CN103173554B - Detection kit for detecting and distinguishing multiple kinds of echinococcus - Google Patents
Detection kit for detecting and distinguishing multiple kinds of echinococcus Download PDFInfo
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- CN103173554B CN103173554B CN201310102181.4A CN201310102181A CN103173554B CN 103173554 B CN103173554 B CN 103173554B CN 201310102181 A CN201310102181 A CN 201310102181A CN 103173554 B CN103173554 B CN 103173554B
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Abstract
The invention discloses a kit for detecting echinococcus granulosus, echinococcus multilocularis and echinococcus shiquicus. Three pairs of specific amplification primers aiming at three pathogens are respectively arranged in the detection kit. By utilizing the three pairs of primers in the kit, a composite polymerase chain reaction (PCR) method can be used for detecting independent or mixed infection of the three pathogens, and whether the detected sample is infected with echinococcus can be determined according to specific strips occurring in electrophoresis of amplification products.
Description
Technical field
The present invention relates to a kind of for detection of with distinguish Echinococcus granulosus, Echinococcus multilocularis and Shiqu echinococcus detection kit.
Background technology
Echinococcosis (echinococcosis) is commonly called as hydatidosis (hydatid disease), is a kind of Zoonosis parasitosis being caused by larva-echinococcus (hydatid cyst) of echinococcus.The echinococcus that at present whole world has been found is adhered to 5 kinds separately, Echinococcus granulosus (
echinococcus granulosus, Eg), Echinococcus multilocularis (
echinococcus mutilocularis, Em), Fu Shi echinococcus (
echinococcus vogeli, Ev), less save echinococcus (
echinococcus oligarthus, Eo) and Shiqu echinococcus (
echinococcus shaquacus, Es).Wherein the larva of Echinococcus granulosus, Echinococcus multilocularis and Fu Shi echinococcus causes respectively people's capsule type echinococcosis (cystic echinococcosis), alveolitoid echinococcosis (alveolar echinococcosis) and many capsules echinococcosis, human health is caused to significant damage, and seriously hinder the development of livestock industry.Only there is three kinds of Echinococcus granulosus, Echinococcus multilocularis and Shiqu echinococcus in China, mainly be distributed in Qinghai, Tibet, Sichuan, Xinjiang, Gansu, the Inner Mongol and Ningxia etc. and economize (district), wherein serious with Echinococcus granulosus harm, popular area is the most extensive, and alveolitoid echinococcosis is the highest to people's lethality rate.Echinococcus Granulosus Cysts can infect the many animals such as people and Niu, sheep, pig, and Canis animals is the final host of Echinococcus granulosus, and adult parasitizes in its small intestine, and worm's ovum is discharged with ight soil, and contaminated food, drinking-water and surrounding environment form main contagium.Canis animals infects containing the animal viscera of echinococcus by searching for food, and eats the morbidity of worm's ovum postoperative infection by mistake people.The intermediate host of Echinococcus multilocularis and Shiqu echinococcus is mainly the grassland such as vole and pika wildlife, and adult main parasitic is in the small intestine of the Canis animalss such as dog, wolf, fox, and worm's ovum excretes with ight soil, infects people and multiple rodent.People's echinococcosis multilocularis harm is serious, and case fatality rate is high, claims again " worm cancer ".In pastoral area, grassland, a large amount of wandering dogs infect Echinococcus multilocularis when movable on grassland, get rid of the ight soil containing worm's ovum in people living quarters, thereby threaten the western numerous herdsman's of China life with healthy.So detect in time, exactly the infection conditions of Endemic Area final host echinococcus and infection animal forced to expelling parasite, to control that popular, the comprehensive measures for the prevention and control of echinococcosis is implemented and the evaluation of prevention effect etc. all tool be of great significance.
Have at present the method that Echinococcus granulosus and echinococcus multilocularis infection dog are carried out to single detection, comprise detection method (the Boufana BS such as conventional sense method (methylarecaidin katharsis and cut open inspection method), DNA-PCR detection method, antigen-ELISA detection method and excrement antigen LAMP
et al. Evaluation of three PCR assays for the identification of the sheep strain (Genotype1) of
echinococcus granulosusin canid feces and parasite tissues[J].
am J Trop Med Hyg, 2008,78 (5): 777-783; Pierangeli NB,
et al. Usefulness and validation of a coproantigen test for dog echinococcosis screening in the consolidation phase of hydatid control in Neuqu é n, Argentina[J].
parasitol Int, 2010,59 (3): 394-399. Dinkel A,
et al. Detection of
echinococcus multilocularisin the de nitive host:coprodiagnosis by PCR as an alternative to Necropsy[J].
j Clin Microbiol, 1998,36 (7): 1871-1876.; Eckert J. Predictive values and quality control of techniques for the diagnosis of
echinococcus multilocularisin definitive hosts.
acta Trop, 2003,85 (2): 157-163).In aforesaid method, conventional sense method has the danger of infecting people and surrounding environment, and other several method all cannot carry out somatotype detection to multiple echinococcus in a reaction, has the problem that testing process is loaded down with trivial details and waste material.
Summary of the invention
The invention provides one and can overcome prior art deficiency, for detection of with distinguish the detection kit of multiple echinococcus.
Of the present invention for detection of including three pairs of special primers with distinguishing in the detection kit of multiple echinococcus, they are respectively: Echinococcus granulosus specificity upstream and downstream primer SEQ ID № 1 and SEQ ID № 2, Echinococcus multilocularis specificity upstream and downstream primer SEQ ID № 3 and SEQ ID № 4, Shiqu echinococcus specificity upstream and downstream primer SEQ ID № 5 and SEQ ID № 6.
For convenience of using, of the present invention for detection of being also placed with distinguishing in the detection kit of multiple echinococcus: Taq archaeal dna polymerase, 10 × PCR Buffer, dNTP (2.5 mmol/ μ L), MgCl
2(25 mmol/ μ L), aseptic double-distilled water (ddH
2o).
The present invention is based upon on cause of disease molecular biology mechanism, based on Echinococcus granulosus, the chondriogen of Echinococcus multilocularis and Shiqu echinococcus, respectively to three kinds of cause of disease design specificity amplification primers, and sets up the independent or polyinfection of composite PCR method for three kinds of cause of diseases of somatotype detection.The multiplex PCR of setting up in the present invention, system design three kinds of echinococcus existing for China design respectively that three pairs of specific detection primers infect for echinococcus separately and compound detection, what three kinds of etiologic typings detected for this reason is pioneering.All advantages that this multiplex PCR comprises regular-PCR technology, comprise that detected result specificity is high, detection reaction sensitivity, detect quick and high throughput testing etc., also there is the advantage such as high efficiency, systematicness and economical and convenient of composite PCR, be applicable to clinical and laboratory and carry out Identification of etiology fast and accurately.
The present invention is that a kind of composite PCR method of applying infects the test kit that carries out somatotype detection to echinococcus, in application, with three pairs of special primers in test kit of the present invention, test sample is carried out to pcr amplification, then the product after amplification is carried out to electrophoresis, whether infected according to whether occurring in map of current that specific band is determined test sample, and infected the concrete kind in these three kinds of parasites of Echinococcus granulosus, Echinococcus multilocularis and Shiqu echinococcus.Adopt this test kit both can detect for intermediate host's infection, also can detect for final host's infection, and can detect the possible multiple infection of final host.Operating process related to the present invention comprises from intermediate host's lesion tissue (packing and content thereof) of doubtful echinococcus infection and final host's ight soil extracts complete genome DNA, preparation is containing the composite PCR reaction system of three pairs of primers and full genomic templates, after carrying out PCR reaction, agarose gel electrophoresis is observed amplification, and judges according to the size of object band the echinococcus type infecting.With three pairs of specific PCRs detection primer of the present invention, the blister band tapeworm that is final host in order to dog (
taenia hydatigena, Th), Taenia Pisiformis (
taenia pisiform, Tp), bull band tapeworm (
taenia multiceps, Tm), diphlidium caninum (
dipylidium caninum, DC) and genomic dna is that template is carried out specificity checking amplification, has been showed no pcr amplification product, showing three pairs, to detect primer specificity high.The three pairs of primers of the present invention use simultaneously and do not interfere with each other in a PCR reaction system, show that it has the advantages such as high efficiency, systematicness and economical and convenient, are applicable to clinical and laboratory and carry out Identification of etiology fast and accurately.
accompanying drawing explanation
Fig. 1 is the PCR result electrophorogram that three kinds of echinococcus specific detection primers of the present invention increase to the common single parasite cause of disease DNA profiling take dog as final host respectively, wherein: 1,2,3,4,5 swimming lanes are respectively Auele Specific Primer SEQ ID № 1 and 2 amplification Echinococcus granulosus (Eg), blister band tapeworms (Th), Taenia Pisiformis (Tp), bull band tapeworm (Tm), PCR result electrophorogram when diphlidium caninum (Dc) genomic dna template; 6,7,8,9,10 swimming lanes are Auele Specific Primer SEQ ID № 3 and 4 amplification Echinococcus multilocularis (Em), blister band tapeworms (Th) respectively, Taenia Pisiformis (Tp), bull band tapeworm (Tm), PCR result electrophorogram when diphlidium caninum (Dc) genomic dna template; 11,12,13,14,15 swimming lanes are respectively Auele Specific Primer SEQ ID № 5 and 6 amplification Shiqu echinococcus (Es), blister band tapeworms (Th), Taenia Pisiformis (Tp), bull band tapeworm (Tm), PCR result electrophorogram when diphlidium caninum (Dc) genomic dna template; M is DNA molecular amount standard DL2000.
Fig. 2 is the PCR results of three kinds of echinococcus specific detection primers of the present invention while increasing with three kinds of echinococcus DNA profilings respectively, wherein: 1, PCR result electrophorogram when 2,3 swimming lanes are Auele Specific Primer SEQ ID № 1 and 2 amplification amplification Echinococcus granulosus (Eg), Echinococcus multilocularis (Em) and Shiqu echinococcus (Es) genomic dna template; PCR result electrophorogram when 4,5,6 swimming lanes are respectively Auele Specific Primer SEQ ID № 3 and 4 amplification Echinococcus granulosus (Eg), Echinococcus multilocularis (Em) and Shiqu echinococcus (Es) genomic dna template; PCR result electrophorogram when 7,8,9 swimming lanes are respectively Auele Specific Primer SEQ ID № 5 and 6 amplification Echinococcus granulosus (Eg), Echinococcus multilocularis (Em) and Shiqu echinococcus (Es) genomic dna template; M is DNA molecular amount standard DL2000.
Fig. 3 is the PCR results of three kinds of echinococcus specific detection primers of the present invention while increasing with three kinds of echinococcus hybrid dna templates respectively, wherein: 1,3,5 are respectively Auele Specific Primer SEQ ID № 1,2, PCR result electrophorogram when SEQ ID № 3,4 and SEQ ID № 5,6 expand Echinococcus granulosus (Eg), Echinococcus multilocularis (Em) and Shiqu echinococcus (Es) the mixed genomic DNA template; 2,4,6 swimming lanes are respectively Auele Specific Primer SEQ ID № 1,2, negative control when SEQ ID № 3,4 and SEQ ID № 5,6 amplification; M is DNA molecular amount standard DL2000.
Fig. 4 is that the mixing of three kinds of echinococcus of the present invention detects the composite PCR result of primer while increasing with three kinds of echinococcus DNA profilings respectively, wherein: 1, composite PCR result when 2,3 mix primer that are respectively SEQ ID № 1, SEQ ID № 2, SEQ ID № 3, SEQ ID № 4, SEQ ID № 5 and SEQ ID № 6 increase respectively Echinococcus granulosus (Eg), Echinococcus multilocularis (Em) and Shiqu echinococcus (Es) genomic dna template; 4 is the negative control in mix primer when amplification of SEQ ID № 1, SEQ ID № 2, SEQ ID № 3, SEQ ID № 4, SEQ ID № 5 and SEQ ID № 6; M is DNA molecular amount standard DL2000.
Fig. 5 is that the mixing of three kinds of echinococcus of the present invention detects the composite PCR result of primer while increasing with three kinds of echinococcus DNA hybrid templates, wherein: 1 be SEQ ID № 1, SEQ ID № 2, SEQ ID № 3, SEQ ID № 4, SEQ ID № 5 and the SEQ ID № 6 mix primer composite PCR result while increasing Echinococcus granulosus (Eg), Echinococcus multilocularis (Em) and Shiqu echinococcus (Es) the mixed genomic DNA template; 2 negative controls while being SEQ ID № 1, SEQ ID № 2, SEQ ID № 3, SEQ ID № 4, SEQ ID № 5 and SEQ ID № 6 mix primer amplification; M is DNA molecular amount standard DL2000.
Embodiment
In the present embodiment, choose wandering dog ight soil and carry out echinococcus infection detection.Get appropriate wandering dog excrement, with worm's ovum and polypide relic in the floating dog excrement of saturated saline floatation, after centrifugal collection, adopt excrement DNA extraction test kit to extract complete genome DNA, for subsequent use after mensuration extraction DNA concentration.In PCR reaction tubes, prepare composite PCR reaction liquid, adopt 50 μ L reaction systems, comprise 10 × PCR Buffer, 5 μ L, MgCl
2(25 mmol/ μ L) 5 μ L, for the upstream primer F1:5'-GGTTTTATCGGTATGTTGGTGTTAGTG-3'(SEQ ID № 1 of Echinococcus granulosus specific detection) 1 μ L(50 μ mol/mL) and downstream primer R1:5'-CATTTCYTGAAGTTAACAGCATCACG-3'(SEQ ID № 2) 1 μ L(50 μ mol/mL), for the upstream primer F2:5'-CATTAATTATGGATGTTTCC-3'(SEQ ID № 3 of Echinococcus multilocularis specific detection) 1 μ L(50 μ mol/mL) and downstream primer R2:5'-GGAAATACCCCACTATCC-3'(SEQ ID № 4) 1 μ L(50 μ mol/mL), for the upstream primer F3:5'-GCTTTAAGTGCGTGACTTTTAATCCC-3'(SEQ ID № 5 of Shiqu echinococcus specific detection) 1 μ L(50 μ mol/mL) and downstream primer R3:5'-CATCAAAACCAGCACT AATACTCA-3'(SEQ ID № 6) 1 μ L(50 μ mol/mL), dNTP (2.5 mmol/ μ L) 1 μ L, Taq archaeal dna polymerase 0.5 μ L, dog excrement complete genome DNA template amount is 100 ng, water is supplied 50 μ L.In thermal cycler, complete PCR reaction process, response procedures is: 95 ℃ of denaturation 4 min, and 94 ℃ of sex change 50 S, 52 ℃ of annealing 40S, 72 ℃ are extended 40 S, 35 circulations, 72 ℃ lengthen extension 10 min, have reacted rear 4 ℃ of preservations.PCR reaction product is carried out agarose gel electrophoresis, gel imaging system is observed amplification, and result is judged and analyzed, the specific amplification stripe size of Echinococcus granulosus, Echinococcus multilocularis and Shiqu echinococcus is respectively 196 bp, 584 bp and 471 bp, object band is carried out to nucleic acid sequencing analysis, show that three object bands adhere to Echinococcus granulosus, Echinococcus multilocularis and Shiqu echinococcus separately.
Concrete testing process of the present invention is as follows:
Obtaining of 1 wandering dog excrement
Fresh dog excrement is collected in the dog zone of action that wanders around, or collects dog excrement after the food catharsis of throwing something and feeding containing Arecoline hydrobromide to wandering dog.
The extraction of 2 wandering dog excrement complete genome DNAs
1) by the dog ight soil obtaining in-70
°frozen 1 week of C.
2) preparation of excrement DNA: extract excrement DNA according to excrement DNA extraction test kit specification sheets.
The foundation of 3 pcr amplification systems
In PCR reaction tubes, prepare composite PCR reaction liquid, adopt 50 μ L reaction systems, add 10 × PCR Buffer, 5 μ L, MgCl
2(25 mmol/ μ L) 5 μ L, primer Eg № 1, Eg № 2, Em № 1, Em № 2, the each 1 μ L(50 μ mol/mL of Es № 1, Es № 2), dNTP (2.5 mmol/ μ L) 1 μ L, Taq archaeal dna polymerase 0.5 μ L, dog excrement complete genome DNA template amount is 100 ng, and water is supplied 50 μ L.
4 pcr amplification conditions
PCR response procedures is: 95 ℃ of denaturation 5 min, and 94 ℃ of sex change 50 S, 52 ℃ of annealing 40 S, 72 ℃ are extended 40 S, 35 circulations, 72 ℃ lengthen extension 10 min, have reacted rear 4 ℃ of preservations.
5 pcr amplification results are observed and are judged
PCR reaction product is carried out electrophoresis in 1.5% (w/v) sepharose (containing 0.5 μ g/mL ethidium bromide), and ultraviolet gel imaging system is observed amplification, according to the size of object band, judges the echinococcus kind infecting.
<110> Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
<120> for detection of with distinguish the detection kit of multiple echinococcus
The number of sequence (as 18) in <160> sequence table (gene order of note: CN1 to CN8 and its aminoacid sequence)
<210> 1
<211> 27
<212> DNA
<213> artificial sequence (Echinococcus granulosus upstream primer)
<400>
ggttttatcg gtatgttggt gttagtg 27
<210> 2
<211> 26
<212> DNA
<213> artificial sequence (Echinococcus granulosus downstream primer)
<400>
catttcytga agttaacagc atcacg 26
<210> 3
<211> 20
<212> DNA
<213> artificial sequence (Echinococcus multilocularis upstream primer)
<400>
cattaattat ggatgtttcc 20
<210> 4
<211> 18
<212> DNA
<213> artificial sequence (Echinococcus multilocularis downstream primer)
<400>
ggaaataccc cactatcc 18
<210> 5
<211> 26
<212> DNA
<213> artificial sequence (Shiqu echinococcus upstream primer)
<400>
gctttaagtg cgtgactttt aatccc 26
<210> 6
<211> 24
<212> DNA
<213> artificial sequence (Shiqu echinococcus downstream primer)
<400>
catcaaaacc agcactaata ctca 24
Claims (2)
- For detection of with distinguish the detection kit of multiple echinococcus, it is characterized in that including in test kit three pairs of special primers, they are respectively: Echinococcus granulosus specificity upstream and downstream primer SEQ ID № 1 and SEQ ID № 2, Echinococcus multilocularis specificity upstream and downstream primer SEQ ID № 3 and SEQ ID № 4, Shiqu echinococcus specificity upstream and downstream primer SEQ ID № 5 and SEQ ID № 6.
- According to claim 1 for detection of with distinguish the detection kit of multiple echinococcus, it is characterized in that also having in detection kit of the present invention: Taq archaeal dna polymerase, 10 × PCR Buffer, dNTP, MgCl 2, aseptic double-distilled water.
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Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103409521B (en) * | 2013-08-08 | 2015-04-15 | 中国农业科学院兰州兽医研究所 | Detection kit used for detecting echinococcus shiquicus (larvae) |
| CN104164512A (en) * | 2014-08-26 | 2014-11-26 | 中国农业科学院兰州兽医研究所 | PCR-RFLP detection kit for authenticating and differentiating infections of echinococcus multilocularis and echinococcosis shiquicus |
| CN107164479B (en) * | 2017-05-27 | 2021-03-30 | 四川省疾病预防控制中心 | Primer pair combination and kit for detecting and identifying human tissue hydatid pathogen |
| CN107365849B (en) * | 2017-08-10 | 2021-02-26 | 西南民族大学 | Kit for canine granulosa and echinococcus multilocularis based on POCKIT Micro fluorescent PCR platform and application |
| CN110527730B (en) * | 2018-05-25 | 2022-06-21 | 中国农业科学院兰州兽医研究所 | Echinococcus shikoensis detection kit based on RPA technology and application thereof |
| CN110846309B (en) * | 2018-08-20 | 2023-03-24 | 中国农业科学院兰州兽医研究所 | Primer pair and primer probe set for identifying echinococcus multilocularis and application of primer pair and primer probe set |
| CN109762910B (en) * | 2019-01-10 | 2023-06-09 | 四川省疾病预防控制中心 | Primer and kit for simultaneously detecting two types of echinococcosis |
| CN111235285A (en) * | 2020-03-04 | 2020-06-05 | 南京亿科人群健康研究院有限公司 | Kit for detecting echinococcosis diagnosis |
| CN111876495B (en) * | 2020-08-24 | 2023-07-14 | 新疆畜牧科学院兽医研究所（新疆畜牧科学院动物临床医学研究中心） | Taqman Real-Time PCR detection kit for echinococcus and application thereof |
| CN112226517B (en) * | 2020-09-22 | 2023-10-20 | 杭州医学院 | Primer group for detecting and distinguishing echinococcosis granulosa and echinococcosis multifilialis through loop-mediated isothermal amplification |
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