CN104774960A - Method for applying dual high-resolution melting curve technology to detect Bartonella - Google Patents
Method for applying dual high-resolution melting curve technology to detect Bartonella Download PDFInfo
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Abstract
The invention provides a method for applying a dual high-resolution melting curve technology to detect Bartonella. By the adoption of the method, Bartonella henselae and Bartonella Quintana can be simultaneously and quickly detected, and effective measures are provided for early-stage rapid diagnosis, monitoring, epidemiological investigation and the like of a series of diseases including endocarditis caused by the Bartonella henselae and Bartonella Quintana.
Description
Technical field
The present invention relates to the molecular Biological Detection field of Bartonella, specifically, relate to a kind of method applying dual high resolving power melting curve technology for detection Bartonella.
Background technology
Infective endocarditis (Infective endocarditis, IE) is a class serious threat life, the extremely complicated and disease that diagnosis is very difficult of clinical manifestation.In recent years, although prevention and therapy level has had large increase, M & M is still higher.Current research finds that infecting the mankind's endocarditic Bartonella (Bartonella spp.) kind gets more and more, 8 kinds of Bartonellas are reported, wherein with bartonella henselae (B.henselae, and trench fever Bartonella (B.quintana Bh), Bq) be main, these two kinds of pathogenic agent are gram's staining feminine gender, oxidase negative, nutritional condition requires harsh, the entozoic aerobasilus of facultative intracellular, main parasitic is people, cat, in the vascular endothelial cell such as dog and rodent and red corpuscle, propagated by the HAEMATOPHAGOUS ARTHROPODS such as flea and body louse, mankind's cat scratch disease can be caused, trench fever, the various diseases such as endocarditis and bacillary angiomatosis.Except Bh and Bq, have report cause humans and animals endocarditic also have B.vinsonii subsp.berkhoffii, B.vinsonii subsp.arupensis, B.clarridgeiae, B.koehlerae and B.alsatica.
At present, to Bh and Bq detection method, mainly separation and Culture, Serologic detection, Standard PCR and real-time fluorescence quantitative PCR detect.Bartonella bacteria growing is slow, nutritional condition requires harsh and is difficult to separation and Culture, the rabbit blood of 5%-10%, sheep blood or horse blood (defiber anti-freezing) is added in general application tryptic soy agar substratum, Columbia medium or brain heart infusion agar, in humid conditions, cultivate 7-14d for 25 DEG C-37 DEG C, the even longer time, bacterium colony does not have characteristic, can not be used for strain identification.Serologic detection detects antibody by IFA method and ELISA method usually, Bh and Bq has commercially available detection kit, and the method has certain subjectivity when sentence read result, and sensitivity and specificity are all not high, Bq and Bh can not be distinguished well, and easily and other microorganisms such as chlamydozoan there is cross reaction.Standard PCR detects to be existed primer and combines and lack specificity, and in laboratory pollution and clinical sample, PCR inhibition exists and causes false negative etc., also needs further agarose gel electrophoresis to check order and verify after gene amplification, relatively consuming time.Real-time fluorescence probe PCR method is according to sequence-specific probes difference species, add specificity and sensitivity, but need expensive probe, day by day popularize on infectious disease pathogens detects in recent years, external report is set up real-time fluorescence probe PCR method by amplification ssrA gene and is detected Bartonella at present, domestic also have report to adopt TaqMan-MGB probe technique to establish detection Bh and Bq substance real time fluorescence quantifying PCR method, but a PCR system can only detect one or a class pathogenic agent, can not detect multiple pathogens simultaneously.High resolving power melting curve analysis technology (High-resolution melting analysis, HRM) be a kind of new technology developed on real-time fluorescence quantitative PCR, overcome unsaturated dyestuff as the reaction of SYBR Green suppression PCR, the shortcomings such as rearrangement can not be there is in dehybridization procedure, the fluorescent signal sent in fusion processes is made to have higher resolving power, reported that adopting SYBR dyestuff to set up detects Bartonella fluorescence quantifying PCR method abroad, latest report has employing saturable dye cyto9 amplification rpoB, gltA, ITS order-checking to detect Bartonella.But there is no the report that the dual HRM technology of application detects these two kinds of pathogenic agent of Bh and Bq simultaneously at present both at home and abroad.
Summary of the invention
The object of the invention is, based on dual high resolving power melting curve technology, to provide a kind of method detecting Bartonella.
In order to realize the object of the invention, first the present invention is provided for the combination of primers that real-time fluorescence quantitative PCR detects Bartonella, described combination of primers comprises for real-time fluorescence quantitative PCR detection bartonella henselae (B.henselae, Bh) primer pair I and the primer pair II for real-time fluorescence quantitative PCR detection trench fever Bartonella (B.quintana, Bq).
Described primer pair I is:
BH14560-F：5’-GAGTCAGATGGAATGGTATTGGTTATC-3’
BH14560-R：5’-CGGTTTATCCCCCACAAGATAGG-3’
Described primer pair II is:
BQ11520-F：5’-GGAACCAGTCCGCCAAAAG-3’
BQ11520-R：5’-ATGGGTTGGTTTCTGCTGAGG-3’
The present invention also provides the test kit detecting Bartonella for real-time fluorescence quantitative PCR containing above-mentioned combination of primers.
The present invention further provides a kind of method applying dual high resolving power melting curve technology for detection Bartonella, comprise the following steps:
1) DNA in sample is extracted;
2) with step 1) in extract DNA be template, utilize aforementioned combination of primers carry out dual high resolving power melting curve real-time fluorescence quantitative PCR reaction;
3) real-time fluorescence quantitative PCR product is analyzed.
Wherein, step 2) the middle reaction system 20 μ L:MeltDoctor adopted
tMhRM MasterKit 10 μ L, upstream and downstream primer (10 μm of ol/L) each 0.6 μ L of primer pair I and primer pair II, DNA profiling 1 μ L, nuclease free water complements to cumulative volume 20 μ L.
Amplification program is: 95 DEG C of 3min; 95 DEG C of 15s, 60 DEG C of 1min, 40 circulations;
Melting program is: 95 DEG C of 30s, 70 DEG C of 30s, then with the ramp to 95 DEG C of 0.2 DEG C/s, and collection first order fluorescence per second.
Dual Real-time High Resolution rate melting curve method high specificity, highly sensitive, good stability that the present invention sets up.The display of specificity experiments result only has corresponding Bartonella to amplify fluorescent signal, the bacterium of other kinds is showed no fluorescent signal, and the melting temperature (Tm) of bartonella henselae and trench fever Bartonella (Melting temperature, Tm) value is respectively 75.60 ± 0.5 DEG C and 79.80 ± 0.5 DEG C; Sensitivity experiments result is presented in the reaction system of 20 μ L, and dual Real-time High Resolution rate melting curve method detects bartonella henselae and trench fever Bartonella minimum detectability is respectively 3.64 × 10
1individual copy and 5.62 × 10
1individual copy, improves 100 times than Standard PCR susceptibility, also demonstrates good linear relationship and amplification efficiency in addition, coefficient R
2be respectively 0.998 and 0.997, E value be respectively 102.8% and 104.7%.CV value in repeated experiment result display group and between group is 0.22%-0.50% and 0.50%-1.2%, in allowed band.Wherein between bartonella henselae group endoporus, CV value is at 0.22%-0.50%, and between group, replication CV value is at 0.50%-0.98%.Between trench fever Bartonella group endoporus, CV value is at 0.24%-0.45%, and between group, replication CV value is at 0.50%-1.20%.Adopt present method simultaneously rapid detection can go out bartonella henselae and trench fever Bartonella, the researchs such as the Rapid&Early diagnosis of a series of diseases such as the endocarditis caused by these two kinds of Bartonellas, monitoring and epidemiology survey provide effective means.
Accompanying drawing explanation
Fig. 1 is the amplification curve of bartonella henselae (A) and trench fever Bartonella (B) different annealing temperature in the embodiment of the present invention 1.
Fig. 2 is the amplification curve of bartonella henselae (A) and trench fever Bartonella (B) different primers concentration in the embodiment of the present invention 1.
Fig. 3 is the melting curve that in the embodiment of the present invention 1, trench fever Bartonella difference melts speed 0.1 DEG C/s (A), 0.2 DEG C/s (B), 0.4 DEG C/s (C), 0.6 DEG C/s (D) and 0.8 DEG C/s (E).
Fig. 4 is amplification curve and the typical curve of bartonella henselae (A) and the sensitivity analysis of trench fever Bartonella (B) substance HRM real-time fluorescence quantitative PCR in the embodiment of the present invention 1.
Fig. 5 is amplification curve and the typical curve of bartonella henselae (A) and the sensitivity analysis of trench fever Bartonella (B) dual HRM real-time fluorescence quantitative PCR in the embodiment of the present invention 1.
Fig. 6 is bartonella henselae (A) and trench fever Bartonella (B) different extent of dilution standard plasmid Standard PCR electrophorogram in the embodiment of the present invention 1.
Fig. 7 is the result of the embodiment of the present invention 1 middle high-resolution melting curve real-time fluorescence quantitative PCR specific detection trench fever Bartonella and bartonella henselae; Wherein, A is melting curve, and B is differentiation curve, and C is homogenization curve.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is experiment condition all conveniently, as Sambrook equimolecular Cloning: A Laboratory Manual (Sambrook J & Russell DW, Molecular cloning:a laboratory manual, 2001) condition of, or according to manufacturer's specification sheets advising.
Embodiment 1 applies the method for dual high resolving power melting curve technology for detection Bartonella
1 materials and methods
1.1 bacterial strains and biological specimen DNA
Bartonella genomic dna comprises bartonella henselae (B.henselae, Bh) ATCC49882, trench fever Bartonella (B.quintana, Bq) ATCC VR-358, Vincent Bartonella wins 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) ATCCVR-152, 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 provide by Inst of Infection Disease Prevention and Control, Chinese Diseases Prevention an's Vector factors watch-keeping cubicle.
Other bacterial genomes DNA comprises streptococcus pneumoniae (Streptococcuspneumoniae), hemophilus influenzae (Haemophilus influenzae), helicobacter pylori (Helicobacter pylori), bacillus canalis capsulatus (Klebsiella pneumoniae), Salmonella typhimurtum (Salmonella typhimurium), shigella flexneri (Shigellaflexneri), legionella pneumophilia (Legionella pneumophila), Rickettsia prowazeki (Rickettsia prowazekii), R. massillia (R.massilliae), Japan's rickettsia (R.japonica), Anaplasma phagocytophila (Anaplasma phagocytophilum), yersinia pestis (Yersinia pestis EV76), Acinetobacter bauamnnii (Acinetobacterbaumanii), Leptospira (Leptospira interrogans) and hedgehog (Erinaceus) provide by Inst of Infection Disease Prevention and Control, Chinese Diseases Prevention an's laboratory.
1.2 key instrument equipment and reagent
Quantitative real time PCR Instrument, CFX96, BioRad; Table model high speed centrifuge, 5804R, Eppendorf; Trace dna concentration determination instrument, NanoDrop-1000, Thermo FisherScientific.
QIAamp DNA Mini Kit (QIAGEN), Fast Cycling PCR Kit (QIAGEN), cloning kit is
zero Cloning Kit, purchased from Beijing Quanshijin Biotechnology Co., Ltd; MeltDoctor
tMhRM Master Kit is purchased from AppliedBiosystems company; Plasmid extraction kit is purchased from Promega company.
The Design and synthesis of 1.3 target gene screenings, primer
Carry out screening according to Bh and Bq gene and the similarity of other bacterial genomes to obtain detecting target candidate gene site.From US National Biotechnology Information center (National Center ofBiotechnology Information, NCBI) downloaded existing Bartonella genome sequence and annotation information in GenBank database, comprised 9 and complete graphic sequence and 30 draft sequence.First ORTHOMCL (V1.4) software is used, homology analysis is carried out to all Bartonella genes obtained, the full gene of Bartonella henselae strain Houston-1 (GenBank database serial number: BX897699.1) and Bartonella quintana str.Toulouse (GenBank database serial number: BX897700.1) is not found homogenic gene, as the specific gene of each species in other kinds; Finally the specific gene recognized is reused sequence Local Alignment Tool (Basic Local Alignment Search Tool, BLAST) blastn in the nucleic acid alignment programs nucleotide blast in software compares, to get rid of the mistake that may occur in predictive genes, obtain the special gene sequence determined.Then suitable Bh and Bq specific gene is therefrom chosen, design suitable two pairs of primer foots combination, and the primer of design is compared with Primer-blast further, ensure that two pairs of combination of primers do not have primer dimer and non-specific amplification, the primer of design is as shown in table 1.Primer is synthesized by Tian Yihuiyuan bio tech ltd, Beijing.
The dual HRM PCR in real time of table 1 two pairs of primer sequences
The preparation of 1.4 standard substance
The DNA extracted with Bh reference culture ATCC 51672TM and Bq reference culture ATCC, for template, is standard PCR amplification object fragment 100bp and 125bp with the primer in table 1 respectively; PCR primer is connected to after reclaiming purifying
on Zero Cloning carrier; Vector introduction competent escherichia coli cell after connecting, screening positive clone, chooses positive bacteria and drops into performing PCR qualification, shake bacterium upgrading grain, as the standard substance drawing quantitative PCR typical curve to the correct bacterium colony of qualification.Carry out PCR qualification and order-checking qualification again to institute's upgrading grain, confirm successfully to construct plasmid standard, compare by the gene order in the recombinant plasmid sequence measured and GenBank, result sequence homology reaches 100%.Plasmid and the positive bacteria containing recon are stored in-20 DEG C and-70 DEG C respectively.
1.5 plasmid copy number concentration conversions
The concentration of Bh and Bq two kinds of plasmids is recorded by nucleic acid concentration determinator and is respectively 81.0ng/ μ L and 125.7ng/ μ L.
the base number of Zero Cloning carrier is 3953bp, and the molecular-weight average of each base is 660 dalton/bp.Prepared plasmid concentration is converted into copy number, wherein detects gene copy number in every μ L sample and estimate according to following formula: samples copy number=concentration ng/L × Avogadro constant × 10
-9/ (660 × recombinant plasmid base number).1.82 × 10 are respectively by calculating Bh and Bq two kinds of copy number concentration
10copy/μ L and 2.81 × 10
10copy/μ L.
1.6 reaction systems and reaction parameter
HRM substance real-time PCR reactions condition: reaction system adopts 20 μ L, MeltDoctor
tMhRM Master Kit is 10 μ L, and upstream and downstream primer (10 μm of ol/L) is respectively 0.6 μ L, DNA profiling DNA 1 μ L, nuclease free water polishing.Amplification program: the first step 95 DEG C of denaturation 3min, a circulation; Second step, 95 DEG C of sex change 15s, 60 DEG C of annealing temperature 1min, 40 circulations.Melting program is: 95 DEG C of sex change 30s, 70 DEG C of cooling 30s, is then warming up to 95 DEG C (0.2 DEG C gathers first order fluorescence).
HRM dual real-time fluorescence quantitative PCR reaction conditions: reaction system adopts 20 μ L, MeltDoctor
tMhRM Master Kit is 10 μ L, and two pairs of primers (10umol/L) are respectively 0.4 μ L, DNA profiling DNA 1 μ L, nuclease free water polishing.Amplification program: the first step 95 DEG C of denaturation 3min, a circulation; Second step, 95 DEG C of sex change 15s, 60 DEG C of annealing temperature 1min, 40 circulations.Melting program is: 95 DEG C of sex change 30s, and 70 DEG C of cooling 30s, are then warming up to 95 DEG C (0.2 DEG C/s gathers first order fluorescence).The software CFX Manager software using Bole CFX96 real-time fluorescence quantitative PCR instrument to carry v.2 with Precision Melt Analysis
tMsoftware analyzes.
Standard PCR reaction system 20 μ L:10 μ L Master Mix, 0.6 μ L upstream and downstream primer (10 μm of ol/L), 2 μ L templates, 2 μ L dyestuffs, deionized water polishing.Amplification program: 95 DEG C of 5min, 1 circulation; 96 DEG C of 5s, 54 DEG C of 5s, 68 DEG C of 4s, 30 circulations; 72 DEG C of 1min.
1.7 reaction condition optimization
1.7.1 annealing temperature optimization: with Bh and Bq positive control plasmid DNA for masterplate, 55-65 DEG C is carried out gradient quantitative PCR, and each gradient temperature arranges 3 multiple holes, arranges 1 negative control hole.According to cycle threshold (Cycle threshold value, the C of amplified reaction
t), the fluorescence signal intensity of amplification curve and the suitableeest annealing temperature of melting curve screening.
1.7.2 primer concentration optimization: primer concentration is 100nmol/L, 200nmol/L, 300nmol/L, 400nmol/L and 500nmol/L, each sample arranges 3 multiple holes, arranges 1 negative control hole.According to the C of amplified reaction
tthe fluorescence signal intensity solubility curve fluorescence of value, amplification curve selects best primer concentration.
1.7.3 melt rate optimized: immobilized primer concentration, select 0.1 DEG C/s, 0.2 DEG C/s, 0.4 DEG C/s, 0.6 DEG C/s and 0.8 DEG C/s, each sample arranges 3 multiple holes, arranges 1 negative control hole.According to the C of amplified reaction
tthe fluorescence signal intensity of value, amplification curve and melting temperature (Tm) peak value select best melting speed.
1.8 sensitivity analysis
With the standard plasmid (10 of 10 × doubling dilution
0-10
7copy/μ L) carry out substance and dual HRM real-time fluorescence quantitative PCR as template, delivery plate 2 μ L, primer concentration are 300nmol/L to detect the susceptibility of the method.Carry out standard PCR amplification with the plasmid of same concentration for template simultaneously, compare the difference of real-time fluorescence PCR and Standard PCR susceptibility.
1.9 specificity analyses
Substance and dual HRM real-time fluorescence probe PCR specificity analyses: the concentration of the genomic dna of the host animals such as non-to Bb, other kind of Bartonella, Agrobacterium tumefaciens etc. Bartonella bacterium and dog, cat, mouse, people and tick and communication media is adjusted to about 1-10ng/ μ L, and delivery plate 1 μ L carries out the specificity of quantitative PCR detection the method.
1.10 repeatability is analyzed
Choose 1 μ L Bh and Bq standard plasmid concentration 10
2copy/μ L, 10
5copy/μ L and 10
8copy/μ L is template, and primer concentration is 300nmol/L, prepares dual HRM quantitative fluorescent PCR reaction system and detects.In reacting with a quantitative PCR, each standard plasmid extent of dilution does 6 repeating holes, analyzes between hole and namely organizes interior difference; Carry out respectively independently repeating experiment for 6 times with above-mentioned the same terms, analyze group difference, calculate its variation coefficient, the variation coefficient (CV)=standard deviation (SD)/mean number (M).
2 results
The optimization of 2.1 reaction conditionss
2.1.1 annealing temperature optimization: annealing temperature is 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 set temperature gradient optimizing annealing temperature, result display is above-mentioned 8 thermogrades from 55 DEG C to 65 DEG C, each temperature is larger on experimental result impact, to produce C
tminimum and the fluorescent absorption value of value is maximum, and both consider for foundation, present " S " type curve of classics, select optimum annealing temperature.Under normal circumstances when the temperature is sufficiently low, can ensure that primer is effectively annealed with aim sequence, but high-temperature that simultaneously also will be enough is to reduce non-specific binding.Consider to make experiment have higher specificity and keeping the best polymerization activity of enzyme, selecting 60 DEG C is annealing temperature, now also obtain desirable expanding effect, and amplification curve presents typically " S " type (Fig. 1).
2.1.2 primer concentration optimization: 100nmol/L, 200nmol/L, 300nmol/L, 400nmol/L and 500nmol/L optimum result is respectively to primer concentration and carries out com-parison and analysis, the C of Bh
tbe worth all between 18.73-19.59, the C of Bq
tvalue is all between 19.84-20.57.When primer concentration is 100nmol/L and 200nmol/L, amplification curve is tending towards smooth, and do not present classical " S " type curve, namely do not see the obvious exponential amplification phase, the peak value of melting temperature (Tm) is lower; Easily occur that when primer concentration is too high non-specific amplification and primer dimer are formed, consider and select best primer concentration to be 300nmol/L, now also obtain desirable expanding effect, amplification curve presents typically " S " type, and fluorescence intensity is higher (Fig. 2) also.
2.1.3 the optimization of speed is melted: with the plasmid of Bq for template carries out the optimization of melting speed (0.1 DEG C/s, 0.2 DEG C/s, 0.4 DEG C/s, 0.6 DEG C/s and 0.8 DEG C/s), each sample arranges 6 multiple holes, arranges 1 negative control hole.Test-results shows, in the temperature interval of four kinds of fluorescent collectings, the resolving power of 0.6 DEG C/s and 0.8 DEG C/s is too poor, the requirement of HRM cannot be reached, resolving power is lower, the scope (M ± SD) of melting temperature (Tm) Tm during 0.6 DEG C/s is 79.8 DEG C ± 0.00, and melting temperature (Tm) Tm during 0.8 DEG C/s is 80.00 DEG C ± 0.00.0.1 DEG C/s, 0.2 DEG C/s and 0.4 DEG C/s tri-melts speed and can obtain specific melting curve and fusing point peak, during 0.1 DEG C/s, melting temperature (Tm) Tm is 80.08 DEG C ± 0.041, and during 0.2 DEG C/s, melting temperature (Tm) Tm is 79.97 DEG C ± 0.082.Melting temperature (Tm) Tm during 0.4 DEG C/s is 80.00 DEG C ± 0.00.0.1 DEG C/s melting curve is steeply sharply to decline, and peak substrate is narrower, and peak value is no significant difference compared with the melting speed of 0.2 DEG C/s, consuming time longer.0.4 DEG C/s is compared with 0.2 DEG C/s, and peak value is lower, and fusing point peak shape is rough, resolving power relative mistake.Therefore the melting speed of 0.2 DEG C/s is adopted to carry out detecting (Fig. 3).
2.2 sensitivity Detection
With the standard plasmid (2.66 × 10 of 10 × doubling dilution
0-2.66 × 10
7copy/μ L) be template, Bh and Bq primer concentration is 300nmol/L, detects the susceptibility of substance and dual HRM real time fluorescence quantifying PCR method.Real-time fluorescence quantitative PCR is carried out in result display, and obtain amplification curve in typical " S " type, meet the standard of real-time fluorescence quantitative PCR amplification curve, typical curve is shown in Fig. 4 and Fig. 5.In the reaction system of 20 μ L, substance and dual HRM real time fluorescence quantifying PCR method detect Bh and Bq minimum detectability and are respectively 3.64 × 10
1individual copy and 5.62 × 10
1individual copy, amplified production forms single peak value, its T
mvalue is respectively 75.60 DEG C ± 0.5 and 79.80 DEG C ± 0.5, and without the appearance of primer dimer and nonspecific products, blank is without amplified signal.In addition, PCR reaction and display goes out good linear relationship and amplification efficiency, and substance HRM real time fluorescence quantifying PCR method detects Bh and Bq coefficient R
2be respectively 0.998 and 0.999, E value be respectively 98.9% and 100.3% (Fig. 4); Dual HRM real time fluorescence quantifying PCR method detects Bh and Bq coefficient R
2be respectively 0.998 and 0.997, E value be respectively 102.8% and 104.7% (Fig. 5).
Standard PCR detects these two kinds of species Monitoring lower-cut and is 10
3copy rank (Fig. 6), when template concentrations is 10
2, 10
1with 10
0do not have discernible object fragment during copy/μ L, blank does not have amplified band, repeats experimental result constant.As can be seen here, substance and dual HRM real time fluorescence quantifying PCR method improve 100 times than Standard PCR susceptibility.
2.3 repeatability are analyzed
CV value in repeated experiment result display group and between group is 0.22%-0.50% and 0.50%-1.2%, all in allowed band.Wherein between Bh group endoporus, CV value is at 0.22%-0.50%, and between group, replication CV value is at 0.50%-0.98%.Between Bq group endoporus, CV value is at 0.24%-0.45%, and between group, replication CV value is at 0.50%-1.20%.
The dual HRM real-time fluorescence quantitative PCR of table 2 detects in bartonella henselae and trench fever Bartonella group repeated between group
Note: Mean is mean value, and SD is standard deviation, and CV is the variation coefficient
2.4 specific detection
Specific outcome shows, and the dual HRM real time PCR detection method that application is set up detects 12 strain Bartonellas and other 27 strain negative control bacterial strains.For positive, all there is specificity melting curve, T in the amplification of Bh and Bq reference culture
mvalue is 75.60 DEG C ± 0.5, and 79.80 DEG C ± 0.5.All there is not non-specific amplification in the bacterial strain of non-targeted bacterial classification, without amplification curve, melting curve and T
mvalue, blank NTC is negative without amplified signal (Fig. 7), shows that this method has high degree of specificity to Bh and Bq detection.Therefore, according to the typicalness of melting curve and the specificity T of bacterial classification
mvalue, can make qualification to unknown sample.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (4)
1. the combination of primers of Bartonella is detected for real-time fluorescence quantitative PCR, it is characterized in that, described combination of primers comprises for real-time fluorescence quantitative PCR detection bartonella henselae (B.henselae, Bh) primer pair I and the primer pair II for real-time fluorescence quantitative PCR detection trench fever Bartonella (B.quintana, Bq);
Described primer pair I is:
BH14560-F：5’-GAGTCAGATGGAATGGTATTGGTTATC-3’
BH14560-R：5’-CGGTTTATCCCCCACAAGATAGG-3’
Described primer pair II is:
BQ11520-F：5’-GGAACCAGTCCGCCAAAAG-3’
BQ11520-R：5’-ATGGGTTGGTTTCTGCTGAGG-3’。
2. the test kit detecting Bartonella for real-time fluorescence quantitative PCR containing combination of primers described in claim 1.
3. apply the method for dual high resolving power melting curve technology for detection Bartonella, it is characterized in that, comprise the following steps:
1) DNA in sample is extracted;
2) with step 1) in extract DNA be template, utilize the combination of primers described in claim 1 carry out dual high resolving power melting curve real-time fluorescence quantitative PCR reaction;
3) real-time fluorescence quantitative PCR product is analyzed.
4. method according to claim 3, is characterized in that, step 2) described in the reaction of dual high resolving power melting curve real-time fluorescence quantitative PCR, its amplification program is: 95 DEG C of 3min; 95 DEG C of 15s, 60 DEG C of 1min, 40 circulations;
Its melting program is: 95 DEG C of 30s, 70 DEG C of 30s, then with the ramp to 95 DEG C of 0.2 DEG C/s, and collection first order fluorescence per second.
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| CN113025754A (en) * | 2021-03-29 | 2021-06-25 | 上海基灵生物科技有限公司 | Nucleic acid composition, kit and detection method for detecting feline anemia pathogens |
| CN114438238A (en) * | 2022-03-04 | 2022-05-06 | 广东省人民医院 | Primer for detecting infectious endocarditis pathogen and digital PCR kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113025754A (en) * | 2021-03-29 | 2021-06-25 | 上海基灵生物科技有限公司 | Nucleic acid composition, kit and detection method for detecting feline anemia pathogens |
| CN114438238A (en) * | 2022-03-04 | 2022-05-06 | 广东省人民医院 | Primer for detecting infectious endocarditis pathogen and digital PCR kit |
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