CN104975084B - Dual real-time fluorescence quantitative PCR detection triggers the kit of endocarditic Bartonella - Google Patents
Dual real-time fluorescence quantitative PCR detection triggers the kit of endocarditic Bartonella Download PDFInfo
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Abstract
The invention provides a kind of method that dual real-time fluorescence quantitative PCR detection triggers endocarditic Bartonella, methods described is using specific primer sets and the probe being used cooperatively, to triggering endocarditic quintan Bartonella and bartonella henselae to carry out TaqMan real-time fluorescence quantitative PCR detections.The primer of the primer sets is as shown in SEQ ID No.1~4, and the probe being used cooperatively is as shown in SEQ ID No.5~6.Dual real-time fluorescence quantitative PCR sonde method high specificity that the present invention establishes, high sensitivity, stability are good.Specificity experiments result shows that only quintan Bartonella and bartonella henselae amplify fluorescence signal, and the bacterium of other kinds is showed no fluorescence signal.
Description
Technical field
The invention belongs to molecular Biological Detection field, specifically, is related to a kind of dual real-time fluorescence quantitative PCR detection
Trigger the kit of endocarditic Bartonella.
Background technology
Endocarditis (IE) is a kind of to seriously endanger that health, clinical manifestation be extremely complex and the disease of detection difficult
Disease, the sick cause of disease is complicated, according to originally clinical manifestation, potential heart disease, be related to pathogenic microorganism and complication exist with
No etc. to show various forms, its treatment needs physician, cardiologist, surgeon, microbiologist, infection
The consultation of doctors and treatment of sick expert, neurosurgeon, neurosurgery scholar, dept. of radiology scholar and virologist etc..Since sulfonamides
Occur with penicillin, IE treats to have obtained very big change, is no longer universal fatefulue disease.In Past 30 Years, prevention and
Although treatment level has large increase, but morbidity and mortality are still higher, per annual morbidity be 3~10/
100000 people, hospital mortality are 9.6%~26.0%.Netzer etc. reports that the IE death rates are about in the 1950s
40.0%~60.0%, drop to about 30.0% the 1970s to the eighties, 1980~nineteen ninety-five is about 16.0%~
27.0%.Slipczuk etc. study nearly 50 years IE epidemic diseases be shown in hospital mortality the 1970s be 24.4%~
36.8%, and it is horizontal to be always held at this afterwards.
IE pathogen is caused to have streptococcus (Streptococcus spp.), staphylococcus (Staphylococcus
Spp.), Ke Kesi bodies (Coxiella spp.), Bartonella (Bartonella spp.), Chlamydia (Chlamydia
Spp.), corynebacteria (Corynebacterium spp.), brucella (Brucella spp.), enterobacteriaceae
(Enterobacteriaceae spp.) and fungi etc..Its diagnostic method Main Basiss microbiological examination, such as blood and valve
Membrane tissue culture of isolated pathogen, serology, histopathology and nucleic acid molecules diagnosis.Report BCNE patient in IE patient about
Bar 2.5%~31.0% is accounted for, requires harsh, slow-growing mainly due to a kind of nutritional condition, be difficult to the pathogen cultivated, i.e.,
You cause entire body with Ke Kesi bodies, and the wherein BE incidences of disease account for 4.5% in IE patient.Slipczuk etc. reports whole world BCNE
Patient accounts for 18% in the 1960s, accounts for 14% the 1970s, accounts for 23% the 1980s, 20th century 90
Age accounts for 20%, and 21 century previous decade accounts for 14%, and last decade is in downward trend.Britain reports London St. Thomas hospital
1975~2000 years 516 parts of endocarditis cases, research show that BCNE patient accounts for 12.2%, the BE incidences of disease in BCNE patient
Account for 11%.30% is accounted in IE patient in the French BE incidences of disease, 3.9% is accounted in BCNE patient in Brazilian BE patient.
It has collected report in the worldwides such as 1993~2013 years Britain, France, the U.S., India, Sweden and Greece
52 documents, 538 BE altogether, wherein 110 patient profiles are described in detail in 47 documents, 5 documents only report BE case loads
Totally 428.Overlapping Case report is rejected during statistics.The comprehensive various method detection infection endocarditic Bartonellas of people mainly have
8 kinds, in 110 case patients, 50.0% (55) are accounted for by the Bq patients infected, Bh accounts for 23.6% (26), in addition Bvb (2
Example), Bva (5), Ba (2), Be (1), Bk (1) and Bm (1), the patient of unassigned species have 17.It was found that Bh and Bq
For main pathogen, it can also cause a variety of diseases such as mankind CSD, trench fever and BA in addition.In addition at least 6 kinds of Ba Er are reported
Entire body can cause zoogenetic infection endocarditis, respectively Bq, Bh, Bvb, Bc, B.bovis (ox Bartonella) and
B.washoensis。
At present, culture primarily discrete to Bh and Bq detection methods, Serologic detection, Standard PCR and real time fluorescent quantitative
PCR is detected.Bartonella bacteria growing slowly, nutritional condition require harsh and be difficult to be separately cultured, growth is relative during Liquid Culture
Than very fast.Serologic detection generally uses IFA methods and ELISA method detection antibody, and as a result interpretation has subjective factor, sensitivity and spy
The opposite sex is not high, it is impossible to distinguishes Bq and Bh very well, and easily cross reaction occurs with other microorganisms such as Chlamydias.Standard PCR is examined
There is primer and cause false negative etc. with reference to PCR mortifiers presence in specific, laboratory pollution and clinical sample is lacked in survey, in base
Electrophoresis sequencing is also needed to verify after gene-amplification, relatively time-consuming, cost is also higher.Real-time fluorescence probe PCR method is according to sequence
Specific probe distinguishes species, adds specificity and sensitivity, but needs synthesising probing needle price somewhat expensive, at present external report
Real-time fluorescence probe PCR method detects Bartonella, and this domestic laboratory has established detection Bvb substance real time fluorescent quantitatives
PCR sonde methods, other laboratories report establishes detection Bh and Bq substance real-time fluorescences using TaqMan-MGB probe techniques to be determined
PCR method is measured, but a PCR system can only detect a kind of or a kind of pathogen, it is impossible to while detect multiple pathogens.In recent years
It is more and more extensive to carry out the application of multiple real time fluorescence quantifying PCR sonde method, the technology is that several fluorescence probes mix, root
Multiple pathogens can be effectively distinguished in single pipe according to respective fluorescence probe color difference, sample is saved, saves reagent, reduces
Cost and Acceleration study process etc..It there is no both at home and abroad using dual probe method at present while detect Bh and Bq report, double PCR
Method has the advantages that high efficiency, saves the time, saves sample, and 50% efficiency etc. is improved on the basis of original substance PCR.Cause
This is necessary to establish dual real-time fluorescence quantitative PCR sonde method to detect cause people's endocarditis common two kinds of Ba Ertong
Body, differentiates for both Bartonellas and differentiation provides effective detection method.
The content of the invention
It is an object of the invention to provide a kind of dual real-time fluorescence quantitative PCR detection to trigger endocarditic Bartonella
The kit of (quintan Bartonella and bartonella henselae).
In order to realize the purpose of the present invention, it is used to detect present invention firstly provides one kind and triggers endocarditic Bartonella
Real-time fluorescence quantitative PCR primer sets, the primer sets include be used for detect the primer pair I of bartonella henselae and for detecting
The primer pair II of quintan Bartonella；
The primer pair I is：
BhF：5’-GAGTCAGATGGAATGGTATTGGTTATC-3’；
BhR：5’-CGGTTTATCCCCCACAAGATAGG-3’；
The primer pair II is：
BqF：5’-TGCCAAGTATGCGAGTTCCTG-3’；
BqR：5’-TCAGAGTTAGGTGCAAGTTCTATGG-3’.
Present invention also offers the probe being used cooperatively with foregoing primer sets, the probe is：
Probe I：
BhT：FAM-CCCTCCCGGTTTTTCTCTGGGCCT-BHQ1；
Probe I I：
BqT：CY5-TCTGCGCCCGGTTCTGAAATGCCT-BHQ2；
Wherein, FAM and CY5 is fluorescent reporter group, and BHQ1 and BHQ2 are fluorescent quenching gene.
Present invention also offers the kit containing foregoing primer sets and foregoing probes.
Further, the kit also includes dNTPs, Taq archaeal dna polymerase, Mg2+, in PCR reaction buffers one
Kind is a variety of.
Further, the job step of the kit is to triggering endocarditis using foregoing primer sets and foregoing probes
Bartonella carry out TaqMan real-time fluorescence quantitative PCR detections.
Further, the job step specifically includes：
1) DNA in sample is extracted；
2) using the DNA of extraction in step 1) as template, dual real-time fluorescence is carried out using foregoing primer sets and foregoing probes
Quantitative PCR reacts；
3) real-time fluorescence quantitative PCR product is analyzed.
Further, the PCR reaction systems of the kit are calculated as with 20 μ l：
Preferably, the concentration of the primer is 10 μm of ol/L, the concentration of the probe is 10 μm of ol/L.
Preferably, the PCR reaction conditions of the kit are：95 DEG C 2 minutes, single loop；95 DEG C 3 seconds, 60 DEG C 30
Second, totally 40 circulations.
The beneficial effects of the present invention are：
Dual real-time fluorescence quantitative PCR sonde method high specificity that the present invention establishes, high sensitivity, stability are good.Specifically
Property experimental result show that only bartonella henselae and quintan Bartonella amplifies fluorescence signal, the bacterium of other kinds is equal
Have no fluorescence signal；Sensitivity experiments result is shown in 20 μ L reaction system, dual real-time fluorescence quantitative PCR probe in real time
It is respectively 3.64 × 10 that method, which detects bartonella henselae and quintan Bartonella minimum detectability,1Individual copy and 3.28 × 101It is individual
Copy, improves 100 times than Standard PCR sensitiveness, also shows that good linear relationship and amplification efficiency, coefficient correlation in addition
R2Respectively 0.994 and 0.998, E value be respectively 100.0% and 103.4%.In repeated experiment result display group between group
CV values are 0.19%-0.53% and 0.49%-1.66%, in allowed band.The Chinese quick detection can be gone out using this method simultaneously
Bartonella and quintan Bartonella are matched, is a series of early stage of diseases such as the endocarditis caused by both Bartonellas
The researchs such as quick diagnosis, monitoring and epidemiology survey provide effective means.
Brief description of the drawings
Fig. 1 is bartonella henselae (A) and quintan Bartonella (B) different annealing temperature in the embodiment of the present invention 1
Amplification curve.
Fig. 2 is bartonella henselae (A) and quintan Bartonella (B) different probe concentration in the embodiment of the present invention 1
Amplification curve.
Fig. 3 is bartonella henselae (A) and quintan Bartonella (B) different primers concentration in the embodiment of the present invention 1
Amplification curve.
Fig. 4 is real-time fluorescence quantitative PCR sonde method specific detection quintan Bartonella and the Chinese in the embodiment of the present invention 1
Match the result of Bartonella.
Fig. 5 is 14 plants of Ba Er of the domestic separation of real-time fluorescence quantitative PCR sonde method specific detection in the embodiment of the present invention 1
The result of entire body (6 plants of Bh and 8 plant of Bq).
Fig. 6 is bartonella henselae substance real-time fluorescence quantitative PCR sonde method sensitivity analysis in the embodiment of the present invention 1
Amplification curve (A) and standard curve (B), its amplification efficiency are 98.1%, coefficient R2For 0.996.
Fig. 7 is quintan Bartonella substance real-time fluorescence quantitative PCR sonde method sensitivity analysis in the embodiment of the present invention 1
Amplification curve (A) and standard curve (B), its coefficient R2For 0.998, amplification efficiency 102.9%.
Fig. 8 is that bartonella henselae and quintan Bartonella dual real-time fluorescence quantitative PCR are visited in the embodiment of the present invention 1
The amplification curve (A) and standard curve (B) of skill of handling needles sensitivity analysis, the wherein phase of bartonella henselae and quintan Bartonella
Close coefficients R2Respectively 0.994 and 0.998, amplification efficiency value is respectively 100.0% and 103.4%.
Fig. 9 is bartonella henselae (A) and the different dilution factor standards of quintan Bartonella (B) in the embodiment of the present invention 1
Plasmid Standard PCR electrophoretogram.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
According to conventional laboratory conditions, 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.
The method that embodiment 1 detects Bartonella using dual real-time fluorescence quantitative PCR sonde method
1 material and method
1.1 bacterial strains and biological specimen DNA
Bartonella genomic DNA includes bartonella henselae (B.henselae, Bh) ATCC49882, quintan Ba Er
Entire body (B.quintana, Bq) ATCC VR-358, Vincent Bartonella win lattice Hough subspecies (B.vinsonii
Subsp.berkhoffii) ATCC 51672, bacillus sample Bartonella (B.bacilliformis, Bb), kirschner Bartonella
(B.clarridgeiae, Bc) ATCC 51734, gram strangle Bartonella (B.koehlerae, Bk) ATCC 700693,
ATCC35685, Vincent Bartonella Vincent subspecies (B.vinsonii subsp.vinsonii, Bvv) ATCC VR-152, Vincent
Bartonella A Lupan subspecies (B.vinsonii subsp.arupensis, Bva) ATCC700727, Elizabethan's Bartonella
(B.elizabethae, Be) ATCC 49927, Lindsey Graham Bartonella (B.grahamii, Bg) ATCC 700132 and road will
Bartonella (B.doshiae, Bd) ATCC 700133；People, cat, rabbit, dog, sheep and tick genomic DNA.
Other bacterial genomes DNA include helicobacter pylori (Helicobacter pylori), shigella flexneri
(Shigella flexneri), Japanese Richettsia (R.japonica), Anaplasma phagocytophila (Anaplasma
Phagocytophilum), yersinia pestis (Yersinia pestis EV76), Acinetobacter bauamnnii
(Acinetobacter baumanii), staphylococcus aureus (Staphyloccocus aureus), Leptospira
(Leptospira interrogans), brucella (Brucella spp.), dung ball intestines bacterium (Enterococcus
Faecium), Listeria monocytogenes (Listeria monocytogenes), Neisseria meningitidis
(Neisseria meninyitidis), streptococcus pneumonia (Streptococcus pneumoniae), haemophilus influenzae
(Haemophilus influenzae), campylobacter jejuni (Campylobacter jejuni), bacillus canalis capsulatus
(Klebsiella pneumoniae), Salmonella typhimurtum (Salmonella typhimurium), legionella pneumophilia
(Legionella pneumophila), comma bacillus (Vibrio cholerae), Rickettsia prowazeki (Rickettsia
Prowazekii), R. massillia (R.massilliae), Borrelia burgdoyferi (Bolrelia burgdorferi) and thorn
Hedgehog (Erinaceus europaeus).
2.2 key instrument equipment and reagent
Real-time fluorescence quantitative PCR instrument, CFX96, BioRad；Table model high speed centrifuge, 5804R, Eppendorf；Micro core
Measuring acid concentration instrument, NanoDrop-1000, Thermo Fisher Scientific；371 type CO2 incubators,
ThermoScientific, the U.S.；Maxwell turbidimetry, BioMerieux, France；
Schneider Insect cellculture liquid, Sigma, the U.S.；Standard hyclone, Tianjin Hao oceans biological products section
Skill Co., Ltd；TSB, BD, the U.S.；TSA, BD, the U.S.；Fast Cycling PCR Kit and QIAamp DNA Mini
Kit is purchased from QIAGEN companies, 2 × TransTaq-T PCR SuperMix and cloning kit-T5Zero
Cloning Kit are purchased from Beijing Quanshijin Biotechnology Co., Ltd；QPCR Master Mix and plasmid extraction examination
Agent box is purchased from Promega companies.
The design and synthesis of the screening of 2.3 target genes, primer and TaqMan probe
Screened to obtain detection target candidate gene position according to the similarity of Bh and Bq genes and other bacterial genomes
Point.Download existing Bartonella genome sequence and annotation information from NCBI Genbank databases, including 9 complete
Into graphic sequence and 30 draft sequences.First by ORTHOMCL (V1.4) software, all Bartonella genes of acquisition are entered
Row homology analysis, by B.henselae strain Houston-1 (GenBank database serial numbers：BX897699.1) and
B.quintana str.Toulouse (GenBank database serial numbers：BX897700.1 full gene) in other kinds not
The gene of homologous gene is found, the specific gene as each species；The specific gene recognized is finally reused into BLAST
The blastn in nucleic acid alignment programs nucleotide blast in software is compared, to exclude to go out in predictive genes
Existing mistake, the special gene sequence determined.Then suitable Bh and Bq specific genes are therefrom chosen and use Beacon
The Software for Design probes of Designer 7 and primer, and the primer and probe of design is further compared with Primer-blast
It is right, ensure not have primer dimer and non-specific amplification between two pairs of primers and two probe combinations.Primed probe such as table 1
It is shown.By Beijing Tian Yihuiyuan bio tech ltd synthetic primer and probe.
The dual real-time fluorescence quantitative PCR sonde method primer and probe of table 1.
2.4 the preparation of standard items
The DNA extracted using BhATCC and BqATCC expands purpose fragment 100bp with above-mentioned primer and probe respectively as template
And 103bp, it is connected to after PCR primer recovery purifyingOn-T5Zero cloning vectors；Vector introduction is big after connecting
Enterobacteria competent cell, screening positive clone, choose positive bacteria and drop into performing PCR identification, to identifying that correct bacterium colony shakes bacterium
Upgrading grain, as the standard items for drawing quantitative PCR standard curve.Enter performing PCR identification and sequencing identification again to institute's upgrading grain, it was demonstrated that
Plasmid standard is successfully constructed, with the recombinant plasmid sequence of measure compared with the gene order in GenBank, as a result sequence
Row homology is up to 100%.Plasmid and positive bacteria containing recon are stored in -20 DEG C and -70 DEG C respectively.
2.5 plasmid copy number concentration conversions
Bh and Bq plasmid concentrations are respectively 81.0ng/uL and 73.1ng/ μ L.- T5Zero Cloning carriers
Base number be 3953bp, the mean molecule quantity of each base is 660 dalton/bp.Prepared plasmid concentration is converted into
Copy number, wherein detection gene copy number is estimated according to below equation in per μ L samples：Samples copy number=concentration ng/L × Ah
Fu Jiadeluo constant × 10-9/ (660 × recombinant plasmid base number).Distinguished by the way that two kinds of copy Particle densities of Bh and Bq are calculated
For 1.82 × 1010Copy/μ L and 1.64 × 1010Copy/μ L.
2.6 reaction system and response parameter
The real-time sonde method reaction system of substance (20 μ L)：10μL PromegaQPCR Master Mix, 0.6
μ L sense primers and anti-sense primer (primer initial concentration is 10 μm of ol/L), (probe initial concentration is 10 μm of ol/ to 0.4 μ L probes
L), the μ L of DNA profiling DNA 1, nuclease-free water polishing.Dual sonde method reaction system (20 μ L) in real time：PromegaQPCR Master Mix are 10 μ L, and two pairs of initial concentrations, 10 μm of ol/L primers are respectively 0.6 μ L, and two probes are dense
10 μm of ol/L of degree are respectively 0.4 μ L, the μ L of DNA profiling DNA 1, nuclease-free water polishing.Amplification program：95℃2min；95 DEG C of 3s,
60 DEG C of 30s, 40 circulations.
The μ L of regular-PCR 20 reaction system：10 μ LMaster Mix, 0.6 μ L upstream and downstream primer (10 μm of ol/L), 1 μ L moulds
Plate, 2 μ L Dye, deionized water polishing.Amplification program：95℃5min；96 DEG C of 5s, 53 DEG C of 5s, 68 DEG C of 5s, 35 circulations；72℃
1min。
2.7 reaction condition optimization
(1) annealing temperature optimizes：To extract plasmid as template, 55 DEG C of -65 DEG C of progress gradient quantitative PCRs, each temperature ladder
Degree sets 3 Duplicate Samples, sets 1 negative control sample.According to amplified reaction Ct, the fluorescence signal intensity of amplification curve and melting
Curve screens optimum annealing temperature.
(2) concentration and probe concentration optimizes：Be 500nmoL/L by primer concentration, concentration and probe concentration be respectively 100nmoL/L,
200nmoL/L, 300nmoL/L, 400nmoL/L and 500nmoL/L, if 3 hole Duplicate Samples, a blank control are detected, root
Optimal concentration and probe concentration is selected according to the Ct of amplified reaction and the fluorescence signal intensity of amplification curve.
(3) primer concentration optimizes：The concentration and probe concentration of selection, primer concentration 100nmoL/L, 200nmoL/L, 300nmoL/
L, 400nmoL/L and 500nmoL/L, each concentration set 3 Duplicate Samples and a blank control, according to the Ct of amplified reaction and
The fluorescence signal intensity selection concentration and probe concentration of amplification curve.
2.8 specificity analyses
The specificity analysis of dual real-time fluorescence probe PCR：By all Bartonellas bought abroad of laboratory storage
Reference culture, the genomic DNA of non-Bartonella negative strain carry out real-time fluorescence sonde method as template and detected.
Verify that this laboratory separates on mouse and monkey at home using the dual real-time fluorescence quantitative PCR sonde method detection of foundation
8 plants of Bq and 6 plant of Bh.
2.9 sensitivity analysis
With the standard plasmid (10 of 10 × doubling dilution7-100Copy/μ L) 2 μ L are template, primer concentration is 300nmoL/
L and concentration and probe concentration are 200nmoL/L to detect the sensitiveness of substance and dual real-time fluorescence probe PCR method, are drawn minimum
Monitoring lower-cut.
2.10 repeatability analysis
It is respectively that 200nmoL/L and 300nmoL/L prepares the reaction of dual real-time fluorescence quantitative PCR by probe and primer concentration
System, repeated detection is carried out by template of Bh and Bq standard plasmids (108,105 and 102 copies/μ L) 2 μ L.With once double
In the PCR reaction amplifications of weight real-time fluorescence probe quantitative, each standard plasmid concentration makees 6 parallel samples, with M, SD in analysis group
And CV；Independently do 6 repetitions, M, SD and CV between analysis group, calculate its coefficient of variation, the coefficient of variation (CV)=standard deviation
(SD)/average (M).
3 results
The optimization of 3.1 reaction conditions
(1) annealing temperature optimizes：Annealing temperature be 55.0 DEG C, 55.7 DEG C, 57.0 DEG C, 59.0 DEG C, 61.4 DEG C, 63.3 DEG C,
64.5 DEG C and 65.0 DEG C, by setting thermograde to optimize annealing temperature, as a result show above-mentioned 8 temperature ladders from 55 DEG C to 65 DEG C
Degree, between 18.29-18.52, Bq Ct values influence very Bh Ct values between 20.39-20.98 on experimental result
It is micro-, consider easily there is non-specific amplification during lower temperature, it is optimum annealing temperature to select 60 DEG C.(as shown in Figure 1)
(2) concentration and probe concentration optimizes：Five concentration and probe concentrations set carry out real-time fluorescence quantitative PCR detection, as a result show Bh
Ct values between 18.45-18.23, influence very little, but when concentration and probe concentration is 100nmoL/L, fluorescence intensity is minimum；Bq's
Ct values do not influence equally substantially between 20.62-21.30, but when concentration and probe concentration is 100nmoL/L, fluorescence intensity is most
It is small；It is very stable to repeat experimental result.Therefore working concentrations (as shown in Figure 2) of the 200nmoL/L for probe is selected
(3) primer concentration optimizes：Experimental result shows Bh Ct values between 17.95-19.03；Bq Ct values exist
Between 20.84-21.39.As 100nmoL/L, fluorescence intensity is too low, and as 200-500nmoL/L, primer concentration influences on Ct
Very little, but easily there is non-specific amplification when generally primer concentration is too high, in addition in view of clinical practice, the reduction that should try one's best tries
Agent wastes, therefore optimal primer concentration is 300nmoL/L, now also obtain preferable expanding effect, and allusion quotation is presented in amplification curve
" S " type (as shown in Figure 3) of type.
3.2 specific detection
Specific outcome is shown, using the dual real-time PCR probe method of foundation to 12 plants of Bartonellas and other 27 plants of the moon
Property control strain is detected.The amplification of Bh and Bq reference cultures is the positive, and the bacterial strain of non-targeted strain and blank control do not go out
Existing non-specific amplification, have no that fluorescence signal without amplification curve (as shown in Figure 4), shows that the method has height to Bh and Bq detections
Specificity.6 plants of bartonella henselaes and the 8 plants of quintan Bartonellas that this experiment separates at home are detected using this method, are examined
It is as shown in Figure 5 to survey result.
3.3 substance real-time fluorescence quantitative PCR sensitivity analyses
With the standard plasmid (10 of 10 × doubling dilution7-100Copy/uL) template 2 μ L, primer concentration 300nmoL/L, are visited
Pin concentration is the sensitiveness that 200nmoL/L detects real-time fluorescence quantitative PCR sonde method.Result of the test shows obtained amplification curve
In typical S types (as shown in Figure 6 and Figure 7).In 20 μ L reaction system, real time fluorescence quantifying PCR method detection Bh plasmids
It is respectively 3.64 × 10 with Bq plasmid minimum detection limits1Individual copy and 3.28 × 101It is individual.Blank control does not have amplified signal, in addition
Also show that the coefficient R of good linear relationship and amplification efficiency, Bh and Bq2Respectively 0.996 and 0.998, E value difference
For 98.1% and 102.9%；
3.4 dual real-time fluorescence quantitative PCR sensitivity analyses
With the standard plasmid (10 of 10 × doubling dilution0-107Copy/μ L) it is template, primer and probe selects what is optimized
Concentration, detect the sensitiveness of multiple real time fluorescence quantifying PCR sonde method.As a result it is in typical S types that display, which obtains amplification curve, symbol
Close the standard of real-time fluorescence quantitative PCR amplification curve.In 20 μ L reaction system, Bh and Bq minimum detectabilities are respectively 3.64
×101Individual copy and 3.28 × 101Individual copy (as shown in Figure 8).In addition, dual real-time fluorescence quantifying PCR method detects Bh and Bq
Coefficient R2Respectively 0.994 and 0.998, E value be respectively 100.0% and 103.4%, it is good to show that PCR reactions are shown
Linear relationship and amplification efficiency.
It is 10 that Standard PCR, which detects both species Monitoring lower-cuts,3Rank is copied, when template concentrations are 102During copy/μ L
The fragment of amplification obscures visible, it is impossible to determines well；When template concentrations are 101With 100There is no purpose fragment during copy/μ L, it is empty
No amplified band is compareed in vain, and it is constant (as shown in Figure 9) to repeat experimental result.As can be seen here, dual real-time fluorescence quantitative PCR probe
Method improves 100 times than Standard PCR sensitiveness.
The repeatability analysis of 3.5 dual real-time fluorescence quantitative PCR sonde methods
In group between hole CV values between 0.19%-0.53%, between group replication CV values 0.49%-1.66% it
Between (as shown in table 2).
Repeatability between group in the dual real-time fluorescence quantitative PCR sonde method of table 2 detection Bh and Bq groups
The present invention choose Bh and Bq specific gene conserved sequences, devise primed probe, by optimize reaction condition and
Amplification system, experimental result show that substance real-time fluorescence quantitative PCR and multiple real time fluorescence are determined with detection specificity well
Measure PCR amplification curves amplification efficiency and linearly dependent coefficient it is all relatively good, detection Bh and Bq minimums be respectively 37 copy and
33 copies, hence it is evident that improve 100 times than Standard PCR.The dual real-time fluorescence quantitative PCR sonde method that this research is established can be
Two pathogen can be detected simultaneously in primary first-order equation, there is higher application value in clinic, while saving sample
Reach economical and practical again, Acceleration study process etc..
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.
Claims (2)
1. trigger the real-time fluorescence quantitative PCR kit of endocarditic Bartonella for detecting, it is characterised in that containing drawing
Thing group and the probe being used cooperatively with primer sets；
The primer sets include being used for the primer pair I for detecting bartonella henselae and the primer for detecting quintan Bartonella
To II；
The primer pair I is：
BhF：5’-GAGTCAGATGGAATGGTATTGGTTATC-3’；
BhR：5’-CGGTTTATCCCCCACAAGATAGG-3’；
The primer pair II is：
BqF：5’-TGCCAAGTATGCGAGTTCCTG-3’；
BqR：5’-TCAGAGTTAGGTGCAAGTTCTATGG-3’；
The probe is：
Probe I：
BhT：FAM-CCCTCCCGGTTTTTCTCTGGGCCT-BHQ1；
Probe I I：
BqT：CY5-TCTGCGCCCGGTTCTGAAATGCCT-BHQ2；
Wherein, FAM and CY5 is fluorescent reporter group, and BHQ1 and BHQ2 are fluorescent quenching gene；
Wherein, the working concentration of probe is 200nmol/L, and the working concentration of primer is 300nmol/L.
2. kit according to claim 1, it is characterised in that the kit also includes dNTPs, Taq DNA and polymerize
Enzyme, Mg2+, one or more in PCR reaction buffers.
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| WO2010039775A1 (en) * | 2008-10-03 | 2010-04-08 | Ibis Biosciences, Inc. | Compositions for use in identification of members of the bacterial class alphaproteobacter |
| CN104087661A (en) * | 2014-06-17 | 2014-10-08 | 中国疾病预防控制中心传染病预防控制所 | Method for detecting B. bacilliformis by uxing TaqMan real-time fluorescence quantification PCR |
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2015
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010039775A1 (en) * | 2008-10-03 | 2010-04-08 | Ibis Biosciences, Inc. | Compositions for use in identification of members of the bacterial class alphaproteobacter |
| CN104087661A (en) * | 2014-06-17 | 2014-10-08 | 中国疾病预防控制中心传染病预防控制所 | Method for detecting B. bacilliformis by uxing TaqMan real-time fluorescence quantification PCR |
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| Molecular Detection of Bartonella Quintana,B. koehlerae, B. henselae,B. clarridgeiae, Rickettsia felis, and Wolbachia pipientis in Cat Fleas, France;Jean-Marc Rolain等;《Emerging Infectious Diseases》;20030331;第9卷(第3期);第339页、表1 * |
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