CN101492741A - Method for quantitative detection of mycoplasma hyopneumoniae - Google Patents
Method for quantitative detection of mycoplasma hyopneumoniae Download PDFInfo
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Abstract
The invention provides a PCR primer for real-time fluorescent quantitative detection of Mycoplasma hyopneumoniae and a quantitative detection method thereof. The Mycoplasma hyopneumoniae is the main pathogen of swine enzootic pneumonia, is difficult to culture, and is difficult to detect quantitatively by microscope as the shape thereof is too small, and the traditional hyopneumoniae CCU detection and CFU detection have long time consumption, high pollution rate, different detection results due to different culture media, operation methods or criteria as well as difficult practical application. The real-time fluorescent quantitative PCR detection method established by the invention adopts the concentration-known standard plasmids containing target genes as reference, has the advantages of fast speed, high sensitivity and precise, and can be used for the quantitative or micro-detection of cultures of Mycoplasma hyopneumoniae and semi-finished products of vaccines.
Description
Technical field
The invention belongs to molecular biology and pathogenic agent quantitative measurement technology field.Particularly, the present invention relates to be used to detect the real-time fluorescence quantitative PCR primer and the probe of mycoplasma hyopneumoniae, and use it to carry out quantitative detection method.More particularly, be PCR primer and probe, and use it to carry out quantitative detection method the real time fluorescent quantitative of mycoplasma pneumoniae p110 gene design.
Background technology
(Mycoplasma hyopneumoniae is to cause epidemic swine pneumonia (SwineEnzootic Pneumoniae, main pathogen EP) Mhp) to mycoplasma hyopneumoniae.Epidemic swine pneumonia is claiming swine enzootic pneumonia again, has high incidence, the characteristics of low actual, chronic dry cough, poor growth, characteristics such as hypoevolutism can appear after the morbidity, can cause after the infection that pig immunity reduces, thereby cause polyinfections such as other cause of diseases such as actinobacillus pleuropneumoniae, porcine reproductive and respiratory syndrome virus, pneumonia pasteurellosis bacillus, cause porcine respiratory syndrome or other diseases, the development of serious harm pig industry.
The Mhp separation difficulty needs the long period, and separation efficiency is lower.Even separate successfully, Mhp thalline feature is not obvious, and the microscopic examination thalline can't obtain clear and definite result.The solid colony diameter of Mhp is 500 μ m only, colony characteristics and the not obvious (Bi Dingren of other mycoplasma colony characteristicses difference, " Mycoplasma reclassifies and up-to-date name in the moccasin body guiding principle ", the academic nd Annual Meeting collection of veterinary microbiology in 1997, the 68-71 page or leaf), thus separation and Culture be difficult to become the main diagnostic method of Mhp.Before molecular biology method attained full development, serodiagnosis was the main diagnostic method of Mhp, still is widely used now, mainly contain: immunofluorescence technique, immunoenzyme technics, agglutination test, the radioimmunity enzyme test, complement fixation test (CFT), indirect hemagglutination test and ELISA.Wherein comparatively commonly used with indirect hemagglutination test, complement fixation test (CFT), ELISA and radioimmunity enzyme test.
Along with the development of molecular Biological Detection technology, mainly concentrate on detection on the dna level at the diagnostic method of Mhp, comprise that nucleic acid probe detects and the PCR detection.(Molecular and Cellular Probes.1989 such as Stemke, 3 (3): 225-232) with behind the disconnected clone at random of the EcoR I enzyme section of Mhp DNA, make probe with recombinant products, though this probe is special to Mhp under stringent hybridization condition, but easy and mycoplasma flocculare produces cross reaction, the DNA of 10pg can be detected, but the Mhp DNA in the disease lung can't be directly detected.Subsequently, have the probe of a series of isotropic substances, digoxigenin labeled to be developed (AhrensP etc., Letters in Applied Microbiology, 1991,12 (6): 249-253); Satoshi Futo etc., Journal of ClinicalMicrobiology, 1992,30 (6): 1509-1513; Kwon D etc., Veterinary Pathology, 1999,36 (4): 308-313).Nucleic acid probe is mainly used in the evaluation of gene clone and clone's product at present, is used for the detection of clinical case, and its susceptibility and specificity are still needed and will further be developed, and probe in detecting step complexity.So, developed afterwards and that sensitivity is higher, more easy PCR detection method (Blanchard etc., Molecular and cellular probes.1996,10 (1): 15-22 of operation; Baumeister etc., Journal of Clinical Microbiology, 1998,36 (7): 1984-1988; Sorensen etc., VeterinaryMicrobiology, 1997,54:23-24).Along with the appearance of nested PCR method, sensitivity and specificity that PCR detects Mhp further improve, and also are extended (Stemke etc., Molecular and CellularProbes.1989,3 (3): 225-232 simultaneously the period detected of clinical sample; Stark etc., Applied and Environmental Microbiology, 1998,64 (2): 543-548; Calasmiglia etc., Veterinary Microbiology, 2000,76 (3): 299-303).
The method that Real Time PCR promptly monitors pcr amplification product and resolves in real time, be nucleic acid quantification technology (Montgomery R A etc., Cytokine, 1997 that on the qualitative technical foundation of PCR, grow up, 9 (10): 717-726), claim quantitative fluorescent PCR again.
Real Time PCR not only is widely used in resolving in genetic expression, also is widely used in the detection of the parsing of SNP somatotype, species evaluation, virus and pathogenic bacteria, the quantitative analysis of genetically modified food, the numerous areas such as parsing of quiding gene copy number.RealTime PCR act on and developed regular-PCR fast, highly sensitive advantage such as detects, having overcome regular-PCR simultaneously can not be accurately quantitatively, deficiency such as pollution easily.It not only realized to dna profiling quantitatively, and has highly sensitive, specificity and characteristics (Zhang Liguo etc. such as reliability is stronger, can realize multiple reaction, level of automation height, nonstaining property, real-time and accuracy, biotechnology, 113 (12): 39-40; Zhang Bei, foreign medical science clinical biochemistry and ecsomatics fascicle, 2003,24 (6): 327-329; Cai Gang, foreign medical science clinical biochemistry and ecsomatics fascicle, 2003,24 (6): 330-332).
The principle of Real Time PCR is to utilize to add fluorescent substance in the PCR reaction system, and by Real Time PCR detection system the fluorescence signal intensity in the PCR reaction process is monitored in real time, finally experimental data is carried out the method for analyzing and processing.Method difference according to introducing fluorescent signal can be divided into Real Time PCR following several: the chimeric method of fluorescence (SYBRGREEN I method); Taq Man probe method; The hybridization probe method; Molecular beacon method (Molecular beacons); The scorpions method; Cycling probe method (Zhang Liguo etc., 2003; Zhang Bei, 2003; Cai Gang, 2003; Yangcheng ripple etc., 2003; Takara, 2006; Wang Xiaohong, 2001; Joseph A.Whelan et al, 2003).At present most widely used is chimeric method of fluorescence and TaqMan probe method.
In recent years, begin to utilize fluorescent quantitative PCR technique to detect mycoplasma both at home and abroad, but only limit to the qualitative detection of Mhp at present, be difficult to still really realize that the Mhp accurate quantification detects.
The inventor is through big quantity research and test repeatedly, and filtering out a pair of is the primer p110realF/R and the probe of target sequence with Mhp p110 gene, set up on this basis a kind of can be to the TaqMan fluorescence probe PCR detection method of Mhp accurate quantification.
Summary of the invention
The object of the invention is to be provided for fluorescence PCR primer and the probe sequence that mycoplasma hyopneumoniae p110 gene quantification detects usefulness, and uses this primer to carry out quantitative detection method.
Purpose of the present invention is achieved through the following technical solutions: according to mycoplasma hyopneumoniae genome p110 (p76) gene order (ACCESSION No.NC_006360) that is published on the GenBank, use its gene order of MegAlign software analysis, according to the principle of design of primer and probe, utilize Primer Expressv2.0 software design fluorescence quantification PCR primer and probe.
Utilize the gene specific oligonucleotide probe of mark fluorescent dyestuff to detect product in the PCR reaction, the most widely used TaqMan probe has used this principle exactly in fluorescence quantifying PCR method at present.The fluorescent probe that adds an a pair of primer and a template specificity during pcr amplification in reaction system, this probe is the oligonucleotide chain of a two ends mark, 5 ' end fluorescence report group of mark and fluorescent quenching group of 3 ' mark, 5 ' end fluorophor was transferred to the 3 ' end fluorescent quenching group that closes on energy after absorbing energy when probe was complete, therefore detected the fluorescence less than this probe 5 ' the end fluorophor sends.But in pcr amplification, after the template sex change low-temperature annealing, the Taq enzyme is under the mediation of primer, extend forward along template, 5 of Taq enzyme '-3 ' 5 prime excision enzyme activity will be combined in the probe cutting on the template already, 5 ' end fluorescence report base is free in the reaction system, breaks away from the shielding of 3 ' end fluorescent quenching group, accepts light stimulus and sends fluorescent signal.Promptly whenever have DNA chain to generate, just receive a fluorescence molecule, it is FAM that the accumulation that has realized fluorescent signal forms corresponding, commonly used fluorophor with the PCR product, TET, VIC, HEX.
The invention provides be used for to mycoplasma hyopneumoniae culture work in-process carry out primer that real-time fluorescence quantitative PCR detects to and probe, wherein said primer and probe are that specificity is at mycoplasma hyopneumoniae p110 gene conservative zone.
In the solution of the present invention, wherein said specifically at the primer of mycoplasma hyopneumoniae p110 gene to and probe, its nucleotides sequence is classified as:
P110realF：AGGATACAAACTGAGAAACCGAGCTA
P110realR：CAAGACCGAGTGGGTATGACCT
Probe: TGGACAGATCGGTGATACAACCCCACA
The accompanying drawing summary
Fig. 1 is illustrated to be the genomic amplification curve result of mycoplasma hyopneumoniae.1, be the amplification curve of template for 10 times with the genome dilution; 2, with genomic 10
-2Amplification curve for template; 3, with genomic 10
-3Amplification curve for template; 4, with genomic 10
-4Amplification curve for template; 5, with genomic 10
-5Amplification curve for template; 6, be the amplification curve of template with DDW;
Fig. 2 is illustrated to be the amplification curve result of standard plasmid.1,10 times (concentration is 10 with the standard plasmid dilution
6Copy/μ l) is the amplification curve of template; 2, with 10 of standard plasmid
-2(concentration is 10
5Copy/μ l) is the amplification curve of template; 3, with 10 of standard plasmid
-3(concentration is 10
4Copy/μ l) is the amplification curve of template; 4, with 10 of standard plasmid
-4(concentration is 10
3Copy/μ l) is the amplification curve of template; 5, with 10 of standard plasmid
-5(concentration is 10
2Copy/μ l) is the amplification curve of template; 6, with 10 of standard plasmid
-6(concentration is 10
1Copy/μ l) is the amplification curve of template; 7, be the amplification curve of template with DDW;
Fig. 3 is illustrated to be the typical curve result of standard plasmid.Transverse axis is represented starting template concentration (log10), and the longitudinal axis is represented the Ct value.Standard plasmid is carried out 10 times of gradient dilutions, get the dilute sample of six concentration and do template, each concentration is done three groups of parallel samples, relation conefficient=0.999, amplification efficiency=98.5%.
Fig. 4 is illustrated to be the specificity test-results.M, DL2000plus; 1, be the PCR result of template with the Mycoplasma orale genome; 2, be the PCR result of template with bacterium BL21 genome; 3, be the PCR result of template with bacterium DH5 α genome; 4, be the PCR result of template with the mycoplasma hyorhinis genome; 5, be the PCR result of template with the mycoplasma hyopneumoniae genome; 6, be the PCR result of template with pig circular ring virus DNA; 7, be the PCR result of template with PRRSV cDNA; 8, be the PCR result of template with the standard plasmid; 9, be the PCR result of template with DDW;
Fig. 5 is illustrated to be 9 sample amplification curve results to be checked.1, with No. 4 sample gene groups amplification curve that is template; 2, with No. 3 sample gene groups amplification curve that is template; 3, with No. 7 sample gene groups amplification curve that is template; 4, with No. 6 sample gene groups amplification curve that is template; 5, with No. 9 sample gene groups amplification curve that is template; 6, with No. 5 sample gene groups amplification curve that is template; 7, with No. 1 sample gene group amplification curve that is template; 8, with No. 2 sample gene groups amplification curve that is template; 9, with No. 8 sample gene groups amplification curve that is template;
It is in order further to understand the present invention better that following embodiment is provided, and never content of the present invention and protection domain is constituted any restriction.
Embodiment
Reagent and equipment
Proteinase K is available from MERCK company; CTAB is available from AMRESCO company; Premix Ex Taq
TM(perfect RealTime PCR) kit is available from the precious biotechnology in Dalian company limited; Other reagent is homemade analytical pure.Real Time PCR instrument (iCycler) is available from U.S. BIO-RAD company; Real Time PCR 8 pipes are available from U.S. BIO-RAD company; Real TimePCR 8 pipe lids are available from U.S. BIO-RAD company.
The foundation of embodiment 1Mhp TaqMan fluorescent quantitative PCR detection method
Present embodiment is intended to use primer that p110realF/R and probe are carried out quantitative fluorescent PCR, has set up Mhp TaqMan fluorescent quantitative PCR detection method.
1.1 the design of primer and probe and synthetic
According to mycoplasma hyopneumoniae genome p110 (p76) gene order (ACCESSION No.NC_006360) that is published on the GenBank, utilize following fluorescence quantification PCR primer of Primer Express v2.0 software design and probe.
P110realF：AGGATACAAACTGAGAAACCGAGCTA
P110realR：CAAGACCGAGTGGGTATGACCT
Probe: TGGACAGATCGGTGATACAACCCCACA
1.2 the genomic extraction of mycoplasma hyopneumoniae
1) with the centrifugal 10min of 1ml Mhp mycoplasma culture 12000rpm room temperature, abandons supernatant;
2) to the precipitation in add 300 μ l Proteinase K cracking buffer systems (10mM Tris-EDTA[pH=7.5], 0.5%[wt/vol] SDS, 100 μ g/ml Proteinase Ks) in, fully suspend back 60 ℃ digestion 1h;
3) add 5M NaCl 50 μ l, and CTAB (10%[wt/vol], 0.7M NaCl) 40 μ l, 65 ℃ of effect 10min behind the mixing;
4) add equal-volume phenol: chloroform, mixing gently, 4 ℃ of centrifugal 5min of 12000rpm;
5) carefully draw 350 μ l upper strata water samples in another sky centrifuge tube, add the dehydrated alcohol that adds 2 times of volume-20 ℃ precoolings behind the 3M NaAC mixing of 1/10 volume again, place 2h or spend the night for 4 ℃;
6) 12000rpm, 4 ℃ of centrifugal 10min;
7) abandon supernatant,, centrifuge tube is inverted on the thieving paper, make the alcohol finish-drying with 700 μ l, 75% alcohol washing precipitation;
8) add 30 μ l-50 μ l sterilization DDW dissolving ,-20 ℃ of preservations after the packing.
1.3 the foundation of fluorescent PCR reaction system and reaction conditions
Press following preparation Real Time PCR reaction system (20 μ l):
Premix?Ex?Taq
TM(2×) 10μl
PCR?Forward?Primer(10μM) 0.4μl
PCR?Reverse?Primer(10μM) 0.4μl
Fluorescent probe solution 0.8 μ l
DNA 2μl
DDW 6.4μl
After reaction solution prepares, instantaneous centrifugal, sample hose put into Real-time PCR instrument after, according to Premix Ex Taq
TMThe test kit specification sheets, adopt following condition to react: 95 ℃ of pre-sex change in 30 seconds, 95 ℃ of sex change in 10 seconds, 60 ℃ of 31 seconds renaturation and extension, sex change and renaturation process repeat totally 40 circulations.According to the amplification curve result of determination.The fluorescent signal of quantitative real time PCR Instrument acquisition and recording 490nm wavelength in the extension process.
1.4TaqMan the foundation of Real Time PCR method
1.4.1Mhp 10 times of gradient dilutions of genome
1) get the 1.5ml centrifuge tube of 7 sterilizations, be numbered 1-7, every pipe adds 90 μ l sterilization DDW;
2) get the Mhp genome 10 μ l of purification, it is added pipe No. 1, change pipettor rifle head;
3) with No. 1 pipe mixing, get 10 μ l and add pipe No. 2, change pipettor rifle head;
4) and the like, to No. 7 pipes, change pipettor rifle head, with No. 7 pipe mixings;
5) genome that will dilute the different concns of getting well is distinguished packing, and-20 ℃ standby.
1.4.2 the foundation of amplification curve
With CCU is 10
8The mycoplasma hyopneumoniae culture carry genome according to 1.2 methods, with the sterilization DDW genome is carried out 10 times of gradient dilutions, be diluted to 10
-7Getting extension rate is 10
-2-10
-7Genome make template DNA, carry out Real Time PCR according to 1.3.According to the variation of fluorescent value in the amplification procedure, go out fluorescence threshold (Thresholdvalue) by computed in software, and calculate Ct (Cycle threshold) value of each bar curve.With the Ct value is X-coordinate, and fluorescent value is an ordinate zou, sets up the amplification curve of quantitative fluorescent PCR.According to the variation of amplification curve shape and Ct value, analyze the credibility of PCR system, the results are shown in Figure 1.
1.4.3 the foundation of typical curve
After amplification curve is set up, calculate the Ct value by the software automatic analyser according to amplification curve; With extension rate is 10
-7, 10
-6... 10
-2Genome concentration be defined as 10 successively
1Copy/μ l, 10
2Copy/μ l ... 10
6Copy/μ l; Logarithmic value with the template initial concentration is a transverse axis, is the longitudinal axis with the Ct value, simulates typical curve by software; Relation conefficient (r according to curve
2) confidence level of the curve that settles the standard; Combined standard slope of a curve (slope) calculates pcr amplification efficient (E) by software or according to formula.
Calculation formula: E=10
[1/slope]-1.
Obtain three groups of Ct values by three groups of amplification curves, simulate three groups of typical curves by software.Correlation coefficient r
2The rectilinearity of reflection typical curve, ideal value should illustrate that near 1 rectilinearity is good more, quantitatively accurate more more greater than 0.98.Ideal pcr amplification efficient should be carried out the TaqMan quantitative fluorescent PCR with p110realF/R primer and probe and set up typical curve in 0.8＜E＜1.2 scopes, and relation conefficient is respectively 0.999, and the rectilinearity of description standard curve is good.Amplification efficiency is respectively 87.5%, and the result shows that primer p110realF/R and probe meet the requirement of fluorescent quantitative PCR technique.
1.5 the structure of standard plasmid
1.5.1 design of primers and the preparation of PCR product
According to mycoplasma hyopneumoniae genome p110 gene order (the ACCESSION NO.NC_006360 that is published on the GenBank, NC_007332, NC_007295), in conjunction with the p110realF/R primer that filters out, use the Primerselect design primer of DNAStar software right:
P110cloneF：GCGGATCCACCAGGTGCATCTTCATC
P110cloneR：GCTCTAGACCGCTGTGGCTAATAGTA
This primer is to synthetic by Shanghai bio-engineering corporation, and above-mentioned p110realF/R is in the sequence of this primer to amplification.To extract mycoplasma hyopneumoniae DNA in 1.2 is template, adds primer P110cloneF/R is set up 50 μ l reaction systems
Sterilization deionized water 33.7 μ l
10×PCR?buffer 5μl
2.5mmol/l?dNTP 4μl
rTaq(5U/μl) 0.3μl
P110cloneF(10pmol/μl) 3μl
P110cloneR(10pmol/μl) 3μl
PCR reaction heat loop parameter is: 95 ℃ of pre-sex change 5min; 45 seconds, 55 ℃ annealing of 95 ℃ of sex change were extended totally 30 circulations 1 minute for 30 seconds, 72 ℃; 72 ℃ are extended 5min then.Get 4 μ l pcr amplification products through 0.8% agarose gel electrophoresis voltage electrophoresis 30min with 5V/cm, behind the EB dyeing 20min, observations under the ultraviolet gel imaging system.
1.5.2 the preparation of recombinant plasmid and evaluation
Use is carried out the PCR product purification available from the EASY PCR purification test kit of full formula King Company according to product description.With the PCR product cloning that reclaims in pEASY-T1, connect product and transform DH5 α competent cell, Xiang Guanzhong adds 800 μ l LB substratum, 150rpm wave and culture 1h in 37 ℃ of shaking tables, get 200 μ l bacterium liquid and coat on the LB flat board that contains AMP (100 μ g/ml) 37 ℃ of overnight incubation.Picking list bacterium colony at random from the flat board is inoculated into 5ml LB (AMP respectively
+, 100 μ g/ml) and carry out 37 ℃ of shaking culture 10h in the liquid nutrient medium, adopt alkaline lysis to prepare plasmid DNA in a small amount, plasmid DNA is dissolved among the 30 μ l sterilization TE, get 4 μ l electrophoresis and identify all the other-20 ℃ of preservations.By the gel imaging system observations, positive recombinant plasmid is selected in preliminary judgement according to the plasmid size behind the electrophoresis.Carry out pcr amplification tentatively to be judged as the male recombinant plasmid, replace outside the mycoplasma hyopneumoniae genomic dna the same 1.5.1 of PCR reaction system and reaction conditions divided by recombinant plasmid.After reaction finishes, get 4 μ l pcr amplification products and carry out agarose gel electrophoresis 30min, observations under the ultraviolet gel imaging system with the voltage of 5V/cm.PCR is identified that the male recombinant plasmid company that delivers checks order, the Mhp p110 gene order of sequencing result and GENBANK announcement is compared, with the consistent positive plasmid of recombinant plasmid of announcement sequence, the recombinant plasmid note that contains the Mhpp110 gene that makes up is standard plasmid pEASY-T1-p110.
1.6 the foundation of the amplification curve of standard plasmid
Standard plasmid is carried out the appropriateness dilution, measure its OD value with ultraviolet spectrophotometer at the 260nm wavelength, the molecular weight of combined standard plasmid calculates plasmid copy number in every μ l solution.
The standard plasmid that calculates copy number is carried out 10 times of gradient dilutions, be diluted to 10 copy/μ l.Getting concentration is 10
6-10
1The standard plasmid of copy/μ l is a template, p110realF/R is a primer, be RealTime PCR with BIO-RAD icycler quantitative real time PCR Instrument, each concentration is made 3 parallel samples, set up amplification curve according to the result, the order that curve is followed from the high density to the lower concentration is arranged, and three parallel sample Ct value differences of each concentration are different not remarkable, as shown in Figure 2.
1.7 the foundation of the typical curve of standard plasmid
With copy number is 10
6-10
1The standard plasmid of/μ l is that template is Real Time PCR, and each concentration is made three parallel samples simultaneously.Reaction finishes the back by the automatic analysis of fluorescence threshold value of software, calculate the Ct value in conjunction with amplification curve, the logarithmic value with the template initial concentration is a transverse axis then, is the longitudinal axis with the fluorescent value, obtain typical curve by the software match, and calculate the relation conefficient and the pcr amplification efficient of curve.
Standard plasmid is carried out carrying out PCR as template behind 10 times of gradient dilutions, and each concentration is made 3 parallel samples, and amplification finishes the back and simulates typical curve, the typical curve correlation coefficient r by computer according to the logarithmic value and the corresponding C t value of starting template concentration
2=0.999, show between the logarithmic value of different gradient quantitative templates and the Ct value to present good linear relationship that the typical curve rectilinearity is good.As calculated, typical curve amplification efficiency E=98.5%, as shown in Figure 3.
1.8TaqMan the optimization of fluorescence quantifying PCR method reaction conditions
With the standard plasmid is template, and p110realF/R is a primer, selects for use different primer concentrations, annealing temperature, extension time to carry out Real Time PCR reaction, according to the PCR reaction result, selects best Real Time PCR reaction conditions.
By repeatedly experiment, the quantitative fluorescent PCR optimum reaction condition of determining is: best primer final concentration is 0.2 μ mol/l;
PCR reaction system following (20 μ l):
Premix?Ex?Taq
TM(2×) 10μl
PCR?Forward?Primer(10μM) 0.4μl
PCR?Reverse?Primer(10μM) 0.4μl
Fluorescent probe solution 0.8 μ l
DNA 2μl
DDW 6.4μl
95 ℃ of pre-sex change in 3 minutes of loop parameter, 95 ℃ of sex change in 5 seconds, 60 ℃ of 30 seconds renaturation and extension, sex change and renaturation process repeat totally 40 circulations.
The specificity test of embodiment 2 mycoplasma hyopneumoniae fluorescence PCR detecting methods
Respectively the common genetic engineering bacterium (bacillus coli DH 5 alpha strain and BL21 strain) in common laboratory, laboratory, Mycoplasma orale and normal and mycoplasma hyopneumoniae polyinfection pathogenic agent (pig circular ring virus, PRRSV and mycoplasma hyorhinis) are control sample, simultaneously with standard plasmid as positive control, as negative control, check is based on the specificity of the fluorescence PCR detecting method on the mycoplasma hyopneumoniae basis with sterilization DDW.
2.1 experiment material
Mycoplasma orale (Mycoplasma orale) CVCC 379 strains of contrast usefulness, mycoplasma hyorhinis (Mycoplasmahyorhinis) CVCC 361 strains are all available from China Veterinery Drug Inspection Office, and other are the laboratory and preserve bacterial classification.
2.2 sample pre-treatments
2.2.1 the acquisition of viral DNA
Extract PRRSV RNA according to Invitrogen company specification sheets.Utilize Oligo (dt) 15Primer (Promega), carry out reverse transcription according to PROMEGA company M-MLV Reverse Transcriptase specification sheets, synthetic cDNA is stored in-20 ℃, as pcr template.PCV utilizes the viral DNA out test kit of day damp genome company to extract DNA, is stored in-20 ℃, as pcr template.
2.2.2 the acquisition of bacterial genomes
According to ordinary method recovery engineering strain E.coli DH5 α, BL21.Above-mentioned bacterial strains bacterium liquid is respectively got 50 μ l respectively, and the bacterial genomes out test kit extraction genome with day damp genome company is stored in-20 ℃, as pcr template.
2.2.3 the genomic acquisition of Mycoplasma orale and mycoplasma hyorhinis
Mycoplasma orale CVCC 379 strains and mycoplasma hyorhinis CVCC 361 pnca gene group extracting method are with 1.2.
2.3 primer is to the specificity test of p110realF/R
DNA, standard plasmid and the DDW that gets the PRRSV cDNA for preparing among the various mycoplasma genomes that prepare among the various bacterial genomes that prepare among the 2.2.2, the 2.2.3, the 2.2.1 and PCV template respectively is PCR.(50 μ l) is as follows for the PCR system:
Sterilization deionized water 31.7 μ l
10xPCR?buffer 5μl
2.5mmol/l?dNTP 4μl
rTaq(5U/μl) 0.3μl
p110realF(10pmol/μl) 3μl
p110realR(10pmol/μl) 3μl
PCR reaction heat loop parameter is:
95 ℃ of pre-sex change 5min
95 ℃ of sex change 30s
60 ℃ of annealing 30s
72 ℃ are extended 30s
Repeat 35 circulations
72 ℃ are extended 5min
The PCR product is respectively got 4 μ l, and with the voltage electrophoresis 30min of 5V/cm, by ultraviolet gel imaging system observations, the result as shown in Figure 1 in 1% sepharose.
As can be seen from Figure 4, positive control and with Mhp be the swimming lane 5 and 8 of template at about 100bp place appearance one specific band, and expect that big or small 117bp meets, the amplified production of all the other swimming lanes is primer dimer.This shows the designed primer of the present invention to can satisfying the needs of specific detection, and with test sample in other microorganism that may exist or not the cross interference phenomenon.
The sensitivity test of embodiment 3 mycoplasma hyopneumoniae fluorescence PCR detecting methods
The standard plasmid that contains goal gene that calculates copy number is done 10 times of gradient dilutions, be diluted to 10 copy/μ l.Getting concentration is 10
6, 10
5, 10
4, 10
3, 10
2, 10
1The plasmid of copy/μ l is that template is Real Time PCR.There is the minimum starting template concentration of product to be the sensitivity of this Real Time PCR.According to the amplification curve analysis, this Real Time PCR method detection sensitivity is 10 copy/μ l or 20 copies/20 μ l reaction systems.See Fig. 2 for details.
4.1 sample to be checked is prepared
Cultivate three generations's mycoplasma hyopneumoniae culture, in per generation, be provided with three parallel samples, cultivates results after 48 hours, is designated as the 1-9 sample respectively, measures the CCU experiment during results immediately, observes the variable color situation every day.
Obtain 9 samples to be checked,, extract genome according to implementing to extract genomic method described in 1.With 1000 times of diluents of 9 samples and 10 times of grade dilutions of standard plasmid is template, makes quantitative fluorescent PCR, according to the typical curve of setting up, calculates the copy number of sample to be checked.
4.2 experimental result
No. 8 CCU value is 10 in 9 samples
-7, other are 10
-8The copy number that the quantitative fluorescent PCR of No. 8 samples calculates is 6.52 * 10
6, other are 4.8 ± 2.5 * 10
7, see Fig. 5, experimental result shows that the fluorescent quantitation method of foundation and traditional CCU method of counting have significant dependency, therefore the fluorescence detection method of setting up can be used for the detection by quantitative of mycoplasma hyopneumoniae culture.
Claims (6)
1. the primer that is used for the mycoplasma hyopneumoniae culture is carried out the real-time fluorescence quantitative PCR detection is to reaching probe, and wherein said primer is to reaching the P110 gene conservative zone of probe specificity at mycoplasma hyopneumoniae.
The described primer of claim 1 to and probe, wherein said primer to and the sequence of probe be:
P110realF：AGGATACAAACTGAGAAACCGAGCTA
P110realR：CAAGACCGAGTGGGTATGACCT
Probe: TGGACAGATCGGTGATACAACCCCACA
3. one kind is carried out the real-time fluorescence PCR detection method of detection by quantitative to mycoplasma hyopneumoniae, and this method comprises the following steps:
1) cultivation of bacterial strain;
2) to the genomic extraction of Mhp;
3) to extract the genome sample add the described primer of claim 1 to and probe, and the Premix ExTaq that uses of pcr amplification
TM(2 *), DDW obtain pcr amplification reaction liquid;
4) the PCR pipe that pcr amplification reaction liquid will be housed places on the fluorescent PCR instrument;
5) according to used fluorescence dye type, select corresponding fluorescence channel, carry out the fluorescent PCR amplification, record respectively detects the pcr amplification cycle number (C of sample
t);
6) according to the reaction C of each sample
tBe worth, judge the copy number of Mhp in the sample to be checked according to typical curve.
4. the described real-time fluorescence PCR detection method of claim 3, this method use the described primer of claim 2 to and probe.The described real-time fluorescence PCR detection method of claim 3, wherein said genome extract and comprise:
1) with the centrifugal 10min of 1ml Mhp mycoplasma culture 12000rpm room temperature, abandons supernatant;
2) in precipitation, add 300 μ L Proteinase K cracking buffer systems (10mM Tris-EDTA[pH=7.5], 0.5%[wt/vol] SDS, 100 μ g/ml Proteinase Ks) in, back 60 ℃ of digestion 1h fully suspend;
3) add 5M NaCl 50 μ L, and CTAB (10%[wt/vol], 0.7M NaCl) 40 μ L, 65 ℃ of effect 10min behind the mixing;
4) add equal-volume phenol: chloroform, mixing gently, 4 ℃ of centrifugal 5min of 12000rpm;
5) carefully draw 350 μ L upper strata water samples in another sky centrifuge tube, add the dehydrated alcohol that adds 2 times of volume-20 ℃ precoolings behind the 3M NaAC mixing of 1/10 volume again, place 2h or spend the night for 4 ℃;
6) 12000rpm, 4 ℃ of centrifugal 10min;
7) abandon supernatant,, centrifuge tube is inverted on the thieving paper, make the alcohol finish-drying with 700 μ L, 75% alcohol washing precipitation;
8) add 30 μ L-50 μ L sterilization DDW dissolving ,-20 ℃ of preservations after the packing.
5. the described real-time fluorescence PCR detection method of claim 3, wherein the fluorescent PCR amplification condition is: 95 ℃ of pre-sex change in 3 minutes, 95 ℃ of sex change in 5 seconds, 60 ℃ of 30 seconds renaturation and extension, sex change and renaturation process repeat totally 40 circulations.
6. claim 1 or 2 described primers are to reaching the purposes of probe in the detection that is used for mycoplasma hyopneumoniae.
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| CN113308561B (en) * | 2021-06-04 | 2022-05-31 | 天康制药(苏州)有限公司 | Primer group for detecting mycoplasma hyopneumoniae, application thereof and real-time fluorescent quantitative PCR detection method |
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