CN107083434A - A kind of fluorescent PCR quantitatively detects the streptococcic Sample pretreatment method of B races and application - Google Patents
A kind of fluorescent PCR quantitatively detects the streptococcic Sample pretreatment method of B races and application Download PDFInfo
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Abstract
The present invention relates to Bacteria Detection technical field, and in particular to a kind of fluorescent PCR quantitatively detects the streptococcic Sample pretreatment method of B races.The method outstanding feature is, tested bacteria need not be washed from sample swab and get off to prepare sample suspension, without carrying out high speed concussion and the nucleic acid extraction process such as boil, but the B races streptococcus lyases directly lysisin situ B races streptococcus on swab is used, it is directly used in fluorescent PCR detection as PCR amplifications template after the obtained of short duration centrifugation of lysate.This method is good to streptococcic lytic effect, nucleic acid extraction efficiency high, the equipment such as oscillator, water-bath and ice bath are not needed, help to save sample process cost, the operation from swab needed for extraction nucleic acid and step can be also greatly simplified simultaneously, and the cracking liquid energy extracted quantitatively detects B races streptococcus for follow-up fluorescent PCR.
Description
Technical field
The present invention relates to Bacteria Detection technical field, and in particular to a kind of fluorescent PCR quantitatively detects that B races locate before streptococcic
Manage method and its application.
Background technology
B races streptococcus (group B streptococcus, GBS) is also referred to as Streptococcusagalactiae, is that one kind parasitizes the mankind and secreted
The conditioned pathogen of urodaeum and lower digestive tract, healthy population bacterial bearing rate is up to 35%, and general health population infection simultaneously will not
Disease.But be colonized in the B races streptococcus of puerpera's genital tract, can in childbirth, vertical transmission is to neonate, and neonatal B race streptococcus
The disease fatal rates such as infection caused sepsis of the newborn, meningitis and pneumonia are high, and can cause nervous system sequelae.According to
The pregnant woman of statistics about 10%~30% has infection GBS, wherein 40%~70% can pass to neonate in the progress of labor.If
Early stage invasive infection occurs with this bacterium, about 1%~3% in neonate, wherein having 5% can cause death.Early in
1970s B race streptococcus is just it is verified that one of important pathogenic bacteria infected for perinatal period mother and baby.The U.S. in 2010
Center for Disease Control formulates《Perinatal period GBS prevention guideline》, it is proposed that pregnant woman carries out GBS examinations in 34~37 weeks in gestation.
At present, the streptococcic diagnostic method of B races mainly has cultivation, immunological method, microbe auto-analysis identification system
System and fluorescence PCR method.Wherein, the specificity and sensitiveness of cultivation are high, but clinical detection overlong time (at least need 24~
48h, or even longer time), process is cumbersome, needs to use special culture medium and is easily influenceed by miscellaneous bacteria, be not suitable for greatly
Range detection.Immunological method, such as：Latex agglutination tests (latex agglutination, LA), EUSA
(enzyme linked immunosorbent assay, ELISA), CA (countercurrent
Immunoelectrophotesis, CoA) and counter immunoelectrophoresis (countercurrent
Immunoelectrophotesis, CIE) etc., although specificity can be up to 95%, but sensitiveness is not enough, recall rate compared with
It is low.U.S. FDA have issued the very high Feasible degree warning of these method false negatives and false positive rate in 1997.Microorganism is certainly
It is dynamic microorganism to be identified, examined with analyzing and identifying system energy comprehensive automation, but time-consuming, qualification process is cumbersome, to work
The skill requirement for making personnel is high, and equipment is expensive, and cost is high, is unfavorable for a wide range of popularization.
And the fluorescence PCR method of rising in recent years has been largely overcoming the shortcoming of the above method.Quantitative fluorescent PCR
Technology is a kind of sensitiveer, more special, the more accurate nucleic acid grown up based on traditional PCR technique and combination spectral technique
Detection technique.Testing result is accurate, and repeatability is high, dynamic reaction patient treat forward and backward pathogen dynamic change and with clinic
Relation, and the problem of normal PCR need to be post-processed is avoided in whole process, reduces pollution.Real-Time Fluorescent Quantitative PCR Technique
Using a kind of PCR amplification instrument with fluorescence detection device, fluorescence detection device can be sent out according to certain routines periodically
Go out the exciting light of specific wavelength, detection fluorescence signal is collected, by detecting that the dynamic change of fluorescence signal reflects that PCR's is every in real time
The level of amplification of individual circulation, can automatically analyze acquisition amplification curve, according to amplification curve and fluorescence by software after off-test
The intersection point (i.e. Ct values) and the shape of amplification curve of threshold line, it can be determined that positive and negative result；If had in same reaction
Know the qualitative reference product or standard items of concentration, then acquisition standard curve can be automatically analyzed by software, is achieved in unknown
The definite value (i.e. quantitative detection) of sample.Compared with traditional PCR, it adds a two ends in reaction system and marked respectively
The probe of fluorescent reporter group and quenching group.When probe structure is complete, the fluorescent energy that fluorescent reporter group is sent is quenched
Group absorptions, are presented quenching effect；If there is the presence of target sequence in amplification procedure, with the extension of target fragment, probe point
Son is gradually hydrolyzed by Taq enzyme and cut off, and fluorescent reporter group is mutually dissociated with quenching group, has blocked fluorescence energy transfer between the two
Effect, the fluorescence signal that fluorescent reporter group is sent is collected by fluorescence detection device.With the progress of amplification, fluorescence signal with
The amplification of purpose fragment and linear enhancing is presented.After off-test, the software that can be carried by fluorescent PCR instrument automatically analyzes number
According to, the definite value result of positive and negative result and concentration of specimens can be obtained, therefore, detection of the technology in target polynucleotide sample
In quantitative analysis, traditional PCR method is gradually replaced, quite varied application is obtained.Studies have shown that real-time fluorescence PCR
The method for cultivation of bacteria of detection method and standard, up to more than 90%, has been obtained in terms of the Sensitivity and Specificity that GBS is detected
The approval of United States food and drag administration (FDA) is simultaneously applied to clinical screening.
Expanded before PCR detections, it is necessary to extract nucleic acid from clinical sample as template.Currently for pregnant woman
GBS examinations in, the collection of sample is typically used on sterile swab, swab head using materials, swab such as terylene, fiber or Buddhist nun's suedes
Handle is plastic material.In sampling, swab head is respectively placed in rotating acquisition secretion in genital tract and rectum.Carrying out in fact
When fluorescent PCR amplification before, it is necessary to swab carry out sample process.The method used at present is first, by the swab after sampling, to be put into
In cleaning fluid, with oscillator, vibration is rather planted at a high speed, and bacterium is released from swab head, sample suspension is prepared into, then enter
The processes such as the follow-up nucleic acid extraction of row, such as by the concentration of sample suspension, washing, then plus lysate, boil, high speed centrifugation etc..Most
Certain supernatant is taken to expand template for PCR afterwards.In practical clinical, the method exposes many weak points：(1) nucleic acid
Extraction process complexity is, it is necessary to a variety of instrument and equipments；(2) time-consuming for sample process, and when handling sample, by being enriched with, cleaning,
Multiple steps such as boiling lysis, high speed centrifugation, DNA losses are serious；(3) steps such as the high-temperature heating being related in sample process,
If lid collapses out, can there are Aerosol Pollution, sample losses equivalent risk.
The content of the invention
For the deficiencies in the prior art, B races are quantitatively detected object of the present invention is to provide a kind of fluorescent PCR
Streptococcic Sample pretreatment method and its application, the method original position directly on swab by using B races streptococcus lyases
B races streptococcus is cracked, obtained lysate can be directly used for fluorescent PCR detection, solve sample process process in the prior art
It is cumbersome, time-consuming, sample contamination risk the problems such as.
The technical proposal of the invention is realized in this way：
A kind of fluorescent PCR quantitatively detects the streptococcic sample swab pre-treating method of B races, to swab to be measured on B is added dropwise
Race's streptococcus cracking enzyme liquid, is stored at room temperature after 5~30min, lysate is directly extruded from swab, and lysate is centrifuged, and is taken
Appropriate supernatant expands template as fluorescent PCR.
Further, described B races streptococcus cracking enzyme liquid includes GBS-PlySb enzyme liquids and B races streptococcus lyases is slow
Fliud flushing.
Further, the amino acid sequence of the GBS-PlySb enzymes is：
MATYQEYKSRSNGNAYDIDGSFGAQCWDGYADYCKYLGLPYANCTNTGYARDIWEQRHENGILNYFDEVEVMQAGDV
AIFMVVDGVTPYSHVAIFDSDAGGGYGWFLGQNQGGANGAYNIVKIPYSATYPTAFRPKVFKNAVTVTGNIGLNKGD
YFIDVSAYQQADLTTTCQQAGTTKTIGSSRSYRETGTMTVTVDALNVRRAPNTSGEIVAVYKRGESFDYDTVIIDVN
GYVWVSYIGGSGKRNYVATGATKDGKRFGNAWGTFK。
Further, the concentration of GBS-PlySb enzymes is 20~200 μ g/mL in the GBS-PlySb enzyme liquids.
Further, described B races streptococcus cracking enzyme buffer liquid include Tris buffer solutions, glycine-NaOH buffer,
One or more in HEPES bufferings, MES buffer solutions.
Further, the concentration of B races streptococcus cracking enzyme buffer liquid is 10~100mmol/L, and pH value is 6~8.
A kind of fluorescent PCR quantitatively detects the application of the streptococcic sample swab pre-treating method of B races, and methods described is applied to
Prepare in detection pregnant woman's genital tract or enteron aisle on the streptococcic fluorescent quantificationally PCR detecting kit of B races.
Beneficial effect：Compared with prior art, the method is by using the direct original position on swab of B races streptococcus lyases
B races streptococcus is cracked, obtained lysate can be directly used for fluorescent PCR detection, solve sample process process in the prior art
It is cumbersome, time-consuming, sample contamination risk the problems such as, and using this method fluorescent PCR quantitatively detect B races streptococcus when, streptococcus
More preferably, nucleic acid extraction is more efficient for lytic effect.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the accompanying drawing used required in technology description to be briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, on the premise of not paying creative work, can be with
Other accompanying drawings are obtained according to these accompanying drawings.
Fig. 1 is that fluorescent PCR of the present invention quantitatively detects B races streptococcus method flow diagram；
Fig. 2 is that the lysate handled in the process of the present invention with commercial kit after sample respectively is template, for fluorescence
The comparative result figure of quantitative PCR detection, wherein curve 1 are the amplification curve obtained by the lysisin situ thalline processing on swab,
Curve 2 is the amplification curve obtained by commercial kit processing sample.
Embodiment
Following instance is only used for illustrating certain embodiments of the present invention, and should not be construed as the model of the limitation present invention
Enclose.Present disclosure can be improved from material, method and reaction condition simultaneously, it is all these to improve, all should
Within the spirit and scope for falling into the present invention.
Embodiment 1：Fluorescent PCR is carried out using the inventive method and quantitatively detects the streptococcic specific implementation step of B races
As shown in figure 1, sampled first with swab, to swab to be measured on B races streptococcus cracking enzyme liquid is added dropwise, be stored at room temperature
After 5-30min, directly from swab extrude lysate (such as by swab head be pressed in centrifuge tube wall on), and by the lysate of extrusion from
The heart, finally takes appropriate supernatant to expand template into PCR system for PCR, collects fluorescence signal.Its distinguishing feature is need not be by
Tested bacteria is washed from swab gets off to prepare sample suspension, but lysate is directly dropped in lysisin situ is carried out on swab head.
Wait after the completion of cracking, a certain amount of lysate, of short duration centrifugation, according to wanting for PCR kit for fluorescence quantitative are extruded from swab head
Ask, take a certain amount of supernatant to go to expand into performing PCR as template.This method can be greatly simplified from swab needed for extraction nucleic acid
Operation and step.
In the advantage of this method for convenience of description, specific embodiment below, using the one plant of agalasisa being clinically separated chain
Coccus uses commercialized B clusters streptococcus kit for detecting nucleic acid (fluorescent PCR method) (Thymopetidum Injection thing science as analog sample
(China) Co., Ltd) as control, tested by its specification, while using the inventive method, using can be quick at normal temperatures
Cracking streptococcic streptococcus cracking enzyme reagent kit, (kit is Wuhan Sai Sirui Microbial Technics Ltd product, product
Numbering：It is fast to split 01), carry out contrast test.For the lysate template of acquisition, using commercialized B clusters streptococcus detection of nucleic acids
PCR reagent in kit (fluorescent PCR method) (Triplex Internat Biosciences (China) Co., Ltd.) carries out augmentation detection.
First, prepared by GBS-PlySb enzymes
The preparation of 1.PlyGBS catalytic domain, PlySs2 cell wall binding domain
Complete sequence synthesizes lyases PlyGBS and lyases PlySs2 genetic fragments (the limited public affairs of Nanjing Jin Sirui biotechnologys
Department), composition sequence is fitted into pUC57 plasmids.Using plyGBS genes as template, GBS180-F/GBS180-R is primer, is expanded
PlyGBS catalytic domain GBS180 sequence, using plySs2 genes as template, PlySb-F/PlySb-R is primer, is expanded
PlySs2 cell wall binding domain.Clone's primer used in building and restriction enzyme site information are as follows：
GBS180-F:5-TTAACCATGGGCATGGCTACCTACCAGG-3
GBS180-R:5-TATAGGATCCGATCGTTTTGGTCGTGC-3
PlySb-F:5-TATAGGATCCTCTCGTTCCTATCGCGAG-3
PlySb-R:5-TATACTCGAGTTTAAATGTACCCCAAG-3
GBS180-F primers underscore mark is restriction enzyme Nco I sites, GBS180-R and PlySb-F primers
Underscore mark is restriction enzyme BamHI sites, and PlySb-R primers underscore mark is restriction enzyme XhoI
Site.
The program of PCR amplification gene fragments is as follows：Using 2 μ L genes as template, each μ g of primer 1 that add enter performing PCR amplification,
PCR reaction conditions are：(1) 94 DEG C of pre-degeneration 5min；(2) 94 DEG C denaturation 30sec, 62 DEG C, 45sec, 72 DEG C, 45sec, 30 are followed
Ring；(3) 72 DEG C of extension 10min.
2. soluble-expression GBS-PlySb protein
By above-mentioned fragment with being connected into after corresponding enzymic digestion with postdigestive pET28b (+) carriers of NcoI and XhoI, obtain
To GBS-PlySb expression vector pET28b-GBS-PlySb.Then e. coli bl21 (DE3) is converted.With GBS180-
F/PlySb-R is that primer pair transformant enters performing PCR checking, the amplifiable GBS-PlySb genes for obtaining total length.
3.GBS-PlySb expression and purification
Bacterial strain BL21 (DE3)/pET28b-GBS-PlySb will be expressed to be expressed with 2mM IPTG low temperature inductions.Collect thalline
Ultrasonication afterwards, takes supernatant to cross Ni affinity columns, 250mM eluting peaks is collected, after dialysed overnight in PBS.
Albumen after dialysis can cross HiTrap Q Sepharose FF further across ion-exchange resin purification
Column (GE Healthcare), collects effluent.Effluent is crossed into HiTrap SP Sepharose FF column,
Then 1M NaCl gradient elutions, Fractional Collections eluting peak are used.Active each pipe is mixed after dialysed overnight in PBS, i.e.,
For enzyme liquid after purification.The enzyme liquid is further added after cryoprotector, you can is prepared as freeze-dried powder by freeze-drying, is easy to
The long-time storage under low temperature and normal temperature, keeps enzymatic activity.
2nd, reagent and swab sample prepare
According to streptococcus cracking enzyme reagent kit explanation, resuscitation fluid therein is added in the lyophilized powder bottle of streptococcus and mixes standby
With；
It is standby that cracking enzyme solutions are put into 4 DEG C of refrigerators；
Illustrate according to commercial kit, take out 10x concentrated cleaning solutions and press 1 with sterilizing pure water：9 (volume ratios) carry out dilute
Release, be put into 4 DEG C of refrigerators standby；
Calculate after the reaction tube number n (number of samples+feminine gender, positive control) for needing to carry out, take out GBS-PCR reaction solutions and enzyme
Deng the μ L GBS-PCR reaction solutions of n x 44.3, the μ LTaq enzyme liquids of n x 0.5, the μ L UNG of n x 0.2 are added into a centrifuge tube
And vibrate after mixing, brief centrifugation, dispense into n PCR reaction tube, often the μ L of pipe 45, cover and sample application zone is transferred to after lid, keep away
It is standby that light is put into 4 DEG C of refrigerators.
Analog sample is prepared and swab sampling：The Streptococcusagalactiae liquid preserved on a small quantity is taken, LB cultures are inoculated into super-clean bench
37 DEG C of incubated overnights in base, take in 5mL to 15mL centrifuge tubes, 10000r/min centrifugation 5min, remove supernatant, take the safe general reagents of 5mL
Cleaning fluid in box is resuspended thalline, then is used as with the bacterium solution after 1000 times of cleaning solution dilution and simulates secretion.Take sterile swab (one
Secondary property uses swab (women swab), Jiangsu Kangjian Medical Apparators Co., Ltd.) 2, swab head is immersed in bacterium solution respectively
5s is taken out, and the sterile swab being collected is put back in sterile swab sleeve pipe, completes sampling.
3rd, sample process：
Method A：By the method in commercial kit specification.
1mL cleaning fluids are added in swab sleeve pipe, sample suspension is made in concussion 2min at a high speed with oscillator.Take out all
Sample is put into 1.5mL centrifuge tubes, and 13000r/min centrifugations 5min removes supernatant, adds 50 microlitres of cleaning fluids and is resuspended.In sample tube
The extraction solid content added in 1 pipe kit, be vortexed at a high speed concussion 5min with strength oscillator (U.S. Vortex-Genie).Will
Sample tube after concussion, 95 DEG C of water-bath 2min, immediately after ice bath 2-5min, 13000r/min centrifugation 1min take 5 μ L of supernatant liquid to arrive
Expanded in PCR system.
Method B：Lysisin situ B races streptococcus carries out fluorescent PCR detection on swab
Race streptococcus cracking enzyme liquid GBS-PlySb enzyme liquid 100 μ L of the concentration for 20 μ g/mL is added dropwise on swab head, will wipe
Son is put back in swab sleeve pipe, is stored at room temperature 10min.Swab is taken out from sleeve pipe, swab head is inserted to 1mL aseptic plastic
In pipettor gun head, about 20-50 μ L lysate is squeezed out into 1.5mL sterile centrifugation tube.By lysate 13000r/
Min centrifuges 1min, takes the μ L of supernatant 5 to be expanded for PCR.
4th, fluorescence real-time quantitative PCR is detected：
1. set PCR programs：
37 DEG C, 2min；
94 DEG C, 2min；
Circulation 10 times：94 DEG C, 20s；55 DEG C, 45s；
Circulation 30 times：94 DEG C, 20s；55 DEG C, 45s.
Using Bio-Rad CFX Connect quantitative real time PCR Instruments, GBS detection fluoresceins are FAM.In fourth stage
55 DEG C, the 45s stages collect fluorescence signal, and resulting amplification curve is as shown in Figure 2.
In this example, the Ct values of the amplification curve obtained by the method for commercial kit are in situ on 16.20, cotton swab
The Ct values of amplification curve obtained by cracking B races streptococcus method are 12.8.From the point of view of qualitative results, both are obtained for positive knot
Really, the interpretation of result is not influenceed.But from the point of view of numerically, the extraction efficiency of lysisin situ B races streptococcus method compares business on swab
The Ct values of industry kit method are small, and lytic effect is relatively preferable, and its reason is to use the side in current commercial kit
Method when handling sample, it is necessary to bacterium is released from swab, the process will not 100% release, and after sample suspension
In the processing such as continuous centrifugal concentrating, a part of bacterium may also lose, and cause the nucleic acid-templated number finally given less.And in this hair
In bright method, bacterium is directly cracked, on swab by added B races streptococcus cracking enzyme liquid in the original location without being discharged from swab
Bacterium energy 100% is cracked, and without subsequent processes, the nucleic acid amount discharged is more.Importantly, relative to
The step of sample processing method of commercial kit, swab lysisin situ method, shortens, and easy to operate, the time only needs to 12min
Left and right, and commercial methods need 25min or so.In addition, in whole processing procedure, the inventive method do not need oscillator,
The equipment such as dry bath pot and ice bath, helps to save sample process cost.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
God is with principle, and any modification, equivalent substitution and improvements made etc. should be included in the scope of the protection.
Claims (7)
1. a kind of fluorescent PCR quantitatively detects the streptococcic sample swab pre-treating method of B races, it is characterised in that：Wiped to be measured
B races streptococcus cracking enzyme liquid is added dropwise on son, is stored at room temperature after 5~30min, lysate is directly extruded from swab, and will cracking
Liquid is centrifuged, and is taken appropriate supernatant as fluorescent PCR and is expanded template.
2. a kind of fluorescent PCR according to claim 1 quantitatively detects the streptococcic sample swab pre-treating method of B races, its
It is characterised by：Described B races streptococcus cracking enzyme liquid includes GBS-PlySb enzyme liquids and B races streptococcus cracking enzyme buffer liquid.
3. a kind of fluorescent PCR according to claim 2 quantitatively detects the streptococcic sample swab pre-treating method of B races, its
It is characterised by：The amino acid sequence of the GBS-PlySb enzymes is：
MATYQEYKSRSNGNAYDIDGSFGAQCWDGYADYCKYLGLPYANCTNTGYARDIWEQRHENGILNYFDEVEVMQ
AGDVAIFMVVDGVTPYSHVAIFDSDAGGGYGWFLGQNQGGANGAYNIVKIPYSATYPTAFRPKVFKNAVTVTGNIGL
NKGDYFIDVSAYQQADLTTTCQQAGTTKTIGSSRSYRETGTMTVTVDALNVRRAPNTSGEIVAVYKRGESFDYDTVI
IDVNGYVWVSYIGGSGKRNYVATGATKDGKRFGNAWGTFK。
4. a kind of fluorescent PCR according to claim 2 quantitatively detects the streptococcic sample swab pre-treating method of B races, its
It is characterised by：The concentration of GBS-PlySb enzymes is 20~200 μ g/mL in the GBS-PlySb enzyme liquids.
5. a kind of fluorescent PCR according to claim 2 quantitatively detects the streptococcic sample swab pre-treating method of B races, its
It is characterised by：Described B races streptococcus cracking enzyme buffer liquid includes Tris buffer solutions, glycine-NaOH buffer, HEPES and delayed
One or more in punching, MES buffer solutions.
6. a kind of fluorescent PCR according to claim 2 quantitatively detects the streptococcic sample swab pre-treating method of B races, its
It is characterised by：The concentration of B races streptococcus cracking enzyme buffer liquid is 10~100mmol/L, and pH value is 6~8.
7. a kind of fluorescent PCR as described in claim 1~6 quantitatively detects the streptococcic sample swab pre-treating method of B races
Application, it is characterised in that：The fluorescent PCR quantitatively detects that the streptococcic sample swab pre-treating method of B races is applied to preparation
Detect the streptococcic fluorescent quantificationally PCR detecting kit of B races in pregnant woman's genital tract or enteron aisle.
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| WO2011065854A1 (en) * | 2009-11-24 | 2011-06-03 | Technophage, Investigação E Desenvolvimento Em Biotecnologia, Sa | Enterococcal phage peptides and methods of use thereof |
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