CN101092646A - Method for diagnosing sporangium and egg capsule molecules of sarcocyst - Google Patents
Method for diagnosing sporangium and egg capsule molecules of sarcocyst Download PDFInfo
- Publication number
- CN101092646A CN101092646A CN 200710065686 CN200710065686A CN101092646A CN 101092646 A CN101092646 A CN 101092646A CN 200710065686 CN200710065686 CN 200710065686 CN 200710065686 A CN200710065686 A CN 200710065686A CN 101092646 A CN101092646 A CN 101092646A
- Authority
- CN
- China
- Prior art keywords
- sarcocystis
- primer
- enzyme
- pcr
- egg capsule
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
This invention relates to a method for detecting sporangium and oocyst of Sarcocystis. The method comprises: (1) mixing a small amount of feces with 37% ZnSO4 solution, centrifuging and standing; (2) detecting floating feces with a microscope, collecting sporangium and oocyst, placing in centrifugal tube, and storing at a low temperature; (3) performing PCR with the primers to amplify 18SrRNA in sporangium and oocyst of Sarcocystis; (4) digesting PCR product with restriction endonuclease, and detecting through gel electrophoresis, or detecting the nucleotide sequence with the primers used before. The primers can also be used as probes in hybridization reactions, or for manufacturing gene chips or microarrays. The method can be used for identifying Sarcocystis with high sensitivity and high specificity.
Description
Technical field
The invention belongs to the diagnosis of parasitosis, particularly the coccidia and the coccidiosis method of utilization gene diagnosis livestock and poultry and human body.
Background technology
Term definition in the following content:
Egg capsule: the stage that sarcocystis detects from ight soil.An egg capsule contains two sporocysts.
Sporocyst: sophisticated sporocyst contains 4 sporozoites.
Packing: sarcocystis parasitizes in the muscle, is the shuttle shape, and the fully-developed packing contains bradyzoite.
The parasite of Miescheria is a special sexual cell endoparasitism protozoon, and be the typical life history of top multiple door polypide its life history, comprises schizogony, gametogony and sporogony.Sarcocystis is the different host's property parasite of obligate, promptly will grow in middle and final host respectively, just is accomplished its life history, and sarcocystis extensively parasitizes fish, reptiles, birds and mammals.Because many kinds of sarcocystis colonize in cat, dog, domestic animals and fowls and human body, cause sarcosporidiasis.Domestic animal and Herbivore be generally as the intermediate host of sarcocystis, intermediate host the eaten food that polluted by sporocyst or water and infected.Monogony is finished in Herbivore or Omnivore intermediate host body usually.Monogony is carried out a generation to the triple-substituted schizogony in animal capillary endothelium and epithelial cell.Last generation schizont forms packing in the Pu of voluntary muscle, central nervous system and heart Ken Shi fiber and muscle bundle.The size of sarcocystis packing and coming in every shape depends on the worm kind.And syngenesis is generally cat, dog and primate only flesh-eater, also comprises in the human body and finishing.As the predation of final host's carnivore or eat to the muscle tissue that contains the sarcocystis packing and nervous tissue and infected, being encapsulated in stomach and small intestine is digested, disengage bradyzoite, bradyzoite is actively movable, invade mucous membrane of small intestine, growth is female greatly, Xiao Xiong's gamont, and then growth is large and small gamete.After fertilization produces wall parcel zygote and forms egg capsule, and egg capsule carries out sporogony at little intestinal lamina propria, and the spore egg capsule that sporogony is finished contains two sporocysts, contains 4 sporozoites in each sporocyst again.Spore egg capsule wall is less than 1 micron, and Chang Yi breaks, so single free sporocyst is released into enteric cavity, is discharged to the external world with final host's ight soil.Time that egg capsule and sporocyst appear at the ight soil first of eating behind the packing generally need 7-14 days from the final host.
The classification criterion of sarcocystis mainly comprises packing, particularly the light microscopic of cyst wall upper process and Electronic Speculum structure; Isozyme; Final host and intermediate host's specificity, the latter particularly, the intermediate host who it is generally acknowledged sarcocystis has very strict specificity, and it is nonsensical on the worm kind is differentiated from the sporocyst and the egg capsule form of final host's discharge, because the packing collection is convenient, can be directly used in the worm kind identifies, therefore, with molecule means research sarcocystis, it all is material with the packing, and egg capsule and sporangial collection difficulty, be difficult to identify the worm kind, so egg capsule and sporangial molecule marking research lag behind packing always, at present nearly all research means all can not be differentiated the worm kind from egg capsule and sporocyst form, especially relatively is difficult to directly detect egg capsule and sporocyst and differentiate the worm kind from ight soil.But, need in epidemiology survey to judge which kind of sarcocystis has infected the final host, and 2004, at Elsheikha HM, Murphy AJ, Mansfield LS.2004.Prevalence of Sarcocystisspecies sporocysts in Northern Virginia opossums (Didelphis virginiana) .Parasitol.Res.93 (5): 427-431. has reported to take from and has cutd open the sporocyst that scrapes thing and the egg capsule of the small intestine epithelium mucous membrane of didelphid extremely, the quantity of its sporocyst and egg capsule is big, is easy to research.Evidently, this method can not apply to the clinical diagnosis of human infection disease.2006, Elsheikha HM, Murphy AJ, Trembley SJ, Mansfield LS, Ghanam MS, el-GarhyMF.2006 Molecular and microscopic techniques for detection of Sarcocystis neuronasporocysts in fecal samples.J Egypt Soc Parasitol.Aug; 36 (2): this research of 713-25. concentrates on a worm kind, egg capsule and the sporocyst collected in the animal excrement of experimental infection, the performing PCR of going forward side by side amplification, experimental infection animal often infective dose is big, can obtain wide variety of materials, yet the sample size after human body or the animal natural infection in ight soil is very rare, and this report does not relate to the evaluation of worm kind.
Summary of the invention
The present invention seeks to change in molecule means research sarcocystis all is the general method of material with the packing, similar on form at Endospore worm's ovum capsule with sporangial stage, volume is small, very rare from the quantity that ight soil is discharged, the worm kind is differentiated very difficult, and the evaluation in this stage has the present situation of important value and urgent need, provides sensitivity and specificity height, clinical detection to make things convenient for, differentiate from molecular conformation the gene diagnosis method of sarcocystis sporocyst and egg capsule worm kind.
The present invention realizes by following steps and content:
1) ight soil that takes a morsel adds an amount of 37%ZnSO
4In the zinc sulfate flotation fluid centrifuge tube, leave standstill after centrifugal;
2) draw with suction pipe or droplet move on slide glass with the supernatant liquid number that transfering loop picks the superiors in the centrifuge tube, with) 100 times microscopy, put into another centrifuge tube cryopreservation with sheet for cleaning sticking sporocyst and egg capsule from slide or centrifuge tube;
3) comprise that to have table 1 be primer, the 18SrRNA constant gene segment C 1500-2000bp Nucleotide of sarcocystis sporocyst that pcr amplification covered and egg capsule or its any a part of nucleotide fragments, or be used for pcr amplification as primer, or be used for hybridization as probe, or preparation gene chip or microarray, perhaps be used for restriction analysis;
Table 1 is used for the PCR and the sequencing primer of sarcocystis 18S rRNA gene
| Primer | Direction | Series 5 '---3 ' | The position |
| 18S?2L 18S?2H | Forward Reverse | GGATAACCTGGTAATTCTATG ACCTGTTATTGCCTCAAACTTC | 158-178* 1530-1551* |
* locate according to the shuttle shape sarcocystis serial position of Holmdahl (1994)
4) to the specific amplified fragments of each worm kind, restriction enzyme and reaction conditions carry out endonuclease reaction in the table 2 with being selected from, and the volume of endonuclease reaction is 10-20ul, result's gel detection,
Table 2. is used for the enzyme and the enzyme tangent condition thereof of sarcocystis 18S rRNA gene restriction analysis
| The enzyme title | The enzyme cut is made condition | |||
| Damping fluid | Time (h) | Enzyme dosage (U) | Temperature (℃) | |
| EcoR?I Pst?I Alu?I Mbo?I Mbo?II Taq?I Hinf?I Bcl?I Rsa?I Ssp?I Acc?I Dra?I Xba?I | H H B C B E B C C E G B D | 8 20 8 10 50 8 8 48 10 16 8 10 8 | 10 5 5 5 5 5 5 5 10 7 5 10 10 | 37 37 37 37 37 65 37 50 37 37 37 37 37 |
Perhaps the primer with table 1 detects nucleotide sequence as sequencing primer.
Described primer is selected from table 3, the different single-minded target region dna fragmentations of the sarcocystis egg capsule that is covered by the pcr amplification primer,
Table 3 is used for the PCR and the sequencing primer of sarcocystis 18S rRNA gene
| Primer | Direction | Series 5 '---3 ' | The position |
| 18S?1L 18S?1H | Forward Reverse | CCATGCATGTCTAAGTATAAGC TATCCCCATCACGATGCATAC | 446-469* 1650-1670* |
| 18S?2L 18S?2H 18S?3L 18S?3H 18S?6H 18S?1OL 18S?10H | Forward Reverse Forward Reverse Reverse Forward Reverse | GGATAACCTGGTAATTCTATG ACCTGTTATTGCCTCAAACTTC CTAGTGATTGGAATGATGGG GGCAAATGCTTTCGCAGTAG CAGAAACTTGAATGATCTATCG CGAACGGATCGCATTATGATC GGCGGACTGATTGCTTCCAG | 156-178* 1530-1551* 566-586* 1040-1070* 348-368* 281-301* 674-693* |
* locate according to the shuttle shape sarcocystis serial position of Holmdahl (1994).
Described primer is optimized in the reaction conditions at pcr amplification, combination and the condition such as the table 4 of the primer of amplification sarcocystis gene:
Table 4. is used for the PCR of sarcocystis 18S rRNA gene and the combination and the condition of sequencing primer
| Combination of primers | Optimum annealing temperature ℃ | Best second extension time | It deny long-time pre-sex change |
| 18S1L6H 18S2L6H 18S2L3H 18S3L3H 18S2L2H 18S3L1H 18S10L10H | 50 50 56 56 53-55 56 56-63 | 30 30 80 60 100 60 30 | No no no |
Annotate: long-time pre-sex change, 97 ℃ of promptly pre-sex change were placed on ice in 5 minutes, just added the archaeal dna polymerase of 1.25U.PCR and sequencing primer that described primer is used for sarcocystis 18S rRNA gene further are the listed primer of table 5:
Table 5 is used for the PCR and the sequencing primer of sarcocystis 18S rRNA gene
| Primer | Direction | Series 5 '---3 ' | The position |
| 18S?2L 18S?2H 18S?3H | Forward Reverse Reverse | GGATAACCTGGTAATTCTATG ACCTGTTATTGCCTCAAACTTC GGCAAATGCTTTCGCAGTAG | 156-178* 1530-1551* 1040-1070* |
In the nest-type PRC reaction, the PCR combination of primers is 18S 2L, 18S 2H for the first time, and the PCR combination of primers is 18S2L, 18S 3H for the second time.
The restriction enzyme of described endonuclease reaction further is the one or more combination of table 6:
Table 6. is used for the enzyme and the enzyme tangent condition thereof of sarcocystis 18S rRNA gene restriction analysis
| The enzyme title | The enzyme cut is made condition | |||
| Damping fluid | Time (h) | Enzyme dosage (U) | Temperature (℃) | |
| Alu?I Mbo?I Mbo?II Taq?I Hinf?I Bcl?I Rsa?I Ssp?I Acc?I Dra?I | B C B E B C C E G 8 | 8 10 50 8 8 48 10 16 8 10 | 5 5 5 5 5 5 10 7 5 10 | 37 37 37 65 37 50 37 37 37 37 |
The effect that the present invention has is: people's meat sample sarcocystis (S.hominis-like), shuttle shape sarcocystis (S.fusiformis) sarcocystis can obtain the sarcocystis of 7 egg capsules, for the PCR-RFLP of total DNA 18SrRNA gene of egg capsule or the PCR-sequencing analysis of 18SrRNA gene, can identify the worm kind.Other is as pig Sarcocystis hominis (S.suihominis), Sarcocystis meischeriana (S.miescheriana), Sarcocystis hominis (S.hominis), hair shape sarcocystis (S.hirsuta), ox Sarcocystis cruzi (S.cruzi), Lai Shi Endospore worm (S.levinei), hair shape sample sarcocystis (S.hirsuta-like), people's meat sample sarcocystis (S.hominis-like), buffalo Sarcocystis cruzi (S.cruzi-like), the Other Meat sporozoite of shuttle shape sarcocystis (S.fusiformis) and this genus thereof should have effect same according to the accumulation of previous work.
The present invention uses concentration method and checks the sarcocystis sporocyst, can collect quantity egg capsule and sporocyst very rare, omission easily in the ight soil; The pcr amplification primer can cover all gene segments of sarcocystis egg capsule with reporting different at present, and the specific restriction enzyme that will identify each worm kind is used for the diagnosis and the evaluation of worm kind thereof of sarcocystis egg capsule, thereby, the completely new approach that provides egg capsule and sporocyst to collect, identify, substantive breakthroughs have been obtained, on the cellular elements biology level, reached from the purpose of sporocyst and egg capsule stage discriminating worm kind, had important public hygienics meaning and economic worth.
Description of drawings
Fig. 1 is for being the glue figure of several sarcocystis egg capsule of primer enzyme endonuclease reaction with 18S2L3H.
Embodiment
(1) diagnostic method of sarcocystis sporocyst and egg capsule molecule
Embodiment one:
Restriction analysis 1 (PCR-RFLP)
1.1. stool examination and collection sporocyst and egg capsule comprise preparation 37%ZnSO
4Zinc sulfate flotation fluid (ZuSO
47Rz03319 adding distil water 1000ml), get excrement 1g and add 37%ZnSO
4Zinc sulfate flotation fluid 100ml adds in the 15ml centrifuge tube.Put under the centrifuge tube from.The supernatant liquid number of drawing the superiors in the centrifuge tube with suction pipe droplet moves on slide glass, with 150 times microscopy.The sporocyst and the egg capsule that arrive for the purpose of sticking with sheet for cleaning.The scraps of paper are put into the 1.5ml centrifuge tube ,-20 ℃ of preservations.
1.2. obtain target DNA fragment by pcr amplification
1.2.1PCR amplification condition
1.2.1.1 every 50ul reaction volume has 10 * Buffer 5ul, dNTPs 2ul, and BSA 2ul, primer 12ul, primer 2 2ul, Taq enzyme 1.25U, total DNA 2ul, surplus is a deionized water.
1.2.1.2 in the PCR reaction conditions, the primer that has needs long-time pre-sex change, promptly pre-sex change 97 degree were placed on ice in 5 minutes, added the archaeal dna polymerase of 1.25U, and the primer that has does not then need this step, as following table 2.
The combination and the amplification condition thereof of the primer of table 2. amplification sarcocystis gene
| Combination of primers | Optimum annealing temperature ℃ | Best second extension time | It deny long-time pre-sex change |
| 18S1L6H 18S2L6H 18S2L3H 18S3L3H 18S2L2H 18S3L1H 18S10L10H | 50 50 56 56 53-55 56 56-63 | 30 30 80 60 100 60 30 | No no no |
Be used for the reaction product that enzyme cuts and derive from the nest-type PRC reaction product, PCR18s 2L, 18s 2H for the first time, PCR 18s 2L, 18s 3H are that combination of primers is a following table for the second time.
Table. the combination and the amplification condition thereof of the primer of amplification sarcocystis gene
| Combination of primers | Optimum annealing temperature ℃ | Best second extension time | It deny long-time pre-sex change |
| 18S2L3H 18S2L2H | 56 53-55 | 80 100 | Do not want |
1.3.PCR the purpose fragment of reaction is used for the digestion with restriction enzyme reaction.
The volume of endonuclease reaction raw material is 10-20ul, wherein contains: enzyme reaction 10 * Buffer of 2ul, and an amount of DNA and 5U enzyme, surplus is supplied with distilled water.With above-mentioned reaction raw materials mixing, according to the optimal reactive temperature condition of different enzymes, put into corresponding incubator, place the regular hour.As following table 3:
Table 3. is used for the enzyme and the enzyme tangent condition thereof of sarcocystis 18S rRNA gene restriction analysis
| The enzyme title | The enzyme cut is made condition | |||
| Damping fluid | Time (h) | Enzyme dosage (U) | Temperature (℃) | |
| EcoR?I Pst?I Alu?I Mbo?I Mbo?II Taq?I Hinf?I Bcl?I Rsa?I Ssp?I Acc?I Dra?I Xba?I | H H B C B E B C C E G B D | 8 20 8 10 50 8 8 48 10 16 8 10 8 | 10 5 5 5 5 5 5 5 10 7 5 10 10 | 37 37 37 37 37 65 37 50 37 37 37 37 37 |
1.4.PCR amplification acquisition target DNA fragment is used for enzyme and cuts the digestion with restriction enzyme reaction result:
By the different big or small dna fragmentations of the molecular weight of not acquisition of the same race, be divided into different types as following table:
| Haplotype | Taxon(sample?size) | Restriction enzyme and electrophoresis form | |||||||||||
| EcoRI | TsqI | DraI | HinfI | AluI | PstI | BclI | AcoI | MboII | SspI | RsaI | MboI | ||
| I | S.hirsuta(3) S.hirsuta-like(9) | A | B | A | B | A | A | C | A | A | B | B | B |
| II | S.fusiformis(11) | A | A | B | D | A | A | A | A | B | B | B | A |
| III | S.mischeriana(10) | A | B | A | B | B | A | A | A | A | B | A | A |
| IV | S.hominis(8) S.hominis-like(14) | A | B | A | C | A | A | B | A | A | A | A | A |
| V | S.suihominis(16) | A | B | A | B | B | A | A | A | A | A | A | A |
| VI | Sarcocystis?sp.(2) | A | B | C | A | A | / | A | A | A | A | C | A |
The glue figure that with 18S2L3H is several sarcocystis egg capsule of primer enzyme endonuclease reaction is an accompanying drawing 1.
Embodiment two:
Restriction analysis 2 (PCR-RFLP)
2.1 stool examination and collection sporocyst and egg capsule comprise preparation 37%ZnSO
4Zinc sulfate flotation fluid (ZuSO
47Hz03319 adding distil water 1000ml), get excrement 1g and add 37%ZnSO
4Zinc sulfate flotation fluid 100ml adds in the 15ml centrifuge tube.Put under the centrifuge tube from.The supernatant liquid number of drawing the superiors in the centrifuge tube with suction pipe droplet moves on slide glass, with 130 times microscopy.The sporocyst and the egg capsule that arrive for the purpose of sticking with sheet for cleaning.The scraps of paper are put into the 1.5ml centrifuge tube ,-20 ℃ of preservations.
2.2 obtain target DNA fragment by pcr amplification
2.2.1PCR amplification condition
2.2.1.1 every 50ul reaction volume has 10 * Buffer 5ul, dNTPs 2ul, and BSA 2ul, primer 12ul, primer 2 2ul, Taq enzyme 1.25U, total DNA 2ul, surplus is a deionized water.
Be used for the reaction product that enzyme cuts and derive from the nest-type PRC reaction product, PCR18s 2L, 18s 2H for the first time, for the second time PCR 18s 3L, 18s 3H be combination of primers be table //.Wherein, long-time pre-sex change, promptly pre-sex change 97 degree were placed on ice in 5 minutes, added the archaeal dna polymerase of 1.25U.With the combination of primers of summary of the invention table 5, the amplification of the condition of according to the form below.
Table //. the combination and the amplification condition thereof of the primer of amplification sarcocystis gene
| Combination of primers | Optimum annealing temperature ℃ | Best second extension time | It deny long-time pre-sex change |
| 18S3L3H 18S2L2H | 56 53-55 | 80 100 | To want |
2.3 the digestion with restriction enzyme reaction, the purpose fragment of PCR reaction is used for enzyme and cuts.
The volume of endonuclease reaction raw material is 10-20ul, wherein contains: enzyme reaction 10 * Buffer of 2ul, and an amount of DNA and 5U enzyme, surplus is supplied with distilled water.With above-mentioned reaction raw materials mixing, according to the optimal reactive temperature condition of different enzymes, put into corresponding incubator, place the regular hour.As following table 6:
Table 6. is used for the enzyme and the enzyme tangent condition thereof of sarcocystis 18S rRNA gene restriction analysis
| The enzyme title | The enzyme cut is made condition | |||
| Damping fluid | Time (h) | Enzyme dosage (U) | Temperature (℃) | |
| Alu?I Mbo?I Mbo?II Taq?I Hinf?I Bcl?I Rsa?I Ssp?I Acc?I Dra?I | B C B E B C C E G B | 8 10 50 8 8 48 10 16 8 10 | 5 5 5 5 5 5 10 7 5 10 | 37 37 37 65 37 50 37 37 37 37 |
Embodiment three:
Be used for the PCR of sarcocystis 18S rRNA gene studies and the primer of order-checking with table 1, check order.
3.1 stool examination and collection sporocyst and egg capsule comprise preparation 37%ZnSO
4Zinc sulfate flotation fluid (ZuSO
47Hz03319 adding distil water 1000ml), get excrement 1g and add 37%ZnSO
4Zinc sulfate flotation fluid 100ml adds in the 15ml centrifuge tube.Put under the centrifuge tube from.The supernatant liquid number of drawing the superiors in the centrifuge tube with suction pipe droplet moves on slide glass, with 200 times microscopy.The sporocyst and the egg capsule that arrive for the purpose of sticking with sheet for cleaning.The scraps of paper are put into the 1.5ml centrifuge tube, and-20 degree are preserved.
3.2 obtain target DNA fragment by pcr amplification
3.2.1PCR amplification condition
3.2.1.1 every 50ul reaction volume has 10 * Buffer 5ul, dNTPs 2ul, and BSA 2ul, primer 12ul, primer 2 2ul, Taq enzyme 1.25U, total DNA 2ul, surplus is a deionized water.
3.2.1.2 in the PCR reaction conditions, the long-time pre-same 1.2.1.2 of sex change, and identical with table 2 in the foregoing invention content.
The combination and the amplification condition thereof of the primer of several amplification of table 2. sarcocystis gene
| Combination of primers | Optimum annealing temperature ℃ | Best second extension time | It deny long-time pre-sex change |
| 18S1L6H 18S2L6H 18S2L3H 18S3L3H 18S2L2H 18S3L1H 18S10L？H | 50 50 56 56 53-55 56 56-63 | 30 30 80 60 100 60 30 | No no no |
3.3PCR the result of reaction, behind the purpose fragment purification, order-checking.
The purpose fragment of pcr amplification-Sarcocystis hominis 28h7ho strain 18SrRNA Gene Partial consecutive nucleotides sequencing result Sarcocystis hominis 28h7ho strain 18SrRNA Gene Partial sequence:
1?tggataaccg?tggtaattct?atggctaata?catgcgcaaa?tactatatct?ttacgatata
61?gtagtgttta?ttagatacag?aaccaacacg?ctcctttttg?ggggtgtcaa?aattaggtga
121?ttcatagtaa?ccgaacggat?cgcattatga?tcatattatt?atgattggcg?atagatcatt
181?caagtttctg?acctatcagc?tttcgacggt?agtgtattgg?actaccgtgg?cagtgacggg
241?taacggggaa?ttagggttcg?attccggaga?gggagcctga?gaaacggcta?ccacatctaa
301?ggaaggcagc?aggcgcgcaa?attacccaat?cctgactcag?ggaggtagtg?acaagaaata
361?acaacactgg?aaattcaatt?tctagtgatt?ggaatgatgg?gaatccaaac?ccctttcaga
421?gtaacaattg?gagggcaagt?ctggtgccag?cagccgcggt?aattccagct?ccaatagcgt
481?atattaaagt?tgttgcagtt?aaaaagctcg?tagttggata?tctgctggaa?gcaatcagtc
541?cgccctattt?tagggtgtgc?acttgatgaa?ttctggcatc?tattattctt?aatataatga
601?ttattgaatt?gattttcaat?aatcawtatt?aggaataata?cagttacttt?gagaaaatta
661?gagtgtttga?agcaggctta?ttgccttgaa?tactgcagca?tggaataaca?atataggatt
721?tcggttctat?tattttgttg?gtttgtagga?ctgaaataat?gattaatagg?gacagttggg
781?ggcattcgta?tttaactgtc?agaggtgaaa?ttcttagatt?tgttaaagac?gaactactgc
841?gaaagcattt?gccaaggatg?ttttcattaa?tcaagaacga?aagttagggg?ctcgaagacg
901?atcagatacc?gtcgtagtct?taaccataaa?ctatgccgac?tagagatagg?aaaatgtcat
961?tatttcgact?tctcctgcac?cttatgagaa?atcaaagtct?ttgggttctg?gggggagtat
1021?ggtcgcaagg?ctgaaactta?aaggaattga?cggaagggca?ccaccaggcg?tggagcctgc
1081?ggcttaattt?gactcaacac?ggggaaactc?accaggtcca?gacatgggaa?ggattgacag
1141?attgatagct?ctttcttgat?tctatgggtg?gtggtgcatg?gccgttctta?gttggtggag
1201?tgatttgtct?ggttaattcc?gttaacgaac?gagaccttaa?cctgctaaat?aggatcaaga
1261?acgcacttcg?ttgtgttttt?gtatcacttc?ttagagggac?tttgcgtgtc?taacgcaagg
1321?aagtttgagg?caataacagg?tctgtgatgc?ccttagatgt?tctgggctgc?acgcgcgcta
1381?cactgatgca?tccaacaagt?tgttgaaaac?cttggccgat?aggtttaggt?aatcttttga
Content of the present invention is not limited to the restriction of the content that embodiment enumerates.
Claims (6)
1. sarcocystis sporocyst and egg capsule molecular diagnosis method may further comprise the steps and content:
1) ight soil of getting 1-2g adds an amount of 37%ZnSO
4In the zinc sulfate flotation fluid centrifuge tube, leave standstill after centrifugal;
2) draw with suction pipe or droplet move on slide glass, with greater than 100 times microscopy, put into another centrifuge tube cryopreservation with sheet for cleaning sticking sporocyst and egg capsule from slide or centrifuge tube with the supernatant liquid number that transfering loop picks the superiors in the centrifuge tube;
3) comprising it, table 1 is arranged is primer, the 18SrRNA constant gene segment C 1500-2000bp Nucleotide of sarcocystis sporocyst that pcr amplification covered and egg capsule or its any a part of nucleotide fragments, or be used for pcr amplification as primer, or be used for hybridization, or preparation gene chip or microarray as probe;
Table 1 is used for the PCR and the sequencing primer of sarcocystis 18SrRNA gene
* locate according to the shuttle shape sarcocystis serial position of Holmdahl (1994)
4) to the specific amplified fragments of each worm kind, or do restriction analysis, or detect nucleotide sequence as sequencing primer with the primer of table 1, when carrying out restriction analysis, restriction enzyme and reaction conditions carry out endonuclease reaction in the table 2 with being selected from, the volume of endonuclease reaction is 10-20ul, result's gel detection
Table 2. is used for the enzyme and the enzyme tangent condition thereof of sarcocystis 18SrRNA gene restriction analysis
2. sarcocystis sporocyst according to claim 1 and egg capsule molecular diagnosis method is characterized in that with the primer that is selected from table 3, and the different single-minded target region dna fragmentations of sarcocystis egg capsule that the pcr amplification primer is covered are crossed in the road,
Table 3 is used for the PCR and the sequencing primer of sarcocystis 18S rRNA gene
* locate according to the shuttle shape sarcocystis serial position of Holmdahl (1994).
3. sarcocystis sporocyst according to claim 1 and egg capsule molecular diagnosis method is characterized in that in the pcr amplification optimization reaction conditions combination and the condition such as the table 4 of the primer of amplification sarcocystis gene:
Table 4. is used for the PCR of sarcocystis 18S rRNA gene and the combination and the condition of sequencing primer
Annotate: long-time pre-sex change, 97 ℃ of promptly pre-sex change were placed on ice in 5 minutes, just added the archaeal dna polymerase of 1.25U.
4. sarcocystis sporocyst according to claim 1 and egg capsule molecular diagnosis method is characterized in that the PCR and the sequencing primer that are used for sarcocystis 18S rRNA gene further are the combination of the listed primer of table 5:
Table 5 is used for the PCR and the sequencing primer of sarcocystis 18S rRNA gene
。
5. Endospore worm sporocyst according to claim 4 and egg capsule molecular diagnosis method is characterized in that the PCR combination of primers is 18S 2L, 18S 2H for the first time in the nest-type PRC reaction, and the PCR combination of primers is 18S 2L, 18S 3H for the second time.
6,, it is characterized in that the restriction enzyme of endonuclease reaction further is the one or more combination of table 6 according to claim 1,2,3,4 or 5 described sarcocystis sporocyst and egg capsule molecular diagnosis methods:
Table 6. is used for the enzyme and the enzyme tangent condition thereof of sarcocystis 18S rRNA gene restriction analysis
。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710065686 CN101092646A (en) | 2007-02-15 | 2007-02-15 | Method for diagnosing sporangium and egg capsule molecules of sarcocyst |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710065686 CN101092646A (en) | 2007-02-15 | 2007-02-15 | Method for diagnosing sporangium and egg capsule molecules of sarcocyst |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101092646A true CN101092646A (en) | 2007-12-26 |
Family
ID=38991081
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200710065686 Pending CN101092646A (en) | 2007-02-15 | 2007-02-15 | Method for diagnosing sporangium and egg capsule molecules of sarcocyst |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101092646A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103224982A (en) * | 2013-02-02 | 2013-07-31 | 中国水产科学研究院淡水渔业研究中心 | Detection and expelling method for coilia ectenes heteromazocraes lingmueni based on molecular marker technology |
| US8778843B1 (en) * | 2011-08-03 | 2014-07-15 | Fry Laboratories, L.L.C. | Semi-pan-protozoal by quantitative PCR |
| CN104531839A (en) * | 2014-11-26 | 2015-04-22 | 中华人民共和国上海出入境检验检疫局 | Rapid and sensitive plasmodium malariae detection method |
| CN110922491A (en) * | 2019-12-17 | 2020-03-27 | 河南科技大学 | Sarcocystis fusion antigen, encoding gene, indirect ELISA antibody detection kit and application thereof |
-
2007
- 2007-02-15 CN CN 200710065686 patent/CN101092646A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8778843B1 (en) * | 2011-08-03 | 2014-07-15 | Fry Laboratories, L.L.C. | Semi-pan-protozoal by quantitative PCR |
| CN103224982A (en) * | 2013-02-02 | 2013-07-31 | 中国水产科学研究院淡水渔业研究中心 | Detection and expelling method for coilia ectenes heteromazocraes lingmueni based on molecular marker technology |
| CN104531839A (en) * | 2014-11-26 | 2015-04-22 | 中华人民共和国上海出入境检验检疫局 | Rapid and sensitive plasmodium malariae detection method |
| CN104531839B (en) * | 2014-11-26 | 2017-12-26 | 中华人民共和国上海出入境检验检疫局 | A kind of malariae detection method of rapid sensitive |
| CN110922491A (en) * | 2019-12-17 | 2020-03-27 | 河南科技大学 | Sarcocystis fusion antigen, encoding gene, indirect ELISA antibody detection kit and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Read et al. | Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR-RFLP | |
| Thompson¹ et al. | Variation in Giardia: implications for taxonomy and epidemiology | |
| JP5317430B2 (en) | Probe set, probe carrier, and fungal discrimination identification method | |
| Le Roux et al. | DNA probes as potential tools for the detection of Marteilia refringens | |
| Parvizi et al. | First detection of Leishmania major in peridomestic Phlebotomus papatasi from Isfahan province, Iran: comparison of nested PCR of nuclear ITS ribosomal DNA and semi-nested PCR of minicircle kinetoplast DNA | |
| Rodrigues et al. | Brazilian isolates of Trypanosoma (Megatrypanum) theileri: diagnosis and differentiation of isolates from cattle and water buffalo based on biological characteristics and randomly amplified DNA sequences | |
| Liu et al. | Molecular identification of hookworms in stray and shelter dogs from Guangzhou city, China using ITS sequences | |
| Danehchin et al. | Isolation and genotyping of Toxoplasma gondii strains in ovine aborted fetuses in Khorasan Razavi Province, Iran | |
| CN105506153B (en) | A kind of kit and method detecting Aeromonas veronii | |
| CN101092646A (en) | Method for diagnosing sporangium and egg capsule molecules of sarcocyst | |
| Ahmed et al. | Molecular basis for identification of species/isolates of gastrointestinal nematode parasites | |
| Rognlie et al. | Myxobolus cerebralis in Tubifex tubifex from a whirling disease epizootic in Montana | |
| CN110093432B (en) | sRNA marker for distinguishing mycobacterium tuberculosis from BCG vaccine and application thereof | |
| Motloang et al. | Prevalence of Theileria equi and Babesia caballi infections in horses belonging to resource-poor farmers in the north-eastern Free State Province, South Africa | |
| Parvizi et al. | Detection, identification and molecular typing of Leishmania major in Phlebotomus papatasi from a focus of zoonotic cutaneous leishmaniasis in central of Iran | |
| CN113025738B (en) | SNP molecular marker related to drug resistance of halofuginone of Eimeria and application thereof | |
| Xenoulis et al. | Detection of Tritrichomonas foetus in cats in Greece | |
| Qin et al. | Morphological and molecular characteristics of a single oocyst for the identification of Eimeria species in domestic rabbits (Oryctolagus cuniculus f. domesticus) | |
| Sardar et al. | Molecular evidence suggests the occurrence of Entamoeba moshkovskii in pigs with zoonotic potential from eastern India | |
| Liang et al. | Occurrence and genetic characterization of GRA6 and SAG2 from Toxoplasma gondii oocysts in cat feces, Kunming, China | |
| MacPherson et al. | Ribosomal RNA sequences for the specific detection of Toxoplasma gondii by hybridization assay | |
| Vassalos et al. | Blastocystosis: an emerging or re-emerging potential zoonosis | |
| Dos Santos et al. | Diagnosis of Pentatrichomonas hominis from domestic cats in Southeastern Brazil | |
| Alsaad et al. | The first record of zoonotic genes of cutaneous leishmaniasis among human, dogs, and sandflies by nested polymerase chain reaction and phylogenetic analyses | |
| Tan et al. | First molecular detection of Tritrichomonas foetus in domestic cats in Klang Valley, Malaysia. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |