CN107653332A

CN107653332A - Primer, kit and method for Chlamydia detection

Info

Publication number: CN107653332A
Application number: CN201711052888.3A
Authority: CN
Inventors: 周继章; 李兆才; 娄忠子; 费媛媛
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2017-10-30
Filing date: 2017-10-30
Publication date: 2018-02-02

Abstract

One kind is based on the animal chlamydia detection method of recombinase polymeric enzymatic amplification technology (RPA), this method is using the specific binding of recombinase and single strand binding protein cooperative achievement primer and template at normal temperatures, and extension primer generates new DNA in the presence of archaeal dna polymerase.It is a feature of the present invention that according to chlamydia trachomatis gene envB sequences Designs and having screened a set of primer detected available for RPA and probe combinations, a kind of animal chlamydia specificity fluorescent RPA detection methods are established.The Chlamydia specificity fluorescent RPA detection methods of the present invention are easy to operate, quick, high specificity, suitable for field quick detection.

Description

Primer, kit and method for Chlamydia detection

Technical field

It is used to detect the primer and unrelated probe of animal chlamydia, and detection kit the present invention relates to a kind of, definitely Say be it is a kind of detect specificity fluorescent recombinase polymeric enzymatic amplification technology (RPA) detection primer of animal chlamydia, kit, this Invention further relates to carry out the detection side of the animal chlamydia disease of non-disease diagnosis with probe or kit using the primer of the present invention Method.

Background technology

Chlamydia (Chlamydia) is the entozoic gramnegative bacterium of strict born of the same parents, 0.2~0.5 μm of diameter, between vertical Gram time between body and virus, it can only can be grown by 0.45 μm of filter membrane in host cell, it is impossible to as most of bacterium It is grown on agar plate.Chlamydia cause of disease has extensive pathogenic, and in recent years, chlamydiosis is more tight in China's prevalence Weight, especially milk cow, yak, sheep, goat, pig etc., seriously hinder the further development of cow estate, Yak production etc., And cause huge economic loss.In addition, as a kind of important zoonosis, the disease can cause that animal and people's is more Kind disease, infection people can cause trachoma, pneumonia, disease in the urological system etc..Therefore, timely, discovery Chlamydia early, and use Sensitive, special diagnostic method, in Accurate Diagnosis animal miscarriage preferendum chlamydiosis, there is provided the diagnosis of science bridle chlamydiosis Foundation, promote aquaculture to develop in a healthy way, ensure food security, protect people's health aspect particularly important.

For many years, mainly resist both at home and abroad for the diagnostic method of chlamydiosis including the detection of CHLA Casset, Chlamydia Physical examination survey and molecular biology for detection.Antigen detection mainly includes the dyeing of Ji's nurse Sa, the inspection-free survey (EIA) of enzyme and separated The inspection-free diagnostic method of surveying of culture, wherein enzyme is applied to batch detection, and its shortcoming is that specificity is bad, and it is still one to be separately cultured Indispensable chlamydia diagnosis method, while it is difficult culture that many bacterial strains, which are, common there is also mycoplasma contamination is asked Topic.Antibody testing method mainly includes indirect hemagglutination test (IHA), complement fixation test (CFT) (CFT), EUSA And micro indirect immunofluorescence assay (MIF) (ELISA).Wherein, indirect hemagglutination diagnostic test and complement fixation test (CFT) this two The features such as kind of method is respectively provided with low sensitivity, poor specificity, result judge strong subjectivity and unsuitable mass detection, limits Their extensive uses in clinic.ELISA diagnosises are with for the more of liopopolysaccharides antigen, but chlamydial LPS class resists Former some epitopes are similar to other gramnegative bacteriums, cross reaction and false positive easily occur.Such as prior art It is to recombinate Chlamydia that CN201110367644.0, which discloses a kind of ELISA kit for detecting swine Chlamydophila abortus antibody, Major outer membrane protein rMOMP is envelope antigen, and in the prior art to recombinate polymorphism outer membrane protein POMP as envelope antigen The swine Chlamydophila abortus disease indirect ELISA antibody detection method of foundation, but these prior arts are to be directed to specific one The ELISA antibody indirects detection method or kit of kind animal exploitation, narrow application range, without versatility, are not used to more The Chlamydia checkout and diagnosis of kind animal, use cost are higher, it is difficult to popularize.Micro indirect immunofluorescence assay is once proposed as examining The most reliable serological method of disconnected CPN, but the diagnostic method technical requirements are high, time-consuming, therefore do not turn into The conventional detection project of CPN.Molecular biology for detection mainly includes nucleic acid hybridization, PCR detection methods and ring Mediated isothermal amplification technology (LAMP).

RPA technologies are by being established with Britain Camb Babraham research institutes and being invented by TwistDx biotech companies (http://www.twistdx.co.uk/).The technology is former needed for reaction using the nucleic acid replication mechanism of T4 bacteriophages as principle Material has restructuring zymoprotein uvsX and uvsY, single strand binding protein gp32, archaeal dna polymerase and the oligonucleotides of T4 phage encodeds Acid；Also need to primer, probe, template, ddH simultaneously₂O and Mg²⁺Deng.The technology is former based on the polymerase-mediated amplification of recombinase Reason, DNA replication dna in organism is simulated, can carry out isothermal duplication to target fragment at normal temperatures, break away to thermal cycler instrument Requirement, purpose fragment can be gone out by rapid amplifying in a short time, have simplicity, quickly, it is sensitive the advantages that.

The content of the invention

The technical problems to be solved by the invention are to provide：One kind can overcome prior art insufficient, for detecting animal clothing The primer pair of substance specific detection；A kind of primer pair sequence fluorescence recombinase polymeric enzymatic amplification for animal chlamydia detection Technology (RPA) detection probe sequence；One kind is used to detect the specific detection kit of animal chlamydia；A kind of method.It is a kind of For animal chlamydia fluorescence recombinase polymeric enzymatic amplification technology (RPA) detection kit；And for animal chlamydia disease Detection method and fluorescence recombinase polymeric enzymatic amplification technology (RPA) detection method.

The primer pair sequence for being used for animal chlamydia detection of the present invention is SEQ ID No.2 and SEQ ID No.3.

The probe sequence for being used for animal chlamydia RPA detections of the present invention, it is characterised in that be SEQ ID No.4.This hair Include the primer pair that sequence is SEQ ID No.2 and SEQ ID No.3 in the bright kit for animal chlamydia detection.

It is SEQ ID No.2 and SEQ ID that the present invention, which is used to include sequence in the kit of animal chlamydia RPA detections, No.3 primer pair, and the probe sequence that sequence is SEQ ID No.4.

The non-disease of the present invention diagnoses animal chlamydia detection method：Genomic DNA by animal sample to be detected is Template, expanded with SEQ ID No.2, SEQ ID No.3, and according to whether there is particular bands determination in amplified production electrophoresis Whether detected animal has choamydiae infection.

The non-disease of the present invention diagnoses animal chlamydia fluorescence recombinase polymeric enzymatic amplification technology (RPA) detection method： It is template by the genomic DNA of animal sample to be detected, is carried out with SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 Amplification, signal detection is carried out using quantitative real time PCR Instrument it is determined that being detected, and determined according to obvious whether change of the upper curvature of detection Whether tested animal has choamydiae infection.

The method of invention has high detection sensitivity, and detection method is relatively simple, especially with the present invention's During animal chlamydia fluorescence recombinase polymeric enzymatic amplification technology (RPA) detection method, with more easy, fast and effectively advantage, Prevention and control for animal chlamydia are significant.

RPA technologies are applied in a series of Molecular Detection of pathogenic microorganisms at present, as mycobacterium tuberculosis, Foot and mouth disease virus, canine parvovirus, African swine fever etc..But have no application of the RPA technologies in Chlamydia detection.Therefore, originally Invention establishes a kind of simplicity, fast and effectively Chlamydia RPA detection methods, the prevention and control for Chlamydia are significant.

Brief description of the drawings

Fig. 1 is that the PCR of primer is verified：M is DL2000 DNA marker；1~6 is the PCR amplifications of 6 pairs of RPA amplimers As a result.

Fig. 2 is that the RPA of primer is verified：M is DL2000DNA marker；1st, 3,6 be 3 pairs of primers Basic kit RPA Amplification；—：For negative control；+：For positive control.

Fig. 3 is the specificity analysis of RPA fluorescence detection methods：A:Negative control；B:Positive control；C、D:Two plants of Chlamydia bases Because of a group amplification；E:Brucella genome testing result；F:Toxoplasma cdna group testing result

Fig. 4 is RPA fluorescence detection method sensitivity analysis：A is that template concentrations are 10⁷Copies/ μ L amplification curve；B It is 10 for template concentrations⁶Copies/ μ L amplification curve；C is that template concentrations are 10⁵Copies/ μ L amplification curve；D is template Concentration is 10⁴Copies/ μ L amplification curve；E is that template concentrations are 10³Copies/ μ L amplification curve；F is that template concentrations are 10²Copies/ μ L amplification curve；G is that template concentrations are 10¹Copies/ μ L amplification curve.

Embodiment

It is below the embodiment of the present invention, main material used in the present invention, reagent, food are as follows：

Bacterial strain, carrier and genome：Competent escherichia coli cell DH5 α, DNA Marker and pMD18-T simple are carried Body is purchased from TaKaRa Biotech companies；Chlamydia, brucella and toxoplasma cdna group DNA are that this laboratory is protected Deposit.

Main agents and kit：Methanol, ethanol, isopropanol and glycerine etc. are purchased from the limited public affairs of Tianjin chemical reagent Department；Sodium chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate etc. are purchased from Beijing Chemical Plant；PCR premix enzymes ExTaq, plasmid extraction reagent Box, PCR purifying QIAquick Gel Extraction Kit, glue reclaim kit, pMD18-T carriers connection kit etc. are purchased from TaKaRa Biotech companies.

Key instrument：Pipettor, PCR instrument are purchased from German Eppendorf companies；Electronic balance is Beijing Sai Duosi instruments System Co., Ltd's product；Electro-heating standing-temperature cultivator is purchased from Shanghai precision experimental facilities company；DYY-6C electrophoresis apparatuses, horizontal shaker It is purchased from Liuyi Instruments Plant, Beijing；Electric heating constant temperature tank is the grand experimental facilities Co., Ltd product of upper Nereid, real-time fluorescence quantitative PCR Instrument is Bio-Rad products.

Concrete operations are as follows：

1st, the sequence information of target gene is obtained

Using the outer membrane protein envB encoding genes envB of Chlamydia elementary body (EB) sequence as testing goal gene, from Chlamydia (NC_004552.2) reference sequences are obtained in GenBank databases, and determine envB genes (NC_004552.2: 199142-200499) (1358bp) is SEQ ID No.1 sequence information.

2nd, the design and synthesis of relevant primer

With reference to the rule of bioinformatics software DNAMAN and RPA design of primers, design gene envB is SEQ ID No.1 RPA amplimers and regular-PCR amplimer, send Hua Da gene to be synthesized.Wherein, regular-PCR amplimer Upstream and downstream primer is respectively designated as SEQ ID No.5 and SEQ ID No.6, and as follows, RPA primers need further to be screened.

envBPCRF：5 '-GTTCATTTGCCAGCGGGAAGATAGAGG-3 ' are SEQ ID No.5

envBPCRR：5 '-AGAACCACGGTTTGTTACACAAATACGG-3 ' are SEQ ID No.6

3rd, RPA primers are verified

3.1 regular-PCRs expand

The RPA primers of synthesis are verified using regular-PCR, pig miscarriage chlamydia trachomatis gene group is this experiment needed for experiment Room preserves, and -20 DEG C freeze.

PCR system is as follows：

PCR amplification programs：95 DEG C first fully denaturation 5min；Then 35 circulations, it is respectively：94 DEG C of denaturation 30s；56℃ Anneal 30s；72 DEG C of extensions 45s, last 72 DEG C of extensions 10min.

3.2 PCR amplifications：

Regular-PCR checking is carried out to 6 pairs of primers of synthesis, by screening, as shown in figure 1,6 pairs of primers expand The band of target sizes has been arrived, wherein the 1st, 3,6 pair of primer does not have miscellaneous band to occur in addition to primer dimer, traveling one can be entered Step screening.

3.3 RPA are verified

The pig preserved using laboratory miscarries chlamydia trachomatis gene group as template, is expanded using RPA kits Basickit Checking.

Detailed operating procedures are as follows：

(1), reaction system is added in 1.5ml centrifuge tubes according to following table

Whirlpool concussion mixes；

(2), 47.5ul reaction systems are added in the freeze-dried reaction reaction tubes in kit, used Pipette blows and beats mixing up and down, and the particle into reaction tube all is resuspended；

(3), 2.5ul 280mM MgAc are added in reaction tube lid, cover tightly rear brief centrifugation and the mixing that is vortexed；

(4) reaction tube, is placed in 37 DEG C of incubation 4min；

(5) reaction tube, is taken out from 37 DEG C of reactors, mixing of turning upside down, 37 DEG C of reactors is replaced in after vortex and are incubated Educate 20min；

(6), amplification is reclaimed using DNA purification kits；

(7), take 5ul recovery products to enter row agarose gel electrophoresis, amplification is analyzed.

3.4 RPA amplifications

To the three pairs of RPA primers filtered out by regular-PCR, amplification checking is carried out using RPA kits Basickit, tied As shown in Fig. 2 three pairs of primers have amplified the tune band of target sizes, can carry out next step as RPA amplimers grinds fruit Study carefully experiment.Wherein, the band brightness of the 6th pair of primer and clip size are optimal, therefore, the 3rd pair of primer are set as originally grinding The target primer studied carefully, i.e. SEQ ID No.2 and SEQ ID No.3.

envBRPAF：5 '-CTTCAGGAGCTACAATTTTAGAAGCCGAGGGAGC-3 ' are SEQ ID No.2.

envBRPAR：5 '-TTTCCAATGGGTTGTAGCTTCTGCACAAGAAGTGC-3 ' are SEQ ID No.3.

Wherein, SEQ ID No.2 and SEQ ID No.3 are that upstream and downstream primer expands to template, obtain 215bp mesh Fragment.

4, the design and synthesis of probe

The rule designed with reference to bioinformatics software DNAMAN and RPA probe, with reference to primer SEQ ID No.2 and SEQ ID No.3 extension increasing sequences, design RPA probes, i.e. SEQ ID No.4.There is fluorophor FAM and quenching in probe sequence Group BHQ1, separated by dSpacer between two groups, 3 ' ends are modified by C3Spacer, by raw work bioengineering (Shanghai) stock The synthesis of part Co., Ltd.

envBprobe：5’-CTGAAATCTGCTGTAACAAAGCTGTATGG[dT-Fam]G[dSpacer]A[dT- BHQ1] CAAAGAGATGTGCCCAG [C3-Spacer] -3 ' is SEQ ID No.4.

5 using Exo kit chlamydia specificity fluorescents RPA detections

5.1 specificity fluorescent RPA are detected

Using SEQ ID No.4 as probe, SEQ ID No.2 and SEQ ID No.3 are primer, two plants preserved with laboratory Chlamydia, brucella and toxoplasma cdna group are detection target, with primer combination of probe provisioned in kit with And DNA is control, is expanded according to kit specification, and signal detection is carried out using quantitative real time PCR Instrument.

Detailed operating procedure is as follows：

Whirlpool concussion mixes；

(4), reaction tube is placed in quantitative real time PCR Instrument, 37 DEG C, 20min, 1min collect first order fluorescence signal, collection Amplification curve.

5.2 using SEQ ID No.4 as probe, and SEQ ID No.2 and SEQ ID No.3 are primer, chlamydia, cloth Lu Shi Bacillus and toxoplasma cdna group carry out RPA detections, as a result as shown in Figure 3:It is bent that two kinds of chlamydia trachomatis gene groups generate amplification Line, brucella and toxoplasma cdna group do not produce amplification curve, illustrate that this method has stronger spy to detection Chlamydia The opposite sex.

6, Chlamydia specificity fluorescent RPA detection sensitivity are analyzed

The structure of 6.1 plasmid standards

(1) gene envB pcr amplification primer thing SEQ ID No.5 and SEQ ID No.6 are used, chlamydia genome enters Performing PCR expands,

PCR system is as follows：

PCR amplification programs：95 DEG C first fully denaturation 5min；Then 35 circulations, it is respectively：94 DEG C of denaturation 30s；56℃ Anneal 30s；72 DEG C of extensions 1min, last 72 DEG C of extensions 10min.

(2) PCR primer purifying recovery and construction recombination plasmid standard items

PCR primer is carried out according to TaKaRa Biotech companies PCR primer purification kit explanation, the PCR primer of acquisition Cloned, be cloned on pMD18-Tsimple carriers by T-A,

Obtain recombinant plasmid pMD18-T-envB, as plasmid standard.

(3) plasmid standard concentration mensuration

PMD18-T-envB concentration is measured with protein nucleic acid analyzer, concentration is 49.3ng/ul after measured.

(4) plasmid standard copy number calculates

PMD18-Tsimple carrier empty carriers size is 2692bp, envB gene 1358bp, therefore, PMD18-T-envB Recombinant plasmid size is 4050bp.Plasmid standard copy number is calculated followed by gene copy number calculation formula, it is as follows It is shown, it is computed, the plasmid standard copy number of this research and establishment is about 1.1 × 10¹⁰copies/μL。

(5) doubling dilution of recombinant plasmid

Recombinant plasmid pMD18-T-envB is subjected to 10 times of doubling dilutions, makes its concentration 10⁷~10¹Copies/ μ L it Between, and as the template of RPA reactions.

6.2 sensitivity analysis

(1) the RPA amplifications based on sensitivity analysis

With concentration 10⁷~10¹The mould that recombinant plasmid pMD18-T-envB between copies/ μ L reacts as fluorescence RPA Plate, using SEQ ID No.4 as probe, SEQ ID No.2 and SEQ ID No.3 are primer, and detailed operating procedure is pressed with 5.1 Expanded according to kit specification, and signal detection is carried out using quantitative real time PCR Instrument.

(2) amplification

Using SEQ ID No.4 as probe, SEQ ID No.2 and SEQ ID No.3 are primer, and concentration is 10⁷~ 10¹The template that recombinant plasmid pMD18-T-envB between copies/ μ L reacts as fluorescence RPA, the clothing established to this research Substance specificity fluorescent RPA detections carry out sensitivity analysis, as a result as shown in figure 4, with the reduction of template copy numbers, curve rises Fly the time and fluorescence signal value gradually declines, be 10 in copy number²When still have amplification curve, when copy number be 10¹When be determined as Do not take off, i.e., without obvious amplification curve.

Result above proves, when being 10 table template²And have an obvious amplification curve when copying above, the departure time with The increase of template copy numbers and shorten, fluorescence intensity strengthens with the increase of template copy numbers, meets the requirement of Site Detection.

Sequence table

<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences

<120>Primer, kit and method for Chlamydia detection

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1358

<212> DNA

<213> NC_004552.2:199142-200499 genes ()

<400> 1

gttcatttgc cagcgggaag atagaggccg ctgctgcaga gtctcttgct acaagattca 60

ttgccagtac cgaaaactca aatgacaatg ttttgcaagc aacagccaag aaagttagat 120

ttggtcgtaa caaaaatcaa agacaagaac aaaaacatac tggcgctttc tgtgataaag 180

aattttatcc ttgcgaaggt ggtcagtgcc aatccgtcga tactacacaa gaatcttgct 240

acggcaaaat gtattgtgtc cgtgttaacg atgactgtaa cgtggaaatt agccaagctg 300

tacctgaata tgcaacagta ggatctcctt atcctattga aattcttgct gtaggtaaaa 360

aagattgcgt taatgttgtg atcactcaac aacttccttg cgaagttgag tttgtcagca 420

gtgatcctgc gacaacacca acctcagata gcaaattaat ctggacaatt gattgcttag 480

gtcaaggtga aaaatgcaaa attaccgttt gggtaaaacc tcttaaagaa ggttgttgct 540

tcaccgcggc tactgtatgc gcttgcccag aacttcgctc ttataccaaa tgcggacaac 600

cagctatttg tattaagcaa gaaggccctg agtgtgcttg cttacgttgc ccagtttgtt 660

acaaaatcga agtttgcaac acaggttctg ctatagcccg taatgttgtc gttgataacc 720

ctgttcccga tggctatact cacgcttcag gacaacgcgt tctttccttt aacttaggag 780

atatgcgccc tggggattct aaatgcttct ctgtggagtt ttgcccgcaa aaaagaggaa 840

aaattactaa cgtagctacc gtatcttact gcggaggaca taaatgttcc gcgaacgtaa 900

ctactgtagt taacgaaccc tgcgtacaag tcaatatctc tggagctgac tggtcttatg 960

tatgtaagcc tgtagaatac actatcgttg tgtccaacct aggggatctt aaactttacg 1020

atgtcgttgt agaagatacc gtaccttcag gagctacaat tttagaagcc gagggagctg 1080

aaatctgctg taacaaagct gtatggtgca tcaaagagat gtgcccagga gagactctac 1140

aatttaaagt tgttgctaaa gcacaaagcc caggtaaatt cacaaatcaa gttgttgtca 1200

aaacaaactc cgattgtgga acttgcactt cttgtgcaga agctacaacc cattggaaag 1260

gtctggcagc tactcatatg tgcgtaatcg ataccaatga tcctatttgc gtaggagaaa 1320

atactgtata ccgtatttgt gtaacaaacc gtggttct 1358

<210> 2

<211> 34

<212> DNA

<213>Artificial sequence (Chlamydia specific amplification sense primer)

<400> 2

tcttatgtat gtaagcctgt agaatacact atcg 34

<210> 3

<211> 34

<212> DNA

<213>Artificial sequence (Chlamydia specific amplification anti-sense primer)

<400> 3

ctttagcaac aactttaaat tgtagagtct ctcc 34

<210> 4

<211> 48

<212> DNA

<213>Artificial sequence (probe sequence)

<400> 4

ctgaaatctg ctgtaacaaa gctgtatggg acaaagagat gtgcccag 48

<210> 5

<211> 208

<212> DNA

<213>Utilize the product () after foregoing Chlamydia specific amplification

<400> 5

tcttatgtat gtaagcctgt agaatacact atcgttgtgt ccaacctagg ggatcttaaa 60

ctttacgatg tcgttgtaga agataccgta ccttcaggag ctacaatttt agaagccgag 120

ggagctgaaa tctgctgtaa caaagctgta tggtgcatca aagagatgtg cccaggagag 180

actctacaat ttaaagttgt tgctaaag 208

<210> 6

<211> 27

<212> DNA

<213>Artificial sequence (commonly expands sense primer)

<400> 6

gttcatttgc cagcgggaag atagagg 27

<210> 7

<211> 28

<212> DNA

<213>Artificial sequence (commonly expands anti-sense primer)

<400> 7

agaaccacgg tttgttacac aaatacgg 28

Claims

1. the primer pair sequence for animal chlamydia detection, it is characterised in that be SEQ ID No.2 and SEQ ID No.3.

2. the probe sequence for animal chlamydia RPA detections, it is characterised in that be SEQ ID No.4.

3. a kind of kit for animal chlamydia detection, it is characterised in that it is SEQ ID to include sequence in kit No.2 and SEQ ID No.3 primer pair.

4. a kind of kit for animal chlamydia RPA detections, it is characterised in that it is SEQ ID to include sequence in kit No.2 and SEQ ID No.3 primer pair, and the probe sequence that sequence is SEQ ID No.4.

5. a kind of animal chlamydia disease detection method of non-disease diagnosis, it is characterised in that by the genome of animal sample to be detected DNA is template, is expanded with SEQ ID No.2, SEQ ID No.3, and according to whether there is specific bar in amplified production electrophoresis Band determines whether tested animal has choamydiae infection.

6. a kind of animal chlamydia detection method of non-disease diagnosis, it is characterised in that by the genome of animal sample to be detected DNA is template, is expanded with SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, is determined it is determined that being detected using fluorescence Measure PCR instrument and carry out signal detection, and determine whether tested animal has choamydiae infection according to obvious whether change of the upper curvature of detection.