CN101353690A - Cryptosporidium and cryptosporidium parvum specific PCR detecting reagent kit and detecting method - Google Patents
Cryptosporidium and cryptosporidium parvum specific PCR detecting reagent kit and detecting method Download PDFInfo
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Abstract
The invention provides a peculiar PCR detection kit of Cryptosporidium and micro Cryptosporidium, comprising a DNA lysis solution, a PCR reaction solution, a peculiar primer of the Cryptosporidium and the micro Cryptosporidium and positive control genomic DNA of the micro Cryptosporidium. According to the diagnosing sequence for analyzing Cryptosporidium and species peculiarities, a Cryptosporidium and species peculiar primer are designed by utilizing a peculiar detection method of PCR, the sensibility of the primer is improved by optimizing and amplifying conditions, the amplifying result can be directly observed by electrophoretic analysis. The kit of the invention has strict design, simple and easy operation, high sensibility and strong peculiarity, can simultaneously identify the Cryptosporidium infection of Cryptosporidium and species peculiarities so as to obtain an accurate and objective judgment result.
Description
Technical field
The present invention relates to the diagnosis and the detection technique of cryptosporidiosis, provided a kind of Cryptosporidium specificity and cryptosporidium parvum kind specific PCR detecting reagent kit and detection method or rather, belong to parasite detection technique field.
Background technology
Cryptosporidiosis (Cryptosporidiosis) is a kind ofly to endanger serious people beast and suffer from protozoal disease altogether by Cryptosporidium is caused, can cause especially ruminating animal and people's serious diarrhoea of Mammals.Named Cryptosporidium to have 27 kinds at present, the Cryptosporidium of energy infected person has multiple, wherein common with the infection of cryptosporidium parvum, the cryptosporidium parvum host range is extensive and harm is serious, therefore Mammalss such as many infected person, domestic animal are the maximum Cryptosporidium kinds of harm in the Cryptosporidium.
The diagnostic method of cryptosporidiosis mainly contains etiological diagnosis, immunology diagnosis and diagnosis of molecular biology three major types.Sick learn the many Ziehi-Neelsen stain inspections of diagnosis by ight soil microscope inspection and improvement, this method waste time and energy and recall rate low.Immunology diagnosis has indirect hemagglutination test (IHA), immunofluorescent test (IFA), enzyme linked immunosorbent assay (ELISA), monoclonal antibody methods such as (McAb), and so these immunological method specificitys and susceptibility are also lower.Therefore, the detection method of developing a kind of special sensitive Cryptosporidium and cryptosporidium parvum kind has been extremely urgent.
Summary of the invention
The invention provides the species specific PCR detection kit of a kind of Cryptosporidium and cryptosporidium parvum, be used for the differential diagnosis of cryptosporidiosis and cryptosporidium parvum kind.
The present invention also provides the detection method of this test kit.
Cryptosporidium of the present invention and cryptosporidium parvum kind specific PCR detecting reagent kit comprise with the lower section:
(1) dna cleavage liquid: the mixing solutions that contains NaCl, Tris-HCl, EDTA, SDS and the Proteinase K of different concns;
Wherein, concentration proportioning is: NaCl 100mM, Tris-HCl (pH 7.5) 20Mm, EDTA25Mm, SDS 2% (w/v) and Proteinase K 0.1 μ g/ μ l;
(2) PCR reaction solution: 4 kinds of dNTPs of each 100-300 μ M of final concentration, primer CF, CR, HF and the HR of each 10-100pmol/ μ l of final concentration, the Mg of 1.5-4.5mM
2+Concentration; The Taq enzyme contains 2.0U by each PCR reaction (20 μ l) and adds, and does not contain the Taq enzyme in the reaction solution;
(3) Cryptosporidium and cryptosporidium parvum species-specific primer:
Belong to the specific diagnosis primer: CF:5 '-ATTGGAGGTTGTTCCTTACTCCT-3 '
CR：5’-AGCCGCCCATAGAATCAAGAA-3’
Specific specificity diagnostic primers: HF:5 '-TAAGAAGCCACCAAGAAGGCG-3 '
HR：5′-TCAATAGGCTTAAATGGGTTCGGGA-3
(4) positive control: positive control is the cryptosporidium parvum genomic dna, and relatively whether PCR product and monitoring PCR operating process be correct.
Test kit of the present invention can also contain agarose (Agarose), tetrabromophenol sulfonphthalein point sample damping fluid and Taq enzyme, and its concentration is preferably ethidium bromide 10 μ g/ μ l; Tetrabromophenol sulfonphthalein point sample damping fluid and Taq enzyme 5U/ μ l.Also can also contain the PCR reaction tubes.
The detection method of test kit of the present invention may further comprise the steps:
1) pre-treatment of sample: this water mixing of taking a sample sieves, and crosses earlier 60 orders, after 100 mesh sieves, and filtrate centrifugation three times repeatedly, precipitation is standby sample;
2) extraction of sample DNA:
(1) above-mentioned precipitation is transferred in the 1.5ml centrifuge tube, added the 1ml lysate, multigelation is three times in liquid nitrogen and 65 ℃ of water-baths;
(2) sample after the freeze thawing is added 5-6 μ l (20mg/ml) Proteinase K, making its final concentration is 100 μ g/ml.In 65 ℃ of water-baths, soak 2h behind the mixing;
(3) use phenol: chloroform: primary isoamyl alcohol (24: 25: 1) carries out extracting twice, 7000r/min centrifugal ten minutes;
3), pcr amplification:
(1) prepare the PCR reaction tubes of sample number to be checked+2, comprise PCR liquid, Taq enzyme etc. in the pipe, cover tight lid, mixing is standby on vortice;
(2) negative and positive control are added respectively in the good pipe of mark, then sample is added successively that also mark is good, place the PCR instrument.The PCR condition is: 95 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, 60 ℃ of annealing 40s, 72 ℃ of extension 30s, and totally 30 circulations, back 72 ℃ are extended 10min;
4), the PCR product is observed:
Take by weighing agarose, in microwave oven, heated three minutes with (0.8%-1.0%) behind the electrophoresis liquid mixing.Deng glue cooling back difference application of sample in well, electrophoresis is 10 minutes under the 100V voltage, observation experiment result under ultraviolet lamp afterwards, infect if exist 424bp and 223bp amplified band just to prove cryptosporidium parvum simultaneously, if having only the 223bp band just to prove the Cryptosporidium spp of other kind.
Test example 1
The composition of test kit
Lysate (NaCl 100mM, Tris-HCl (pH 7.5) 20Mm, EDTA 25Mm, SDS 2% (w/v) and Proteinase K 0.1 μ g/ μ l) 20ml; PCR reaction solution 10 pipe (20 μ l system), wherein the concentration of dNTPs is that the final concentration of 250 μ M, primer HF, HR and CF, CR respectively is 0.5pmol/ μ l, Mg separately
2+Final concentration is 1.5mM; The 4g agarose; 50 μ l ethidium bromides (20 μ g/ μ l); 0.5% tetrabromophenol sulfonphthalein point sample damping fluid, 50 μ l; 10 μ l Taq enzymes (5U/ μ l); 1 cryptosporidium parvum genomic dna positive control.
Experimental example 2
The experiment of test kit specificity
The DNA that gets 10 samples such as sheep cryptosporidium parvum, cryptosporidium andersoni, Cryptosporidium baileyi, pig Cryptosporidium, people source giardia lamblia, dog source giardia lamblia, chicken eimeria tenella, mouse eimeria tenella, toxoplasma gondii, trichomonas is a template, respectively it is increased with the amplification system of setting up.
Amplification condition is:
Amplified production carries out electrophoretic analysis with 0.8% agarose, observations on gel imaging system.
The result shows (seeing accompanying drawing 2,3): plant special primer H and only obtained amplification in ox and sheep cryptosporidium parvum sample, other sample standard deviation does not have amplification, and amplified band is 424bp, meets kind of a special examination criteria.Belong to special primer C and all obtained amplification respectively in cryptosporidium parvum, cryptosporidium andersoni, Cryptosporidium baileyi, pig Cryptosporidium sample, amplified band is 223bp, and other sample standard deviation does not have amplification, meets to belong to special examination criteria.
Among the figure, Lane1-10: ox cryptosporidium parvum, sheep cryptosporidium parvum, cryptosporidium andersoni, Cryptosporidium baileyi, pig Cryptosporidium, people source giardia lamblia, dog source giardia lamblia, chicken eimeria tenella, mouse eimeria tenella, toxoplasma gondii, trichomonas.
Experimental example 3
The experiment of test kit susceptibility
The total nucleic acid that extracts is quantitative on the foranalysis of nucleic acids instrument, be 100ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng, 0.0001ng with the content in concentration dilution to the reaction system respectively.The reaction conditions homospecificity detects embodiment, establishes negative control simultaneously.Product carries out electrophoretic analysis with 0.8% agarose, observations on gel imaging system.The result shows that the detection threshold of H primer is 0.01ng (10pg), and the detection threshold of C primer is 0.001ng (1pg), and the threshold value that double PCR detects also is respectively H (10pg) and C (1pg), meets the sensitivity Detection standard fully.
See accompanying drawing 3,4,5,6:Lane1-7: the template concentrations gradient is made as: 100ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng, 0.0001ng.H and C primer lowest detection threshold value are respectively as seen from the figure: H (10pg) and C (1pg).
Experimental example 4
The stability of test kit and repeated experiment
Positive template, the PCR reaction solution, EX Taq enzyme is stored under-20 ℃ of conditions, and 4 ℃ of preservations of other article such as lysate, tetrabromophenol sulfonphthalein get final product.When being 30 days, 60 days, 100 days, 150 days, 200 days, period of storage takes out each component, with known PCR male sample detection test kit stability.In addition, 15 parts of PCR positive sample are with this test kit revision test 3 times, 15 parts of same repetitions 3 times of PCR negative sample, and amplification condition is as previously mentioned.The result shows that the component of taking out at above day part has all obtained two bright special purpose bands in known PCR positive and 15 parts of PCR positive sample, and blank and 15 parts of negative samples all do not have any amplified band, so test kit stability and repeatability are good.
The quality guaranteed period test of test kit
The test kit of storing 1 month, 3 months, 5 months, 7 months and 10 months 4 ℃ and-20 ℃ is respectively taken out and known positive is detected.Amplification and inspection method are the same, and the result shows that the test kit of preserving for October still can amplify purpose band clearly, does not have assorted band under 4 ℃ and-20 ℃ of conditions.
Positively effect of the present invention is: utilize the method for detecting specificity of PCR, belong to special and species specific diagnosing sequence design genus and plant special primer according to analyzing, improve its susceptibility, electrophoretic analysis direct viewing amplification by optimizing amplification condition.Test kit design of the present invention is rigorous, simple to operation, and susceptibility is strong, and high specificity can identify simultaneously to belong to special and the special Cryptosporidium spp of kind that the result judges accurately objective.
Description of drawings
Fig. 1: double PCR primer (H and C) concentration proportioning is groped;
Fig. 2,3: two primer specificity are groped;
Fig. 4,5,6: two primers reach double PCR system susceptibility separately and grope;
Embodiment
For the ease of understanding the present invention, especially exemplified by following examples.Its effect is understood that it is to explaination of the present invention but not to any type of restriction of the present invention.
Cryptosporidium specific diagnostic sequence: the 18SrRNA gene (this gene be each kind of Cryptosporidium common and be kind between a fragment gene of genovariation minimum) conservative part in the sequence, comparison is total for a plurality of Cryptosporidium kinds in NCBI, and homology meets and belongs to special examination criteria greater than 98%.
attggaggttgttccttactccttcagcaccttatgagaaatcaaagtctttgggttctagggggagtat
ggtcgcaaggctgaaacttaaaggaattgacggaagggcaccaccaggagtggagcctgcggcttaatttg
actcaacacgggaaaactcaccaggtccagacataggaaggattgacagattgatagctccttcttgatt
ctatgggcggct
Cryptosporidium parvum kind specific diagnostic sequence: one section finger-like U1 small nuclear ribonucleoprotein gene, comparison only is that people source and cryptosporidium parvum are common among the NCBI, homology is 100%, meets kind of a special detection research standard.
taagaagccaccaagaaggcggaaggcacaactataatttaagaaagttgttgaaaagtgaaaactatagggaactacag
aagaaaaaagatgaggaagaggtccgaaaggagtttatgaagttacaaggggtcgagcaaacttctaatcgtgaaaagaaaa
gaatcaaactgatagataaggaagtcagatcaggaagttctaccacttttggagataatgttttcacagccaattatacctttgatg
attcaaagaatatttttgaaaaaaaagatcataatgaaagccatgacaaaacgaacgaaatgggaattattgggaaatgggaag
aagttaaagagtctgaatctgcctttttaggaaatattaccttagaacaacaagatcatactcaaatcccgaacccatttaag
cctattga
Special as follows according to the Cryptosporidium of embodiment 1 with kind specific diagnostic sequences Design specific diagnosis primer:
Belong to the specific diagnosis primer: CF:5 '-ATTGGAGGTTGTTCCTTACTCCT-3 '
CR：5’-AGCCGCCCATAGAATCAAGAA-3’
Specific specificity diagnostic primers: HF:5 '-TAAGAAGCCACCAAGAAGGCG-3 '
HR：5’-TCAATAGGCTTAAATGGGTTCGGGA-3
More than two kinds of primers (double PCR) in same system can amplify kind special simultaneously and belong to a special diagnostic gene segment, the purpose band is bright, does not have non-specific assorted band.Realize detection of cryptosporidium and small purpose of concealing sub-worm simultaneously.
Cryptosporidium and cryptosporidium parvum PCR detection kit, it is as follows to form structure:
(1) dna cleavage liquid: the mixing solutions that contains NaCl, Tris-HCl, EDTA, SDS and the Proteinase K of different concns.Each concentration proportioning is: NaCl 100mM, Tris-HCl (pH 7.5) 20Mm, EDTA 25Mm, SDS 2% (w/v) and Proteinase K 0.1 μ g/ μ l.The effect of lysate is with egg capsule cracking and digestion protein wherein, discharges genomic dna.
(2) PCR reaction solution: 4 kinds of dNTPs of each 100-300 μ M of final concentration, primer CF, CR, HF and the HR of each 10-100pmol/ μ l of final concentration, the Mg of 1.5-4.5mM
2+Concentration.The Taq enzyme contains 2.0U by each PCR reaction (20 μ l) and adds, and does not contain the Taq enzyme in the reaction solution.
(3) Cryptosporidium and cryptosporidium parvum species-specific primer
(4) positive control: the used positive control of test kit of the present invention is the cryptosporidium parvum genomic dna, and its effect is whether comparison PCR product and monitoring PCR operating process be correct.
In addition, test kit of the present invention can also contain agarose (Agarose), tetrabromophenol sulfonphthalein point sample damping fluid and Taq enzyme, and its concentration is preferably ethidium bromide 10 μ g/ μ l; Tetrabromophenol sulfonphthalein point sample damping fluid and Taq enzyme 5U/ μ l.Can also contain the PCR reaction tubes.
The optimizing process of test kit PCR reaction conditions of the present invention is:
Two pairs of primer optimizations of concentration separately: primer is set in the final concentration gradient of system is: 0.5 μ M, 1 μ M, 1.5 μ M, 2 μ M, 2.5 μ M, 3 μ M, attempt all being increased accordingly with this interval primer, wherein working as the primer amount is 10pmol (the storage concentration of primer is 20pmol/ μ l, gets 0.5 μ l) best results when final concentration is 0.5pmol/ μ l.
Two pairs of primer optimizations of annealing temperature separately: the annealing temperature that primer is set is respectively: 47 ℃, 49 ℃, 52 ℃, 54 ℃, 56 ℃, 59 ℃, 61 ℃, 63 ℃, on the grads PCR instrument, increase, and result's two primers in the time of 59 ℃ all have optimized amplification.
Two pairs of primer optimizations of dNTPs concentration separately: every kind of dNTPs (each) concentration gradient is set is: 50 μ M, 100 μ M, 250 μ M, 350 μ M, 400 μ M., in this concentration section, all obtain good amplification, wherein two primers all have best amplification when concentration is 250 μ M (each).
The two pairs of primers are Mg separately
2+The optimization of degree: Mg is set
2+The final concentration gradient is: 1nM, 1.5nM, 2nM, 3nM, 4nM, 5nM, wherein concentration two primers when 1.5mM all have best amplification.
Groping of the best enzyme concn of two primers: the enzyme concn gradient is set is: 0.5U, 1U, 2U, 3U, 4U, and two primers have good amplification at this concentration Duan Shijun, but two primers all obtain optimized amplification when 2U.
Groping of two-fold PCR system condition: the Mg of double PCR system
2+Final concentration is 3mM, and the final concentration of dNTPs (each) is 500 μ M, and enzyme concn is 2U, and annealing temperature is 59 ℃, and the concentration of primer C is 0.5pmol/ μ l, and the final concentration of primer H is 0.5pmol/ μ l.
See that each concentration proportioning gradient of accompanying drawing 1:Lane1-6:H and C primer is: 0.5/0.5 μ M, 0.6/0.5 μ M, 0.7/0.5 μ M, 0.8/0.5 μ M, 0.9/0.5 μ M, 1/0.5 μ M.Just can bring into play best expanding effect when by last figure as seen, two primer concentration proportionings are 0.5/0.5 μ M.
Gather the geographic 35 parts of cow dung samples in Changchun and 50 parts of sheep excrement samples respectively, do contrast with classical modified acid fast stain method and sample is detected in conjunction with double PCR method.
(1) modified acid fast stain method
Each excrement sample is got 20g, adds 5 times of water and stirs evenly with glass stick, filters with 60 order nylon mesh earlier, filter with 100 order nylon mesh again, with the filtrate smear, seasoning or dry on spirit lamp, an amount of azaleine dye liquor is covered above the filtrate that fixes flowing water flushing after 7-8 minute.Drip 10% sulfuric acid and cover above the painted filtrate of azaleine, flowing water flushing after 5-6 minute.Drip Victoria Green WPB above sample, 2min after washing, 10 * 100 times of oily mirror microscopies after the seasoning.
Attached: the acid-fast stain dye liquor preparation method
1. azaleine dye liquor: basic fuchsin 4g, 95% alcohol 20ml, phenylic acid 8ml, distilled water 100ml.2.10% sulfuric acid: 98% vitriol oil 10ml, distilled water 90ml.
3. Victoria Green WPB is redyed: 0.2g Victoria Green WPB, distilled water 100ml.
(2) PCR detects
1, the pre-treatment of sample
This 20g water mixing of taking a sample sieves, and crosses earlier 60 orders, after 100 mesh sieves, and the flat whizzer 3000r/min of filtrate water centrifugation three times repeatedly, precipitation is standby sample.
2, the extraction of sample DNA
(1) above-mentioned precipitation is transferred in the 1.5ml centrifuge tube, added the 1ml lysate, multigelation is three times in liquid nitrogen and 65 ℃ of water-baths.
(2) sample after the freeze thawing is added 5-6 μ l (20mg/ml) Proteinase K, making its final concentration is 100 μ g/ml.In 65 ℃ of water-baths, soak 2h behind the mixing.
(3) use phenol: chloroform: primary isoamyl alcohol (24: 25: 1) carries out extracting twice, 7000r/min centrifugal ten minutes.
(4) in the EP pipe that is drawn to sterilization that top water is careful, note not drawing the egg white layer of medial degeneration.
(5) add 3M sodium-acetate (PH5.2) 60-70 μ l at aqueous phase, the cold ethanol that adds 2 times of volumes again precipitates-20 ℃ of refrigerator overnight.
(6) next day, will precipitate sample centrifugal 30min in 4 ℃ of refrigerated centrifuges completely, abandon behind the supernatant again with 75% alcohol washing one time.
(7) abandon freeze-drying in freeze drier behind the alcohol, with TE liquid or sterilize and 4 steam water dissolution and preserve.
3, pcr amplification:
(1) get PCR reaction solution, Taq enzyme etc. by the PCR reaction tubes of sample number to be checked+2, mix in a pipe, set up 20 μ l systems, cover tight lid, mixing is standby on vortice.
(2) get negative and each 1 μ l of positive control adds respectively in the good pipe of mark, each 1 μ l of sample thief adds successively that also mark is good then, places the PCR instrument.
(3) the pcr amplification condition is:
4, the PCR product is observed:
Take by weighing agarose, the sepharose of preparation 0.8% adds E.B. by 0.5 μ g/ml, heats three minutes in microwave oven with (0.8%-1.0%) behind the electrophoresis liquid mixing.Deng glue cooling back difference application of sample in well, electrophoresis is 10 minutes under the 100V voltage, observation experiment result under ultraviolet lamp afterwards, infect if exist 424bp and 223bp amplified band just to prove cryptosporidium parvum simultaneously, if having only the 223bp band just to prove the Cryptosporidium spp of other kind.
(3), modified acid fast stain and double PCR detected result
Find that through modified acid fast stain and double PCR detection the modified acid fast stain method detects 3 routine cryptosporidium parvums and 18 routine cryptosporidium andersonis in 35 parts of cow dung samples, two-fold PCR method detects 4 routine cryptosporidium parvums and 20 routine cryptosporidium andersonis, the modified acid fast stain method detects 6 routine cryptosporidium parvums and 0 routine cryptosporidium andersoni in 50 parts of sheep excrement samples, and double PCR method detects 8 routine cryptosporidium parvums and 0 routine cryptosporidium andersoni.
Claims (3)
1, a kind of Cryptosporidium and cryptosporidium parvum kind specific PCR detecting reagent kit comprise with the lower section:
(1) dna cleavage liquid: the mixing solutions that contains NaCl, Tris-HCl, EDTA, SDS and the Proteinase K of different concns; Each concentration proportioning is: NaCl 100mM, Tris-HCl (pH7.5) 20Mm, EDTA25 Mm, SDS 2% (w/v) and Proteinase K 0.1 μ g/ μ l;
(2) PCR reaction solution: 4 kinds of dNTPs of each 100-300 μ M of final concentration, primer CF, CR, HF and the HR of each 0.5-3pmol/ μ l of final concentration, the Mg of 1.5-4.5mM
2+Concentration; The Taq enzyme contains 2.0U by each PCR reaction (20 μ l) and adds, and does not contain the Taq enzyme in the reaction solution;
(3) Cryptosporidium and cryptosporidium parvum species-specific primer:
Belong to the specific diagnosis primer: CF:5 '-ATTGGAGGTTGTTCCTTACTCCT-3 '
CR：5’-AGCCGCCCATAGAATCAAGAA-3’
Specific specificity diagnostic primers: HF:5 '-TAAGAAGCCACCAAGAAGGCG-3 '
HR：5′-TCAATAGGCTTAAATGGGTTCGGGA-3
(4) positive control: the used positive control of test kit of the present invention is the cryptosporidium parvum genomic dna, and its effect is whether comparison PCR product and monitoring PCR operating process be correct.
2, test kit according to claim 1, wherein the final concentration of 4 kinds of dNTPs is each 250 μ M in the PCR reaction solution, primer HF, HR and CF, CR and final concentration respectively are 0.5pmol/ μ l, Mg
2+Final concentration is 1.5mM.
3, the detection method of the described test kit of claim 1 may further comprise the steps:
1) pre-treatment of sample: this water mixing of taking a sample sieves, and crosses earlier 60 orders, after 100 mesh sieves, and filtrate centrifugation three times repeatedly, precipitation is standby sample;
2) extraction of sample DNA:
(1) above-mentioned precipitation is transferred in the 1.5ml centrifuge tube, added the 1ml lysate, multigelation is three times in liquid nitrogen and 65 ℃ of water-baths;
(2) sample after the freeze thawing is added 5-6 μ l (20mg/ml) Proteinase K, making its final concentration is 100 μ g/ml.In 65 ℃ of water-baths, soak 2h behind the mixing;
(3) use phenol: chloroform: primary isoamyl alcohol (24: 25: 1) carries out extracting twice, 7000r/min centrifugal ten minutes;
3), pcr amplification:
(1) prepare the PCR reaction tubes of sample number to be checked+2, comprise PCR liquid, Taq enzyme etc. in the pipe, cover tight lid, mixing is standby on vortice;
(2) negative and positive control are added respectively in the good pipe of mark, then sample is added successively that also mark is good, place the PCR instrument; The PCR condition is: 95 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, 60 ℃ of annealing 40s, 72 ℃ extend 30s, establish 30 circulations altogether since second step, 72 ℃ are extended 10min then;
4), the PCR product is observed:
Take by weighing agarose, in microwave oven, heated three minutes with (0.8%-1.0%) behind the electrophoresis liquid mixing.Deng glue cooling back difference application of sample in well, electrophoresis is 10 minutes under the 100V voltage, observation experiment result under ultraviolet lamp afterwards, infect if exist 424bp and 223bp amplified band just to prove cryptosporidium parvum simultaneously, if having only the 223bp band just to prove the Cryptosporidium spp of other kind.
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2008
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| CN102168137A (en) * | 2011-02-16 | 2011-08-31 | 中国疾病预防控制中心寄生虫病预防控制所 | Primer set for detecting oocyst deoxyribonucleic acid (DNA) of Cryptosporidium by using loop-mediated isothermal amplification technology, kit, detection method and application |
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| CN103898203B (en) * | 2012-12-28 | 2016-09-28 | 中国科学院动物研究所 | The detection method of Cryptosporidum parvum and detection kit |
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| CN106319046A (en) * | 2016-08-22 | 2017-01-11 | 吉林大学 | Nested PCR (polymerase chain reaction) detection kit for 11 types of rabbit Eimeria |
| CN107365843A (en) * | 2017-07-31 | 2017-11-21 | 北京莱博泰克生物技术有限公司 | For detecting LAMP primer composition and its application of two kinds of main parasitic worms for causing calf diarrhea |
| CN109234420A (en) * | 2018-10-09 | 2019-01-18 | 河南省奶牛生产性能测定中心 | Detect fluorescence quantification PCR primer, probe, kit and the method for Cryptosporidum parvum |
| CN111118189A (en) * | 2020-01-07 | 2020-05-08 | 西北农林科技大学 | Bovine cryptosporidium parvum specific primer and PCR detection method and application thereof |
| CN111607656A (en) * | 2020-06-28 | 2020-09-01 | 哈密市动物疫病预防控制中心 | Triple PCR detection primer, method and kit for tapeworm and sporozoon in canine feces |
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