CN106222256A - A kind of Multiple immunizations fluorescence analysis method detecting chicken virus mycoplasma and chicken Mycoplasma synoviae - Google Patents
A kind of Multiple immunizations fluorescence analysis method detecting chicken virus mycoplasma and chicken Mycoplasma synoviae Download PDFInfo
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Abstract
The invention discloses a kind of multi-fluorescence immune analysis method for detecting chicken virus mycoplasma and chicken Mycoplasma synoviae.The present invention is simple to operate, obtains target amplification fragment by PCR, then amplified production, fluorescence-encoded micro-beads and Streptavidin phycoerythrin is hybridized, and then reads MFI value by detector, offers an explanation different types of cause of disease.The inventive method, it is possible to detection and identification chicken virus mycoplasma and chicken Mycoplasma synoviae simultaneously, and have high specificity, highly sensitive, the advantage such as reproducible, the multiple different molecules of interest that can realize in same sample detect simultaneously.Motility of the present invention is good, can add and subtract the kind of detection cause of disease the most on this basis.
Description
Technical field
The invention belongs to the Pathogen test field of aquaculture, be specifically related to a kind of detection chicken virus mycoplasma and chicken synovial fluid rami capsulares
The multi-fluorescence immune analysis method of substance.
Background technology
Mycoplasma gallinarum disease is the infectious disease caused by mycoplasma, based on directly contact through respiratory tract infection with through egg infection
Want route of transmission, the most common are chicken virus mycoplasma (MG) and Mycoplasma synoviae (MS).Chicken virus mycoplasma mainly causes chicken slow
Property respiratory symptom, airsacculitis, egg drop reduction, be that aviculture causes in mycoplasma serious financial consequences person；Chicken synovial capsule
Mycoplasma mainly causes the arthropathy of chicken and subclinical breathing
Road symptom, airsacculitis and arthropathy cause the discarded rate of broiler chicken ketoboidies substantially to increase.Often occur that both are same clinically
Time mixed infection situation.Chicken group is by after mycoplasma infection, and the immunologic function of its body reduces, cause to animal infectious diease because of
Susceptibility increases, and causes mortality rate to increase, and production performance reduces and causes serious economic loss.In recent years, along with intensive
The development of poultry husbandry, in chicken group, mycoplasma infection is the most universal, and some chicken group's mycoplasma infection rates have been up to 56.9 %, seriously
Hinder the sound development of poultry husbandry.
At present the routine diagnostic method of MG and MS is mainly taked serology and 2 kinds of methods of separation and Culture.Serology side
Method is owing to often occurring that nonspecific cross reaction affects the accuracy of diagnosis；And separation and Culture, owing to it is to required condition
Require harshness, waste time and energy, be not suitable with the needs of modern intensive aviculture development.
It is widely used for this situation molecular biology method, establishes PCR/ multiplex PCR, fluorescence the most both at home and abroad fixed
The detection methods such as amount PCR.The advantage that although round pcr has high specificity, sensitivity is high, but result judges to need electrophoresis, takes
Time laborious, and product easily produces pollution and causes false positive.Fluorescent quantitative PCR technique has merged the multiple advantage of PCR,
Quantitative to molecules of interest is realized, it is not necessary to electrophoresis detection by the change of fluorescence signal in directly detection PCR course of reaction, and
And the operation of whole process complete stopped pipe type, pollution probability reduces, it is to avoid the false positive issue that Standard PCR easily causes.Relatively
Standard PCR, quantitative fluorescent PCR has advantage at aspects such as sensitivity, specificity and speed, but in actual applications, works as sample
When measuring the biggest, substance fluorescent PCR is at cost and exists for certain inferior position in terms of the time, and sets up a multiple fluorescence PCR
Method is more more complex than substance, and it is higher to the requirement of reagent and primer, simultaneously need to
Ensure that, without interfering between the fluorophor of different probe institute labelling, the quantitative real time PCR Instrument of use has the most
Individual sense channel, makes experiment difficulty strengthen.At present, yet there are no application Multiple immunizations fluorescence analysis method to chicken virus mycoplasma and
Chicken Mycoplasma synoviae carries out the report detected.
Summary of the invention
In order to solve the deficiency in the presence of prior art, it is an object of the present invention to provide a kind of detection chicken poison and prop up
The multi-fluorescence immunoassay primer sets of substance and chicken Mycoplasma synoviae；
Further object is that a kind of multi-fluorescence detecting chicken virus mycoplasma and chicken Mycoplasma synoviae of offer is exempted from
Epidemic disease assay kit；
Further object is that a kind of multi-fluorescence detecting chicken virus mycoplasma and chicken Mycoplasma synoviae of offer is exempted from
Epidemic disease analyzes method.
The technical solution used in the present invention is:
One is used for detecting chicken virus mycoplasma (Mycoplasma gallisepticum, MG) and chicken Mycoplasma synoviae
The multi-fluorescence immunoassay primer sets of (Mycoplasma synoviae, MS), comprising: for detecting chicken virus mycoplasma
Primer is to G1 and G2, for detecting the primer of chicken Mycoplasma synoviae to S1 and S2, it is characterised in that: described for detecting chicken poison
The conserved sequence design to being PVPA gene according to chicken virus mycoplasma of the primer of mycoplasma；Described for detecting chicken synovial capsule
The conserved sequence design to being vlhA gene according to chicken Mycoplasma synoviae of the primer of mycoplasma.
As preferably, the nucleotide sequence of the described primer pair for detecting chicken virus mycoplasma is as follows:
MG primer G1:5 '-CTTACAGATTACAGGAGCAG-3 ' (SEQ ID NO.1),
MG primer G2:5 '-AAGCATTTCATTTGTTTTAC-3 ' (SEQ ID NO.2)；
As follows for detecting the nucleotide sequence of the primer pair of chicken Mycoplasma synoviae:
MS primer S1:5 '-ATTAGCAGCTAGTGCAGTGG-3 ' (SEQ ID NO.3),
MS primer S2:5 '-TTTGAGGATTATCAGTATTTG-3 ' (SEQ ID NO.4).
Preferably, the 5 ' ends of primer G1 and S1 are added with tag sequence also by spacer sequence.
Preferably, the tag sequence that the 5 ' of primer G1 and S1 are held is respectively as follows:
The tag sequence of primer G1 is: 5 '-CACTACACATTTATCATAACAAAT-3 ' (SEQ ID NO.5)；
The tag sequence of primer S1 is: 5 '-CTTTCTTAATACATTACAACATAC-3 ' (SEQ ID NO.6).
Preferably, the 5 ' ends of primer G2 and S2 have been also added with biotin labeling.
A kind of multi-fluorescence immunoassay kits for detecting chicken virus mycoplasma and chicken Mycoplasma synoviae, its feature
Being, described test kit includes: (1) above-described multi-fluorescence immunoassay primer sets；(2) 2 kinds of different fluorescence of coding
The fluorescence-encoded micro-beads including anti-tag sequence of color, described anti-tag sequence can correspondingly be divided with multi-fluorescence immunity
Tag complementary pairing on the forward primer of analysis primer sets；(3) SA-PE complex.
A kind of multi-fluorescence immune analysis method for detecting chicken virus mycoplasma and chicken Mycoplasma synoviae, including as follows
Step:
1) from sample, extract DNA, with DNA as template, add the primer sets described in any of the above item and carry out PCR amplification；
2) fluorescence-encoded micro-beads by amplified production, including anti-tag sequence and SA-PE hybridization；Miscellaneous
After knot bundle, utilize instrument to be analyzed, determine the type of its mycoplasma；
Preferably, step 2) in fluorescence-encoded micro-beads on be coated with special anti-tag sequence, described anti-tag sequence
Can correspondingly match with the tag complementary in claim 3.
Preferably, in step 1), the reaction system of PCR amplification is:
2×Multiplex PCR Master Mix 10ul
Forward primer mixed liquor 1ul(0.2uM, final conc.)
Downstream primer mixed liquor 1ul(0.2uM, final conc.)
Template 1ul(＜ 500ng)
ddH2O 7ul
Total 20ul
Preferably, in step 1), the response procedures of PCR amplification is: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 60 DEG C of annealing
90s, 72 DEG C extend 20s；Circulate 35 times；72 DEG C extend 10min eventually.
Preferably, step 2) in amplified production, fluorescence-encoded micro-beads and SA-PE hybridization system
For:
Fluorescence-encoded micro-beads 20 L
SA-PE 75 L
Amplified production 5 L
Total 100µL
37 DEG C of reaction 30min.
The invention has the beneficial effects as follows:
The specific tag sequence that the forward primer 5 ' of the present invention is held, can be coated with 2 kinds of special anti-tag sequence
Fluorescence-encoded micro-beads combines, and the Biotin labelling that downstream primer 5 ' is held, can be with SA-PE (SA-PE)
Hybridize.When chicken virus mycoplasma and chicken Mycoplasma synoviae are detected by the inventive method simultaneously, obtain target by PCR
Amplified fragments, then hybridizes amplified production, fluorescence-encoded micro-beads and SA-PE (SA-PE), then
When reading MFI value by detector, offer an explanation different types of mycoplasma.
Multi-fluorescence immunoassay (MFIA) and TAG technology are combined by the method for the present invention, and TAG technology uses
The universal tag that Luminex is proprietary, by the specificity complementary pairing of sequence label Yu anti-sequence label, carries out nucleic acid experiment excellent
Change, product development and molecular diagnostic assay.TAG technology can guarantee that identical renaturation temperature and hybridization efficiency, and is prevented effectively from not
With crisscrossing between the microsphere of detectable substance labelling.
The inventive method, it is possible to simultaneously chicken virus mycoplasma and chicken Mycoplasma synoviae are accurately detected, and special
Property strong, highly sensitive, reproducible.Compared with traditional detection method, it is multiple that the inventive method achieves in same sample
Different molecules of interest detect simultaneously, and sample consumption is few, simple to operate, quick, can be substantially reduced testing cost.
Accompanying drawing explanation
Fig. 1 is chicken virus mycoplasma and the electrophoretogram of 2 kinds of cause of diseases PCR of chicken Mycoplasma synoviae；
Fig. 2 is the multi-fluorescence immune analysis method test experience result of chicken virus mycoplasma and 2 kinds of cause of diseases of chicken Mycoplasma synoviae
Figure；
Fig. 3 is that the multi-fluorescence immune analysis method detection specificity of chicken virus mycoplasma and 2 kinds of cause of diseases of chicken Mycoplasma synoviae is real
Test result figure；
Fig. 4 is that the multi-fluorescence immune analysis method detection sensitivity of chicken virus mycoplasma and 2 kinds of cause of diseases of chicken Mycoplasma synoviae is real
Test result figure；
Fig. 5 is the multi-fluorescence immune analysis method Clinical detection experiment of chicken virus mycoplasma and 2 kinds of cause of diseases of chicken Mycoplasma synoviae
Result figure.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but is not limited thereto.
Embodiment 1 design of primers
Design one group for detecting the multi-fluorescence immunoassay primer of chicken virus mycoplasma and chicken Mycoplasma synoviae wherein, described
For detecting the conserved sequence design to being PVPA gene according to chicken virus mycoplasma of the primer of chicken virus mycoplasma；Described for
The conserved sequence design to being vlhA gene according to chicken Mycoplasma synoviae of the primer of detection chicken Mycoplasma synoviae.Pass through
After repetition test and screening, obtain following primer sequence:
As follows for detecting the nucleotide sequence of the primer pair of chicken virus mycoplasma:
MG primer G1:5 '-CTTACAGATTACAGGAGCAG-3 ' (SEQ ID NO.1),
MG primer G2:5 '-AAGCATTTCATTTGTTTTAC-3 ' (SEQ ID NO.2)；
As follows for detecting the nucleotide sequence of the primer pair of chicken Mycoplasma synoviae:
MS primer S1:5 '-ATTAGCAGCTAGTGCAGTGG-3 ' (SEQ ID NO.3),
MS primer S2:5 '-TTTGAGGATTATCAGTATTTG-3 ' (SEQ ID NO.4).
5 ' the ends of primer G1 and S1 are added with tag sequence also by spacer sequence, and tag sequence is respectively as follows:
The tag sequence of primer G1 is: 5 '-CACTACACATTTATCATAACAAAT-3 ' (SEQ ID NO.5)；
The tag sequence of primer S1 is: 5 '-CTTTCTTAATACATTACAACATAC-3 ' (SEQ ID NO.6).
Embodiment 2: for detecting the foundation of the multi-fluorescence immunoassay kits of MS, MG
Test kit includes following components:
(1) 2 weight fluorescence immunoassay primer sets designed by embodiment 1；
The fluorescence-encoded micro-beads including anti-tag sequence of (2) 2 kinds of different iridescent of coding, described anti-tag sequence energy
Correspondingly match with the tag complementary on multi-fluorescence immunoassay primer sets forward primer；Two kinds of microspheres are purchased from
Luminex company, fluorescence-encoded micro-beads number the most corresponding for concrete MS with MG is MTAG-A025 and MTAG-042.
(3) SA-PE complex；
(4) pcr amplification reaction system.
The foundation of the multi-fluorescence immune analysis method detection method of embodiment 3 MS, MG
1, the structure of plasmid
Automatically extract instrument with the nucleic acid of sky root and extract the DNA of MG, MS cause of disease, respectively corresponding the drawing designed by embodiment 1 respectively
Thing, carries out PCR amplification, amplified production carries out agarose gel electrophoresis detection respectively and cuts glue purification.With TaKaRa company
CDNA after purification is connected in pMD-19T carrier by test kit, will connect product and convert to DH5a competent cell, and select list
Clone, carries out bacterium colony PCR qualification, the bacterium colony being accredited as positive bacteria is carried out plasmid extraction, send order-checking.
2, plasmid PCR amplification
Substance, double PCR amplification is carried out with specific amplification MS, MG primer.
The preparation of forward primer mixed liquor: G1 and S1 is mixed with 1:1 ratio；The preparation of downstream primer mixed liquor:
G2 and S2 is mixed with 1:1 ratio.Utilize the specific template of two kinds of cause of diseases, and the double template of MS and MG, to above-mentioned
The atopy region of two kinds of cause of diseases expands.The preparation of the most double template: plasmid two-by-two is mixed with the ratio of 1:1
Close.
Pcr amplification reaction system is as follows:
2×Multiplex PCR Master Mix 10ul
Forward primer mixed liquor 1ul(0.2uM, final conc.)
Downstream primer mixed liquor 1ul(0.2uM, final conc.)
Template 1ul(＜ 500ng)
ddH2O 7ul
Total 20ul
The response procedures of amplification is:
94 DEG C of denaturations 5min；
94 DEG C of degeneration 30s, 60 DEG C of annealing 90s, 72 DEG C extend 20s；Circulate 35 times；
72 DEG C extend 10min eventually.
PCR primer carries out agarose gel electrophoresis analysis, and its electrophoretogram is as shown in Figure 1.1:100bp DNA marker, 2:
MG, 3:MS, 4:MS+MG, 5:PCR blank.
From figure 1 it appears that the amplified production size that the amplified production size of MG is about 133bp, MS is about 141bp,
Owing to the amplified production size of both cause of diseases is close, so the electrophoretic band of double PCR amplified production cannot be differentiated.
3, PCR primer and fluorescence-encoded micro-beads working solution, the hybridization of Streptavidin phycoerythrin (SA-PE) working solution, bag
Include following steps:
Be coated with 2 kinds of microspheres of special anti-tag sequence respectively, wherein anti-tag sequence can correspondingly with MS and MG two
Plant the tag complementary pairing on cause of disease primer.Two kinds of microspheres are purchased from luminex company, and concrete MS with MG is the most corresponding
Fluorescence-encoded micro-beads number be MTAG-A025 and MTAG-042.
The preparation of fluorescence-encoded micro-beads working solution: by 2500/μ l fluorescence-encoded micro-beads with 1.1 × Tm
Hybrdization Buffer is diluted to 1 μ l and about contains 125/kind fluorescence-encoded micro-beads.
Prepared by SA-PE working solution: 1mg/ml SA-PE 1 × Tm Hybrdization Buffer is diluted to 10 μ g/ μ
l。
Abundant resuspended fluorescence-encoded micro-beads working solution, each sample well and background hole add microsphere working solution 20 μ l, sample
Hole adds 5 μ l PCR primer, background hole adds 5 μ l PCR blank products, adds the SA-PE working solution of 75 μ l, fill
Divide mixing, in metal heater, hatch 30min for 37 DEG C.
According to the explanation of detector Luminex 200 instrument, the 50 above-mentioned reactant liquors of μ l after hybridization are detected, result
As shown in Figure 3, although the amplified production of double template cannot be differentiated with electrophoresis, but read with detector Luminex 200 instrument
During MFI value, hence it is evident that offer an explanation different types of cause of disease.
Result criterion (result criterion also can be adjusted by note: this criterion is only for reference) is as follows:
The determination of lowest detection threshold value (cutoff value): choose 10 healthy chicken tissue samples (each sample parallel is repeated 3 times),
Read MFI value respectively and calculate its meansigma methods and standard deviation.The MIF value adding 3 times of standard deviations with meansigma methods sets it as cutoff
Value.The present invention obtained cutoff value is 278.5, therefore the cutoff value of the present invention is set to 300.Only detect sample
When MFI value is higher than 300, this experimental data just can effectively be analyzed.
The analysis of sample to be tested judges: 1) as the MFI value ＞ 300 of sample to be tested, it is judged that for positive sample；2) test sample is treated
During this MFI value≤300, it is judged that for feminine gender, need to carry out repeating test or take other detection methods to verify further.
The multi-fluorescence immunoassay detection specificity experiments of embodiment 4 MS and MG
Respectively with chicken Mycoplasma synoviae (MS), chicken virus mycoplasma (MG), infectious laryngotracheitis virus (ILTV), avian infectious
Anemia virus (CAV), Marek’s disease poison (MDV) carry out multi-fluorescence immunoassay detection as template.Experimental result such as Fig. 4 institute
Show, only Mycoplasma synoviae, chicken virus mycoplasma, be positive, other be feminine gender, illustrate that detection system specificity is good.
The multi-fluorescence immunoassay detection sensitivity experiment of embodiment 5:MS and MG
The plasmid of preparation is carried out quantitatively, use 10 times of dilution methods to be diluted, be diluted to 101Copies/ μ l, uses above-mentioned foundation
Multi-fluorescence immune analysis method detect.The multi-fluorescence immunoassay detection sensitivity experimental result of MS and MG such as figure
Shown in 5, test result indicate that, the sensitivity detection of MG, MS is limited to 102copies/ul。
From the lung of chicken house collection, trachea, kidney, liver sample, automatically extract instrument with sky root nucleic acid and extract DNA, use Qiagen
Multiplex PCR Plus kit expand, using DNA as template, use the multi-fluorescence immunity of above-mentioned MS, MG to divide
Analysis detection method detects, amplified production and fluorescence-encoded micro-beads and SA-PE hybridization, reads on Luminex 200 detector
Number.Concrete steps are with reference to embodiment 3, and experimental result is as shown in Figure 5.
Showing through regular-PCR test experience result, sample 1,2,4,5,6,7,8,11,12,14,15,16,18,19,20 is
Negative sample；3,10 is Mycoplasma synoviae sample；9,13,17 is chicken virus mycoplasma sample.By sequencing analysis, as a result one
Cause.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-described embodiment
Limit, the change made under other any spirit without departing from the present invention and principle, modify, substitute, combine, simplify,
All should be the substitute mode of equivalence, within being included in protection scope of the present invention.
<110>Experimental Animals Supervising Station, Guangdong Prov.
<120>a kind of Multiple immunizations fluorescence analysis method detecting chicken virus mycoplasma and chicken Mycoplasma synoviae
<130>
<160> 6
<170> PatentIn version 3.5
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Claims (10)
1. for detecting a multi-fluorescence immunoassay primer sets for chicken virus mycoplasma and chicken Mycoplasma synoviae, comprising:
For detecting the primer of chicken virus mycoplasma to G1 and G2, for detecting the primer of chicken Mycoplasma synoviae to S1 and S2, its feature
It is: the conserved sequence design to being PVPA gene according to chicken virus mycoplasma of the described primer for detecting chicken virus mycoplasma
's；The described primer for the detecting chicken Mycoplasma synoviae conserved sequence to being vlhA gene according to chicken Mycoplasma synoviae
Design.
Multi-fluorescence immunoassay primer sets the most according to claim 1, it is characterised in that be used for detecting chicken virus mycoplasma
The nucleotide sequence of primer pair as follows:
MG primer G1:5 '-CTTACAGATTACAGGAGCAG-3 ' (SEQ ID NO.1),
MG primer G2:5 '-AAGCATTTCATTTGTTTTAC-3 ' (SEQ ID NO.2)；
As follows for detecting the nucleotide sequence of the primer pair of chicken Mycoplasma synoviae:
MS primer S1:5 '-ATTAGCAGCTAGTGCAGTGG-3 ' (SEQ ID NO.3),
MS primer S2:5 '-TTTGAGGATTATCAGTATTTG-3 ' (SEQ ID NO.4).
Primer the most according to claim 1 and 2, it is characterised in that: the 5 ' ends of primer G1 and S1 are also by spacer sequence
It is added with tag sequence.
Primer the most according to claim 3, it is characterised in that: the tag sequence that the 5 ' of primer G1 and S1 are held is respectively as follows:
The tag sequence of primer G1 is: 5 '-CACTACACATTTATCATAACAAAT-3 ' (SEQ ID NO.5)；
The tag sequence of primer S1 is: 5 '-CTTTCTTAATACATTACAACATAC-3 ' (SEQ ID NO.6).
Primer the most according to claim 1 and 2, it is characterised in that: the 5 ' ends of primer G2 and S2 have been also added with biotin mark
Note.
6., for detection chicken virus mycoplasma and a multi-fluorescence immunoassay kits for chicken Mycoplasma synoviae, it is special
Levying and be, described test kit includes: the primer sets described in (1) any one of claim 1-5；(2) 2 kinds of different iridescent of coding
The fluorescence-encoded micro-beads including anti-tag sequence, described anti-tag sequence can correspondingly with in claim 3 or 4
Tag complementary matches；(3) SA-PE complex.
7. for detecting a multi-fluorescence immune analysis method for chicken virus mycoplasma and chicken Mycoplasma synoviae, including walking as follows
Rapid:
From sample, extract DNA, with DNA as template, add the primer sets described in any one of claim 1-5 and carry out PCR amplification；
By affine to amplified production, the fluorescence-encoded micro-beads including anti-tag sequence of 2 kinds of different iridescent of coding and strepto-
Element-phycoerythrin hybridization；After hybridization terminates, utilize instrument to be analyzed, determine the type of its cause of disease.
Method the most according to claim 7, it is characterised in that: step 2) in fluorescence-encoded micro-beads on be coated with special
Anti-tag sequence, described anti-tag sequence can correspondingly be matched with the tag complementary in claim 3.
Method the most according to claim 7, it is characterised in that: in step 1), the reaction system of PCR amplification is:
2×Multiplex PCR Master Mix 10ul
Forward primer mixed liquor 1ul
Downstream primer mixed liquor 1ul
Template 1ul
ddH2O 7ul
Total 20ul
The response procedures of PCR amplification is: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 60 DEG C of annealing 90s, 72 DEG C extend 20s；Follow
Ring 35 times；72 DEG C extend 10min eventually.
Method the most according to claim 7, it is characterised in that: step 2) in amplified production, fluorescence-encoded micro-beads and strepto-
The system of Avidin-phycoerythrin hybridization is:
Fluorescence-encoded micro-beads 20 L
SA-PE 75 L
Amplified production 5 L
Total 100µL
37 DEG C of reaction 30min.
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Cited By (4)
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| CN109971872A (en) * | 2019-03-29 | 2019-07-05 | 南阳师范学院 | A kind of the polymerase spiral augmentation detection primer sets and kit of fowl Mycoplasma synoviae |
| CN110195120A (en) * | 2019-06-21 | 2019-09-03 | 青岛英赛特生物科技有限公司 | Duplex PCR primer sets and kit a kind of while that detect MS-H vaccine strain and versatility |
| CN111537736A (en) * | 2020-05-18 | 2020-08-14 | 中国农业大学 | Indirect ELISA (enzyme-linked immunosorbent assay) detection kit and detection method for mycoplasma gallisepticum antibody |
| CN114703302A (en) * | 2022-03-29 | 2022-07-05 | 广西壮族自治区兽医研究所 | Method for detecting mycoplasma synoviae and reagent used by method |
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| CN114703302A (en) * | 2022-03-29 | 2022-07-05 | 广西壮族自治区兽医研究所 | Method for detecting mycoplasma synoviae and reagent used by method |
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