CN101565705A - Schistosoma japonicum microsatellite locus and application thereof - Google Patents
Schistosoma japonicum microsatellite locus and application thereof Download PDFInfo
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Abstract
The invention discloses a microsatellite locus and an application thereof in schistosome biometrical genetics. The microsatellite locus is provided with a nucleotide sequence selected form SEQ ID NO: 1-17. The microsatellite locus provides a genetic mark which can effectively apply the schistosome biometrical genetics research. The invention also provides a primer of a microsatellite locus sequence by special augmentation. The primer has favorable augmentation effect and unique augmentation product.
Description
Technical field
The invention belongs to biotechnology and genetics field, more specifically, the present invention relates to a class japonicum microsatellite DNA, and the application in the Schistosoma japonicum population genetics.
Background technology
SSR (Simple Sequence Repeat) claim microsatellite DNA again, it is a kind of emerging molecule marker, it is the tandem repetitive sequence that reaches tens Nucleotide that repeating unit forms by 2-5 Nucleotide, and it is distributed widely on the different seats of whole eukaryotic gene group.The repetition number of the same seat of Different Individual repeating unit may be inequality, thereby form polymorphism, i.e. SSR molecule marker.SSR is compared with other molecule marker such as RFLP and RAPD, has the polymorphism height, analyze easy, easily be automated high flux screening, low cost and other advantages, known its sequence after, as long as synthetic primer, doing a PCR reaction and just can obtain the result, is a kind of ideal molecule marker, and the genetic map ground that has been applied to the mankind and other species is set up.
In recent years, the research of excavating microsatellite locus from the full genome of Schistosoma japonicum more and more causes people's attention, and many scholars have done a large amount of research.But owing to lack the full genome database of Schistosoma japonicum, the site that many institutes adopt all is microsatellite locus of Schistosoma mansoni, and this gives research and used the work zone problem.
Summary of the invention
The object of the present invention is to provide a class japonicum microsatellite DNA.
Another object of the present invention is to provide primer corresponding to described japonicum microsatellite DNA.
Another object of the present invention is to provide described japonicum microsatellite DNA application in the Schistosoma japonicum population genetics.
In a first aspect of the present invention, a kind of isolating polynucleotide are provided, described polynucleotide have and are selected from the nucleotide sequence of SEQ ID NO:1-17 shown in arbitrary.
A second aspect of the present invention provides the purposes of described polynucleotide, and it is used for as little satellite mark:
Determining of schistosomicide genetic affinity; The location of japonicum gene; The conservative property analysis of various or each subspecies of schistosomicide; Bilharzial evolutionary analysis; Or bilharzial kind or the evaluation of region population.
In another preference, described schistosomicide is Schistosoma japonicum (Schistosoma japonicum).
In a third aspect of the present invention, provide the primer that a kind of specific amplification is selected from the microsatellite locus sequence of SEQ ID NO:1-17 shown in arbitrary (it also can increase and be present in (promptly identical with this microsatellite locus locus) on this corresponding microsatellite locus, but the sequence of simple repeated sequence number different with the simple repeated sequence number of the microsatellite locus shown in this SEQ ID NO:1-17 (length that is sequence can be different)) (to).
In another preference, described primer is that primer is right, described primer to have be selected from following sequence to shown in sequence:
SEQ ID NO:18 and SEQ ID NO:19; SEQ ID NO:20 and SEQ ID NO:21; SEQID NO:22 and SEQ ID NO:23; SEQ ID NO:24 and SEQ ID NO:25; SEQ ID NO:26 and SEQ ID NO:27; SEQ ID NO:28 and SEQ ID NO:29; SEQ ID NO:30 and SEQ IDNO:31; SEQ ID NO:32 and SEQ ID NO:33; SEQ ID NO:34 and SEQ ID NO:35; SEQ ID NO:36 and SEQ ID NO:37; SEQ ID NO:38 and SEQ ID NO:39; SEQ IDNO:40 and SEQ ID NO:41; SEQ ID NO:42 and SEQ ID NO:43; SEQ ID NO:44 and SEQ ID NO:45; SEQ ID NO:46 and SEQ ID NO:47; SEQ ID NO:48 and SEQ IDNO:49; Or SEQ ID NO:50 and SEQ ID NO:51.
In a fourth aspect of the present invention, the purposes of described primer is provided, whether be used for measuring the japonicum gene group exists corresponding to described microsatellite locus and (has the sequence identical with this microsatellite locus, perhaps be positioned at this corresponding site (promptly identical), but simple repeated sequence number different with the simple repeated sequence number of described microsatellite locus (length that is sequence can be different) with this microsatellite locus locus) polynucleotide.
In a fifth aspect of the present invention, a kind of method of determining the genetic affinity between the schistosomicide in two or more samples is provided, described method comprises:
(1) serves as a mark with described microsatellite locus sequence;
(2) primer of the microsatellite locus sequence of usefulness specific amplification (1) increases to bilharzial genomic dna in described two or more samples respectively, thereby obtains corresponding amplified production;
(3) compare the similarities and differences of bilharzial amplified production in two or more samples, thereby determine the genetic affinity between the schistosomicide in two or more samples.
In another preference, by the band number and/or the situation of electrophoretic analysis amplified production, the band number and/or the position consistency of amplified production are high more, represent that the genetic affinity between two or more schistosomicide is near more; The number of entries of amplified production and/or position difference are big more, represent that the genetic affinity between two or more schistosomicide is far away more.
In a sixth aspect of the present invention, provide a kind of and be used for determining schistosomicide genetic affinity, location japonicum gene, schistosomicide is various or each subspecies conservative property is analyzed, the detection kit of schistosomicide evolutionary analysis or schistosomicide cultivar identification, contain in the described test kit: specific amplification be selected from the microsatellite locus sequence of SEQ ID NO:1-17 shown in arbitrary primer (to).
In another preference, also contain the material that is selected from down group in the described test kit: pcr amplification reagent, electrophoresis reagent, or sequence analysis software.
In a seventh aspect of the present invention, a kind of polynucleotide collection that is used for genetic analysis is provided, described polynucleotide collection comprises the microsatellite locus sequence shown in the SEQ ID NO:1-17.
In another preference, described polynucleotide collection is used for as the set of little satellite mark: the determining of schistosomicide genetic affinity; The location of japonicum gene; The conservative property analysis of various or each subspecies of schistosomicide; Bilharzial evolutionary analysis; Or bilharzial cultivar identification.
On the other hand, provide the microsatellite locus sequence that the described polynucleotide of a class specific amplification concentrate primer (to).
In another preference, described primer is that primer is right, described primer to have be selected from following sequence to shown in sequence:
SEQ ID NO:18 and SEQ ID NO:19; SEQ ID NO:20 and SEQ ID NO:21; SEQID NO:22 and SEQ ID NO:23; SEQ ID NO:24 and SEQ ID NO:25; SEQ ID NO:26 and SEQ ID NO:27; SEQ ID NO:28 and SEQ ID NO:29; SEQ ID NO:30 and SEQ IDNO:31; SEQ ID NO:32 and SEQ ID NO:33; SEQ ID NO:34 and SEQ ID NO:35; SEQ ID NO:36 and SEQ ID NO:37; SEQ ID NO:38 and SEQ ID NO:39; SEQ IDNO:40 and SEQ ID NO:41; SEQ ID NO:42 and SEQ ID NO:43; SEQ ID NO:44 and SEQ ID NO:45; SEQ ID NO:46 and SEQ ID NO:47; SEQ ID NO:48 and SEQ IDNO:49; Or SEQ ID NO:50 and SEQ ID NO:51.
On the other hand, provide a kind of method of determining the genetic affinity between the schistosomicide in two or more samples, described method comprises:
(1) concentrates from the described polynucleotide that are used for the analysis of molecule linkage inheritance and select one or more microsatellite locus sequences and serve as a mark;
(2) use primer, respectively bilharzial genomic dna in described two or more samples is increased, thereby obtain corresponding amplified production corresponding to corresponding microsatellite locus sequence;
(3) compare the similarities and differences of bilharzial amplified production in two or more samples, thereby determine the genetic affinity between the schistosomicide in two or more samples.
On the other hand, a kind of be used for determining schistosomicide genetic affinity, location japonicum gene, schistosomicide is various or each subspecies conservative property analysiss, schistosomicide evolutionary analysis, schistosomicide kind or region population are identified detection kit or system are provided, contain in described test kit or the system: the primer of the concentrated one or more microsatellite locus sequences of the described polynucleotide of specific amplification (to).
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 .PCR amplified production electrophoretogram.Wherein, M:DNA mark, the single sample in popular district, 1-96:96 Wuhan on the sjpn8 of site pcr amplification product.
Fig. 2. heterozygote sample genescan traces figure.
Fig. 3. homozygote sample genescan traces figure.
Fig. 4. the genetic distance between each sample of popular district, Jiangxi Province.
The analysis of the hereditary difference (AMOVA) between the individual different popular districts (Tongling, Guichi, Duchang, Changde, Yueyang, Shashi, Xichang) of Fig. 5 .7 Schistosoma japonicum sample population.
The UPGMA pedigree chart of Fig. 6 .7 different popular districts (Tongling, Guichi, Duchang, Changde, Yueyang, Shashi, Xichang) population.
Embodiment
The inventor is through extensive and deep research, develop a class and be applicable to the polynucleotide collection that the schistosomicide sample is carried out functional genome research, the assignment of genes gene mapping, relationship evaluation or cultivar identification, described polynucleotide are concentrated and are included a plurality of simple repeated sequences (microsatellite locus sequence, SSR), utilize the polymorphism that can detect schistosomicide population gene corresponding to the Auele Specific Primer of these simple repeated sequences.Therefore, one or more simple repeated sequences of concentrating based on these polynucleotide can be analyzed bilharzial genetic affinity etc.The inventor has also designed specific primer based on described simple repeated sequence, and described primer amplification is respond well, and amplified production is unique.Finished the present invention on this basis.
As used herein, term " contain " or " comprising " comprised " comprising ", " mainly by ... constitute ", " basically by ... constitute " and " by ... constitute ".
As used herein, described " simple repeated sequence ", " tandem repetitive sequence ", " SSR site (sequence) ", " microsatellite sequence ", " microsatellite locus ", " microsatellite DNA " is used interchangeably, and all is meant to have to be selected from the nucleotide sequence of SEQ ID NO:1-17 shown in arbitrary.
As used herein, described " polynucleotide collection " is a kind of set of polynucleotide, wherein contains the polynucleotide shown in the SEQID NO:1-17.When being used for genetic analysis, can select one or more described polynucleotide from described polynucleotide, as the SSR marker.
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
Simple repeated sequence
As used herein, described " simple repeated sequence (SSR) " be called again " tandem repetitive sequence (and ShortTandem Repeats; STR); or " microsatellite DNA (Microsatellite DNA) "; be meant a kind of short sequence; for example a kind of single, two, three, four or five-Nucleotide, it repeats once in a certain specific nucleotide sequence at least.
For adopting the SSR site is the population genetics research of genetic marker, selects suitable SSR site and seems particularly important.At first, its modular construction requires simple, because complex structure (the multiple base is many, and the insertion of its non-repetition base is perhaps arranged) will bring certain deviation to subsequent analysis; Secondly, the position at microsatellite locus place should be away from gene coding region, if be in or near gene coding region, these sites just may be lost in the process that goes down to posterity of population, can not reflect the hereditary feature of colony truly; The 3rd, selected microsatellite locus original series will have the specificity of height.In addition, very high requirement is arranged also in the selection that is studied sample size,, may lose some allelotrope, thereby bring deviation for the calculating of hereditary difference between the sample if the sample size of choosing is too little.Therefore, the selection in suitable SSR site need be passed through big quantitative analysis and test work.
Each SSR site of the present invention has and is selected from the nucleotide sequence of SEQ ID NO:1-17 shown in arbitrary.The set of these nucleotide sequences constitutes described polynucleotide collection.
SSR of the present invention site can effectively reflect certain population exclusive genetics feature, it has following feature: described site all is the tumor-necrosis factor glycoproteins of 3-4 base and does not comprise complex construction; It is special that the original series in described site is the schistosoma japonicum gene group; The multiplicity of little satellite in described site is at 10-25 time; And described site all is in position in the full genome away from the coding region.
Primer
Although each SSR site that described polynucleotide are concentrated can be different (the repetition number differences that promptly contain simple repeated sequence) in different populations, but mostly its two terminal sequence is the single-copy sequence guarded, therefore can be according to a pair of special primer of the sequences Design at these two ends, by round pcr core microsatellite DNA sequence amplification is therebetween come out, utilize technology such as order-checking or electrophoretic analysis just can obtain its length polymorphism.
Therefore, the invention provides the primer in the described SSR site that can be used for increasing.Described primer designs according to the conserved sequence at these two ends, SSR site.The method that the primer design method is well known to those skilled in the art.Usually, can according to its two ends sequences Design primer not of the same race, determine only primer at a SSR site by the PCR test.These primers more particularly, when the design primer, adopt close Tm temperature, so that can be used in high-throughout amplification and detection.In addition, the uniqueness of guaranteeing the site of primer amplification is necessary.Generally speaking, the Oligonucleolide primers of 18-26bp has specificity preferably.
Adopt described primer, can amplify corresponding SSR site in schistosomicide (particularly Schistosoma japonicum) genome.And, adopt identical primer right, be template with the bilharzial genomic dna of different sources, can obtain to comprise the polynucleotide of polymorphism SSR structure (not equal) as SSR multiplicity difference or length.
Use
Simple repeated sequence of the present invention and corresponding primer thereof serve many purposes.Include but not limited to be used for: the determining of schistosomicide genetic affinity; The location of japonicum gene; The conservative property analysis of various or each subspecies of schistosomicide; Bilharzial evolutionary analysis; Or bilharzial cultivar identification.
Each worm kind of schistosomicide: by increased in these Schistosoma japonicum SSR sites the site that searching can be increased in other schistosomicide beyond the Schistosoma japonicum in other kind schistosomicide.If can successfully be increased in the site, illustrate that this site exists jointly in multiple schistosomicide, this site just can be utilized by other blood suction.
Bilharzial evolutionary analysis: microsatellite marker can be used as the means of relation research between species, carries out the evolutionary analysis between species.Determine evolutionary relationship between species according to genetic distance.
Bilharzial cultivar identification: the SSR mark that utilizes the inventor to develop, schistosomicide is carried out pcr amplification, seek the pairing varietY specificity SSR of each kind mark, just can realize that the schistosomicide kind carries out cultivar identification under the prerequisite of morphological specificity binding molecule mark.
As a kind of optimal way of the present invention, SSR of the present invention site can be applied to carrying out the comparison of schistosomicide population between the popular epidemic-stricken area of different schistosomicide, analyzes the hereditary difference between each schistosomicide population, or sets up the pedigree graph of a relation between these populations.Perhaps, be applied to schistosomicide kind or region population and identify as molecule marker.
As a kind of optimal way of the present invention, SSR of the present invention site can be applied on the epidemiology.Its application method is for example: certain zone (this area there is no the record-newly-increased epidemic-stricken area of schistosomicide in the past) outburst schistosomicide, can be at random in the human body that infects or animal body, obtain Schistosoma japonicum adult sample (forming a new population), utilize microsatellite locus of the present invention, above-mentioned sample is carried out high-throughout genescan, analyze its genetics feature.Again by its contrast with the population structure feature of having understood, judge the source of the schistosomicide population in this newly-increased epidemic-stricken area, by cutting off this source or propagate bilharzial host's (as positive oncomelania) the path of migrating, thereby can reach the monitoring of this newly-increased epidemic-stricken area schistosomiasis japanica and the purpose of control.
Genetic analysis method
After getting cicada each SSR site provided by the invention or its corresponding Auele Specific Primer, those skilled in the art can adopt multiple technology known in the art to determine bilharzial genetic affinity (or sibship), locate bilharzial gene, various or each subspecies carries out the conservative property analysis to schistosomicide, carry out the schistosomicide evolutionary analysis, or carry out bilharzial cultivar identification, or schistosomicide region population is identified.Because the pcr amplification in SSR site is a specific amplification, its stability and circulation ratio are all better.
The method that detects the product behind little satellite pcr amplification can adopt technology well known in the art, and the easiest the most frequently used method is to adopt agarose gel electrophoresis technology.By number and/or the big or small difference of determining amplified production that compares amplified band.This method is simple to operate, and is with low cost.
Another kind of more meticulous method is the genescan by sequenator, thereby can tell the difference of 1-2 base on the PCR product size well.Genescan can adopt the more known softwares of those skilled in the art, for example GenMapper 4.0 softwares.Through a large amount of verification experimental verifications, the microsatellite locus of invention can satisfy the requirement of high-throughput genescan.
Detection kit or system
The present invention also provides a kind of and has been used for determining schistosomicide genetic affinity, location japonicum gene, schistosomicide is various or the detection kit or the system of each subspecies conservative property analysiss, schistosomicide evolutionary analysis, schistosomicide kind or the evaluation of region population, contains in described test kit or the system: but specific amplification be selected from the microsatellite locus sequence of SEQ IDNO:1-17 shown in arbitrary primer (to).Described primer (to) the different microsatellite locus of repetition number that also can increase and be on the same locus of microsatellite locus of SEQ ID NO:1-17 shown in arbitrary and contain simple repeated sequence.
In addition, also contain in the described test kit various analysis schistosomicide genetic affinities, location japonicum gene, schistosomicide is various or each subspecies conservative property is analyzed, schistosomicide evolutionary analysis or other required material of schistosomicide cultivar identification.For example can comprise the material that is selected from down group: pcr amplification reagent, electrophoresis reagent, PCR product purification reagent, or sequence analysis software.These reagent all can be the conventional uses of those skilled in the art.
In addition, the working instructions that also can contain in the described test kit so that the using method of described test kit to be described, provide more suitable working conditions.
Advantage of the present invention and effect:
1. develop a class and be applicable to the microsatellite locus that the schistosomicide sample is carried out functional genome research, the assignment of genes gene mapping or cultivar identification, described microsatellite locus can effectively must be illustrated the hereditary difference between the Schistosoma japonicum population.
2. found suitable primer at each microsatellite locus, described primer amplification is respond well, and amplified production is unique.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
The acquisition of embodiment 1SSR and design of primers
1. the extracting of adult sample DNA
Adopt phenol extracting and the ethanol precipitation of DNA:
1) get the wall scroll worm and insert 200 μ l extraction damping fluid washing polypide 3 times, the centrifugal 2min of 2000g separates washing lotion;
2) blot washings, add 20 μ l extraction buffers, with the sterilization of the RNAase-free rod of milling for a short time, the polypide of milling is to homogenate;
3) add the extraction damping fluid to final volume 50 μ l;
4) add 0.5 μ l RNAase (10ug/ μ l), 5 μ l SDS (10%), 0.5 μ l protein digestion enzyme is with rifle mixing repeatedly;
5) 110 rev/mins 56 ℃ digestion 1h in compound constant temperature water bath vibrator;
6) add equal-volume 50 μ l phenol: chloroform: Virahol is 25: 24: 1 a mixed solution, mixing, 10000g, centrifugal 5min;
7) get supernatant, add 50 μ l chloroforms: Virahol is 24: 1 a mixed solution, mixing, 10000g, centrifugal 5min;
8) get supernatant, add 80 μ l dehydrated alcohols, mixing ,-30 ℃ are spent the night;
9) add 1ml 70% ethanol, 10000g, centrifugal 15min;
10) take off layer solution and add 10 μ lddH
2O, stand-by.
Or employing protease K digesting method:
1) washs polypide 3 times with 200 μ l GNT damping fluids, 2000g, centrifugal 2min separating, washing liquid;
2) blot washings, add 20 μ l GNT damping fluids, with the sterilization of the RNAase-free rod of milling for a short time, the polypide of milling is to homogenate;
3) add 40 μ l GNT damping fluids, rotation 10s;
14) add 1 μ l protease digestion enzyme, with pipettor mixing repeatedly, in compound constant temperature water bath vibrator 110 rev/mins in 56 ℃ of digestion 1h;
5) 10000g, centrifugal 5min, room temperature is placed, and shifts in supernatant to the clean 1.5ml EP pipe;
6) add 50 μ l NID damping fluids, vibration 30s puts room temperature 3min;
7) rotation mixes 30s, 95 ℃ of 15min inactivated proteases digestive ferments;
8) 10000g, centrifugal 5min gets in the clean 1.5ml EP pipe of supernatant to, and is stand-by.
2. the acquisition of the screening of microsatellite locus and primer
Obtain primary data from genome database, and the screening of the following principle of process:
A) 3-4 base repeat (CGG for example, TACC); B) each site does not comprise complex construction; C) sequence is that the schistosoma japonicum gene group is special; D) multiplicity of little satellite is at 10-25 time; E) residing position, site should be away from the coding region; F) sequence of site flank is single in the full genome of Schistosoma japonicum.The inventor is that the sequence of TAA is screened at repeating unit, obtains the original series in a qualified site surplus in the of 50.Process is further analyzed, is tested and selects, and obtains 17 effective microsatellite sequences with typicalness at last, difference called after Sjpn1-Sjpn17, and their sequence is successively shown in SEQ ID NO:1-SEQ ID NO:17.
Adopt Primer 5.0 software design primers, and preliminary PCR screening, finally determine 17 pairs of effective primers, as table 1.
The primer sequence and the annealing temperature thereof of 17 microsatellite locus of table 1 Schistosoma japonicum
| Little satellite | Primer sequence | SEQ ID NO： | Annealing temperature (℃) |
| Sjpn1 | F：TGAGCACAACTGTATATCCCAAA R：TGGGCAGACATACCAGGTTC | 18 19 | F：59.53 R：60.38 |
| Sjpn2 | F：ATCAATACCGTTCCCAGTGTTT R：CCCACGGTGAATTCTTCATT | 20 21 | F：59.35 R：59.79 |
| Sjpn3 | F：TGGCATTGACTACAGCGTTC R：TTGAATGAAAAAGGCTGTTACAAA | 22 23 | F：59.87 R：59.45 |
| Sjpn4 | F：ACAAGCTCCAATCGTCTCTGA R：GAATACTGCCGCCCTTGTAA | 24 25 | F：59.11 R：60.10 |
| Sjpn5 | F：TGGTGCAAAAATTAACCAACG R：TTCGATAGTACTGCGTCAATCTG | 26 27 | F：60.74 R：59.83 |
| Sjpn6 | F：CGCTATTTATTACTCGGCGTTC R：CGGTCACCAACTCCAAGAAG | 28 29 | F：60.13 R：59.96 |
| Sjpn7 | F：GAGGGGGAGAAGATTAGACCA R：TTCACATACCTCCACCTCACC | 30 31 | F：59.52 R：59.78 |
| Sjpn8 | F：ATGCACGTAAAGAAAAGGGTAAA R：TGATCTCCTACTGCGTTTCTGA | 32 33 | F：58.85 R：60.02 |
| Sjpn9 | F：GATGAAACAGATACCCAGCAC | 34 | F：59.95 |
| R：TGCATGTAAAAATGGCTTGC | 35 | R：59.71 | |
| Sjpn10 | F：TTTGTGCCATGTTGTGTACG R：ACCGGGCTGAGTTTCATCAT | 36 37 | F：59.82 R：61.82 |
| Sjpn11 | F：CACCATTCCCAACAGACACA R：CAGTGTATCCATGATTTACTCGAATC | 38 39 | F：60.42 R：60.15 |
| Sjpn12 | F：CGCTTCAGTGAATTGAAGTGTT R：TTTCAGACAAAGTAAATGACCTCAG | 40 41 | F：59.42 R：58.92 |
| Sjpn13 | F：GTGATTGAGGGAAATGGATGA R：CAATTTGTTTCCTCGCTTTCTT | 42 43 | F：59.74 R：59.77 |
| Sjpn14 | F：AAATTAACGCACGGACATCA R：AGAATATTGGGACCGGATCA | 44 45 | F：59.04 R：59.20 |
| Sjpn15 | F：TGATCACAAATACGAAACTAGCC R：GCATTCACAATGGGCAACTA | 46 47 | F：58.34 R：59.55 |
| Sjpn16 | F：TTGGCTATGGTTCTTTTGTGG R：TTGTAACCTGTAGGCTGCTGAA | 48 49 | F：59.98 R：59.94 |
| Sjpn17 | F：GGGTCGAAAGTGTGTGTGTG R：CAAGGTGAATGAAAGCGAAAT | 50 51 | F：60.05 R：58.89 |
Designed primer is to all proving conclusively by BLAST.With Sjpn10 is example, and designed primer is Blast at the full genome database of Schistosoma japonicum, and by Blast result as can be known: the Sjpn10 site is positioned at Contig position SJC_C00056977, and site sequence has uniqueness in the full genome of Schistosoma japonicum.
Embodiment 2. is the genetic analysis of example with site sjpn1
1. based on Sjpn 1 sequence, design corresponding primer, as follows:
F：TGAGCACAACTGTATATCCCAAA
R：TGGGCAGACATACCAGGTTC
2.PCR reaction amplifies the corresponding target fragment on the complete genome DNA of Schistosoma japonicum sample.
The PCR cycling condition is: 95 ℃ of 5min; 94 ℃ of 45s, 55 ℃ of 45s, 72 ℃ of 45s, 30 circulations; 72 ℃ of 10min.The PCR reaction system is: the 25ng genomic dna, and the SBS Taq polysaccharase of 2 units, each 10pm of two-way primer, 1.25mM MgCl2,1 μ l, 10 * reaction buffer, 0.5 μ l dNTPs (2.5mM, TaKaRa).
3. ordinary method is carried out purifying to the PCR product.
4. genetics signature analysis is for example analyzed the genetics feature in a plurality of (as Tongling, Guichi, Duchang, Changde, Yueyang, Shashi, Xichang).Selectable genetic analysis method is as follows:
(1). the PCR product is carried out electrophoresis identify
By the band number and/or the situation of electrophoretic analysis amplified production, the band number and/or the position consistency of amplified production are high more, represent that the genetic affinity between two kinds of schistosomicide is near more; The number of entries of amplified production and/or position difference are big more, represent that the genetic affinity between two kinds of schistosomicide is far away more.
(2). the genescan of sample
The PCR product separates with ABI 3730 XL automatic sequencers, the definite length of deciding the dna fragmentation of PCR product with ABI Genescan-500LIZ (Applied Biosystems) intramolecularly mapping.And carry out primary data with ABI 3730 XL and GenMapper 4.0 softwares (Applied Biosystems) and handle.
The genetic analysis method and the sjpn1 in other site are similar.
The Preliminary detection and the genescan of embodiment 3.PCR amplified production
1.PCR the Preliminary detection of amplified production
The PCR product is carried out genescan (Genscan) before, institute's amplification PCR products is being detected, guaranteeing the success of genescan experiment.Institute's amplification PCR products should have male purpose band, and the banding pattern of purpose band is single, and phenomenons such as traction must not be arranged.Fig. 1 is the electrophorogram of the pcr amplification product of 96 single samples on the sjpn8 of site in popular district, Wuhan, and the single purpose band all appears in each swimming lane, and size is about 250bp.
2.GeneMapper 4.0 operation
According to the different loci PCR product size that increases out different control panel (Panel) is set, and operation GeneMapper 4.0, described operation can be with reference to working instructions.
3. genotype (Genetypes)
Because Schistosoma japonicum is a diplont, so the genotype of its single sample has two kinds: heterozygote is two different allelotrope; Homozygote only has only an allelotrope.
(i). heterozygote
As shown in Figure 2: three samples are heterozygote, and two allelic peak figure clearly appear in each sample standard deviation.
(ii). homozygote
As shown in Figure 3: two samples are homozygote, and single clearly allelic peak figure appears in each sample standard deviation.
Save as the Excel form by GenMapper 4.0 derived datas, benchmark (i.e. the size of the flanking sequence of these little satellite repeating part both sides) according to each microsatellite locus calculates the length scale of little satellite repeating part, and then calculates the number of times of little satellite TAA repeating unit.The number of times of TAA repeating unit is used for follow-up genetic analysis.
Embodiment 4. statistical analysis
1. the allelotrope number is observed heterozygosity and expectation heterozygosity
(Ho Observedheterozgosity) is the index that is used to analyze polymorphism for allelic number (Na, Number of allele) and observation heterozygosity.Heritable variation degree between the disparity range reflection population of Na and Ho.Analyzed 7 population samples, found that diplontic Na is 5～20; Ho is 0.138～1.000 (table 2), differs greatly, and has reflected to exist between population on high-caliber heritable variation and each site to have a plurality of allelotrope.According to the value that heterozygosity is observed in each site, can utilize software GenAlEx6 to calculate the expectation heterozygosity He (Expected heterozgosity He) in each site, the expectation heterozygosity in each site and observed heterozygosity there was no significant difference.
The feature of 17 new microsatellite DNA polymorphic sites of table 2 China's Mainland Schistosoma japonicum
| Microsatellite locus | The polymorphism index | Tongling, Anhui | The Guichi, Anhui | The Jiangxi Duchang | Changde, hunan | Yueyang, hunan | Shashi, Hubei | Xichang, Sichuan |
| Sjpn1 | *Na Ho He | 11 0.696 0.834 | 11 1.000 0.867 | 15 0.869 0.879 | 13 1.000 0.867 | 13 1.000 0.903 | 9 0.966 0.844 | 11 0.946 0.854 |
| Sjpn2 | Na Ho He | 15 0.957 0.893 | 14 0.867 0.886 | 15 0.803 0.838 | 9 0.818 0.805 | 11 0.667 0.845 | 10 1.000 0.856 | 10 0.429 0.502 |
| Sjpn3 | Na Ho He | 13 0.957 0.875 | 11 0.822 0.863 | 18 0.820 0.918 | 14 1.000 0.885 | 15 0.786 0.887 | 13 1.000 0.880 | 10 0.607 0.827 |
| Sjpn4 | Na Ho He | 12 1.000 0.864 | 11 0.911 0.867 | 15 0.967 0.864 | 10 0.964 0.851 | 17 0.810 0.871 | 6 0.621 0.659 | 11 0.875 0.877 |
| Sjpn5 | Na Ho He | 12 0.870 0.878 | 11 0.822 0.890 | 13 0.721 0.866 | 10 1.000 0.842 | 12 0.857 0.897 | 8 0.931 0.865 | 10 0.589 0.854 |
| Sjpn6 | Na Ho He | 12 1.000 0.913 | 15 0.889 0.893 | 18 0.885 0.893 | 12 1.000 0.851 | 14 0.762 0.882 | 10 0.931 0.904 | 12 0.929 0.856 |
| Sjpn7 | Na Ho He | 15 0.522 0.898 | 13 0.931 0.897 | 12 0.426 0.885 | 10 0.500 0.834 | 14 0.595 0.905 | 8 0.138 0.757 | 11 0.464 0.857 |
| Sjpn8 | Na Ho He | 14 0.957 0.903 | 12 0.889 0.900 | 19 0.705 0.929 | 9 0.636 0.839 | 17 0.786 0.921 | 10 0.966 0.873 | 13 0.875 0.884 |
| Sjpn9 | Na Ho He | 14 0.174 0.875 | 11 0.533 0.875 | 14 0.492 0.902 | 7 0.400 0.769 | 14 0.381 0.896 | 8 0.793 0.796 | 8 0.732 0.815 |
| Sjpn10 | Na Ho He | 12 0.957 0.867 | 8 0.578 0.849 | 14 0.869 0.891 | 7 0.836 0.793 | 10 0.833 0.865 | 11 0.643 0.807 | 9 0.804 0.846 |
| Sjpn11 | Na Ho He | 11 0.652 0.838 | 8 0.222 0.816 | 14 0.590 0.886 | 8 0.727 0.797 | 12 0.643 0.877 | 5 0.241 0.758 | 12 0.661 0.778 |
| Sjpn12 | Na Ho He | 11 0.913 0.854 | 11 0.933 0.860 | 16 0.918 0.905 | 13 1.000 0.870 | 17 0.905 0.879 | 7 0.828 0.724 | 10 0.911 0.802 |
| Sjpn13 | Na Ho He | 12 0.913 0.873 | 13 0.756 0.815 | 14 0.787 0.874 | 10 0.909 0.851 | 15 0.810 0.900 | 10 1.000 0.846 | 10 0.750 0.832 |
| Na | 12 | 12 | 12 | 10 | 14 | 13 | 7 |
| Sjpn14 | Ho He | 0.783 0.819 | 1.000 0.880 | 0.869 0.881 | 0.982 0.797 | 0.952 0.888 | 0.862 0.873 | 0.821 0.741 |
| Sjpn15 | Na Ho He | 13 0.826 0.888 | 11 0.733 0.870 | 15 0.525 0.903 | 11 0.836 0.846 | 14 0.619 0.893 | 9 0.655 0.776 | 14 0.661 0.857 |
| Sjpn16 | Na Ho He | 12 0.652 0.827 | 11 0.733 0.838 | 13 0.885 0.847 | 10 0.982 0.841 | 13 0.690 0.869 | 10 1.000 0.808 | 9 1.000 0.822 |
| Sjpn17 | Na Ho He | 16 0.870 0.915 | 12 0.933 0.887 | 20 0.885 0.921 | 12 1.000 0.848 | 20 0.738 0.913 | 11 0.793 0.862 | 15 0.750 0.817 |
| Mean (on average) | Na Ho He | 12.824 0.806 0.872 | 10.882 0.797 0.868 | 15.118 0.766 0.887 | 10.294 0.858 0.834 | 14.235 0.755 0.888 | 9.529 0.780 0.817 | 10.706 0.753 0.813 |
Annotate: * Na: allelic number; Ho: observe heterozygosity; He: expectation heterozygosity.
Existing report, (Observed heterozgosity is 0.33～1.00 Ho) to the observation heterozygosity of 11 microsatellite locus on the Schistosoma mansoni genome, and allelic number is 2～6; The average Ho of existing report Schistosoma japonicum is 0.05～0.82, and average allelic number is 2.75～10.75.
The Ho of 17 new microsatellite locus that the present invention adopts is 0.138～1.000, allelic number is 5～20, the result shows the allelotrope number of Schistosoma japonicum allelotrope number considerably beyond Schistosoma mansoni, also there are certain difference in the scope of heterozygosity and average heterozygosity, illustrate that these 17 new microsatellite locus can disclose more heritable variation information, the Schistosoma japonicum of China's Mainland and Schistosoma mansoni have visibly different hereditary feature.Point out its reason be Schistosoma japonicum and Schistosoma mansoni in very long evolutionary process, be subjected to the influence of different ecological environment, produced tangible evolution difference.
2. the hereditary difference in the same population between the sample
Utilize GenClone 1.0 softwares (referring to Arnaud-Haond S etc., GENCLONE:a computerprogram to analyze genotypic data, test for clonality and describe spatial clonalorganization[J] .Mol Ecol Notes, 2007,7 (1): 15-17) all samples of the popular district of Duchang, the most tangible Jiangxi of hereditary difference in the population are analyzed, what GenClone 1.0 calculated is the difference between the allelotrope between any 2 samples, so (Schistosoma japonicum is a diplont among 17 sites, to 2 allelotrope should be arranged, heterozygote is 2 different allelotrope, homozygote is 2 identical allelotrope), genetic distance maximum between any 2 samples is 34.The result shows: the genetic distance between most two samples is distributed between 25～32, wherein genetic distance be 30 frequency up to 70% (Fig. 4), show that the hereditary difference between each sample is obvious in the Duchang population of Jiangxi.Hereditary difference in other populations between each sample is also bigger.
3. the analysis of hereditary difference between the population
The analysis of hereditary difference (AMOVA) is F statistics (F
ST: a kind of instrument F-statistic), it is analyzed the genetic distance matrix between codominant allele, and the hereditary difference that the result is used for weighing between population accounts for the ratio of whole hereditary difference, thereby the degree of hereditary difference between the population is described.Result to 7 population analysiss shows: the ratio that the interior generation of population hereditary difference accounts for whole hereditary difference is 90%, and it is higher to produce the shared ratio of difference between the population, is 10% (Fig. 5), has confirmed that the difference between the population is comparatively obvious.
Wherein, Permutation (displacement number of times)=9999; Rst is 0.103556 (the P value is 0.000＜＜0.05).
D. be used to oncomelania number, the infection rate of this positive oncomelania in popular district and the analysis of efficient gene type number and single multiple site genotype (MLG) number of the infection studied
In the Duchang population of Jiangxi, its single multiple site genotype number is 61 (hero is 28, and female is 33), and the oncomelania number that is collected in this popular district only is 11; And in the population of Shashi, Hubei, its single multiple site genotype (MLG) number is 29 (hero is 15, and female is 14), and the oncomelania number that is collected in this popular district is 30 (table 3).
The oncomelania number that table 3 infects, the infection rate of this positive oncomelania in popular district and efficient gene type number and single multiple site genotype (MLG) number
| The place | Positive oncomelania infection rate | The quantity of the positive oncomelania of gathering | The effective sample that is used to analyze is counted * | Single site MLG number (male, female) |
| TN (Tongling) | 3.25％ | 10 | 59 | 23(12，11) |
| GC (Guichi) | 0.14％ | 7 | 58 | 45(27，18) |
| DC (Duchang) | 6.5％ | 11 | 62 | 61(28，33) |
| CD (Changde) | 0.93％ | 21 | 72 | 55(21，34) |
| YY (Yueyang) | 0.02％ | 29 | 44 | 42(25，17) |
| SH (Shashi) | ＜0.01％ | 30 | 41 | 29(15，14) |
| XC (Xichang) | 0.14％ | 15 | 67 | 56(33，23) |
Annotate: each popular district all branch sex analyzes, and used software is GENALEx.
* remove all missing datas
2Only analyze 16 sites (not comprising the P7 site).
The above results points out single multiple site genotype (MLG) number relevant with the infection rate of the positive oncomelania in popular district, and the infection rate of the positive oncomelania in the popular district of Jiangxi Duchang is up to 6.5%, and the infection rate of the positive oncomelania in popular district, Shashi, Hubei is far smaller than 0.01%.High infection rate may cause leveling off to most in this population the mode of inheritance of random mating.
E. UPGMA pedigree analysises of seven popular district populations
The UPGMA pedigree chart is used for illustrating the genetic distance between each population.The UPGMA pedigree chart of 7 populations shows: the Jiangxi Duchang, and Tongling, Anhui, Shashi, Hubei and changde, hunan are cluster, its genetic distance is at 0.0178～0.0363; Guichi, Anhui and yueyang, hunan are other cluster, and its genetic distance is 0.0247; And Xichang, Sichuan is separately cluster, and Xichang, Sichuan and other genetic distances of two bunches are at 0.0192～0.0693 (Fig. 6).The UPGMA pedigree chart has shown between each popular district and has existed hereditary difference that wherein popular district, Sichuan and other popular district hereditary differences are particularly evident.
The explanation of hereditary difference between 5. 7 popular districts of embodiment population
The inventor also finds to have certain hereditary difference between the popular district of 7 of the China's Mainland population, and wherein popular district, Sichuan population obviously is different from other population.This difference can be explained with geographical position and ecological shielding.Popular district, Sichuan is positioned at the upstream, Three Gorges, and therefore the migrating and mix the restriction that is subjected to the geographical position of its positive oncomelania, may cause some gene in its population to be difficult for genetic heterozygosis with area, downstream population.Relatively, other popular districts all are positioned at the lake country, and migrating of positive oncomelania is relatively easy, and its genotype will be abundanter.In addition, the selective pressure that spiral shell produces of going out also may cause genotypic change.In addition, the tangible genetic heterozygosis of the open-air sample of the Schistosoma japonicum of China's Mainland has confirmed that in the side there is gene diversity at genome in Schistosoma japonicum with transcribing on the group.
The present invention has analyzed microsatellite DNA as the application of genetic marker in the research of Schistosoma japonicum population genetics, has set up a kind of population genetics research that is suitable for, simple and rapid single Schistosoma japonicum adult sample genomic dna extractive method.Utilize 17 new microsatellite locus to analyze for 7 Schistosoma japonicum population genetic diversity for genetic marker, illustrate the China's Mainland Schistosoma japonicum and had the obvious genetic diversity, between the different popular districts of China, may have gene pleiomorphism, there is complicated population heritable variation, thereby provides support for the monitoring of schistosomiasis japanica and the research of vaccine from now on.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Prevention ﹠ Control Station of Parasitic Disease, China Diseases Prevention ﹠ C, Nanfang Research Centre, State Human Gene Group, East China University of Science
＜120〉Schistosoma japonicum microsatellite locus and application thereof
<130>082356
<160>51
<170>PatentIn version 3.3
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＜213〉Schistosoma japonicum (Schistosoma japonicum)
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＜213〉Schistosoma japonicum (Schistosoma japonicum)
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＜213〉Schistosoma japonicum (Schistosoma japonicum)
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＜213〉Schistosoma japonicum (Schistosoma japonicum)
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tattttactt tctctgtaaa aaataaataa caatgagatt ttaacatgaa ctaagtgaat 420
aagtagtttc atattataaa ctattgtcac attaatt 457
<210>16
<211>442
<212>DNA
＜213〉Schistosoma japonicum (Schistosoma japonicum)
<400>16
tcatttgttc aatgctgtat ttatatattt gttaaaaata atccatatga taacgtatta 60
tagtttgttg gctatggttc ttttgtggca aatcttcaga tgattctatt gtcatctatg 120
tttcgtgaat gtttcaagaa atttgaaata actgttagaa aatacattta aattgtagct 180
tacttaagca tatttaggaa taataataat aataataata ataataataa taataataat 240
aatagtaagt aactttctaa tatagttgtg tcaatgacaa actatgtaac cgaaaatttt 300
cagcagccta caggttacaa ctgattttaa tgacaatatt tttctactag tacatgaatg 360
agtaatttca tactgcattc tagacacttt tattttgtac agttggagaa tctaactttt 420
acttttatcc cgcttgtttg tc 442
<210>17
<211>454
<212>DNA
＜213〉Schistosoma japonicum (Schistosoma japonicum)
<220>
<221>misc_feature
<222>(79)..(79)
<223>n is a，c，g，or t
<400>17
gtgactggta ttgattaatt ggtagataaa taaaaaaaaa tatttgtata ggggtcgaaa 60
gtgtgtgtgt gtgtgtggng gggggatttt aataatagtt gcatttgacc cttatatgta 120
caattatgcg taaactgttt taccttaatg atcgtgcatt tataatgtag acttaggatt 180
atgatgcttt atatcagtta taataataat aataataata ataataataa taataataat 240
aataataata ataaacgtat ttcgctttca ttcaccttgg ttattttcag tgtctagaaa 300
attagtgaaa caaatacatt tcgtaatttg caaaggacag aaaacatgat ctagattcta 360
tgtgataatc aacgttgaga tatagttaaa gtatagacag taaactactc acgtgaatag 420
gcaactttat aaattggtgt agcacatgaa tcaa 454
<210>18
<211>23
<212>DNA
＜213〉primer
<400>18
tgagcacaac tgtatatccc aaa 23
<210>19
<211>20
<212>DNA
＜213〉primer
<400>19
tgggcagaca taccaggt tc 20
<210>20
<211>22
<212>DNA
＜213〉primer
<400>20
atcaataccg ttcccagtgt tt 22
<210>21
<211>20
<212>DNA
＜213〉primer
<400>21
cccacggtga attcttcatt 20
<210>22
<211>20
<212>DNA
＜213〉primer
<400>22
tggcattgac tacagcgttc 20
<210>23
<211>24
<212>DNA
＜213〉primer
<400>23
ttgaatgaaa aaggctgtta caaa 24
<210>24
<211>21
<212>DNA
＜213〉primer
<400>24
acaagctcca atcgtctctg a 21
<210>25
<211>20
<212>DNA
＜213〉primer
<400>25
gaatactgcc gcccttgtaa 20
<210>26
<211>21
<212>DNA
＜213〉primer
<400>26
tggtgcaaaa attaaccaac g 21
<210>27
<211>23
<212>DNA
＜213〉primer
<400>27
ttcgatagta ctgcgtcaat ctg 23
<210>28
<211>22
<212>DNA
＜213〉primer
<400>28
cgctatttat tactcggcgt tc 22
<210>29
<211>20
<212>DNA
＜213〉primer
<400>29
cggtcaccaa ctccaagaag 20
<210>30
<211>21
<212>DNA
＜213〉primer
<400>30
gagggggaga agattagacc a 21
<210>31
<211>21
<212>DNA
＜213〉primer
<400>31
ttcacatacc tccacctcac c 21
<210>32
<211>23
<212>DNA
＜213〉primer
<400>32
atgcacgtaa agaaaagggt aaa 23
<210>33
<211>22
<212>DNA
＜213〉primer
<400>33
tgatctccta ctgcgtttct ga 22
<210>34
<211>21
<212>DNA
＜213〉primer
<400>34
gatgaaacag atacccagca c 21
<210>35
<211>20
<212>DNA
＜213〉primer
<400>35
tgcatgtaaa aatggcttgc 20
<210>36
<211>20
<212>DNA
＜213〉primer
<400>36
tttgtgccat gttgtgtacg 20
<210>37
<211>20
<212>DNA
＜213〉primer
<400>37
accgggctga gtttcatcat 20
<210>38
<211>20
<212>DNA
＜213〉primer
<400>38
caccattccc aacagacaca 20
<210>39
<211>26
<212>DNA
＜213〉primer
<400>39
cagtgtatcc atgatttact cgaatc 26
<210>40
<211>22
<212>DNA
＜213〉primer
<400>40
cgcttcagtg aattgaagtg tt 22
<210>41
<211>25
<212>DNA
＜213〉primer
<400>41
tttcagacaa agtaaatgac ctcag 25
<210>42
<211>21
<212>DNA
＜213〉primer
<400>42
gtgattgagg gaaatggatg a 21
<210>43
<211>22
<212>DNA
＜213〉primer
<400>43
caatttgttt cctcgctttc tt 22
<210>44
<211>20
<212>DNA
＜213〉primer
<400>44
aaattaacgc acggacatca 20
<210>45
<211>20
<212>DNA
＜213〉primer
<400>45
agaatattgg gaccggatca 20
<210>46
<211>23
<212>DNA
＜213〉primer
<400>46
tgatcacaaa tacgaaacta gcc 23
<210>47
<211>20
<212>DNA
＜213〉primer
<400>47
gcattcacaa tgggcaacta 20
<210>48
<211>21
<212>DNA
＜213〉primer
<400>48
ttggctatgg ttcttttgtg g 21
<210>49
<211>22
<212>DNA
＜213〉primer
<400>49
ttgtaacctg taggctgctg aa 22
<210>50
<211>20
<212>DNA
＜213〉primer
<400>50
gggtcgaaag tgtgtgtgtg 20
<210>51
<211>21
<212>DNA
＜213〉primer
<400>51
caaggtgaat gaaagcgaaa t 21
Claims (10)
1. isolating polynucleotide is characterized in that, described polynucleotide have and are selected from the nucleotide sequence of SEQ ID NO:1-17 shown in arbitrary.
2. the purposes of the described polynucleotide of claim 1 is characterized in that, described polynucleotide are used for as little satellite mark:
Determining of schistosomicide genetic affinity; The location of japonicum gene; The conservative property analysis of various or each subspecies of schistosomicide; Bilharzial evolutionary analysis; Bilharzial cultivar identification; Or schistosomicide region population is identified.
3. a specific amplification is selected from the primer of the microsatellite locus sequence of SEQ ID NO:1-17 shown in arbitrary.
4. primer as claimed in claim 3 is characterized in that, described primer is that primer is right, described primer to have be selected from following sequence to shown in sequence:
SEQ ID NO:18 and SEQ ID NO:19; SEQ ID NO:20 and SEQ ID NO:21; SEQID NO:22 and SEQ ID NO:23; SEQ ID NO:24 and SEQ ID NO:25; SEQ ID NO:26 and SEQ ID NO:27; SEQ ID NO:28 and SEQ ID NO:29; SEQ ID NO:30 and SEQ IDNO:31; SEQ ID NO:32 and SEQ ID NO:33; SEQ ID NO:34 and SEQ ID NO:35; SEQ ID NO:36 and SEQ ID NO:37; SEQ ID NO:38 and SEQ ID NO:39; SEQ IDNO:40 and SEQ ID NO:41; SEQ ID NO:42 and SEQ ID NO:43; SEQ ID NO:44 and SEQ ID NO:45; SEQ ID NO:46 and SEQ ID NO:47; SEQ ID NO:48 and SEQ IDNO:49; Or SEQ ID NO:50 and SEQ ID NO:51.
5. the purposes of the arbitrary described primer of claim 3-4 is characterized in that, is used for measuring the japonicum gene group and whether has polynucleotide corresponding to the described microsatellite locus of claim 1.
6. method of determining the genetic affinity between the schistosomicide in two or more samples is characterized in that described method comprises:
(1) serves as a mark with the described microsatellite locus sequence of claim 1;
(2) primer of the microsatellite locus sequence of usefulness specific amplification (1) increases to bilharzial genomic dna in described two or more samples respectively, thereby obtains corresponding amplified production;
(3) compare the similarities and differences of bilharzial amplified production in two or more samples, thereby determine the genetic affinity between the schistosomicide in two or more samples.
7. method as claimed in claim 6 is characterized in that, by the band number and/or the situation of electrophoretic analysis amplified production, the band number and/or the position consistency of amplified production are high more, represent that the genetic affinity between two or more schistosomicide is near more; The number of entries of amplified production and/or position difference are big more, represent that the genetic affinity between two or more schistosomicide is far away more.
One kind be used for determining schistosomicide genetic affinity, location japonicum gene, schistosomicide is various or the detection kit of each subspecies conservative property analysiss, schistosomicide evolutionary analysis, schistosomicide cultivar identification or the evaluation of schistosomicide region population, it is characterized in that, contain in the described test kit: specific amplification is selected from the primer of the microsatellite locus sequence of SEQ ID NO:1-17 shown in arbitrary.
9. detection kit as claimed in claim 8 is characterized in that, also contains the material that is selected from down group in the described test kit: pcr amplification reagent, electrophoresis reagent, or sequence analysis software.
10. a polynucleotide collection that is used for genetic analysis is characterized in that, described polynucleotide collection comprises the microsatellite locus sequence shown in the SEQ ID NO:1-17.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880726A (en) * | 2010-07-23 | 2010-11-10 | 华南农业大学 | Primer for detecting schistosoma japonicum Yunnan geographical strains, kit containing same and detection method |
| CN101993955A (en) * | 2010-12-09 | 2011-03-30 | 上海海洋大学 | Method for quickly and accurately identifying genotype of hyriopsis cumingii |
| CN104988148A (en) * | 2015-07-20 | 2015-10-21 | 广西壮族自治区水牛研究所 | Swamp type buffalo SSR primer and application thereof |
| CN105986016A (en) * | 2015-02-05 | 2016-10-05 | 中国疾病预防控制中心寄生虫病预防控制所 | Schistosoma japonicum katsurada mitochondrial molecular marker and application thereof |
| CN106811511A (en) * | 2015-11-27 | 2017-06-09 | 复旦大学 | Schistosoma japonicum region specificity correlation SNP and its application |
| CN110763795A (en) * | 2019-04-04 | 2020-02-07 | 中山大学 | Serum biomarker for early diagnosis of schistosomiasis japonica, screening method and application |
| CN112195255A (en) * | 2020-11-06 | 2021-01-08 | 苏州大学 | Multiple PCR primer combination, kit and method for determining individual genotype of schistosoma japonicum |
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2008
- 2008-04-22 CN CN2008100364229A patent/CN101565705B/en active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880726A (en) * | 2010-07-23 | 2010-11-10 | 华南农业大学 | Primer for detecting schistosoma japonicum Yunnan geographical strains, kit containing same and detection method |
| CN101993955A (en) * | 2010-12-09 | 2011-03-30 | 上海海洋大学 | Method for quickly and accurately identifying genotype of hyriopsis cumingii |
| CN105986016A (en) * | 2015-02-05 | 2016-10-05 | 中国疾病预防控制中心寄生虫病预防控制所 | Schistosoma japonicum katsurada mitochondrial molecular marker and application thereof |
| CN104988148A (en) * | 2015-07-20 | 2015-10-21 | 广西壮族自治区水牛研究所 | Swamp type buffalo SSR primer and application thereof |
| CN104988148B (en) * | 2015-07-20 | 2017-12-15 | 广西壮族自治区水牛研究所 | A kind of swamp type water buffalo SSR primers and its application |
| CN106811511A (en) * | 2015-11-27 | 2017-06-09 | 复旦大学 | Schistosoma japonicum region specificity correlation SNP and its application |
| CN106811511B (en) * | 2015-11-27 | 2021-10-29 | 复旦大学 | Japanese blood fluke region specificity relative mononucleotide polymorphism and its application |
| CN110763795A (en) * | 2019-04-04 | 2020-02-07 | 中山大学 | Serum biomarker for early diagnosis of schistosomiasis japonica, screening method and application |
| CN110763795B (en) * | 2019-04-04 | 2021-06-25 | 中山大学 | Serum biomarker for early diagnosis of schistosomiasis japonica, screening method and application |
| CN112195255A (en) * | 2020-11-06 | 2021-01-08 | 苏州大学 | Multiple PCR primer combination, kit and method for determining individual genotype of schistosoma japonicum |
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| Publication number | Publication date |
|---|---|
| CN101565705B (en) | 2012-11-28 |
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