CN101962679A - Kit for detecting fasciola hepatica by using PCR specificity - Google Patents
Kit for detecting fasciola hepatica by using PCR specificity Download PDFInfo
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- CN101962679A CN101962679A CN 201010297132 CN201010297132A CN101962679A CN 101962679 A CN101962679 A CN 101962679A CN 201010297132 CN201010297132 CN 201010297132 CN 201010297132 A CN201010297132 A CN 201010297132A CN 101962679 A CN101962679 A CN 101962679A
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Abstract
The invention discloses a kit for detecting fasciola hepatica by using PCR specificity. The kit comprises DNA cracking solution, PCR reaction solution and fasciola hepatica, fasciola gigantica and middle fluke DNA positive contrast solution. The invention also provides a method for detecting the fasciola hepatica by using a specific PCR method. The method can rapidly, specifically and flexibly diagnose and detect fasciola hepatica cercaria in snails and fasciola hepatica ova in excrements of infected animals. A rapid, specific and sensitive PCR method is established for detecting the imagoes, the cercaria and the ova of the fasciola hepatica, the fasciola gigantica and the middle fluke, so that the imagoes, the cercaria and the ova of the fasciola hepatica, the fasciola gigantica and the middle fluke can be accurately differentiated. The kit of the invention has the advantage of simple and programmed operation, and the method has the advantages of high specificity, high sensitivity and objective result judgment and can be used for rapidly differentiating the fasciola hepatica.
Description
Technical field
The invention belongs to field of biological detection, relate in particular to a kind of test kit, particularly the specific PCR of liver fluke, fasciola gigantica, osculant fluke adult, cercaria, worm's ovum detects, and is the test kit of the special detection fasciola of a kind of PCR of employing specifically.
Background technology
Fascioliasis is to parasitize domestic animal liver, bile duct and a kind of verminosis of causing by Fasciolidae (Fasciolidae) Fasciola (Fasciola) fluke (liver fluke, fasciola gigantica, osculant fluke).Mainly betide ruminating animal (ox, buffalo, sheep, goat, camel etc.), it mainly is the malnutrition and caused chronic the becoming thin and depletion of poisoning that symptom is examined by association; Pathological characters is chronic cholangitis and hepatitis.The people also can infect fasciola.This disease pathogen is mainly liver-plate shape fluke and two kinds of big fasciolas, also finds osculant fluke infection animal and human's report at present in Vietnam.Three kinds of fluke adult forms are similar substantially, and polypide is flat, is the willow leaf shape.The long 20-35 millimeter of liver fluke, wide 5-13 millimeter, look reddish brown, is flat foliated lamellar, and the polypide shoulder is wide and obvious; The long 33-76 millimeter of fasciola gigantica, wide 5-12 millimeter, shoulder is not obvious, the blunt circle in rear end.The form of middle fluke is difficult to definite description, the form of some osculant fluke is similar to liver fluke, the approximate again fasciola gigantica of the form of some osculant fluke, so, aspect the classification location of osculant fluke, the difficult differentiation osculant of traditional morphology fluke.
In recent years, based on the molecular biology method of polymerase chain reaction (PCR) in the widespread use of parasitology field.Advantages such as PCR is easy, quick, special with it, sensitivity have been used in the diagnosis of people and animal parasitosis, and the diagnostic method of fascioliasis mainly is the worm's ovum of discharging by morphologic detection adult and infection animal.The key of setting up fasciola specific PCR diagnostic method is to seek responsive, special genetic marker.
In sum, existing detection method exists that recall rate is not high, false positive is more, wastes time and energy, and occurs mistaken diagnosis easily and fails to pinpoint a disease in diagnosis, problem such as susceptibility is limited, it is a kind of easy to use, highly sensitive to press for discussion, and is suitable for the method for quick of cercaria and worm's ovum.
Summary of the invention
The object of the present invention is to provide the test kit of the special detection fasciola of a kind of PCR of employing, the test kit of the special detection fasciola of described this employing PCR will solve that the method recall rate that detects fasciola in the prior art is not high, false positive is more, waste time and energy, occur mistaken diagnosis easily and fail to pinpoint a disease in diagnosis the technical problem that susceptibility is limited.
The invention provides a kind of test kit that adopts specific PCR to detect fasciola, contain dna cleavage liquid, PCR reaction solution and liver fluke, fasciola gigantica, osculant fluke DNA positive control solution, in the described dna cleavage liquid by cell pyrolysis liquid, EDTA, Proteinase K and RNase A solution are formulated, in described dna cleavage liquid, the final concentration of described cell pyrolysis liquid is 7-10 μ M, the final concentration of described EDTA in described dna cleavage liquid is 0.5M, the final concentration of described Proteinase K in described dna cleavage liquid is 18-20mM, the final concentration of described RNase A in described dna cleavage liquid is 3-5uM, in the described PCR reaction solution by dATP, dTTP, dGTP, dCTP, the downstream primer of amplification liver fluke, the downstream primer of amplification fasciola gigantica, the shared upstream primer and the MgCl of amplification liver fluke and fasciola gigantica
2Formulated, the sequence of the downstream primer of described amplification liver fluke is 5 ' CCAATGACAAAGTGACAGCG-3 ', the downstream primer of described amplification fasciola gigantica is 5 ' CCAATGACAAAGTAACAGCA-3 ', the sequence of the shared upstream primer of amplification liver fluke and fasciola gigantica is 5 ' ATATTGCGGCCATGGGTTAG-3 ', in described PCR reaction solution, described dATP, dTTP, the final concentration of dGTP and dCTP is 200 μ M, the downstream primer of described amplification liver fluke, the final concentration of the downstream primer of amplification fasciola gigantica and the shared upstream primer of amplification liver fluke and fasciola gigantica is 50pmol/ μ L, described MgCl
2Final concentration be 2mM.
Further, also contain the Taq archaeal dna polymerase in the described test kit.
Further, the concentration of described liver fluke, fasciola gigantica and osculant fluke DNA positive control solution is 33.0-42ng/ μ l.
Further, the concentration of described Taq archaeal dna polymerase is 6U/ μ L.
Further, the volume of described Taq archaeal dna polymerase is 25 μ L.
The present invention also provides a kind of method that adopts specific PCR method detection lug type fluke, comprises the steps:
(1) adult, cercaria, the worm's ovum to unknown fluke carries out the extraction of DNA;
(2) with the DNA of said extracted as template, utilize the PCR reaction solution to increase, in the described PCR reaction solution by the shared upstream primer and the MgCl of downstream primer, amplification liver fluke and the fasciola gigantica of the downstream primer of dATP, dTTP, dGTP, dCTP, amplification liver fluke, amplification fasciola gigantica
2Formulated, the sequence of the downstream primer of described amplification liver fluke is 5 ' CCAATGACAAAGTGACAGCG-3 ', the downstream primer of described amplification fasciola gigantica is 5 ' CCAATGACAAAGTAACAGCA-3 ', and the sequence of the shared upstream primer of amplification liver fluke and fasciola gigantica is 5 ' ATATTGCGGCCATGGGTTAG-3 ';
(3) amplified production detects through agarose gel electrophoresis, and ultraviolet lamp is observed down.
Further, the pcr amplification condition is: 92-95 ℃ of pre-sex change 4-6min; 92-95 ℃ sex change 25-35sec, 50-60 ℃ annealing is extended 25-35sec, 30-40 circulation for 25-35sec, 70-74 ℃; Extend 4-6min after 70-74 ℃.
Further, in described PCR reaction solution, the final concentration of described dATP, dTTP, dGTP and dCTP is 200 μ M, the final concentration of the downstream primer of the downstream primer of described amplification liver fluke, amplification fasciola gigantica and the shared upstream primer of amplification liver fluke and fasciola gigantica is 50pmol/ μ L, described MgCl
2Final concentration be 2mM.
The present invention provides the using method of described test kit simultaneously, may further comprise the steps:
(1) extraction of DNA: the polypide material places the centrifuge tube of 1.5mL, blows and beats flushing 3 times repeatedly with distilled water, removes the water in the centrifuge tube, adds dna cleavage liquid digesting protein and discharges genomic dna, 56 ℃ of digested overnight 15-18h; The polypide suspension that digestion is good is pressed the Promega test kit
SV Genomic DNA purification system operation instruction is extracted polypide DNA, directly use or standby in-20 ℃ of refrigerators preservations.
The extracting method of cercaria DNA is crushed it with two slide glasss for host's spiral shell is washed 5 times repeatedly with the sterilization distilled water, removes the spiral shell shell of outside, and the extracting method of cercaria is with reference to the extracting method of polypide in the spiral shell meat.
The extracting method of worm's ovum DNA is also improved with reference to the method for M ü ller (2007) purification worm's ovum, get the positive ight soil of 0.1 gram in 1.5ml Eppendorf pipe, add under the solution 1ml room temperature of the potassium hydroxide contain 2% Triton X-100 and 0.1M and act on 10min, constantly shake up therebetween.The centrifugal 2min of 10000rpm removes supernatant then, repeats the step of front and cleans twice.Centrifugal going adds the saturated sulfo-sodium sulfide solution of 1.2ml, mixing, the centrifugal 5min of 14000rpm behind the supernatant.This moment, worm's ovum mainly was distributed in liquid level, and small portion is distributed in the liquid.Draw 200 μ l supernatants respectively in another 1.5ml Eppendorf pipe, add the 1.2ml distilled water, the centrifugal 2min of 12000rpm.Remove supernatant, keep 100 μ l left and right sides liquid, make precipitation and liquid mixing with pipettor piping and druming.Then the liquid of each pipe is all moved on in the same pipe, fill it up with distilled water in the centrifuge tube, the centrifugal 2min of 12000rpm.Remove supernatant, add the 1.2ml distilled water again, so repeat 3～5 times.The last centrifugal supernatant that goes keeps 30 μ l liquid.
In the microscopically operation, from the worm's ovum of purifying, draw 1,3,5 worm's ovums respectively in the 1.5ml Eppendorf pipe that contains 30 μ l distilled waters with pipettor, carry out mark, to detect the susceptibility of worm's ovum specific PCR detection method.
Add half granulated glass sphere of liquid volume, instantaneous centrifugal behind the vortex concussion 1min, boil 5min in the boiling water, the last centrifugal 1min of 10000rpm gets 3 μ l supernatant pcr amplifications.
(2) pcr amplification: sample number n (n=sample number+2) gets the PCR reaction solution, the TaqDNA enzyme is mixed in the centrifuge tube, mixing, packing according to increasing; Positive and negative contrast liquid is added respectively in the branch tubulature, and the DNA that gets each sample adds in the corresponding reaction tubes; Centrifugal mixing behind each reaction tubes mark places on the PCR instrument and reacts;
(3) the PCR product is observed: get product application of sample after the amplification on 1.0% sepharose, electrophoresis is placed on observations and photographic analysis under the ultraviolet transilluminator.
The described TaqDNA enzyme of step (2) contains 1.25U by each PCR reaction and adds.
The described pcr amplification condition of step (2) is: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30sec, 55 ℃ of annealing 30sec, 72 ℃ of extension 30sec, 35 circulations; Extend 5min after 72 ℃.
The optimizing process of test kit PCR reaction conditions of the present invention is:
Mg
2+Concentration gradient between 1.5mM to 3.0mM, annealing temperature adjusts between 60 ℃ at 50 ℃, to obtain the best Mg with the ITS-2PCR amplification
2+Concentration and optimum annealing temperature.Definite through testing, optimum annealing temperature is 60 ℃; MgCl
2Concentration be 2.5mM; Cycle number is chosen as 35 circulations.
The present invention is particularly useful for the detection of the worm's ovum of intermediate host's spiral shell mesocercaria and infection animal discharge.
The present invention is by carrying out rapid detection to coming from the whole world different popular districts (China, the Niger, France, the U.S., Spain) fasciola adult strain isolated, cercaria, worm's ovum.
The present invention adopts round pcr, utilize the increased difference of rrna internal transcribed spacer district-2 gene (The second internal transcribed spacers (ITS-2) ofnuclear ribosomal DNA) of three kinds of fasciolas to design two pairs of special primers, found the parasite of differentiation liver fluke, fasciola gigantica, osculant fluke and other common infected person that this fragment gene can be special.The applicant designs the method that a kind of fasciola specific PCR detects according to the characteristic of fasciola ITS-2 gene and in conjunction with the detection characteristics of round pcr.
The invention has the beneficial effects as follows:
(1) the present invention is by carrying out Genetic Variation Analysis to the fasciola strain isolated that comes from different popular districts, the ITS-2 sequence total length of finding fasciola gigantica by order-checking is 361bp, the ITS-2 sequence total length of liver fluke is 362bp, and the variant sites of two worm kinds has 6, but all disperses very much.Design has selected during primer sequence between the ITS-2 sequence 270bp-290bp as downstream primer, contains two variant sites, and the base A mispairing at 271bp place is become G.The applicant designs a kind of specific reaction system according to above-mentioned two variant sites, has set up quick, special, the sensitive detecting method that are used to differentiate liver fluke, fasciola gigantica, osculant fluke.
(2) the present invention is by optimizing reaction system and reaction conditions, develop a kind of specific detection test kit of differentiating cloud liver fluke, fasciola gigantica, osculant fluke, test kit sequencing simple to operate, the method high specificity, the susceptibility height, the result judges objective, not only can be used for the diagnosis and the epidemiology survey of humans and animals fascioliasis, also can be used for studying scientific research purposes such as the exploitation of fasciola Molecular Identification mark and analysis, further study for fascioliasis diagnosis and Prevention Technique etc. and lay the foundation.
Description of drawings
Fig. 1 is a liver fluke specificity electrophoretogram; Wherein, M, DL2000marker; The 1-8 representative: liver fluke, fasciola gigantica, the osculant fluke, Schistosoma japonicum is with dish fluke, eurytrema pancreaticum, Toxocara vitulorum, buffalo DNA; 9 negative contrasts.
Fig. 2 is for being fasciola gigantica specificity electrophorogram; M, DL2000marker; The 1-8 representative: liver fluke, fasciola gigantica, the osculant fluke, Schistosoma japonicum is with dish fluke, eurytrema pancreaticum, Toxocara vitulorum, buffalo DNA; 9 negative contrasts.
Fig. 3 is a liver fluke specific PCR susceptibility electrophoretogram; 1 representation DNA concentration is: 44.16ng (liver fluke); 2-7 representation DNA extension rate is as follows: 25,50,100,200, and 400,800 times; 8 negative contrasts.
Fig. 4 is a fasciola gigantica specific PCR susceptibility collection of illustrative plates; 1 representation DNA concentration is: 140.96ng (fasciola gigantica); 2-7 representation DNA extension rate is as follows: 25,50,100,200, and 400,800 times; 8 negative contrasts.
Fig. 5 carries out the electrophoretogram that specific PCR detects for adopting special primer to liver fluke; 1 is fasciola gigantica adult positive control, 2 is the fasciola gigantica of Guizhou buffalo, 3 is the fasciola gigantica of Guangxi buffalo, 4 is the liver fluke from the Guizhou goat, 5 is the osculant fluke from the Heilungkiang milk cow, 6-7 is the liver fluke from the Sichuan goat, 8 is the liver fluke from the Gansu goat, 9 is the liver fluke from the Nanjing goat, 10 is the liver fluke from French goat, 11 is the liver fluke from Niger ox, and 12 is the fasciola gigantica from Niger sheep, 13 negative contrasts.
The electrophoretogram of Fig. 6 for adopting special primer that the fasciola gigantica specific PCR is detected; 1 is fasciola gigantica adult positive control, 2 is the fasciola gigantica of Guizhou buffalo, 3 is the fasciola gigantica of Guangxi buffalo, 4 is the liver fluke from the Guizhou goat, 5 is the osculant fluke from the Heilungkiang milk cow, 6-7 is the liver fluke from the Sichuan goat, 8 is the liver fluke from the Gansu goat, 9 is the liver fluke from the Nanjing goat, 10 is the liver fluke from French goat, 11 is the liver fluke from Niger ox, and 12 is the fasciola gigantica from Niger sheep, 13 negative contrasts.
Fig. 7 is for adopting special primer to carry out the electrophoretogram that specific PCR detects from the worm's ovum in the buffalo ight soil of natural infection fasciola gigantica; 1 represents fasciola gigantica adult sample (FgGXB1); 2 represent liver fluke adult sample (FhCMA); 3-11 represents the worm's ovum sample in the buffalo ight soil that has infected fasciola gigantica; 12-17 represents single fasciola gigantica worm's ovum; The ight soil of the buffalo of fasciola gigantica is not infected in 18 representatives.
Fig. 8 carries out the electrophoretogram that specific PCR detects for adopting special primer to intermediate host's spiral shell of natural infection fasciola gigantica cercaria; 1 represents fasciola gigantica adult sample (FgGXB1); 2 represent liver fluke adult sample (FhCMA); 3-18 represents single spiral shell sample (infecting the fasciola gigantica cercaria); Intermediate host's spiral shell sample of fasciola gigantica cercaria is not infected in 19 representatives.
Embodiment
A kind of test kit that adopts specific PCR to detect fasciola of the present invention, contain dna cleavage liquid, PCR reaction solution and liver fluke, fasciola gigantica, osculant fluke DNA positive control solution, in the described dna cleavage liquid by cell pyrolysis liquid, EDTA, Proteinase K and RNase A solution are formulated, in described dna cleavage liquid, the final concentration of described cell pyrolysis liquid is 7-10uM, the final concentration of described EDTA in described dna cleavage liquid is 0.5M, the final concentration of described Proteinase K in described dna cleavage liquid is 18-20mM, the final concentration of described RNase A in described dna cleavage liquid is 3-5uM, in the described PCR reaction solution by dATP, dTTP, dGTP, dCTP, the downstream primer of amplification liver fluke, the downstream primer of amplification fasciola gigantica, the shared upstream primer and the MgCl of amplification liver fluke and fasciola gigantica
2Formulated, the sequence of the downstream primer of described amplification liver fluke is 5 ' CCAATGACAAAGTGACAGCG-3 ', the downstream primer of described amplification fasciola gigantica is 5 ' CCAATGACAAAGTAACAGCA-3 ', the sequence of the shared upstream primer of amplification liver fluke and fasciola gigantica is 5 ' ATATTGCGGCCATGGGTTAG-3 ', in described PCR reaction solution, described dATP, dTTP, the final concentration of dGTP and dCTP is 200 μ M, the downstream primer of described amplification liver fluke, the final concentration of the downstream primer of amplification fasciola gigantica and the shared upstream primer of amplification liver fluke and fasciola gigantica is 50pmol/ μ L, described MgCl
2Final concentration be 2mM.
Further, also contain the Taq archaeal dna polymerase in the described test kit.
Further, the concentration of described liver fluke, fasciola gigantica and osculant fluke DNA positive control solution is 33.0-42.0ng/ μ l.
Further, the concentration of described Taq archaeal dna polymerase is 6U/ μ L.
Further, the volume of described Taq archaeal dna polymerase is 25 μ L.
2 one preferred test kits of embodiment
Test kit includes dna cleavage liquid, and the mixing solutions that wherein contains (final concentration 0.5M) EDTA, Proteinase K (final concentration 20mM) and the RNase A solution (final concentration 5 μ M) of cell pyrolysis liquid (7 μ M), pH 8.0 is total to 100ml; 100 reactions of PCR reaction solution (25 μ L/ reaction): be that final concentration is dATP, dTTP, dGTP, the dCTP of 200 μ M, final concentration is that primer, the final concentration of the amplification liver fluke of 50pmol/ μ L is that downstream primer, the final concentration of the amplification fasciola gigantica of 50pmol/ μ L is that the amplification liver fluke of 50pmol/ μ L and shared upstream primer, the final concentration of fasciola gigantica are the MgCl of 2mM
2Mixing solutions, 25 μ L Taq archaeal dna polymerases (6U/ μ L); Liver fluke, fasciola gigantica, osculant fluke DNA positive control solution.
The test of embodiment 2 test kit specificitys
With liver fluke, fasciola gigantica, each 1 μ L of osculant fluke control sample DNA through the DNA validation verification is template, carries out pcr amplification according to the reaction conditions of test kit, establishes the contrast of blank and test kit positive and negative simultaneously.
Table 1PCR amplification system
The pcr amplification condition:
94 ℃ of sex change 5min
72 ℃ are extended 30sec
72 ℃ are extended 5min
The PCR product in the 1.0%TBE sepharose behind the electrophoresis, observations under the ultraviolet transilluminator, gel imaging system shooting.
Test kit can expand the band (Fig. 1 and Fig. 2) that 300bp respectively from liver fluke, fasciola gigantica, osculant fluke (two kinds of primers all can expand) sample DNA as a result.
The sensitivity test of embodiment 3 test kits
Dilute after at first the fasciola adult being extracted DNA, vortex vibration mixing, the working specification of pressing Eppendorf Biophotometer nucleic acid-protein determinator detects its total dna content.Liver fluke is 44.16ng, and fasciola gigantica is 140.96ng.By 25,50,100,200,400,800 times of dilution DNA, the pcr amplification condition is the same, establishes blank simultaneously.The PCR product detects through 1.0% agarose gel electrophoresis, to determine its susceptibility.Test-results shows this PCR detection method susceptibility height, and low energy detects the liver fluke of 0.11ng, the fasciola gigantica of 0.35ng (Fig. 3 and Fig. 4).
With liver fluke, fasciola gigantica, each 1 μ L of osculant fluke (from China, the Niger, France, the U.S., Spain) sample DNA through the DNA validation verification is template, reaction conditions according to test kit carries out pcr amplification, establishes the contrast of blank and test kit positive and negative simultaneously.
The PCR product in the 1.0%TBE sepharose behind the electrophoresis, observations under the ultraviolet transilluminator, gel imaging system shooting.
Test kit can expand the band (Fig. 5 and Fig. 6) that 300bp respectively from liver fluke, fasciola gigantica, osculant fluke (two kinds of primers all can expand) sample DNA as a result.Separately Fig. 5 band that comes out represents that promptly this fasciola is a liver fluke; Separately Fig. 6 band that comes out represents that then it is a fasciola gigantica; And two figure come out together, represent that then it is the osculant fluke.
The preservation period test of embodiment 5 test kits
To detect known sample with-20 ℃ of test kits of preserving 1 month, 3 months, 6 months, 9 months at 4 ℃, amplification condition is with aforementioned.The result shows that test kit can be in 4 ℃ (except Bst enzymes ,-20 ℃ of preservations of palpus) and-20 ℃ of preservation prolonged preservation, and in 9 months of test period, positive all can amplify the purpose bright band under suitable preservation condition, and negative control does not have the appearance of purpose bright band.
1.DNA sample
15 samples of the worm's ovum of fasciola gigantica, 16 samples of cercaria sample, 70% alcohol is preserved standby.
2.DNA extraction
The extracting method of cercaria DNA is crushed it with two slide glasss for host's spiral shell is washed 5 times repeatedly with the sterilization distilled water, removes the spiral shell shell of outside, and the extracting method of cercaria is with reference to the extracting method of adult in the spiral shell meat.
The extracting method of worm's ovum DNA is also improved with reference to the method for M ü ller (2007) purification worm's ovum, get the positive ight soil of 0.1 gram in 1.5ml Eppendorf pipe, add under the solution 1ml room temperature of the potassium hydroxide contain 2% Triton X-100 and 0.1M and act on 10min, constantly shake up therebetween.The centrifugal 2min of 10000rpm removes supernatant then, repeats the step of front and cleans twice.Centrifugal going adds the saturated sulfo-sodium sulfide solution of 1.2ml, mixing, the centrifugal 5min of 14000rpm behind the supernatant.This moment, worm's ovum mainly was distributed in liquid level, and small portion is distributed in the liquid.Draw 200 μ l supernatants respectively in another 1.5ml Eppendorf pipe, add the 1.2ml distilled water, the centrifugal 2min of 12000rpm.Remove supernatant, keep 100 μ l left and right sides liquid, make precipitation and liquid mixing with pipettor piping and druming.Then the liquid of each pipe is all moved on in the same pipe, fill it up with distilled water in the centrifuge tube, the centrifugal 2min of 12000rpm.Remove supernatant, add the 1.2ml distilled water again, so repeat 3～5 times.The last centrifugal supernatant that goes keeps 30 μ l liquid.
In the microscopically operation, from the worm's ovum of purifying, draw 1,3,5 worm's ovums respectively in the 1.5ml Eppendorf pipe that contains 30 μ l distilled waters with pipettor, carry out mark, to detect the susceptibility of worm's ovum specific PCR detection method.
Add half granulated glass sphere of liquid volume, instantaneous centrifugal behind the vortex concussion 1min, boil 5min in the boiling water, the last centrifugal 1min of 10000rpm gets 3 μ l supernatant pcr amplifications.
3.PCR amplification
At first use test kit above-mentioned extractive genomic dna is carried out pcr amplification, do blank, negative control and positive control simultaneously.
4. agarose electrophoretic analysis
The PCR product is electrophoresis in the 1.0%TBE sepharose, observations under the ultraviolet transilluminator, gel imaging system shooting.
5. result and analysis
Prior liver fluke, fasciola gigantica, osculant fluke adult strain isolated, cercaria, the worm's ovum sample of identifying through morphology carried out the PCR reaction, above sample standard deviation can be detected (Fig. 7 and Fig. 8) fast, proved that the specific PCR method of being set up can detect liver fluke, fasciola gigantica, osculant fluke infection fast and effectively, also can be used for the epidemiological investigation of fascioliasis.
Claims (8)
1. test kit that adopts specific PCR to detect fasciola, it is characterized in that: contain dna cleavage liquid, PCR reaction solution and liver fluke, fasciola gigantica, osculant fluke DNA positive control solution, in the described dna cleavage liquid by cell pyrolysis liquid, EDTA, Proteinase K and RNase A solution are formulated, in described dna cleavage liquid, the final concentration of described cell pyrolysis liquid is 7-10uM, the final concentration of described EDTA in described dna cleavage liquid is 0.5M, the final concentration of described Proteinase K in described dna cleavage liquid is 18-20mM, the final concentration of described RNase A in described dna cleavage liquid is 3-5uM, in the described PCR reaction solution by dATP, dTTP, dGTP, dCTP, the downstream primer of amplification liver fluke, the downstream primer of amplification fasciola gigantica, the shared upstream primer and the MgCl of amplification liver fluke and fasciola gigantica
2Formulated, the sequence of the downstream primer of described amplification liver fluke is 5 ' CCAATGACAAAGTGACAGCG-3 ', the downstream primer of described amplification fasciola gigantica is 5 ' CCAATGACAAAGTAACAGCA-3 ', the sequence of the shared upstream primer of amplification liver fluke and fasciola gigantica is 5 ' ATATTGCGGCCATGGGTTAG-3 ', in described PCR reaction solution, described dATP, dTTP, the final concentration of dGTP and dCTP is 200 μ M, the downstream primer of described amplification liver fluke, the final concentration of the downstream primer of amplification fasciola gigantica and the shared upstream primer of amplification liver fluke and fasciola gigantica is 50pmol/ μ L, described MgCl
2Final concentration be 2mM.
2. a kind of test kit that adopts specific PCR to detect fasciola according to claim 1 is characterized in that: also contain the Taq archaeal dna polymerase in the described test kit.
3. a kind of test kit that adopts specific PCR to detect fasciola according to claim 1 and 2, it is characterized in that: the concentration of described liver fluke, fasciola gigantica and osculant fluke DNA positive control solution is 33.0-42.0ng/ μ l.
4. a kind of test kit that adopts specific PCR to detect fasciola according to claim 2, it is characterized in that: the concentration of described Taq archaeal dna polymerase is 6U/ μ L.
5. a kind of test kit that adopts specific PCR to detect fasciola according to claim 2, it is characterized in that: the volume of described Taq archaeal dna polymerase is 25 μ L.
6. a method that adopts specific PCR method detection lug type fluke is characterized in that comprising the steps:
(1) adult, cercaria, the worm's ovum to unknown fluke carries out the extraction of DNA;
(2) with the DNA of said extracted as template, utilize the PCR reaction solution to increase, in the described PCR reaction solution by the shared upstream primer and the MgCl of downstream primer, amplification liver fluke and the fasciola gigantica of the downstream primer of dATP, dTTP, dGTP, dCTP, amplification liver fluke, amplification fasciola gigantica
2Formulated, the sequence of the downstream primer of described amplification liver fluke is 5 ' CCAATGACAAAGTGACAGCG-3 ', the downstream primer of described amplification fasciola gigantica is 5 ' CCAATGACAAAGTAACAGCA-3 ', and the sequence of the shared upstream primer of amplification liver fluke and fasciola gigantica is 5 ' ATATTGCGGCCATGGGTTAG-3 ';
(3) amplified production detects through agarose gel electrophoresis, and ultraviolet lamp is observed down.
7. according to claim 6 a kind of adopt specific PCR method detection lug type fluke, method, it is characterized in that: the pcr amplification condition is: 92-95 ℃ of pre-sex change 4-6min; 92-95 ℃ sex change 25-35sec, 50-60 ℃ annealing is extended 25-35sec, 30-40 circulation for 25-35sec, 70-74 ℃; Extend 4-6min after 70-74 ℃.
8. according to claim 6 a kind of adopt specific PCR method detection lug type fluke, method, it is characterized in that: in described PCR reaction solution, the final concentration of described dATP, dTTP, dGTP and dCTP is 200 μ M, the final concentration of the downstream primer of the downstream primer of described amplification liver fluke, amplification fasciola gigantica and the shared upstream primer of amplification liver fluke and fasciola gigantica is 50pmol/ μ L, described MgCl
2Final concentration be 2mM.
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| CN 201010297132 Pending CN101962679A (en) | 2010-09-29 | 2010-09-29 | Kit for detecting fasciola hepatica by using PCR specificity |
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Cited By (5)
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| CN103175967A (en) * | 2013-03-05 | 2013-06-26 | 广西大学 | Double-antibody sandwich enzyme-linked immunosorbent assay kit used for detecting fasciola gigantica antigen, and preparation method thereof |
| CN109609669A (en) * | 2019-01-18 | 2019-04-12 | 浙江大学 | A kind of sheep liver fascioliasis PCR quick detection kit and application based on ITS-2 gene |
| CN109750112A (en) * | 2019-03-15 | 2019-05-14 | 扬州大学 | Detect the detection kit and its preparation method and application of sea whelk shape fluke infection |
| CN110564859A (en) * | 2019-06-13 | 2019-12-13 | 中国农业科学院兰州兽医研究所 | Marker and kit for fasciola hepatica infection detection |
| CN116411088A (en) * | 2023-03-31 | 2023-07-11 | 黑龙江八一农垦大学 | PCR-RFLP primer and method for distinguishing schistosoma japonicum from schistosoma japonicum |
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| CN101586161A (en) * | 2009-06-19 | 2009-11-25 | 华南农业大学 | Primer, method and kit for detecting animal clonorchiasis sinensis specificity |
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| CN103175967A (en) * | 2013-03-05 | 2013-06-26 | 广西大学 | Double-antibody sandwich enzyme-linked immunosorbent assay kit used for detecting fasciola gigantica antigen, and preparation method thereof |
| CN109609669A (en) * | 2019-01-18 | 2019-04-12 | 浙江大学 | A kind of sheep liver fascioliasis PCR quick detection kit and application based on ITS-2 gene |
| CN109750112A (en) * | 2019-03-15 | 2019-05-14 | 扬州大学 | Detect the detection kit and its preparation method and application of sea whelk shape fluke infection |
| CN109750112B (en) * | 2019-03-15 | 2021-10-19 | 扬州大学 | Detection kit for detecting spiroschistosoma infection and preparation method and application thereof |
| CN110564859A (en) * | 2019-06-13 | 2019-12-13 | 中国农业科学院兰州兽医研究所 | Marker and kit for fasciola hepatica infection detection |
| CN110564859B (en) * | 2019-06-13 | 2022-06-24 | 中国农业科学院兰州兽医研究所 | Marker and kit for fasciola hepatica infection detection |
| CN116411088A (en) * | 2023-03-31 | 2023-07-11 | 黑龙江八一农垦大学 | PCR-RFLP primer and method for distinguishing schistosoma japonicum from schistosoma japonicum |
| CN116411088B (en) * | 2023-03-31 | 2024-05-24 | 黑龙江八一农垦大学 | PCR-RFLP primer and method for distinguishing schistosoma japonicum from schistosoma japonicum |
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