CN107557443A - Group B streptococcus fluorescence quantitative PCR detection kit - Google Patents
Group B streptococcus fluorescence quantitative PCR detection kit Download PDFInfo
- Publication number
- CN107557443A CN107557443A CN201711030047.2A CN201711030047A CN107557443A CN 107557443 A CN107557443 A CN 107557443A CN 201711030047 A CN201711030047 A CN 201711030047A CN 107557443 A CN107557443 A CN 107557443A
- Authority
- CN
- China
- Prior art keywords
- gbs
- actin
- probe
- pcr
- kit
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
the invention belongs to the technical field of bacteria detection, and particularly discloses a group B streptococcus fluorogenic quantitative PCR detection kit, which comprises a group B streptococcus fluorogenic quantitative PCR detection primer and a group B streptococcus fluorogenic quantitative PCR detection probe, wherein the primer and the probe comprise GBS amplification primer pairs GBS-F and GBS-R, GBS detection probe GBS-P, internal reference primer pairs β -actin-F and β -actin-R, and internal reference probe β -actin-P, and nucleotide sequences of GBS-F, GBS-R, GBS-P, β -actin-F, β -actin-R and β -actin-P are respectively and sequentially shown as SEQ ID NO 1-SEQ ID NO 6.
Description
Technical field
The present invention relates to Bacteria Detection technical field, in particular it relates to which a kind of B races streptococcus fluorescence quantitative PCR detection is tried
Agent box.
Background technology
B races streptococcus（Group B streptococcus, GBS）Scientific name Streptococcusagalactiae, it is aerobic Gram-positive chain
Coccus, vagina and rectum are normally lodged in, belong to conditioned pathogen.Because polysaccharide material belongs to antigen construct classification in cell membrane
In B races, therefore general at present Streptococcusagalactiae original name is substituted using B races streptococcus.In the 1970s, GBS has been demonstrate,proved
Actually one of Main Pathogenic Bacteria of perinatal period mother and baby infection, occupies very important status, while it is also in perinatal medicine
Infantile Septicemia and the most common reason of meningitis.The bacterial bearing rate of gravid woman about 5%~30% according to statistics, wherein 40%~70%
Neonate can be passed in the progress of labor.If neonate, with this bacterium, about 1%~3% occurs that early stage is invasive
Infection, wherein thering is 5% can cause death.Since nineteen ninety, the Center for Disease Control（CDC）Start formulation childbirth GBS prevention
Property examination, first time in 1996 are issued《Perinatal period GBS prevention guideline》, and 2 have been carried out to guide in the ensuing more than ten years
Secondary revision, neonate's early onset GBS infection rates are made to reduce 70%.The Center for Disease Control newly revises issue within 2010《Enclose
Term GBS prevention guideline》It is recommended that to all pregnant woman gestation 35~37 weeks when take vagina and rectal secretions to make GBS detections, tie
Fruit positive carries out prophylactic treatment.
The domestic screening method to GBS mainly has bacterial cultivation, amynologic diagnostic method and fluorescent PCR to detect at present
Method.（1）Bacterial cultivation：Due to needing to carry out Bacteria Culture, to identify the positive at least needs 2d, if identification is negative at least
3d is needed, and the immunology sensitiveness of Bacteria Culture can be influenceed by amount of bacteria, if amount of bacteria deficiency is difficult to detect
Go out the GBS positives, therefore phenomenon is failed to pinpoint a disease in diagnosis in clinical detection and is happened occasionally.（2）Immunological method：Although this method specificity is higher,
But sensitiveness is inadequate, recall rate is relatively low, can not meet the requirement of quick early diagnosis.（3）Quantitative fluorescent PCR technology：PCR
Nucleic acid amplification detection technique is after improving from initial etiologic diagnosis transition sxemiquantitative till now, quantitatively examine for many years
It is disconnected, it is constantly progressive with nucleic acid fluorescent labelling techniques, normal PCR technology combination spectral technique grown up it is a kind of it is sensitiveer,
More special, more accurate nucleic acid detection technique, i.e. quantitative fluorescent PCR technology.This method testing result is accurate, and repeatability is high, dirty
Dye rate is low, and generally all auxiliary detection can be carried out by fluorescent quantitative PCR technique in the streptococcic clinical diagnosis of B races.It is but existing
There is fluorescent quantitative PCR technique to still have the deficiencies such as false positive, Stability and veracity are low, sensitivity is low, limit it and facing
Application on bed.Contain complicated ingredient in addition, working as in sample to be detected（Such as whole blood, mucoprotein, urine, excrement, human genome
DNA, clotrimazole suppository, Miconazole suppository, nystatin ointment, imodium or Titanoreine etc.）When, available reagent box
It is often not satisfactory to the accuracy and sensitivity of GBS detections.
The content of the invention
The present invention is in order to overcome the above-mentioned deficiency of prior art, there is provided one group of B races streptococcus fluorescence quantitative PCR detection is drawn
Thing and probe.
It is a further object to provide a kind of B races streptococcus fluorescent quantificationally PCR detecting kit, the kit energy
Realization is to sample DNA specificity and high-sensitivity detection, so that clinical research uses, available for the streptococcic generaI investigation of B races and in advance
It is anti-.
To achieve these goals, the present invention is achieved by following scheme：
One group of B races streptococcus fluorescence quantitative PCR detection primer and probe, including：GBS amplimers are to GBS-F and GBS-R, GBS
Detection probe GBS-P, internal control primer is to β-actin-F and β-actin-R, internal reference probe β-actin-P；Described GBS-F, GBS-
R, GBS-P, β-actin-F, β-actin-R and β-actin-P nucleotide sequence are respectively successively such as SEQ ID NO:1~SEQ
ID NO:Shown in 6.
The present invention is using quantitative fluorescent PCR and binding specificity primer and Taqman probe techniques, and there is provided the house keeper of people
Gene β-actin are used as internal reference, and its amplification situation can prompt the accuracy of clinical sampling and the quality of the DNA profiling of addition,
The situation of DNA cloning in each reacting hole can be monitored.
The nucleotide sequence 5 ' of GBS detection probes GBS-P and internal reference probe the β-actin-P holds equal mark fluorescent report
Group, 3 ' ends mark non-fluorescence quenching group NFQ and are connected with modification group MGB；Wherein, the fluorescent reporter group is selected from
Any of FAM, HEX, VIC, TET, JOE, ROX or CY3.
The quenching group of probe provided by the invention does not produce fluorescence in itself using non-fluorescence quenching group NFQ, NFQ, can
To substantially reduce the intensity of background signal, the accuracy of detection is improved；In addition, modification group MGB is connected with probe（Minor
Groove Binder）, MGB is minor groove binding, can be by the melting temperature of probe（Tm）Value improves 10 DEG C or so, in order to obtain
Same Tm values, MGB probes can be designed to shorter than general T aqMan probes, both reduced synthesis cost, and also caused spy
The success rate of pin design greatly improves；TaqMan-MGB probes can both carry out gene quantification analysis, can realize gene mutation again
（SNP）Analysis.
Preferably, the nucleotide sequence 5 ' of the GBS detection probes GBS-P holds flag F AM fluorescent reporter groups, described interior
Join the probe β-actin-P end of nucleotide sequence 5 ' mark HEX or VIC fluorescent reporter groups.
The present invention is also claimed any of the above-described described primer and probe and is preparing B race's streptococcus detection reagents or reagent
Application in terms of box.
The present invention is also claimed any of the above-described described primer and probe and prepared for B races chain in detection of complex sample
Application in terms of the reagent or kit of coccus.The complex samples refer in testing sample containing whole blood, mucoprotein, urine,
In excrement, human gene group DNA, clotrimazole suppository, Miconazole suppository, nystatin ointment, imodium or Titanoreine
Any one or several impurity.
A kind of B races streptococcus fluorescent quantificationally PCR detecting kit, determine comprising any of the above-described described B races streptococcus fluorescence
Measure PCR detection primers and probe.
Similarly, the kit can be used for detecting the B races streptococcus in above-mentioned complex samples, and therefore, the kit is
The streptococcic kit of B races in a kind of detection of complex sample.
Preferably, the volume ratio of the GBS amplimers pair and GBS detection probes is GBS-F:GBS-R:GBS-P=1:1:
1.5~2.5；The internal control primer pair and the volume ratio of internal reference probe are β-actin-F:β-actin-R:β-actin-P=1:1:
1.5~2.5.
It is highly preferred that the volume ratio of the GBS amplimers pair and GBS detection probes is GBS-F:GBS-R:GBS-P=1:
1:2；The internal control primer pair and the volume ratio of internal reference probe are β-actin-F:β-actin-R:β-actin-P=1:1:2.
The kit also includes DNA extract solutions, PCR reaction solutions, positive control solution and negative controls；The DNA is carried
Liquid is taken by 1 × Tris-EDTA buffer solutions and Triton X-100（Triton X-100）Composition, the two volume ratio according to
Secondary is 99:1；The positive control solution is the colibacillus deactivating containing target fragment or the B races streptococcus culture fluid of inactivation；Institute
Negative controls are stated not contain the streptococcic solution of B races, nuclease free water or physiological saline.
, can further validating DNA extract solution using containing the colibacillus deactivating of target fragment as positive control solution
Validity, avoid because DNA extraction failure caused by false negative；Fracture caused by can effectively avoiding plasmid possible when preserving simultaneously
And degraded, target fragment is better met in quality and quantitative demand.
Preferably, the PCR reaction solutions into being grouped into：PCR- fluorescence probe methods premix system 8~12 μ L；GBS expands
Increase primer pair GBS-F and GBS-R and GBS detection probe GBS-P, volume ratio GBS-F:GBS-R:GBS-P=1:1:1.5~
2.5；For internal control primer to β-actin-F and β-actin-R and internal reference probe β-actin-P, volume ratio is β-actin-F:β-
actin-R:β-actin-P=1:1:1.5~2.5；Add nuclease free water to 20 μ L.
It is highly preferred that the volume ratio of the GBS amplimers pair and GBS detection probes is GBS-F:GBS-R:GBS-P=1:
1:2；The internal control primer pair and the volume ratio of internal reference probe are β-actin-F:β-actin-R:β-actin-P=1:1:2.
Preferably, the reaction condition of the PCR reaction solutions is：
UNG enzyme reactions：50 DEG C 2 minutes；
Taq archaeal dna polymerases activate：95 DEG C 10 minutes；
Denaturation：95 DEG C 15 minutes,
Annealing, extension and fluorescent collecting：60 DEG C 30 seconds, 45 circulation.
Preferably, the PCR- fluorescence probe methods premix system includes Taq archaeal dna polymerases, PCR buffer solutions, dNTPs
（DTTP is all substituted by dUTP）, uracil-N-glycosylase（UNG enzymes）And MgCl2。
Preferably, the application method of the kit, i.e., the method that GBS is detected using the kit, is comprised the following steps：
S1. sample DNA is extracted；
S2. it is loaded：Sample DNA, negative controls, positive control solution are corresponded to respectively and add the PCR reactions equipped with PCR reaction solutions
In pipe/plate, cumulative volume is respectively 20 μ L, obtains corresponding sample P CR reaction systems, negative PCR reaction systems and positive PCR reactions
System；
S3.PCR is expanded：Sample P CR reaction systems, negative PCR reaction systems and positive PCR reaction systems obtained by S2 are put respectively
In on quantitative real time PCR Instrument, response procedures are set, enter performing PCR amplification；Wherein fluorescence detection channel selection be：Select FAM passages
B races streptococcus is detected, selects HEX/VIC Air conduct measurement internal references.
After S4.PCR amplified reactions terminate, judged whether to infect B races streptococcus according to fluorescence curve.
Compared with prior art, the invention has the advantages that：
（1）Kit provided by the present invention devises specific primer and Taqman-MGB probe, detection accuracy is higher,
Sensitivity is stronger, and stability is good, has preferable antijamming capability, can detect GBS from the sample of complicated component.
（2）Kit provided by the present invention is significantly improved using the combination of unique PCR buffer systems with thermal starting enzyme
PCR amplification efficiency, fluorescence signal is stronger, and sensitivity is higher, can detect the template singly copied, obtain wider array of linear model
Enclose, it is quantitatively more accurate to target gene.
（3）Kit provided by the present invention uses dUTP-UNG decontamination systems, greatly reduces due to amplified production
False positive caused by pollution.
Brief description of the drawings
Fig. 1 is the amplification curve that kit A detects sample in embodiment 2, and wherein abscissa represents period, ordinate table
Show fluorescence intensity.
Fig. 2 is the amplification curve of kit A specificity experiments in embodiment 3, and wherein abscissa represents period, ordinate
Represent fluorescence intensity.
Embodiment
The present invention is made with reference to Figure of description and specific embodiment and further being elaborated, the embodiment
It is served only for explaining the present invention, is not intended to limit the scope of the present invention.Test method used in following embodiments is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
Embodiment 1
1st, design 4 groups of GBS amplimers to, GBS detection probes, internal control primer pair and internal reference probe, the nucleotide sequence such as institute of table 1
Show, study the detection case of different primers and probe to complex sample.
The nucleotide sequence of the primer and probe of table 1
2nd, 4 kinds of kits are prepared, every kind of kit only includes one group in 4 groups of primer and probes of A, B, C, D, except primer and spy
Outside pin, remaining composition all same, kit A, B, C, D are respectively obtained.
The preparation process of the kit is as follows：
A kind of B races streptococcus fluorescent quantificationally PCR detecting kit, including one group of B races streptococcus fluorescence quantitative PCR detection primer and
Probe, DNA extract solutions, PCR reaction solutions, positive control solution and negative controls.Wherein described B races streptococcus quantitative fluorescent PCR
Detection primer and probe include：GBS amplimers are to GBS-F and GBS-R, GBS detection probe GBS-P, internal control primer to β-
Actin-F and β-actin-R, internal reference probe β-actin-P.
The nucleotide sequence 5 ' of the GBS detection probes GBS-P holds flag F AM fluorescent reporter groups, the internal reference probe
β-actin-P the end of nucleotide sequence 5 ' mark HEX or VIC fluorescent reporter groups.Wherein, the fluorescent reporter group can also
For with any of TET, JOE, ROX or CY3.
The end of nucleotide sequence 3 ' of GBS detection probes GBS-P and internal reference probe the β-actin-P marks non-fluorescence quench
Go out and group NFQ and be connected with modification group MGB.
The volume ratio of the GBS amplimers pair and GBS detection probes is GBS-F:GBS-R:GBS-P=1:1:2；It is described
Internal control primer pair and the volume ratio of internal reference probe are β-actin-F:β-actin-R:β-actin-P=1:1:2.
The DNA extract solutions are by 1 × Tris-EDTA buffer solutions and Triton X-100（Triton X-100）
Composition, the two volume ratio are followed successively by 99:1；The positive control solution is the colibacillus deactivating containing target fragment or the B of inactivation
Race's streptococcus culture fluid；The negative controls are not contain the streptococcic solution of B races, nuclease free water or physiological saline.
The PCR reaction solutions into being grouped into：PCR- fluorescence probe methods premix system 8~12 μ L；GBS amplimers pair
GBS-F and GBS-R and GBS detection probe GBS-P, volume ratio GBS-F:GBS-R:GBS-P=1:1:2；Internal control primer to β-
Actin-F and β-actin-R and internal reference probe β-actin-P, volume ratio are β-actin-F:β-actin-R:β-actin-P=
1:1:2；Add nuclease free water to 20 μ L.
The reaction condition of the PCR reaction solutions is：
UNG enzyme reactions：50 DEG C 2 minutes；
Taq archaeal dna polymerases activate：95 DEG C 10 minutes；
Denaturation：95 DEG C 15 minutes,
Annealing, extension and fluorescent collecting：60 DEG C 30 seconds, 45 circulation.
The PCR- fluorescence probe methods premix system includes Taq archaeal dna polymerases, PCR buffer solutions, dNTPs（DTTP is whole
Substituted by dUTP）, uracil-N-glycosylase（UNG enzymes）And MgCl2。
The application method of the kit, i.e., the method that GBS is detected using the kit, is comprised the following steps：
S1. sample DNA is extracted；
S2. it is loaded：Sample DNA, negative controls, positive control solution are corresponded to respectively and add the PCR reactions equipped with PCR reaction solutions
In pipe/plate, cumulative volume is respectively 20 μ L, obtains corresponding sample P CR reaction systems, negative PCR reaction systems and positive PCR reactions
System；
S3.PCR is expanded：Sample P CR reaction systems, negative PCR reaction systems and positive PCR reaction systems obtained by S2 are put respectively
In on quantitative real time PCR Instrument, response procedures are set, enter performing PCR amplification；Wherein fluorescence detection channel selection be：Select FAM passages
B races streptococcus is detected, selects HEX/VIC Air conduct measurement internal references.
After S4.PCR amplified reactions terminate, judged whether to infect B races streptococcus according to fluorescence curve.
3rd, detection of 4 kinds of kits to complex sample
Through cultivation test positive and two groups of negative samples, it is 2%~10% that volume ratio is separately added into sample for selection
Whole blood, 0.05%~5% mucoprotein, 2%~10% urine, 2%~10% excrement, 10~20ng/mL human gene group DNA and
10~100 mg/mL Common drugs（Clotrimazole suppository, Miconazole suppository, nystatin ointment, imodium, compound angle dish acid
Ester bolt, woman's health bolt, Compound Dexamethasone Acetate）, GBS detections are carried out to sample according to the application method of mentioned reagent box, every group of sample repeats 10
It is secondary, record testing result.
As a result it is as shown in table 2.Contain whole blood, mucoprotein, urine, excrement, people's base in sample it can be seen from the result of table 2
During because of a group DNA, clotrimazole suppository, Miconazole suppository, nystatin ointment, imodium, Titanoreine, to kit A
Testing result have no significant effect, still there is higher sensitivity and accuracy；But work as in sample and contain woman's health bolt and dermatitis
Usually, testing result is had a significant impact, Detection accuracy can reduce, therefore when using kit A detection GBS, should avoid
Use woman's health bolt and Compound Dexamethasone Acetate.
When the composition of sample to be tested is more complicated（Contain whole blood, mucoprotein, urine, excrement, human gene group DNA, clotrimazole
Suppository, Miconazole suppository, nystatin ointment, imodium, Titanoreine）, kit B, C, D testing result by
Considerable influence, sensitivity and accuracy are relatively low.
The kit interference laboratory test results of table 2
Result above shows that the primer and probe contained in kit A is to the sensitivity detected of GBS in complex samples and accuracy
It is higher, therefore the primer and probe using A groups primer and probe as final reagent preparation box, GBS-F, GBS-R, GBS- in A groups
P, β-actin-F, β-actin-R and β-actin-P nucleotide sequence are respectively successively such as SEQ ID NO:1~SEQ ID NO:
Shown in 6.
Embodiment 2
The B races streptococcus in clinical sample is detected using kit A, step is as follows：
First, sample DNA is extracted
1. the preparation before detection
In advance 15~30min by reagent from -20 DEG C taking-up, defrosting reagent, by each component be vortexed concussion 20~40s, and it is of short duration from
2~6s of the heart；Prepare water-bath, be 100 DEG C by temperature setting.
The preparation of 2.DNA extract solutions
The formula of 1mL DNA extract solutions：1×Tris-EDTA Buffer 990μL+Triton X-100 10μL.
Above-mentioned 1 × Tris-EDTA Buffer and Triton X-100 are mixed after 30~40 DEG C of heating, while magnetic force
15~30min of stirring is completely dissolved to blob of viscose shape.Wherein Triton X-100 are very sticky liquid, should be stirred to fully molten
Solution, it is well mixed, preparation should be completed in 1h.Obtained DNA extract solutions are the liquid of achromatism and clarity, and pH value is 7.5~8.2,
It is stored in cold storage refrigerator（2~8 DEG C）.
3. pregnant 34~37 weeks pregnant woman's genital tracts of extraction or intestinal secretion thing sample DNA
The swab for gathering sample is shaken into rinsing 20~60s, 8000~16000rmp with 1mL physiological saline and centrifuges 2~10min,
Supernatant is abandoned, is precipitated（As unobvious can repeat centrifugation once）30~100 μ L DNA extract solutions are added fully to mix, 80~
100 DEG C of water-baths 5~15min, 8000~16000rmp centrifuge 5~15min, and supernatant is the DNA discharged, draws supernatant extremely
1.5mL centrifuge tubes preserve.
4. extract GBS negative controls
Using nuclease free water as negative controls, the preparation of nuclease free water uses the synergy ultra-pure waters of Millipore companies
Instrument；Take 20~80 μ L GBS blank controls product to add in 1.5mL centrifuge tubes, 1mL is complemented to sterile saline, 8000~
16000rmp centrifuges 2~10min, abandons supernatant, and precipitation adds 30~100 μ L DNA extract solutions and fully mixed, 80~100 DEG C of water
5~15min is bathed, 8000~16000rmp centrifuges 5~15min, and supernatant is the DNA discharged, draws supernatant to 1.5mL centrifuge tubes
Preserve.
5. extract GBS positive reference substances
Positive reference substance is by GBS-cfb clone E. colis stoste, H-actin clone E. colis stoste and physiological saline group
Into its volume ratio is 1:1:8；Take 2~8 μ L GBS positive reference substances to add in 1.5mL centrifuge tubes, supplied with sterile saline
2~10min is centrifuged to 1mL, 8000~16000rmp, abandons supernatant, it is fully mixed that precipitation adds 30~100 μ L DNA extract solutions
Even, 80~100 DEG C of water-bath 5~15min, 8000~16000rmp centrifuge 5~15min, and supernatant is the DNA discharged, in absorption
Preserved to 1.5mL centrifuge tubes clearly.
2nd, PCR reaction solutions are prepared
According to the μ L PCR reaction solutions of 3 recipe configuration of table 500：
The PCR of table 3 reacts formula of liquid（500μL）
Wherein, PCR- fluorescence probe methods premix system concentration be 2 ×, include Taq DNA polymerases, PCR buffer solutions, dNTPs
（DTTP is all substituted by dUTP）, uracil-N-glycosylase（UNG enzymes）And MgCl2.The premixed material has used dUTP-UNG
Decontamination system, dUTP is added in PCR reaction system process for preparation, therefore be formed the amplification production containing dU bases
Thing, and before this product can enter performing PCR reaction in next time, eliminated by the UNG ferment treatments in PCR system, thus effectively gone
Except the residual contamination of PCR primer, the false positive caused by amplified production pollutes is greatly reduced.UNG enzymes are in PCR cycle
In pre-degeneration step can be inactivated, therefore do not interfere with the formation of the new PCR primer of base containing dU.Taq in premixed material
Archaeal dna polymerase is a kind of through chemical modification, new high efficiency thermal starting enzyme, at normal temperatures without polymerase activity, is effectively avoided
Caused non-specific amplification, enzyme swash by primer and template non-specific binding or primer dimer under normal temperature condition
Work must be incubated 10 minutes at 95 DEG C.The combination of unique PCR buffer systems with thermal starting enzyme, significantly improves PCR amplification
Efficiency, fluorescence signal is stronger, and sensitivity is higher, can detect the template singly copied, obtains the wider array of range of linearity, to purpose base
Because quantitative more accurate.
3rd, B races streptococcus is detected
1. 10 cultures of pair clinical acquisitions are detected as negative, 10 gestation pregnant woman's reproductions in 34~37 weeks for cultivating test positive
Road or intestinal secretion thing swab sample, sample DNA is extracted, and prepare positive control and negative control.
2. sample-adding：According to sample quantity n to be measured（Negative control, positive control need to be included）PCR reaction solutions are dispensed, according to 15
~19 μ L/ pipes are dispensed into PCR reaction tubes, are transferred to sample process area；By the sample DNA extracted, negative control, positive control
Respectively it is separately added into PCR reaction tubes, final total volume is 20 μ L, and the of short duration centrifugation several seconds makes all reagents be concentrated to reaction tube bottom
Portion, after covering tightly lid or sealing film, it is transferred to nucleic acid amplification area and enters performing PCR reaction.
3.PCR is expanded：PCR reaction tubes are placed in the sample cell of quantitative real time PCR Instrument, it is positive right to be set by corresponding title
According to, negative control and unknown sample, and sample ID, detection target title are set.
（1）Fluorescence detection channel selects：Select FAM Air conduct measurements GBS；Select HEX/VIC Air conduct measurement internal references.
（2）Response procedures set as follows：
UNG enzyme reactions：50 DEG C 2 minutes；
Taq archaeal dna polymerases activate：95 DEG C 10 minutes；
Denaturation：95 DEG C 15 minutes,
Annealing, extension and fluorescent collecting：60 DEG C 30 seconds, 45 circulation.
（3）It is 20 μ L to select reaction system.
（4）Setting completed, preserves file, runs response procedures.
4. result judgement：After reaction terminates, instrument automatically saves result, and the software carried using instrument is divided automatically
Analysis, or initial value, end value and the threshold line value of regulation baseline are analyzed manually, record sample Ct（That is cycle
Threshold, refer to the fluorescence signal in PCR reaction tubes and reach the cycling numerical value undergone during the threshold value of setting）It is worth result, judges
Testing result.Result judgement standard is as shown in table 4：
The result judgement standard of table 4
Even sample GBS（FAM passages）There is amplification curve, then it is positive for B races streptococcus；If sample GBS（FAM passages）Without amplification
Curve, and internal standard（HEX/VIC passages）There is amplification curve, then it is negative for B races streptococcus；If without amplification curve, testing result
It is invalid.
5. testing result：Testing result is as shown in table 5 and Fig. 1.Kit A identifies 13 parts of positive sample, is reflected than culture
It is higher to determine result positive rate, and the used time is shorter.Result above shows, kit A has that quick, convenient, the degree of accuracy is high, operable
The advantages that property is strong.
The comparison of the B races streptococcus sample reagent box testing result of table 5 and culture testing result
Embodiment 3
Kit A specific detection：
The clinical common all kinds of pathogen B races streptococcus of selection, streptococcus pneumonia, micrococcus scarlatinae, streptococcus thermophilus, variation chain
Coccus, streptococcus pyogenes, lactobacillus acidophilus, lactobacillus reuteri, MRSE, EHEC DH5 α, white read ball
Bacterium, extract DNA according to the method for embodiment 2 respectively, prepare PCR reaction solutions, and according to the streptococcic detection of B races in embodiment 2
Method detects to each bacterial strain respectively, and using B races streptococcus reference culture as positive control.
As a result as shown in Fig. 2 only B group streptococcus reference culture and clinical strains are positive, show that kit A has very
With clinical common all kinds of pathogen cross reaction will not occur for high specificity, kit A.
Embodiment 4
Kit detects GBS contrast test：
To 60 gestation, 34~37 weeks pregnant woman's genital tracts or intestinal secretion thing swab sample of clinical acquisitions, using kit A, purchase
The kit 1 bought [is purchased from Thymopetidum Injection thing science（China）Co., Ltd, state tool note standard 20153400272], the kit of purchase
2 is [sincere purchased from Bo Er（Beijing）Science and Technology Ltd., state's tool note standard 20163402238], carry out GBS according to the method for embodiment 2
Detection, while sample is detected using cultivation, each sample parallel test 3 times, records the test result of sample, such as
Shown in table 6.Kit A detects 19 GBS positives in 60 samples, and compared to the existing kit 1 and 2 in market, it is detected
Positive rate it is higher, specificity and accuracy are more preferable, can detect the GBS of lower concentration, and parallel test result is consistent three times,
The stability of detection is good, therefore kit A can be applied to the streptococcic clinical generaI investigation of B races and prevention well.
The kit A of table 6, kit 1, kit 2 and cultivation testing result
Sequence table
<110>Guangzhou Sai Zhe biotech inc
<120>A kind of B races streptococcus fluorescent quantificationally PCR detecting kit
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213>B races streptococcus (group B streptococcus)
<400> 1
aaatcataaa ctgtctcagg gttgg 25
<210> 2
<211> 25
<212> DNA
<213>B races streptococcus (group B streptococcus)
<400> 2
gtgacaactc cacaagtggt aaatc 25
<210> 3
<211> 32
<212> DNA
<213>B races streptococcus (group B streptococcus)
<400> 3
aactgtctca gggttggcac gcaatgaagt ct 32
<210> 4
<211> 22
<212> DNA
<213>B races streptococcus (group B streptococcus)
<400> 4
tgtgcccatc tacgaggggt at 22
<210> 5
<211> 22
<212> DNA
<213>B races streptococcus (group B streptococcus)
<400> 5
ctccttaatg tcacgcacga tt 22
<210> 6
<211> 28
<212> DNA
<213>B races streptococcus (group B streptococcus)
<400> 6
ccctccccca tgccatcctg cgtctgga 28
Claims (10)
1. one group of B races streptococcus fluorescence quantitative PCR detection primer and probe, it is characterised in that including：GBS amplimers pair
GBS-F and GBS-R, GBS detection probe GBS-P, internal control primer is to β-actin-F and β-actin-R, internal reference probe β-actin-
P；GBS-F, GBS-R, GBS-P, β-actin-F, β-actin-R and β-actin-P nucleotide sequence are distinguished successively such as
SEQ ID NO:1~SEQ ID NO:Shown in 6.
2. primer and probe according to claim 1, it is characterised in that the GBS detection probes GBS-P and internal reference probe
β-actin-P nucleotide sequence 5 ' holds equal mark fluorescent reporter group, and 3 ' ends mark non-fluorescence quenching group NFQ and connection
There is modification group MGB；Wherein, any of the fluorescent reporter group in FAM, HEX, VIC, TET, JOE, ROX or CY3
Kind.
3. primer and probe according to claim 2, it is characterised in that the nucleotides sequence of the GBS detection probes GBS-P
Row 5 ' hold flag F AM fluorescent reporter groups, and the end of nucleotide sequence 5 ' of the internal reference probe β-actin-P marks HEX or VIC glimmering
Light reporter group.
4. any described primer and probe of claims 1 to 3 answering in terms of B races streptococcus detection reagent or kit is prepared
With.
5. any described primer and probe of claims 1 to 3 is being prepared for the streptococcic reagent of B races in detection of complex sample
Or the application in terms of kit.
6. application according to claim 5, it is characterised in that the complex samples refer in testing sample containing whole blood,
Mucoprotein, urine, excrement, human gene group DNA, clotrimazole suppository, Miconazole suppository, nystatin ointment, imodium or compound
Any of angle dish acid esters bolt or several impurity.
A kind of 7. B races streptococcus fluorescent quantificationally PCR detecting kit, it is characterised in that the kit include claim 1~
3 any described primer and probes.
8. kit according to claim 7, it is characterised in that the GBS amplimers pair and the body of GBS detection probes
Product ratio is GBS-F:GBS-R:GBS-P=1:1:1.5~2.5；The internal control primer pair and the volume ratio of internal reference probe be β-
actin-F:β-actin-R:β-actin-P=1:1:1.5~2.5.
9. kit according to claim 7, it is characterised in that the kit also includes DNA extract solutions, PCR reacts
Liquid, positive control solution and negative controls；The DNA extract solutions are by 1 × Tris-EDTA buffer solutions and Triton X-100 groups
Into the two volume ratio is followed successively by 99:1；The positive control solution is the B races of the colibacillus deactivating containing target fragment or inactivation
Streptococcus culture fluid；The negative controls are not contain the streptococcic solution of B races, nuclease free water or physiological saline；
The PCR reaction solutions into being grouped into：PCR- fluorescence probe methods premix system 8~12 μ L；GBS amplimers are to GBS-
F and GBS-R and GBS detection probe GBS-P, volume ratio GBS-F:GBS-R:GBS-P=1:1:1.5~2.5；Internal control primer pair
β-actin-F and β-actin-R and internal reference probe β-actin-P, volume ratio are β-actin-F:β-actin-R:β-actin-P
=1:1:1.5~2.5；Add nuclease free water to 20 μ L；The reaction condition of the PCR reaction solutions is：50 DEG C 2 minutes；95 DEG C 10 points
Clock；95 DEG C 15 minutes, 60 DEG C 30 seconds, 45 circulation.
10. kit according to claim 9, it is characterised in that the PCR- fluorescence probe methods premix system includes Taq
Archaeal dna polymerase, PCR buffer solutions, dNTPs, UNG enzyme and MgCl2；DTTP in the dNTPs is all substituted by dUTP.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711030047.2A CN107557443A (en) | 2017-10-27 | 2017-10-27 | Group B streptococcus fluorescence quantitative PCR detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711030047.2A CN107557443A (en) | 2017-10-27 | 2017-10-27 | Group B streptococcus fluorescence quantitative PCR detection kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107557443A true CN107557443A (en) | 2018-01-09 |
Family
ID=61032012
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711030047.2A Pending CN107557443A (en) | 2017-10-27 | 2017-10-27 | Group B streptococcus fluorescence quantitative PCR detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107557443A (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108441542A (en) * | 2018-04-17 | 2018-08-24 | 广州市妇女儿童医疗中心 | Streptococcusagalactiae visualization kit for detecting nucleic acid based on recombinase polymeric enzymatic amplification technology and method |
| CN108531627A (en) * | 2018-04-27 | 2018-09-14 | 杭州德同生物技术有限公司 | One kind is for detecting the streptococcic RPA fluorescent quantitations primer pair of B races, probe, kit and detection method |
| CN108866164A (en) * | 2018-07-05 | 2018-11-23 | 南方医科大学 | The streptococcic detection method of B race and kit |
| CN109136395A (en) * | 2018-08-27 | 2019-01-04 | 郑州安图生物工程股份有限公司 | A kind of B race streptococcus kit for detecting nucleic acid |
| CN109825616A (en) * | 2019-03-18 | 2019-05-31 | 广州微远基因科技有限公司 | For detecting streptococcic primer sets of B race and application thereof |
| CN111850143A (en) * | 2020-04-21 | 2020-10-30 | 天津金域医学检验实验室有限公司 | GBS DNA nucleic acid detection method |
| CN112226522A (en) * | 2019-07-15 | 2021-01-15 | 中生方政生物技术股份有限公司 | Primer, probe, kit and detection method for detecting group B streptococcus |
| CN112609012A (en) * | 2020-12-10 | 2021-04-06 | 北京华瑞康源生物科技发展有限公司 | Real-time fluorescent PCR (polymerase chain reaction) primer, probe and kit for detecting group B streptococcus nucleic acid |
| CN112654721A (en) * | 2018-06-13 | 2021-04-13 | 简·探针公司 | Compositions and methods for detecting group B streptococcal nucleic acids |
| CN112824541A (en) * | 2019-11-21 | 2021-05-21 | 利多(香港)有限公司 | Methods, oligonucleotides and kits for detecting streptococci |
| CN116590439A (en) * | 2023-04-21 | 2023-08-15 | 四川恒元信生物医药科技有限公司 | RT-PCR detection primer for group B streptococcus 16S rRNA, probe, kit and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1712546A (en) * | 2004-06-25 | 2005-12-28 | 吴秉铨 | Primer for detecting B-family chain coccus special sequence, short-handled gyrate probe, reagent box and method thereof |
| CN103409509A (en) * | 2013-07-02 | 2013-11-27 | 江苏硕世生物科技有限公司 | Group B streptococcus fluorescence PCR detection kit |
| CN105400883A (en) * | 2015-12-15 | 2016-03-16 | 武汉海吉力生物科技有限公司 | Primers, Taqman probe and kit for detection of STMN1 mRNA expression quantity |
| CN106048051A (en) * | 2016-07-25 | 2016-10-26 | 泰普生物科学（中国）有限公司 | Candida krusei fluorescence PCR detection kit |
| CN106148506A (en) * | 2015-04-27 | 2016-11-23 | 重庆贝羿生物科技有限公司 | The antenatal detection kit of a kind of B race streptococcus quantitative fluorescent PCR and application thereof |
| CN106967839A (en) * | 2017-05-27 | 2017-07-21 | 济凡生物科技（北京）有限公司 | Primer, probe, kit and the method for B races streptococcus fluorescence quantitative PCR detection |
-
2017
- 2017-10-27 CN CN201711030047.2A patent/CN107557443A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1712546A (en) * | 2004-06-25 | 2005-12-28 | 吴秉铨 | Primer for detecting B-family chain coccus special sequence, short-handled gyrate probe, reagent box and method thereof |
| CN103409509A (en) * | 2013-07-02 | 2013-11-27 | 江苏硕世生物科技有限公司 | Group B streptococcus fluorescence PCR detection kit |
| CN106148506A (en) * | 2015-04-27 | 2016-11-23 | 重庆贝羿生物科技有限公司 | The antenatal detection kit of a kind of B race streptococcus quantitative fluorescent PCR and application thereof |
| CN105400883A (en) * | 2015-12-15 | 2016-03-16 | 武汉海吉力生物科技有限公司 | Primers, Taqman probe and kit for detection of STMN1 mRNA expression quantity |
| CN106048051A (en) * | 2016-07-25 | 2016-10-26 | 泰普生物科学（中国）有限公司 | Candida krusei fluorescence PCR detection kit |
| CN106967839A (en) * | 2017-05-27 | 2017-07-21 | 济凡生物科技（北京）有限公司 | Primer, probe, kit and the method for B races streptococcus fluorescence quantitative PCR detection |
Non-Patent Citations (3)
| Title |
|---|
| CAMPOS,J.等: "《CP020432.1-GenBank》", 29 March 2017 * |
| 徐力等: "《分子生物学检验与新技术分册》", 31 January 2007 * |
| 温旺荣等: "《临床分子诊断学》", 30 April 2015 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108441542A (en) * | 2018-04-17 | 2018-08-24 | 广州市妇女儿童医疗中心 | Streptococcusagalactiae visualization kit for detecting nucleic acid based on recombinase polymeric enzymatic amplification technology and method |
| CN108531627A (en) * | 2018-04-27 | 2018-09-14 | 杭州德同生物技术有限公司 | One kind is for detecting the streptococcic RPA fluorescent quantitations primer pair of B races, probe, kit and detection method |
| CN112654721A (en) * | 2018-06-13 | 2021-04-13 | 简·探针公司 | Compositions and methods for detecting group B streptococcal nucleic acids |
| CN108866164A (en) * | 2018-07-05 | 2018-11-23 | 南方医科大学 | The streptococcic detection method of B race and kit |
| CN109136395A (en) * | 2018-08-27 | 2019-01-04 | 郑州安图生物工程股份有限公司 | A kind of B race streptococcus kit for detecting nucleic acid |
| CN109825616A (en) * | 2019-03-18 | 2019-05-31 | 广州微远基因科技有限公司 | For detecting streptococcic primer sets of B race and application thereof |
| CN112226522A (en) * | 2019-07-15 | 2021-01-15 | 中生方政生物技术股份有限公司 | Primer, probe, kit and detection method for detecting group B streptococcus |
| CN112824541A (en) * | 2019-11-21 | 2021-05-21 | 利多(香港)有限公司 | Methods, oligonucleotides and kits for detecting streptococci |
| CN111850143A (en) * | 2020-04-21 | 2020-10-30 | 天津金域医学检验实验室有限公司 | GBS DNA nucleic acid detection method |
| CN111850143B (en) * | 2020-04-21 | 2023-02-07 | 天津金域医学检验实验室有限公司 | GBS DNA nucleic acid detection method |
| CN112609012A (en) * | 2020-12-10 | 2021-04-06 | 北京华瑞康源生物科技发展有限公司 | Real-time fluorescent PCR (polymerase chain reaction) primer, probe and kit for detecting group B streptococcus nucleic acid |
| CN116590439A (en) * | 2023-04-21 | 2023-08-15 | 四川恒元信生物医药科技有限公司 | RT-PCR detection primer for group B streptococcus 16S rRNA, probe, kit and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107557443A (en) | Group B streptococcus fluorescence quantitative PCR detection kit | |
| CN103361434B (en) | Multiple fluorescence PCR detection kit and detection method for clostridium difficile toxin genes | |
| CN102112625B (en) | Method of distinguishing inflammatory pathogen causing acute respiratory infection | |
| CN107937613A (en) | Respiratory system common seven kinds of influenza virus pathogen real-time fluorescence multiple PCR primer probes and kit | |
| CN104846097B (en) | Helicobacter pylori parting and drug resistant mutant genes detection kit | |
| Kimura et al. | Rapid and reliable loop-mediated isothermal amplification method for detecting Streptococcus agalactiae | |
| CN103409509A (en) | Group B streptococcus fluorescence PCR detection kit | |
| CN110373485A (en) | A kind of ureaplasma urealyticum, three joint inspection kit of chlamydia trachomatis and gonococcus | |
| CN105779625B (en) | It is a kind of to detect that Streptococcus suis is universal and streptococcus suis 2-type double fluorescent quantitative PCR primer, kit and method simultaneously | |
| CN109988854A (en) | For detecting oligonucleotide combinatorial, method and the kit of pathogenic Bao Te bacillus | |
| CN106967839A (en) | Primer, probe, kit and the method for B races streptococcus fluorescence quantitative PCR detection | |
| US6355435B1 (en) | Methods for detecting and enumerating Campylobacter jejuni in environmental samples and for identifying antibiotic-resistant strains | |
| CN105368946A (en) | Group B streptococcus nucleic acid assay kit based on PCR (polymerase chain reaction) fluorescent probe method | |
| CN101381767B (en) | Universal real time fluorescent PCR detection method of trichinella | |
| CN104342487B (en) | Mycoplasma nucleic acid constant-temperature amplification method | |
| CN105567802A (en) | Fluorescence PCR (polymerase chain reaction) detection kit for Chlamydia pneumoniae | |
| CN110734988A (en) | methicillin-resistant staphylococcus aureus (MRSA) nucleic acid isothermal amplification method | |
| RU2435853C1 (en) | TEST SYSTEM FOR QUANTITATIVE DETERINATION OF Streptococcus agalactiae IN BIOLOGICAL MATERIAL | |
| CN111647686A (en) | Enterovirus EV71, CA16 and EV universal nucleic acid detection reagent | |
| CN110468223A (en) | Primer, probe, kit and the method for mycoplasma pneumoniae and Bao Te Pseudomonas detection of nucleic acids based on dUTP/UNG method | |
| CN110656188A (en) | Primer and/or probe composition for detecting bacillus causing bloodstream infection and application thereof | |
| CN103725761B (en) | Group B streptococcus (GBS) nucleic acid detection kit and detection method | |
| CN104561341B (en) | Primers and kit for detecting macrolide drug-resistance genes of bacteria | |
| CN114438238A (en) | Primer for detecting infectious endocarditis pathogen and digital PCR kit | |
| Kukla et al. | Improved laboratory diagnostics of Streptococcus pneumoniae in respiratory tract samples through qPCR |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180109 |
|
| RJ01 | Rejection of invention patent application after publication |