacttgcta atcaaattta gacattatct agtgaatttc tgtgacgata aagattaaag 15120

aacactctcc actgccacct ctcctgcatt tctattctga aggcattggg gagctaggaa 15180

tgatgttaaa cagataagtc tcaggatgat atctgtgttt tagaaaggtc aattcagaag 15240

cattgtgcgg ggtgggttga atgagcggaa atagatgtag ggtaaactag atggtagatg 15300

gcatgggttt gccctaaggc ataagttctg ttaaccaagg gaaaggaaaa catgtcagag 15360

agaattggga actgaatgag taaggctgga gaatgggtgc cctgataatt tactttgcca 15420

aacataacca ggcaacaata ttttcataag gaccctaaat ctagagagtt gtgaatcagc 15480

atctcaatct agaaaaaaaa atcttgaatg caattcccaa agaataaaat ttttaaaatt 15540

aaaagaaaca tccattacag tttgggctta gggtttaatt ctaggggtgc aagggtggtt 15600

aattaaaagg gaagcctatt agcacttcaa tttaatgccc ctttccattt ctgaaatact 15660

gaaatcttcc ttacctttgc aaagagtatc tattctaaag caagagctaa cattttatat 15720

cagtgtgaaa cactggagtt ccccagaaag tctaaagaaa atcaaagtgc tttaaaagcc 15780

caatattatc attacttcta ttgcaacttt tacatacata tattgttttt gttttccaga 15840

gagattagtt tataaaacaa agcaaacaaa accctacaga agggtttgta ttctggtctt 15900

gtagagcagc aaggaagggg gctctcagga gtctgtggca ctttgcagcc tggtttaagg 15960

gtaaggaccc cggggacaga gtgctggagg cagctactct aggtcagtca gatcccaggc 16020

agtgcagagg caaaaaattg gggtttctga acacctccat tactcattag tgaatttgtg 16080

cagagtaatg cgtacttgta aagaaatctg ttgaactggt gagaggaagc cccagcagga 16140

gaatttggtg accaaaactg tccccagcat tagaggggtt tcacattttc atctcctgaa 16200

ggaatggctg ctgtgactcc ctggctggat tctaggggca ccctgatctt cacagccatc 16260

ctcgccaact tatgtgtagt ccttttcttc ttgagccaag aaaagattgg tagcatcttt 16320

tcagacatat taaatcaaga cacgacattt ttaaaataaa aatattttta tagacacata 16380

tatggcatag atgcagttat ttctatattt aatgttattt tgtttctgaa gtaatgtatt 16440

tgttatttat cctcctactc actacctgac tctgtgaagg agttggcatg ttttacaata 16500

aaagacagat gctatagaaa cattaagata aagaaaaaga acacagtccg tgaactgaat 16560

ttcaggagag aagagaagga attcaaagat agctgctaga gctgaggaca aaaaaacaaa 16620

acaaaacaaa acaaaaaaaa cgcacaaaaa aaaaccagac acacaaaaaa atagaacaaa 16680

acaaaacaaa aactctgaaa ttctttggaa gcaagtaaaa aaagaaattt gctattatat 16740

gataaaataa aatttaccag tttctcaaga gataaatgtc agaaaagttt acagagcata 16800

ccatttttta agccaaaaaa gaaaaaaaaa agtgttgact gatgtctggt cacaggttgt 16860

tagaccacag agttctccag acacacagac agaagcttct ctcctagtct ctcttggttc 16920

cctgtgttaa aagcactgcc tctcaccggg caggtgagtg tgctgctgca gtagagtctg 16980

gagagatgct tacaggttcc agctgattag cagagcaggg ataattgccg ttccttatgc 17040

taactaggga ccacattctc cctggctcaa atcatcagct ttcctgctgg ccccagggga 17100

ctctcaactc ctttcttggg tgcactattt ggagatgtga ccttggacaa cagagctccc 17160

aaaagtcaaa aggctgcttg catgtgttga aattatttgt gcaatatcat gtgggaattg 17220

tctacatggg aacacagttt tgtagaaaga agatatacag tttttattat gttttcaagt 17280

gagctcataa cttcaaaaac ataaggataa ttaatgtagg attctagcac tacatctgtg 17340

atattgcata agtttcaccg tgaagtaaca tgcattgaag tagggtggtc ttcattccac 17400

cttgaccctt ggactatacc ttctacaact actgctcagg ataggcaggg acccttcctc 17460

atagtgtctc tagccccttc tctgctcttt tctgtaatgt caaatcatca taaaaccaca 17520

gtgaatatat ctatagtgat ttatgattta taaagttcat gtaaatatat cattaatgta 17580

agcttcacaa tgactctggg aggtaagcac tgtagcctcc attttacaga tgagaaaact 17640

caagttcaga gattttccat gaatggttca aggtctcact agaggtagta gattccggat 17700

ccgtgaggga acctgaattc tgtgatatgt ttacagtgta ttcttcccac tagaagccat 17760

gatgaggttg gaaaaggtgg actttccaca tagctgaaac catcccaagc cactaatacc 17820

tcttcctgtt agctcacttg atgcgctgaa atgtgttctc agctgctcag tatatacatc 17880

ataaatttct gcttacagca tctatagaaa aggcgatttt gctagatggt agctcaagac 17940

attagcatat gtgacgtcta ttaaaccttg gatacataga ctcctgtcct ggtcacagag 18000

ttacccacaa tggcaagtac aatgggggtg gggtgatgct ggtgggaagg ggagtttcct 18060

ttattttgga tgggctttag cagctttcga tcatttgcac aaaaggattt gactctgccc 18120

atcagaaagc acttcttctg aatttcccca cataggaggc ctatgagaca cgtgaagata 18180

tcatcaggct ggtgggggag ctggtgagca tcgttggggc tgtgatcatc ctgctcctag 18240

aggtgagggg caggcggggt ccaggctaat cccatcccat ctaaggccca gactgtaccg 18300

cagtccacat tgtatttttg ctttgccgtc ctctccccag attccagaca tcttcagggt 18360

tggtgcctct cgctattttg gaaagacgat tcttgggggg ccattccatg tcatcatgtg 18420

agtatcccct cattccagcc ccctgccttt ccttaccaga atcttttcct gtctccagct 18480

cactctctga gttctaggcc tcaggtcact ctcactccaa ggacttctct ccagtggact 18540

tgtaggtgta actgtatgtg cgttttaggt gtaaagacta agcctgagtg tgtggatacc 18600

accccggtca ccactatgct gtagggacct aaaatagttc cagcttgtct ctcactctct 18660

gctttctgct cccccagcat cacctatgcc tccctggtgc tggtgaccat ggtgatgcgg 18720

ctcaccaaca ccaatgggga ggtggtgccc atgtcctttg ccctggtgct gggctggtgc 18780

agtgtcatgt atttcactcg aggattccag atgctgggtc ccttcaccat catgatccag 18840

aaggtcagtg ctttcccgaa gcttcctccg cctcaggcac aagaccttca cagacagagg 18900

gatggaggta gaatggagct ggtagaacta tgggggcctc ttgatctgtt agcgttagct 18960

gctcaaaagc agtggaaaag gaatcatccc accctttctt ccttctgttt cctgcagatg 19020

atttttggag acctaatgcg tttctgctgg ctgatggctg tggtcatctt gggatttgcc 19080

tccggtaagg caccatacac tcctctacta gatcagcaga tctagttctc cagaaagttc 19140

actctcattc ttgctctcag ttctcatttg cacttcattt tgctttcttt ttttttgttt 19200

gttttttgtt ttgttttttc ctgttggaag gtaagtctaa aagcacaccc atatttatag 19260

gaccaggggc cactcaggtt ggcctggaac gtaaagccat gtgttcaggg ccaataagat 19320

ttatgttcac taactgcctt gcaagatctt taaaggagag acaggaaaag acttttggtg 19380

agaacagggc attaaacagg aaagactggc cagtcatgga tctcagaaaa ccatgtggac 19440

aaacagagat agtgccatgc ttaactctcc tatgtttaca attcagaaga ggaaacctaa 19500

agcactcaac aacatataac ttgctttttt tctatatgtg gtcatttaaa gctgagaaaa 19560

atatgaaaga attagaaaaa ggaggtgcag aaaaacagct tggacagttt gaagatgaca 19620

gagaaaacaa aaaagaagag ttgtgaagag agactgggag aacaggaaca cacaggcacc 19680

cagtcaaggc acagccagtg aaagtgccag ggagcaaaag aaaggaacgg cacagcagga 19740

aaacacaggc aggtggaaac agccagaggg aaggagaggg tgggtggagg agcagaagaa 19800

agtccttagg gaggggcaga aagaggaagg cccaggtctg aaaagcaaaa ggccagtagc 19860

tcaaattcag tcctcagtgt ctgcaactga ccatctatta gacaagaaga ggcactagaa 19920

ggatcccccc atctctaaaa agcaatgact cagtgcagag gactctatta ctgtcctcag 19980

ggatttcccg ttcaatgggg attcccacaa aacaacagac caaaacaaaa aacaaacaaa 20040

acaaaacaac aacaacaaaa cccccaccaa aatctgaaat gtgaaagagt aaaaccatgg 20100

cgttctgcct gtttggagtt ttccacccca agcccttatg taccctttcc tcccactccc 20160

tggccatcta catctataca taacacagat tacaaaatga ggtgatggtc ccagagtggc 20220

tttctgtaga tgtcctctat ccagaactct ctaaatgttc acttcttggc cattgcactt 20280

gcttttctct taaataaata aggaggcaat gctttgtcat agaccctgtt tctgctctca 20340

gaatgtgctt cagctcccta tgcagtgagg aggctttcaa gcagtccagc gctgtcctga 20400

agttccctta cgctcactgc tatccagtag gctccaccct aagcaggaat ccgtttccta 20460

tgttggttca ggttcactca gggaaggtca gagtgatgga gggttatcta gaggccatca 20520

agattaaaaa aggggctctt cttttagcct gttcatgtgt tctgttcttt aaaaattgtt 20580

gaggattcca tacattcttg taataacttg ccttttaagt atttggtttt ctaaaatgca 20640

aaaaccagtt aggtgaccta ttatccatta tccatacaaa cacacacgct gcacacacaa 20700

ttttgttgtt gttaaatgac tattaccatg accattaccc agagtggtga acttaatcat 20760

aggacatctt agggttagac agttccacag gaaactacat ttattagtat gtaattatct 20820

gtgcatgtgt gcaagtgtat ttagatagat agatagatag atagatagat agatagatag 20880

atagatcaga gatagaataa gtgctaacca tgtccatgct tattgtgata actccattat 20940

cccatctcca tgtcctcttc ctgggcggag cagcgttcta tatcattttc cagacagagg 21000

acccaaccag tctggggcaa ttctatgact accccatggc actgttcacc acctttgagc 21060

tttttctcac tgttattgat gcacctgcca actacgacgt ggacttgccc ttcatgttca 21120

gcattgtcaa cttcgccttc accatcattg ccacactgct catgctcaac ttgttcatcg 21180

ccatgatggg cgacacccac tggagggtgg cccaggagag ggatgagctc tggagggccc 21240

aggtgagttt atttgtgtta gtcttgctaa gaagcagggg ttggagagaa aaatcaaagt 21300

ggaaaatgtt atggtggatc attcttaaaa gagtcacaga aaaacccagt gaggctgtca 21360

cctcagaaga ggtttgtgta aacctgaggt acatgtggat aaatggaagg gacagaagga 21420

ggattaaatg gacgatagtg gcaagaggtg gggttgttgc tcacagggga aaagtagacc 21480

taggattgat ccttgagtcc agtgggaact agaagtgacc ttgttctggt atattccagt 21540

tttcaatttg gatcttatct tctatttgct tctgtaaatc cctgtttctc tatgatcttt 21600

tctttatatc accatagact gataaatatt agaattagaa gtgaccctca atggccctca 21660

agttgacctc ctacttaaca taataatcat ttattggtca gaatgtcatt caatatcttc 21720

tctaatgctg attagtacct tattatcttg caaattcagg tagaggttgg gaggaaggga 21780

gttttcccat atttttaaag attttgttat tagacattta tgtcgcattg agctaaaacc 21840

tatcttctta aaatttcatt ctttggcgcc agtctttccc tctcatatat cacagaacac 21900

attcttccat ataagcacca ttcaaatatt tgaatatagc catcattttt cccttagact 21960

tttcattcat ttaatgatga tcatttagac cctgctatgt gttgacaaat gcctaggagc 22020

tagggacatg agcataaaca caacagacac catatgtgcc ctcatggagc tgacggttta 22080

gttggaaaga cgggcatgaa gtgaatgact tcacaaaaag ctgtaaaatt acagcagcaa 22140

aagtgctata gagaatgtaa ggtgctaaga ggaggataca acagaggaat cggactggga 22200

agtgaagaca tgctttcctg agggagcaac atttaagatg agaacccaaa ggtgagcaag 22260

aactaggtag ttaaaaggaa ggagggaaaa ggatgaaggt caaggttagt agcaattagg 22320

actcttggct acagggacag aaaaccaaac tcaaacagac ttgcagattt ttaaacaggg 22380

ggaagaggat gtgttagtcc tcataaatgg gaagtttaga gaattaaact aactttagag 22440

gtatctggcc agaatgcaga tccacatctt atacattaca tccattcctc tgttcggctc 22500

tcttttgacc tctcttctaa ggcaagctct cccatcgtgt ttgtgagatc atttctagaa 22560

gctccaaact ccaaaaagaa tgaggatgtg caatgcaaca cgcagctccg agggccccac 22620

tcacgttgta ttctgtgagg ctcctggtgc caggcaaaac tgcaggcttt aaaagaacat 22680

ctttccccct gccaattcct tccatgttcc atctcatggg aacacattcg ggtcataggc 22740

acatcgttga atctaaggaa agaccaagga ctagatagtg gaggtgttgg tgatggtggt 22800

ggaggggagg tttgggggaa aatagattaa ttaagcacaa ctgtgtgaag cccagtccac 22860

tcaaaccaat gcactaaaag tgatgaggga gcaattccct aaatgaaaaa tgggtgctgt 22920

tacccgagga aggacggggc tgcatgatgg ctcaggagga acaataaaca tttctttcaa 22980

ggcaaagaag aacagctatc cagggtaaga gcacagaatg actggaagtc ctgagctagg 23040

agggaacttg gtgcaattaa gaaactgaaa gaagtgtgga gtaatgagga agatagggag 23100

tagaaaaaca tgaggtgaag aatttgctat tttgaagatt gcactccttc aattaggaag 23160

ccttcctggt tctgatctat tctgattgca cccttttgtc catgctctaa tagatcatgg 23220

tttctttgaa actagaatag tccccagtat gattagggca atgcggtaca aagcgggact 23280

atgcctttcc ttacttggac ataatgctaa gggtacctaa agctgagtca ggtcttttag 23340

caaatataca aatggccctt attaagcttg tgatcagcta gcactcccac acctttgttc 23400

tatgactcac ttatctaagc ttcctaaatg taaatatagg accttagatt tggtttactt 23460

aaatttcttg ttaatgattt tacaccagca ttccaatcta gcgagataat tttatattgc 23520

ttctgttgcc ccacatatta catatccctc ccagttttgt gtcacaggca tatttgataa 23580

gcttgtttcc catgtttcat ctaagtaatt aatgagggct gagcaaagga cagaacgtgt 23640

ggtggacttg ctaccagaaa tttccctgcc aacttgcagt cattaatcaa cccttttcct 23700

tcctaagatg tggatattta atttgctgaa aattccctga acagtaacag tatcaattca 23760

tccccaagat cgtagagatt ctctttaatc attttattta aatcgagata attggtctgt 23820

ggcttttata tgaaaaagaa gttgagatta gtttggagtg actcattctc catgagtgaa 23880

tagtaatgta agggcaacta ttttattcaa aacactcaca aaaaatcctc tctcatttct 23940

tgatgagtga agctggttgc atggtgcctg gcacaggctg tggggtgaat aggtggctgt 24000

tctcaccact gacctctagt gtcctatctc acagattgcc tttggttccc tttatcagtg 24060

gcctcagtgc ctctttgaac ctcatggctc tttcagagtt cttcctgtat ctcctgcctc 24120

cttccaggtc gtggccacca cagtgatgct ggagcggaag ctgcctcgct gcctgtggcc 24180

tcgctccggg atctgtgggt gcgaattcgg gctgggggac cgctggttcc tgcggtgagt 24240

gatggtgtgc acatgcaaaa ctgcagatgt gtataaggcc acggaagcaa gggtgaccga 24300

caggcgtcct ggagcctaca ttagcctgtc taggggaggg ggctggagct gaagtggagg 24360

ggtacgaaga atgctgtgtg tacctcaggg cagccacgtg taaaaggttg tgtgcatgtg 24420

tccatgctca gacgtcagca tgggttacca gacttgctgt aaccttgttc tgagctgacc 24480

ttcatctcca gtgtactttt ggcttggatc taacttcctc cttcatattc tgccctcatc 24540

tcccggcgtg actctcctct ctcctcaaat ttacttcctg aattgcatct cgtcatcctt 24600

gtgacacgct tatctattag ttggctctgt tttctctttc acattcctac gtattagtaa 24660

cacacaaaca cacacacacc tgtgtgcgtc ctaccaccct gcaagccctg attgctcagc 24720

ctccattatc tctatcccag gttagcctgc cctccatcac ctcccttcac ttgatcccag 24780

atactcttcc tgtttcaact ctgctgcctt atttctgctt ccctcttgac cctgctttct 24840

aaatggtcac ctctacttct cccctgccca tgtctcccca cctggattga gtcctttccc 24900

ctttcccctt atcagggttg agaaccacaa tgatcagaat cctctgcgag tgcttcgcta 24960

tgtggaagtg ttcaagaact cagacaagga ggatgaccag gagcatccat ctgagaaaca 25020

gccctctggg gctgagagtg ggactctagc cagagcctct ttggctcttc caacttcctc 25080

cctgtcccgg accgcgtccc agagcagcag tcaccgaggc tgggagatcc ttcgtcaaaa 25140

caccctgggg cacttgaatc ttggactgaa ccttagtgag ggggatggag aggaggtcta 25200

ccatttttga ttaacatcgc tatcactctt gaccttactc ccggttggcc tgggggcggg 25260

gacagagacg gagacctctg cctatgcaag tgtctaactt ctgtgcctgt taatcatggg 25320

agggtgagac agaacaatcc ctaaagggtc atgcctcaca cttcacatca gaatttctgg 25380

caatgggcaa tggtcatcga ttgtctcacg tattttctgg gctcttgcaa gtcacccatc 25440

tcaggaaaaa ggaggttggc aactaaagac atgaggcagg gatgctagat taatgtcagg 25500

accatttctc ttctgcccca cgcagcccct agaaagtagt aagctgtgag gcttttctgg 25560

ctccccaggg cttacgtggg aagagccagg catggcatag aggttgtggc ccttcttttt 25620

ttc 25623

Claims (3)

1. The application of the reagent for detecting the SNP locus related to the susceptibility of the heavy metal poisoning in the preparation of products for detecting the susceptibility of the heavy metal poisoning of a subject is characterized in that,

the SNP locus is 103 th nucleotide C > T of TRPV5 gene;

the sequence of the TRPV5 gene is shown as SEQ ID No. 10;

the heavy metal is lead;

the reagent comprises a PCR primer pair for amplifying the SNP locus and a single base extension primer for amplifying the SNP locus, wherein an upstream primer of the PCR primer pair is shown as SEQ ID No.1, a downstream primer of the PCR primer pair is shown as SEQ ID No.2, and the single base extension primer is shown as SEQ ID No. 7.

2. The use according to claim 1, wherein the reagents further comprise PCR reaction reagents, and/or alkaline phosphatase and its reaction buffer, and/or single base extension reaction reagents, and/or desalting resin;

the PCR reaction reagent comprises: dNTP, Mg²⁺PCR reaction premix and DNA polymerase;

the single base extension reaction reagent comprises: ddNTP, single-base extension reaction buffer solution, single-base extension reaction stop solution and single-base extension enzyme.

3. The use of claim 1, wherein the reagent for detecting SNP sites associated with susceptibility to heavy metal poisoning can also be used in combination with other products for detecting susceptibility to heavy metal poisoning in a subject.