CN102676653A - Non-diagnostic method for detection of O1 group and O139 group of vibrio cholerae through double TaqMan probe fluorescence RT-PCR (Reverse Transcription-Polymerase Chain Reaction) - Google Patents
Non-diagnostic method for detection of O1 group and O139 group of vibrio cholerae through double TaqMan probe fluorescence RT-PCR (Reverse Transcription-Polymerase Chain Reaction) Download PDFInfo
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Abstract
The invention discloses a non-diagnostic method for detection of O1 group and O139 group of vibrio cholerae through double TaqMan probe fluorescence RT-PCR (Reverse Transcription-Polymerase Chain Reaction). A primer and a probe are designed aiming at an O antigen synthetic gene rfb gene of O1 group and O139 group of vibrio cholerae, and real-time quantitative detection is performed on a sample to be detected; the method is extremely sensitive to an rfb gene fragment of the O1 group and O139 group of vibrio cholerae; and the detection lower limit of the double real-time PCR is 100 times higher than that of the normal PCR and can reach 1.0*10<2> copies per milliliter. Through the double real-time PCR, two genes or two kinds of different bacteria can be simultaneously detected in the reaction system, so that experimental steps, experimental time and reagent consumption are greatly reduced, and the non-diagnostic method has extremely high application value in actual application, particularly during epidemic situation outbreak.
Description
Technical field
The present invention relates to the non-diagnostic methods that a kind of dual TaqMan fluorescence probe quantitative RT-PCR detects O1 crowd and O139 group cholera vibrio.
Background technology
Cholera is a kind of ancient and popular transmissible disease of severe intestinal widely that is caused by vibrio cholerae (Vibrio cholerae); Once caused repeatedly in the world and be very popular; Patient's clinical symptom shows as violent vomiting more, and a large amount of thin rice gruel shapes are drained, and can cause rapid dehydration; As treat untimelyly, and can cause hypovolemic shock, oxypathy etc., in addition dead.
Vibrio cholerae is separated in 1854 by Italian anatomist Filippo Pacini first, belongs to vibrionaceae (Vibrionaceae) Vibrio (Vibrio), aerobic or facultative anaerobe; Gram-negative, thalline is short and small, and is crooked slightly; Curved or funny point-like, thalline tail end amphitrichous, motion is active.Vibrio cholerae has a plurality of serotypes, can cause that wherein the cholera popular mainly contains two kinds: O1 crowd and O139 crowd.Since 1817, it all is to be caused by the O1 group cholera vibrio that the cholera of 7 global ranges is very popular; It is popular that the cholera that non-O1 group cholera vibrio causes has been broken out first in India in 1992 and Bangladesh, involves Asia, U.S.A, three continents, Europe at present successively, constitute overstep national boundaries, the situation that is very popular on circle, continent.
In view of the cholera serious threat public's life security, it is most important therefore to detect vibrio cholerae fast, accurately and delicately.Cholera is effectively controlled in China basically, but generation or popular potential threat still exist.Get the upper strata culture after traditional vibrio cholerae detection method needs in basic protein peptone nutrient solution, to carry out strain culturing earlier and do the vibrios isolation identification; The required cycle of this detection method is long, workload is big; And be subject to environment, culture condition and subjective factor influence, be unfavorable for the quick diagnosis of vibrio cholerae.Therefore set up a kind of fast, responsive, preventing and controlling have crucial meaning to special detection method to cholera.With hemolysin gene (hlyA) is the fluorescence quantitative PCR detection O1 crowd of probe foundation and the method for O139 group cholera vibrio, and its specific degree is not high, and can detect non-O1 crowd and non-O139 group cholera vibrio simultaneously.
Based on the double fluorescent PCR method specific detection O1 crowd of SYBR Green and the method for O139 group cholera vibrio; Need to distinguish specific amplification and non-specific amplification by melting curve; This makes at the purpose amplified fragments of bacterial strain to be checked because of occurring being difficult to judge through melting curve that using in the evaluation of O1 crowd and O139 group cholera vibrio has certain limitation when base mutation influences product melting temperature (Tm) (Tm value).
Summary of the invention
To the problem that prior art exists, the object of the present invention is to provide the non-diagnostic methods of the dual TaqMan fluorescence probe RT-PCR detection of good, highly sensitive detection O1 crowd of a specific specificity and O139 group cholera vibrio.
For realizing above-mentioned purpose, a kind of dual TaqMan fluorescence probe RT-PCR of the present invention detects the non-diagnostic methods of O1 crowd and O139 group cholera vibrio, and this non-diagnostic methods comprises:
O antigen synthetic gene rfb gene design primer and probe to O1 crowd and O139 group cholera vibrio:
Further, 5 ' the end institute mark fluorescent element of said probe O1-P and O139-P is respectively FAM and HEX, and probe 3 ' end all connects quenching group.
Further, said this quenching group is the non-fluorescent quenching group of BHQ1.
Further, said this non-diagnostic methods comprises step:
1) extracts bacterial chromosomal dna with test kit;
2) real-time fluorescence RT-PCR reacts, and prepares 20 μ L PCR reaction systems of certain concentration of component, and of short duration centrifugal branch installs in the PCR pipe behind the mixing;
3) testing sample is joined in the reaction system, increase;
4) collect fluorescent signal at annealing stage, detect the Ct value.
Further, the system component and the volume thereof of said real-time fluorescence RT-PCR reaction are following:
Cycling condition:
Further, the concrete steps of said O1 crowd and O139 group cholera vibrio standard substance preparation comprise:
1) according to O1 crowd who is designed and O139 group cholera vibrio primer, the check order full pure culture liquid dna profiling of bacterial strain N16961 and MO45 of increase respectively O1 crowd and O139 group cholera vibrio;
2) reclaim test kit according to glue and reclaim the purifying target DNA fragment;
3) preparation O1 crowd and O139 group cholera vibrio rfb gene clone;
4) insert segmental restricted enzyme cutting analysis behind extraction O1 crowd and the O139 group cholera vibrio plasmid, get the endonuclease reaction result who is positive and carry out cloned plasmids order-checking experiment;
5) after the concentration conversion of O1 crowd and O139 group cholera vibrio DNA was copy number, it is subsequent use that plasmid is diluted to several concentration gradients.
Further, the check order full amplification condition of pure culture liquid dna profiling of bacterial strain N16961 and MO45 of said O1 crowd and O139 group cholera vibrio is:
Further, said dna fragmentation with glue recovery purifying prepares O1 crowd and O139 group cholera vibrio rfb gene clone, and the ligation system of preparation is:
Further, during the preparation of said O1 crowd and O139 group cholera vibrio rfb clone, with O1F/O1R and O139F/O139R to the bacterium colony of growing on the nutrient agar plate that scribbles Amp 100 evaluation of increasing.
Further, said endonuclease reaction system is following:
Further, after said double digestion is identified the positive, machine order-checking on the cloned plasmids, sequence results and known aim sequence are compared.
Further, the detection lower limit to O1 crowd and O139 group cholera vibrio plasmid is 1.0 * 10 in the system of said real-time fluorescence RT-PCR reaction
2Copy/μ l.
Beneficial effect of the present invention is: (retro-transcript-PCR RT-PCR) has very high sensitivity and specific degree to dual PCR in real time.This method is very sensitive to the rfb gene fragment of O1 crowd and O139 group cholera vibrio, and the detection lower limit of dual PCR in real time is higher 100 times than regular-PCR, can reach 1.0 * 10
2Copy/μ l.Dual PCR in real time can detect two genes or two kinds of different bacteriums simultaneously in a reaction system, significantly reduced experimental procedure, experimental period and to the consumption of reagent, in practice, very high using value is arranged especially during the epidemic situation outburst.
Description of drawings
Fig. 1 a is the screening figure of O1 group cholera vibrio clone;
Fig. 1 b is the screening figure of O139 group cholera vibrio clone;
Fig. 2 is O1 crowd and O139 group cholera vibrio plasmid double digestion figure as a result;
Fig. 3 a is O1 group cholera vibrio plasmid amplification sequencing fragment figure;
Fig. 3 b is O139 group cholera vibrio plasmid amplification sequencing fragment figure;
Fig. 4 a is O1 group cholera vibrio sensitivity figure as a result;
Fig. 4 b is O139 group cholera vibrio sensitivity figure as a result;
Fig. 5 a is O1 group cholera vibrio specific degree figure as a result;
Fig. 5 b is O139 group cholera vibrio specific degree figure as a result;
Fig. 6 a is that O1 group cholera vibrio substance PCR in real time detects lower limit figure;
Fig. 6 b is that O139 group cholera vibrio substance PCR in real time detects lower limit figure;
Fig. 7 a is an O1 group cholera vibrio substance PCR in real time canonical plotting;
Fig. 7 b is an O139 group cholera vibrio substance PCR in real time canonical plotting;
Fig. 8 is that the dual PCR in real time of O1 crowd and O139 group cholera vibrio detects lower limit figure;
Fig. 9 is the dual PCR in real time canonical plotting of O1 crowd and O139 group cholera vibrio;
Annotate: M is a dna molecular amount standard among Fig. 1 a and Fig. 1 b; A1-8 is O139 group cholera vibrio clone; B1-8 is O1 group cholera vibrio clone; O1 group cholera vibrio clone son 1～8 is all positive; O139 group cholera vibrio clone son 1～8 is all positive;
M is a dna molecular amount standard among Fig. 2, DL2000, and 1 cuts for O1 group cholera vibrio plasmid Sal I and EcoR I enzyme, and 2 cut for O139 group cholera vibrio plasmid Sal I and EcoR I enzyme;
Fig. 3 a is an O1 group cholera vibrio plasmid sequencer map, and Fig. 3 b is an O139 group cholera vibrio plasmid sequencer map;
The negative contrast of N among Fig. 4 a, Fig. 4 b;
The positive contrast of P among Fig. 5 a, Fig. 5 b;
The concentration of the plasmid template of curve 1-9 is followed successively by 1.0 * 10 among Fig. 6 a, Fig. 6 b
8Copy/μ l～1.0 * 10
0Copy/μ l; N: negative control; A detects lower limit for the O1 group cholera vibrio, and B detects lower limit for the O139 group cholera vibrio; X-coordinate is a cycle number; Ordinate zou is a fluorescent value;
X is that lg value, the y of plasmid concentration are the Ct value among Fig. 7 a, Fig. 7 b;
The concentration of curve 1～9 plasmid template is followed successively by 1.0 * 10 among Fig. 8
8Copy/μ l～1.0 * 10
0Copy/μ l; N: negative control; X-coordinate is a cycle number; Ordinate zou is a fluorescent value;
A is an O1 group cholera vibrio typical curve among Fig. 9; B is an O139 group cholera vibrio typical curve; X is the lg value of plasmid concentration; Y is the Ct value.
Embodiment
Different according to O antigen, vibrio cholerae can be divided into more than 200 serotype, and wherein only O1 crowd and O139 group cholera vibrio product strain can cause the popular of cholera.Other serotype can cause diseases such as human gastrointestinal inflammation, but never causes the popular of cholera.Therefore, O1 crowd and O139 group cholera vibrio have the public hygienics meaning with respect to other vibrio cholerae.
The present invention has set up based on the O1 group cholera vibrio of TaqMan probe and the dual real time PCR detection method of O139 group cholera vibrio.The goal gene that detects is the O antigen synthetic gene rfb gene fragment of O1 group cholera vibrio and O139 group cholera vibrio.The rfb gene fragment of the real-time PCR method amplification that the present invention sets up finds that only the rfb gene fragment with O1 crowd and O139 group cholera vibrio has good homology after the correlated series comparison in Blast and GenBank.
According to the O antigen synthetic gene rfb gene of O1 crowd and O139 group cholera vibrio, with dual PCR in real time primer of Beacon Designer 7.0 software designs and probe:
The position of the sequence of amplified production and primer and probe is following:
Concrete experimental implementation step is following:
1. extract bacterial chromosomal dna with test kit;
1) according to O1 crowd who is designed and O139 group cholera vibrio primer, the check order full pure culture liquid dna profiling of bacterial strain N16961 and MO45 of increase respectively O1 crowd and O139 group cholera vibrio.
Amplification condition is following:
2) reclaim test kit according to glue and reclaim the purifying target DNA fragment;
3) preparation O1 crowd and O139 group cholera vibrio rfb gene clone;
The DNA of gained in the last step is reclaimed product prepares following ligation system:
Prepare O1 crowd and O139 group cholera vibrio rfb clone according to the step of recommending in the TaKaRa pMD18-T Vector test kit then.Select the bacterium colony of on the nutrient agar plate that scribbles Amp 100, growing, identify with O1F/O1R and O139F/O139R amplification.Qualification result is seen Fig. 1 a and Fig. 1 b.Select PCR and identify that positive bacterium colony is inoculated in the 5ml LB liquid nutrient medium that contains 5 μ l Amp 100 37 ℃ of shaking table overnight cultures.
4) plasmid extraction kit according to QIAgen extracts O1 crowd and O139 group cholera vibrio plasmid, and concrete steps are the same.Be respectively 90ng/ μ l and 58ng/ μ l with the O1 crowd of NanoDrop ultraviolet spectrophotometer mensuration gained and the concentration of O139 group cholera vibrio plasmid.Select Sal I and two kinds of restriction endonuclease of EcoR I that O1 crowd and O139 group cholera vibrio plasmid are inserted segmental restricted enzyme cutting analysis.The endonuclease reaction system is following:
Enzyme is cut product and is identified with 2% agarose gel electrophoresis.Qualification result is as shown in Figure 2;
After double digestion is identified the positive, machine order-checking on the cloned plasmids.O1 crowd and O139 group cholera vibrio rfb gene amplification sequencing fragment result see Fig. 3 a and Fig. 3 b successively.Sequence results and known aim sequence carry out BLAST relatively, and the result proves that the recombinant plasmid sequence is correct, no base sudden change.
5) concentration conversion of O1 crowd and O139 group cholera vibrio DNA is that copy number is respectively: 2.6 * 10
10Copy/μ l and 1.9 * 10
10Copy/μ l.With EasyDilution above-mentioned plasmid is diluted to 1.0 * 10 successively
10Copy/μ l～1.0 * 10
0Copy/μ l, totally 11 concentration gradients are subsequent use.
2. real-time fluorescence RT-PCR reacts, and prepares 20 μ L PCR reaction systems of certain concentration of component, and of short duration centrifugal branch installs in the PCR pipe behind the mixing;
3. testing sample is joined in the reaction system, increase;
4. collect fluorescent signal at annealing stage, detect the Ct value.
The system component and the volume thereof of real-time fluorescence RT-PCR reaction are following:
Cycling condition:
5.O1 crowd and O139 group cholera vibrio substance TaqMan real-time PCR reactions Parameter Optimization
1) optimization of primer concentration
To O1 crowd and O139 group cholera vibrio reaction system, the concentration of upstream and downstream primer is got 10 concentration gradients of 100～1000nmol/L respectively, and concentration and probe concentration remains on 100nmol/L.Each concentration gradient is done 8 parallel appearance.CFX96 Real Time pcr amplification appearance with Bio-Rad increases.Reaction system and reaction conditions are the same.To the Ct value of each concentration gradient of obtaining, carry out the variance analysis of completely random design data with SPSS 15.0 softwares.
To O1 group cholera vibrio substance TaqMan real-time PCR reactions system; Find through variance analysis; The Ct value difference of 10 groups of concentration gradients is different to have statistical significance (table 1) on the whole; Through relatively finding in twos, preceding 3 groups of different not statistically significants of Ct value difference, preceding 3 groups have a statistical significance on the whole with other Ct value differences of 7 groups are different.Combine preceding 3 groups standard deviation simultaneously, the concentration of selected primer is 200nmol/L.
The comparison of 10 groups of primer concentration gradients of table 1 PCR in real time Ct value
To O139 group cholera vibrio substance TaqMan real-time PCR reactions system; Find through variance analysis; The Ct value difference of 10 groups of concentration gradients is different to have statistical significance (table 2) on the whole; Through relatively finding in twos, preceding 3 groups of different not statistically significants of Ct value difference, preceding 3 groups have a statistical significance on the whole with other Ct value differences of 7 groups are different.Combine preceding 3 groups standard deviation simultaneously, the concentration of selected primer is 200nmol/L.
The comparison of 10 groups of primer concentration gradients of table 2 PCR in real time Ct value
2) optimization of concentration and probe concentration
To O1 crowd and O139 group cholera vibrio reaction system, the concentration of upstream and downstream primer remains on 200nmol/L, and the concentration of probe gets 100,200,300,400 and 5 concentration gradients of 500nmol/L respectively, and each concentration gradient is done 8 parallel appearance.CFX96 Real Time pcr amplification appearance with Bio-Rad increases, and reaction system and reaction conditions are the same.Ct value to each concentration gradient of obtaining is carried out the variance analysis of completely random design data with SPSS 15.0 softwares.
To O1 group cholera vibrio substance TaqMan real-time PCR reactions system; When the concentration of probe reaches 400nmol/L and 500nmol/L; The variation of Ct value increases between 8 parallel kind, and standard deviation reaches 0.63 and 0.74, shows the instability that PCR becomes when being reflected at this concentration and probe concentration.So, when analyzing, cast out these two groups, only analyze preceding 3 groups.Through variance analysis, preceding 3 groups Ct value difference is different to have statistical significance (table 3) on the whole.In conjunction with comparing in twos, selecting the concentration of probe is 200nmol/L.
The comparison of table 3 PCR in real time 3 mandrin concentration gradient Ct values
To O139 group cholera vibrio substance TaqMan real-time PCR reactions system, the difference of 5 groups of Ct values has statistical significance (table 4) on the whole.Find relatively that in twos the 1st group Ct value and other difference of four groups have statistical significance, its Ct value is greater than all the other 4 groups.Combine all the other standard deviations of four groups simultaneously, the concentration of selected probe is 200nmol/L.
The comparison of 5 groups of concentration and probe concentration gradients of table 4 PCR in real time Ct value
In sum, the O1 group cholera vibrio TaqMan real-time PCR reactions condition of optimization is:
Cycling condition is following:
Detect fluorescence at annealing stage.
The O139 group cholera vibrio TaqMan real-time PCR reactions condition of optimizing is:
Cycling condition is following:
Detect fluorescence at annealing stage.
6. substance TaqMan PCR in real time detection architecture laboratory inner evaluation
1) sensitivity evaluation
Detect 132 strain O1 group cholera vibrios and 126 strain O139 group cholera vibrios with optimizing good O1 group cholera vibrio and O139 group cholera vibrio substance TaqMan real-time PCR reactions system respectively.
As a result, to O1 group cholera vibrio substance TaqMan PCR in real time, except that negative control, 132 strain O1 group cholera vibrios are all positive; To O139 group cholera vibrio substance TaqMan PCR in real time, except that negative control, 126 strain O139 group cholera vibrios all positive (Fig. 4 a and Fig. 4 b).
So the sensitivity of O1 group cholera vibrio and O139 group cholera vibrio substance TaqMan real-time PCR reactions system is 100%.
2) specific degree evaluation
Detect O139 group cholera vibrio/O1 group cholera vibrio, non-O1 crowd and non-O139 group cholera vibrio, Vibrio mimicus, Vibrio parahaemolyticus, Vibrio vulnificus, vibrio alginolyticus, Aeromonas hydrophila, Maxwell vibrios, vibrio fluvialis, Vibrio furnissii, maiden's vibrio piscium, Plesiomonas shigelloides, Escherichia coli O 157, enterotoxigenic E.Coli, Salmonella bacterium and Shigella bacterium to optimize good O1 crowd and O139 group cholera vibrio substance TaqMan real-time PCR reactions system, respectively with N16961 and the positive contrast of MO45.
(Fig. 5 is crowd and O139 (Fig. 5 b) group cholera vibrio substance TaqMan PCR in real time a), and except that positive control, all the other bacterial strains all are negative to O1.So the specific degree of O1 crowd and O139 group cholera vibrio substance TaqMan real-time PCR reactions system is 100%.
3) evaluation of detection lower limit
With O1 crowd and the O139 group cholera vibrio plasmid series concentration gradient (1.0 * 10 that obtains
8Copy/μ l～1.0 * 10
0Copy/μ l) be template, each concentration gradient is got 2 μ l, carries out PCR in real time respectively according to reaction system and the reaction conditions optimized, and each concentration gradient is done 8 parallel appearance.Carry out the amplification of regular-PCR simultaneously.
I. detect confirming of lower limit
For O1 group cholera vibrio PCR in real time, when plasmid concentration is 1.0 * 10
8Copy/μ l～1.0 * 10
2During copy/μ l, all amplification is positive for 8 parallel appearance of each concentration gradient, when plasmid concentration is 1.0 * 10
1Copy/μ l and 1.0 * 10
0During copy/μ l, the parallel appearance of 8 of each concentration gradient has 4 not occur increasing with 6 samples respectively.
So, be limited to 1.0 * 10 under the detection of O1 group cholera vibrio substance TaqMan PCR in real time detection architecture
2(Fig. 6 a) is limited to 1.0 * 10 under the detection of regular-PCR to copy/μ l
4Copy/μ l.
For O139 group cholera vibrio PCR in real time, when plasmid concentration is 1.0 * 10
8Copy/μ l～1.0 * 10
2During copy/μ l, all amplification is positive for 8 parallel appearance of each concentration gradient, when plasmid concentration is 1.0 * 10
1Copy/μ l and 1.0 * 10
0During copy/μ l, the parallel appearance of 8 of each concentration gradient has 3 not occur increasing with 7 samples respectively.
So, be limited to 1.0 * 10 under the detection of O139 group cholera vibrio substance TaqMan PCR in real time detection architecture
2Copy/μ l (Fig. 6 b) is limited to 1.0 * 10 under the detection of regular-PCR
4Copy/μ l.
Ii. typical curve
(Fig. 7 a) crowd is an X-coordinate with the lg value of O139 (Fig. 7 b) group cholera vibrio plasmid concentration, is ordinate zou with corresponding C t value, drafting O1 crowd and O139 group cholera vibrio substance PCR in real time typical curve with O1 respectively.The result sees table 5, table 6.
The different O1 group cholera vibrio of table 5 plasmid concentration gradient Ct value
The different O139 group cholera vibrio of table 6 plasmid concentration gradient Ct value
The lg value (x) of O1 group cholera vibrio plasmid concentration is expressed with the available following functional expression of the relation of Ct value (y) among Fig. 7 a:
Y=-3.28x+41.6, both coefficient R
2=0.999.It is thus clear that the concentration of working as O1 group cholera vibrio plasmid is 1.0 * 10
8Copy/μ l～1.0 * 10
2In the time of in this scope of copy/μ l, the logarithmic value of plasmid concentration and Ct value have extraordinary dependency.
The lg value (x) of O139 group cholera vibrio plasmid concentration is expressed with the available following functional expression of the relation of Ct value (y) among Fig. 7 b:
Y=-3.35x+41.082, both coefficient R
2=0.999.It is thus clear that the concentration of working as O139 group cholera vibrio plasmid is 1.0 * 10
8Copy/μ l～1.0 * 10
2In the time of in this scope of copy/μ l, the logarithmic value of plasmid concentration and Ct value have extraordinary dependency.
Iii. amplification efficiency analysis
The O1 group cholera vibrio PCR in real time detection architecture of setting up among the present invention, when the concentration of O1 group cholera vibrio plasmid 1.0 * 10
8Copy/μ l～1.0 * 10
2In the time of in this scope of copy/μ l, amplification efficiency is 101.7%.
O139 group cholera vibrio PCR in real time detection architecture, when the concentration of O139 group cholera vibrio plasmid 1.0 * 10
8Copy/μ l～1.0 * 10
2In the time of in this scope of copy/μ l, amplification efficiency is 98.9%.
7.O1 crowd and the dual TaqMan PCR in real time of O139 group cholera vibrio
Dual TaqMan real-time PCR reactions system is to join in the hybrid reaction system optimizing the ratio of good substance reaction system according to 1: 1.With O1 crowd and O139 group cholera vibrio plasmid series concentration gradient is template (1.0 * 10
8Copy/μ l～1.0 * 10
0Copy/μ l), carry out dual TaqMan real-time PCR reactions according to following reaction system, each concentration gradient is done 8 parallel appearance, and reaction conditions is the same.
1) detects confirming of lower limit
To O1 crowd and O139 group cholera vibrio plasmid, dual PCR in real time does not have raising not reduce their detection lower limit yet.Dual real-time PCR reactions system still is 1.0 * 10 to both detection lower limits
2Copy/μ l (Fig. 8).
2) typical curve
Lg value with O1 crowd and O139 group cholera vibrio plasmid concentration is an X-coordinate respectively, is that ordinate zou is drawn dual PCR in real time typical curve with corresponding C t value.The result sees table 7, Fig. 9.
Table 7O1 crowd and the different plasmid concentration gradient of the dual PCR in real time of O139 group cholera vibrio Ct value
3) amplification efficiency
The dual TaqMan PCR in real time of O1 crowd and O139 group cholera vibrio, when the concentration of template 1.0 * 10
8Copy/μ l～1.0 * 10
2In the time of in this scope of copy/μ l, the amplification efficiency of O1 template and O139 template is respectively 101.4% and 99.9%.
In sum, in O1 crowd and the dual TaqMan real-time PCR reactions of O139 group cholera vibrio system, serious dimer and mismatching phenomenon do not take place in the existence of two kinds of primers and probe, do not have to reduce detection lower limit and the amplification efficiency to two kinds of templates.
Above-mentioned pathogenic bacteria DNA extracts from test kit; Detection method disclosed by the invention is not to be object with human body and animal, just in order to detect whether have pathogenic bacteria, imports and exports article etc. as detecting powder, food, customs; Not to be diagnosed as purpose, not belong to the diagnostic method of disease.
Only if specifically defined, it is known term in the relevant technologies field that the present invention describes used term.The chemical symbol of standard and dummy suffix notation can exchange with its full name and use.
Only if special indicating, the present invention uses but clearly sets forth or simple technology and the method for setting forth is meant normally used technology in present technique field and method, can carry out according to technology well known in the art and method.The use of test kit is to carry out according to the specification sheets that manufacturers or supplier provide.
Claims (10)
1. a dual TaqMan fluorescence probe RT-PCR detects the non-diagnostic methods of O1 crowd and O139 group cholera vibrio, it is characterized in that this non-diagnostic methods comprises:
O antigen synthetic gene rfb gene design primer and probe to O1 crowd and O139 crowd's arc vibrio cholerae:
2. non-diagnostic methods as claimed in claim 1 is characterized in that, 5 ' the end institute mark fluorescent element of said probe O1-P and O139-P is respectively FAM and HEX, and probe 3 ' end all connects quenching group; Said this quenching group is the non-fluorescent quenching group of BHQ1.
3. non-diagnostic methods as claimed in claim 1 is characterized in that, said this non-diagnostic methods comprises step:
1) extracts bacterial chromosomal dna;
2) real-time fluorescence RT-PCR reacts, and prepares 20 μ L PCR reaction systems of certain concentration of component, and of short duration centrifugal branch installs in the PCR pipe behind the mixing;
3) testing sample is joined in the reaction system, increase;
4) collect fluorescent signal at annealing stage, detect the Ct value.
4. non-diagnostic methods as claimed in claim 3 is characterized in that, the system component and the volume thereof of said real-time fluorescence RT-PCR reaction are following:
Cycling condition:
5. non-diagnostic methods as claimed in claim 3 is characterized in that, the concrete steps of said O1 crowd and the preparation of O139 crowd's arc vibrio cholerae standard substance comprise:
1) according to the O1 crowd who is designed and O139 crowd's arc vibrio cholerae primer, the check order full pure culture liquid dna profiling of bacterial strain N16961 and MO45 of increase respectively O1 crowd and O139 crowd's arc vibrio cholerae;
2) reclaim test kit according to glue and reclaim the purifying target DNA fragment;
3) preparation O1 crowd and O139 crowd's arc vibrio cholerae rfb gene clone;
4) insert segmental restricted enzyme cutting analysis behind extraction O1 crowd and the O139 crowd's arc vibrio cholerae plasmid, get the endonuclease reaction result who is positive and carry out cloned plasmids order-checking experiment;
5) after the concentration conversion of O1 crowd and O139 crowd's arc vibrio cholerae DNA was copy number, it is subsequent use that plasmid is diluted to several concentration gradients.
6. non-diagnostic methods as claimed in claim 5 is characterized in that, the check order full amplification condition of pure culture liquid dna profiling of bacterial strain N16961 and MO45 of said O1 crowd and O139 crowd's arc vibrio cholerae is:
7. non-diagnostic methods as claimed in claim 5 is characterized in that, said dna fragmentation with glue recovery purifying prepares O1 crowd and O139 crowd's arc vibrio cholerae rfb gene clone, and the ligation system of preparation is:
8. non-diagnostic methods as claimed in claim 7 is characterized in that, during the preparation of said O1 crowd and O139 crowd's arc vibrio cholerae rfb clone, with O1F/O1R and O139F/O139R to the bacterium colony of growing on the nutrient agar plate that the scribbles Amp100 evaluation of increasing.
9. non-diagnostic methods as claimed in claim 5 is characterized in that, said endonuclease reaction system is following:
10. non-diagnostic methods as claimed in claim 5 is characterized in that, said double digestion is identified machine order-checking on the cloned plasmids that is positive, and sequence results and known aim sequence are compared; Detection lower limit to O1 crowd and O139 crowd's arc vibrio cholerae plasmid in the system of said real-time fluorescence RT-PCR reaction is 1.0 * 10
2Copy/μ l.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104032009A (en) * | 2014-06-12 | 2014-09-10 | 江崇才 | Detecting method of vibrio cholera |
| CN105087569A (en) * | 2015-09-07 | 2015-11-25 | 南开大学 | Specific nucleotides for Vibrio cholerae O18, O19, O23 and O12 and applications of specific nucleotides |
| CN111197094A (en) * | 2018-11-16 | 2020-05-26 | 深圳市疾病预防控制中心 | Compositions, kits and methods for genotyping vibrio parahaemolyticus |
| CN111996268A (en) * | 2020-08-31 | 2020-11-27 | 福建农林大学 | Vibrio alginolyticus double TaqMan probe real-time fluorescent quantitative PCR (polymerase chain reaction) detection kit and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101113476A (en) * | 2007-05-30 | 2008-01-30 | 中国疾病预防控制中心传染病预防控制所 | Pathogenic microorganism DNA detecting chip and preparation method and application thereof |
| CN101768636A (en) * | 2009-01-06 | 2010-07-07 | 蔡剑平 | Composition and kit for detecting vibrio cholerae and detection method |
| CN102367475A (en) * | 2011-09-20 | 2012-03-07 | 麻丽丹 | M-PCR (multiplex-polymerase chain reaction) detection kit for different serotype vibrio cholerae and detection method thereof |
-
2012
- 2012-03-14 CN CN2012100671806A patent/CN102676653A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101113476A (en) * | 2007-05-30 | 2008-01-30 | 中国疾病预防控制中心传染病预防控制所 | Pathogenic microorganism DNA detecting chip and preparation method and application thereof |
| CN101768636A (en) * | 2009-01-06 | 2010-07-07 | 蔡剑平 | Composition and kit for detecting vibrio cholerae and detection method |
| CN102367475A (en) * | 2011-09-20 | 2012-03-07 | 麻丽丹 | M-PCR (multiplex-polymerase chain reaction) detection kit for different serotype vibrio cholerae and detection method thereof |
Non-Patent Citations (3)
| Title |
|---|
| 徐海滨等: "霍乱弧菌多重PCR检测方法的建立和运用", 《海峡预防医学杂志》, vol. 15, no. 02, 31 December 2009 (2009-12-31), pages 52 - 54 * |
| 杨辉等: "产毒型O139群霍乱弧菌特异性基因序列实时荧光双重TaqMan聚合酶链反应快速检测体系的建立", 《中国卫生检验杂志》, vol. 19, no. 04, 30 April 2009 (2009-04-30), pages 742 - 745 * |
| 胡继红等: "产毒型O1群霍乱弧菌实时荧光双重TaqMan PCR的临床应用前研究", 《中国医药生物技术》, vol. 4, no. 01, 28 February 2009 (2009-02-28), pages 14 - 18 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104032009A (en) * | 2014-06-12 | 2014-09-10 | 江崇才 | Detecting method of vibrio cholera |
| CN105087569A (en) * | 2015-09-07 | 2015-11-25 | 南开大学 | Specific nucleotides for Vibrio cholerae O18, O19, O23 and O12 and applications of specific nucleotides |
| CN105087569B (en) * | 2015-09-07 | 2017-08-25 | 南开大学 | To vibrio cholerae O 18, O19, O23 and the special nucleotides of O12 and its application |
| CN111197094A (en) * | 2018-11-16 | 2020-05-26 | 深圳市疾病预防控制中心 | Compositions, kits and methods for genotyping vibrio parahaemolyticus |
| CN111197094B (en) * | 2018-11-16 | 2021-02-23 | 深圳市疾病预防控制中心 | Compositions, kits and methods for genotyping vibrio parahaemolyticus |
| CN111996268A (en) * | 2020-08-31 | 2020-11-27 | 福建农林大学 | Vibrio alginolyticus double TaqMan probe real-time fluorescent quantitative PCR (polymerase chain reaction) detection kit and preparation method thereof |
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