CN102146446A - Method and kit for quickly detecting clonorchiasis sinensis metacercaria in freshwater fish - Google Patents
Method and kit for quickly detecting clonorchiasis sinensis metacercaria in freshwater fish Download PDFInfo
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- CN102146446A CN102146446A CN2011100047729A CN201110004772A CN102146446A CN 102146446 A CN102146446 A CN 102146446A CN 2011100047729 A CN2011100047729 A CN 2011100047729A CN 201110004772 A CN201110004772 A CN 201110004772A CN 102146446 A CN102146446 A CN 102146446A
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Abstract
The invention provides a method for quickly detecting clonorchiasis sinensis metacercaria in freshwater fish, which comprises the following steps of: treating an in vitro freshwater fish sample; extracting DNA of the sample; performing polymerase chain reaction (PCR) amplification on the DNA of the sample; and performing electrophoresis observation on the PCR amplification product. In the step of treating the freshwater fish sample, the sample to be detected is added into buffer solution and broken by using glass beads for homogenizing, and then the homogenized solution is sucked and dripped on a filter paper (FTA) card; and in the step of extracting the DNA of the sample, the FTA card is naturally dried, and then the FTA card is washed by using pure terephthalic acid (PTA) card lotion. The invention also provides a kit for quickly detecting the clonorchiasis sinensis metacercaria in the freshwater fish. One clonorchiasis metacercaria in each gram of freshwater fish can be detected by using the method; quantitative PCR technology can distinguish three different metacercaria; and the method is simple, convenient and quick, has high sensitivity, and is suitable for food safety inspection and customs epidemic prevention inspection.
Description
Technical field
The present invention relates to bioengineering field, relate in particular to the detection method of clonorchis sinensis, the method and the test kit thereof of encysted metacercaria of clonorchis sinensis in particularly a kind of rapid detection fresh-water fishes.
Background technology:
Clonorchiasis sinensis (Clonorchiasis sinensis) claim rot again, is to colonize in people's the hepatic duct caused liver and bladder disease by clonorchis sinensis to become main a kind of food source property people beast and suffer from parasitosis altogether.Once the report that infects at the state-owned clonorchis sinensis of China, Japan, Korea S, Philippines, Singapore and Malaysia, Russia etc.In recent years, because the introducing of the variation of food and external food habits, this disease progressively increases at the infection rate of China, and the touching rate of crowd does not wait from 1～30%.Show according to whole nation parasitosis investigation first, national infection rate average out to 0.356%, in prediction on such basis, nearly 4,700,000 the infecteds in the whole nation.Its major cause be the food living fish custom due to.Survey data according to 1988 shows that the geographic pseudorasbora parva infection rate in Jiamusi, Heilungkiang also is 100%; Area, Sun-moon Lake, Taiwan Province, pseudorasbora parva, Ke Shi minnow infect the infection rate of encysted metacercaria of clonorchis sinensis up to 100%.Encysted metacercaria of clonorchis sinensis mainly is distributed in the various piece of fish body, and is as muscle, head, skin, fin and squama etc., generally maximum with the flesh of fish and a fish meat.Except that fresh-water fishes, fresh water shrimp, as caridina nilotica gracilipes (Caridina nilotica gracilipes), Macrobrachium superbum (Macrobrachium superbum) etc. the bladder worm parasitism is arranged also, therefore, the detection of bladder worm has become the matter of utmost importance of food safety in fishery products, the particularly fresh-water fishes.
Encysted metacercaria of clonorchis sinensis ovalize (as shown in Figure 1), big or small 92 μ m～110 μ m * 100 μ m～140 μ m, cyst wall divides two-layer, and outer wall is thicker, and inwall is thinner.One larva is arranged, larva tool mouth, ventral sucker, intestinal tube and contain the calcareous particulate excretory vesicle of black in the capsule.
Behind the human infection encysted metacercaria of clonorchis sinensis, can cause body maldigestion, diarrhoea, stomachache, liver severe pain, hepatomegaly, become thin, nanism.Patient late period can concurrent obstructive jaundice, biliary colic, cholangitis, cholecystitis and primary bile duct liver cancer.The fluke bladder worm is directly threatening the people's safe diet, therefore, need a kind of fast, responsive, simply detect that the encysted metacercaria of clonorchis sinensis method shows very urgent and necessary especially in the fresh-water fishes.
The FTA technology comprises a kind of special dry chemistry reagent mixture that is embedded in protein denaturant, sequestrant and free radical scavenger in the filter substrate.It is nontoxic to the people, can preserve under room temperature and high humidity environment with the nucleic acid of FTA technology collection and degrade for many years and not.The principle of work of FTA card is infectious pathogen cracking inactivation when contact FTA in the sample, and the nucleic acid in the pathogenic agent then is fixed.By a simple step, elute DNA and just can be applied to the downstream and increase to test from blocking purifying.The FTA technology has: the cost of 1. saving low temperature transportation sample and preserving the cryogenic refrigerator of sample.2. make the organism rapid deactivation, avoided the risk of sample contamination in the operating process.3. handle sample, separate and to obtain DNA and only need 15-30 minute, shorten, reduce separation steps (4-16 hour).4. sample preparation only needs the process of a simple eluted dna, has saved the cost of using purification kit.5. the sample demand minimizes (each sample collecting district 12-40 μ l) and therefore, has been widely used in the extraction of biological DNA such as virus, microorganism, parasite.
Summary of the invention
The object of the present invention is to provide the method and the test kit thereof of encysted metacercaria of clonorchis sinensis in a kind of rapid detection fresh-water fishes, the method for encysted metacercaria of clonorchis sinensis and test kit thereof will solve method complexity and the not high technical problem of sensitivity that detects encysted metacercaria of clonorchis sinensis in the fresh-water fishes in the prior art in the described this rapid detection fresh-water fishes.
The invention provides the method for encysted metacercaria of clonorchis sinensis in a kind of rapid detection fresh-water fishes, comprise a step, the step of an extraction sample DNA, step and step of the product of pcr amplification being carried out electrophoresis observation that a DNA to sample carries out pcr amplification stripped fresh-water fishes sample preparation; In described step to stripped fresh-water fishes sample preparation, sample to be detected is added damping fluid glass strain homogenate smash, draw homogenate then and drop on the FTA card; In the step of described extraction sample DNA,, then the FTA card is washed with FTA card washing lotion the seasoning of FTA card; Carry out in the step of pcr amplification at described DNA sample, FTA card after the washing is put into the PCR reaction tubes after drying carry out pcr amplification, the upstream primer that amplification is adopted is CGAGGGTCGGCTTATAAAC, downstream primer is GGAAAGTTAAGCACCGACC, and the product to amplification carries out electrophoresis and observation then.
Further, comprise that also one is carried out the step of DNA extraction and pcr amplification to the male reference substance, described male reference substance is clonorchis sinensis adult or bladder worm.
Further, carry out in the step of electrophoresis observation at described product pcr amplification, the electrophoretic band of sample to be detected and the electrophoretic band of positive control are compared, if the length of amplified production is 315bp, then represent to contain in the sample encysted metacercaria of clonorchis sinensis, otherwise, then represent not contain in the sample encysted metacercaria of clonorchis sinensis.
Further, carry out in the step of pcr amplification, carry out pcr amplification putting into the PCR reaction tubes after the cutting of FTA card at described DNA to sample.
Further, described FTA card washing lotion is the aqueous solution of being made up of Tris-HCl and EDTA, and the concentration of described Tris-HCl in FTA card washing lotion is 0.01mol/L, and the concentration of described EDTA in FTA card washing lotion is 0.1mmol/L, and pH is 8.0.
Further, the reaction system of described pcr amplification is: 2 * Taq PCR Master Mix50 μ L, and 1%BSA 4 μ L, 10 μ mol/L upstream primers and downstream primer are respectively 3 μ L, ddH2O40 μ L.
Further, the PCR reaction conditions is 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 1min, 62 ℃ of annealing 1min, 72 ℃ are extended 1min, 40 circulations; 72 ℃ are extended 10min.
The present invention also provides the test kit of encysted metacercaria of clonorchis sinensis in a kind of rapid detection fresh-water fishes, described test kit contains detection primer CS1 and detects primer CS2, the sequence of described CS1 is CGAGGGTCGGCTTATAAAC, and the sequence of described CS2 is GGAAAGTTAAGCACCGACC.
Further, also contain positive reference substance, described positive reference substance is the sample of clonorchis sinensis adult or bladder worm.
Further, also contain the PCR reaction solution in the described test kit,
Principle of work of the present invention is: the present invention at first adopts the FTA method to extract DNA in the detected sample fast, with the DNA pcr amplification that extracts, amplified production with positive reference substance and detected sample carries out electrophoresis then, observe electrophoretic length, if the length of amplified production is 315bp, then represent to contain in the sample encysted metacercaria of clonorchis sinensis, otherwise, then represent not contain in the sample encysted metacercaria of clonorchis sinensis.
The present invention compares with prior art, and its effect is actively with tangible.The invention provides encysted metacercaria of clonorchis sinensis method in the rapid detection fresh-water fishes, can detect 1 fluke bladder worm in every g fresh-water fishes with method of the present invention, quantitative PCR technique can be distinguished 3 kinds of different bladder worms in the fresh-water fishes, have easy, quick and the high advantage of susceptibility, suitable food safety, customs's epidemic prevention check.
Description of drawings:
Fig. 1 is that encysted metacercaria of clonorchis sinensis just cuts open bladder worm identification of morphology figure under the mirror.
Fig. 2 is the schema of the method for encysted metacercaria of clonorchis sinensis in a kind of rapid detection fresh-water fishes of the present invention.
4. " Fig. 3 is the electrophoresis comparison diagram that detects encysted metacercaria of clonorchis sinensis in the fresh-water fishes.Contain 1 China in " 1 " expression 1 gram sample among the figure and prop up the scrotum larva of a tapeworm or the cercaria of a schistosome, contain 3 China in " 3 " expression 1 gram sample and prop up the scrotum larva of a tapeworm or the cercaria of a schistosome, and the like.
Embodiment:
The reagent that the present invention adopts is: FTA card (Whatman company); FTA card washing lotion TE buffer:0.01mol/L Tris-HCl, pH 8.0; 0.1mmol/L EDTA, (Whatman company); TAE electrophoretic buffer (giving birth to worker's biological products company limited) (storing solution 50 *): Tris alkali 242g, Glacial acetic acid 57.1mL, 0.5mol/L EDTA (pH 8.0) 100mL available from Shanghai, be settled to 1 with distilled water, 000mL, mixing, room temperature preservation, get 2mL 50 * TAE electrophoretic buffer during use, dilute for 200mL working fluid (0.5 *) and get final product.Water: the PCR water meets the specification of one-level water among the GB/T 6682 and uses the DEPC water treatment; 2 * Taq PCR Master Mix (contains 0.1U Taq polysaccharase/L, 500mol/L dNTPeach, 20mmol/L Tris-HCl (pH8.3), 100mmol/L KCl, 3mmol/L MgCL2, other stablizer and toughener, (giving birth to worker's biological products company limited available from Shanghai); Marker2000 (takara company); 0.1mmol/L EDTA (pH 8.0) (giving birth to worker's biological products company limited) available from Shanghai.
The method of encysted metacercaria of clonorchis sinensis in embodiment a kind of rapid detection fresh-water fishes of the present invention
1. the extraction of flesh of fish sample DNA:
After fish gone dace; get the back of the body, tail fin portion muscle; take by weighing the flesh of fish 1 gram, add PH 8.0 damping fluids (0.1mmol/L EDTA) again and smash, draw homogenate 20 μ L and drop on the FTA card; DNA is directly extracted in seasoning (or 56 ℃ of 5min) back;, wash 3 times with FTA card washing lotion, at every turn 5min; shred filter paper behind the airing in the PCR reaction tubes, be that template is carried out pcr amplification with the DNA of extraction with the filter membrane sheet of a diameter 6mm of tapping and plugging machine punching system with micro glass beads homogenate.
2.PCR detection step
Pcr amplification: cumulative volume is 100 μ L.Reaction system is that dna profiling shreds (the diameter 6mmFTA filter membrane sheet a slice that makes), 2 * Taq PCR Master Mix, 50 μ L; 1%BSA 4 μ L; Each 3 μ L of 10 μ mol/L primers; DdH2O 40 μ L, the upstream primer that amplification is adopted is CGAGGGTCGGCTTATAAAC, downstream primer is GGAAAGTTAAGCACCGACC.
Reaction conditions: 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 1min, 62 ℃ of annealing 1min, 72 ℃ are extended 1min, 40 circulations; 72 ℃ are extended 10min.
3. electrophoresis:
Amplified production length is respectively 315bp.Whether agarose gel electrophoresis check PCR product has feature band (adopting the ethidium bromide colour developing).
If the length of amplified production is 315bp, then represent to contain in the sample encysted metacercaria of clonorchis sinensis, otherwise, then represent not contain in the sample encysted metacercaria of clonorchis sinensis.(as shown in Figure 3)
The test kit method that embodiment 2 is traditional
Test kit method key step is:
1. with the super clean bench alcohol disinfecting, ultraviolet is sterilized; Tweezers and scissors sterilization; Get sterilization 1.5ml centrifuge tube, add polypide (or bladder worm), fully wash with single water that steams repeatedly, at last washing lotion is discarded.
2. mix up thermostatted in advance: 55 degrees centigrade; Sample hose adds: Nuclei lysis solution 200 microlitres; 0.5M EDTA (PH8.0 50 microlitres; Proteinase K (20mg/ml) 20 microlitres; RNase A Solution (4mg/ml) 5 microlitres.
3. put into 55 degrees centigrade of heating 16-18 hour, add 250 microlitre lysis Buffer, the whirlpool concussion, horse back moves among the minicolumns and (is prepared in advance).
4.13000 change, centrifugal, 3 minutes 30 seconds, outwell the liquid in the collection tube, pillar is relay the recovery collector.
5. add 650 microlitre wizard SV wash solution, 13000 left the heart 1 minute and 40 seconds, discarded waste liquid, and 13000 leave the heart, 2 minutes and 30 seconds.
6. pillar is moved into new 1.5ml centrifuge tube, add 30 microlitre Nuclease-Free Water, room temperature was put 2 minutes.
7.13000 left the heart 1 minute 30 seconds, centrifugal back adds 30 microlitre Nuclease-Free Water, room temperature was put 2 minutes.
8. centrifugal again 13000 change, and 2 minutes 30 seconds, remove micro-column, centrifugal liquid in pipe is required DNA.
9. increase then and electrophoresis,, then represent to contain in the sample encysted metacercaria of clonorchis sinensis if the length of amplified production is 315bp, otherwise, then represent not contain in the sample encysted metacercaria of clonorchis sinensis (as shown in Figure 3).
As shown in Figure 3, the result of test kit method and FTA method is consistent, and is long but traditional test kit method is handled sample, separation obtains the DNA time, and complex steps, and cost is also than higher.
Claims (10)
1. the method for encysted metacercaria of clonorchis sinensis in the rapid detection fresh-water fishes comprises that a step to the fresh-water fishes sample preparation that exsomatizes, one extract the step of sample DNA, step and step of the product of pcr amplification being carried out electrophoresis observation that a DNA to sample carries out pcr amplification; It is characterized in that: in described step stripped fresh-water fishes sample preparation, sample to be detected is added damping fluid to be smashed with glass strain homogenate, drawing homogenate then drops on the FTA card, in the step of described extraction sample DNA, with the seasoning of FTA card, then the FTA card is washed with FTA card washing lotion, carry out in the step of pcr amplification at described DNA sample, FTA card after the washing is put into the PCR reaction tubes after drying carry out pcr amplification, the upstream primer that amplification is adopted is CGAGGGTCGGCTTATAAAC, downstream primer is GGAAAGTTAAGCACCGACC, and the product to amplification carries out electrophoresis and observation then.
2. the method for encysted metacercaria of clonorchis sinensis in the rapid detection fresh-water fishes as claimed in claim 1, it is characterized in that: comprise that also one is carried out the step of DNA extraction and pcr amplification to the male reference substance, described male reference substance is clonorchis sinensis adult or bladder worm.
3. the method for encysted metacercaria of clonorchis sinensis in the rapid detection fresh-water fishes as claimed in claim 1, it is characterized in that: carry out in the step of electrophoresis observation at described product to pcr amplification, the electrophoretic band of sample to be detected and the electrophoretic band of positive control are compared, if the length of amplified production is 315bp, then represent to contain in the sample encysted metacercaria of clonorchis sinensis, otherwise, then represent not contain in the sample encysted metacercaria of clonorchis sinensis.
4. the method for encysted metacercaria of clonorchis sinensis in the rapid detection fresh-water fishes as claimed in claim 1 is characterized in that: carry out in the step of pcr amplification at described DNA to sample, carry out pcr amplification with putting into the PCR reaction tubes after the cutting of FTA card.
5. the method for encysted metacercaria of clonorchis sinensis in the rapid detection fresh-water fishes as claimed in claim 1, it is characterized in that: the aqueous solution of described FTA card washing lotion for forming by Tris-HCl and EDTA, the concentration of described Tris-HCl in FTA card washing lotion is 0.01mol/L, the concentration of described EDTA in FTA card washing lotion is 0.1mmol/L, and pH is 8.0.
6. the method for encysted metacercaria of clonorchis sinensis in the rapid detection fresh-water fishes as claimed in claim 1, it is characterized in that: the reaction system of described pcr amplification is: 2 * Taq PCR Master Mix, 50 μ L, 1%BSA 4 μ L, 10 μ mol/L upstream primers and downstream primer are respectively 3 μ L, ddH
2O 40 μ L.
7. the method for encysted metacercaria of clonorchis sinensis in the rapid detection fresh-water fishes as claimed in claim 1 is characterized in that: the PCR reaction conditions is 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 1min, 62 ℃ of annealing 1min, 72 ℃ are extended 1min, 40 circulations; 72 ℃ are extended 10min.
8. the test kit of encysted metacercaria of clonorchis sinensis in the rapid detection fresh-water fishes, it is characterized in that: contain in the described test kit and detect primer CS1 and detect primer CS2, the sequence of described CS1 is CGAGGGTCGGCTTATAAAC, and the sequence of described CS2 is GGAAAGTTAAGCACCGACC.
9. the test kit of encysted metacercaria of clonorchis sinensis in the rapid detection fresh-water fishes as claimed in claim 8 is characterized in that: also contain positive reference substance, described positive reference substance is the sample of clonorchis sinensis adult or bladder worm.
10. the test kit of encysted metacercaria of clonorchis sinensis in the rapid detection fresh-water fishes as claimed in claim 8 is characterized in that: also contain the PCR reaction solution in the described test kit.
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| CN105132539A (en) * | 2015-08-19 | 2015-12-09 | 舟山出入境检验检疫局综合技术服务中心 | Ribosomal DNA ITS1 gene-based polymerase chain reaction (PCR) amplification kit for detecting clonorchis sinensis metacercaria and amplification primer |
| CN105132538A (en) * | 2015-08-19 | 2015-12-09 | 舟山出入境检验检疫局综合技术服务中心 | Dual-polymerase chain reaction (PCR) amplification kit for detecting simple anisakis and clonorchis sinensis metacercaria and amplification primer thereof |
| CN105132414A (en) * | 2015-08-19 | 2015-12-09 | 舟山出入境检验检疫局综合技术服务中心 | PCR amplification kit for detecting clonorchis sinensis metacercaria on basis of plastosome COI genes and amplification primer |
| CN106771171A (en) * | 2017-02-17 | 2017-05-31 | 中山大学 | A kind of fresh-water fishes encysted metacercaria of clonorchis sinensis infection colloidal gold fast detecting test paper strip and preparation method thereof |
| CN109355395A (en) * | 2018-09-13 | 2019-02-19 | 侯美如 | Primer is used in three kinds of fluke bladder worm multi-PCR detection methods and detection in fresh-water fishes |
| CN117568494A (en) * | 2024-01-17 | 2024-02-20 | 黑龙江八一农垦大学 | Multiple PCR (polymerase chain reaction) detection primer group, kit and detection method for zoonotic metacercaria in freshwater fish |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105132539A (en) * | 2015-08-19 | 2015-12-09 | 舟山出入境检验检疫局综合技术服务中心 | Ribosomal DNA ITS1 gene-based polymerase chain reaction (PCR) amplification kit for detecting clonorchis sinensis metacercaria and amplification primer |
| CN105132538A (en) * | 2015-08-19 | 2015-12-09 | 舟山出入境检验检疫局综合技术服务中心 | Dual-polymerase chain reaction (PCR) amplification kit for detecting simple anisakis and clonorchis sinensis metacercaria and amplification primer thereof |
| CN105132414A (en) * | 2015-08-19 | 2015-12-09 | 舟山出入境检验检疫局综合技术服务中心 | PCR amplification kit for detecting clonorchis sinensis metacercaria on basis of plastosome COI genes and amplification primer |
| CN106771171A (en) * | 2017-02-17 | 2017-05-31 | 中山大学 | A kind of fresh-water fishes encysted metacercaria of clonorchis sinensis infection colloidal gold fast detecting test paper strip and preparation method thereof |
| CN109355395A (en) * | 2018-09-13 | 2019-02-19 | 侯美如 | Primer is used in three kinds of fluke bladder worm multi-PCR detection methods and detection in fresh-water fishes |
| CN109355395B (en) * | 2018-09-13 | 2022-04-05 | 黑龙江八一农垦大学 | Multiplex PCR detection method for three fluke metacercaria in freshwater fish and primers for detection |
| CN117568494A (en) * | 2024-01-17 | 2024-02-20 | 黑龙江八一农垦大学 | Multiple PCR (polymerase chain reaction) detection primer group, kit and detection method for zoonotic metacercaria in freshwater fish |
| CN117568494B (en) * | 2024-01-17 | 2024-03-29 | 黑龙江八一农垦大学 | Multiple PCR (polymerase chain reaction) detection primer group, kit and detection method for zoonotic metacercaria in freshwater fish |
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Application publication date: 20110810 |