CN109929937A - The thermophilic skin bacterium specificity identification primer of tortoise, kit and its identification method - Google Patents
The thermophilic skin bacterium specificity identification primer of tortoise, kit and its identification method Download PDFInfo
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- CN109929937A CN109929937A CN201910257052.XA CN201910257052A CN109929937A CN 109929937 A CN109929937 A CN 109929937A CN 201910257052 A CN201910257052 A CN 201910257052A CN 109929937 A CN109929937 A CN 109929937A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses the thermophilic skin bacterium specificity identification primer of tortoise, kit and its identification methods.The specificity identification primer includes primer AC1 or AC2 or AC3.Using the thermophilic skin bacterium specificity identification primer of 3 pairs of tortoises of the invention, only can determine that with regular-PCR reagent and electrophoresis method as a result, easy to operate, it is low in cost.By the verifying with other bacterial strains, without non-specific amplification, illustrate that this 3 pairs of primer specificities are strong, and it is applied to the identification of the thermophilic skin bacterium of tortoise in crocodile lizard skin granuloma tubercle, have the characteristics that specific height, high sensitivity, at low cost, easy to operate, can be applied to animal, in microorganism or environmental samples the thermophilic skin bacterium of tortoise specific detection.
Description
Technical field
The present invention relates to microbial moleculars to identify field, and in particular to the thermophilic skin bacterium specificity identification primer of tortoise, kit
And its identification method.
Background technique
The main safeguard measure of the conservation of wildlife is habitat protection, but the disease research of wild animal also should be desirable
Extinction because serious disease is possible to cause endangered wildlife population quantity acutely wavy, or even is improved in the field of concern
Risk, if frog chytrid disease has been acknowledged as causing the driving force of field amphibian animal population sharply reduction, mycoplasma infection
Upper respiratory disease once caused the desert cave tortoise population in the U.S. sharply to reduce, and them is promoted to be listed in animals on the brink of extinction.
Chitinophile fungi has been reported that in mammality, birds, reptiles, the mankind, is Zoonosis skin disease, mainly dynamic
It is propagated in object, once leads to domestic animal huge economic losses.
The pathogen of chitinophile fungi first is that the thermophilic skin bacterium of tortoise (Austwickia chelonae), belong to Dermatophilaceae actinomyces,
It is a kind of Filamentous, gram-positive bacteria, is named as Dermatophilus chelonae in initial document, at 2010
A.chelonae is named as by the category new as one.Chitinophile fungi caused by the thermophilic skin bacterium of tortoise mainly in reptiles discovery compared with
It is more, it is initially to be found in a kind of Australian crocodile tortoise；It is also split into king snake (Ophiophagus hannah)；
It has been reported that the concurrent infection of Bearded Dragon (Pogona vitticeps) tortoise thermophilic skin bacterium and frog virus has been broken out in Japan；In recent years at me
The artificial rescue population of state first class of protection animal --- crocodile lizard has also broken out serious skin granulomatosis, and main pathogens are just
It is the thermophilic skin bacterium of tortoise；It finds in an experiment, Chinese skink can also be infected.In addition to reptiles, the thermophilic skin bacterium infection of tortoise is also in bird
It is reported in class and mammality, is such as preced with carrion crow meat (Corvus corone cornix) and ox.The injection thermophilic skin bacterium of tortoise can also lead
Cause the chitinophile fungi of sheep, rabbit and cavy.According to the Source Study of the pathogen to crocodile lizard skin granulomatosis, the thermophilic skin bacterium of tortoise may
In the water and soil of the environment of crocodile lizard life.For the body that finds the cause of disease in time, transmission of pathogen is cut off, avoids suffering a loss, compeled
Be essential the method for wanting the thermophilic skin bacterium of Rapid identification tortoise, especially early stage identification or environmental samples identification.Traditional identification method master
Will be by the means such as culture of isolated, pathological section, microexamination mycelia, time and effort consuming, and specificity and sensitivity are inadequate.Have
Report designs quantification PCR primer using 16S rRNA gene order, in amplification dermatophilus congolensis (Dermatophilus
Congolensis tortoise thermophilic skin bacterium is amplified while), the two is distinguished by solution temperature, to reach Rapid identification
Purpose.16S rRNA gene order conservative is high, therefore, is usually used in Bacteria Identification, still, also just since its conservative is strong,
It is easy to amplify the sequence of close bacterial strain simultaneously.On the other hand, quantitative fluorescent PCR reagent and instrument price are high, at high cost.Compel
Be essential the method for identifying the thermophilic skin bacterium of tortoise for wanting specificity good, at low cost.
Summary of the invention
The first purpose of the invention is to provide a kind of thermophilic skin bacterium specificity identification primers of tortoise comprising primer AC1 or AC2
Or AC3,
The primer AC1 includes: AC1-F:5'-TTTTGGCGAGTTTTGTACCCT-3'(SEQ ID NO.1)
AC1-R:5'-TCCCACAGCGATTAATTGACA-3'；(SEQ ID NO.2)
The primer AC2 includes: AC2-F:5'-CGGGAACTGGTCAAACCTT-3'(SEQ ID NO.3)
AC2-R:5'-CACAATAAGCGGACTAACCAA-3'；(SEQ ID NO.4)
The primer AC3 includes: AC3-F:5'-CCGTCGTCAAGCCCATCGTA-3'(SEQ ID NO.5)
AC3-R:5'-CGTGATCGGTTATGCACCCTA-3'(SEQ ID NO.6).
A second object of the present invention is to provide a kind of for identifying that the kit of the thermophilic skin bacterium of tortoise, the kit include above-mentioned
The thermophilic skin bacterium specificity identification primer AC1 or AC2 or AC3 of tortoise.
It is thermophilic using the above-mentioned thermophilic skin bacterium specificity identification primer progress tortoise of tortoise that third object of the present invention is to provide a kind of
The method (diagnostic and therapeutic method of non-disease) of the skin dientification of bacteria comprising following steps:
(1) total DNA of sample to be tested is extracted；
(2) using the total DNA of sample to be tested as template, using the above-mentioned thermophilic skin bacterium specificity identification primer AC1 of tortoise or
AC2 or AC3 carries out PCR amplification；
(3) amplified production is subjected to agarose gel electrophoresis analysis and gel imaging system test strip, if amplifying tortoise
The specific fragment of thermophilic skin bacterium then proves that sample to be tested contains the thermophilic skin bacterium of tortoise.
The PCR uses 25 μ L reaction systems, comprising: 2 × Taq PCR Mix, 12.5 μ L, 1.0 μ L of DNA profiling, mould
Plate usage amount can be adjusted according to the concentration of DNA, 0.5 μ L of forward and reverse primer, and end of the primer in PCR reaction system is dense
Degree is 0.2 μm of ol/L, uses ddH2O polishing is to 25 μ L.
The PCR response procedures are as follows: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 1min, 55-71 DEG C of annealing 1min, 72 DEG C
Extend 1-2min, total 30-35 circulation；Last 72 DEG C of extensions 8min.Wherein annealing temperature can be according to primer AC1, AC2 or AC3
Optimum annealing temperature be adjusted, when being expanded using primer AC1,72 DEG C of extension of time can use 1min.
Fourth object of the present invention is to provide a kind of characteristic nucleotide sequence for identifying the thermophilic skin bacterium of tortoise, this feature core
Nucleotide sequence is shown in SEQ ID NO.7 or SEQ ID NO.8 or SEQ ID NO.9.
Fifth object of the present invention is to provide the thermophilic skin bacterium specificity identification primers of above-mentioned tortoise or above-mentioned for identifying
The application of the kit of the thermophilic skin bacterium of tortoise thermophilic skin bacterium of tortoise in identification animal, microorganism or environmental samples.
Compared with prior art, the invention has the following beneficial effects:
The present invention is based on the whole genome sequences of the thermophilic skin bacterium of tortoise, develop the PCR primer of the thermophilic skin bacterium of 3 pairs of Rapid identification tortoises,
It only can determine that with regular-PCR reagent and electrophoresis method as a result, easy to operate, it is low in cost.By the verifying with other bacterial strains,
It is being compared in the nucleic acid database of NCBI the results show that the amplified production of 3 pairs of primers is not 16S without non-specific amplification
The conserved sequence of Bacteria Identification is used for known to rRNA gene, Cox1 gene etc., in the nucleic acid database of NCBI, with AC1 primer
The most like sequence of amplified production be Kytococcus sedentarius DSM 20547, similarity is only 76.31%, with
The most like sequence of the amplified production of AC2 primer is Arsenicicoccus sp.oral taxon 190, and similarity is only
84.98%, most like sequence is Nocardiodes dokdonensis FR1436 with the amplified production of AC3 primer, like degree
Only 75.12%, and the amplified production of 3 pairs of primers is and dermatophilus congolensis is without similitude, illustrates that this 3 pairs of primer specificities are strong,
And 3 pairs of primers are applied to the identification of the thermophilic skin bacterium of tortoise in crocodile lizard skin granuloma tubercle, there is specificity height, sensitivity
Feature high, at low cost, easy to operate, can be applied to animal, in microorganism or environmental samples the thermophilic skin bacterium of tortoise specific detection.
Detailed description of the invention
Fig. 1 is the specificity verification result of the thermophilic skin bacterium AC1 primer (A) of tortoise, AC2 primer (B) and AC3 primer (C).M:
Marker；1: positive control (Austwickia chelonae LK16-18)；2:Paenibacillus sp.LK16-15；3:
Salmonella enterica LK18-19；4:Pseudomonas protegens Exi5-13；5:Pseudomonas
putida DG56-2；6:Aeromonas sp.Exi4-46；7:Morganella morganii DG56-16；8:
Acinetobacter sp.Exi5-53；9:Acinetobacter sp.LK16-25；10:Glutamicibacter
sp.Exi1-5；11:Leucobacter sp.Exi1-11；12:Paenarthrobacter sp.Exi3-13；13:
Chryseobacterium sp.Ex1-9；14:Elizabethkingia sp.Exi2-17；15:Bacillus sp.Exi3-
7；16: hybrid dna；17: negative control (water).
Fig. 2 is the validity check result of the thermophilic skin bacterium AC1 primer (A) of tortoise, AC2 primer (B) and AC3 primer (C).M:
Marker；1: positive control (Austwickia chelonae LK16-18)；2:L.LK16；3:L.LK17；4:L.LK19；5:
L.LK20；6:L.LK21；7:L.LK22；8:L.LK07；9:L.LK13；10: negative control (water).
Fig. 3 is that the optimum annealing temperature of the AC1 primer (A) of the thermophilic skin bacterium of tortoise, AC2 primer (B) and AC3 primer (C) is groped to tie
Fruit.(A) M:Marker；1-11:55 DEG C -65 DEG C, it is spaced 1 DEG C；12: water.(B) M:Marker；1-12:60 DEG C -71 DEG C, interval 1
℃.(C) M:Marker；1-17:55 DEG C -71 DEG C, it is spaced 1 DEG C.
Specific embodiment
The following examples are further illustrations of the invention, rather than limiting the invention.
The common general T aq enzyme of pcr amplification reaction applied molecular biology, customary systems and program in following embodiments
Amplifiable, the present invention is matched using the Easy Taq PCR mixed liquor of Beijing Quanshijin Biotechnology Co., Ltd by following system
System:
Embodiment 1
(1) design of primers: graphic sequence is completed according to the thermophilic skin bacterium LK16-18 genome of tortoise, is designed within the scope of full-length genome
Special primer.
(2) specificity verification: tortoise Dermatophilus is in actinomyces door (Actinobacteria), gram-positive bacteria, therefore,
14 plants of selection separating from crocodile lizard lesion, belongs to fellow disciple, Gram-negative or the positive other bacteriums and mixing
DNA carries out the specificity verification of primer, and using the thermophilic skin bacterium LK16-18 of tortoise as positive control, water is as negative control.Mixing
DNA is mixed by the DNA of the thermophilic skin bacterium LK16-18 of tortoise and other the 14 plants bacteriums for verifying.Bacterial strain such as table 1 for verifying
It is shown.
All PCR use 25 μ L reaction systems, comprising: 2 × Taq PCR Mix, 12.5 μ L, DNA profiling 1.0 μ L, it is positive and negative
To 0.5 μ L of primer, final concentration of 10 μm ol/L, ddH of the primer in system2O 10.5μL。
The wherein PCR response procedures of AC1 primer are as follows: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 1min, 65 DEG C of annealing 1min,
72 DEG C of extension 1min, total 30-35 circulation；Last 72 DEG C of extensions 8min.
The PCR response procedures of AC2 primer and AC3 primer are as follows: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 1min, 65 DEG C of annealing
1min, 72 DEG C of extension 2min, total 30-35 circulation；Last 72 DEG C of extensions 8min.
It is verified by PCR, has 3 pairs of primers that can amplify the specific fragment of the thermophilic skin bacterium of tortoise, be named as AC1, AC2 respectively
And AC3, target fragment size are respectively 912bp, 1846bp and 1853bp.Primer sequence is as shown in table 2, and AC1 primer amplification produces
The sequence of object such as SEQ ID NO.7, the sequence of AC2 primer extension product such as SEQ ID NO.8, the sequence of AC3 primer extension product
Column are as shown in SEQ ID NO.9.Shown in the specificity verification the result is shown in Figure 1 of AC1 primer, AC2 primer and AC3 primer.By Fig. 1's
(A)-(C) can amplify the special of the thermophilic skin bacterium of very bright tortoise it is found that respectively using AC1, AC2, AC3 primer progress PCR amplification
Property segment, for all verifying bacterial strains without non-specific amplification, success amplifies the segment of the thermophilic skin bacterium of tortoise, sequencing knot from hybrid dna
Fruit shows that target fragment sequence is correct, illustrates that this 3 pairs of primer specificities are strong.
Table 1 is used for the bacterial strain of primer specificity verifying
The unique identification primer of the thermophilic skin bacterium (A.chelonae) of 2 tortoise of table
The amplified production of AC1, AC2, AC3 primer is compared in the nucleic acid database of NCBI respectively it is found that AC1, AC2,
The amplified production of this 3 pairs of primers of AC3 is not the conservative sequence that Bacteria Identification is used for known to 16S rRNA gene, Cox1 gene etc.
Column, most like sequence is Kytococcus sedentarius DSM20547 with the amplified production of AC1 primer, and similarity is
76.31%, most like sequence is Arsenicicoccus sp.oral taxon 190, phase with the amplified production of AC2 primer
It is 84.98% like degree, most like sequence is Nocardiodes dokdonensis with the amplified production of AC3 primer
FR1436 is 75.12% like degree, and the amplified production of 3 pairs of primers is and dermatophilus congolensis is without similitude.
(3) AC1, AC2, AC3 primer validation verification: are respectively used for amplifying to the crocodile lizard granulomatosis lesion of different samples
Total DNA, to verify the validity of primer, and using the thermophilic skin bacterium LK16-18 of tortoise as positive control, water is as negative control.It is different
The lesion DNA title of sample be respectively L.LK16, L.LK17, L.LK19, L.LK20, L.LK21, L.LK22, L.LK07,
L.LK13.The validity check result of the thermophilic skin bacterium AC1 primer of tortoise, AC2 primer and AC3 primer is as shown in Figure 2.By (A)-of Fig. 2
(C) it is found that carrying out PCR amplification using the crocodile lizard granulomatosis lesion of AC1, AC2, AC3 primer pair difference sample respectively, can have
Effect amplifies the specific fragment of the thermophilic skin bacterium of tortoise, and sequencing result shows that target fragment sequence is correct.
All PCR use 25 μ L reaction systems, comprising: 2 × Taq PCR Mix, 12.5 μ L, DNA profiling 1.0 μ L, it is positive and negative
To 0.5 μ L of primer, final concentration of 10 μm ol/L, ddH of each primer in system2O 10.5μL。
The wherein PCR response procedures of AC1 primer are as follows: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 1min, 57 DEG C of annealing 1min,
72 DEG C of extension 1min, total 30-35 circulation；Last 72 DEG C of extensions 8min.
The PCR response procedures of AC2 primer and AC3 primer are as follows: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 1min, 65 DEG C of annealing
1min, 72 DEG C of extension 2min, total 30-35 circulation；Last 72 DEG C of extensions 8min.
(4) optimum annealing temperature determines:
The optimum annealing temperature of thermophilic skin bacterium unique identification primer AC1, AC2, the AC3 of tortoise is explored using grads PCR.All PCR
Using 25 μ L reaction systems, comprising: 2 × Taq PCR Mix, 12.5 μ L, 1.0 μ L of DNA profiling, 0.5 μ L of forward and reverse primer, respectively
Final concentration of 10 μm ol/L, ddH of the primer in system2O 10.5μL。
PCR response procedures are as follows: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 1min, anneal (temperature gradient) 1min, 72 DEG C of extensions
1-2min (AC1 primer extend 1min, AC2 primer, AC3 primer extend 2min), total 30-35 circulation；Last 72 DEG C of extensions
8min.Wherein AC1 primer is expanded under 55 DEG C of -65 DEG C of annealing conditions, and AC2 primer expands under 60 DEG C of -67 DEG C of annealing conditions
Increase；AC3 primer expands under 55 DEG C of -71 DEG C of annealing conditions.
The optimum annealing temperature of the AC1 primer of the thermophilic skin bacterium of tortoise, AC2 primer and AC3 primer gropes result and sees Fig. 3.
Temperature gradient PCR is the results show that AC1 primer can amplify purpose band (figure under 55 DEG C of -65 DEG C of annealing conditions
3A), 63-65 DEG C when weak non-specific amplification is appeared below in 750bp, it is big with purpose band difference in size, and purpose band
It is very bright, the judgement of result is not influenced.AC2 primer can amplify target fragment under 60 DEG C of -67 DEG C of annealing conditions, and 66 DEG C of whens go out
The non-specific amplification band of an existing 1000bp or so, it is of different sizes with purpose band, therefore, the judgement (figure of result is not influenced
3B).The optimum annealing temperature of AC2 primer is 64 DEG C and 65 DEG C.
AC3 primer can amplify purpose band under 55 DEG C of -71 DEG C of annealing conditions, and 1000bp occur in 70 DEG C -71 DEG C whens
The non-specific amplification band of left and right, but it is of different sizes with purpose band, the judgement (Fig. 3 C) of purpose band is not influenced.AC3 primer
Optimum annealing temperature be 63 DEG C.
The above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as pair
Limitation of the invention, protection scope of the present invention should be defined by the scope defined by the claims..For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, these change
It also should be regarded as protection scope of the present invention into retouching.
Sequence table
<110>Guangdong Province's living resources Applied Research Laboratory
<120>the thermophilic skin bacterium specificity identification primer of tortoise, kit and its identification method
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ccgcatcact cccgaggtgt acgacttcaa cccgcgcgcc cgccatgtct acgagaagat 900
cggcttcgtg tacgagggcg ccggccgcga ggctcagcga tcacagccgt gatccctcta 960
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tgaaccccgg aaccgtgagc cccggcgccc gtccgaggac ctcggctgcg ccactgttca 1140
cgccagctcg cctggtcgcc gcgctccctc ccacttcagc gatgaagagc ggagttactt 1200
cggacgacgg agcggaaccg gttcgagctc aggaatcgtg aaaatgcggt cacagtgatc 1260
ggccacggta tcgctgtggg tcgccacgat gacggcacgt cctccgtcgg ccagttcacg 1320
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cacgatgatg cggctcggtt tgaccaggag acgcgccaag gccactcgtt gctgctcacc 1440
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cgaaccgctg tcaggttgct caagaagtcc gatgcagtgc aggagggtgg attttccact 1740
tccgctgacc cctctgacgg caagcatctc tcctgcaccg acttggagtt cgatatcaga 1800
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ccttgttttt cgaggtgata aaccacgtgc tcggctccca cgaggcgggc tttacccgca 480
aaggccggcg ccgctttacc caggtcatgg accgaggcaa cccaggcaaa aagagacttg 540
gccagccttt cagtaccgcc gagttcagca gccacccttc ttcgcacgat ccgaggaatc 600
cagtgatccc ataagtgatc ggcaacggcg gccgtgtcct ccaggtggac caccaacggc 660
agataggagc catcctctcg gctcagcttc ccccacaagg cccaggccct gtcagaccat 720
cctggaatcc tcgacatgag gccctcccca gtcgtcggca atgcttcgtc gcgccgcctc 780
tcgccttcga caatacctgt caacacacac cacatcaaac cctcaaactc cccatcgaga 840
acacaattta tattttttta aggatccaag gagtgcggac agcaaaatct tctcaggccc 900
acacagagga gaagctatcc ccgcaggtgc ggggcgcaca tactttcagg ggcttcccca 960
tcgagaacac agggttcatc cccgccgatg cggggcatca tcgcgatgag ccgacagcgc 1020
aagaagccat gacagtctac gcccccagca aactcgaaca aatttgtccc acagccaagt 1080
tcatggaacg agccaatgac aaaacccctg tcggccacca catatccgga atgctcggaa 1140
catgcacgac ggctggcctc agacctgtag aaccgcaccg ctcaaccgtc gcgcctcacc 1200
gcacgtaccg tttggtccac gcccgtcccg cagcatccac caccgttccg cgcgcgccca 1260
cgacgagcag gcacttcatg tcgatgctcc gcgggtccag ggtgcccagc gtggtcaccg 1320
tcagcgattc ctcggcccgg ccgacgtccc ggccgaccac caccacgcac tctgcgcccc 1380
gctcggccag aagcacctcg tgggcgcgca cgatctgttc ggtgcgggtc cgtgacgccg 1440
ggttgtagat cgccaccacc aggtcggcac ggctcaccgc ccgcagccgc tcctccacga 1500
cctcccacgg tttgagccgg tcggacagtg agatcaccgc gtagtccccg cccagtgggg 1560
cacctgctcg tgccgcgacc gcctgcgcag cggtcacccc cggcagcacc cgcaccggca 1620
cccggctgta cggcgtgccg tccgcggcac gaccttcggc ggcctcgaag accgccgagg 1680
ccatcccgaa gactcccgcg tccccgccgg agaccactgc gaccttctct cccgccaatg 1740
ccaggtccaa cgccagacgg gcccggtcga cctcgacggt gttcccgctg gcgtgccggg 1800
tcagcccggt ccgctgcggc acccggtcca cgtagggtgc ataaccgatc acg 1853
Claims (7)
1. the thermophilic skin bacterium specificity identification primer of tortoise, which is characterized in that including primer AC1 or AC2 or AC3,
The primer AC1 includes: AC1-F:5'-TTTTGGCGAGTTTTGTACCCT-3'
AC1-R:5'-TCCCACAGCGATTAATTGACA-3'；
The primer AC2 includes: AC2-F:5'-CGGGAACTGGTCAAACCTT-3'
AC2-R:5'-CACAATAAGCGGACTAACCAA-3'；
The primer AC3 includes: AC3-F:5'-CCGTCGTCAAGCCCATCGTA-3'
AC3-R:5'-CGTGATCGGTTATGCACCCTA-3'.
2. a kind of for identifying the kit of the thermophilic skin bacterium of tortoise, which is characterized in that the kit includes described in claim 1
The thermophilic skin bacterium specificity identification primer of tortoise.
3. a kind of method of the identification thermophilic skin bacterium of tortoise of diagnostic and therapeutic method of non-disease, which comprises the following steps:
(1) total DNA of sample to be tested is extracted；
(2) using the total DNA of sample to be tested as template, the thermophilic skin bacterium specificity identification primer of tortoise described in claim 1 is utilized
AC1 or AC2 or AC3 carries out PCR amplification；
(3) amplified production is subjected to agarose gel electrophoresis, gel imaging system test strip, if amplifying the spy of the thermophilic skin bacterium of tortoise
Heteroleptic then proves that sample to be tested contains the thermophilic skin bacterium of tortoise.
4. according to the method described in claim 3, it is characterized in that, the PCR uses 25 μ L reaction systems, comprising: 2 ×
12.5 μ L of TaqPCR Mix, 1.0 μ L of DNA profiling, 0.5 μ L of forward and reverse primer, final concentration of 0.2 μ of each primer in system
Mol/L, ddH2O 10.5μL。
5. according to the method described in claim 3, it is characterized in that, the PCR response procedures are as follows:
95 DEG C of initial denaturation 3min；95 DEG C of denaturation 1min, 55-71 DEG C of annealing 1min, 72 DEG C of extension 1-2min, total 30-35 recycles；
Last 72 DEG C of extensions 8min.
6. a kind of characteristic nucleotide sequence for identifying the thermophilic skin bacterium of tortoise, which is characterized in that including SEQ ID NO.7 or SEQ ID
Nucleotide sequence shown in NO.8 or SEQ ID NO.9.
7. the thermophilic skin bacterium specificity identification primer or as claimed in claim 2 for identifying the thermophilic skin bacterium of tortoise of tortoise described in claim 1
Kit thermophilic skin bacterium of tortoise in identification animal, microorganism or environmental samples application.
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