CN109097484A - It is a kind of for detecting the primer and fluorescent quantitative PCR detection method of source of fish Vibrio vulnificus metalloprotease gene - Google Patents
It is a kind of for detecting the primer and fluorescent quantitative PCR detection method of source of fish Vibrio vulnificus metalloprotease gene Download PDFInfo
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Abstract
The present invention provides a kind of for detecting the primer and fluorescent quantitative PCR detection method of source of fish Vibrio vulnificus metalloprotease gene, and the nucleotide sequence of the primer is as follows: upstream primer VVPF:CCTGGTTTCTCGGGTGCTG；Downstream primer VVPR:TGGTATGCCGTTGACTCTTTGA.Primer and fluorescent quantitative PCR detection method of the present invention for detecting source of fish Vibrio vulnificus metalloprotease gene is the early detection and Rapid identification, tissue tropism research, epidemiological survey etc. of Vibrio vulnificus infection, provides a kind of special, sensitive and lower-cost detection method.Early diagnosis, Rapid identification and clinical application for the infection of source of fish Vibrio vulnificus lay the foundation.
Description
Technical field
The invention belongs to molecular biology fields, more particularly, to one kind for detecting source of fish Vibrio vulnificus metalloproteinases
The primer and fluorescent quantitative PCR detection method of gene.
Background technique
The virulence factor of Vibrio vulnificus has synocytotoxin, metalloproteinases, capsular polysaccharide, flagellum and RTX toxin, with
Thallus is pathogenic closely related.Many scholars conduct extensive research (Oliver et to their pathogenic mechanism
al.1986).In having been reported, each virulence factor plays a significant role in Vibrio vulnificus course of infection, passes through different ways
Diameter plays its pathogenic effects, damages that (force waits 2015 quietly to host；Wang Mingyi and Hu Chengjin 2017).
All the time, Vibrio vulnificus is many in the report of aquaculture industry, and the animal having been reported has haliotis discus hannai Ino (horse
The strong people are equal 1996), stone sole (Shen Zhi by force equal 2001), cobio (letter record normal etc. 2003), turbot (in Lan Ping etc. 2008) and
Synechogobius hasta (Ma Aimin etc. 2008), spotted maigre (finish can right 2011), Tilapia mossambica (Li Jiong etc. 2011) and lance tail to answer shrimp brave
Fish (2011 can so be waited by finishing) etc..Vibrio vulnificus disease shows symptom different, no typical pathologic variation due to kind difference, therefore gives
Clinical diagnosis causes many puzzlements.Vibrio vulnificus endangers seriously culture fishery, causes biggish economic loss,
It has been subjected to the particular concern and attention of vast aquatic products disease research person.
Metalloproteinases (Metalloprotease) i.e. VVP is that bacterial strain is uniquely secreted to extracellular protease, is one
The stronger virulence factor (FAN et al.2011) of kind.Major part protein intracorporal to people has extensive proteinase activity,
Collagen and elastin laminin (Miyoshi et al.1995) can be decomposed and then lead to tissue necrosis and skin lesion, it can
Bradykinin in activation blood plasma causes vasopermeability to enhance in turn, local edema or bleb occurs.The production of bradykinin is also
One of important feature sex expression of body (Wang et al.2008) after Vibrio vulnificus infection.
Aquatic pathogenic bacterium separation identification mainly uses traditional Physiology and biochemistry identification method, molecular biology method and immunology
Method is detected.Wherein Physiology and biochemistry identification method cooperation molecular biology method is still important morbid vibrio detection means.
Serologic detection is that serological reaction occurs using specific antibody and antigen, to determine the type of pathogen.Indirect fluorescent is anti-
The technology (Qin Qiwei etc. 1995) and double crush syndrome detection method (Wang Chongming etc. 1999) both methods of body are all immunologys
Based on the practical application that is detected in Vibrio vulnificus of serological detection method, detection sensitivity is high, and minimum Bacteria Detection number is 1
×104CFU/mL.But above method program is complicated, and time-consuming is taken a lot of work.
In aquatic products quarantine in recent years, TaqMan probe method, which has been obtained, to be widely applied, Vibrio vulnificus vvhA gene
The primer and probe of sequence design carries out real-time fluorescence quantitative PCR detection, and Pan Junhang etc. (2010) has been set up detection wound
Vibrios TaqMan probe method, sensitivity reach 1.4CFU/mL.But previous work is complicated, needs to design the TaqMan probe of specificity,
It is expensive.
Real-Time Fluorescent Quantitative PCR Technique (Real-time Fluorescent Quantitative PCR, RT-PCR/q-
It PCR) is a kind of new nucleic acid quantitation technique.Cheng Xiaoyan etc. (2010) has built up qPCR detection prawn white spot using the dyestuff
Syndrome virus method, and special elaboration has been carried out to the specificity of test result.But to source of fish Vibrio vulnificus metal egg
White enzyme gene, q-PCR technology is there is not yet related application.
Real-time fluorescence quantitative PCR test method is divided into two kinds: SYBRGreen I method and two kinds of TaqMan probe method.
SYBRGreen is a kind of dyestuff that fluorescence can be issued under the irradiation of 497nm light source, only (the light source irradiation hair in conjunction with DNA double chain
Fluorescence out), DNA dissociates away (light source irradiation does not issue fluorescence) after becoming single-stranded.To the power and double-stranded DNA quantity of fluorescence
It is related.This method can be applied to it is a variety of test and it is cheap.SYBR Green quantitative fluorescent PCR has rapid reaction, repeats
Property good, high sensitivity, clear result the advantages that, be widely used in various bacteria and virus Rapid identification.TaqMan probe method
Accuracy and specificity two be higher than I method of SYBRGreen, test result is significant, but previous work is complicated, needs to design specificity
TaqMan probe, it is expensive.
Summary of the invention
In view of this, the present invention is directed to propose it is a kind of for detect the primer of source of fish Vibrio vulnificus metalloprotease gene with
And fluorescent quantitative PCR detection method, to provide a kind of special, sensitive and lower-cost detection method.
In order to achieve the above objectives, the technical scheme of the present invention is realized as follows:
It is a kind of for detecting the primer of source of fish Vibrio vulnificus metalloprotease gene, the nucleotide sequence of the primer is as follows
It is shown:
Upstream primer VVPF:CCTGGTTTCTCGGGTGCTG；
Downstream primer VVPR:TGGTATGCCGTTGACTCTTTGA.
It is a kind of for detecting the fluorescent quantitative PCR detection method of source of fish Vibrio vulnificus metalloprotease gene, including it is as follows
Step:
S1: Vibrio vulnificus DNA in the source of fish is extracted using bacterium water-boiling method (95 DEG C of 5min)；
S2: using the DNA that step S1 is extracted as DNA profiling, DNA profiling is pressed into 10 doubling dilutions, is diluted to various concentration
Quantitative PCR amplified reaction is carried out using primer described in claim 1 as amplimer；
S3: the pcr amplification product obtained to step S2 carries out agarose gel electrophoresis detection, by gel imaging system into
The analysis of row electrophoretic image.
Further, the quantitative pcr amplification system includes: I 10 μ L, 10 μ of SYBR Premix Ex Taq in terms of 20 μ L
Each 0.4-0.8 μ L of mol/L upstream and downstream primer, 2 μ L of DNA profiling, remaining uses ddH2O complements to 20 μ L.
Further, the program of the quantitative pcr amplification are as follows:
(1) in 95 DEG C of progress DNA profiling initial denaturation reactions, reaction time 3min；
(2) in 95 DEG C of progress DNA profiling reactions of degeneration (RD), reaction time 10s；
(3) it anneals in 58~63 DEG C, time 30s；
(4) circulation step (1)-(4) 40 times；
(5) in 72 DEG C of extension 2min；
(6) fluorescence signal acquisition is set when annealing.
Preferably, the volume of 10 μm of ol/L upstream and downstream primers is respectively 0.4 μ L, 0.8 μ L in the quantitative pcr amplification system
Or 0.6 μ L.
Preferably, the annealing temperature in the step (3) in the program of the quantitative pcr amplification be 62.1 DEG C, 58.3 DEG C or
60.5℃。
Compared with the existing technology, it is of the present invention for detect the primer of source of fish Vibrio vulnificus metalloprotease gene with
And fluorescent quantitative PCR detection method has the advantage that
The present invention establishes the fluorescence quantitative PCR detection side of Vibrio vulnificus according to Vibrio vulnificus VVP gene order design primer
Method detects its sensitivity and specificity, and is evaluated the stability of this method.Because quantitative fluorescent PCR is easy to operate, inspection
It surveys the advantages that period is short, sensitivity is high and reproducible and is widely used.There has been no SYBR Green qPCR detections to create at present
Hurt the report of vibrios.This technology uses fluorescent dye SYBR Green I, the virulence VVP gene order design based on Vibrio vulnificus
Primer, optimizing reaction system and condition verify specificity, sensibility and repeatability, establish qPCR detection method.
For the early detection and Rapid identification of Vibrio vulnificus infection, tissue tropism research, epidemiological survey etc., one kind is provided
Specifically, sensitive and lower-cost detection method.It lays the foundation for the early diagnosis and Rapid identification of source of fish Vibrio vulnificus infection.
Detailed description of the invention
Fig. 1 is pcr amplification product agarose gel electrophoresis figure in embodiment 1；
Fig. 2 is the real time fluorescent quantitative datagram of the amplified production of different primers concentration；
Fig. 3 is the melting curve of VVP gene primer；
Fig. 4 is the standard curve of VVP gene primer；
Fig. 5 is the special linearity curve of VVP gene primer；
Fig. 6 is the sensitivity curves of VVP gene primer.
Specific embodiment
In addition to being defined, technical term used in following embodiment has universal with those skilled in the art of the invention
The identical meanings of understanding.Test reagent used in following embodiment is unless otherwise specified conventional biochemical reagent；It is described
Experimental method is unless otherwise specified conventional method.
Below with reference to examples and drawings, the present invention will be described in detail.
Embodiment 1
(1) culture and extracting genome DNA of bacterium
Vibrio vulnificus is incubated in 2216E fluid nutrient medium (production of Qingdao GaoKeYuan Hai Bo Bioisystech Co., Ltd)
Culture extracts DNA of bacteria using bacterium water-boiling method.
Water-boiling method proposes DNA of bacteria specific steps are as follows:
In centrifuge tube, 12,000rpm centrifugation 2min are abandoned bacterium solution 2ml Vibrio vulnificus after 28 DEG C of shaken cultivation 18-24h
Supernatant, is added 500 μ L ultrapure waters, and vortex oscillation 30s is placed in 95 DEG C of water-baths 5min, 12,000rpm centrifugation 2min and takes supernatant
(350 μ L) is DNA profiling.
(2) design and synthesis of primer:
It is searched in Genbank and downloads the highly conserved virulence VVP gene order of Vibrio vulnificus, with primer5.0 primer
Design software design primer is as shown in table 1, and it is storing liquid, -20 DEG C of guarantors that resulting primer, which is diluted to 10 μm of ol/L with ultrapure water,
It deposits.
The primer of the amplification virulence gene of table 1
(3) regular-PCR expands
DNA profiling is pressed into 10 doubling dilutions, various concentration is diluted to and carries out carrying out common PCR reaction using above-mentioned primer,
System is 25 μ L.Electrophoresis is carried out to regular-PCR amplified production, the size of target fragment is observed by gel imaging system.
Specific PCR program is as follows:
95 DEG C of initial denaturation 5min, 94 DEG C of denaturation 30s, 58.3 DEG C of annealing 30s, 72 DEG C of extension 1min, 72 DEG C extend eventually
10min, 30 circulations；
Reaction system: 25uL, 10uL Mix (Beijing Tiangeng biochemical technology Co., Ltd production), 11uL water, upstream and downstream is drawn
Each 1uL of object, 2uLDNA template；
Virulence gene VVP specific primer is detected through PCR amplification and agarose gel electrophoresis, obtains expected purpose segment
(as shown in Figure 1), it is similar to design of primers primer size prediction result (182bp or so).
(4) quantitative pcr amplification system is established
20 μ L quantitative PCR reaction systems are as follows: SYBR Premix Ex Taq I 10 μ L, 10 μm each point of ol/L upstream and downstream primer
Not Wei 0.4 μ L, 0.8 μ L and 0.6 μ L, DNA of bacteria be 2 μ L of template, remaining uses ddH2O complements to 20 μ L.
PCR cycle parameter are as follows: 95 DEG C of initial denaturations 3min, 95 DEG C of denaturation 10s, 58-63 DEG C of annealing 30s, after 40 circulations,
72 DEG C of extension 2min.
Reaction condition: 95 DEG C of initial denaturation 3min, 95 DEG C of denaturation 10s are moved back respectively at 62.1 DEG C, 58.3 DEG C and 60.5 DEG C
Fiery 30s repeats 40 circulations.Fluorescence signal acquisition is set when annealing.
Fig. 2 is the real time fluorescent quantitative datagram of the amplified production of different primers concentration.Fig. 3 is 65.0 DEG C of 5s, 95 DEG C of 5s
The melting curve figure of acquisition, the melting curve after VVP gene magnification is shown only unimodal as seen from Figure 3, illustrates primer free
Dimer and nonspecific products, and for the tool specificity of primer designed by VVP gene.
DNA profiling is subjected to 10 times of gradient dilutions, draws standard curve, as shown in Figure 4.It can be seen that by canonical plotting,
For the amplification efficiency E value of VVP gene between 90~100%, coefficient R 2 is all larger than 0.995, illustrates this fluorescent quantitation
PCR data is reliable.The examination criteria curve of linear gradient is in isometry and collimation, has preferable linear relationship.
2 specific test of embodiment
With Vibrio harveyi, Vibrio anguillarum, vibrio parahaemolytious, Aeromonas veronii, Aeromonas hydrophila, (Tianjin water is generated
State and cultivation key lab save) DNA be used as template, as negative control, progress quantitative fluorescent PCR reaction, to test
Demonstrate,prove the specificity of primer.
Vibrio harveyi, Vibrio anguillarum, vibrio parahaemolytious, Aeromonas veronii, thermophilic is had detected with the detection method of embodiment 1
Hydrophila, the results show that only specific amplification occur in 3 plants of Vibrio vulnificus, and other bacterium and negative control are bent without amplification
Line, as shown in Figure 5.
3 sensitivity test of embodiment
Sensitivity technique uses optimum reaction condition, to dose known amounts (1.88 × 108) Vibrio vulnificus extract DNA carry out
10 times of gradient dilutions go out 6 each gradients as DNA profiling, carry out quantitative fluorescent PCR reaction, measure the minimum inspection of institute's method for building up
Limit is surveyed, then calculates the minimum biomass that this method can detect.
Using the standard items of 10 times of gradient dilutions as template, qPCR detection is carried out, as the result is shown the qPCR Monitoring lower-cut of VVP point
The diluted concentration gradient for not being is 10-6, i.e., corresponding bacterial concentration is 1.88 × 102CFU/mL, maximum Ct value are
34.87, the quantitative fluorescent PCR method sensibility for showing that the present invention establishes is higher, as shown in Figure 6.
4 repetitive test of embodiment
Respectively using the DNA of different extension rates as template, 10 repetitions are set, carry out quantitative fluorescent PCR reaction detection
Repeatability analyzes Ct value, calculates its coefficient of variation.QPCR as the result is shown VVP each group repeat test the coefficient of variation 0.04%
~0.5%, it is repeated well to show that the method established has, specific test result is shown in Table 2:
The repetitive test of 2 VVP gene of table
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (6)
1. a kind of for detecting the primer of source of fish Vibrio vulnificus metalloprotease gene, it is characterised in that: the nucleosides of the primer
Acid sequence is as follows:
Upstream primer VVPF:CCTGGTTTCTCGGGTGCTG；
Downstream primer VVPR:TGGTATGCCGTTGACTCTTTGA.
2. a kind of for detecting the fluorescent quantitative PCR detection method of source of fish Vibrio vulnificus metalloprotease gene, it is characterised in that:
Include the following steps:
S1: Vibrio vulnificus DNA in the source of fish is extracted using bacterium water-boiling method；
S2: using the DNA that step S1 is extracted as DNA profiling, DNA profiling is pressed into 10 doubling dilutions, is diluted to various concentration to weigh
Benefit require 1 described in primer as amplimer carry out quantitative pcr amplification reaction；
S3: agarose gel electrophoresis detection is carried out to the pcr amplification product that step S2 is obtained, electricity is carried out by gel imaging system
Swimming imaging analysis.
3. according to claim 2 for detecting the fluorescence quantitative PCR detection of source of fish Vibrio vulnificus metalloprotease gene
Method, it is characterised in that: the quantitative pcr amplification system includes: I 10 μ L, 10 μ of SYBR Premix Ex Taq in terms of 20 μ L
Each 0.4-0.8 μ L of mol/L upstream and downstream primer, 2 μ L of DNA profiling, remaining uses ddH2O complements to 20 μ L.
4. according to claim 2 for detecting the fluorescence quantitative PCR detection of source of fish Vibrio vulnificus metalloprotease gene
Method, it is characterised in that: the program of the quantitative pcr amplification are as follows:
(1) in 95 DEG C of progress DNA profiling initial denaturation reactions, reaction time 3min；
(2) in 95 DEG C of progress DNA profiling reactions of degeneration (RD), reaction time 10s；
(3) it anneals in 58~63 DEG C, time 30s；
(4) circulation step (1)-(4) 40 times；
(5) in 72 DEG C of extension 2min；
(6) fluorescence signal acquisition is set when annealing.
5. according to claim 2 for detecting the fluorescence quantitative PCR detection of source of fish Vibrio vulnificus metalloprotease gene
Method, it is characterised in that: the volume of 10 μm of ol/L upstream and downstream primers is respectively 0.4 μ L, 0.8 μ L in the quantitative pcr amplification system
Or 0.6 μ L.
6. according to claim 2 for detecting the fluorescence quantitative PCR detection of source of fish Vibrio vulnificus metalloprotease gene
Method, it is characterised in that: the annealing temperature in step (3) in the program of the quantitative pcr amplification be 62.1 DEG C, 58.3 DEG C or
60.5℃。
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Cited By (3)
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| CN111172302A (en) * | 2019-12-27 | 2020-05-19 | 天津农学院 | Providencia retx gene primer, fluorescent quantitative PCR detection method and application |
| CN111534608A (en) * | 2019-11-01 | 2020-08-14 | 广州微芯生物科技有限公司 | Fluorescent quantitative PCR method for detecting toxigenic vibrio vulnificus and corresponding kit |
| CN111676302A (en) * | 2020-06-05 | 2020-09-18 | 江苏海洋大学 | Establishment and application of vibrio vulnificus RPA-LFS rapid detection method |
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Application publication date: 20181228 |