CN105018613A - Method for detecting Echinococcus multilocularis from fox excrement - Google Patents
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Abstract
The invention discloses a method for detecting Echinococcus multilocularis pathogen from fox excrement. The method includes: placing the collected Tibetan fox excrement sample into 70% ethanol, and performing cryopreservation at minus 80 DEG C for more than three weeks; using double-layer sterilizing gauze to enrich excrement sample Echinococcus multilocularis eggs, using a tissue crusher to crush egg embryonic membranes in a mechanical oscillation manner, using an excrement DNA extracting kit to extract the DNA of the Echinococcus multilocularis egg in the excrement sample, and using nest PCR amplification to detect the Echinococcus multilocularis eggs. The method is simple to operate, capable of fast detecting whether Echinococcus multilocularis infection exists in the fox excrement or not, high in sensitivity and high in specificity.
Description
Technical field
The invention belongs to molecular Biological Detection field, relate to the discriminating that alveolar hydatid disease in faecal samples is former.
Background technology
Echinococcus multilocularis hydatidosis (alveolar echinococcosis, AE) is a kind of lethality parasitic zoonoses caused by Echinococcus multilocularis (Echinococcus multilocularis, Em).Because this disease there is no the methods for the treatment of of special efficacy, be therefore called as the cancer of parasitosis.AE is in the animal husbandry of China's Major Epidemic in the northwestward, Ban Nongban grazing district, and wherein, East of Tibetan Plateau pastoral area is epidemic-stricken area the most serious in the world.At present, AE has become one of the most serious public health security problem in pastoral area, China plateau.
Hide fox (Vulpes ferrilata) and domesticated dog is the main final host of Zang Dong pastoral area Em, however due to domesticated dog freely movable in extensive range, and keep close contacting with the mankind, existing research mainly concentrates in the approach of domesticated dog propagation AE.But, undeniable be domesticated dog or fox all using the wild small-sized mammalian as Em intermediate host for food, correlative study finds that the infection rate of dog Em is 17.0% (52/305) in Xinjiang, 5.1% (3/59) in Qinghai, 12.0% (45/371) (Chai et al., 2003 in Sichuan; Wang Hu etc., 2000; Budke et al., 2005), and to be presented at Shiqu County Sichuan Province to the investigation result of wild fox Em infection rate be 59.1% (13/22), be 33.3% (4/12) (Qiu Jiamin etc., 1995 in Chengduo County, Qinghai Province and Oenothera littaralis; Wang Hu etc., 2000), the Em infection rate of fox is all higher than domesticated dog.Therefore, in the research of China AE, be necessary very much to start with from wild Vulpes species, the epidemiology of research AE in wildlife and ecological characteristics.Such research will for starting with from transmission of disease source, wildlife epidemic disease source, the angle controlled from bionomic control and species controls the source pathophoresis of wildlife epidemic disease provides important Research foundation and theoretical foundation, really realizes the comprehensive effectively control of the western pastoral area AE of China.
But, existing detection method is as (the Dinkel A such as pathogeny detection method (methylarecaidin katharsis and cut open inspection method), ELISA detection method, LAMP detection technique of routine, et al.Detection of Echinococcusmultilocularis in the definitive host:coprodiagnosis by PCR as an alternative toNecropsy [J] .J Clin Microbiol, 1998,36 (7): 1871-1876.; Eckert J.Predictivevalues and quality control of techniques for the diagnosis of Echinococcusmultilocularis in definitive hosts.Acta Trop, 2003,85 (2): 157-163.; Ni XM.Loop-Mediated Isothermal Amplification (LAMP) assay for the identification ofEchinococcus multilocularis infections in canine definitive hosts.Particle andFibre Toxicology, 2014,7 (1): 35-42.) all there is certain shortcoming, Sensitivity and Specificity is all lower, and wherein conventional detection method also has the danger infecting people and surrounding environment.Echinococcus tapeworm worm kind is differentiated, the DNA analysis method more effective and special (Craiget al., 2007 that extensively adopt at present compared to these technology; Nakao et al., 2007; 2010).Faeces DNA detection technique is a kind of molecular analysis methods of non-damage, is applicable to diagnose whether infect echinococcus (Mathiset al., 1996 by the ight soil of final host; Dinkel et al., 1998; 2011; Stefanic et al., 2004; Trachsel et al., 2007; H ü ttner et al., 2009).
Summary of the invention
The object of this invention is to provide one and can overcome prior art deficiency, from fox ight soil, detect the method for Echinococcus multilocularis.
The invention provides a kind of efficient faeces DNA analytical procedure, to detect the alveolar hydatid disease hidden in fox ight soil former, first we use the cytochrome b of Mitochondrial DNA (Cytb) gene fragment whether to distinguish ight soil from Tibetan fox, then detected and qualification Em by the method for the specific fragment of worm's ovum DNA in nested PCR amplification excrement sample.
The invention provides a kind of method detecting Echinococcus multilocularis from fox excrement, the method comprises the following steps:
One. hide fox fecal sample, be kept in 70% ethanol, be placed in-80 DEG C freezing more than 3 weeks.
Two. use dual-layer sterilization gauze and historrhexis's instrument enrichment and the worm's ovum vibrated in destruction step one gained excrement sample, extract test kit by faeces DNA and extract worm's ovum DNA in excrement sample.
Three. the worm's ovum DNA that nested PCR amplification step 2 obtains also identifies worm's ovum.During nested PCR amplification, four special primers are upstream outer primer respectively: F1:5 '-TTGAATTTGCCACGTTTGAATGC-3 ' (SEQ ID NO.1); Downstream outer primer: R1:5 '-GAACCTAACGACATAACATAATGA-3 ' (SEQ ID NO.2); Upstream internal primer: F2:5 '-GTCATATTTGTTTAAGTATAAGTGG-3 ' (SEQ ID NO.3); Downstream inner primer: R2:5 '-CACTCTTATTTACACTAGAATTAAG-3 ' (SEQ ID NO.4).
Wherein step one hides fox fecal sample is that field is collected, concrete collection method is: between in July, 2010 to August, we lay line-transect at Shiqu County Sichuan Province cloud ripple ditch by systematic sampling, adopt clean-up method of sampling Bian collection to 184 parts, excrement sample, all samples all have recorded the GPS site of sampling point.Divided grade according to the color of sample, shape, moisture and smell to its freshness, 1 grade is the fresh excrement sample on the same day, and namely there are moisture and gloss in surface; 2 grades is the comparatively fresh excrement sample in a few days, and surface color is comparatively dark, and appearance profile is obvious; 3 grades is excrement sample in 1 week, and color is greyish white, and appearance profile is obvious; 4 grades is older excrement sample, more difficult identification.We only adopt the fresh class sample of 1 grade and 2 grades in principle, but when really can not find fresh excrement sample in line-transect region, and our class that also Bian collection is older just.Save the sample of for some time in the wild in physical environment, although quality is not as fresh sample, also therefrom can extract the DNA that substantially can meet research and require.The excrement sample that we collect mainly is distributed on thick grass, fox hole, flag, on limit, water source and mount.Time in the wild during sampling, for security consideration, we pick up sample with disposable bamboo chopsticks, are kept at 50ml and are equipped with in the centrifuge tube of 70% ethanol, behind time laboratory, and-80 DEG C of freezen protective more than 3 weeks.
The Tibetan fox fecal sample wherein collected in the wild in step one uses cytochrome b (Cytb) gene fragment of Mitochondrial DNA whether to distinguish ight soil from Tibetan fox, concrete steps can with reference to (Jiang WB, WangXM, Li M, Wang ZH* (2011) Identification of the Tibetan fox (Vulpes ferrilata) and the red fox (Vulpes vulpes) by copro-DNAdiagnosis, Molecular EcologyResources, 11 (1): 206-210.)
Wherein faeces DNA extracts test kit is QIAamp DNAStool Mini kit, purchased from the nested PCR amplification described in German Qiagen company, for the ordinary skill in the art, can with reference to such as Publication about Document (Nakao M, Sako Y, Yokoyama N, Fukunaga M, Ito A (2000) Mitochondrial geneticcode in cestodes.Mol Biochem Parasitol, 111:415-424.Nonaka N, Hirokawa H, Inoue T, Nakao R, Ganzorig S, Kobayashi F, Inagaki M, Egoshi K, Kamiya M, OkuY (2010) The first instance of a cat excreting Echinococcus multilocularis eggs inJapan.Parasitol Int, 57:519-520.)
Above-mentioned Echinococcus multilocularis specifically refers to Echinococcus multilocularis hydatidosis (alveolar echinococcosis, AE).
Another object of the present invention is to provide a kind of from fox ight soil, detect the former test kit of alveolar hydatid disease, it is characterized in that, it is upstream outer primer respectively that this test kit comprises four special primers: F1:5 '-TTGAATTTGCCACGTTTGAATGC-3 ' (SEQ ID NO.1); Downstream outer primer: R1:5 '-GAACCTAACGACATAACATAATGA-3 ' (SEQ ID NO.2); Upstream internal primer: F2:5 '-GTCATATTTGTTTAAGTATAAGTGG-3 ' (SEQ ID NO.3); Downstream inner primer: R2:5 '-CACTCTTATTTACACTAGAATTAAG-3 ' (SEQ IDNO.4).
Above-mentioned a kind of test kit detecting Echinococcus multilocularis from fox ight soil, is characterized in that, this test kit comprises faeces DNA and extracts test kit.
Beneficial effect
The present invention adopts the specific amplified of nest-type PRC in the former process of alveolar hydatid disease detecting from fox ight soil, have easy, quick, cheap and that accuracy is high feature.
Worm's ovum number in ight soil compares much less with bacterium with virus, and oncosphere by embryophoric membrane around, to the cracking of many chemical reagent and enzyme, there is height resistivity.Therefore, fecal bacteria and viral DNA extracting method and be not suitable for worm's ovum.So, be the detection sensitivity improving worm's ovum in excrement sample, to the enrichment of worm's ovum and the fragmentation of chorion particularly important.The traditional method of echinococcus worm's ovum enrichment utilizes the continuous method crossing hoof and the broken floating combination of chlorination, and the method repeatedly extruding the excrement slag of two-layer sterile gauze parcel that we adopt is relatively more efficient, compared with first two method, also reduce the possibility of crossed contamination.In addition, we adopt the broken worm's ovum embryophoric membrane of historrhexis instrument mechanical oscillation to discharge DNA wherein, although deposit the possibility that worm's ovum and Tao Zhu collision rift discharge at high speeds genomic dna also has fracture, but compared with the chorion breaking methods such as other such as ultrasonic, liquid nitrogen multigelation, this method uses manpower and material resources sparingly, and completely airtight experimental state also ensure that sample is not contaminated.The nest-type PRC that we introduce in an experiment, carries out two-wheeled amplification, compared to traditional single-wheel PCR, adds amplified production amount, improves the sensitivity and specificity that detect.
Accompanying drawing explanation
Fig. 1 is that Echinococcus multilocularis worm's ovum is broken the Photomicrograph (× 400) of embryophoric membrane, wherein: before the process of (A) mechanical oscillation, the tapeworm egg (B) of the hard thick stock film of band to vibrate worm's ovum 15s with rotating speed 5500r/min with historrhexis's instrument.(C) repeatedly vibrate 2 times with rotating speed 5500r/min with historrhexis's instrument, often vibrate 15s, and suspend 15s, worm's ovum embryophoric membrane is completely broken.
Fig. 2 is the electrophoresis of laboratory sample detected result, wherein: M is DNA standard molecule dL1000 (Takara); 1-12 swimming lane hides fox faeces DNA sample, and through qualification, 9,10 and 12 road samples exist Em to be infected, and Em+ is the tissue DNA sample of Em, and "-" is negative control.
Fig. 3 is the electrophoresis of nest-type PRC primer specificity evaluation result, and M is DNA standard molecule dL1000 (Takara); Be seven kinds of parasite body tissue DNA afterwards successively, respectively: Ts is taeniasis suis, Sm is Spirometra mansoni, Dt is fish tapeworm, and Dc is diphlidium caninum, and Eg is Echinococcus granulosus, Es is Shiqu echinococcus, and Em is Echinococcus multilocularis, and "-" is negative control.
Embodiment
Below in conjunction with accompanying drawing, the present invention is further described.The one of following embodiment just preferably in embodiment, not limitation of the present invention.Change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify, all should be the substitute mode of equivalence, be included within protection scope of the present invention.Do not make experiment reagent and the method for specified otherwise, all refer to conventional reagent and method.
Embodiment 1
The collection of 1 Tibetan fox fecal sample: hiding fox fecal sample is (Thomas township of the Shiqu County Sichuan Province Russia cloud ripple ditch (N 33 ° 10 ' collected within the scope of the family territory of the Tibetan fox of 7 radiotrackings, E 97 ° 39 ')), all sample standard deviations are kept in 70% ethanol, for security consideration ,-80 DEG C of freezen protective more than 3 weeks before experiment.
2 excrement sample worm's ovum enrichments: get 2g excrement sample and be placed in 150ml beaker, add 100ml deionized water.80 DEG C of water-bath 10min, scissors rubs, mixing.100 mesh filter screens filter dirty solution, and filtrate is kept in 50ml test tube, fall filtrate in being laid on the dual-layer sterilization gauze in plate, and extruding gauze for several times, makes wherein turbid liquid ooze out, is collected in 50ml test tube.The centrifugal 30min of 3600g, supernatant discarded.
The fragmentation of 3 worm's ovum embryophoric membranes: add 600ul ASL, with rifle head, itself and precipitation are mixed, 70 DEG C hatch 10min after be drawn into one in the lump and be placed with equivalent Tao Zhu (diameter is 0.5mm, Peqlab Biotechnology, Erlangen, Germany) tissue homogenate pipe in.Homogenate tube is placed on historrhexis's instrument, the embryophoric membrane that the broken worm's ovum of mechanical oscillation is hard thick, historrhexis's instrument parameter setting: rotating speed is 5500r/m; Cycle number is 2 times; Vibration 15s; Suspend 15s.Result as Fig. 1 (wherein: the tapeworm egg (B) of the hard thick stock film of band to vibrate worm's ovum 15s with rotating speed 5500r/min with historrhexis's instrument before the process of (A) mechanical oscillation.(C) repeatedly vibrate 2 times with rotating speed 5500r/min with historrhexis's instrument, often vibrate 15s, suspends 15s, and worm's ovum embryophoric membrane is completely broken).
4 excrement sample DNA extraction: extract test kit (Qiagen, QIAamp DNAStool Mini kit) specification sheets according to faeces DNA and extract host DNA and worm's ovum DNA in excrement sample.
5 nested PCR amplification qualification worm's ovums: four special primers are upstream outer primer respectively: F1:5 '-TTGAATTTGCCACGTTTGAATGC-3 '; Downstream outer primer: R1:5 '-GAACCTAAC GACATAACATAATGA-3 '; Upstream internal primer: F2:5 '-GTCATATTTGTTTAAGTATAAGTGG-3 '; Downstream inner primer: R2:5 '-CACTCTTATTTACACTAGAATTAAG-3 '.Nest-type PRC medial and lateral primer pair amplifies system is 20 μ L, comprise 50-100ng template DNA (second take turns pcr amplification template be 2 μ l), 5 ' end primer and 3 ' end each 0.2 μM of primer, each 200 μMs of dNTP, 10 × buffer is (containing 15mmol/L MgC1
2) 2 μ l, 1U Taq enzyme and 2%BSA0.5 μ l.; Establish the positive (Echinococcus multilocularis adult DNA) and negative control simultaneously.Two-wheeled pcr amplification condition is 95 DEG C of 5min, 94 DEG C of 30s, 52 DEG C of 45s, 72 DEG C of 90s, totally 35 circulations, 72 DEG C of 10min.PCR primer, through 2% agarose gel electrophoresis, judges according to result the genetic material that whether there is Echinococcus multilocularis in excrement sample, result as Fig. 2 (wherein: M is DNA standard molecule dL1000 (Takara); 1-12 swimming lane hides fox faeces DNA sample, and through qualification, 9,10 and 12 road samples exist Em to be infected, and Em+ is the tissue DNA sample of Em, and "-" is negative control).We are extracted taeniasis suis (Taenia solium), Spirometra mansoni (Spirometramansoni), fish tapeworm (Diphyllobothrium latum), diphlidium caninum (Dipylidiumcaninum), Echinococcus granulosus (E.granulosus) the parasite DNA different with Shiqu echinococcus (E.shiquicus) seven kinds, adopt identical nested PCR detection method, by the analysis to experimental result, inquire into the specificity of the method, result is as Fig. 3.
The present invention adopts the specific amplified of nest-type PRC in the former process of alveolar hydatid disease detecting from fox ight soil, have easy, quick, cheap and that accuracy is high feature.
Embodiment 2
We devise the Echinococcus multilocularis genetic material that labelled by nested-PCR method comes to exist in examination fox excrement.Outside primer selects band section universal primer (F1, R1) to increase line grain DNA Terminal oxidase I subunit gene (COI) partial sequence, and length is 874bp.Inner primer Primer Premier 5.0 software designs, and product length is 243bp.
The effect assessment of embodiment 3 nest-type PRC
We evaluate set up faeces DNA nest-type PRC diagnostic method by random selecting 72 increment product from the 184 parts of excrement samples altogether collected for 2010.Wherein successfully extract 48 parts of sample DNAs, through species identification, all from Tibetan fox.Tapeworm worm's ovum carries out (Fig. 1) after enrichment and broken process in excrement sample, we use nest-type PRC and Xiao (Xiao N, Nakao M, Qiu JM, Budke CM, Giraudoux P, Craig PS, Ito A (2006a) Short Report:Dual infection of animal hosts with differentspecies of Echinococcus in the eastern Qinghai-Tibetan Plateau region of China.Am J Trop Med Hyg, the Em genetic material that PCR-RFLP 75:292-294.) set up two kinds of methods exist in examination fox excrement respectively, find that the detected result of two kinds of methods exists significant difference <0.001, the Wilcoxon sign test of paired sample).In 48 excrement samples, nest-type PRC detects 9 (19%) individual samples and infects Em, and PCR-RFLP only detects that 3 (6%) individual samples infect sense Em.In addition, allly in PCR-RFLP method, detect that the sample standard deviation be positive is confirmed (table 1) in nest-type PRC.
Table 1 two kinds of methods compare echinococcus worm's ovum detected result in the fox ight soil of Tibetan
Claims (6)
1. from fox excrement, detect a method for Echinococcus multilocularis, the method comprises the following steps:
A. hide fox fecal sample, be kept in 70% ethanol, be placed in-80 DEG C freezing more than 3 weeks;
B. use dual-layer sterilization gauze carry out the worm's ovum enrichment of excrement sample and utilize historrhexis's instrument to carry out the fragmentation of worm's ovum embryophoric membrane, extract test kit by faeces DNA and extract worm's ovum DNA in excrement sample;
C. the worm's ovum DNA that obtains of nested PCR amplification step B identify worm's ovum, during nested PCR amplification, four special primers are that upstream outer primer: F1 is as shown in SEQ ID NO.1 respectively; Downstream outer primer: R1 is as shown in SEQ ID NO.2; Upstream internal primer: F2 is as shown in SEQ ID NO.3; Downstream inner primer: R2 is as shown in SEQ ID NO.4.
2., very according to a kind of method detecting Echinococcus multilocularis from fox excrement according to claim 1, it is characterized in that, described Echinococcus multilocularis specifically refers to Echinococcus multilocularis hydatidosis.
3. very according to a kind of method detecting Echinococcus multilocularis from fox excrement according to claim 1, it is characterized in that, the acquisition method hiding fox fecal sample in steps A is: field is collected and hidden fox fecal sample, line-transect is laid by systematic sampling, adopt clean-up method of sampling Bian collection to excrement sample, all samples all records the GPS site of sampling point; According to the color of sample, shape, moisture and smell, its freshness is classified: 1 grade is the fresh excrement sample on the same day, and namely there are moisture and gloss in surface; 2 grades is the comparatively fresh excrement sample in a few days, and surface color is comparatively dark, and appearance profile is obvious; 3 grades is excrement sample in 1 week, and color is greyish white, and appearance profile is obvious; 4 grades is older excrement sample, more difficult identification; Only adopt the fresh class sample of 1 grade and 2 grades in principle, but when really can not find fresh excrement sample in line-transect region, also can the older class of Bian collection just; The excrement sample collected mainly is distributed on thick grass, fox hole, flag, on limit, water source and mount; Time in the wild during sampling, pick up sample with disposable bamboo chopsticks.
4. very according to a kind of method detecting Echinococcus multilocularis from fox excrement according to claim 3, it is characterized in that, the Tibetan fox fecal sample that field is collected uses the cytochrome b gene fragment of Mitochondrial DNA whether to distinguish ight soil from Tibetan fox.
5. from fox ight soil, detect the former test kit of alveolar hydatid disease, it is characterized in that, this test kit comprises four special primers, is that upstream outer primer: F1 is as shown in SEQ ID NO.1 respectively; Downstream outer primer: R1 is as shown in SEQ ID NO.2; Upstream internal primer: F2 is as shown in SEQ ID NO.3; Downstream inner primer: R2 is as shown in SEQ ID NO.4.
6. a kind of test kit detecting Echinococcus multilocularis from fox ight soil according to claim 5, is characterized in that, this test kit comprises faeces DNA and extracts test kit.
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