CN102191324A - Trypanosoma evansi and trypanosoma lewisi dual-polymerase chain reaction (PCR) assay kit and preparation method thereof - Google Patents
Trypanosoma evansi and trypanosoma lewisi dual-polymerase chain reaction (PCR) assay kit and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a trypanosoma evansi and trypanosoma lewisi dual-polymerase chain reaction (PCR) assay kit, which is used for quick PCR detection for trypanosoma evansi and trypanosoma lewisi. The invention also provides a preparation method of the kit. A primer is designed according to species specific gene sequences of the trypanosoma evansi and the trypanosoma lewisi by using a dual PCR specificity detection method; the sensitivity of the kit is improved by optimizing a PCR reaction system and a PCR reaction condition; and the result is observed after an amplified product is subjected to agarose gel electrophoresis. The trypanosoma evansi and the trypanosoma lewisi can be quickly and accurately detected and identified through the kit at the same time. The invention is characterized in that: the kit is simple and convenient, high in sensitivity and specificity and the like.
Description
Technical field
The present invention discloses a kind of Trypanosoma evansi and trypanosoma lewisi two-fold PCR detection kit, is used for Trypanosoma evansi and trypanosoma lewisi fast PCR and detects, and the present invention also provides the preparation method of this test kit, belongs to parasite detection technique field.
Background technology
Trypanosome (Trypanosoma) is a kind of common flagellate in the mammalian, is the infecting both domestic animals and human parasitic protozoa that a class has extensive host, is popular in each continent of Asia and Africa Latin America.Popular types is also different in different continents, in the Asia and main popular Trypanosoma evansi of China (Trypanosoma evansi) and trypanosoma lewisi (Trypanosoma lewisi).The Trypanosoma evansi route of transmission is mainly through hematophagous bug mechanical transmission and placental infection.In livestock breeding industry, can cause the dehab of domestic animal, cause domestic animal day by day to become thin, in addition dead.There is the case of infected person in report India such as Joshi.In addition, trypanosoma lewisi is also comparatively general in rodentine infection all over the world, and main parasitic is propagated by the media flea under the natural condition in the brown rat body, existing abroad human infection case report, and 2007 have report Thailand confirmed cases is that trypanosoma lewisi infects; Also there is similar report in India.Trypanosoma lewisi antibody positive among the area crowd of investigation report China northeast is arranged, may have subclinical or missed case among the crowd, also brought new threat at present China's livestock industry and public health.
Summary of the invention
The invention provides a kind of Trypanosoma evansi and trypanosoma lewisi two-fold PCR detection kit, differentiate fast and detection when can be used for Trypanosoma evansi and trypanosoma lewisi.
The present invention further provides the preparation method of mentioned reagent box.
Multiple important water people beast of the present invention suffers from protozoon detection kit simultaneously altogether, comprises with the lower section:
(1) sample lysate and DNA extraction reagent
The mixing solutions of sample lysate: NaCl, Tris-HCl, EDTA, SDS and Proteinase K; Wherein, concentration proportioning is: NaCl 100mM, Tris-HCl (pH 7.5) 20Mm, EDTA 25 Mm, SDS 2%(w/v) and Proteinase K 0.1 μ g/ μ l;
DNA extraction reagent is phenol: chloroform: primary isoamyl alcohol (24:25:1).
(2) PCR reaction solution: contain 4 kinds of dNTPs of each 100-300 μ M of reaction final concentration, the primer of each 10-100pmol/ μ l of reaction final concentration, the Mg2+ concentration of 1.5-4.5mM; Archaeal dna polymerase, sterile purified water.
(3) double PCR primer:
Trypanosoma evansi primer TE-a and TE-s, trypanosoma lewisi primer TL-a and TL-s;
(4) positive control: positive control is Trypanosoma evansi DNA and trypanosoma lewisi DNA.
(5) negative control: get the negative check sample of sterile purified water.
Test kit of the present invention can contain agarose Agarose(99.0%), tetrabromophenol sulfonphthalein point sample damping fluid (5U/ μ L) and Taq enzyme (5U/ μ L), ethidium bromide (10 μ g/ μ L); Can also contain the PCR reaction tubes.
The preparation method of test kit of the present invention may further comprise the steps:
(1) sample lysate and DNA extraction reagent
Sample lysate: NaCl, Tris-HCl, EDTA, SDS and Proteinase K are made mixing solutions; Wherein, concentration proportioning is: NaCl 100mM, Tris-HCl (pH 7.5) 20Mm, EDTA 25 Mm, SDS 2%(w/v) and Proteinase K 0.1 μ g/ μ l;
DNA extraction reagent is phenol: chloroform: primary isoamyl alcohol (25:24:1).
(2) double PCR primer:
Trypanosoma evansi and trypanosoma lewisi specific diagnostic gene are chosen in comparison on GeneBank, respectively RoTat 1.2 VSG genes and 18S rRNA gene.
Trypanosoma evansi (the amplified fragments size is 482bp):
TE-a：5'CGGGTCGTCTGCTAAAGT3'
TE-s：5'GCCCGCAGTTGCCTAT3'
Trypanosoma lewisi (the amplified fragments size is 261bp):
TL-a：5'GAGCTCAAGCGGCAGGTTA3'
TL-s：5'?TGCGCGAGAAGAGAGGACT?3'
(3) PCR reaction solution contains 4 kinds of dNTPs that react each 100-300 μ M of final concentration, the primer of each 10-100pmol/ μ l of reaction final concentration, the Mg2+ concentration of 1.5-4.5mM; Archaeal dna polymerase, the sterilization distilled water;
(4) positive control is Trypanosoma evansi DNA and trypanosoma lewisi DNA, and relatively whether PCR product and monitoring PCR operating process be correct;
(5) negative control: get the negative check sample of sterile purified water, purpose in getting rid of reaction solution pollution of nucleic acid and the false positive problem in the operating process.
Positively effect of the present invention is: utilize double PCR method for detecting specificity, according to Trypanosoma evansi and trypanosoma lewisi kind special gene sequence design primer, improve its susceptibility by optimizing PCR reaction system and condition, amplified production is observations behind agarose gel electrophoresis.The present invention can be simultaneously detects fast and accurately and differentiates Trypanosoma evansi and trypanosoma lewisi, has characteristics such as simple and convenient, highly sensitive, high specificity.
Description of drawings
Fig. 1 is the present invention's two-fold PCR specificity proof diagram;
Fig. 2 detects the Trypanosoma evansi sensitivity map for the present invention's two-fold PCR;
Fig. 3 detects the trypanosoma lewisi sensitivity map for the present invention's two-fold PCR.
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.
1, double PCR differentiates the design of primers of detection kit
It is as follows to utilize biosoftware to design double PCR primer according to the specific gene of choosing:
Trypanosoma evansi:
TE-a：5'CGGGTCGTCTGCTAAAGT3'
TE-s：5'GCCCGCAGTTGCCTAT3'
Trypanosoma lewisi:
TL-a：5'GAGCTCAAGCGGCAGGTTA3'
TL-s：5'?TGCGCGAGAAGAGAGGACT?3'
More than the two pairs of primers in same system (double PCR reaction system) simultaneously augmentation detection go out Trypanosoma evansi and trypanosoma lewisi specific gene segment, can realize detecting simultaneously and differentiating the purpose of Trypanosoma evansi and trypanosoma lewisi.
, sample lysate and DNA extraction reagent preparation
According to following concentration proportioning NaCl 100mM, Tris-HCl (pH 7.5) 20Mm, EDTA 25 Mm, SDS 2%(w/v) and Proteinase K 0.1 μ g/ μ l mix preparation sample lysate; DNA extraction reagent: volume ratio is the phenol of 25:24:1: chloroform: primary isoamyl alcohol.
, the PCR reaction solution preparation
4 kinds of dNTPs that contain each 100-300 μ M of reaction final concentration, the primer of each 10-100pmol/ μ l of reaction final concentration, the Mg2+ concentration of 1.5-4.5mM; Archaeal dna polymerase, the sterilization distilled water;
4, contrast
Positive control is Trypanosoma evansi DNA and trypanosoma lewisi DNA; Negative control is the sterilization distilled water;
5, reaction system optimization
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 when the primer amount be that the storage concentration of 10pmol(primer is 20pmol/ μ L, get 0.5 μ L) best results when final concentration is 0.5 pmol/ μ L.
Two pairs of primer optimizations of annealing temperature separately: the annealing temperature that primer is set is respectively: 47 ℃, 50 ℃, 53 ℃, 55 ℃, 57 ℃, 59 ℃, 61 ℃, 63 ℃, on the grads PCR instrument, increase, and result's two primers in the time of 55 ℃ all have optimized amplification.
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.
Experimental example 1, double PCR differentiate the experiment of detection method specificity
Get 1 ng Schistosoma japonicum, the graceful protozoon of Du Shi Li Shi and Taylor Se Shi worm and increase according to this PCR reaction system and amplification condition, verify its specificity as negative template.
The result shows that check sample does not all have amplification, meets to belong to special examination criteria, Fig. 1.
Experimental example 2
Two-fold PCR differentiates the experiment of detection method susceptibility
Blood sampling contains trypanosome polypide several 1 * 10 with every mL blood behind counting
5Individual, 1 * 10
4Individual, 1 * 10
3Individual, 1 * 10
2Individual, 10,1,0.5 concentration is diluted with the PBS damping fluid, extracts genome according to the template treatment process, and the performing PCR augmentation detection of going forward side by side is verified its sensitivity.The result shows that trypanosoma lewisi and Trypanosoma evansi two-fold PCR detection method lowest detectable limit can reach 1 polypide/reaction, belongs to high-sensitivity detecting method, meets the sensitivity Detection standard, sees Fig. 2-3.
Experimental example 3
The stability of test kit and repeated experiment
Positive template, PCR reaction solution can be under-20 ℃ of conditions long storage, dissolve 4 ℃ of preservations of back 4 ℃ of conditions and get final product, should avoid multigelation.When being 30 days, 60 days, 100 days, 150 days, 200 days, period of storage takes out each component, according to detection architecture detection kit stability.Get 15 parts of positive sample with this test kit revision test 3 times, 15 parts of same repetitions 3 times of PCR negative sample.The result shows in component non-false positive and the false negative result appearance in stability and replica test that above day part takes out, so test kit stability and repeatability are good.
Experimental example 4
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, known sample is detected according to detection architecture.The result shows that the test kit of preserving for October still can be made accurately sample and detects under 4 ℃ and-20 ℃ of conditions.
Test example 1
Gather 16 parts of Trypanosoma evansi infecting mouse blood samples and 19 parts of trypanosoma lewisi infected rats blood samples respectively, do contrast with classical Wright's staining method and sample is detected in conjunction with double PCR method.
(1) Wright Stain
Each sample is doubly dripped sheet in the back with PBS dilution 3-5, and the dropping Wright's stain makes dye liquor be paved with blood and is coated with face, dyeing 1min; Softly wash surperficial 5min with distilled water, dry and be placed on microscopically and observe microscopy.
(2) double PCR detects
1, the pre-treatment of sample
This 0.5mL that takes a sample, horizontal centrifuge 3000g centrifugation.
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 1h behind the mixing.
(3) use phenol: chloroform: primary isoamyl alcohol (24:25:1) carries out extracting twice, 12000g 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 1h in-20 ℃ of refrigerators.
(6) will precipitate sample centrifugal 30min of 12000g in 4 ℃ of refrigerated centrifuges completely, abandon behind the supernatant again with 75% alcohol washing one time.
(7) drying after abandoning alcohol, with the TE damping fluid or sterilize and 4 steam water dissolution and preserve, to be checked.
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, 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: 95 ℃ of pre-sex change 5min, and 95 ℃ of sex change 30s, 55 ℃ of annealing 40s, 72 ℃ are extended 30s, 30 circulations of increasing, 72 ℃ of final 10min that extend.
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 2 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, and observation experiment result under ultraviolet lamp infects if the 482bp amplified band proves Trypanosoma evansi afterwards; If proving trypanosoma lewisi, infects the 261bp amplified band.
(3), Wright Stain and double PCR detected result
Detect relatively through Wright Stain and double PCR, find that Wright Stain detects 14 routine Trypanosoma evansis in 16 parts of Trypanosoma evansi blood samples, double PCR method detects 16 routine Trypanosoma evansis; Wright Stain detects 18 routine trypanosoma lewisis in 19 parts of trypanosoma lewisi blood samples, and double PCR method detects 19 routine trypanosoma lewisis.Wright Stain fails Trypanosoma evansi and trypanosoma lewisi are made discriminating accurately simultaneously, and double PCR detection method can be distinguished the two.
Sequence table
＜110〉Jilin University
＜120〉Trypanosoma evansi and trypanosoma lewisi two-fold PCR differentiates detection kit and preparation method
<210> 1
<211> 18
<212> DNA
＜213〉synthetic
<220>
＜221〉primer (primer)
<222> (1)..(18)
<223>
<400> 1
cgggtcgtctgctaaagt 18
<210> 2
<211> 16
<212> DNA
＜213〉synthetic
<220>
＜221〉primer (primer)
<222> (1)..(16)
<223>
<400> 2
gcccgcagttgcctat 16
<210> 3
<211> 19
<212> DNA
＜213〉synthetic
<220>
＜221〉primer (primer)
<222> (1)..(19)
<223>
<400> 3
gagctcaagcggcaggtta 19
<210> 4
<211> 19
<212> DNA
＜213〉synthetic
<220>
＜221〉primer (primer)
<222> (1)..(19)
<223>
<400> 4
tgcgcgagaagagaggact 19
Claims (3)
1. Trypanosoma evansi and trypanosoma lewisi two-fold PCR detection kit comprises with the lower section:
(1) sample lysate and DNA extraction reagent
The mixing solutions of sample lysate: NaCl, Tris-HCl, EDTA, SDS and Proteinase K; Wherein, concentration proportioning is: NaCl 100mM, Tris-HCl (pH 7.5) 20Mm, EDTA 25 Mm, SDS 2%(w/v) and Proteinase K 0.1 μ g/ μ l;
DNA extraction reagent is phenol: chloroform: primary isoamyl alcohol (24:25:1);
(2) PCR reaction solution: contain 4 kinds of dNTPs of each 100-300 μ M of reaction final concentration, the primer of each 10-100pmol/ μ l of reaction final concentration, the Mg2+ concentration of 1.5-4.5mM; Archaeal dna polymerase, sterile purified water;
(3) double PCR primer:
Trypanosoma evansi primer TE-a and TE-s,
Trypanosoma lewisi primer TL-a and TL-s;
(4) positive control: positive control is Trypanosoma evansi DNA and trypanosoma lewisi DNA;
(5) negative control: get the negative check sample of sterile purified water.
2. test kit according to claim 1 is characterized in that:
Can also contain agarose (99.0%), tetrabromophenol sulfonphthalein point sample damping fluid (5U/ μ L) and Taq enzyme (5U/ μ L), ethidium bromide (10 μ g/ μ L); Can also contain the PCR reaction tubes.
3. the preparation method of the described test kit of claim 1 may further comprise the steps:
(1) sample lysate and DNA extraction reagent
Sample lysate: NaCl, Tris-HCl, EDTA, SDS and Proteinase K are made mixing solutions; Wherein, concentration proportioning is: NaCl 100mM, Tris-HCl (pH 7.5) 20Mm, EDTA 25 Mm, SDS 2%(w/v) and Proteinase K 0.1 μ g/ μ l;
DNA extraction reagent is phenol: chloroform: primary isoamyl alcohol (25:24:1)
(2) double PCR primer:
Trypanosoma evansi and trypanosoma lewisi specific diagnostic gene are chosen in comparison on GeneBank, respectively RoTat 1.2 VSG genes and 18S rRNA gene;
Trypanosoma evansi (the amplified fragments size is 482bp):
TE-a：5'CGGGTCGTCTGCTAAAGT3'
TE-s：5'GCCCGCAGTTGCCTAT3'
Trypanosoma lewisi (the amplified fragments size is 261bp):
TL-a：5'GAGCTCAAGCGGCAGGTTA3'
TL-s：5'?TGCGCGAGAAGAGAGGACT?3'
(3) PCR reaction solution contains 4 kinds of dNTPs that react each 100-300 μ M of final concentration, the primer of each 10-100pmol/ μ l of reaction final concentration, the Mg2+ concentration of 1.5-4.5mM; Archaeal dna polymerase, the sterilization distilled water;
(4) positive control is Trypanosoma evansi DNA and trypanosoma lewisi DNA, and relatively whether PCR product and monitoring PCR operating process be correct;
(5) negative control: get the negative check sample of sterile purified water;
(6) assembling test kit.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103278642A (en) * | 2013-05-15 | 2013-09-04 | 广西大学 | Double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting trypanosoma evansi and preparation method thereof |
| CN106868121A (en) * | 2017-02-15 | 2017-06-20 | 中山大学 | A kind of primer pair and kit for detecting trypanosoma lewisi |
| CN107058502A (en) * | 2017-02-15 | 2017-08-18 | 中山大学 | A kind of primer pair and kit for detecting mouse trypanosome |
| CN107217093A (en) * | 2017-05-22 | 2017-09-29 | 中山大学 | A kind of primer pair and its detection method and kit for detecting Trypanosoma evansi |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101935716A (en) * | 2010-09-10 | 2011-01-05 | 南京农业大学 | Double detection method of porcine reproductive and respiratory syndrome virus and bovine viral diarrhea virus from pig |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101935716A (en) * | 2010-09-10 | 2011-01-05 | 南京农业大学 | Double detection method of porcine reproductive and respiratory syndrome virus and bovine viral diarrhea virus from pig |
Non-Patent Citations (2)
| Title |
|---|
| IMADELDIN E ARADAIB ECT.AL: "A simple and rapid method for detection of trypanosoma evansi in the dromedary camel using a nested polymerase chain reaction", 《KINETOPLASTID BIOLOGY AND DISEASE》, 20 March 2006 (2006-03-20), pages 1 - 6 * |
| 石玉良等: "二重PCR快速检测牛伊氏锥虫和牛巴斯虫的研究", 《广西农业科学》, 28 February 2010 (2010-02-28), pages 163 - 166 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103278642A (en) * | 2013-05-15 | 2013-09-04 | 广西大学 | Double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting trypanosoma evansi and preparation method thereof |
| CN106868121A (en) * | 2017-02-15 | 2017-06-20 | 中山大学 | A kind of primer pair and kit for detecting trypanosoma lewisi |
| CN107058502A (en) * | 2017-02-15 | 2017-08-18 | 中山大学 | A kind of primer pair and kit for detecting mouse trypanosome |
| CN107058502B (en) * | 2017-02-15 | 2020-07-03 | 中山大学 | Primer pair and kit for detecting mouse trypanosomes |
| CN106868121B (en) * | 2017-02-15 | 2020-11-06 | 中山大学 | Primer pair and kit for detecting trypanosoma lewisi |
| CN107217093A (en) * | 2017-05-22 | 2017-09-29 | 中山大学 | A kind of primer pair and its detection method and kit for detecting Trypanosoma evansi |
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