CN107385057A - Detect the RPA IAC primers and method of comma bacillus - Google Patents
Detect the RPA IAC primers and method of comma bacillus Download PDFInfo
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The invention provides primer pair and amplification of internal standard sequence, the primer pair includes SEQ ID NO：Nucleotide sequence and SEQ ID NO shown in 1：Nucleotide sequence shown in 2, the amplification of internal standard sequence include SEQ ID NO：Nucleotide sequence shown in 3.Present invention also offers the application of described primer pair and amplification of internal standard sequence in the product for preparing detection or auxiliary detection comma bacillus；And the method for the detection comma bacillus based on RPA IAC technologies.It the experiment proved that, the present invention RPA IAC primer specificities are good, high sensitivity, detection time is short, does not need special instrument and applied widely, and based on the primer and coordinate the RPA IAC detection methods of the comma bacillus of amplification interior label foundation sensitive, accurate, easy and quick, there is directive significance to inlet and outlet complementary goods and product quarantine.
Description
Technical field
The present invention relates to method, primer and the kit of field of biological detection, more particularly to detection comma bacillus.
Background technology
Comma bacillus is a kind of Gram-negative facultative Bacteroides nodosus, aquatic dynamic in the shellfishes such as oyster, shrimp, crab and shell-fish
It is widely distributed in thing, it is one of popularity degree, extent of injury highest food-borne pathogens.The mankind are cholera under field conditions (factors)
Unique susceptible person of vibrios, the mainly water source by pollution or food peroral infection, main clinical manifestation are violent vomiting, abdomen
Rush down, dehydration, the death rate is high.Since 1817, seven pandemic reports in the cholera whole world occur altogether in history, have caused hundreds of
The death of ten thousand people, therefore cholera belongs to international quarantine infectious disease, and category A infectious disease is also listed in China.South China cholera is relative
It is multiple, substantial amounts of human and material resources need to be put into every year and carry out prevention and control, therefore the detection to comma bacillus has ten in public health
Divide important meaning.
At present, the detection to comma bacillus still relies primarily on conventional method, i.e., increases bacterium first with selective medium, enter
And combine biochemical and serological method and identified.Traditional technique in measuring result is accurate, but exist detection efficiency it is low, detection mesh
Mark the deficiencies of single, sensitivity is low and time-consuming, cumbersome.To meet the requirement of pathogenic bacteria quick detection, develop enzyme-linked
Immunofluorescence assay (VIDAS-CHL), enzyme immunoassay (EIA), PCR (PCR), colloidal gold strip method,
The methods of API biochemical identification test strips method, loop-mediated isothermal amplification (LAMP) technology and real-time fluorescence PCR.Wherein, VIDAS-
There is the defects such as poor specificity, sensitivity is low in CHL methods, EIA methods, colloid gold test paper method and API methods, without obtaining extensively should
With；LAMP methods, as a kind of emerging detection method, although drastically increasing detection efficiency, testing cost is reduced again,
It is that caused false positive rate is higher.Round pcr obtains as a kind of High sensitivity, fast and convenient detection method in numerous areas
Extensive use, revolutionary achievement is especially achieved in terms of pathogen detection, turns into a goldstandard of nucleic acid quick detection,
And DNA probe technology, real-time fluorescence PCR technology and PCR combination denaturing high-performance chromatographies are developed again on basis herein
(DHPLC) technology etc., and PCR primer is designed to restrict the key factor of such detection method success or failure, conventional PCR
Design of primers, not only need to compare the specificity of primer repeatedly, and need the parameters and reaction condition of optimizational primer, especially
It is that annealing temperature is even more to be optimized repeatedly, to prevent non-specific amplification, the primer chosen under the restriction of these conditions
Easily influence the validity and specificity of amplification.In addition, the universal price of fluorescence detection device is higher, also limit to a certain extent
The popularization and application of this kind of detection method.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of sensitive, accurate, easy and be quickly based on RPA-IAC technologies
Detection comma bacillus method, present invention also offers specific good, high sensitivity, the detection time for this method it is short,
Special instrument and RPA primers applied widely and amplification of internal standard sequence and corresponding detection kit are not needed.
In order to solve the above-mentioned technical problem, the present invention is achieved through the following technical solutions:
First aspect present invention provides primer pair and amplification of internal standard sequence, and the primer pair includes SEQ ID NO：1 institute
The nucleotide sequence and SEQ ID NO shown：Nucleotide sequence shown in 2, the amplification of internal standard fragment include SEQ ID NO：3 institutes
The nucleotide sequence shown.
Second aspect of the present invention provides described primer pair and amplification of internal standard sequence and is preparing detection or auxiliary detection suddenly
Application in the product of random vibrios；
In a preference, the product for preparing detection or aiding in detection comma bacillus includes：Prepare detection or auxiliary
Whether detection biological sample infects the product of comma bacillus, and prepares detection or auxiliary detection pathogen is or candidate is cholera
The product of vibrios.
Third aspect present invention provides a kind of kit, including described primer pair and includes described amplification of internal standard
The plasmid of sequence.
In a preference, described kit also includes RPA constant-temperature amplification reagents.
In a preference, the RPA constant-temperature amplifications reagent includes the RPA amplification kits from TwistDX companies
The reagent that TwistAmp Basic kits are included.
In a preference, in addition to DNA extracts reagents.
In a preference, the DNA extracts reagents are DP432 including the article No. from Tiangeng biochemical technology Co., Ltd
The reagent that is included of Animal genome DNA extraction kit.
In a preference, it is DP302 that the DNA extracts reagents, which are also included from Tiangeng biochemical technology Co., Ltd article No.,
The reagent that is included of bacterial genomes DNA extraction kit.
In a preference, in addition to positive control and negative control.
Fourth aspect present invention provides described kit and is detecting or aiding in detection comma bacillus, or prepare detection or
Application in the product of auxiliary detection comma bacillus.
In a preference, the detection or auxiliary detection comma bacillus include：Detection or auxiliary detection biological sample are
No infection comma bacillus, and detection or auxiliary detection pathogen whether be or candidate is comma bacillus.
In a preference, the product for preparing detection or aiding in detection comma bacillus includes：Prepare detection or auxiliary
Whether detection biological sample infects the product of comma bacillus, and prepares detection or auxiliary detection pathogen is or candidate is cholera
The product of vibrios.
Fifth aspect present invention provides a kind of RPA-IAC methods for detecting or aiding in detection comma bacillus, including the use of
The step of described primer pair and the plasmid for including described amplification of internal standard sequence or kit.
In a preference, the detection or auxiliary detection comma bacillus include：Detection or auxiliary detection biological sample are
No infection comma bacillus, and detection or auxiliary detection pathogen whether be or candidate is comma bacillus.
In a preference, described method comprises the following steps：
1) RPA-IAC is expanded
Described amplification of internal standard as template and described primer pair and is included using the DNA that biological sample or pathogen contain
The plasmid of sequence or the kit carry out RPA amplifications.
2) judge whether biological sample infects comma bacillus according to the RPA-IAC results expanded, or pathogen whether be or
Candidate is comma bacillus.
Optional, the step of of extracting DNA is additionally included in biological sample or pathogen before step 1).
In a preference, the DNA that extracted in biological sample or pathogen is to use the limited public affairs of Tiangeng biochemical technology
The Animal genome DNA extraction kit or Tiangeng biochemical technology Co., Ltd article No. that the article No. of department is DP432 are the thin of DP302
Bacterium genome DNA extracting reagent kit is carried out.
In a preference, the standard that the result of the RPA-IAC amplifications judges is：
If the amplified production that the RPA-IAC expands to obtain only has the target gene fragment that size is 280bp, or gathers around simultaneously
The amplification of internal standard fragment and target gene fragment for having size to be respectively 337bp, 280bp, then it is determined as that biological sample infects cholera
Vibrios, or pathogen is or candidate is comma bacillus；If it is 337bp's that the amplified production that the RPA expands to obtain, which only has size,
Amplification of internal standard fragment, not containing the target gene fragment that size is 280bp, then it is determined as that biological sample is uninfected by comma bacillus,
Or pathogen is not or candidate is not comma bacillus；If the amplified production that the RPA expands to obtain is not 337bp's containing size
Amplification of internal standard fragment and not containing size be 280bp target gene fragment, be determined as reaction for false negative, it is necessary to re-start
Detection.
In a preference, the reaction system of the RPA-IAC amplifications is as follows：
0.2mL TwistAmp reaction tubes containing lyophilized enzyme powder
The μ L of rehydration buffer solution 29.5
SEQ ID NO：1 and SEQ ID NO：Each 2 μ L of primer shown in 2, the final concentration of primer is 0.4 μm of ol/L
Include SEQ ID NO：The plasmid 1.81 × 10 of amplification of internal standard sequence shown in 33copies
Template DNA 50ng
Magnesium acetate solution 2.5 μ L, concentration 280mmol/L
Deionized water complements to 50 μ L；
In a preference, the reaction condition of the RPA-IAC amplifications is as follows：The temperature of the RPA-IAC amplifications is 37
DEG C, time 40min.
Sixth aspect present invention provides a kind of system for screening comma bacillus, including：
Nucleic acid-extracting apparatus, the nucleic acid-extracting apparatus are used to extract the nucleic acid sample in the biological sample or pathogen
This；
RPA-IAC amplification devices, the RPA-IAC amplification devices are connected with the nucleic acid-extracting apparatus, suitable for described
Primer pair or the kit to the sample of nucleic acid carry out RPA-IAC amplifications；
Judgment means, the judgment means are connected with the RPA-IAC amplification devices, so as to what is expanded based on RPA-IAC
As a result, judge whether the biological sample infects comma bacillus, or pathogen whether be or candidate is comma bacillus.
In a preference, the reaction system of the RPA-IAC amplifications is as follows：
0.2mL TwistAmp reaction tubes containing lyophilized enzyme powder
The μ L of rehydration buffer solution 29.5
SEQ ID NO：1 and SEQ ID NO：Each 2 μ L of primer shown in 2, the final concentration of primer is 0.4 μm of ol/L
Include SEQ ID NO：The plasmid of amplification of internal standard sequence shown in 3,1.81 × 103copies
Template DNA 50ng
Magnesium acetate solution 2.5 μ L, concentration 280mmol/L
Deionized water complements to 50 μ L；
Optional, the reaction condition of the RPA-IAC amplifications is as follows：The temperature of the RPA-IAC amplifications is 37 DEG C, the time
For 40min.
The biological sample of the present invention is for blood, cell, tissue etc., or has mixed the food of blood, cell, tissue etc. etc.
Deng；And pathogen is then the mixing of a kind of pure culture or at least more than two kinds of strain.
" amplification of internal standard fragment " of the present invention is added in PCR reaction systems to indicate the one of false negative phenomenon
The conserved genetic sequences of the artificial constructed DNA sequence dna of section either one section of pathogenic bacteria.Amplification interior label effect cardinal principle be：It will expand
Increase internal standard to be added in PCR reaction systems, be allowed to carry out coamplification with target gene, if existed in reaction system
Restraining factors, then the amplified reaction of amplification interior label and target gene will all be suppressed, so as to reach instruction PCR reaction false negative
Purpose.
Beneficial effects of the present invention include：
(1) present invention designs specific RPA-IAC primers for comma bacillus, establishes comma bacillus RPA-IAC detections
Method, qualitative detection can be carried out to comma bacillus.
(2) amplification interior label sequence and cholera vibrio gene group and human genome of the invention are non-homogeneous, can ensure
Effectively evade the generation of detection false negative and operating personnel's individual nucleic acid Interference Detection while target sequence amplification efficiency；.
(3) present invention only devises pair of primers for comma bacillus can be completed to expand, and the primer for eliminating complexity is set
Meter process；The primer amplification of the present invention only needs isothermal reaction at 37 DEG C, it is not necessary to special heat circulating equipment；During reaction
Between only need 40min, detection time is short；Unlike the dispersion plating of LAMP products, RPA-IAC amplified productions have according to design of primers site
There is the band of particular size, its result is easy to judge.
(4) RPA-IAC amplimers specificity of the invention is good, high sensitivity and applied widely.
(5) the comma bacillus RPA-IAC detection methods that the present invention establishes, it is sensitive, accurate, easy and quick, to inlet and outlet
Complementary goods and examination and test of products quarantine have directive significance.
Brief description of the drawings
Fig. 1 is the specific test electrophoresis detection result of RPA-IAC primer pairs in the embodiment of the present invention 4.Wherein M is
Marker DL1000,1：Comma bacillus；2：Vibrio fluvialis；3：Vibrio parahaemolytious；4：Vibrio mimicus；5：Vibrio alginolyticus；6：Wound
Vibrios；7：Vibrio harveyi；8：Salmonella；9：Escherichia coli；10：Staphylococcus aureus；11：Listeria monocytogenes.
Fig. 2 is the sensitivity test electrophoresis detection result that RPA-IAC is expanded in the embodiment of the present invention 5.Wherein M is marker
DL1000,1-6：Comma bacillus template amount is followed successively by 100ng, 10ng, 1ng, 0.1ng, 0.01ng and 0.001ng, amplification interior label
Dosage be 1.81 × 103copies。
Fig. 3 is the sensitivity test electrophoresis detection result that RPA is expanded in the embodiment of the present invention 5.Wherein M is marker
DL1000,1-6：Comma bacillus template amount is followed successively by 100ng, 10ng, 1ng, 0.1ng, 0.01ng and 0.001ng.
Embodiment
Unless specifically indicated, term used herein has the general sense in art of the present invention.
Below with reference to specific embodiments and the drawings, the present invention will be described, it is necessary to which explanation, these embodiments are only
It is illustrative, and is not considered as limiting the invention.Unreceipted particular technique or condition in embodiment, according to normal
Experiment condition is advised, such as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW, Molecular
cloning:A laboratory manual, 2001), or the condition according to manufacturer's specification suggestion.Agents useful for same or instrument
The unreceipted production firm person of device, being can be by the conventional products of acquisition purchased in market.
The vibrio parahaemolytious that is used in following examples, comma bacillus, vibrio alginolyticus, Vibrio vulnificus, vibrio mimicus, river
Vibrios, Vibrio harveyi, Escherichia coli, staphylococcus aureus, salmonella and Listeria monocytogenes are entered and left the border by Shantou
Inspection and quarantine bureau provides.
Embodiment 1
Present embodiments provide RPA primer pairs and amplification of internal standard sequence and its application.
The screening technique of RPA primer pairs is：For comma bacillus owpW genes (GenBank ID:CP013316.1), if
Count a plurality of RPA primers and carry out a large amount of screenings, the effect between comprehensive its specificity, sensitivity, primer and primer, and
The suitability of used each primer and RPA amplification kits, it is good, reproducible and high sensitivity finally to filter out specificity
The RPA primer pairs of following detectable comma bacillus：
owpW-F：5′-GAAGGTGACTTTATTGTGCGCGCGGGTATTGCC-3′(SEQ ID NO：1)；
owpW-R：5′-CACCAAAGTAGTATTGGACCATAAAGGTAGGT-3′(SEQ ID NO：2)；
The design and synthesis of amplification of internal standard sequence：To avoid the individual nucleic acid of the homologous interference of close bacterium and testing staff
Pollution, is considered by comprehensive analysis, using highly conserved pig detection β-actin partial nucleic acid sequence as base
Plinth, both ends connect primer sequence owpW-F and owpW-R respectively, form the amplification of internal standard sequence that sequence size as follows is 337bp
Row：
GAAGGTGACTTTATTGTGCGCGCGGGTATTGCCgttcgagaccttcaacaccccagccatgtacgtggccatccagg
cggtgctgtccctgtacgcctctggccgcaccactggcatcgtgatggactccggagacggggtcacccacacggtg
cccatctacgaggggtacgccctgccccacgccatcctgcgtctggacctggctggccgggacctgaccgactacct
catgaagatcctgacggagcggggctacagcttcaccaccacggccgagcgggagatcgtgcgggacatcaaggACC
TACCTTTATGGTCCAATACTACTTTGGTG(Seq ID NO：3)
Above-mentioned Seq ID NO：In 3, big write sequence is the owpW-F original series and owpW-R reverse complemental of addition
Sequence, small write sequence 272bp are DQ452569.1 and entitled Sus scrofa beta from Genbank accession number
489-760 one section of sequence in actin (ACTB) gene, partial cds, and the homology checking Jing Guo theory and practice.
The synthetic work of above-mentioned RPA-IAC primer pairs and amplification of internal standard sequence, the limited public affairs of work biotechnology are given birth to by Shanghai
Department completes.
Upper in application, above-mentioned primer pair and amplification of internal standard sequence can be applied to prepare detection or auxiliary detects comma bacillus
Product, such as amplification of internal standard sequence is prepared into the plasmid containing this amplification of internal standard sequence, then with primer pair or other combinations
Get up to turn into the product of detection or auxiliary detection comma bacillus, be used directly to detect or aid in detecting whether biological sample infects suddenly
Random vibrios.
Embodiment 2
A kind of kit and its application are present embodiments provided, the kit includes：
(1)SEQ ID NO：1、SEQ ID NO：2 (coming from embodiment 1), and contain such as SEQ ID NO：Internal standard shown in 3
The plasmid of extension increasing sequence；
(2) DNA extracts reagents；
(3) tube cell containing lyophozyme；
(4) rehydration buffer solution；
(5) magnesium acetate solution.
Above-mentioned tube cell containing lyophozyme, rehydration buffer solution (Rehydration Buffer) and magnesium acetate solution
(280mmol/L) is (TwistDX companies, article No. are from RPA amplification kit TwistAmp Basic kits
TABAS03KIT the reagent in).
The Animal genome DNA for the Tiangeng biochemical technology Co., Ltd that it is DP432 from article No. that the DNA extracts reagents, which are,
Reagent in extracts kit and the bacterial genomes DNA extractions from the Tiangeng biochemical technology Co., Ltd that article No. is DP302
The reagent that kit is included.
It is described to contain such as SEQ ID NO：The structure of the plasmid of amplification of internal standard sequence shown in 3：The internal standard that embodiment 1 is synthesized
Extension increasing sequence is connected on universal support PUC57, is obtained containing such as SEQ ID N O：The positive matter of amplification of internal standard sequence shown in 3
Grain.
The construction work of above-mentioned amplification of internal standard sequence plasmid, general load is connected to using conventional T4 phage DNAs ligase
Body PUC57, and positive plasmid freeze-dried powder is prepared into, it is to be completed by Shanghai Sheng Gong Bioisystech Co., Ltd in the present invention.Press
According to quality (g/ μ L)/(650g/mol × base number) × (6.02 × 10 of copy number N copies/ μ L=PCR fragments23) calculate,
Positive plasmid dry powder is diluted to 1.81 × 103copies/μL
Utilize SEQ ID NO：1、SEQ ID NO：2 and above-mentioned contain such as SEQ ID NO：Amplification of internal standard sequence shown in 3
Plasmid carries out RPA-IAC amplifications for comma bacillus, obtained RPA-IAC target genes amplified production and amplification interior label product
Size is respectively 280bp, 337bp.
It is demonstrated experimentally that above-mentioned DNA extracts reagents, tube cell containing lyophozyme, rehydration buffer solution, magnesium acetate solution, Yi Jiqi
With SEQ ID NO：1、SEQ ID NO：2 and contain such as SEQ ID NO：The plasmid of amplification of internal standard sequence shown in 3 is used in combination,
Effect is more superior, is in particular in that high specificity, reproducible, high sensitivity and Quality Control effect are good.
Upper in application, mentioned reagent box can be applied to detect or aid in detecting on comma bacillus, such as detection or auxiliary inspection
Survey whether biological sample infects comma bacillus, or detection or auxiliary detect biological sample to be measured whether be or candidate is cholera arc
Bacterium；It can also be used to the product for preparing detection or auxiliary detection comma bacillus, such as prepare detection or auxiliary detection biological sample
The product of comma bacillus whether is infected, or prepares detection or aids in the product that detection pathogen is or candidate is comma bacillus.
Embodiment 3
A kind of method for detecting or aiding in detection comma bacillus is present embodiments provided, such as detects or aid in detection biological
Whether sample infects comma bacillus, or whether detection or auxiliary detection pathogen are or candidate is comma bacillus that this method uses
The primer pair of embodiment 1 or the kit of embodiment 2.
The above method comprises the following steps：
Step 1：RPA-IAC is expanded
Using biological sample or the DNA of pathogen as template, add and contain such as SEQ I D NO in embodiment 2：Internal standard shown in 3
The plasmid of extension increasing sequence, using SEQ ID NO：1 and SEQ ID NO：The primer pair of 2 compositions carries out RPA-IAC amplifications, obtains
RPA-IAC amplified productions, while blank control (template is ultra-pure water) is set.
The compound method of RPA-IAC amplification systems is as follows：Into the 0.2mL TwistAmp reaction tubes containing lyophilized enzyme powder
Add rehydration buffer solution (Rehydration Buffer) 29.5 μ L, the upstream and downstream primer (owpW-F and owpW- of embodiment 1
R) each 2 μ L (final concentration of primer is 0.4 μm of ol/L), contain such as SEQ ID NO：The μ L of plasmid 1 of amplification of internal standard sequence shown in 3
(1.81×103Copies), template DNA 1ul (50ng), the μ L (280mmol/L) of magnesium acetate solution 2.5 is finally added, are spent
Ionized water complements to 50 μ L.
RPA-IAC amplification reaction conditions：Above-mentioned RPA-IAC amplification systems are fully mixed, are placed on 37 DEG C of metal bath
40min is reacted, obtains RPA-IAC amplified productions.
Step 2：The electrophoresis detection of RPA-IAC amplified productions
After RPA-IAC reactions terminate, 50 μ L phenol/chloroforms (1 are added into above-mentioned amplified production:1) solution, fully mix
12000rpm centrifuges 2min afterwards, takes 5 μ L of supernatant liquid to observe result on gel imaging system in 1.5% agarose gel electrophoresis,
And RPA-IAC amplified productions are sequenced and (sequence verification have been carried out during method for building up, repeat no more herein).
The standard that the result of RPA-IAC amplification judges is：
If the amplified production that the RPA-IAC expands to obtain only has the target gene fragment that size is 280bp, or gathers around simultaneously
The amplification of internal standard fragment and target gene fragment for having size to be respectively 337bp, 280bp, then it is judged to, containing comma bacillus, prompting
The biological sample infects comma bacillus, or pathogen is or candidate is comma bacillus；If the RPA-IAC expands obtained expansion
Volume increase thing only has the amplification of internal standard fragment that size is 337bp, not containing the target gene fragment that size is 280bp, is then determined as
Comma bacillus is not contained, prompts the biological sample to be uninfected by comma bacillus, or pathogen is not or candidate is not comma bacillus；
If amplification of internal standard fragment that the amplified production that the RPA-IAC expands to obtain is not 337bp containing size and not being containing size
280bp target gene fragment, it is judged to reacting false negative, prompts needs to re-start detection.
If plant to be measured or pathogen to be measured do not extract DNA also, before above method step (1) RPA-IAC amplifications also
Can be including the use of Animal genome DNA extraction kit (being purchased from Tiangeng biochemical technology Co., Ltd, article No. DP432) or thin
Bacterium genome DNA extracting reagent kit (being purchased from Tiangeng biochemical technology Co., Ltd, article No. DP302) is according to kit specification pair
The step of DNA in biological sample or pathogen to be measured is extracted.
Embodiment 4
The present embodiment has carried out validity and specificity verification to the primer pair and amplification of internal standard sequence of embodiment 1, used
Material to be tested it is as follows：Vibrio parahaemolytious, Vibrio vulnificus, vibrio alginolyticus, comma bacillus, vibrio mimicus, vibrio fluvialis, Kazakhstan Vickers
Vibrios, Escherichia coli, staphylococcus aureus, salmonella and Listeria monocytogenes, these bacterial strains are for purchase and through strictly testing
The reference culture of card, it is maintained in Shantou Entry-Exit Inspection and Quarantine Bureau inspection and quarantine technique center.
Step 1：The extraction of genomic DNA
The article No. of TIANGEN Biotech (Beijing) Co., Ltd. is used as DP302 bacterial genomes DNA extraction kit
Extract vibrio parahaemolytious, Vibrio vulnificus, vibrio alginolyticus, comma bacillus, vibrio mimicus, river arc respectively according to kit specification
Bacterium, Vibrio harveyi, Escherichia coli, staphylococcus aureus, the genomic DNA of salmonella and Listeria monocytogenes.
Step 2：RPA-IAC is expanded
Using various genomic DNA templates：
Group 1 is using the genomic DNA pools of comma bacillus as template (positive control).
Group 2 is using the genomic DNA of vibrio fluvialis as template.
Group 3 is using the genomic DNA of vibrio parahaemolytious as template.
Group 4 is using the genomic DNA of vibrio mimicus as template.
Group 5 is using the genomic DNA of vibrio alginolyticus as template.
Group 6 is using the genomic DNA of Vibrio vulnificus as template.
Group 7 is using the genomic DNA of Vibrio harveyi as template.
Group 8 is using the genomic DNA of salmonella as template.
Group 9 is using the genomic DNA of Escherichia coli as template.
Group 10 is using the genomic DNA of staphylococcus aureus as template
Group 11 is using the genomic DNA of Listeria monocytogenes as template.
Be respectively template with a group 1- groups 11, carry out RPA-IAC amplifications, RPA-IAC amplification method with embodiment 3 the step of
One.
Step 3：The electrophoresis detection of RPA-IAC amplified productions
The method of the electrophoresis detection of RPA-IAC amplified productions is the same as the step of embodiment 3 two.
Interpretation of result：
As a result as shown in figure 1, group 1- groups 11 correspond to swimming lane 1-11 respectively, swimming lane 1, which is shown at 337bp and 280bp, to be had
Amplified band, according to the criterion of embodiment 3, for the positive, i.e., smoothly detection comma bacillus, remaining swimming lane 2- swimming lanes 11 are shown
Only there is amplified band at 337bp, according to the criterion of embodiment 3, this eliminates false negative possibility, further carry
The high reliability of result.To sum up, it is above-mentioned it is positive judge and the amplification interior label of accurate and all group of negative result of determination into
Work(expands, and thus proves that the primer pair of the embodiment of the present invention 1 and amplification of internal standard sequence have validity, high specific and good instruction
Property；Further, the detection or auxiliary detection cholera arc that the present invention is established based on primer pair and amplification of internal standard sequence and kit
The method of bacterium can be detected accurately to comma bacillus.
Embodiment 5
The present embodiment has carried out sensitivity checking to the primer pair and amplification of internal standard sequence of embodiment 1, and with not adding expansion
Increase internal standard (not adding and contain such as SEQ ID NO in embodiment 2：The plasmid of amplification of internal standard sequence shown in 3) it is compared, it is used
Material to be tested it is as follows：Comma bacillus, this bacterial strain are purchase and the reference culture through strictly verifying, are stored in Shantou entry and exit inspection
Test Quarantine Bureau's inspection and quarantine technique center.
Step 1：The extraction of genomic DNA
The article No. of TIANGEN Biotech (Beijing) Co., Ltd. is used as DP302 bacterial genomes DNA extraction kit
Extract the genomic DNA of comma bacillus respectively according to kit specification, and 10 times of gradients of obtained genomic DNA progress are dilute
Release, the genomic DNA of the comma bacillus of various concentrations is prepared.
Step 2：RPA is expanded and RPA-IAC amplifications
Using various concentrations comma bacillus genomic DNA as template：
Group 1：With 100ng/ μ L cholera vibrio gene group DNA (1 μ L) for template.
Group 2：With 10ng/ μ L cholera vibrio gene group DNA (1 μ L) for template.
Group 3：With 1ng/ μ L cholera vibrio gene group DNA (1 μ L) for template.
Group 4：With 0.1ng/ μ L cholera vibrio gene group DNA (1 μ L) for template.
Group 5：With 0.01ng/ μ L cholera vibrio gene group DNA (1 μ L) for template.
Group 6：With 0.001ng/ μ L cholera vibrio gene group DNA (1 μ L) for template.
It is respectively template with a group 1- groups 6, carries out RPA amplifications and RPA-IAC amplifications respectively, the method for RPA-IAC amplifications is same
The step of embodiment 3 one, RPA amplifications are distinguished as in amplification system not adding in embodiment 2 containing such as with RPA-IAC amplifications
SEQ ID NO：The plasmid of amplification of internal standard sequence shown in 3.
Step 3：The electrophoresis detection of RPA amplified productions and RPA-IAC amplified productions
The method of the electrophoresis detection of the two amplified production is the same as the step of embodiment 3 two.
Interpretation of result：
The result of RPA-IAC amplifications is as shown in Fig. 2 group 1-4 has band at 280bp and group 2-6 has bar at 337bp
Band, this explanation group 1-6 result effectively and organizes 5- groups 6 without false negative possibility, while also illustrates drawing for the embodiment of the present invention 1
Thing is to higher sensitivity, can detect that in sample the only minim DNA containing 0.1ng；Result such as Fig. 3 institutes of RPA amplifications
Show, sensitivity results expand with RPA-IAC.Amplification interior label is added in summary does not have the sensitivity for reducing detection.
Comparative example 1
In fact, before the primer pair to the present invention is being sought, that has attempted the present invention includes amplification of internal standard sequence
Plasmid (coming from embodiment 2) combines with very more primer pairs, only won in this comparative example it is wherein some be illustrated, its
Remaining it will not go into details, is specially：
Divide using the primer pair of embodiment 1 and with the Software for Design of Primer Premier 5.0 and selection wherein row forward
Primer pair, with embodiment 3 establish detection or auxiliary detect comma bacillus method to 50 parts outlet marine products examined
Survey, with reference to microbiological test of food hygiene national standard (standard No.：GB4789.7-2013 to this 50 portions of marine products in)
Point discrete system biochemical identification of comma bacillus is carried out, the sampling of sensitivity test and template concentrations gradient are set according to reality
Apply example 5 to carry out, testing result is as shown in table 2.
Table 2
As a result show：In view of the presence of the detection knot of primer pair of the present invention in the case of the plasmid for including amplification of internal standard sequence
Fruit and the testing result of normative reference and published detection method are completely the same, this explanation primer pair high specificity of the present invention,
High sensitivity and repeatability is very well, and the combination of the several primer randomly selected using primer-design software is then more or less
In the presence of specificity is not strong, sensitivity is not high, repeatability is bad and disturbs the various defects such as amplification interior label.
Comparative example 2
This comparative example provides the primer pair (coming from embodiment 1) of different reagents and the present invention, includes amplification of internal standard
The plasmid (coming from embodiment 1) of sequence combines testing result contrast during detection comma bacillus.
Various reagents and primer pair of the invention and the plasmid combinations for including amplification of internal standard sequence:
Combination 1：Primer pair of the present invention and plasmid+certain the commercially available Animal genome DNA extraction for including amplification of internal standard sequence
The commercially available RPA amplification kits 1 of kit 1+
Combination 2：Primer pair of the present invention and plasmid+certain the commercially available Animal genome DNA extraction for including amplification of internal standard sequence
The commercially available RPA amplification kits 2 of kit 2+
Combination 3：Primer pair of the present invention and plasmid+certain the commercially available Animal genome DNA extraction for including amplification of internal standard sequence
The commercially available RPA amplification kits 3 of kit 3+
Present invention combination：Primer pair of the present invention and include the plasmid of amplification of internal standard sequence+have from Tiangeng biochemical technology
The reagent that limit company article No. is included by the DP432 Animal genome DNA extraction kit+article No. from TwistDX companies
The reagent included by TABAS03KIT RPA amplification kit TwistAmp Basic kits.
During using the invention described above combine detection comma bacillus, its method uses the method in embodiment 3 to carry out.Sensitivity
The sampling of experiment and the setting of template concentrations gradient are carried out according to embodiment 5.
It is every to use commercial reagent box during using combinations thereof 1, combination 2 and the detection comma bacillus of combination 3, according to it
Kit specification carries out operation progress, and remaining condition is the same as present invention combination.Be marked with simultaneously accurate published control methods (see
Comparative example 1) detected.
By sample product with 50 portions of outlet marine products in comparative example 1.
Testing result is as shown in table 3.
Table 3
As a result show：Primer pair of the present invention, the inspection for including the plasmid of amplification of internal standard sequence and the combination of each particular agent
Survey result and the testing result of EU criteria and published detection method is completely the same, this explanation primer pair of the present invention, internal standard
The kit that the combination of extension increasing sequence and each particular agent is formed has high specificity, high sensitivity and reproducible excellent
Gesture, and in the case where amplification of internal standard sequence be present using primer pair of the present invention and the combination of other some reagents randomly selected
Then more or less presence specificity is strong, the not high and repeated bad various defects of sensitivity.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
SEQUENCE LISTING
<110>Inspection and Quarantine Technic Center, Guangdong Entry-Exit Inspection and Qu；Shantou Entry-Exit Inspection and Quarantine Bureau inspection and quarantine skill
Art center
<120>Detect the RPA-IAC primers and method of comma bacillus
<130> 17CN
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 33
<212> DNA
<213> Artificial
<220>
<223> SEQ ID NO ：1
<400> 1
gaaggtgact ttattgtgcg cgcgggtatt gcc 33
<210> 2
<211> 32
<212> DNA
<213> Artificial
<220>
<223> SEQ ID NO ：2
<400> 2
caccaaagta gtattggacc ataaaggtag gt 32
<210> 3
<211> 334
<212> DNA
<213> Artificial
<220>
<223> SEQ ID NO ：3
<400> 3
gaaggtgact ttattgtgcg cgcgggtatt gccgttcgag accttcaaca ccccagccat 60
gtacgtggcc atccaggcgg tgctgtccct gtacgcctct ggccgcacca ctggcatcgt 120
gatggactcc ggagacgggg tcacccacac ggtgcccatc tacgaggggt acgccctgcc 180
ccacgccatc ctgcgtctgg acctggctgg ccgggacctg accgactacc tcatgaagat 240
cctgacggag cggggctaca gcttcaccac cacggccgag cgggagatcg tgcgggacat 300
caaggaccta cctttatggt ccaatactac tttggtg 337
Claims (10)
1. primer pair and amplification of internal standard sequence, it is characterised in that the primer pair includes SEQ ID NO：Nucleotides sequence shown in 1
Row and SEQ ID NO：Nucleotide sequence shown in 2, the amplification of internal standard fragment include SEQ ID NO：Nucleotides sequence shown in 3
Row.
2. primer pair according to claim 1 and amplification of internal standard sequence are preparing detection or the production of auxiliary detection comma bacillus
Application in product；
Optional, the product for preparing detection or aiding in detection comma bacillus includes：Prepare detection or auxiliary detects biological sample
Whether product infect the product of comma bacillus, and prepare detection or aid in the production that detection pathogen is or candidate is comma bacillus
Product.
3. a kind of kit, it is characterised in that including the primer pair described in claim 1 and include described in claim 1
The plasmid of amplification of internal standard sequence.
4. kit according to claim 3, it is characterised in that also including RPA constant-temperature amplification reagents；
Optional, the RPA constant-temperature amplifications reagent includes the RPA amplification kits TwistAmp from TwistDX companies
The reagent that Basic kits are included；
Optional, in addition to DNA extracts reagents；
Optional, the DNA extracts reagents include the animal gene that the article No. from Tiangeng biochemical technology Co., Ltd is DP432
The reagent that group DNA extraction kit is included.
Optional, the DNA extracts reagents are also included from the bacterial gene that Tiangeng biochemical technology Co., Ltd article No. is DP302
The reagent that group DNA extraction kit is included；
Optional, in addition to positive control and negative control.
5. the kit described in claim 3 or 4 is detecting or aided in detection comma bacillus, or prepares detection or auxiliary detection suddenly
Application in the product of random vibrios；
Optional, the detection or auxiliary detection comma bacillus include：Whether detection or auxiliary detection biological sample infect cholera
Vibrios, and detection or auxiliary detection pathogen whether be or candidate is comma bacillus；
Optional, the product for preparing detection or aiding in detection comma bacillus includes：Prepare detection or auxiliary detects biological sample
Whether product infect the product of comma bacillus, and prepare detection or aid in the production that detection pathogen is or candidate is comma bacillus
Product.
A kind of 6. RPA-IAC methods for detecting or aiding in detection comma bacillus, it is characterised in that including the use of claim 1 institute
The primer pair stated and include any one of the plasmid or claim 3-4 of amplification of internal standard sequence described in claim 1
The step of kit；
Optional, the detection or auxiliary detection comma bacillus include：Whether detection or auxiliary detection biological sample infect cholera
Vibrios, and detection or auxiliary detection pathogen whether be or candidate is comma bacillus.
7. according to the method for claim 6, it is characterised in that：Comprise the following steps：
1) RPA-IAC is expanded
Claim 1 as the primer pair described in template and claim 1 and is included using the DNA that biological sample or pathogen contain
Kit any one of the plasmid or claim 3-4 of described amplification of internal standard sequence carries out RPA amplifications；
2) judge whether biological sample infects comma bacillus according to the RPA-IAC results expanded, or pathogen whether be or candidate
For comma bacillus；
Optional, the step of of extracting DNA is additionally included in biological sample or pathogen before step 1)；
It is optional, it is described extracted in biological sample or pathogen DNA be use the article No. of Tiangeng biochemical technology Co., Ltd for
DP432 Animal genome DNA extraction kit or Tiangeng biochemical technology Co., Ltd article No. is DP302 bacterial genomes
DNA extraction kit is carried out；
Optional, the standard that the result of the RPA-IAC amplifications judges is：
If the amplified production that the RPA-IAC expands to obtain only has the target gene fragment that size is 280bp, or possesses simultaneously big
Small is respectively 337bp, 280bp amplification of internal standard fragment and target gene fragment, then is determined as that biological sample infects comma bacillus,
Or pathogen is or candidate is comma bacillus；If the amplified production that the RPA expands to obtain only has the internal standard that size is 337bp to expand
Increase fragment, not containing the target gene fragment that size is 280bp, be then determined as that biological sample is uninfected by comma bacillus, or cause of disease
Bacterium is not or candidate is not comma bacillus；If the amplified production that the RPA expands to obtain does not expand containing the internal standard that size is 337bp
Increase fragment and not containing size be 280bp target gene fragment, be determined as reaction for false negative, it is necessary to re-start detection.
8. according to the method for claim 7, it is characterised in that：The reaction system of the RPA-IAC amplifications is as follows：
Optional, the reaction condition of the RPA-IAC amplifications is as follows：The temperature of RPA-IAC amplification is 37 DEG C, and the time is
40min。
A kind of 9. system for screening comma bacillus, it is characterised in that including：
Nucleic acid-extracting apparatus, the nucleic acid-extracting apparatus are used to extract the sample of nucleic acid in the biological sample or pathogen；
RPA-IAC amplification devices, the RPA-IAC amplification devices are connected with the nucleic acid-extracting apparatus, suitable for using right
It is required that kit carries out RPA-IAC expansions to the sample of nucleic acid any one of primer pair or claim 3-4 described in 1
Increase；
Judgment means, the judgment means are connected with the RPA-IAC amplification devices, so as to the result expanded based on RPA-IAC,
Judge whether the biological sample infects comma bacillus, or pathogen whether be or candidate is comma bacillus.
10. system according to claim 9, it is characterised in that：
The reaction system of the RPA-IAC amplifications is as follows：
Optional, the reaction condition of the RPA-IAC amplifications is as follows：The temperature of RPA-IAC amplification is 37 DEG C, and the time is
40min。
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| CN114507745A (en) * | 2022-03-17 | 2022-05-17 | 广州市微生物研究所有限公司 | RPA-IAC primer for detecting vibrio alginolyticus, kit, application and method thereof |
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