CN103374615A - PCR (Polymerase Chain Reaction) detection kit for cystic echinococcosis of dog - Google Patents
PCR (Polymerase Chain Reaction) detection kit for cystic echinococcosis of dog Download PDFInfo
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- CN103374615A CN103374615A CN 201210120092 CN201210120092A CN103374615A CN 103374615 A CN103374615 A CN 103374615A CN 201210120092 CN201210120092 CN 201210120092 CN 201210120092 A CN201210120092 A CN 201210120092A CN 103374615 A CN103374615 A CN 103374615A
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Abstract
The invention discloses a PCR (Polymerase Chain Reaction) detection kit for cystic echinococcosis of a dog. According to the method, a pair of primers is designed according to a 12S rDNA (ribosome Deoxyribose Nucleic Acid) sequence conserved region of pathogene echinococcus granulosus of the cystic echinococcosis, a PCR reaction is carried out with the DNA contained in the wastes of an animal to be detected serving as a template, and then a comparison is carried out relative to an attached negative control and a positive control, in order to determine whether the detected animal infects the echinococcosis. The detection kit is simple and easy to use, and high in sensitivity and stability and can be used for monitoring the health condition of a pet dog.
Description
Technical field
The invention belongs to zoonotic diagnostic techniques, relate to the detection method to the dog echinococcosis granulosa.
The invention provides a kind of universal PCR test kit that detects the dog echinococcosis granulosa.Content of the present invention relates to a pair of PCR primer that is used for detecting the dog echinococcosis granulosa, and a cover has comprised the PCR detection kit that PCR reaction mix (containing primer), positive control, negative control, tetrabromophenol sulfonphthalein sample-loading buffer form and detected accordingly schedule of operation.
Background technology
Echinococcosis (Echinococcosis) claims again hydatidosis (hydatid disease), the larva by Echinococcus (Echinococcus) tapeworm---the infecting both domestic animals and human parasitosis that a class due to echinococcus (echinococcus orhydatidcyst) parasitizes in animal (the comprising the people) body can the serious harm HUMAN HEALTH all has distribution all over the world.Sour jujube ball (tapeworm) belongs in classification and is under the jurisdiction of Platyhelminthes, Cyclophyllidea, band section.Present confirmed Echinococcus tapeworm has 5 kinds: Echinococcus granulosus (E.granulosus), Echinococcus multilocularis (E.multilocularis), save echinococcus (E.oligarthrus), Fu Shi echinococcus (E.vogeli) and Shiqu echinococcus (E.shiquicus) less.The adult of echinococcus not of the same race and larva thereof all have difference at form, biological property, the aspect such as pathogenic.Wherein, save less echinococcus and the Fu Shi echinococcus mainly is distributed in the central and south america, save less the definite report that echinococcus there is no human infection, the larva of Fu Shi echinococcus can cause many capsules property echinococcosis at human body once in a while.And the larva of Echinococcus granulosus and Echinococcus multilocularis causes respectively echinococcosis granulosa (cystic echinococcosis, CE) and echinococcosis multilocularis (alveolar echinococcosis, AE) in host.China is the echinococcosis district occurred frequently, deposits popular three kinds of Echinococcus granulosus, Echinococcus multilocularis and the Shiqu echinococcus that have in China at present, especially with Echinococcus granulosus the most extensively and serious.
If the final host of Echinococcus tapeworm is take Canis animals as main.Its adult parasitizes in host's small intestine, and worm's ovum is discharged with ight soil, contaminated food, drinking-water and surrounding enviroment.The final host who infects echinococcus is the important contagium of echinococcosis, so detect in time, exactly the infection conditions of Endemic Area final host echinococcus and infection animal forced expelling parasite, popular, the comprehensive measures for the prevention and control of control echinococcosis is implemented and prevention effect etc. all has great importance.
The people is returned polluted source or food by the echinococcus ovum edible behind discharge host enteron aisle.The people namely can infect after the water after careless edible pollution the or food.The course of disease of echinococcosis granulosa is slow, and latent period is long, about 1～30 year.Symptom is very hidden, and is often subtle.Along with expanding gradually of the tumour of infection site, occupancy pressure symptom and the systemic toxic symptoms of parasitic site are obvious gradually.The internal organs parasitic according to echinococcus can be divided into hydatidosis hepatic echinococcosis, echinococcosis pulmonum, cerebral echinococcosis, osseous hydatid disease etc.Pet main cause pet dog is arranged in the recent period and infect echinococcosis granulosa and cause the report of osseous hydatid disease.Initial infection, sour jujube ball col parasitizes in the medullary space, then spreads along spongy bone and bone hole, destroys gradually sclerotin, finally can cause fracture.If tumour is worn out cortex of bone and invaded surrounding soft tissue, can form huge enclosed mass.If cause the skin ulceration, then can form the fistula of not healing for a long time, fester and Echinococcus hydatid cyst chip can be discharged thus, and can the secondary chronic suppurative osteomyelisis.If involve the joint, also can cause luxatio pathologica.Pathology initial stage non-evident sympton along with the development of the state of an illness, pain, numbness, limb muscle atrophy can occur.But the tumour pressuring nerve that vertebra, rumpbone etc. are located produces the sings and symptoms of neurothlipsis, even paraplegia.In addition, worm's ovum also can infect other positions of apparatus urogenitalis official rank such as eyes, kidney, bladder, ureter, prostate gland, spermatic cord, ovary, uterine tube, uterus and vagina, also there is the report of echinococcus parasitism at the positions such as heart, spleen, muscle, pancreas, its symptom is similar to innocent tumour, simultaneously can also cause multiple complications, such as accompanying infection, break, anaphylactic shock etc., endanger huge.
Because this sick latent period is long, and Symptom hiding is large, infecting the early stage discovery that is difficult for, therefore strengthen dog detection only, stop to infect being only prevention and control originally from the source.
What echinococcus infected makes a definite diagnosis and can detect according to adult, specific antigens or specific DNA molecule in enteron aisle or the ight soil.Usually the detection of the final host echinococcus being infected is relatively more difficult with diagnosis, comparatively ideal method is not yet arranged, its reason is: (1) echinococcosis Endemic Area Canis animals except infecting echinococcus, infect other Taeniidaes toward contact, the echinococcus worm's ovum is difficult to distinguish with other band tapeworm worm's ovums in form; (2) in ight soil, may lack echinococcus characteristic serobila fragment; (3) echinococcus polypide fragment is little, is difficult for seeing in ight soil or easily out in the cold.Arecoline hydrobromide (or arecoline Specia)) purgative therapy is the method for widely using clinically with cuing open the inspection method; Recently, along with the development of biotechnology, the novel method of a series of Canis animals echinococcus Infect And Diagnoses and detection has appearred, researching and developing such as excrement antigen ELISA method, excrement DNA detection method etc., and obtained initial success, help to provide clinically and detect and diagnosis efficiency.
The reported first such as Bretagne detect echinococcus multilocularis infection with round pcr from fox excrement DNA, hereafter, utilize molecular biology for detection (excrement polypide DNA detection method) to obtain very big improvement.Zhang Yalou etc. have set up the PCR method that detects Echinococcus granulosus DNA in the domesticated dog ight soil.
PCR can hypersensitivity, the rapid detection target gene DNA of high specific, even also can detect when containing trace Echinococcus granulosus DNA in the sample.Though have at present the report to the PCR detection method of dog echinococcosis granulosa, the report of correlation detection test kit do not arranged.
Summary of the invention
The object of the present invention is to provide a kind of PCR test kit and corresponding operating program that detects the dog echinococcosis granulosa.Utilize that the primer of high degree of specificity can efficiently and accurately from being amplified the purpose fragment the animal excrement of echinococcus granulosus infection or the secretory product, rapidly and efficiently, recall rate is high, the result is accurate, cost is controlled.
Technical essential of the present invention is the design primer, and the purpose fragment of specific amplification provides foundation for clinical diagnosis, and can judge accurately cause of disease by order-checking when being necessary.For achieving the above object, the technical solution used in the present invention is as follows:
According to Echinococcus granulosus 12S rDNA sequence conserved regions design pair of primers, primer sequence is Eg F 5 '-TTTTCTTTGCTTTTATGTGG-3 ' and Eg R, 5 '-TCAATAACAACCGAGGG-3 ', and its sequence is seen respectively sequence 1 and the sequence 2 in the sequence table.PCR product length is 475bp, and its sequence is seen the sequence 3 in the sequence table.
A kind of dog echinococcosis granulosa PCR detection kit, comprising: (1) 2 * PCR reacts mix.Contain the PCR reaction buffer among the mix, dNTP, primer Eg F and Eg R; Archaeal dna polymerase; (2) positive control (recombinant plasmid that contains Echinococcus granulosus 12S rDNA sequence conserved regions); (3) negative control (Healthy Cats or dog are without the excrement sample filtrate of poisoning the asepticize processing); (4) tetrabromophenol sulfonphthalein sample-loading buffer; (5) 1.5ml centrifuge tube; (6) sampling swab; (7) sample preparation damping fluid.
The use flow process of one this test kit of cover comprises: (1) sample preparation.Scrape the animal excreta sample to be checked (or urine, nasal secretions etc.) that takes a morsel with sampling swab and place the 1.5ml centrifuge tube, add 1ml sample preparation damping fluid, fully vibration makes the excrement sample abandon swab after swab comes off, centrifugal 1 minute of 12000g, it is resuspended to get precipitation adding 1ml sample preparation damping fluid, boiling water bath ten minutes, and 12000g is centrifugal ten minutes after the cooling, draw supernatant and change in another cleaning sterile centrifuge tube ,-20 ℃ save backup; (2) PCR.Reaction system is 20 μ l.In three 0.2ml EP pipes, add respectively 10 μ l PCR mix, add 8 μ l aseptic deionized waters, 2 μ l samples or the positive and negative control, the rearmounted PCR instrument of mixing increases.The PCR reaction conditions is: 95 ℃ of denaturation 10min, and 94 ℃ of sex change 30sec, 55 ℃ of annealing 30sec, 72 ℃ are extended 30sec, circulate 30 times.72 ℃ of polishings extend the end of 10min afterreaction after the loop ends.(3) agarose electrophoretic analysis is identified the PCR product.With sample and positive and together electrophoresis evaluation of negative control, after finishing, through observing under ultraviolet lamp behind the ethidium bromide staining, each swimming lane electrophoretic band situation relatively is to judge that sample is as being subjected to the sample product whether with Echinococcus granulosus DNA with gel.
That uses that universal PCR test kit of the present invention both can quickness and high efficiency detects animal and whether infects Echinococcus granulosus, and can be by order-checking to determine the cause of disease kind, for the clinical diagnosis treatment facilitate under the condition of necessity.
Description of drawings
Accompanying drawing 1: a doubtful trouble hydatidosis dog PCR result.The purpose clip size is 475bp (together lower).
The 1st swimming lane, DL2000 marker, stripe size (bp) is respectively 2000,1000,750,500,250,100 (together lower) from top to bottom; The 2nd swimming lane, positive control; The 3rd swimming lane, sample P CR result; The 4th swimming lane, negative control.
Accompanying drawing 2: a doubtful trouble hydatidosis dog PCR result
The 1st swimming lane, DL2000 marker; The 2nd swimming lane, positive control; The 3rd swimming lane, sample P CR result; The 4th swimming lane, negative control.
Accompanying drawing 3: a doubtful trouble hydatidosis dog PCR result
The 1st swimming lane, DL2000 marker; The 2nd swimming lane, positive control; The 3rd swimming lane, sample P CR result; The 4th swimming lane, negative control.
Embodiment
The invention will be further described below in conjunction with embodiment, but the present invention is not subjected to the restriction of embodiment.
Molecular biology working method among all embodiment is familiar with by these those skilled in the art, can be with reference to (the laboratory manual such as Sambrook " molecular cloning ", the cold spring port, 1989) reach " fine works molecular biology experiment guide " (work such as U.S./F. Ao Sibai, Yan Ziying etc. translate, Beijing, Science Press, 1998).
Embodiment one
Get one of certain doubtful trouble hydatidosis dog, scraping ight soil a little, press test kit and use flow operations, the result as shown in Figure 1.Judge that according to the result this dog has only infected hydatidosis.Detection paper result is also positive, and it is correct to have confirmed the PCR detected result.
Embodiment two
Get one of certain doubtful trouble hydatidosis dog, scraping ight soil a little, press test kit and use flow operations, the result as shown in Figure 2.Judge that according to the result this dog has only infected hydatidosis.Detection paper result is also positive, and it is correct to have confirmed the PCR detected result.
Embodiment three
Get one of certain doubtful trouble hydatidosis dog, collect ight soil a little, press test kit use flow operations, the result as shown in Figure 3.Judge that according to the result this dog does not only infect hydatidosis.Detection paper result is negative, and it is correct to have confirmed the PCR detected result.
Claims (3)
1. dog echinococcosis granulosa PCR detection kit, this test kit comprises: a pair of Auele Specific Primer, PCR reacts mix, positive control dna and negative control.
2. according to claim 1, specific primer sequence is Eg F:5 '-TTTTCTTTGCTTTTATGTGG-3 ' and Eg R, 5 '-TCAATAACAACCGAGGG-3 '.
3. according to claim 1, positive control dna is to contain the recombinant plasmid that length is 475bp Echinococcus granulosus 12S rDNA sequence conserved regions fragment, and the negative control sample refers to that the nothing of Healthy Cats or dog poisons the excrement sample that asepticize is processed.
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| CN 201210120092 CN103374615A (en) | 2012-04-20 | 2012-04-20 | PCR (Polymerase Chain Reaction) detection kit for cystic echinococcosis of dog |
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| CN 201210120092 CN103374615A (en) | 2012-04-20 | 2012-04-20 | PCR (Polymerase Chain Reaction) detection kit for cystic echinococcosis of dog |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104059973A (en) * | 2014-06-16 | 2014-09-24 | 浙江大学 | Echinococcus granulosus detection kit and application thereof |
| CN106888985A (en) * | 2015-12-18 | 2017-06-27 | 英业达科技有限公司 | The outer display information of pet is monitored to judge the system and method for pet health state |
| CN107365849A (en) * | 2017-08-10 | 2017-11-21 | 西南民族大学 | The kit of dog particulate, Echinococcus multilocularis based on POCKIT Micro fluorescent PCR platforms and application |
| CN109762910A (en) * | 2019-01-10 | 2019-05-17 | 四川省疾病预防控制中心 | It is a kind of for detecting the primer and kit of amphitypy echinococcosis simultaneously |
| CN109890985A (en) * | 2017-07-02 | 2019-06-14 | 中南大学湘雅医院 | Detect the method and kit of parasitic infection |
| 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 |
-
2012
- 2012-04-20 CN CN 201210120092 patent/CN103374615A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104059973A (en) * | 2014-06-16 | 2014-09-24 | 浙江大学 | Echinococcus granulosus detection kit and application thereof |
| CN104059973B (en) * | 2014-06-16 | 2016-05-18 | 浙江大学 | A kind of Echinococcus Granulosus Cysts detection kit and application |
| CN106888985A (en) * | 2015-12-18 | 2017-06-27 | 英业达科技有限公司 | The outer display information of pet is monitored to judge the system and method for pet health state |
| CN109890985A (en) * | 2017-07-02 | 2019-06-14 | 中南大学湘雅医院 | Detect the method and kit of parasitic infection |
| US11702705B2 (en) | 2017-07-02 | 2023-07-18 | Xiangya Hospital Central South University | Method for detecting parasitic infection and kit |
| CN107365849A (en) * | 2017-08-10 | 2017-11-21 | 西南民族大学 | The kit of dog particulate, Echinococcus multilocularis based on POCKIT Micro fluorescent PCR platforms and application |
| 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 |
| CN109762910A (en) * | 2019-01-10 | 2019-05-17 | 四川省疾病预防控制中心 | It is a kind of for detecting the primer and kit of amphitypy echinococcosis simultaneously |
| 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 |
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Application publication date: 20131030 |