CN105506121A - Nucleotide sequence used for vibrio parahaemolyticus and vibrio vulnificus detection - Google Patents
Nucleotide sequence used for vibrio parahaemolyticus and vibrio vulnificus detection Download PDFInfo
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Abstract
The invention discloses a primer sequence used for MERT-LAMP amplification. The primer sequence comprises primers F3, primers B3, primers EFIP, primers BIP, primers LF and primers LB for the vibrio parahaemolyticus and the vibrio vulnificus respectively, fluorophores different from one another are marked at the 5' ends of the EFIP primers, and fluorescence quenching groups are marked at other positions of the primers. The invention further discloses application of the primers to vibrio parahaemolyticus and vibrio vulnificus detection. By the adoption of the primer sequence, multiple constant-temperature detection is achieved through a one-step method, operation is easy and convenient, specificity is high, sensitivity is high, and detection speed is high.
Description
Technical field
The present invention relates to the application of MERT-LAMP technology in Bacteria Detection, particularly differentiate the application in detection Vibrio parahemolyticus and Vibrio vulnificus, belong to molecular biology and microbiological art.
Background technology
Vibrio parahemolyticus (Vibrioparahaemolyticus) and Vibrio vulnificus (Vibriovulnificus) are two kinds of important food origin disease pathogenic agent, are extensively to be present in the Gram-negative bacteria in river mouth, coastal and ocean environment.Vibrio parahemolyticus and Vibrio vulnificus are usually separated from sea-food and clinical samples, cause the enteric fever of sea-food source property, and clinical symptom shows as heating, diarrhoea.According to WHO data statistics, Vibrio parahemolyticus is considered to cause the topmost factor of sea-food source property disease, and Vibrio vulnificus is considered to cause the topmost factor of sea-food source property disease death, and its mortality ratio reaches 95%.The whole world has 70,000 patients to die from sea-food source property diarrhoea every year, and the diarrhoea case of most sea-food sources property is that Vibrio parahemolyticus and Vibrio vulnificus cause, thus is subject to showing great attention to of countries in the world, becomes the great public health problem of various countries.Therefore, treat quickly and accurately to give clinical patient, carry out the epidemiology survey of sea-food monitoring and Vibrio parahemolyticus and Vibrio vulnificus, research and develop the detection method that saves time, laborsaving and specificity is higher, can detect simultaneously and identify that Vibrio parahemolyticus and Vibrio vulnificus necessitate.
Detection at present for Vibrio parahemolyticus and Vibrio vulnificus depends on traditional Zengjing Granule and biochemical identification.About 5 to 7 days consuming time of the method, comprises and increases bacterium, selects to cultivate and follow-up biochemical identification, and its inferior position takes time and effort, and the interpretation of chemical result depends on the subjective judgement of people, causes result poor repeatability, easily misjudges.Along with the fast development of nucleic acid diagnostic techniques, some diagnostic techniquess based on PCR are (as regular-PCR technology, Fluorescence PCR assay) be used to the rapid detection of Vibrio parahemolyticus and Vibrio vulnificus, but these methods depend on expensive plant and instrument, need follow-up electrophoretic procedures, expensive probe synthesis, and skilled operator.The laboratory fallen behind for some cannot be carried out, and limits the utilization of these technology.The PCR method of these detection techniques of current utilization diagnosis Vibrio parahemolyticus and Vibrio vulnificus and Real-timePCR method, detection sensitivity is poor, and testing process is consuming time longer, is unfavorable for rapid detection and Emergent detection.
Loop-mediated isothermal amplification technique (Loop-mediatedisothermalamplification, LAMP) be a kind of new nucleic acid specificity amplification technique set up by Notomi etc., the advantage such as there is high specificity, sensitive height, simple to operate, product easily detects.This technology is widely used in molecular diagnosis field.LAMP designs 4 core primers for 6 specific regions of target sequence, utilizes the new chain synthesis of BstDNA polysaccharase catalysis under constant temperature (60 ~ 65 DEG C) with strand-displacement activity, thus makes target sequence efficient amplification.Article 4, among core primers 2 be inner primer, i.e. FIP (Forwardinnerprimer, FIP) and BIP (Backwardinnerprimer, BIP).FIP comprises Flc and F2 (complementary sequence in F2c region), i.e. 5 '-Flc-F2; BIP comprises B1c (complementary sequence in B1 region) and B2, i.e. 5 '-Blc-B2.All the other two core primers are outer primer is F3 and B3.Two ring primers (Loopprimes, LF and LB) are in addition added in reaction system accelerates LAMP reaction.LAMP reacts the target DNA that can increase special, efficiently, fast, makes the amount of product reach 10 within an hour
9individual copy.
After LAMP amplification, the detection of its product can be observed by agarose electrophoresis poststaining.Comparatively easy method directly adds SYBRGreen I dyeing in the product, and presenting green is positive reaction, and orange red is negative reaction.Also can be judged by the turbidity of amplification by product magnesium pyrophosphate precipitation, liquid is muddy, and what centrifugal or adularescent precipitated is positive reaction, and without this phenomenon is then negative reaction.More simple method adds visible dyes in the reactive mixture now, and the color of positive reaction pipe becomes green from light gray, and negative reaction pipe then keeps original light gray.But whether these methods all can only detect LAMP reaction and carry out, and can not identify the specific amplification for a certain target sequence, cause LAMP technology can only detect single purpose sequence, limit the utilization of LAMP technology.
In order to overcome traditional LAMP method technical bottleneck, the applicant disclosed a kind of new MERT-LAMP (MultipleEndonucleaseRestrictionReal-TimeLoop-MediatedIso thermalAmplification) amplification technique (CN104962607A) in 2015, that is, the real-time loop-mediated isothermal amplification technique of many restriction enzymes.Correlation technique content disclosed in this patent documentation is as among the description quoting technical literature and be incorporated into the application.
MERT-LAMP amplification technique is based on LAMP reaction, in conjunction with digestion with restriction enzyme and fluoroscopic examination principle, restriction enzyme site sequence is increased by FIP and/or the BIP primer 5 ' end in LAMP amplification, and mark fluorescent group thereon, and quencher is marked in primer other parts, amplified production can specific identification, cutting by the restriction enzyme in reaction system, fluorophor and quencher separate by this process, and the fluorescent signal of release can be detected by fluorimetric detector.Different fluorescent signals represents different target sequences, thus the detection of can once increase single or multiple goal gene fragment.Thus realize the multiple Constant Temperature Detection of single stage method.The method is easy and simple to handle, high specificity, highly sensitive, detection speed are fast, therefore has wide practical use in diagnostic detection field.
The present invention for the specific gene toxR of Vibrio parahemolyticus and the specific gene rpoS gene design of Vibrio vulnificus two cover MERT-LAMP amplimers, is intended to quick, the responsive and special detection of nucleic acids system set up for Vibrio parahemolyticus and Vibrio vulnificus pathogenic agent respectively.
Summary of the invention
Based on above-mentioned purpose, the present invention provide firstly one group of primer sequence for MERT-LAMP amplification, it is characterized in that, described sequence comprises: the primer V.p-F3 shown in SEQIDNO:1, primer V.p-B3 shown in SEQIDNO:2, the primer V.p-EFIP shown in SEQIDNO:4, the primer V.p-BIP shown in SEQIDNO:5, primer V.p-LF shown in SEQIDNO:6, the primer V.p-LB shown in SEQIDNO:7; And/or
Primer V.v-F3 shown in SEQIDNO:8, the primer V.v-B3 shown in SEQIDNO:9, the primer V.v-EFIP shown in SEQIDNO:11, primer V.v-BIP shown in SEQIDNO:12, primer V.v-LF shown in SEQIDNO:13, the primer V.v-LB shown in SEQIDNO:14
Wherein, be respectively marked with a fluorophor different from each other at the 5 ' end of primer V.p-EFIP and primer V.v-EFIP, and have a quenching of fluorescence group at other position mark of described primer.
Preferably, described sequence also comprises the primer V.p-FIP shown in SEQIDNO:3, and/or the use of the primer V.v-FIP shown in SEQIDNO:10, FIP is in order to provide more EFIP binding site, thus forms more enzyme simple stage property end.
In a preferred technical scheme, the fluorophor that primer V.p-EFIP marks is HEX, and the fluorophor of described V.v-EFIP mark is Cy5.
More preferably, the quenching of fluorescence group that described primer V.p-EFIP marks is BHQ-1, and the quenching of fluorescence group of described V.v-EFIP mark is BHQ-2.
The most preferably, the quenching of fluorescence group mark position of described primer V.p-EFIP is positioned at 5 ' end the 13rd bit base, and the quenching of fluorescence group mark position of described primer V.v-EFIP is positioned at 5 ' end the 11st bit base.
Present invention also offers the application of above-mentioned sequence in Vibrio parahemolyticus and/or Vibrio vulnificus detect, described detection adopts MERT-LAMP amplification technique.
Preferably, the temperature of reaction of described MERT-LAMP amplification is 60-64 DEG C.
In a preferred technical scheme, described application adopts single pattern detection pattern, and the primer used is:
Primer V.p-F3 shown in SEQIDNO:1, primer V.p-B3 shown in SEQIDNO:2, the primer V.p-EFIP shown in SEQIDNO:4, the primer V.p-BIP shown in SEQIDNO:5, primer V.p-LF shown in SEQIDNO:6, the primer V.p-LB shown in SEQIDNO:7; Or
Primer V.v-F3 shown in SEQIDNO:8, primer V.v-B3 shown in SEQIDNO:9, the primer V.v-EFIP shown in SEQIDNO:11, the primer V.v-BIP shown in SEQIDNO:12, primer V.v-LF shown in SEQIDNO:13, the primer V.v-LB shown in SEQIDNO:14.
Preferably, described sequence also comprises the primer V.p-FIP shown in SEQIDNO:3, or the primer V.v-FIP shown in SEQIDNO:10.
In another preferred technical scheme, described application adopts multiplicity sampling detecting pattern, and the primer used is:
Primer V.p-F3 shown in SEQIDNO:1, primer V.p-B3 shown in SEQIDNO:2, the primer V.p-EFIP shown in SEQIDNO:4, the primer V.p-BIP shown in SEQIDNO:5, primer V.p-LF shown in SEQIDNO:6, the primer V.p-LB shown in SEQIDNO:7; With
Primer V.v-F3 shown in SEQIDNO:8, primer V.v-B3 shown in SEQIDNO:9, the primer V.v-EFIP shown in SEQIDNO:11, the primer V.v-BIP shown in SEQIDNO:12, primer V.v-LF shown in SEQIDNO:13, the primer V.v-LB shown in SEQIDNO:14.
Preferably, described sequence also comprises the primer V.p-FIP shown in SEQIDNO:3, and the primer V.v-FIP shown in SEQIDNO:10.
The present invention applies above-mentioned primer can detect Vibrio parahemolyticus or Vibrio vulnificus separately respectively by MERT-LAMP, and a step simultaneously can detect and differentiate Vibrio parahemolyticus and Vibrio vulnificus accurately, there is easy and simple to handle, high specificity, the advantage that highly sensitive, detection speed is fast.The Monitoring lower-cut of diagnosis Vibrio parahemolyticus is 250fgDNA/ reaction tubes, and the shortest detection time consumed is approximately 19 minutes, and the DNA detecting lowest detectable limit level also only consumes about 30 minutes.The Monitoring lower-cut of Diagnosis of Trauma vibrios is 125fgDNA/ reaction tubes, and the shortest detection time consumed is approximately 19 minutes, and the DNA detecting lowest detectable limit level also only consumes about 28 minutes.The present invention applies above-mentioned primer can detect and differentiate Vibrio parahemolyticus and Vibrio vulnificus by MERT-LAMP simultaneously exactly, the positive is to the detection of 27 strain Vibrio parahemolyticus and 12 strain Vibrio vulnificus, there is not positive findings in the non-Vibrio parahemolyticus of 73 strain, non-wound vibrios, shows good specificity.
Accompanying drawing explanation
Fig. 1. design of primers position and direction schematic diagram;
Fig. 2 .MERT-LAMP amplification fluorescent visual detection and electrophoresis detection result judge collection of illustrative plates;
The optimal reaction temperature of Fig. 3 .MERT-LAMP technology detects collection of illustrative plates;
Fig. 4 .MERT-LAMP detects the sensitivity technique collection of illustrative plates of single sample;
Fig. 5 .LAMP detects the sensitivity technique collection of illustrative plates of single sample;
Fig. 6 .MERT-LAMP detects the sensitivity technique collection of illustrative plates of multiplicity sampling;
The specific detection collection of illustrative plates of Fig. 7 .MERT-LAMP technology.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to protection scope of the present invention.
The object of the present invention is to provide one to be applied to Bacteria Detection, particularly the detection sequence of constant temperature technology, further provide MERT-LAMP amplification technique to detect the method for Vibrio parahemolyticus and Vibrio vulnificus, and be applied to the test kit of the method.
MERT-LAMP amplimer is designed according to the specific gene toxR (Genbankaccessionno.L11929) of Vibrio parahemolyticus and the specific gene rpoS (Genbankaccessionno.AY187681) of Vibrio vulnificus, toxR gene is present in all Vibrio parahemolyticus, its specificity is good, Vibrio parahemolyticus and the nearer bacterial classification of other sibships and Pseudomonas can be distinguished.RpoS gene is present in all Vibrio vulnificus, and its specificity is good, Vibrio vulnificus and the nearer bacterial classification of other sibships and Pseudomonas can be distinguished.Utilize Multiple Sequence Alignment software ClustalX1.83 and primer-design software PrimerPremier5.0 to design MERT-LAMP and react primer, and the Auele Specific Primer of acquisition is carried out sequence alignment analysis in ncbi database, mate to get rid of non-specific that primer and other species sequence may exist, finally obtain after optimizing two cover MERT-LAMP amplimers.Fig. 1 (Figure 1A is the sequence for Vibrio parahemolyticus amplification, and Figure 1B is the sequence for Vibrio vulnificus amplification) is seen in the position of design of primers and direction, and sequence is in table 1.
Table 1 primer information
V.p, Vibrioparahaemolyticus, Vibrio parahemolyticus;
V.v, Vibriovulnificus, Vibrio vulnificus;
F, forward, forward;
B, backward, oppositely;
FIP, forwardinnerprimer, forward inner primer;
EFIP, thenovelforwardinnerprimer, enzyme target forward inner primer;
BIP, backwardinnerprimer, reverse inner primer;
LF, loopforwardprimer, ring-type forward primer;
LB, loopbackwardprimer, ring-type reverse primer;
Probe, probe.
Wherein, V.p-EFIP is marked with fluorophor HEX (chlordene-6-methyl fluorescein) and quenching group BHQ-1 (Blackholequencher1, fluorescence quenching 1), and concrete mark position is:
5'-HEX-TGCAATG-TGAGAT(BHQ1)TCCGCAGGGTTTGTAATTATTTTTGGCACTATTACTACCG-3'；
V.v-EFIP is marked with fluorophor Cy5 (Cy5 fluorescein) and quenching group BHQ-2 (Blackholequencher2, fluorescence quenching 2), and concrete mark position is:
5'-Cy5-TGCAATG-CTCT(BHQ-2)TCCAGCGTTGAAGGCTCTTTTCCGAAACAAAAAGAAGTGTTGG-3'；
V.p-Probe is marked with fluorophor FAM (6-Fluoresceincarboxylic acid) and quenching group BHQ-1, and concrete mark position is:
5’-FAM-ACGCCAAACCGAACCTTTCTGCTGA-BHQ1-3′；
V.v-Probe is marked with fluorophor HEX and quenching group BHQ-1, and concrete mark position is: 5 '-HEX-CCGTTAACCGAACCACCCGCAA-BHQ-1-3 '.
The reagent using and relate in the present invention:
LoopampKit (EikenChemicalCo.Ltd., Tokyo, Japan) is purchased from Japanese Rong Yan company.DNA extraction kit (QIAampDNAminikits; Qiagen, Hilden, Germany) be purchased from German Qiagen company.QPCR reaction system mixture Premix (TakaraBio, Inc., Otsu, Japan, dNTP and damping fluid) is purchased from Takara bio tech ltd, Beijing.PCR reaction system mixture M IX (Taq DNA polymerase, dNTP and damping fluid) is purchased from Beijing CoWin Bioscience Co., Ltd..DL50DNAMarker and DL50DNAMarker is purchased from precious biotechnology (Dalian) company limited.All the other reagent are commercially available analytical pure product.
The key instrument using and relate in the present invention's experiment: Loopamp real-time turbidimeter LA-320C (EikenChemicalCo., Ltd, Japan) is purchased from Japanese Rong Yan company.Real-time fluorescence detector is Rotor-GeneQReal-TimeSystem (Qiagen), German Qiagen product; Electrophoresis equipment is Jun Yi east, Beijing electrophoresis equipment company limited product; PCR instrument is SensoquestLabcycler, German Sensoquest product; Gel imaging system is Bio-RadGelDoxXR, U.S. Bio-Rad product.
Vibrio parahemolyticus, the extraction of the genomic dna of Vibrio vulnificus and other kind bacterium.
Genome extracts: the extraction of bacterial genomes uses the DNA extraction kit (QIAampDNAminikits of Qiagen company; Qiagen, Hilden, Germany), operate to specifications.Utilize ultraviolet spectrophotometer to measure the concentration of genomic dna and purity, Vibrio parahemolyticus and vibrio vulnficus gene group DNA are with GE buffer serial-dilution (from 2.5ng, 250pg, 25pg, 2.5pg, 250fg, 125fg, 62.5fg to 31.25fg).The all a small amount of packing of various genomic dna ,-20 DEG C save backup.
Embodiment 1.MERT-LAMP standard reaction system
The reaction system of LAMP is as follows: the concentration of primer EFIP and FIP is respectively 20pmol, the concentration of primer BIP is that 40pmol is (in order to obtain better technique effect, also primer EBIP can be introduced, namely increase the primer BIP of restriction enzyme site sequence, at this moment, the concentration of primer BIP, EBIP is respectively 20pmol), the concentration of primers F 3 and B3 is 5pmol, the concentration of primer LF and BF is the MgSO of the Betain of 20pmol, 10mM, 6mM
4, the dNTP of 1mM, 10 × BstDNA polymerase buffer of 2.5 μ L, the strand displacement archaeal dna polymerase of 8U, the DNA restriction enzyme of 15U, the template of 1 μ L, adds deionized water to 25 μ L.Whole reaction is carried out in fluorescence detector (Rotor-GeneQRealTimeSystem, Qiagen), isothermal duplication 60-64 DEG C 1h, 80 DEG C of 5min termination reactions.
Embodiment 2.MERT-LAMP multiple reaction system
The reaction system of MERT-LAMP is as follows: the concentration of primer V.v-EFIP and V.v-FIP is respectively the concentration of 10pmol, primer V.v-BIP is 20pmol, and the concentration of primer V.v-LF and V.v-LB is 10pmol, and the concentration of primer V.v-F3 and V.v-B3 is 10pmol; The concentration of primer V.p-EFIP, V.p-FIP is respectively the concentration of 30pmol, primer V.p-BIP is 60pmol, and the concentration of primer V.p-LF and V.p-LB is 30pmol, and the concentration of primer V.p-F3 and V.p-B3 is 10pmol; The MgSO of the Betain of 10mM, 6mM
4, the dNTP of 1mM, 10 × BstDNA polymerase buffer of 12.5 μ L, the strand displacement archaeal dna polymerase of 8U, the DNA restriction enzyme of 15U, the template of 1 μ L, adds deionized water to 25 μ L.Whole reaction is carried out in fluorescence detector (Rotor-GeneQRealTimeSystem, Qiagen), isothermal duplication 60-64 DEG C 1h, 80 DEG C of 5min termination reactions.
Embodiment 3.LAMP standard reaction system
The reaction system of LAMP is as follows: the concentration of primers F IP is respectively the concentration of 40pmol, primer BIP is 40pmol, and the concentration of primers F 3 and B3 is 5pmol, and the concentration of primer LF and BF is the MgSO of the Betain of 20pmol, 10mM, 6mM
4, the dNTP of 1mM, 10 × BstDNA polymerase buffer of 2.5 μ L, the strand displacement archaeal dna polymerase of 8U, the template of 1 μ L, adds deionized water to 25 μ L.Whole reaction is carried out in the real-time turbidimeter LA-320C (EikenChemicalCo., Ltd, Japan) of Loopamp, isothermal duplication 60-64 DEG C 1h, 80 DEG C of 5min termination reactions.
Embodiment 4.Real-timeRCR method reaction system
Wherein, the upstream primer sequence of the qPCR system of Vibrio parahemolyticus is V.p-FSEQID:15, and downstream primer sequence is V.p-RSEQIDNO:16, and probe sequence is V.p-ProbeSEQIDNO:17.
The upstream primer sequence of Vibrio vulnificus is V.v-FSEQID:18, and downstream primer sequence is V.v-RSEQIDNO:19, and probe sequence is V.v-ProbeSEQIDNO:20.
Amplification also measurement result on real-time fluorescence quantitative PCR instrument:
At fluorescence detector (Rotor-GeneQRealTimeSystem, Qiagen) Real-time reaction is carried out in, after amplification terminates, get same analysis of threshold data after deduction background fluorescence signal, determine Ct (cyclethreshold) value of this reaction.
The common RCR reaction system of embodiment 5.
PCR reaction conditions:
Wherein, the upstream primer sequence of Vibrio parahaemolyticus is V.p-FSEQID:15, and downstream primer sequence is V.p-RSEQIDNO:16.
The upstream primer sequence of Vibrio vulnificus is V.v-FSEQID:18, and downstream primer sequence is V.v-RSEQIDNO:19.
Reaction terminate after, get 10 μ lPCR products and carry out electrophoresis on the sepharose of 2.5%, and on gel imaging system observation analysis result.
The feasibility checking of embodiment 6.MERT-LAMP
Standards system is for verifying the feasibility of MERT-LAMP primer, under standards system condition, add the MERT-LAMP primer of a set of correspondence and corresponding DNA profiling, confirm MERT-LAMP technical feasibility by visible color method of changing, electrophoresis assays and fluorescence detection method.
Visible color method of changing: MERT-LAMP also produces a large amount of pyrophosphate ions while synthetic DNA, and this ion can capture the mn ion be combined with fluorexon, makes fluorexon recover unbound state and fluoresce.The magnesium ion that this light-emitting admixture can produce in reaction is combined, and fluorescence is enhanced.Can pass through fluorescence visual detection colour-change sentence read result, positive reaction pipe becomes green from light gray, and negative reaction keeps light grey constant, sees Fig. 2 A (Vibrio parahemolyticus) and Fig. 2 B (Vibrio vulnificus).
Electrophoresis assays: due to MERT-LAMP reaction with LAMP reacting phase seemingly, its product is also the loop-stem structure of target sequence formation and the DNA fragmentation mixture of many rings Cauliflower spline structure of a series of inverted repeat, on gel, the staged collection of illustrative plates of different size fragment zone composition is manifested after electrophoresis, can judge according to clip size, see Fig. 2 C (Vibrio parahemolyticus) and 2D (Vibrio vulnificus).
Real-time fluorescence detection method: under standards system condition, in reaction tubes, add a set of primer and corresponding template, the fluorescence that enzyme cuts rear generation is received by fluorimetric detector, obtains stable fluorescence curve.
Embodiment 7. measures the optimal reaction temperature of MERT-LAMP technology
Under standards system condition, add the MERT-LAMP primer of Vibrio vulnificus and the DNA profiling of Vibrio vulnificus, its template concentrations is 25pg/ μ l.Reaction is carried out (60-67 DEG C) under constant temperature, and application of results real-time fluorescence instrument detects, and obtains two groups of different dynamic curve diagrams at different temperature, sees Fig. 3.The 60-64 DEG C of recommended optimal reaction temperature as MERT-LAMP technology.
Embodiment 8.MERT-LAMP detects the sensitivity of single sample
Under standards system condition, doubling dilution template DNA (2.5ng, 250pg, 25pg, 2.5pg, 250fg, 125fg, 62.5fg and 31.25fg/ microlitre), the each concentration template of 1 microlitre adds in reaction system, is detected, obtain stable fluoroscopic examination figure, see Fig. 4 by real-time fluorescence.MERT-LAMP detects single target sequence, and the Monitoring lower-cut of diagnosis Vibrio parahemolyticus is 250fgDNA/ reaction tubes, and the shortest detection time consumed is approximately 19 minutes, and the DNA detecting lowest detectable limit level also only consumes about 30 minutes.When in reaction system, genomic templates amount is reduced to below 250fg, there is not positive amplification in MERT-LAMP reaction, sees Fig. 4 A.When product carries out electrophoresis detection, its Monitoring lower-cut is also 250fgDNA/ reaction tubes, sees Fig. 4 C.
MERT-LAMP detects single target sequence, and the Monitoring lower-cut of Diagnosis of Trauma vibrios is 125fgDNA/ reaction tubes, and the shortest detection time consumed is approximately 19 minutes, and the DNA detecting lowest detectable limit level also only consumes about 28 minutes.When in reaction system, genomic templates amount is reduced to below 125fg, there is not positive amplification in MERT-LAMP reaction, sees Fig. 4 B.When product carries out electrophoresis detection, its Monitoring lower-cut is also 125fgDNA/ reaction tubes, sees Fig. 4 D.
Embodiment 9. compares MERT-LAMP, and LAMP, qPCR and PCR detect the sensitivity of single sample
Under standard LAMP system condition, doubling dilution template DNA (2.5ng, 250pg, 25pg, 2.5pg, 250fg, 125fg, 62.5fg and 31.25fg/ microlitre), the each concentration template of 1 microlitre adds in reaction system, by real-time Turbidity measurement, obtain stable real-time Turbidity measurement figure, see Fig. 5.LAMP detects single target sequence, and the Monitoring lower-cut of diagnosis Vibrio parahemolyticus is 250fgDNA/ reaction tubes, and the shortest detection time of consumption is approximately 19 minutes, and the DNA detecting lowest detectable limit level also only consumes about 30 minutes.When in reaction system, genomic templates amount is reduced to below 250fg, there is not positive amplification in LAMP reaction, sees that Fig. 5 A is (in Fig. 5 A, the DNA concentration that real-time Haze curve is from left to right corresponding in turn to is 2.5ng, 250pg, 25pg, 2.5pg, 250fg, 125fg and 62.5fg/ microlitre).When product carries out electrophoresis detection, its Monitoring lower-cut is also 250fgDNA/ reaction tubes, sees Fig. 5 C.
LAMP detects single target sequence, and the Monitoring lower-cut of Diagnosis of Trauma vibrios is 125fgDNA/ reaction tubes, and the shortest detection time of consumption is approximately 19 minutes, and the DNA detecting lowest detectable limit level also only consumes about 28 minutes.When in reaction system, genomic templates amount is reduced to below 125fg, there is not positive amplification in LAMP reaction, sees Fig. 5 B, (in Fig. 5 B, the DNA concentration that real-time Haze curve is from left to right corresponding in turn to is 2.5ng, 250pg, 25pg, 2.5pg, 250fg and 125fg/ microlitre).When product carries out electrophoresis detection, its Monitoring lower-cut is also 125fgDNA/ reaction tubes, sees Fig. 5 D.
Under the qPCR reaction conditions of standard, doubling dilution template DNA (2.5ng, 250pg, 25pg, 2.5pg, 250fg, 125fg, 62.5fg and 31.25fg/ microlitre), 1 microlitre each concentration template adds anti-qPCR and answers in system, detected by real-time fluorescence, obtain stable real-time fluorescence test pattern, in table 2.QPCR detects single target sequence, and the Monitoring lower-cut of diagnosis Vibrio parahemolyticus is 2.5pgDNA/ reaction tubes, and when in reaction system, genomic templates amount is reduced to below 2.5pg, qPCR reaction does not occur positive amplification, in table 2.QPCR detects single target sequence, and the Monitoring lower-cut of Diagnosis of Trauma vibrios is 2.5pgDNA/ reaction tubes, and when in reaction system, genomic templates amount is reduced to below 2.5pg, qPCR reaction does not occur positive amplification, in table 2.
Under the PCR reaction conditions of standard, doubling dilution template DNA (2.5ng, 250pg, 25pg, 2.5pg, 250fg, 125fg, 62.5fg and 31.25fg/ microlitre), 1 microlitre each concentration template adds anti-qPCR and answers in system, and PCR detects single target sequence, the Monitoring lower-cut of diagnosis Vibrio parahemolyticus is 25pgDNA/ reaction tubes, when in reaction system, genomic templates amount is reduced to below 25pg, there is not positive amplification, in table 2 in PCR reaction.PCR detects single target sequence, and the Monitoring lower-cut of Diagnosis of Trauma vibrios is 25pgDNA/ reaction tubes, and when in reaction system, genomic templates amount is reduced to below 25pg, PCR reaction does not occur positive amplification, in table 2.
Table 2.MERT-LAMP, LAMP, qPCR and PCR detect the remolding sensitivity of single sample comparatively
Embodiment 10. verifies Multiple detection ability and the sensitivity of MERT-LAMP
In order to obtain stable multi-fluorescence test pattern, adaptation Multiple detection being optimized to standards system, setting up Multiple detection system.We add the template of two cover primers and correspondence in multiple reaction mixture simultaneously, and (doubling dilution template DNA is to 2.5ng, 250pg, 25pg, 2.5pg, 250fg, 125fg, 62.5fg and 31.25fg/ microlitre, 1 microlitre each concentration template adds in multiple MERT-LAMP reaction system).Detected by fluorimetric detector, obtain stable multi-fluorescence detection figure, see Fig. 6.MERT-LAMP reaction system can carry out the detection of two target sequences simultaneously, demonstrate the Multiple detection ability of MERT-LAMP technology, when application MERT-LAMP detects multiple target sequence, its detection time and detection sensitivity and simple sequence detect the change not having matter, and the shortest detection time is about 19 minutes.
When multiple MERT-LAMP detects multiple target sequence simultaneously, the Monitoring lower-cut of diagnosis Vibrio parahemolyticus is 250fgDNA/ reaction tubes, and the shortest detection time consumed is approximately 19 minutes, and the DNA detecting lowest detectable limit level also only consumes about 30 minutes.When in reaction system, genomic templates amount is reduced to below 250fg, there is not positive amplification in MERT-LAMP reaction, sees Fig. 6 A.The Monitoring lower-cut of Diagnosis of Trauma vibrios is 125fgDNA/ reaction tubes, and the shortest detection time consumed is approximately 19 minutes, and the DNA detecting lowest detectable limit level also only consumes about 28 minutes.When in reaction system, genomic templates amount is reduced to below 125fg, there is not positive amplification in MERT-LAMP reaction, sees Fig. 6 B.
The evaluation of embodiment 11. detection specificity
With common pathogenic bacterium and conditioned pathogen DNA (Vibrio parahaemolyticus, Vibrio vulnificus, shigella, salmonella, Listeria monocytogenes, vibrio cholerae, listeria ivanovii, Bacillus cereus, enteropathogenic Escherichia coli, enterotoxigenic E.Coli, enteroinvasive E.Coli etc.) evaluate the specificity of MERT-LAMP reaction system for template, bacterial strain information is in table 3.MERT-LAMP amplification technique can detect and differentiate Vibrio parahemolyticus and Vibrio vulnificus simultaneously accurately, and non-Vibrio parahemolyticus, non-wound vibrios do not occur positive findings, illustrates that the specificity of MERT-LAMP method is good, sees Fig. 7.
Table 3. bacterial strain information
Note: ATCC, American Type Culture Collecti; ICDC, Inst of Infection Disease Prevention and Control, Chinese Diseases Prevention an.
Claims (11)
1. one group is used for the primer sequence of MERT-LAMP amplification, it is characterized in that, described sequence comprises: the primer V.p-F3 shown in SEQIDNO:1, primer V.p-B3 shown in SEQIDNO:2, primer V.p-EFIP shown in SEQIDNO:4, primer V.p-BIP shown in SEQIDNO:5, the primer V.p-LF shown in SEQIDNO:6, the primer V.p-LB shown in SEQIDNO:7; And/or
Primer V.v-F3 shown in SEQIDNO:8, the primer V.v-B3 shown in SEQIDNO:9, the primer V.v-EFIP shown in SEQIDNO:11, primer V.v-BIP shown in SEQIDNO:12, primer V.v-LF shown in SEQIDNO:13, the primer V.v-LB shown in SEQIDNO:14
Wherein, be respectively marked with a fluorophor different from each other at the 5 ' end of primer V.p-EFIP and primer V.v-EFIP, and have a quenching of fluorescence group at other position mark of described primer.
2. sequence according to claim 1, is characterized in that, described sequence also comprises the primer V.p-FIP shown in SEQIDNO:3, and/or the primer V.v-FIP shown in SEQIDNO:10.
3. sequence according to claim 1, is characterized in that, the fluorophor that primer V.p-EFIP marks is HEX, and the fluorophor of described V.v-EFIP mark is Cy5.
4. sequence according to claim 3, is characterized in that, the quenching of fluorescence group that described primer V.p-EFIP marks is BHQ-1, and the quenching of fluorescence group of described V.v-EFIP mark is BHQ-2.
5. sequence according to claim 4, is characterized in that, the quenching of fluorescence group mark position of described primer V.p-EFIP is positioned at 5 ' end the 13rd bit base, and the quenching of fluorescence group mark position of described primer V.v-EFIP is positioned at 5 ' end the 11st bit base.
6., according to the application of described sequence arbitrary in claim 1-5 in Vibrio parahemolyticus and/or Vibrio vulnificus detect, described detection adopts MERT-LAMP amplification technique.
7. application according to claim 6, is characterized in that, the temperature of reaction of described MERT-LAMP amplification is 60-64 DEG C.
8. application according to claim 6, is characterized in that, described application adopts single pattern detection pattern, and the primer used is:
One group of primer sequence being used for MERT-LAMP and increasing, it is characterized in that, described sequence comprises: the primer V.p-F3 shown in SEQIDNO:1, primer V.p-B3 shown in SEQIDNO:2, primer V.p-EFIP shown in SEQIDNO:4, primer V.p-BIP shown in SEQIDNO:5, the primer V.p-LF shown in SEQIDNO:6, the primer V.p-LB shown in SEQIDNO:7; Or
Primer V.v-F3 shown in SEQIDNO:8, primer V.v-B3 shown in SEQIDNO:9, the primer V.v-EFIP shown in SEQIDNO:11, the primer V.v-BIP shown in SEQIDNO:12, primer V.v-LF shown in SEQIDNO:13, the primer V.v-LB shown in SEQIDNO:14.
9. application according to claim 8, is characterized in that, described sequence also comprises the primer V.p-FIP shown in SEQIDNO:3, or the primer V.v-FIP shown in SEQIDNO:10.
10. application according to claim 6, is characterized in that, described application adopts multiplicity sampling detecting pattern, and the primer used is:
Primer V.p-F3 shown in SEQIDNO:1, primer V.p-B3 shown in SEQIDNO:2, the primer V.p-EFIP shown in SEQIDNO:4, the primer V.p-BIP shown in SEQIDNO:5, primer V.p-LF shown in SEQIDNO:6, the primer V.p-LB shown in SEQIDNO:7; With
Primer V.v-F3 shown in SEQIDNO:8, primer V.v-B3 shown in SEQIDNO:9, the primer V.v-EFIP shown in SEQIDNO:11, the primer V.v-BIP shown in SEQIDNO:12, primer V.v-LF shown in SEQIDNO:13, the primer V.v-LB shown in SEQIDNO:14.
11. application according to claim 10, is characterized in that, described sequence also comprises the primer V.p-FIP shown in SEQIDNO:3, and the primer V.v-FIP shown in SEQIDNO:10.
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