CN108715898A - Aeromonas veronii detection primer, kit, detection method and its development approach based on specific sequence - Google Patents
Aeromonas veronii detection primer, kit, detection method and its development approach based on specific sequence Download PDFInfo
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Abstract
The present invention relates to the primer pairs for detecting Aeromonas veronii, the nucleotide sequence of the forward primer of the primer pair is selected from SEQ ID NO.1 or the nucleotide sequence shown in SEQ ID NO.1 is substituted, lacks and/or increases the nucleotide sequence generated after one or several nucleotide, and the nucleotide sequence of the reverse primer of the primer pair is selected from SEQ ID NO.2 or the nucleotide sequence shown in SEQ ID NO.2 is substituted, lacks and/or increases the nucleotide sequence generated after one or several nucleotide.The invention further relates to reagent, kit and its applications in detecting Aeromonas veronii comprising the primer pair or primer pair combination.The method of primer the invention further relates to exploitation for detecting Aeromonas veronii.
Description
Technical field
The present invention relates to a kind of detection primer of the Aeromonas veronii based on specific sequence, include the examination of the primer
Agent, kit, the detection method of Aeromonas veronii and develop Aeromonas veronii detection primer method, belong to molecule
Field of biology.
Background technology
Aeromonas veronii (Aeromonas veronii) is otherwise known as Wei Luona Aeromonas, Velon Aeromonas, all
Grand Aeromonas is a kind of Gram-negative bacillus, is belonged to without gemma amphimicrobian type.The bacterium is dispersed in distribution, generally existing
In fresh water, sewage, soil or even seawater, both ends blunt circle, size is about 1.2-2.5 μm of 0.3-0.7 μ ms, has flagellum, can transport
Dynamic (" the Lateral flagella such as Kirov SM, Tassell BC, Semmler AB, O'Donovan LA, Rabaan AA
and swarming motility in Aeromonas species”[J].J Bacteriol.2002,184(2):547-
555)。
Aeromonas veronii belongs to strong viral disease opportunistic pathogen, pathogenic course include mainly stick-invade-host's Colonization inside plants and
During which the processes such as toxin secretion generate a series of virulence factors, such as gas lysin (aerolysin), enterotoxin (enterotoxin)
With adhesion factor (adherence factor) etc., these virulence factors play in aquatic livestock, the mankind even livestock and poultry infection
(Yuan Danyi, Wang Kaiyu, Chen Defang, Huang Lingyuan, " Aeromonas veronii is in artificial challenge's spot by king's equalization for very important effect
Tissue positioning in fork tail Channel-catfish bodies and DYNAMIC DISTRIBUTION research " [C] southern china fishery forums and the 29th academic conference opinion
Collected works Beihai Fisheries Base Guangxi Provinces, 2013).
It is very extensive that the bacterium infects host range.First, which is known most ancient one of fish-pathogenic bacteria, and infection is more
Kind freshwater fish, seawater fish etc., bring to aquatic products cultured fishes and wild fish and seriously threaten (Bossi-K ü pfer
M,Genini A,Peduzzi R,Demarta A.“Tracheobronchitis caused by Aeromonas veronii
biovar sobria after near-drowning”[J].J Med Microbiol.2007,56(Pt 11):1563-
1564).For example, grass carp infect Aeromonas veronii when local hemorrhage, have the symptoms such as ascites, spleen enlargement (Wang Hao, Quan Keyan,
Xu Lijuan, Li Mengying, Lv Li groups of " Enrofloxacin controls the therapeutic regimen of grass carp Aeromonas veronii " [J] fresh water fisherys,
2013,43(2):47-53)。
Meanwhile Aeromonas veronii can also infect arthropod and reptile, as (room is extra large, old for Eriocheir sinensis
It is emerald green precious, Zhang Xiaojun, Gong Yuanfang, Pueraria lobota curtain Hunan " inspection of Eriocheir sinensis cause of disease Aeromonas veronii " [J] China Zoonosis
Sick journal .2008,24 (1):45-49), Shelled Turtle Trionyx Sinensis (money obviously " separation of Shelled Turtle Trionyx Sinensis group death disease pathogen identify and its
The research of prevention " [D] Hebei Normal University Master's thesis, 2010) etc..In addition, the bacterium can result in mouse and normal person and
Immunologic hypofunction population infection complication causes to include arthritis, wound infection, enterogastritis, bacteremia, meningitis and the heart
Multi-infection illness (Lye DJ. " Gastrointestinal colonization rates for including intimitis etc.
human clinical isolates of Aeromonas veronii using a mouse model”[J].Curr
Microbiol.2011,63(4):332-336)。
In recent years, increasing case shows that Aeromonas veronii has become a kind of important people-fish and suffers from altogether and causes a disease
Bacterium, it is widely distributed, it is pathogenic relatively strong, and very important status is occupied in food security aspect, Countries are by it
It is defined as quarantine object (Wu Tonglei, Dan Xiaofeng, Meng Qingfeng, Guo Weisheng, Wang Weili, " the Vickers gas such as Qian Aidong of food security
Monad progress " [J] China veterinary drug magazine, 2011 (07):41-44).
Currently, the identification of Aeromonas veronii and detection method rely primarily on the nutritional need and metabolite according to bacterium
Difference and the customary physiological biochemistry detection that designs, but this method is time-consuming and laborious, and result unreliable (LamyB, Laurent
The such as F, Verdier L " Accuracy of 6Commercial Systems forIdentifying Clinical
Aeromonas Isolates”[J].Diagnostic Microbiology and Infectious Disease,2010,
67:9-14).Also have and rely on the general bacteriological method such as apparent pathological observation and bacterium separation identification to detect Aeromonas veronii
, but the pathological phenomenon of Aeromonas veronii initiation disease and other Aeromonas are very much like, it is difficult to naked eyes judge, and it is conventional
Bacterium separation identification again take time and effort.In addition, also can applied molecular biology and immunological method.Molecular biology method base
In specific conserved sequence, such as 16srDNA (" the A DNA such as Chac ó n M R, Castro-Escarpulli G, Soler L
Probe Specific for Aeromonas Colonies”[J].Diagnostic Microbiology and
Infectious Disease,2002,44:221-225) or the intergenic region of 16S-23S rRNA (ISR) (Laganowska
M,Kaznowski A.“Restriction Fragment Length Polymorphism of 16S-23S rDNA
Intergenic Spacer of Aeromonas spp”[J].System ApplMicrobiol,2004,27:549-557)
Length and sequence variation carry out the discriminating and classification of bacterium, but the general probe false positive rate that this method uses is higher, valence
Lattice are expensive.
In addition, the ISR types of Aeromonas veronii are more, it is unfavorable for detecting, also some researches show that Aeromonas veroniis
Very high (LAGANOWSKA M, KAZNOWSKI A. " the Restriction fragment length of ISR similitudes
polymorphism of 16S-23SrDNA intergenic spacer of aeromonasspp”[J]Syst Appl
Microbiol,2004,27(5):549-557), disagreement is not yet unified.
As it can be seen that the molecular biological variety identification method of Aeromonas veronii is still immature.Immunological method passes through specificity
The special antigen protein of enzyme-linked immunosorbent assay (ELISA) detection bacterium, also research is insufficient, and not yet identifying can be special
A large amount of antigens (Arora S, Agarwal R K, Bist B. " Comparison of ELISA of different identification Aeromonas veronii
and PCR vis-à-vis Cultural Methods for Detecting Aeromonas spp.in Foods
ofAnimal Origin”[J].International Journal of Food Microbiology,2006,106:177-
183)
Therefore, in general, the detection method of Aeromonas veronii is still in the lag phase, so that aquatic livestock is special
Mistaken diagnosis frequent occurrence when being human infection pathogenic bacteria, often causes serious consequence --- Aquatic farming animals mortality and disease
People's state of an illness continues even dead.
Therefore, develop a kind of method that can quickly, easily detect Aeromonas veronii, be from now on to Vickers gas unit cell
Microbial disease carries out the key and emphasis of prevention and treatment.
Invention content
The purpose of the present invention is to provide a kind of detection primer pair of Aeromonas veronii, primer pair high specificity, the spirits
Sensitivity is high, can quickly and accurately detect Aeromonas veronii.
It is a further object to provide a kind of reagent or kit of detection Aeromonas veronii, sensitivity
It is high, quick, easy, at low cost.
It is a further object to provide a kind of method of detection Aeromonas veronii, this method uses the present invention's
Above-mentioned primer pair, reagent or kit can quickly, easily and accurately identify the presence of Aeromonas veronii.
It is being prepared alternatively, another object of the present invention is to provide the above-mentioned primer pair of the present invention, reagent or kit for detecting
Purposes in the existing detection agent of Aeromonas veronii, so as to quickly, easily and accurately identification Aeromonas veronii
In the presence of.
A kind of method of primer it is also another object of the present invention to provide exploitation for detecting Aeromonas veronii, to
High specificity, high sensitivity are provided, the primer of Aeromonas veronii can be quickly and accurately detected.
The first aspect of the present invention provides the primer pair for detecting Aeromonas veronii, wherein：
(1) nucleotide sequence of the forward primer of the primer pair is selected from SEQ ID NO.1 or shown in SEQ ID NO.1
Nucleotide sequence be substituted, lack and/or increase the nucleotide sequence generated after one or several nucleotide；And
(2) nucleotide sequence of the reverse primer of the primer pair is selected from SEQ ID NO.2 or shown in SEQ ID NO.2
Nucleotide sequence be substituted, lack and/or increase the nucleotide sequence generated after one or several nucleotide.
In one embodiment, primer pair of the present invention is WP_082182332.1 primer pairs, forward primer
Nucleotide sequence as shown in SEQ ID NO.1, the nucleotide sequence of reverse primer is as shown in SEQ ID NO.2.
In one embodiment, a primer in above-mentioned primer pair is detectably labeled.Labeling method is ability
Known to domain.It is described label include it is those of commonly used in the art, for example, it is described label including but not limited to radioactive isotope,
Enzyme, fluorescent material, luminescent substance or biotin etc..Preferably, the label is fluorescent marker, it is highly preferred that the fluorescence mark
Note includes but not limited to the fluorescent dyes such as FAM, HEX, TAMRA, ROX.In a further embodiment, in the primer pair just
It is labeled to the reverse primer during primer is labeled or the primer pair.In another embodiment, the primer can be through
Arbitrary modification means well known by persons skilled in the art are modified and are changed, to improve the specificity of the primer
And/or facilitate the detection of the primer.
The second aspect of the present invention provides the reagent for detecting Aeromonas veronii, and the reagent includes in the present invention
Arbitrary primer described in text.In one embodiment, the every pair of primers for including in reagent of the invention is individually packed.
The third aspect of the present invention provides the kit for detecting Aeromonas veronii, and the kit includes this hair
Bright arbitrary primer or reagent described above.
In one embodiment, kit of the present invention also includes at least one fluorescence probe, such as TaqMan
Probe, FRET hybridization probes (fluorescence resonance energy transmission probe) and molecular beacon.In a preferred embodiment, described
Fluorescence probe is the TaqMan probe designed between primer of the present invention based on SEQ ID NO.3.TaqMan is designed to visit
The method of needle is known to the skilled in the art.
In a further embodiment, 5 ' ends of the TaqMan probe are marked with fluorescent reporter group, such as FAM, VIC
Deng, 3 ' end be marked with fluorescent quenching group, such as TAMRA.
In one embodiment, kit of the present invention also include 2 × PCR master Mix, positive control with
And 10 × PCR bacterium colony reinforcing agents.In one embodiment, the positive control includes nucleotide shown in SEQ ID NO.3
Sequence.Preferably, the positive control is nucleotide sequence shown in SEQ ID NO.3.
The fourth aspect of the present invention is provided detects Vickers using arbitrary primer of the present invention to, reagent or kit
The method of Aeromonas.Alternatively, the fourth aspect of the present invention provides arbitrary primer of the present invention to, reagent or kit
Purposes in preparing the detection agent for detecting Aeromonas veronii.
In one embodiment, detection Aeromonas veronii includes the following steps：
A. using sample genomic dna to be detected or single bacterium colony as pcr template, PCR amplification, amplified production are carried out using primer
There is same strap to be determined as the positive through electrophoretic analysis and positive control, wherein the positive control is shown in SEQ ID NO.3
Nucleotide sequence.
In a further embodiment, the above method of the invention is further comprising the steps of：
B. positive amplification product is recycled, carries out sequencing analysis, and be compared with sequence shown in SEQ ID NO.3；With
C. be based on comparison result, by sequence homology up to 99% or more (such as 99%, 99.5%, 100% or appointing therebetween
What numerical value) detection sample be determined as the Aeromonas veronii positive, that is, detect the presence of Aeromonas veronii.
In a further embodiment, the PCR reaction systems in the step a are：
Detected sample DNA (200ng/ul)：1μl；
Forward and reverse primer (10 μM)：Each 1 μ l；
2×PCR master Mix：25μl；
Distilled water：22μl；
Amount to 50 μ l.
In a further embodiment, the positive control is the sun for including nucleic acid sequence shown in SEQ ID NO.3
Property the reference material or nucleic acid sequence shown in SEQ ID NO.3 forms or the positive control is that identified wild type is tieed up
Family name Aeromonas.
In a further embodiment, wherein the PCR amplification program in the step a is：
94 DEG C of pre-degeneration 5min；
94 DEG C of denaturation 30s；
58 DEG C of annealing 30s；
72 DEG C of extension 30s；
Totally 25 cycles；
72 DEG C are continued to extend 7min.
In one embodiment, method or purposes of the present invention are for detecting the mankind, mouse, livestock and poultry, segmental appendage
The presence of Aeromonas veronii in the species such as animal, reptile (such as Eriocheir sinensis, Shelled Turtle Trionyx Sinensis etc.), aquatic livestock,
In particular for Aeromonas veronii in the species (such as Tilapia mossambica, loach, fancy carp, channel catfish etc.) such as detection aquatic livestock
Presence, it is especially useful in detection fungus mixture in Aeromonas veronii presence.
In a further embodiment, method or purposes of the present invention are for detecting Aeromonas veronii bacterial strain
It is one or more in TH0426, AVNIH1, B565 or CB51.Preferably, method or purposes of the present invention are for examining
Survey Aeromonas veronii bacterial strain TH0426.
The fifth aspect of the present invention provides a kind of method for developing the primer for detecting Aeromonas veronii, the side
Method includes the following steps：
1) bioinformatics means are used, by writing python procedure scripts, analyze all proteins included in NCBI
Sequence, to obtain Aeromonas veronii specific nucleotide sequences；
2) the Aeromonas veronii specific nucleotide sequences obtained in step 1) are based on, for each section of specific sequence point
At least pair of primers is not designed；With
3) primer that design obtains in step 2) is utilized, PCR amplification is carried out for Aeromonas veronii genomic DNA, from
In filter out the primer for capableing of specific amplification Aeromonas veronii specific sequence.
In one embodiment, the method further includes step 4)：The nucleic acid sequence of target PCR product is determined by sequencing
Row.
In one embodiment, above-mentioned steps 1) include the following steps (with reference to figure 1)：
(a) data subset 1 and data subset 2 are obtained from ncbi database, wherein the data subset 1 includes NCBI data
Gas unit cell category (level of category) all protein sequences that library is included, but do not include any egg of Aeromonas veronii (level of kind)
Bai Xulie, the data subset 2 include all protein sequences for the Aeromonas veronii (level of kind) that ncbi database is included；
And the sequence in data subset 1 and data subset 2 is subjected to Blast comparisons, to obtain sequence subset A and sequence subset a (these
The horizontal screen being equivalent in category selects species specific protein sequence), wherein the sequence subset A is by cannot be in data subset 2
1 matched sequence of data subset forms, and the sequence subset a is by can be with 1 matched sequence group of data subset in data subset 2
At；
(b) data subset 3 is obtained from ncbi database, wherein the data subset 3 includes the institute that ncbi database is included
There is the protein sequence of species, but does not include any protein sequence of Aeromonas veronii category (level of category)；And by the sequence
Subset A carries out Blast with the sequence in data subset 3 and compares, and to obtain sequence subset B and sequence subset b, (this is equivalent to
All species horizontal screens select the specific proteins sequence of Aeromonas veronii species), wherein the sequence subset B is by sequence subset
It cannot be formed with 3 matched sequence of data subset in A, the sequence subset b is by can be 3 with data subset in sequence subset A
The sequence composition matched；
(c1) when the number of sequence in sequence subset B is not zero, the sequence in sequence subset B is converted into nucleic acid sequence
Row, to generate sequence subset B ', and the property that all nucleic acid sequences in sequence subset B ' are included with ncbi database
The nucleic acid sequence of kind carries out Blast comparisons, and (this is equivalent to the nucleic acid that Aeromonas veronii species specificity is screened in species level
Sequence), to filter out the protein sequence X1 of Aeromonas veronii (species level) specificity；Or
(c2) when the number of sequence in sequence subset B is zero, then data subset 4, the number are obtained from ncbi database
Include gas unit cell section (level of section) all protein sequences that ncbi database is included according to subset 4, but does not include Vickers gas unit cell
Any protein sequence of Pseudomonas (level of category)；And the sequence in the sequence subset a and data subset 3 is subjected to Blast ratios
Right, to obtain sequence subset C and sequence subset c, (this is equivalent to the specific protein belonged in section's level screening Aeromonas veronii
Bai Xulie), wherein the sequence subset C in sequence subset a with 4 matched sequence of data subset by cannot form, the sequence
Subset c in sequence subset a with 4 matched sequence of data subset by can form；
(d2) data subset 5 is obtained from ncbi database, wherein the data subset 5 includes the institute that ncbi database is included
There is the protein sequence of species, but does not include any protein sequence of Aeromonas veronii section (level of section)；And by the sequence
Subset C carries out Blast with the sequence in data subset 5 and compares, and to obtain sequence subset D and sequence subset d, (this is equivalent to
All species horizontal screens select the specific proteins sequence that Aeromonas veronii belongs to), wherein the sequence subset D is by sequence subset C
In cannot be formed with 5 matched sequence of data subset, the sequence subset d in sequence subset C with data subset 5 by can match
Sequence composition；
(e2) when the number of sequence in sequence subset D is not zero, the sequence in sequence subset D is converted into nucleic acid sequence
Row, to generate sequence subset D ', and the property that all nucleic acid sequences in sequence subset D ' are included with ncbi database
The nucleic acid sequence of kind carries out Blast comparisons, and (this is equivalent to the nucleic acid sequence that Aeromonas veronii species specificity is screened in category level
Row), to filter out the protein sequence X2 that Aeromonas veronii belongs to specificity；Or
(e3) when the number of sequence in sequence subset D is zero, then enter third round and recycle, phase is set according to aforementioned thinking
It is special to start the protein sequence in the specificity of purpose horizontal screen elective course, the horizontal screening purpose in guiding principle one by one for the data subset answered
Foreign preteins, the horizontal specific proteins sequence for screening guiding principle in door, and each comfortable property kind level is further screened,
Finally screen graduate from old-type opera school, protein sequence X3, X4, X5 or X6 of mesh, guiding principle or door specificity.
By above-mentioned complete cycle, best situation is exactly that can filter out Aeromonas veronii by first round cycle
The protein sequence X1 of species specificity；Most complicated situation is exactly by being screened after kind, category, section, mesh, guiding principle, the wheel cycle of door six
Totally six sections of specific sequences for representing different classifications level, common combination are used to identify the presence of Aeromonas veronii to X1-X6.?
In the present invention, inventor has fortunately screened the protein sequence of Aeromonas veronii species specificity by first round cycle
X1。
In one embodiment, above-mentioned steps 1) further include Aeromonas veronii (species level) specificity that will be obtained
Protein sequence and NCBI nucleic acid databases in sequence carry out TBLASTn (protein → nucleotide, i.e., will be in library
Nucleic acid sequence translates into protein sequence, then makees the comparison of albumen and albumen with looked into sequence) it compares, to further determine that
To protein sequence be specificity be present in Aeromonas veronii.Further, the step 1) further includes choosing Blast
Nucleic acid sequence of the one section of nucleic acid sequence that detected matching degree is 100% as Aeromonas veronii (species level) specificity
Row.
In one embodiment, above-mentioned steps 2) include being carried out using Primer Premier 5.0 and DNAMAN softwares
Design of primers.
In one embodiment, above-mentioned steps 3) in PCR reaction systems further comprise as the other thin of negative control
The genomic DNA of bacterium.Further, other bacteriums are selected from bacillus subtilis (Bacillus subtilis), secondary haemolysis
Vibrios (Vibrio parahaemolyticus), Edwardsiella tarda (Edwardsiella tarda), vibrio alginolyticus
(Vibrio alginolyticus), Escherichia coli (Escherichia coli), pseudomonas aeruginosa (Pseudomonas
Aeruginosa) or it is arbitrarily combined.
In preferred embodiments, method of the present invention screens to have obtained Vickers gas shown in SEQ ID NO.4
Aeromonas veronii (species level) shown in the protein sequence or SEQ ID NO.3 of monad (species level) specificity is special
Anisotropic nucleic acid sequence, the protein and nucleic acid sequence identity are as shown in table 1 below.
The feature of 1. Aeromonas veronii specific protein of table and its corresponding nucleic acid sequence
It is highly preferred that method of the present invention obtains the primer sequence shown in SEQ ID NO.1-2, the primer
Sequence information is as shown in table 2 below.
The primer sequence information shown in SEQ ID NO.1-2 of table 2.
In one embodiment, PCR is carried out by primer WP_082182332.1F and WP_082182332.1 R to react
The product arrived has the nucleic acid sequence as shown in SEQ ID NO.3.
In the present invention, unless otherwise indicated by context, otherwise term " primer " and " primer pair " are used interchangeably.Term
" dimension bacterium " and " Aeromonas veronii " may be used interchangeably, and refer both to the Vickers gas unit cell of the entitled Aeromonas veronii of Latin
Bacterium bacterium.
Foregoing teachings are only schematical and to be never intended to be restrictive.In addition to above-mentioned schematic aspect, implementation
Mode and feature, by reference to following detailed description, further aspect, embodiment and feature will be more readily understood.
Description of the drawings
By reference to following attached drawings, further aspect of the invention, feature will be more readily understood.People in the art
Member is it should be understood that these attached drawings only symbolically elaborate according to certain embodiments of the present invention, and should not be taken as
Limitation of the scope of the invention.
Fig. 1 shows to find the logic chart of Aeromonas veronii species specificity sequence.
Fig. 2 shows the protein sequence SEQ ID of Aeromonas veronii (species level) specificity that the present invention obtains
NO.4 carries out the result of TBLASTn comparisons in NCBI nucleic acid databases.
Fig. 3 A show the specific PCR testing result of WP_082182332.1 primer pairs, wherein M：DNA Marker
DL2000；1：Bacillus subtilis；2：Vibrio parahaemolytious；3：Aeromonas veronii；4：Edwardsiella tarda；5：Molten algae arc
Bacterium；6：Escherichia coli；7：Pseudomonas aeruginosa.
Fig. 3 B show the specific PCR testing result of 16s rDNA universal primers pair, wherein M：DNA Marker
DL2000；1：Bacillus subtilis；2：Vibrio parahaemolytious；3：Aeromonas veronii；4：Edwardsiella tarda；5：Molten algae arc
Bacterium；6：Escherichia coli；7：Pseudomonas aeruginosa.
Specific implementation mode
Hereinafter, certain exemplary embodiments are simply just described.As one skilled in the art will recognize that
Like that, without departing from the spirit or scope of the present invention, described embodiment can be changed by various different modes.
Therefore, attached drawing and description are considered essentially illustrative rather than restrictive.
Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art；
Unless otherwise specified, agents useful for same is commercially available in embodiment.
The excavation of 1. Aeromonas veronii species level specific sequence of embodiment
Using bioinformatics means, by writing python procedure scripts, according to flow shown in Fig. 1, analyze in NCBI
The all proteins sequence included.
Flow shown in Fig. 1 includes the following steps：
(a) data subset 1 and data subset 2 are obtained from ncbi database, wherein the data subset 1 includes NCBI data
Gas unit cell category (level of category) all protein sequences that library is included, but do not include any egg of Aeromonas veronii (level of kind)
Bai Xulie, the data subset 2 include all protein sequences for the Aeromonas veronii (level of kind) that ncbi database is included；
And the sequence in data subset 1 and data subset 2 is subjected to Blast comparisons, to obtain sequence subset A and sequence subset a (these
The horizontal screen being equivalent in category selects species specific protein sequence), wherein the sequence subset A is by cannot be in data subset 2
1 matched sequence of data subset forms, and the sequence subset a is by can be with 1 matched sequence group of data subset in data subset 2
At；
(b) data subset 3 is obtained from ncbi database, wherein the data subset 3 includes the institute that ncbi database is included
There is the protein sequence of species, but does not include any protein sequence of Aeromonas veronii category (level of category)；And by the sequence
Subset A carries out Blast with the sequence in data subset 3 and compares, and to obtain sequence subset B and sequence subset b, (this is equivalent to
All species horizontal screens select the specific proteins sequence of Aeromonas veronii species), wherein the sequence subset B is by sequence subset
It cannot be formed with 3 matched sequence of data subset in A, the sequence subset b is by can be 3 with data subset in sequence subset A
The sequence composition matched；
(c1) when the number of sequence in sequence subset B is not zero, the sequence in sequence subset B is converted into nucleic acid sequence
Row, to generate sequence subset B ', and the property that all nucleic acid sequences in sequence subset B ' are included with ncbi database
The nucleic acid sequence of kind carries out Blast comparisons, and (this is equivalent to the nucleic acid that Aeromonas veronii species specificity is screened in species level
Sequence), to filter out the protein sequence X1 of Aeromonas veronii (species level) specificity；Or
(c2) when the number of sequence in sequence subset B is zero, then data subset 4, the number are obtained from ncbi database
Include gas unit cell section (level of section) all protein sequences that ncbi database is included according to subset 4, but does not include Vickers gas unit cell
Any protein sequence of Pseudomonas (level of category)；And the sequence in the sequence subset a and data subset 3 is subjected to Blast ratios
Right, to obtain sequence subset C and sequence subset c, (this is equivalent to the specific protein belonged in section's level screening Aeromonas veronii
Bai Xulie), wherein the sequence subset C in sequence subset a with 4 matched sequence of data subset by cannot form, the sequence
Subset c in sequence subset a with 4 matched sequence of data subset by can form；
(d2) data subset 5 is obtained from ncbi database, wherein the data subset 5 includes the institute that ncbi database is included
There is the protein sequence of species, but does not include any protein sequence of Aeromonas veronii section (level of section)；And by the sequence
Subset C carries out Blast with the sequence in data subset 5 and compares, and to obtain sequence subset D and sequence subset d, (this is equivalent to
All species horizontal screens select the specific proteins sequence that Aeromonas veronii belongs to), wherein the sequence subset D is by sequence subset C
In cannot be formed with 5 matched sequence of data subset, the sequence subset d in sequence subset C with data subset 5 by can match
Sequence composition；
(e2) when the number of sequence in sequence subset D is not zero, the sequence in sequence subset D is converted into nucleic acid sequence
Row, to generate sequence subset D ', and the property that all nucleic acid sequences in sequence subset D ' are included with ncbi database
The nucleic acid sequence of kind carries out Blast comparisons, and (this is equivalent to the nucleic acid sequence that Aeromonas veronii species specificity is screened in category level
Row), to filter out the protein sequence X2 that Aeromonas veronii belongs to specificity；Or (e3) when the number of sequence in sequence subset D
When being zero, then enters third round and recycle, set corresponding data subset according to aforementioned thinking, start screened in purpose level one by one
The specific protein of the protein sequence of the specificity of section, the horizontal screening purpose specific proteins in guiding principle, the horizontal screening guiding principle in door
Bai Xulie, and each comfortable property kind level is further screened, finally screen graduate from old-type opera school, the egg of mesh, guiding principle or door specificity
Bai XulieX 3, X4, X5 or X6.
By above-mentioned complete cycle, best situation is exactly that can filter out Aeromonas veronii by first round cycle
The protein sequence X1 of species specificity；Most complicated situation is exactly by being screened after kind, category, section, mesh, guiding principle, the wheel cycle of door six
Totally six sections of specific sequences for representing different classifications level, common combination are used to identify the presence of Aeromonas veronii to X1-X6.
In the present embodiment, due to including the protein sequence shown in SEQ ID NO.4 in the sequence subset B that screens
Row, that is, the number of sequence is not zero in sequence subset B, and therefore, above-mentioned analytic process only relates to step (a) to (c1).
The protein sequence of Aeromonas veronii (species level) specificity that above-mentioned steps (c1) are obtained and NCBI nucleic acid
Sequence in database carries out TBLASTn comparisons, and the protein sequence further determined that is that specificity is present in Vickers gas list
In born of the same parents bacterium, comparison result is as shown in Figure 2.Then, one section of nucleic acid sequence that the detected matching degrees of Blast are 100% is chosen
Arrange the nucleic acid sequence of (as shown in SEQ ID NO.3) as Aeromonas veronii (species level) specificity.The protein and
Nucleic acid sequence identity is as shown in table 1.
2. test material of embodiment, test method and equipment
1. test material and equipment
Bacterial strain：
Bacillus subtilis (Bacillus subtilis), vibrio parahaemolytious (Vibrio parahaemolyticus),
Aeromonas veronii (Aeromonas veronii), Edwardsiella tarda (Edwardsiella tarda), vibrio alginolyticus
(Vibrio alginolyticus), Escherichia coli (Escherichia coli), pseudomonas aeruginosa (Pseudomonas
Aeruginosa it) is preserved by this laboratory.
Culture medium and reagent：
Bacterial genomes DNA rapid extraction kits (article No. N1152) and PCR reaction reagents win biological section purchased from new east station of Guangzhou
Skill Co., Ltd (Dongsheng Biotech Co., Ltd)；
Tryptone, yeast extract, agar powder, agarose are purchased from the good biological Co., Ltd of Guangdong prestige；
1) LB liquid medium contains following substance：Tryptone 1.0g, it yeast extract 0.5g, NaCl 0.5g, steams
Distilled water 100mL is set 4 DEG C and is saved backup with 10mol/L NaOH tune pH to 7.2,121 DEG C of high pressure sterilization 20min.
2) LB solid mediums contain following substance：Tryptone 1.0g, yeast extract 0.5g, NaCl 0.5g, fine jade
Lipolysaccharide 1.5g, distilled water 100mL are cooled to about 50 DEG C with 10mol/L NaOH tune pH to 7.2,121 DEG C of high pressure sterilization 20min
When pour plate, save backup.
3) 100mg/ml Amp (ampicillin)
Amp 100mg are dissolved in 1ml sterile waters, with 0.22 μm of filter filtration sterilization of diameter, -20 DEG C of preservations.
4) 25mg/ml Cam (kanamycins)
Cam 50mg are dissolved in 2ml ethanol solutions, are distributed into every part of 0.5ml, -20 DEG C of preservations.
5) agarose gel electrophoresis reagent
50 × TAE buffer solutions：Tris alkali 242g, Na2EDTA·2H2O 37.2g, glacial acetic acid 57.1ml add deionized water
Fully it is dissolved to 1000ml, room temperature preservation.
6) 1% agarose solution：0.2g agaroses are weighed, are added into 20ml TAE buffer solutions, are dissolved by heating.
Instrument and equipment
QYC-2102C is complete, and temperature culture shaking table is purchased from new Jiangnan Instrument Ltd. of Ningbo Science and Technology Park area；HH-2 digital displays are permanent
Warm water bath is purchased from Changzhou Guohua Electric Appliance Co., Ltd.；LDZX-50KB vertical pressure steam sterilizers are purchased from the peace medical treatment of Shanghai Shen
Instrument factory；HPX-9272MBE digital display electric heating incubators are purchased from Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd.；WH-861 vortexs
Mixer is purchased from Taicang Hua Lida experimental facilities Co., Ltd, and the double single side clean work stations of SW--CJ--2D are net purchased from Suzhou
Change equipment Co., Ltd；Centrifuge5417R table model high speed centrifuges are purchased from Eppendorf companies of Germany；
Nanodrop2000 nucleic acid-protein detectors are purchased from Thermo companies of the U.S.；CP214 electronic balances purchased from Ao Haosi instruments (on
Sea) Co., Ltd；22331 Hamburg type PCR amplification instruments of AG are purchased from Eppendorf companies of Germany；1600R fully automatic digitals
Labworks image acquisition and analysis software is purchased from Shanghai Tian Neng Science and Technology Ltd.s.
2. test method
(1) genomic DNA template extracts
Using bacterial genomes DNA rapid extraction kits (article No. N1152), all fungus strains are extracted according to specification instruction
Genomic DNA.DNA mass is determined by Ago-Gel, and measures DNA using Nanodrop2000 nucleic acid-protein detectors
Concentration.
(2) prepared by bacterium colony pcr template
A small amount of bacterium colony is scraped, 100uL sterile waters are added and is blown and beaten into uniform bacterium solution, boiling water bath 5min with pipettor, it
12,000rpm centrifuges 1min afterwards, and supernatant can be used as the template of bacterium colony PCR.
(3) PCR amplification of specific fragment product
Using bacterial genomes DNA or single bacterium colony crude extract as template, amplifying specific sequence product is distinguished with primer.
5 μ l pcr amplification products are taken, are separated by electrophoresis with 30g/L Ago-Gels, are made with DNA marker DL2000
For molecular weight marker, after voltage 5V/cm, electrophoresis 20min, carries out analysis with gel images scanner and take pictures.
(4) PCR reaction systems and response procedures.
PCR reaction systems (50 μ l)：
PCR response procedures：25 cycles
Embodiment 3. is based on Aeromonas veronii species level specific sequence design primer and primer screening
Using Primer Premier 5.0 and DNAMAN softwares, the specific nucleic acid sequence obtained for embodiment 1 is set
Multigroup primer is counted, the primer of design is synthesized by Shanghai life work biology Co., Ltd.
Using Aeromonas veronii genomic DNA as template, the different primers pair synthesized with design, respectively according to embodiment 2
Method carry out PCR reactions.Pcr amplification product is analyzed by 30g/L agarose gel electrophoresis.
According to Gel electrophoresis results (not shown), clear, the single primer pair of amplified band is chosen, further to carry out
Primer specificity is analyzed.Pair of primers is obtained, sequence is shown in SEQ ID No.1-2, the primer sequence information such as table 2
It is shown.Further amplified production is sequenced, determines that target fragment is consistent with sequence shown in SEQ ID NO.3.
The verification of 4. Aeromonas veronii specific primer of embodiment
The primer pair obtained using embodiment 3, with bacillus subtilis, vibrio parahaemolytious, Aeromonas veronii, slow love
Moral Fahrenheit bacterium, vibrio alginolyticus, Escherichia coli, pseudomonas aeruginosa gene group DNA are template, are distinguished according to the method for embodiment 2
Carry out PCR reactions.Pcr amplification product is analyzed by 30g/L agarose gel electrophoresis.
Gel electrophoresis results show that primer pair of the invention is only capable of amplifying the mesh of expected size in Aeromonas veronii
Segment, and production concentration is high, and target fragment (Fig. 3 A) can not be then obtained in other negative control bacterial strains.As internal reference,
Using 16s rDNA universal primers to SEQID NO.5 and SEQ ID NO.6, distinguish by template of above-mentioned same genomic DNA
PCR amplification is carried out, the size product consistent with concentration is can get in each sample, further proves the primer pair of the present invention
Have specific (Fig. 3 B) for Aeromonas veronii.
The segment that amplification obtains further is sequenced, through sequence alignment analysis, confirms that the segment that amplification obtains is dimension
The specific nucleic acid sequence of family name Aeromonas.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any
Those familiar with the art in the technical scope disclosed by the present invention, can readily occur in its various change or replacement,
These should be covered by the protection scope of the present invention.Therefore, protection scope of the present invention should be with the guarantor of the claim
It protects subject to range.
Sequence table
<110>University Of Hainan
<120>Aeromonas veronii detection primer, kit, detection method and its development approach based on specific sequence
<141> 2018-3-18
<130>No. 5 sequences
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213>Artificial sequence（Artificial sequence）
<400> 1
aagtgtgctc ggtttgccag tt 22
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence（Artificial sequence）
<400> 2
ctggagcgag catggaaagc 20
<210> 3
<211> 183
<212> DNA
<213>Aeromonas veronii (Ameromonas veronii)
<400> 3
aagtgtgctc ggtttgccag ttagaatttt gaataaaggc agatcacaac cctcataaca 60
gctctcatcc cctaaattga ttagccgaac tgtgaactcc atcacccatt tattcgcctt 120
atcgccatcg atagcagtag acacctcacg ctttctggat acagctttcc atgctcgctc 180
cag 183
<210> 4
<211> 61
<212> PRT
<213>Aeromonas veronii (Ameromonas veronii)
<400> 4
Met Glu Arg Ala Trp Lys Ala Val Ser Arg Lys Arg Glu Val Ser Thr
1 5 10 15
Ala Ile Asp Gly Asp Lys Ala Asn Lys Trp Val Met Glu Phe Thr Val
20 25 30
Arg Leu Ile Asn Leu Gly Asp Glu Ser Cys Tyr Glu Gly Cys Asp Leu
35 40 45
Pro Leu Phe Lys Ile Leu Thr Gly Lys Pro Ser Thr Leu
50 55 60
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence（Artificial sequence）
<400> 5
agagtttgat catggctcag 20
<210> 6
<211> 22
<212> DNA
<213>Artificial sequence（Artificial sequence）
<400> 6
tagggttacc ttgttacgac tt 22
Claims (13)
1. the primer pair for detecting Aeromonas veronii, wherein：
(1) nucleotide sequence of the forward primer of the primer pair is selected from SEQ ID NO.1 or the core shown in SEQ ID NO.1
Nucleotide sequence is substituted, lacks and/or increases the nucleotide sequence generated after one or several nucleotide；And
(2) nucleotide sequence of the reverse primer of the primer pair is selected from SEQ ID NO.2 or the core shown in SEQ ID NO.2
Nucleotide sequence is substituted, lacks and/or increases the nucleotide sequence generated after one or several nucleotide.
2. primer pair as described in claim 1 a, wherein primer in the primer pair is detectably labeled.
3. primer pair as claimed in claim 2, wherein the label is selected from radioactive isotope, enzyme, fluorescent material, shiner
Matter or biotin.
4. primer pair as described in any one of claims 1-3, wherein：
The primer pair is primer pair WP_082182332.1, the nucleotide sequence of forward primer as shown in SEQ ID NO.1,
The nucleotide sequence of its reverse primer is as shown in SEQ ID NO.2.
5. the reagent for detecting Aeromonas veronii, the reagent includes primer pair according to any one of claims 1-4.
6. a kind of kit, the kit includes described in primer pair according to any one of claims 1-4 or claim 5
Reagent.
7. kit as claimed in claim 6, wherein the kit includes also at least one TaqMan probe, to carry out reality
When quantitative fluorescent PCR.
8. kit as claimed in claim 6, wherein the kit also includes 2 × PCR master Mix, positive control
And 10x PCR bacterium colony reinforcing agents.
9. kit as claimed in claim 8, wherein the positive control includes nucleotides sequence shown in SEQ ID NO.3
Row.
10. a kind of reagent or right using described in primer pair according to any one of claims 1-4 or claim 5 is wanted
The method for seeking 7-9 any one of them kits detection Aeromonas veronii, the described method comprises the following steps：
A. using sample genomic dna to be detected or single bacterium colony as pcr template, PCR amplification, amplified production are carried out using the primer
There is same strap to be determined as the positive through electrophoretic analysis and positive control.
11. method as claimed in claim 10, further comprises the steps：
B. positive amplification product is recycled, carries out sequencing analysis, and be compared with sequence shown in SEQ ID NO.3；
C. it is based on comparison result, sequence homology is determined as the Aeromonas veronii positive up to 99% or more detection sample, that is,
Detect the presence of Aeromonas veronii.
12. a kind of method of primer of exploitation for detecting Aeromonas veronii, the method includes following step：
1) bioinformatics means are used, by writing python procedure scripts, analyze all proteins sequence included in NCBI,
To obtain Aeromonas veronii specific nucleotide sequences；
2) the Aeromonas veronii specific nucleotide sequences obtained in step 1) are based on, are set respectively for each section of specific sequence
Meter at least pair of primers；With
3) primer that design obtains in step 2) is utilized, PCR amplification is carried out for Aeromonas veronii genomic DNA, therefrom sieves
Select the primer for capableing of specific amplification Aeromonas veronii specific sequence.
13. method as claimed in claim 12, the primer wherein obtained in step 3) is primer described in claim 1.
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Non-Patent Citations (2)
| Title |
|---|
| KANG，Y.等: "Aeromonas veronii strain TH0426，complete genome，ACCESSION CP012504.1", 《GENBANK》 * |
| 陈万义: "副溶血弧菌高通量分子检测方法的建立与基因分型研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
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