CN104164512A - PCR-RFLP detection kit for authenticating and differentiating infections of echinococcus multilocularis and echinococcosis shiquicus - Google Patents
PCR-RFLP detection kit for authenticating and differentiating infections of echinococcus multilocularis and echinococcosis shiquicus Download PDFInfo
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Abstract
The invention discloses a PCR-RFLP detection kit for authenticating and differentiating the infections of echinococcus multilocularis (Em) and echinococcosis shiquicus (Es). The kit comprises an Em and Es universal primer shown in SEQ ID NO. 1 and 2, restriction endonuclease EcoR I and/or Ssp I, a PCR reagent, and echinococcus multilocularis and echinococcosis shiquicus DNA template reference substances. The purpose of authenticating and differentiating the infections of echinococcus multilocularis and echinococcosis shiquicus can be achieved by extracting the DNA of the single cyst of the sample to be detected as a template, carrying out PCR amplification by using the universal primer, purifying a PCR product, carrying out enzyme digestion by using EcoR I or Ssp I incision enzyme, and then carrying out electrophoresis on an enzyme digestion product. According to the kit disclosed by the invention, the time consumption and money consumption due to sequencing, and the cumbersome process of data processing are omitted, a complex operation system is not required, and infection of echinococcus multilocularis or echinococcosis shiquicus to wild rodents can be rapidly, conveniently and economically differentiated and authenticated.
Description
Technical field
The present invention relates to parasitic detection, particularly a kind of PCR-RFLP detection kit of identifying and distinguishing that many rooms echinococcus and Shiqu echinococcus infect.
Background technology
Echinococcus multilocularis (Echinococcus multilocularis, Em) larva and Shiqu echinococcus (Echinococcus shiquicus, Es) larva all can parasitize rodent body (as Ochotona curzoniae, Qinghai vole etc.) in, and both packing profiles are extremely similar, in long-time, be all considered to the larva of Echinococcus multilocularis, until found Shiqu echinococcus adult in fox (Vuipes ferrilata) enteron aisle of Tibetan, just clearly Shiqu echinococcus and Echinococcus multilocularis are made a distinction, and then definite Shiqu echinococcus novel species that is Echinococcus.The worm's ovum of Echinococcus multilocularis adult infects people and multiple rodent, causes echinococcosis multilocularis, and people's echinococcosis multilocularis harm is serious, and case fatality rate is high, claims again " worm cancer ", and the western numerous herdsman's of serious threat China life is with healthy.The larva main infection rodent of Shiqu echinococcus, there is no the report of people's cases of infection at present.The investigation that rodent echinococcus larva is infected can be used as one of index of a certain regional Epidemic Conditions of Hydatid Disease.Therefore, set up detection kit that a kind of quick, economic method distinguishes these two kinds of echinococcuss to carrying out echinococcosis epidemiology survey, prevention and control the popular of echinococcosis and have great importance.
Qualification is at present distinguished two species and is mainly relied on the difference between its genetic material, this method relates to order-checking, there is no the grass-roots unit of sequenator, relatively consuming time, consumption money seems, simultaneously due to series processing, relate to relevant information biology instrument than equity, during to investigation in enormous quantities, can cause the loaded down with trivial details or situation such as easily make mistakes of data processing.
Along with in succession completing of the examining order of the full gene of plastosome of Echinococcus multilocularis and Shiqu echinococcus, after its plastosome whole genome sequence of comparison, find to exist the number of same restriction enzyme site in two segment DNA sequences different with distribution, this,, for utilizing restriction endonuclease to cut stripe size provide feasibilities different from number are provided fragment same in two kinds of echinococcus Mitochondrial Genome Overview, that length is consistent, just can distinguish these two kinds of echinococcus intuitively according to electrophorogram.This method has advantages of quick, easy than order-checking and consumption money is few, does not need the PCR instrument that checks order required, and the method only needs thermostat water bath and two kinds of restriction endonucleases to complete, and wherein glue recovery test kit is not essential.Up to the present, there is no adopting said method distinguishes wild rodent and infects the bibliographical information of many rooms echinococcus and Shiqu echinococcus.
Summary of the invention
The shortcoming that the object of the invention is to overcome prior art, with not enough, provides a kind of PCR-RFLP method and detection kit of identifying fast, easily and distinguishing many rooms of wild rodent echinococcus and the infection of Shiqu echinococcus.
Object of the present invention realizes by following technological method:
Identify and distinguish the PCR-RFLP method that many rooms echinococcus and Shiqu echinococcus infect, the primer of pcr amplification is Em and Es universal primer, and restriction enzyme is EcoR I and/or Ssp I.Described Em and Es universal primer are:
F：5’-TAA?GWT?RAG?TGT?GTG?TGT?TGG?T-3’，
R：5’-TAA?RCA?AAC?CTC?TCA?ACG?AGA?C-3’；
Wherein, W=A/T, R=A/G.
Utilizing the Em DNA sequencing fragment length that universal primer amplifies is 1417bp, and Es DNA sequencing fragment length is 1426bp.
EcoR I and the Ssp I restriction enzyme site position in Em and Es fragment respectively as depicted in figs. 1 and 2.The Em DNA fragmentation that uses universal primer amplification 261 and 757bp place have Ssp I restriction enzyme site; The Es DNA fragmentation of amplification has EcoR I restriction enzyme site at 254bp place, have Ssp I restriction enzyme site at 1175bp place.
Identify and distinguish the PCR-RFLP detection kit that many rooms echinococcus and Shiqu echinococcus infect, comprise above-mentioned universal primer, EcoR I and/or Ssp I.
Preferably, described qualification and distinguish many rooms echinococcus and Shiqu echinococcus infect PCR-RFLP detection kit, also comprise the PCR reagent for DNA cloning.
Preferred, described test kit also comprises many rooms echinococcus and Shiqu echinococcus DNA profiling standard substance.
Described qualification and distinguish many rooms echinococcus and the preparation method of PCR-RFLP detection kit that Shiqu echinococcus infects is preferably as follows:
(1) design of universal primer and the selection of restriction enzyme site
According to the completed Echinococcus multilocularis of submitting in NCBI (accession number: AB018440) and Shiqu echinococcus (accession number: AB208064) Mitochondrial Genome Overview sequence, the restriction enzyme site that utilizes DNAMAN software to find out respectively to contain in two Mitochondrial DNAs and the position at place thereof, the result of comparing in conjunction with ClustalW software again, find out while or some section of DNA sequences that possesses a certain restriction endonuclease sites on two species Mitochondrial DNAs, with reference to TaKaRa company restriction enzyme products catalogue and quotation information, finally determine that two sections of sequences of the upper 7123-8539 of being set to of Echinococcus multilocularis Mitochondrial Genome Overview and the upper 7160-8585 of being set to of Shiqu echinococcus Mitochondrial Genome Overview are as the reference sequences of qualification, design upstream and downstream degenerate primer, select two kinds of restriction enzyme EcoR I and Ssp I to cut result for mutually verifying enzyme.
(2) selection of archaeal dna polymerase and restriction enzyme
In line with economical, easy, efficient and principle accurately, 2 of archaeal dna polymerase employing TransGen (Beijing) company ×
pCR SuperMix, this product comprises
the reaction buffer of archaeal dna polymerase, dNTPs and optimization, concentration is 2 ×; EcoR I restriction endonuclease, by TaKaRa (Dalian) company is produced, comprises EcoR I restriction endonuclease (10000U) and 10 × EcoR I Buffer; Ssp I restriction endonuclease, by TaKaRa (Dalian) company is produced, comprises Ssp I restriction endonuclease (500U) and 10 × Ssp I Buffer.
(3) preparation of many rooms echinococcus and Shiqu echinococcus DNA profiling standard substance
The rodent lungs packing of field acquisition or liver packing, single packing is separated with tweezers, then extract the DNA of single packing according to tissue DNA extraction test kit specification sheets, the cox1 gene fragment increasing respectively in its Mitochondrial Genome Overview, send after the order-checking of order-checking company, according to after the cox1 gene comparison of submitted to two kinds of echinococcus, determine its worm kind, and the DNA of corresponding single packing or by the goal gene plasmid DNA of recombinating as DNA profiling standard substance.
Described qualification and distinguish many rooms echinococcus and the using method of PCR-RFLP detection kit that Shiqu echinococcus infects comprises the steps:
(1) DNA of the single packing of extraction detected sample.
(2), taking the DNA that extracts as template, use PCR reagent to carry out pcr amplification by following condition: 94 DEG C of 4min; 94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 2min, 35 circulations; 72 DEG C of 10min.
(3) PCR product is carried out to purifying.
(4) re-use the PCR product of EcoR I or Ssp I endonuclease digestion purifying.
(5) enzyme is cut to product and carry out electrophoresis qualification.
Use test kit of the present invention and method can be fast, convenient, economic differentiation qualification wild rodent infects many rooms echinococcus and still infects Shiqu echinococcus, consuming time, the consumption money that having saved that order-checking causes and the complicated processes of data processing, do not need complicated operating system simultaneously, just can carry out in common laboratory, therefore have potential experimental study and the clinical value that has potentiality.
Brief description of the drawings
Fig. 1 is the position view of EcoR I restriction enzyme site in Em and Es fragment.
Fig. 2 is the position view of Ssp I restriction enzyme site in Em and Es fragment.
Fig. 3 is the agarose gel electrophoresis figure of embodiment 2DNA template standard thing pcr amplification product; Wherein, M:DNA standard molecule DL2000, EM:Em DNA profiling standard substance; ES:Es DNA profiling standard substance.
Fig. 4 is that embodiment 2DNA template standard thing pcr amplification product enzyme is cut the agarose gel electrophoresis figure of qualification; Wherein, M:DNA standard molecule DL2000; Left figure is EcoR I endonuclease digestion result, 1: the Em DNA profiling standard substance that enzyme is not cut, 2: the Em DNA profiling standard substance after enzyme is cut, 3: the EsDNA template standard thing that enzyme is not cut, 4: the Es DNA profiling standard substance after enzyme is cut; Right figure is Ssp I endonuclease digestion result, 1: the Em DNA profiling standard substance that enzyme is not cut, 2: the Em DNA profiling standard substance after enzyme is cut, 3: the Es DNA profiling standard substance that enzyme is not cut, 4: the Es DNA profiling standard substance after enzyme is cut.
Fig. 5 is the agarose gel electrophoresis figure of embodiment 3 clinical sample pcr amplification products; Wherein, M:DNA standard molecule DL2000; 1-3 is respectively the strain of Echinococcus multilocularis Dari County, cajaput strain and Jiuzhi County strain; 4-9: the Jiuzhi County strain of Shiqu echinococcus, Dari County strain (5).
Fig. 6 is that embodiment 3 clinical sample pcr amplification product enzymes are cut the agarose gel electrophoresis figure of qualification; Wherein, M:DNA standard molecule DL2000; Left figure is EcoR I endonuclease digestion result, 1: the Em DNA profiling standard substance that enzyme is not cut, 2-4: the strain of Echinococcus multilocularis Dari County, cajaput strain and Jiuzhi County strain enzyme are cut result, 5: the Es DNA profiling standard substance that enzyme is not cut, 6-11: the Jiuzhi County strain of Shiqu echinococcus and Dari County strain (5) enzyme is cut result; Right figure is Ssp I endonuclease digestion result, 1: the Em DNA profiling standard substance that enzyme is not cut, 2-4: the strain of Echinococcus multilocularis Dari County, cajaput strain and Jiuzhi County strain enzyme are cut result, 5: the Es DNA profiling standard substance that enzyme is not cut, 6-11: the Jiuzhi County strain of Shiqu echinococcus and Dari County strain (5 genotype) enzyme is cut result.
Embodiment
Below in conjunction with specific embodiment, the present invention is done to further detailed description.Should be understood that the following examples are only not used in and limit the scope of the invention for the present invention is described.
Embodiment 1
Identify and distinguish the PCR-RFLP detection kit that many rooms echinococcus and Shiqu echinococcus infect, comprise: 2 of TransGen (Beijing) company ×
the DNA profiling standard substance of PCR SuperMix, TaKaRa (Dalian) EcoR of company I and Ssp I restriction endonuclease, universal primer and many rooms echinococcus and Shiqu echinococcus.
The sequence of universal primer is:
F：5’-TAA?GWT?RAG?TGT?GTG?TGT?TGG?T-3’，
R：5’-TAA?RCA?AAC?CTC?TCA?ACG?AGA?C-3’，
Wherein, W=A/T, R=A/G.
Embodiment 2 identifies DNA profiling standard substance
(1) DNA profiling standard substance pcr amplification
With the DNA profiling standard substance of the PCR reagent providing in the test kit in embodiment 1, universal primer, two kinds of echinococcus, do respectively pcr amplification.For there is the situation of Echinococcus multilocularis and the polyinfection of Shiqu echinococcus under checking field conditions, separately increase the PCR reaction of a standard form half and half.PCR reaction system is as follows:
Carry out PCR reaction by following condition: 94 DEG C of 4min; 94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 2min, 35cycles; 72 DEG C of 10min.
Get PCR reaction product 5 μ L in 1.0% (w/v) sepharose, under 120 volts of voltages, carry out electrophoretic analysis, result as shown in Figure 3, use universal primer, amplify respectively the Em DNA fragmentation of 1417bp and the Es DNA fragmentation of 1426bp taking Em DNA profiling standard substance and Es DNA profiling standard substance as template.
(2) PCR product purification
Remaining 20 μ L PCR products are carried out to purifying and can adopt following two kinds of methods:
Method one: add 60 μ L dehydrated alcohols respectively in PCR reaction stoste, making system is that volume fraction is 75% alcohol concn, be placed on-70 DEG C of refrigerator overnight, after taking-up, be placed on ice chest, then use 4 DEG C of whizzer 12000r/min centrifugal 10min at full speed, outwell the liquid in pipe, add again 75% ethanolic soln of 50 μ L, after mixing, be placed on 4 DEG C, the centrifugal 10min of 12000r/min, outwell liquid, get rid of gently the liquid remaining on tube wall, last, be placed on stink cupboard or super clean bench is air-dry, for subsequent use.
Method two: remaining PCR reaction stoste, with after 1.0% (w/v) agarose gel electrophoresis, is cut to object band, reclaim test kit with glue and reclaim, when washing, add elutriant 17 μ L, for subsequent use.
(3) enzyme is cut qualification
The PCR product of purifying is done to enzyme and cut qualification, according to the requirement on two kinds of restriction enzyme working instructions, Substrate DNA is whole PCR products of purifying, and the need of method one purifying are added 17 μ L ddH
2o, method two purifying only need add restriction enzyme and corresponding reaction Buffer, it is as follows that enzyme is cut system:
Endonuclease reaction temperature is 37 DEG C, and the reaction times is controlled in 1～2h, adds 10 × Loadding Buffer2 μ L termination reaction.Get 10 μ L enzymes and cut product in 1.0% (w/v) sepharose, under 120 volts of voltages, carry out electrophoretic analysis, as shown in Figure 4, Em DNA fragmentation obtains 3 fragments after cutting with Ssp I enzyme to result, and size is respectively 261bp, 496bp and 660bp; Es DNA fragmentation obtains 251bp and two fragments of 1175bp after cutting with EcoR I enzyme, after cutting, obtain 254bp and two fragments of 1172bp with Ssp I enzyme.
The application that embodiment 3 detects clinical sample
Clinical sample is respectively Echinococcus multilocularis Dari County strain (be that Dari County separates the Echinococcus multilocularis obtaining, other strains illustrate herewith), cajaput strain and Jiuzhi County strain and the Jiuzhi County strain of Shiqu echinococcus, Dari County strain (Dari County strain 5 genotype: N6/C7, N3/C3, N2/C6, N4/C4 and N5/C5; N represents nad1 gene, and C represents cox1 gene).
(1) prepare clinical sample DNA
Isolate respectively the packing of clinical collection, then extract the DNA of single packing according to tissue DNA extraction test kit specification sheets.
(2) clinical sample is carried out to pcr amplification
Reaction system and condition are with embodiment 2.Get PCR product 5 μ L in 1.0% (w/v) sepharose, under 120 volts of voltages, carry out electrophoretic analysis, result is as Fig. 5.
(3) PCR product purification
PCR product purification adopts glue to reclaim test kit method.
(4) the PCR product of purifying is carried out to enzyme and cut qualification
PCR product to purifying, cuts system with reference to the enzyme of embodiment 2 respectively, utilizes EcoR I and Ssp I restriction endonuclease to carry out enzyme and cuts qualification, and qualification result as shown in Figure 6, shows that the present invention can accurately distinguish and identify many rooms echinococcus and Shiqu echinococcus.
Claims (5)
1. identify and distinguish the PCR-RFLP method that many rooms echinococcus and Shiqu echinococcus infect, it is characterized in that: the primer of pcr amplification is Em and Es universal primer, restriction enzyme is EcoR I and/or Ssp I; Described Em and Es universal primer are:
F：5’-TAA?GWT?RAG?TGT?GTG?TGT?TGG?T-3’，
R：5’-TAA?RCA?AAC?CTC?TCA?ACG?AGA?C-3’，
Wherein, W=A/T, R=A/G.
2. identify and distinguish the PCR-RFLP detection kit that many rooms echinococcus and Shiqu echinococcus infect, it is characterized in that: comprise Em claimed in claim 1 and Es universal primer, EcoR I and/or Ssp I.
Qualification according to claim 2 and distinguish many rooms echinococcus and Shiqu echinococcus infect PCR-RFLP detection kit, it is characterized in that: comprise PCR reagent.
According to the qualification described in claim 2 or 3 and distinguish many rooms echinococcus and Shiqu echinococcus infect PCR-RFLP detection kit, it is characterized in that: comprise many rooms echinococcus and Shiqu echinococcus DNA profiling standard substance.
5. qualification claimed in claim 2 and distinguish many rooms echinococcus and the using method of PCR-RFLP detection kit that Shiqu echinococcus infects, is characterized in that comprising the steps:
(1) DNA of the single packing of extraction detected sample;
(2), taking the DNA that extracts as template, use PCR reagent to carry out pcr amplification by following condition: 94 DEG C of 4min; 94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 2min, 35 circulations; 72 DEG C of 10min;
(3) PCR product is carried out to purifying;
(4) re-use the PCR product of EcoR I or Ssp I endonuclease digestion purifying;
(5) enzyme is cut to product and carry out electrophoresis qualification.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399549A (en) * | 2016-10-28 | 2017-02-15 | 青海大学 | Method for PCR (polymerase chain reaction) detection of and detection kit |
| CN107326082A (en) * | 2017-08-01 | 2017-11-07 | 中国农业科学院兰州兽医研究所 | A kind of universal primer, nucleotide sequence and its discrimination method of three-type-person source tapeworm ep45 genes |
| CN110527730A (en) * | 2018-05-25 | 2019-12-03 | 中国农业科学院兰州兽医研究所 | It is a kind of based on the Shiqu echinococcus detection kit of RPA technology and its application |
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| CN103173554A (en) * | 2013-03-27 | 2013-06-26 | 中国农业科学院兰州兽医研究所 | Detection kit for detecting and distinguishing multiple kinds of echinococcus |
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| CN103173554A (en) * | 2013-03-27 | 2013-06-26 | 中国农业科学院兰州兽医研究所 | Detection kit for detecting and distinguishing multiple kinds of echinococcus |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399549A (en) * | 2016-10-28 | 2017-02-15 | 青海大学 | Method for PCR (polymerase chain reaction) detection of and detection kit |
| CN107326082A (en) * | 2017-08-01 | 2017-11-07 | 中国农业科学院兰州兽医研究所 | A kind of universal primer, nucleotide sequence and its discrimination method of three-type-person source tapeworm ep45 genes |
| CN107326082B (en) * | 2017-08-01 | 2020-12-15 | 中国农业科学院兰州兽医研究所 | Universal primer of human cestode ep45 gene and identification method thereof |
| CN110527730A (en) * | 2018-05-25 | 2019-12-03 | 中国农业科学院兰州兽医研究所 | It is a kind of based on the Shiqu echinococcus detection kit of RPA technology and its application |
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Application publication date: 20141126 |