CN106319046A

CN106319046A - Nested PCR (polymerase chain reaction) detection kit for 11 types of rabbit Eimeria

Info

Publication number: CN106319046A
Application number: CN201610695420.5A
Authority: CN
Inventors: 宫鹏涛; 林永超; 张西臣; 李巍; 李建华; 杨举; 李�赫; 李棕松
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2016-08-22
Filing date: 2016-08-22
Publication date: 2017-01-11

Abstract

The invention aims to disclose a nested PCR (polymerase chain reaction) detection kit for 11 types of rabbit Eimeria. The nested PCR detection kit is characterized in that with an ITS-1 sequence of 18S rRNA gene of the Rabbit Eimeria as a target sequence, a pair of genus-specific nested PCR primers are designed, 11 pairs of species-specific nested PCR primers are designed for each Eimeria body, and a nested PCR detection method is established and used for the detection of 11 types of rabbit Eimeria in a clinical fecal sample. The nested PCR detection kit is rigorous in design, easy to operate, high in flexibility, high in specificity, convenient to operate in batch, capable of identifying infections of specific types of rabbit Eimeria and accurate and objective in result judgment.

Description

A kind of for 11 kinds of rabbit eimeria tenella nest type PCR detection reagents

Technical field

The present invention provides a kind of for 11 kinds of rabbit eimeria tenella nest type PCR detection reagents, for 11 kinds of variety classeses Rabbit eimeria tenella infect detection, the invention also discloses above-mentioned 11 kinds of rabbit eimeria tenella nest type PCR detection reagents Preparation method, belong to technical field of molecular biological detection.

Background technology

The features such as rabbit is little due to volume, and reproductive capacity is strong are frequently as laboratory animal grinding for veterinary and physianthropy Study carefully.It cultivates one of most commonly seen disease is exactly coccidiosis, the most especially infects most commonly seen with eimeria tenella.In the world, rabbit The kind of eimeria tenella has 11 kinds more than, and is easy to produce new drug resistance strain, and therefore this is also that rabbit coccidiosis is difficult to control Treating and prevention, morbidity is not easy to the main cause controlled.Life cycle with other coccidiosiss is the same, and rabbit coccidiosis is divided into non-Sporulated ovum Capsule and Sporulated Oocysts.The detection method of rabbit coccidiosis has multiple, as Microscopical Method For Detection, ELISA(include Salmonella and indirectly ELISA), Standard PCR detection etc..

But above method otherwise observation difficulty, be not easy to batch experiment (such as Microscopical Method For Detection), or the element to operator Matter requirement is higher, intuitive is strong (such as ELISA), or the susceptiveness of detection, specificity the highest (Standard PCR).

Excrement sample genome is one of sample being easiest to collection, and genomic DNA stability is strong, polymerase chain reaction (Polymerase Chain Reaction, PCR) is the most highly sensitive and easily operated DNA detection technique.Due to Fecal sample complicated component, wherein contains a large amount of antibacterial, uses regular-PCR method often to have non-specific bar when detecting Band or false positive results occur.The most general feces genome extracts test kit, in the process coccidiosis Sporangium of sample DNA extraction the most thorough.

Summary of the invention

It is an object of the invention to open a kind of for 11 kinds of rabbit eimeria tenella nest type PCR detection reagents, with rabbit Amy Your coccidiosis 18S rRNA gene ITS-1(internal transcribed space-1) sequence is target sequence, designs a pair Belong to specificity nested PCR primer, then for each polypide design 11 to species specificity nested PCR primer, set up Nested PCR detection method, the detection of 11 kinds of rabbit eimeria tenellas in clinical excrement sample.

Firstly for coccidiosis genome due to the existence of its egg capsule, it is the most inadequate that common genomic kit extracts situation Preferable.Therefore for its feature, experimental implementation is improved, in fecal specimens, i.e. adds the bead of 0.4-0.6mm, And utilize liquid nitrogen to carry out multigelation 4-6 time in sample after adding lysate.

11 kinds of rabbit eimeria tenella Chao Shi PCR detection kit disclosed by the invention, may be used for industrialized production.

11 kinds of rabbit eimeria tenella test kits of the present invention are mainly made up of following different materials and reagent:

(1) diameter 0.4-0.6mm bead；

(2) PCR reactant liquor: 2.5mM dNTPs, the forward primer of final concentration of 10pmol/ μ L and downstream primer, 10 × PCR Buffer, rTaq DNA polymerase, DNA template and ddH₂O.In addition to DNA profiling, it is frozen that other reagent can be formulated as Mix liquid In-20 DEG C of long-term storage；

(3) 11 kinds of rabbit eimeria tenella specific primers:

First round PCR primer:

ITS-1F:GGGAAGTTGCG；

ITS-1R: CTGCGTCCTTCATCGAT；

Second to take turns PCR primer as follows:

Table 2

(4) comparison: positive control is Si Shi eimeria tenella worm kind, negative is ddH for comparison₂O, be used for comparing PCR primer with And monitoring PCR operating process is the most correct.

11 kinds of rabbit eimeria tenella nest type PCR detection reagents of the present invention can also contain agarose (Agarose), bromine Phenol indigo plant spotting buffer and rTaq enzyme, its concentration is preferably ethidium bromide 10 μ g/ μ l；Bromophenol blue spotting buffer.The most all right Containing PCR reaction tube.

The detection method of 11 kinds of rabbit eimeria tenella nest-type PRC detection kit of the present invention, including following step Rapid:

1) genome extracts, and in addition to normal use feces genomic kit, also tackles every part of sample and adds 200mg bead So that coccidiosis Sporangium is crushed, and adding make after GSL (DNA lysate) liquid sample at liquid nitrogen and boiling water anti- Multiple freeze thawing more than 5 times.

2) PCR amplification:

(1) preparing PCR reaction tube the difference labelling of measuring samples number+2, (DNA template is divided to add PCR reactant liquor in pipe Wei measuring samples, negative control and positive control), brief centrifugation after vortex mixing, stand-by.

(2) first round PCR reaction condition is: 95 DEG C of denaturations 5min, 94 DEG C of degeneration 1min, 44 DEG C of annealing 30s, 72 DEG C extend 1min, totally 33 circulations, rear 72 DEG C extension 8min.

(3) can be as all second reaction template taking turns PCR using first round PCR product, second takes turns PCR reaction condition For: 95 DEG C of denaturations 5min, 94 DEG C of degeneration 1min, corresponding annealing temperature (being shown in Table) annealing 30s, 72 DEG C of extension 1min, Totally 35 circulations, latter 72 DEG C extend 10min.

(table one)

Worm kind	Stripe size (bp)	Annealing temperature (DEG C)
			E.sti	217	54
E.fla	196	53
			E.med	154	50
E.coe	256	53
			E.int	241	54
E.irr	226	39
			E.mag	218	46
E.per	157	44
			E.vej	166	49
E.piri	289	42
			E.exi	280	39

3) PCR product is observed:

Preparing 2% agarose gel, electrophoresis 20-30 min under 100V-120V voltage after sample-adding, afterwards at uviol lamp or gel Observation experiment result under imaging system, if the amplified band of the corresponding size of sample existence turns out this sample and has infected accordingly Rabbit eimeria tenella (the worm kind obtained is shown in accompanying drawing 1～Figure 11).

The positive effect of the present invention is: first on genome extracts, due to the use of bead and multigelation Application, is greatly improved the extraction efficiency of coccidiosis genome.The 11 kinds of rabbit eimeria tenella ITS-1 gene diagnosises additionally provided Sequence, according to this primers, improves its sensitivity by optimized expansion condition, and amplification knot is directly observed in electrophoretic analysis Really.11 kinds of rabbit eimeria tenella nest type PCR detection reagent designs of the present invention are rigorous, and simple to operation, susceptiveness is high, specificity By force, and being easy to batched operation, can identify the specific kind of infection of rabbit eimeria tenella, it is the most objective that result judges.

Accompanying drawing explanation

Fig. 1～Figure 11: the 11 kinds of eimeria tenellas second respectively detected take turns the specific band of PCR primer；

Figure 12: Si Shi eimeria tenella specificity verification；

Figure 13: Si Shi eimeria tenella sensitivity checking.

Detailed description of the invention

The following example is further intended to illustrate rather than limit the present invention.It will be appreciated by those skilled in the art that Arriving, on the premise of without departing substantially from the spirit and principles in the present invention, any parallel change and change to the present invention fall within this In the scope of the appended claims of invention.

Embodiment 1 rabbit eimeria tenella specific gene selects

Rabbit eimeria tenella worm kind specific diagnostic sequence: select the ITS-1 gene sequence with well-conserved 18S rRNA Row, in NCBI, it is peculiar for eimeria tenella in comparison, and various coccidiosis has diversity in this section, meets the special inspection of kind Survey research standard.

Embodiment 2.nestPCR specificity is tested:

The purpose one of experiment is to distinguish the difference of rabbit eimeria tenella and other pathogen infections, and it two is to examine respectively Measure different 11 kind eimeria tenella.Therefore common parasitic worm DNA sample (giardia lamblia, neospora, and arch are chosen Worm) carry out specific detection as comparison.As a example by Si Shi eimeria tenella, result is as follows: method can accurately detect rabbit Eimeria tenella, and be Si Shi eimeria tenella, other worm kinds (giardia lamblia G, neospora N, isospora I, grow by toxoplasma speed Sub-G.t, toxoplasma bradyzoite G.b) utilize this detection method can not show particular bands.

The sensitivity test of embodiment 3.nestPCR:

Calculate every μ l gained coccidiosis number, final result below figure by Maxwell counting method, i.e. at least contain 100 at every μ l The existence of rabbit eimeria tenella can be successfully be detected in the case of coccidiosis.

The optimization of example 4.PCR reaction condition

The first round arrange the annealing temperature of primer be respectively as follows: 40 DEG C, 41 DEG C, 42 DEG C, 43 DEG C, 44 DEG C, 45 DEG C, 46 DEG C, 47 DEG C, 48 DEG C, 49 DEG C, 50 DEG C, experiment find 44 DEG C of annealing temperatures optimal.

E.sti second take turns arrange primer annealing temperature be respectively as follows: 50 DEG C, 51 DEG C, 52 DEG C, 53 DEG C, 54 DEG C, 55 DEG C, 56 DEG C, 57 DEG C, 58 DEG C, 59 DEG C, 60 DEG C, experiment finds 54 DEG C of (≈ (Tm_Upstream+Tm_{Lower downstream})/2-5 DEG C) annealing temperature is optimal.

Second annealing temperature taking turns primer calculated according to the first round, set respectively according to upstream and downstream primer Tm meansigma methods-5 DEG C Put other kind eimeria tenellas second takes turns PCR annealing temperature.

Example 5. sample detection

The fecal specimens gathering the 154 portions of rabbit in In Changchun County detects.It is sampled detection, at mirror with saturated saline floatation 20 parts of samples of inspection find 11 parts of typical coccidiosis structures.

Use nested PCR method to detect whole samples, remove 5 parts and extract failed sample, remain 149 parts of samples and be found that 85 parts of positive.

Test example 1

The composition of test kit

Conventional excrement sample genome extracts reagent, bead；PCR reactant liquor 10 is managed (20 μ l system), wherein the concentration of respective dNTPs Being 250 μMs, forward primer, downstream primer, inner side forward primer, the final concentration of inner side downstream primer is respectively 10 pmol/ μ l, Mg²⁺ Final concentration of 1.5 mM；4g agarose；50 μ l ethidium bromides (20 μ g/ μ l)；0.5% bromophenol blue spotting buffer 50 μ l；10μl Taq enzyme (5U/ μ l)；1 part of rabbit eimeria tenella genomic DNA positive control.

Test 2

Test kit specific test

Take Si Shi eimeria tenella (Eimeria stidia), Lan Shi giardia lamblia(Giardia lamblia), neospora(Neospora caninum), isospora (Isospora), bow Shape worm(Toxoplasma gondii) tachyzoite (tachyzoite) and bradyzoite (bradyzoite), Respectively it is expanded with the amplification system set up.

Negative and positive control are separately added in the pipe that labelling is good, then sample are sequentially added into and labelling is good, be placed in In PCR instrument.First round PCR condition is: 95 DEG C of denaturations 5min, 94 DEG C of degeneration 30s, 44 DEG C of annealing 30s, 72 DEG C of extension 30s, Totally 30 circulations, latter 72 DEG C extend 10min；Second takes turns PCR condition is: 95 DEG C of denaturations 5min, 94 DEG C of degeneration 30s, according to table In lattice, annealing temperature sets corresponding 30s, 72 DEG C of extensions 30s, totally 30 circulations, and latter 72 DEG C extend 10min. amplified productions use The agarose of 2% carries out electrophoretic analysis, observed result on gel imaging system.

Result shows (see accompanying drawing 12): only have Si Shi eimeria tenella can present respective strap, other sample standard deviation without Amplification, meets specific detection standard.

Test 3

Test kit sensitivity tests

Maxwell counting method is used to collect Si Shi eimeria tenella 5.75 × 10⁶/mL.Extract 1ml coccidiosis all DNA, and use 57.5 μ The TE of L collects.It is 1.6 × 10 by the content in concentration dilution to first reaction system respectively⁴、8000、4000、2000、 1000、100、10、1.Reaction condition homospecificity detection embodiment, sets negative control simultaneously.Product is carried out with the agarose of 2% Electrophoretic analysis, observed result on gel imaging system.Result shows that threshold value is 100, meets sensitivity Detection standard (see accompanying drawing 13).

Test example 4

The stability of test kit and replica test

Positive template, PCR reactant liquor, rTaq enzyme stores under the conditions of-20 DEG C, the 4 DEG C of preservations of other article such as lysate, bromophenol blue ?.When period of storage be 30 days, 60 days, 100 days, 150 days, 200 days time take out each component, with the positive sample of known PCR Detect 11 kinds of rabbit eimeria tenella nest type PCR detection reagent stability.It addition, 10 parts of PCR positive sample are with 11 kinds of rabbit Amys You repeat to test 3 times by coccidiosis nest type PCR detection reagent, and 15 parts of PCR negative sample are repeated 3 times equally, the most front institute of amplification condition State.Result shows that the component taken out at above day part has all obtained list in known PCR positive and 15 parts of PCR positive sample The purpose band that one becomes clear special, and the negative sample of blank is all without any amplified band, so stabilization of kit and weight Renaturation is good.

Example 5 is executed in examination

The shelf-life test of test kit

11 kinds of rabbit Amys thats of 1 month, 2 months, 3 months, 4 months, 5 months and 6 months will be stored respectively at 4 DEG C and-20 DEG C Coccidiosis nest type PCR detection reagent takes out and detects known positive.Amplification and inspection method are the same, and result shows Under the conditions of 4 DEG C and-20 DEG C, save to 11 kinds of rabbit eimeria tenella nest type PCR detection reagents as long as October still can expand Go out purpose band clearly, without miscellaneous band.

Sequence table 1

<110>Jilin University

<120>

<140>

<160> 1

<210> 1

<211> 217

<212> DNA

<213>Si Shi eimeria tenella (E.stiedai)

<400> 1

1 GTGGGTTTTC TGTGCCCTCC CCCACCATGG GTCGGTTCGG TCACCTCTGC ATTTTCCAAC

61 CTTTGAATCT TTTCTCACTC ACTCTACAAC GGATTTCTTG TAAGGCGGCT ATTTTTTATG

121 TGGTCTGTCT TTCTTTCTGC ATTGTTTTTG TTCCAGTAAA TTGGAACGAA AGTGAGGAGG

181 CGGATGGACG TGCTGAATGA AGCAAAGCAG CAGCCTT

Sequence table 2

<110>Jilin University

<120>

<140>

<160> 1

<210> 1

<211> 196

<212> DNA

<213>yellow eimeria tenella (E.flavescens)

<400> 1

1 GAATATTGTT GCAGTTTACC ACCAATGGGG TTACTTTGTC ACCTTTCTCAA CTCTAGAATC

61 TGTTTTTTTC TTTTCTCCAA CGACGCTTTT TCTTTTTCTC ATTGAAGTTTT TTTTTTCCTC

121 TTCCAACCGC ATAAACTTTT TTTTGTTCCA TCATTCCGAT GGAATAAGGGG AAGATTATGA

181 AGAACGGTTG TTGAGG

Sequence table 3

<110>Jilin University

<120>

<140>

<160> 1

<210> 1

<211> 154

<212> DNA

<213>medium-sized eimeria tenella (E.media)

<400> 1

1 GATTTTTTTC CACTGCGTCC CTGTTTTTCA GTATAGTGG GAAAGAGGTG AGGCAGTTGG

61 GTGTGTAAAA GCATCTTTCG CCCATAGGTC ACCACCACC TGTTGGTGGT GATACTTGGG

121 TGATAGAAGC TTTTTTTACC TTTTCTGTTA TGAA

Sequence table 4

<110>Jilin University

<120>

<140>

<160> 1

<210> 1

<211> 256

<212> DNA

<213>caecum eimeria tenella (E.coecicola)

<400> 1

1 AGCTTGGTGG GTTCTTATTA TTGTACTCTA TCTATCCCAC CACTACTACT ACGGTTCATC

61 ATATTCACCT CCTGCTCTCC ATTTTCCAAC TTTTGAATCT CTCTTTTCAC TTCACAACGA

121 TTTTTTTATA GAAGAAGCCT TGTTATTTGG TTTCTTTCTT CTACTTTTAT GTCTGCATTC

181 TTTTCTTTCC AATAAATTAT TGGGATGAAA TTGAGGCAGA TATGGTATGA TGATATGGAT

241 TTGTTGAAGC AACTAG

Sequence table 5

<110>Jilin University

<120>

<140>

<160> 1

<210> 1

<211> 241

<212> DNA

<213>intestinal eimeria tenella (E.intestinalis)

<400> 1

1 TGTTTGTACC ACCGAGGGAA TAACCTTTGC ATTTTCGAAC TCTTGAATCT TTTTTTCACT

61 CTACAACGAT TTTCGAAAGT ATGCTATTTG ATCTAATTTC TCCTTCTGCA CACTTTTTTT

121 TTCCAGTAAA ATGGAATAAT GGAAGTGTGG CAGGTGGATG TATTGAGAAG CGACAGACTC

181 GCCCGGGAAT AACCAGCGTC AAAATGCTGG TTTTGCTGGA TGAGGAGGGT AGCTTAATGT

241 T

Sequence table 6

<110>Jilin University

<120>

<140>

<160> 1

<210> 1

<211> 226

<212> DNA

<213>without residual eimeria tenella (E.irresidua)

<400> 1

1 TTTGGTGGGA AAAGATGATT CTACTACACT TAAAAGGCCA AAAGTTCCTT CTACTTTTGG

61 AGCTGTGGTA GGGTCGTCAG AGTACCGCCG GTGAGATCAC CTCGATATCA CTTCCAACTC

121 TTGAATCCTT TCTCCAACCT CCTCAACGTG TTCTCTTTTA CTTTAATTAA TATTAAGGTA

181 TTCATTTACC TTGATGCCAT TTTGAATGGG TTAAAAATAA TGCAAA

Sequence table 7

<110>Jilin University

<120>

<140>

<160> 1

<210> 1

<211> 218

<212> DNA

<213>large-scale eimeria tenella (E.magna)

<400> 1

1 TTTACTTATC ACCGAGGGTT GATCACCTAT GCATTTTGCC ACACTTTTGA ATCTTTTTCC

61 ATTCCACAAC GATTTTAAGG CCCACTGCAT CCTTCTTTCC CTCAGTACAC AGTGGAATAG

121 AAGTGAGGTA GCTGGGTGTG TAAAAGCATC TTTTGCCCGC AAGTAACCGC CTGTTGGCGG

181 TGAAAGGTGG GTGGCGGTGG TAAGCTTTAC CTTTCTCG

Sequence table 8

<110>Jilin University

<120>

<140>

<160> 1

<210> 1

<211> 157

<212> DNA

<213>perforation eimeria tenella (E.perforans)

<400> 1

1 TTTTATTTCA TTCCCATTTG CATCCCTTTT TTTCAGTATA TTGATTGGAA AAGAGGTGAG

61 GCAGTTGGGT GTGTGAAAGC ATCTCTCACC CAAAGGTGAC CTCCTGTCGG TGGTGACAGT

121 TGGGTGACAG AAGCTTGACC TTTTCTGTTA TGAAAAG

Sequence table 9

<110>Jilin University

<120>

<140>

<160> 1

<210> 1

<211> 166

<212> DNA

<213>Vickers eimeria tenella (E.vejdovskyi)

<400> 1

1 GTGCTGCCAC AAAAGTCACC GCTGAATTTT CCAACTTTTG AATGTTTTTT CACCCTACAA

61 CGATTTTCTA AGCTCTGCTA TTTGATTGGT TTTCTGTTCT ACCTGTGTCC TCTTTTTTTT

121 CCAGTAACTT GGAATGAAAG GTGAGGCGGG CGGAATGAAT TGTAGC

Sequence table 10

<110>Jilin University

<120>

<140>

<160> 1

<210> 1

<211> 289

<212> DNA

<213>pyriform eimeria tenella (E.piriformis)

<400> 1

1 ACGAATACAT CCCTCTGCCT TACCCTTTAA TTGGCTGAAA GCAGCTTCTA TTTTGAGGTG

61 TGGCTTTCTG GGGCCGGTTG GGGTATGGGT GGGATTGTTG CAGTGTACCA CCTCTATGGG

121 GTTACTTTGT CACCTTTCTC AACACTAGAA TCTGTTTTCT TTTTCTCTCC ATCGACGCGT

181 TTTCTTTTTC TCAATGAAGT TTTTTTTTTT CTCTTCAAAC CGCATAAACT TTTGTCTTCC

241 ATTACTCCGA TGGAATAAGT GGAAGAATGG TTGTGCAGGG GGAGACAAT

Sequence table 11

<110>Jilin University

<120>

<140>

<160> 1

<210> 1

<211> 280

<212> DNA

<213>small-sized eimeria tenella (E.exigua)

<400> 1

1 GAATAAGTTC TGCCTAAAGA GAGCCTTCTA ATTGGACGAA AGTTCTACAC TAGTTGGTGT

61 GGGTCTTTTG GGGCTGATTG GGGCTTTGGG GCTGGGGCTT GTTCGACGAT CACCCTATCA

121 CAATTCTCAA CTTTTGAATC GTTTTTTCTT TTCTCCATCG ACGTATTTTT CTTTGATTTT

181 AGCGATAATT TTCCTTTTTT CTCTTGGAAC CGTTAAAGAT CTTTTCGCCC AAGTCAAAAT

241 ACCAACTGGT TTTTGAGGTG GGTTGGGGAT GGTCTATATA。

Claims

1. one kind is used for 11 kinds of rabbit eimeria tenella nest type PCR detection reagents, it is characterised in that be made up of following raw material:

1) diameter 0.4-0.6mm bead；

2) PCR reactant liquor: 2.5mM dNTPs, the forward primer of final concentration of 10pmol/ μ L and downstream primer, 10 × PCR Buffer, rTaq archaeal dna polymerase, DNA profiling and ddH₂O；In addition to DNA profiling, other reagent can be formulated as Mix liquid frozen in- 20 DEG C of long-term storage；

3) 11 kinds of rabbit eimeria tenella specific primers:

First round PCR primer:

ITS-1F:GGGAAGTTGCG；

ITS-1R:CTGCGTCCTTCATCGAT；

Second to take turns PCR primer as follows:

4) comparison: positive control is Si Shi eimeria tenella worm kind, negative is ddH for comparison₂O, be used for comparing PCR primer and Monitoring PCR operating process is the most correct.

11 kinds of rabbit eimeria tenella nest type PCR detection reagents the most as claimed in claim 1, it is characterised in that: can also contain Having agarose (Agarose), bromophenol blue spotting buffer and rTaq enzyme, its concentration is preferably ethidium bromide 10 μ g/ μ l；Bromophenol blue Spotting buffer.

The detection method of 11 kinds of rabbit eimeria tenella nest type PCR detection reagents the most as claimed in claim 1, including following step Rapid:

1) genome extracts, and in addition to normal use feces genomic kit, also tackles every part of sample and adds 200mg bead So that coccidiosis Sporangium is crushed, and adding make after GSL (DNA lysate) liquid sample at liquid nitrogen and boiling water anti- Multiple freeze thawing more than 5 times；

2) PCR amplification:

(1) preparing PCR reaction tube the difference labelling of measuring samples number+2, (DNA profiling is respectively to add PCR reactant liquor in pipe Measuring samples, negative control and positive control), brief centrifugation after vortex mixing, stand-by.

(2) first round PCR reaction condition is: 95 DEG C of denaturations 5min, 94 DEG C of degeneration 1min, 44 DEG C of annealing 30s, 72 DEG C of extensions 1min, totally 33 circulations, latter 72 DEG C extend 8min.

(3) can be as all second reaction template taking turns PCR using first round PCR primer, second takes turns PCR reaction condition is: 95 DEG C Denaturation 5min, 94 DEG C of degeneration 1min, corresponding annealing temperature (being shown in Table) annealing 30s, 72 DEG C of extensions 1min, totally 35 circulations, Latter 72 DEG C extend 10min.

Worm kind Stripe size (bp) Annealing temperature (DEG C) E.sti 217 54 E.fla 196 53 E.med 154 50 E.coe 256 53 E.int 241 54 E.irr 226 39 E.mag 218 46 E.per 157 44 E.vej 166 49 E.piri 289 42 E.exi 280 39

3) PCR primer is observed:

Prepare 2% agarose gel, electrophoresis 20-30min under 100V-120V voltage after sample-adding, become at uviol lamp or gel afterwards As observation experiment result under system, if the amplified band of the corresponding size of sample existence turns out this sample and infected corresponding rabbit Eimeria tenella.