CN102676658B - Nested PCR (polymerase chain reaction) detection method for mycoplasma hyorhinis - Google Patents
Nested PCR (polymerase chain reaction) detection method for mycoplasma hyorhinis Download PDFInfo
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Abstract
The invention relates to a nested PCR (polymerase chain reaction) detection method for mycoplasma hyorhinis causing polyserositis, arthritis, otitis and the like of swines. The method includes the main steps: firstly, extracting DNA (deoxyribonucleic acid) from a mycoplasma hyorhinis sample, and keeping the DNA at a low temperature for later use; secondly, performing primary PCR amplification for the mycoplasma hyorhinis sample by the aid of a pair of specific primers of Mhr P01 and Mhr P02; thirdly, performing secondary PCR amplification for a primary PCR product of the mycoplasma hyorhinis sample by the aid of another pair of specific primers of Mhr P03 and Mhr P04; and fourthly, performing electrophoresis detection for an amplified product, and if a specific band of 346bp exists, determining the detected pathogenic bacteria as the mycoplasma hyorhinis. By the method, the mycoplasma hyorhinis can be accurately detected in the presence of trace pathogenic bacteria, and can be detected only in the presence of 102CCU/mL of the pathogenic bacteria. The nested PCR detection method for the mycoplasma hyorhinis has the advantages of high detecting speed, high specificity, high sensitivity and easiness to popularize.
Description
Technical field
The nested PCR detection method that the present invention relates to the detection method, particularly mycoplasma hyorhinis of the mycoplasma hyorhinis of a kind of polyserositis that causes pig, sacroiliitis, otitis etc., belongs to biological technical field.
Background technology
Mycoplasma hyorhinis (
mycoplasma hyohinis) be a kind of pathogenic mycoplasma that can cause the illnesss such as pig polyserositis, sacroiliitis, otitis, pneumonia.As a kind of encountered pathogenic bacteria, mycoplasma hyorhinis is transmitted to piggy by sow or large pig conventionally.Ross and Spear (1993) have confirmed from the nasal secretion of 10% sow and 30%～40% weanling pig, to isolate this mycoplasma.Once infect, this mycoplasma is in upper respiratory tract bamboo telegraph and can be separated to from the lungs of infected pigs and ductus nasopharyngeus.
In the chronic hyopneumoniae case of UK and USA, the existence of mycoplasma hyorhinis more than 50% can be detected; In breaking out group, region pneumonia can both be separated to ninety-nine times out of a hundred mycoplasma hyorhinis simultaneously.Although it is very high to separate the ratio of mycoplasma hyorhinis from the sick lung of porcine enzootic pneumonia, most of scholar does not think that this microorganism is the cause of disease of porcine enzootic pneumonia in the past, and reason is also can detect and be separated to mycoplasma hyorhinis in normal health porcine respiratory.Until the series of experiments that the seventies, investigator carried out starts progressively to have confirmed that the generation of mycoplasma hyorhinis and hyopneumoniae has direct dependency.Can bring out hyopneumoniae (Gois etc. 1968,1971 from the isolated a few strain mycoplasma hyorhinises of Czechoslovakia, Denmark, Germany and Britain by respiratory tract approach; Gois and Valicek etc. 1968; Martin etc. 1968; Friis etc. 1971; Poland etc. 1971).Three strain Czechoslovakia isolate (Tr32, Ploand etc. 1971 of mycoplasma hyorhinis; Ne 110 and Ni5, Gois etc. 1971) and a strain Britain isolate (S218 Gois etc. 1971), can induce aseptic piggy generation pneumonia.MeyLing (1971) has checked the little hyopneumoniae of natural occurrence, finds the mycoplasma hyorhinis positive and mycoplasma pneumoniae is negative.Domestic Ningxia agricultural college once, with the doubtful mycoplasma hyorhinis of two strains (Ning Nong 3 and Ning Nong 5) culture collunarium inoculation piggy, brings out slight pneumonia.Taiwan Lin Junhong (2006) can bring out part with mycoplasma hyorhinis isolate A TIT-1,3,7 mixture intratracheal injections attacks malicious pig typical porcine mycoplasmal pneumonia pathology occurs, and success is attacked malicious pig lung and is separated to mycoplasma hyorhinis from major part.
Up to now, mycoplasma hyorhinis is detected mainly take separation and Culture as main, the needed time is longer, needs badly and sets up the high method for quick of a kind of molecular biological susceptibility.
Summary of the invention
The object of the invention is, in order to overcome the existing defect that obviously has length detection time that mycoplasma hyorhinis detected take separation and Culture as master, provides the nested PCR detection method of a kind of energy mycoplasma hyorhinis quick, sensitive, that detect exactly.
The nested PCR detection method of mycoplasma hyorhinis of the present invention, mainly comprises the following steps:
1) DNA of extraction mycoplasma hyorhinis sample, cryopreservation is for subsequent use;
2) with a pair of Auele Specific Primer Mhr P01, Mhr P02, mycoplasma hyorhinis sample is carried out to first round pcr amplification;
Wherein: the sequence of upstream primer Mhr P01 is: 5 '-GCATCTATATTTTCGCCAATAGC-3 ';
The sequence of downstream primer Mhr P02 is: 5 '-AGCTAGAGTTTCATCATTACC-3 ';
Described first round pcr amplification method is: the reaction solution cumulative volume in PCR Reagent Tube I is 20 μ l, comprising: testing sample mycoplasma hyorhinis DNA profiling extracting solution 0.5 ~ 1.0 μ l, 10 × PCR Buffer (Mg
2+free) 1.0 ~ 2.5 μ l, 25mM MgCl
21.0 ~ 2.0 μ l, 2.5mM dNTPs 1.0 ~ 3.0 μ l, 10pmol/ μ l upstream primer Mhr P01 0.5 ~ 1.0 μ l, 10pmol/ μ l downstream primer Mhr P02 0.5 ~ 1.0 μ l, 5 units/μ l TaqDNA polysaccharase, 0.2 ~ 0.5 μ l, sterilizing ultrapure water is mended to cumulative volume 20 μ l, and 3000 ~ 5000g is centrifugal, and mix 2 ~ 5 seconds, is placed in amplified reaction in PCR instrument; Reaction conditions is: unwinds 2 ~ 5 minutes by 94 ~ 96 ℃, and then 94 ~ 96 ℃ of sex change 40 ~ 60 seconds, 50 ~ 56 ℃ of renaturation 60 seconds, 72 ℃ are extended 40 ~ 90 seconds, 25 ~ 35 circulations, last 72 ℃ are extended 6 ~ 10 minutes, obtain mycoplasma hyorhinis first round PCR product;
3) with another, Auele Specific Primer Mhr P03, Mhr P04 are carried out to second to mycoplasma hyorhinis first round PCR product and take turns pcr amplification;
Wherein: the sequence of upstream primer Mhr P03 is: 5 '-GTAGTCAAGCAAGAGGATGT-3 ';
The sequence of downstream primer Mhr P04 is: 5 '-GCTGGAGTTATTATACCAGGA-3 ';
Described second takes turns pcr amplification method is: in PCR Reagent Tube II, add mycoplasma hyorhinis first round PCR product to react, its reaction solution cumulative volume is 20 μ l, comprise: mycoplasma hyorhinis first round PCR product 0.5 ~ .1.0 μ l, 10 × PCR Buffer (Mg
2+free) 1.0 ~ 2.5 μ l, 25mM MgCl
21.0 ~ 2.0 μ l, 2.5mM dNTPs 1.0 ~ 3.0 μ l, 10pmol/ μ l upstream primer Mhr P03 0.5 ~ 1.0 μ l, 10pmol/ μ l downstream primer Mhr P04 0.5 ~ 1.0 μ l, 5 units/μ l TaqDNA polysaccharase, 0.2 ~ 0.5 μ l, sterilizing ultrapure water is mended to cumulative volume 20 μ l, and 3000 ~ 5000g is centrifugal, and mix 2 ~ 5 seconds, is placed in amplified reaction in PCR instrument; Reaction conditions is: unwinds 2 ~ 5 minutes by 94 ~ 96 ℃, and then 94 ~ 96 ℃ of sex change 40 ~ 60 seconds, 46 ~ 50 ℃ of renaturation 60 seconds, 72 ℃ are extended 60 ~ 90 seconds, 25 ~ 30 circulations, last 72 ℃ are extended 6 ~ 10 minutes, obtain mycoplasma hyorhinis second and take turns PCR product;
4) electrophoresis detection
Get mycoplasma hyorhinis second and take turns PCR product 5 ~ 10 μ l, with 2 ~ 6 μ l 0.25%(mass/volume) tetrabromophenol sulfonphthalein sample-loading buffer mixes, containing 0.8 ~ 2%(mass/volume) 0.7 ~ 1%(mass/volume of Goldview) sepharose electrophoresis 20 ~ 30 minutes under voltage 80 ~ 100V, the detection of taking pictures under ultraviolet transmissive lamp in gel imaging system, it is the positive that has mycoplasma hyorhinis to infect or pollute that sample well has the nucleic acid band of a 346bp, can determine and in testing sample, contain pathogenic bacteria mycoplasma hyorhinis, otherwise not have.
Above-mentioned dNTPs is the mixture that dTTP and dATP, dCTP, tetra-kinds of concentration of dGTP are respectively 2.5mM Nucleotide.
Further, the best composition of the reaction solution in the PCR reaction tubes I of described first round pcr amplification is: testing sample mycoplasma hyorhinis DNA profiling extracting solution 1.0 μ l, 10 × PCR Buffer (Mg
2+free) 2.0 μ l, 25mM MgCl
22.0 μ l, 2.5mM dNTPs 2.0 μ l, 10pmol/ μ l upstream primer Mhr P01 0.5 μ l, 10pmol/ μ l downstream primer Mhr P02 0.5 μ l, 5 units/μ l TaqDNA polysaccharase, 0.2 μ l, sterilizing ultrapure water is mended to cumulative volume 20 μ l.
Further, the described second best composition of taking turns the reaction solution in the PCR reaction tubes II of pcr amplification is: mycoplasma hyorhinis first round PCR product 1.0 μ l, 10 × PCR Buffer (Mg
2+free) 2.0 μ l, 25mM MgCl
22.0 μ l, 2.5mM dNTPs 2.0 μ l, 10pmol/ μ l upstream primer Mhr P03 0.5 μ l, 10pmol/ μ l downstream primer Mhr P04 0.5 μ l, 5 units/μ l TaqDNA polysaccharase, 0.2 μ l, sterilizing ultrapure water is mended to cumulative volume 20 μ l.
The present invention designs special primer successfully set up mycoplasma hyorhinis nest-type PRC detection technique, has advantage and the effects such as high specificity, highly sensitive and quarantine cycle be short:
1. current, for the detection of mycoplasma hyorhinis, be all the PCR detection method of common two primers, the present invention uses nest-type PRC to realize the high specificity of this cause of disease, sensitivity up to 10 first
2cCU/ml, and the sensitivity of common double primer PCR is only 10
6cCU/mL.
2. the present invention carries out DNA extraction and Molecular Detection to the tissue of possibility mycoplasma hyorhinis, save the time of traditional separation and Culture, greatly shorten in time the quarantine cycle, only needed 10 ~ 12h to complete from the collection of sample to having detected, be easy to promote.
Accompanying drawing explanation
Fig. 1 is that mycoplasma hyorhinis nest-type PRC second is taken turns PCR specificity gel electrophoresis figure;
M in Fig. 1 is Marker; 1 is mycoplasma hyorhinis positive control; 2 is blank; 3 negative contrasts; 4 is pig mycoplasma flocculare; 5 is mycoplasma hyosynoviae; 6 is mycoplasma hyopneumoniae; 7 is chicken virus mycoplasma; 8 is actinobacillus pleuropneumoniae; 9 is Pestivirus suis; 10 is swine influenza virus; 11 is pig circular ring virus.
Fig. 2 is mycoplasma hyorhinis nest-type PRC first round PCR susceptibility gel electrophoresis figure;
In Fig. 2, M is 2000bp DNA marker, and 1-6 represents take different content DNA as template first round PCR product gel electrophorogram, wherein 1 be 10
7cCU/mL sample, 2 is 10
6cCU/mL sample, 3 is 10
5cCU/mL sample, 4 is 10
4cCU/mL sample, 5 is 10
3cCU/mL sample, 6 is 10
2cCU/mL sample; 7 negative contrasts.
Fig. 3 is that mycoplasma hyorhinis nest-type PRC second is taken turns PCR susceptibility gel electrophoresis figure;
In Fig. 3, M is 2000bp DNA Marker; 1-12 represents to take turns take the DNA of different content as second of template the gel electrophoresis figure of PCR product, and wherein 1 is 10
9cCU/mL sample, 2 is 10
8cCU/mL sample, 3 is 10
7cCU/mL sample, 4 is 10
6cCU/mL sample, 5 is 10
5cCU/mL sample, 6 is 10
4cCU/mL sample, 7 is 10
3cCU/mL sample, 8 is 10
2cCU/mL sample, 9 is 10
1cCU/mL sample, 10 is 10
0cCU/mL sample, 11 is 10
-1cCU/mL sample, 12 is 10
-2cCU/mL sample, 13 is blank; 14 negative contrasts.
Fig. 4 is the gel electrophoresis figure that the mycoplasma hyorhinis nest-type PRC second of the clinical sample of 21 parts of pig farms collections is taken turns PCR product.
M in Fig. 4: be 2000bp DNA Marker; 1 is. positive control; 2. be blank; 3. negative contrast; The mycoplasma hyorhinis nest-type PRC second of the clinical sample that 21 parts of pig farms of 4-24. gather is taken turns PCR product.
Embodiment
Below in conjunction with specific embodiments and the drawings, the invention will be further described.
1) DNA of extraction mycoplasma hyorhinis sample, cryopreservation is for subsequent use;
The DNA of the mycoplasma hyorhinis sample that the present invention uses, can extract according to a conventional method, and concrete grammar is to carry out in the steps below after the pig lung tissue that contains mycoplasma hyorhinis in extraction again:
1) add 100mg and organize to 600 μ l protein lysates, then add Proteinase K 3 μ l(20mg/ml), RNase inhibitory enzyme 3 μ l(10 mg/ml), mix, 55 ℃ of water-bath 2-4 hour, jolting is during this time for several times.
2) add isopyknic phenol: chloroform: primary isoamyl alcohol (25:24:1), the 10min that gently shakes, the centrifugal 5min of 1,2000rpm.
3) (approximately 300 μ l), add chloroform 600 μ l, the centrifugal 5min of 1,2000 rpm to get supernatant.
4) (approximately 200 μ l), add chloroform 600 μ l, the centrifugal 5min of 1,2000 rpm to get supernatant.
5) (approximately 200 μ l), add the dehydrated alcohol of 2 times, and the ammonium acetate of the 5mol/L of 0.4 times, puts upside down and mix gently, place 20min. to get supernatant
6) the centrifugal 5min of 1,2000 rpm, abandons supernatant, adds 70% ethanol, washes 2 times (the centrifugal 1min of 1,2000 rpm).
7) add sterilizing deionized water or the TE solution of 50 μ l.Obtain the DNA of the mycoplasma hyorhinis sample that will use required for the present invention.
2) with a pair of Auele Specific Primer Mhr P01, Mhr P02, mycoplasma hyorhinis sample is carried out to first round pcr amplification;
Wherein: the sequence of upstream primer Mhr P01 is: 5 '-GCATCTATATTTTCGCCAATAGC-3 ';
The sequence of downstream primer Mhr P02 is: 5 '-AGCTAGAGTTTCATCATTACC-3 ';
Above-mentioned primer Mhr P01 and Mhr P02 are respectively by the synthetic DNA fragmentation of base sequence by DNA synthesizer;
Be the based composition difference (number of logging in: X14140.1, M37339.1, CP002669.1, CP00323.1, CP002170.1, GU722587.1) of announcing the P37 sequence of mycoplasma hyorhinis according to GeneBank, utilize primer-design software (Oligo 7) design.
Described first round pcr amplification method is: the reaction solution cumulative volume in PCR Reagent Tube I is 20 μ l, comprising: testing sample mycoplasma hyorhinis DNA profiling extracting solution 0.5 ~ 1.0 μ l, 10 × PCR Buffer (Mg
2+free) 1.0 ~ 2.5 μ l, 25mM MgCl
21.0 ~ 2.0 μ l, 2.5mM dNTPs 1.0 ~ 3.0 μ l, 10pmol/ μ l upstream primer Mhr P01 0.5 ~ 1.0 μ l, 10pmol/ μ l downstream primer Mhr P02 0.5 ~ 1.0 μ l, 5 units/μ l TaqDNA polysaccharase, 0.2 ~ 0.5 μ l, sterilizing ultrapure water is mended to cumulative volume 20 μ l, and 3000 ~ 5000g is centrifugal, and mix 2 ~ 5 seconds, is placed in amplified reaction in PCR instrument; Reaction conditions is: unwinds 2 ~ 5 minutes by 94 ~ 96 ℃, and then 94 ~ 96 ℃ of sex change 40 ~ 60 seconds, 50 ~ 56 ℃ of renaturation 60 seconds, 72 ℃ are extended 40 ~ 90 seconds, 25 ~ 35 circulations, last 72 ℃ are extended 6 ~ 10 minutes, obtain mycoplasma hyorhinis first round PCR product;
3) with another, Auele Specific Primer Mhr P03, Mhr P04 are carried out to second to mycoplasma hyorhinis first round PCR product and take turns pcr amplification;
Wherein: the sequence of upstream primer Mhr P03 is: 5 '-GTAGTCAAGCAAGAGGATGT-3 ';
The sequence of downstream primer Mhr P04 is: 5 '-GCTGGAGTTATTATACCAGGA-3 ';
Above-mentioned primer Mhr P03, Mhr P04 are respectively by the synthetic DNA fragmentation of base sequence by DNA synthesizer; Can be according to document Caron J et al., 2000, Diagnosis and Differentiation of
mycoplasma hyopneumoniaeand
mycoplasma hyorhinisinfections in Pigs by PCR Amplification of the p36 and p46 Genes sequence is synthesized.
Described second takes turns pcr amplification method is: in PCR Reagent Tube II, add mycoplasma hyorhinis first round PCR product to react, its reaction solution cumulative volume is 20 μ l, comprise: mycoplasma hyorhinis first round PCR product 0.5 ~ .1.0 μ l, 10 × PCR Buffer (Mg
2+free) 1.0 ~ 2.5 μ l, 25mM MgCl
21.0 ~ 2.0 μ l, 2.5mM dNTPs 1.0 ~ 3.0 μ l, 10pmol/ μ l upstream primer Mhr P03 0.5 ~ 1.0 μ l, 10pmol/ μ l downstream primer Mhr P04 0.5 ~ 1.0 μ l, 5 units/μ l TaqDNA polysaccharase, 0.2 ~ 0.5 μ l, sterilizing ultrapure water is mended to cumulative volume 20 μ l, and 3000 ~ 5000g is centrifugal, and mix 2 ~ 5 seconds, is placed in amplified reaction in PCR instrument; Reaction conditions is: unwinds 2 ~ 5 minutes by 94 ~ 96 ℃, and then 94 ~ 96 ℃ of sex change 40 ~ 60 seconds, 46 ~ 50 ℃ of renaturation 60 seconds, 72 ℃ are extended 60 ~ 90 seconds, 25 ~ 30 circulations, last 72 ℃ are extended 6 ~ 10 minutes, obtain mycoplasma hyorhinis second and take turns PCR product;
4) electrophoresis detection
Get mycoplasma hyorhinis second and take turns PCR product 5 ~ 10 μ l, with 2 ~ 6 μ l 0.25%(mass/volume) tetrabromophenol sulfonphthalein sample-loading buffer mixes, containing 0.8 ~ 2%(mass/volume) 0.7 ~ 1%(mass/volume of Goldview) sepharose electrophoresis 20 ~ 30 minutes under voltage 80 ~ 100V, the detection of taking pictures under ultraviolet transmissive lamp in gel imaging system, it is the positive that has mycoplasma hyorhinis to infect or pollute that sample well has the nucleic acid band of a 346bp, can determine and in testing sample, contain pathogenic bacteria mycoplasma hyorhinis, otherwise not have.
Above-mentioned dNTPs is the mixture that dTTP and dATP, dCTP, tetra-kinds of concentration of dGTP are respectively 2.5mM Nucleotide.
The concrete component of the reaction solution in the PCR reaction tubes I of described first round pcr amplification can be respectively:
1, testing sample mycoplasma hyorhinis DNA profiling extracting solution 0.5 μ l, 10 × PCR Buffer (Mg
2+free) 1.0 μ l, 25mM MgCl
21.0 μ l, 2.5mM dNTPs 1.0 μ l, 10pmol/ μ l upstream primer Mhr P01 0.5 μ l, 10pmol/ μ l downstream primer Mhr P02 0.5 μ l, 5 units/μ l TaqDNA polysaccharase, 0.2 μ l, sterilizing ultrapure water is mended to cumulative volume 20 μ l.
2, testing sample mycoplasma hyorhinis DNA profiling extracting solution 0.5 μ l, 10 × PCR Buffer (Mg
2+free) 2.5 μ l, 25mM MgCl
22.0 μ l, 2.5mM dNTPs 3.0 μ l, 10pmol/ μ l upstream primer Mhr P01 1.0 μ l, 10pmol/ μ l downstream primer Mhr P02 1.0 μ l, 5 units/μ l TaqDNA polysaccharase, 0.5 μ l, sterilizing ultrapure water is mended to cumulative volume 20 μ l.
3, testing sample mycoplasma hyorhinis DNA profiling extracting solution 1.0 μ l, 10 × PCR Buffer (Mg
2+free) 1.0 μ l, 25mM MgCl
21.0 μ l, 2.5mM dNTPs 1.0 μ l, 10pmol/ μ l upstream primer Mhr P01 0.5 μ l, 10pmol/ μ l downstream primer Mhr P02 0.5 μ l, 5 units/μ l TaqDNA polysaccharase, 0.2 μ l, sterilizing ultrapure water is mended to cumulative volume 20 μ l.
4, testing sample mycoplasma hyorhinis DNA profiling extracting solution 1.0 μ l, 10 × PCR Buffer (Mg
2+free) 2.5 μ l, 25mM MgCl
22.0 μ l, 2.5mM dNTPs 3.0 μ l, 10pmol/ μ l upstream primer Mhr P01 1.0 μ l, 10pmol/ μ l downstream primer Mhr P02 1.0 μ l, 5 units/μ l TaqDNA polysaccharase, 0.5 μ l, sterilizing ultrapure water is mended to cumulative volume 20 μ l.
5, testing sample mycoplasma hyorhinis DNA profiling extracting solution 1.0 μ l, 10 × PCR Buffer (Mg
2+free) 2.0 μ l, 25mM MgCl
22.0 μ l, 2.5mM dNTPs 2.0 μ l, 10pmol/ μ l upstream primer Mhr P01 0.5 μ l, 10pmol/ μ l downstream primer Mhr P02 0.5 μ l, 5 units/μ l TaqDNA polysaccharase, 0.2 μ l, sterilizing ultrapure water is mended to cumulative volume 20 μ l.
In sum, the component of the reaction solution in the PCR reaction tubes I of first round pcr amplification is all instead just successes in following ranges, that is: testing sample mycoplasma hyorhinis DNA profiling extracting solution 0.5 ~ 1.0 μ l, 10 × PCR Buffer (Mg
2+free) 1.0 ~ 2.5 μ l, 25mM MgCl
21.0 ~ 2.0 μ l, 2.5mM dNTPs 1.0 ~ 3.0 μ l, 10pmol/ μ l upstream primer Mhr P01 0.5 ~ 1.0 μ l, 10pmol/ μ l downstream primer Mhr P02 0.5 ~ 1.0 μ l, 5 units/μ l TaqDNA polysaccharase, 0.2 ~ 0.5 μ l, sterilizing ultrapure water is mended to cumulative volume 20 μ l.
The described second concrete component of taking turns the reaction solution in the PCR reaction tubes II of pcr amplification can be respectively:
1, mycoplasma hyorhinis first round PCR product 0.5 μ l, 10 × PCR Buffer (Mg
2+free) 1.0 μ l, 25mM MgCl
21.0 μ l, 2.5mM dNTPs 1.0 μ l, 10pmol/ μ l upstream primer Mhr P03 0.5 μ l, 10pmol/ μ l downstream primer Mhr P04 0.5 μ l, 5 units/μ l TaqDNA polysaccharase, 0.2 μ l, sterilizing ultrapure water is mended to cumulative volume 20 μ l,
2, mycoplasma hyorhinis first round PCR product 0.5. μ l, 10 × PCR Buffer (Mg
2+free) 2.5 μ l, 25mM MgCl
22.0 μ l, 2.5mM dNTPs 3.0 μ l, 10pmol/ μ l upstream primer Mhr P03 1.0 μ l, 10pmol/ μ l downstream primer Mhr P04 1.0 μ l, 5 units/μ l TaqDNA polysaccharase, 0.5 μ l, sterilizing ultrapure water is mended to cumulative volume 20 μ l,
3, mycoplasma hyorhinis first round PCR product 1.0 μ l, 10 × PCR Buffer (Mg
2+free) 1.0 μ l, 25mM MgCl
21.0 μ l, 2.5mM dNTPs 1.0 μ l, 10pmol/ μ l upstream primer Mhr P03 0.5 μ l, 10pmol/ μ l downstream primer Mhr P04 0.5 μ l, 5 units/μ l TaqDNA polysaccharase, 0.2 μ l, sterilizing ultrapure water is mended to cumulative volume 20 μ l.
4, mycoplasma hyorhinis first round PCR product 1.0 μ l, 10 × PCR Buffer (Mg
2+free) 2.5 μ l, 25mM MgCl
22.0 μ l, 2.5mM dNTPs 3.0 μ l, 10pmol/ μ l upstream primer Mhr P03 1.0 μ l, 10pmol/ μ l downstream primer Mhr P04 1.0 μ l, 5 units/μ l TaqDNA polysaccharase, 0.5 μ l, sterilizing ultrapure water is mended to cumulative volume 20 μ l.
5, mycoplasma hyorhinis first round PCR product 1.0 μ l, 10 × PCR Buffer (Mg
2+free) 2.0 μ l, 25mM MgCl
22.0 μ l, 2.5mM dNTPs 2.0 μ l, 10pmol/ μ l upstream primer Mhr P03 0.5 μ l, 10pmol/ μ l downstream primer Mhr P04 0.5 μ l, 5 units/μ l TaqDNA polysaccharase, 0.2 μ l, sterilizing ultrapure water is mended to cumulative volume 20 μ l.
In sum, second takes turns component all instead just successes in following ranges of the reaction solution in the PCR reaction tubes II of pcr amplification, that is: mycoplasma hyorhinis first round PCR product 0.5 ~ 1.0 μ l, 10 × PCR Buffer (Mg
2+free) 1.0 ~ 2.5 μ l, 25mM MgCl
21.0 ~ 2.0 μ l, 2.5mM dNTPs 1.0 ~ 3.0 μ l, 10pmol/ μ l upstream primer Mhr P03 0.5 ~ 1.0 μ l, 10pmol/ μ l downstream primer Mhr P04 0.5 ~ 1.0 μ l, 5 units/μ l TaqDNA polysaccharase, 0.2 ~ 0.5 μ l, sterilizing ultrapure water is mended to cumulative volume 20 μ l.
1. measure by the variable color unit of mycoplasma hyorhinis, the content that records mycoplasma hyorhinis is 10
9cCU/mL;
2. by above-mentioned mycoplasma hyorhinis thalline stoste as gradient dilution be: 10
7cCU/mL, 10
6cCU/mL, 10
5cCU/mL, 10
4cCU/mL, 10
3cCU/mL, 10
2cCU/mL, extract the DNA of each thalline, get 1 μ l
For template, only carry out pcr amplification one time with Auele Specific Primer Mhr P01/P02 respectively, amplification rear electrophoresis the results are shown in Figure 2.Through increasing and all obtain equifinality in triplicate.
In Fig. 2, M is 2000bp DNA marker; 1-6 represents that DNA profiling is respectively 10
7cCU/mL, 10
6cCU/mL, 10
5cCU/mL, 10
4cCU/mL, 10
3cCU/mL, 10
2a pcr amplification product of the mycoplasma hyorhinis of CCU/mL.Fig. 2 demonstration, is used separately Auele Specific Primer Mhr P01/P02 to carry out pcr amplification one time, and the minimum pathogenic bacteria detecting is 10
6cCU/mL.
Be 10 by mycoplasma hyorhinis thalline stoste as gradient dilution
9cCU/mL, 10
8cCU/mL, 10
7cCU/mL, 10
6cCU/mL, 10
5cCU/mL, 10
4cCU/mL, 10
3cCU/mL, 10
2cCU/mL, 10
1cCU/mL, 10
0cCU/mL, 10
-1cCU/mL, 10
-2cCU/mL.Extract after DNA, get 1 μ l
Make template, carry out successively the first round, second by the step 3 of embodiment 1 and step 4 and take turns two-wheeled amplification.
Electrophoresis result through the product of two-wheeled amplification gained is shown in Fig. 3, and in figure, M is 2000bp DNA Marker; 1-12 represents that DNA profiling is respectively 10
9cCU/mL, 10
8cCU/mL, 10
7cCU/mL, 10
6cCU/mL, 10
5cCU/mL, 10
4cCU/mL, 10
3cCU/mL, 10
2cCU/mL, 10
1cCU/mL, 10
0cCU/mL, 10
-1cCU/mL, 10
-2the amplified production of the mycoplasma hyorhinis of CCU/mL, 13 is blank, 14 negative contrasts.Fig. 3 demonstration, through two-wheeled pcr amplification, the minimum pathogenic bacteria detecting is 10
2cCU/mL.
Compared with Fig. 2 of embodiment 3, visible Nested PCR of the present invention adopts twice PCR amplification, and the sensitivity of its detection is used separately special primer Mhr P01/P02 to carry out the detection sensitivity of a pcr amplification far above embodiment 3.Mycoplasma hyorhinis is detected to advantage and the effect such as there is high specificity, highly sensitive and quarantine cycle is short.
21 parts of the clinical samples gathering from pig farm, get 30mg tissue, extract DNA by embodiment 1 step 1, carry out successively the first round and second take turns two-wheeled amplification by the step 2 of embodiment 1 and step 3.
Electrophoresis result after two-wheeled pcr amplification is shown in Fig. 4, visible in figure, in 21 increments bases, has 11 duplicate samples to occur specificity 346bp, and positive rate is 52.4%.Can find in time that mycoplasma hyorhinis infects.
EQUENCE LISTING
<110> Jiangsu Province Agriculture Science Institute
<120> mycoplasma hyorhinis nested PCR detection method
<130> specification sheets
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 23
<212> DNA
<213> artificial sequence
<400> 1
gcatctatat tttcgccaat agc
23
<210> 2
<211> 21
<212> DNA
<213> artificial sequence
<400> 2
agctagagtt tcatcattac c
21
<210> 3
<211> 20
<212> DNA
<213> artificial sequence
<400> 3
gtagtcaagc aagaggatgt
20
<210> 4
<211> 21
<212> DNA
<213> artificial sequence
<400> 4
gctggagtta ttataccagg a
21
Claims (2)
1. a mycoplasma hyorhinis nest-type PRC primer pair, it is characterized in that by first round PCR Auele Specific Primer to and second take turns PCR Auele Specific Primer to forming, described second takes turns PCR Auele Specific Primer to the right amplified production of first round PCR Auele Specific Primer is carried out to pcr amplification;
Described first round PCR Auele Specific Primer is as follows to nucleotide sequence:
Upstream primer is 5 '-GCATCTATATTTTCGCCAATAGC-3 ',
Downstream primer is 5 '-AGCTAGAGTTTCATCATTACC-3 ';
Described second to take turns PCR Auele Specific Primer as follows to nucleotide sequence:
Upstream primer is 5 '-GTAGTCAAGCAAGAGGATGT-3 ',
Downstream primer is 5 '-GCTGGAGTTATTATACCAGGA-3 '.
2. mycoplasma hyorhinis nest-type PRC primer pair as claimed in claim 1 is for the preparation of the purposes detecting in mycoplasma hyorhinis reagent.
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| CN1676612A (en) * | 2004-03-29 | 2005-10-05 | 武汉市第一医院 | Method for detecting and identifying mycoplasma |
| CN101580867A (en) * | 2008-05-14 | 2009-11-18 | 北京泰格瑞分子检验有限公司 | Reporting gene amplification kit for detecting mycoplasma |
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| CN1676612A (en) * | 2004-03-29 | 2005-10-05 | 武汉市第一医院 | Method for detecting and identifying mycoplasma |
| CN101580867A (en) * | 2008-05-14 | 2009-11-18 | 北京泰格瑞分子检验有限公司 | Reporting gene amplification kit for detecting mycoplasma |
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| Title |
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| Diagnosis and Differentiation of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis Infections in Pigs by PCR Amplification of the p36 and p46 Genes;J. CARON, et al.;《JOURNAL OF CLINICAL MICROBIOLOGY》;20000430;第38卷(第4期);第1391页表2，第1393页左栏倒数第1段-右栏第1段 * |
| J. CARON, et al..Diagnosis and Differentiation of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis Infections in Pigs by PCR Amplification of the p36 and p46 Genes.《JOURNAL OF CLINICAL MICROBIOLOGY》.2000,第38卷(第4期),第1391页表2，第1393页左栏倒数第1段-右栏第1段. |
| 李菁竹.支原体污染的巢式PCR检测法的摸索及其初步验证.《中国优秀硕士学位论文全文数据库，医药卫生科技辑》.2010,第2页第1段第6行、第2段第1-4行，第3页第2段第2行. * |
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