CN106191286A - Brucellar detection method, test kit and application thereof - Google Patents
Brucellar detection method, test kit and application thereof Download PDFInfo
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- CN106191286A CN106191286A CN201610617030.6A CN201610617030A CN106191286A CN 106191286 A CN106191286 A CN 106191286A CN 201610617030 A CN201610617030 A CN 201610617030A CN 106191286 A CN106191286 A CN 106191286A
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- brucella
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Abstract
This application discloses brucellar detection method, comprise the steps: to use centrifugal column method to obtain brucella DNA from serum specimen, it is thus achieved that sample to be tested；Pcr amplification reaction；Use quantitative real time PCR Instrument detection reaction result, be set as the fluorescein that Taqman fluorescence probe BKV FP 5 ' end fluorophor is corresponding during fluorescence signal collection, collect fluorescence signal at 60 DEG C；To interpretation of result.The application also provides for the brucellar test kit of a kind of detection.The detection method of the present invention, test kit sensitivity are high, and minimum detection can reach 1000copies/mL；Meanwhile, the specificity of the detection method of the present invention is fine, other the antibacterial generation cross reaction of getting along well, such as escherichia coli, Salmonella enteritidis, hepatitis B virus, ebb virus, human cytomegalic inclusion disease virus etc..
Description
Technical field
The present invention relates to the detection method of a kind of antibacterial, test kit and application thereof, specifically, relate to a kind of brucellar
Detection method, test kit and application thereof.
Background technology
Brucellosis (Brucellosis) is called for short brucellosis, also known as sea remittent fever, Malta fever or brucellosis in underground heat,
It is commonly called as " sluggard is sick ", is to be infected by the bacterial a kind of Zoonosis of endotrophic Brucella (Brucella)
Allergic disease, brucellosis is distributed widely in all over the world.
Be currently known more than 60 kind of domestic animal, poultry, wild animal are brucellar hosts.The source of infection relevant with the mankind
Mainly sheep, cattle and pig, next to that dog.The secretions of ill domestic animal, Excreta, apoblema and breast class contain a large amount of pathogenic bacteria, animal
Between propagate, cause and carry disease germs or fall ill.Animal, the milk product of edible brucella pollution and the reality that people is infected by contact brucellosis
Testing the circulation ways such as room contact causes cloth bacterium to infect.
Brucella is bead bacillus, Gram-negative, and atrichia is formed without spore, general without pod membrane, virulence
Bacterial strain can have meagre pod membrane.After this bacterium invades human body, arrive lymph node with lymph fluid, swallowed by phagocyte.Such as cloth bacterium not
Killed by phagocyte, then antibacterial is at intracellular growth and breeding, forms local primary lesion.This stage has person to be that lymph source property is moved
Moving the stage, be equivalent to incubation period, incubation period is 7-60 days, average two weeks, and small number of patients was up to several months or more than 1 year.Antibacterial exists
In phagocyte, amount reproduction causes phagocyte to rupture, and a large amount of antibacterials enter lymph fluid and blood circulation forms bacteremia therewith.
In blood, antibacterial is swallowed by hemophagocyte again, and with blood flow band to whole body, liver, spleen, lymph node, bone marrow etc.
Mononuclear phagocyte system in breeding, form multiple focus, cause the most not only have bacteremia, septicemia, and
Also has toxemic performance.After the regular period, the bacterial growth breeding of focus of infection enters blood again, causes palindromia.Tissue
Pathology damage is extensive.Clinical manifestation also just variation.So repeatedly becoming chronic infection, chronic phase, invades and spinal column and high point more
Joint, show as long-term fever (including low grade fever), hyperhidrosis, arthralgia hold concurrently or the suspect such as liver, spleen, lymph node and swollen testis and
Sign.
Brucella common detection methods mainly has hemoculture method and rose bengal precipitation test (RBPT), serum agglutination
The immunological methods such as test (SAT), complement fixation test (CFT), elisa (ELISA).Hemoculture separates
Remain " goldstandard " of brucellosis diagnosis to brucella, but the sensitivity of brucella separation and Culture is low, time-consuming, take
Power, and there is bigger biological safety harm.Immunological method has been used to the monitoring of brucellosis and popular at present
In sick investigation, but owing to infecting in early days, body does not also produce the antibody of associated antibodies or generation and also not up to detects
Limit, be easy to get false negative result；And between brucella and some antibacterial, there is cross reaction, the false positive that is easy to get is tied
Really, have impact on the specificity of detection.
Along with the development of Protocols in Molecular Biology, PCR method has been used for auxiliary diagnosis and the pathogen of brucellosis
Detection and identification in.But regular-PCR also exists complex operation step, easily cause pollution, it is difficult to accurate quantitative analysis detection etc. are no
Foot.Therefore, in Infected with Brucella is in early days or to pharmaceutical biological product during brucella detection, be badly in need of setting up a kind of quickly,
Special, the sensitive and brucellar method of high throughput testing.
Fluorescent quantitative PCR technique be a kind of integrate round pcr, fluorescence signal detection and the nucleic acid quantification of data analysis
Detection technique.Quantitative fluorescent PCR employs specific probe, it is possible to target sequence is carried out specific identification, has primer
Probe two ore control, and combine fluorescent labelling techniques, laser measuring technology, digital imaging technology, carry out in the augmentation index phase
Quantitatively, there is the features such as the highest specificity, susceptiveness, accuracy and false positive rate be low.
This product uses Real-Time Fluorescent Quantitative PCR Technique, the brucella in detection by quantitative human serum sample
DNA.With brucella conserved genetic sequences for testing goal fragment, this detection must not individually be evaluated as conditions of patients and refer to
Mark, it is necessary to combine clinical manifestation and conditions of patients is evaluated, also by patient Bu Lu by other laboratory parameters
Salmonella DNA baseline values and the monitoring of situation of change, for assessing response and the therapeutic effect monitoring of anti-bacterial therapies.This reagent
It is not used as brucellar blood source examination.
At present, domestic and international clinical practice does not the most use fluorescence quantifying PCR method to detect brucella.Use the present invention
In be previously mentioned brucella nucleic acid detection kit (PCR-fluorescence probe method), due to use centrifugal column method, can effectively carry out
Nucleic acid extraction, in this test kit, plasmid standards for quantitation that we use and the positive that positive quality control product is inactivated strain or inactivation
Sample, can extract with sample to be tested simultaneously and expand, such that it is able to complete monitoring specimen extracts amplification.
Summary of the invention
The subject matter that the application solves is to provide a kind of brucellar detection method and test kit, cannot be real with solution
Existing technical problem.
In order to solve above-mentioned technical problem, the invention discloses brucellar detection method, comprise the steps:
Sample to be tested obtains:
Centrifugal column method is used to obtain brucella DNA from serum specimen, it is thus achieved that sample to be tested；
Pcr amplification reaction:
The brucella DNA of extraction is added in PCR reaction mixture, carries out PCR amplification；
Described PCR reaction mixture, including: PCR reactant liquor, brucella primed probe mixed liquor,
Described brucella primed probe mixed liquor, including, forward primer Bcsp-F, downstream primer Bcsp-R, Taqman
Fluorescent probe Bcsp-FP, purified water；
Described forward primer Bcsp-F comprises the base sequence as shown in SEQ ID NO:1；
Described downstream primer Bcsp-R comprises the base sequence as shown in SEQ ID NO:2；
Described Taqman fluorescence probe Bcsp-FP comprises the base sequence as shown in SEQ ID NO:3, and described Taqman is glimmering
Light probe BKV-FP5 ' end has fluorescent reporter group, 3 ' ends to have fluorescent quenching group；
Result detects:
Use quantitative real time PCR Instrument detection reaction result, during fluorescence signal collection, be set as Taqman fluorescence probe BKV-
The fluorescein that FP 5 ' end fluorophor is corresponding, collects fluorescence signal at 60 DEG C；To interpretation of result.
Further, the condition of described pcr amplification reaction is: 37 DEG C of 2min；95℃3min；94 DEG C of 15s, 60 DEG C of 35s, 10
Individual circulation；94 DEG C of 15s, 60 DEG C of 35s fluorescence, 35 circulations；25℃1min.
Further, described BKV primed probe mixed liquor, also include: internal standard forward primer, internal standard downstream primer, internal standard
Probe.
Further, described pcr amplification reaction, also include: the Brucella feminine gender quality-control product that be arranged in parallel, Brucella face
Boundary's positive quality control product, Brucella positive quality control product and Brucella plasmid standards for quantitation I～IV reaction group, described Brucella is cloudy
Property control product are negative serum, the critical positive quality control product of described Brucella, Brucella positive quality control product template to be measured for going out
Live strain or inactivation positive sample, the template to be measured of described Brucella plasmid standards for quantitation I～IV is inactivated strain or inactivation sun
Property specimen.
Further, the fluorescent reporter group 6-CF 5(6)-Carboxyfluorescein of described Taqman fluorescence probe BKV-FP, chlordene-
6-methyl fluorescein, VIC fluorescent dye, four chloro-6-CF 5(6)-Carboxyfluorescein, carboxy-X-rhodamine, 6-carboxyl tetramethylrhodamine,
Sulforhodamine, 6-carboxyl-4 ', 5-bis-chloro-2 ', 7 '-dimethoxyfluorescein succinimide ester, Hua Jing 3, Hua Jing 5 or flower
At least one in cyanines 5.5；(4-dimethylamino benzene is even selected from 6-carboxyl tetramethylrhodamine, 4-for described fluorescent quenching group
Nitrilo) benzoic acid, black hole quencher 1, at least one in black hole quencher 2 or black hole quencher 3.
Further, described PCR reactant liquor, including the anti-pollution system of UDG enzyme, the described anti-pollution system of UDG enzyme, including:
UDG enzyme and dUTP.
The application also provides for detecting brucellar test kit, it is characterised in that include that the upstream described in claim 1 is drawn
Thing Bcsp-F, downstream primer Bcsp-R, Taqman fluorescence probe Bcsp-FP, purified water；
Described forward primer Bcsp-F comprises the base sequence as shown in SEQ ID NO:1；
Described downstream primer Bcsp-R comprises the base sequence as shown in SEQ ID NO:2；
Described Taqman fluorescence probe Bcsp-FP comprises the base sequence as shown in SEQ ID NO:3.
Further, this test kit also includes: internal standard forward primer, internal standard downstream primer, internal standard probe, Brucella are cloudy
Property quality-control product, Brucella positive quality control product, the critical positive quality control product of Brucella and Brucella plasmid standards for quantitation I～IV,
Described Brucella feminine gender quality-control product is negative serum, the critical positive quality control product of described Brucella, Brucella positive quality control
Product are inactivated strain or inactivation specimen, and described Brucella plasmid standards for quantitation I～IV is inactivated strain or inactivation positive sample, institute
State Brucella plasmid standards for quantitation I～IV, including Brucella plasmid standards for quantitation I, Brucella plasmid standards for quantitation II,
Brucella plasmid standards for quantitation III, Brucella plasmid standards for quantitation IV.
Further, described test kit includes: brucella primed probe mixed liquor, PCR reactant liquor, Brucella are quantitative
Standard substance, Brucella positive quality control product, the critical positive quality control product of Brucella, Brucella feminine gender quality-control product, Brucella
Internal standard quality-control product, each constituent content is:
The brucellar test kit of detection that the application provides is for brucellar detection.
Compared with prior art, brucellar detection method of the present invention, test kit, reach following effect:
1. test kit involved in the present invention arranges critical positive quality control product, positive quality control product, plasmid standards for quantitation equal
For inactivated strain or inactivation specimen, participate in nucleic acid extraction and amplification, the truest reflection nucleic acid extraction and amplification.
2. the present invention uses uridnine enzyme (UDG) anti-pollution system, and the heated U-DNA that can optionally degrade, to prevent
The previously pollution of pcr amplification product.
3. the present invention uses internal standard system of quality control, is used for monitoring reaction system restraining factors that may be present.Internal standard template
With target gene without homology, what internal standard probe selected is another sense channel not conflicted with target gene probe.
4. the detection method of the present invention, test kit sensitivity height, minimum detection can reach 1000copies/mL；Meanwhile, originally
The specificity of the detection method of invention is fine, other the antibacterial generation cross reaction of getting along well, such as escherichia coli, enteritis sramana
Bacterium, hepatitis B virus, ebb virus, human cytomegalic inclusion disease virus etc..
5. the detection method reaction of the present invention quickly, i.e. can get PCR reaction result, and cost for general 1.5 to 2 hours
Low, non-false positive, is suitable for larger scale clinical and carries out.Thus realize to brucellar quickly, the most quantitatively examine
Survey, thus can guarantee that case diagnosis and treatment timely and therapeutic effect monitoring.
Accompanying drawing explanation
Accompanying drawing described herein is used for providing a further understanding of the present invention, constitutes the part of the present invention, this
Bright schematic description and description is used for explaining the present invention, is not intended that inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 is brucella qualitative detection result figure described in the embodiment of the present invention 2；
Fig. 2 is brucella quantitative measurement standard product testing result figure described in the embodiment of the present invention 3；
Fig. 3 is brucella quantitative measurement standard product described in the embodiment of the present invention 3 and testing sample testing result figure.
Detailed description of the invention
As employed some vocabulary in the middle of description and claim to censure specific components.Those skilled in the art should
It is understood that hardware manufacturer may call same assembly with different nouns.This specification and claims are not with name
The difference claimed is used as distinguishing the mode of assembly, but is used as the criterion distinguished with assembly difference functionally.As logical
" comprising " mentioned in the middle of piece description and claim is an open language, therefore should be construed to " comprise but do not limit
In "." substantially " referring in receivable range of error, those skilled in the art can solve described in the range of certain error
Technical problem, basically reaches described technique effect.Description subsequent descriptions is to implement the better embodiment of the application, so described
For the purpose of description is the rule so that the application to be described, it is not limited to scope of the present application.The protection domain of the application
When being as the criterion depending on the defined person of claims.
Below in conjunction with accompanying drawing, the application is described in further detail, but not as the restriction to the application.Hereafter in order to
Narration is convenient, hereinafter alleged " left " " right " " on " D score etc. is left and right with accompanying drawing itself, upper and lower etc., and direction is consistent.
Embodiment 1
The present embodiment provides the method that sample to be tested obtains, and sample to be tested nucleic acid extraction can use the centrifugal column of commercialization
Extraction method, the magnetic bead extraction method of commercialization or the boiling lysis of commercialization.
The present invention provides employing centrifugal column method to obtain brucella DNA from serum specimen, it is thus achieved that the method for sample to be tested.
Concrete grammar is as follows:
1) use in forward direction buffer GD and add 17mL dehydrated alcohol, in rinsing liquid PW, add 60mL dehydrated alcohol, shake
Even.
2) taking test serum sample 1ml, 13,000rpm are centrifuged 10min, and exhaust supernatant as far as possible.
3) adding 200 μ l buffer GA in bacterial sediment, vibration to thalline thoroughly suspends.
4) in pipe, 20 μ l Proteinase K, 5 μ l Brucella internal standard quality-control product solution are added, mixing.
5) adding 220 μ l buffer GB, vibrate 15sec, places 10min for 70 DEG C, and solution strain is limpid, and brief centrifugation is to go
The globule except cap wall.
6) add 220 μ l dehydrated alcohol, fully vibration mixing 15sec, now it is possible that flocculent deposit, brief centrifugation with
Remove the globule of cap wall.
7) previous step gained solution and flocculent deposit are all added in an adsorption column CB3 that (adsorption column puts into collecting pipe
In), 12,000rpm (～13,400 × g) centrifugal 30sec, outwells waste liquid, is put in collecting pipe by adsorption column CB3.
8) in adsorption column CB3, add 500 μ l buffer GD (the most first check whether before use and added dehydrated alcohol),
12,000rpm (～13,400 × g) centrifugal 30sec, outwells waste liquid, is put in collecting pipe by adsorption column CB3.
9) in adsorption column CB3, add 600 μ l rinsing liquid PW (the most first check whether before use and added dehydrated alcohol),
12,000rpm (～13,400 × g) centrifugal 30sec, outwell waste liquid, adsorption column CB3 puts in collecting pipe.
10) repetitive operation step is 9..
11) adsorption column CB3 is put back in collecting pipe, 12,000rpm (～13,400 × g) centrifugal 2min, outwells waste liquid.Will
Adsorption column CB3 is placed in room temperature and places several minutes, thoroughly to dry rinsing liquid remaining in adsorbing material.
12) being proceeded to by adsorption column CB3 in a clean centrifuge tube, to the middle part of adsorbed film, unsettled dropping 50 μ l washes
De-buffer TE, room temperature placement 2-5min, 12,000rpm (～13,400 × g) centrifugal 2min, collects centrifuge tube by solution
In, it is and extracts nucleic acid DNA.
Embodiment 2
Brucellar qualitative detection
The present embodiment 2 provides a kind of brucellar qualitative checking method:
1, brucellar pcr amplification reaction
1) the pcr amplification reaction agents useful for same of brucella DNA and composition
(1) brucella primed probe mixed liquor:
Brucella primed probe mixed liquor particular make-up composition is forward primer Bcsp-F (50 μMs) 0.6 μ L, and downstream is drawn
Thing Bcsp-R (50 μMs) 0.6 μ L, Taqman fluorescence probe Bcsp-FP (20 μMs) 0.5 μ L, purified water 3.3 μ L.
The preferably component of brucella primed probe mixed liquor is: forward primer Bcsp-F (50 μMs) 0.6 μ L, downstream primer
Bcsp-R (50 μMs) 0.6 μ L, Taqman fluorescence probe Bcsp-FP (20 μMs) 0.5 μ L, internal standard forward primer (20 μMs) 0.5 μ L, interior
Mark downstream primer (20 μMs) 0.5 μ L, internal standard probe (10 μMs) 0.375 μ L, purified water 1.925 μ L.
Described forward primer Bcsp-F comprises the base sequence as shown in SEQ ID NO:1；
Described downstream primer Bcsp-R comprises the base sequence as shown in SEQ ID NO:2；
Described Taqman fluorescence probe Bcsp-FP comprises the base sequence as shown in SEQ ID NO:3, and described Taqman is glimmering
Light probe Bcsp-FP 5 ' end has fluorescent reporter group, 3 ' ends to have fluorescent quenching group.
The fluorescent reporter group of described Taqman fluorescence probe is selected from 6-CF 5(6)-Carboxyfluorescein (6-
Carboxyfluorescein, 6-FAM), chlordene-6-methyl fluorescein, VIC fluorescent dye, four chloro-6-CF 5(6)-Carboxyfluorescein, carboxylic
Base-X-rhodamine, 6-carboxyl tetramethylrhodamine, Sulforhodamine, 6-carboxyl-4 ', 5-bis-chloro-2 ', 7 '-dimethoxy fluorescence
At least one in element succinimide ester, Hua Jing 3, Hua Jing 5 or flower cyanines 5.5；Described fluorescent quenching group is selected from 6-carboxyl four
Rhodamine, 4-(4-dimethylamino-phenylazo) benzoic acid, black hole quencher 1 (Black Hole Quencher 1,
BHQ1), at least one in black hole quencher 2 or black hole quencher 3；
Preferably, when fluorescent quenching group is selected from 4-(4-dimethylamino-phenylazo) benzoic acid, fluorescence report base
Group is selected from 6-CF 5(6)-Carboxyfluorescein, four chloro-6-CF 5(6)-Carboxyfluorescein, 6-carboxyl-4 ', 5 '-two chloro-2 ', 7 '-dimethoxyfluorescein ambers
At least one in amber imide ester, chlordene-6-methyl fluorescein, Hua Jing 3；When fluorescent quenching group is selected from 6-carboxyl tetramethyl
During rhodamine, fluorescent reporter group is selected from 6-CF 5(6)-Carboxyfluorescein, four chloro-6-CF 5(6)-Carboxyfluorescein, 6-carboxyl-4 ', 5 '-two chloro-2 ',
At least one in 7 '-dimethoxyfluorescein succinimide ester or chlordene-6-methyl fluorescein；
When fluorescent quenching group is selected from black hole quencher 1, fluorescent reporter group is selected from 6-CF 5(6)-Carboxyfluorescein, four chloro-6-
CF 5(6)-Carboxyfluorescein, 6-carboxyl-4 ', 5 '-two chloro-2 ', 7 '-dimethoxyfluorescein succinimide esters, chlordene-6-methyl fluorescence
At least one in element or flower cyanines 3；
When fluorescent quenching group is selected from black hole quencher 2, fluorescent reporter group is selected from 6-carboxyl tetramethylrhodamine, flower
At least one in cyanines 3, carboxy-X-rhodamine or Sulforhodamine；
When fluorescent quenching group is selected from black hole quencher 3, fluorescent reporter group is selected from flower cyanines 5 or spends in cyanines 5.5
Kind.
Most preferably, described fluorescent reporter group is 6-FAM；Described fluorescent quenching group is BHQ1.
(2) PCR reactant liquor:
PCR reactant liquor consists of: 10 × reaction buffer 5 μ L, 10mM dNTP Mix 4 μ L, Taq DNA
Polymerase 1.2 μ L, UDG enzyme 0.2 μ L, 10mM dUTP 1 μ L, purified water 13.6 μ L.
2) brucella DNA (sample to be tested) PCR reaction
(1) preparation of PCR reaction mixture and sample-adding are carried out in the following proportions: PCR reactant liquor 25 μ L, brucella primer is visited
Pin mixed liquor 5 μ L is added in PCR pipe, vibration mixing, is added in above-mentioned PCR pipe by the sample to be tested 20 μ L extracted, carries out PCR
Amplification.
(2) reaction condition of PCR reaction is set to: 37 DEG C of 2min；95℃3min；94 DEG C of 15s, 60 DEG C of 35s, 10 are followed
Ring；94 DEG C of 15s, 60 DEG C of 35s fluorescence (phosphor collection), 35 circulations；25℃1min.
2, PCR reaction result interpretation (brucellar qualitative detection interpretation of result)
The present embodiment 2 uses optimal case, i.e. brucella primed probe mixed liquor containing the internal standard, and described Taqman fluorescence is visited
The fluorescent reporter group of pin Bcsp-FP is 6-FAM；Fluorescent quenching group is BHQ1.
The present embodiment 2 uses ABI7500 quantitative real time PCR Instrument, is set as corresponding fluorescein, this reality during fluorescence signal collection
Executing example and be set as FAM, VIC fluorescein, fluorescence signal collection is located at 60 DEG C.
Brucella primed probe mixed liquor, with or without internal standard, does not affect the response of measuring samples channel plot, i.e. cloth Lu Shi
Bacterium detection is for time positive, and measuring samples respective channel there will be S type curve；When brucella detection is for feminine gender, measuring samples pair
Passage is answered to occur without S type curve.
This enforcement 2 uses brucella primed probe mixed liquor containing the internal standard, described Taqman fluorescence probe Bcsp-FP glimmering
Light reporter group is 6-FAM；Fluorescent quenching group is the scheme of BHQ1, and therefore testing result can comprise internal standard channel plot:
If S type curve occurs in internal standard passage, FAM Air conduct measurement is without S type curve simultaneously, it is determined that negative for BKV；If it is interior
There is S type curve in mark passage, and FAM Air conduct measurement has S type curve simultaneously, it is determined that positive for BKV.Utilize said method to 12 example cloth
Shandong Salmonella serum sample, 5 example Healthy Human Serum samples, 2 example escherichia coli serum samples, 2 example Salmonella enteritidis serum samples, 2
Example hepatitis B virus serum sample, 2 example ebb virus's serum samples, 2 example human cytomegalic inclusion disease virus serum samples, mark
This source XXX hospital 27 parts of samples altogether detect, result as shown in Figure 1:
Wherein, 12 example brucella serum sample detections are the positive, and S type curve, FAM passage simultaneously occurs in internal standard passage
Detection has S type curve；Healthy Human Serum sample, escherichia coli serum sample, Salmonella enteritidis serum sample, hepatitis B virus
Serum sample, ebb virus's serum sample, human cytomegalic inclusion disease virus serum sample, detection is feminine gender, and internal standard passage goes out
Existing S type curve, FAM Air conduct measurement is without S type curve simultaneously.
The present embodiment 2 uses internal standard system of quality control, and internal standard participates in sample extraction and amplification, and being used for monitoring reaction system may
The restraining factors existed.Internal standard template and target gene are without homology, and what internal standard probe selected is not conflict with target gene probe
Another sense channel.
Embodiment 3
The present embodiment 2 provides a kind of brucellar quantitative detecting method:
1, the pcr amplification reaction of brucella DNA (sample to be tested)
1) the pcr amplification reaction agents useful for same of brucella DNA and composition
(1) brucella primed probe mixed liquor:
The component of brucella primed probe mixed liquor is: forward primer Bcsp-F (50 μMs) 0.6 μ L, downstream primer
Bcsp-R (50 μMs) 0.6 μ L, Taqman fluorescence probe Bcsp-FP (20 μMs) 0.5 μ L, internal standard forward primer (20 μMs) 0.5 μ L, interior
Mark downstream primer (20 μMs) 0.5 μ L, internal standard probe (10 μMs) 0.375 μ L, purified water 1.925 μ L.
Described forward primer Bcsp-F comprises the base sequence as shown in SEQ ID NO:1；
Described downstream primer Bcsp-R comprises the base sequence as shown in SEQ ID NO:2；
Described Taqman fluorescence probe Bcsp-FP comprises the base sequence as shown in SEQ ID NO:3, and described Taqman is glimmering
Light probe Bcsp-FP 5 ' end has fluorescent reporter group, 3 ' ends to have fluorescent quenching group.Described fluorescent reporter group is 6-FAM；
Described fluorescent quenching group is BHQ1.
(2) BKV PCR reactant liquor:
PCR reactant liquor consists of: 10 × reaction buffer 5 μ L, 10mM dNTP Mix 4 μ L, Taq DNA
Polymerase 1.2 μ L, UDG enzyme 0.2 μ L, 10mM dUTP 1 μ L, purified water 13.6 μ L.
(3) Brucella internal standard quality-control product:
Brucella internal standard quality-control product is the recombiant plasmid of external structure.
(4) Brucella feminine gender quality-control product:
Brucella feminine gender quality-control product is negative serum.
(5) the critical positive quality control product of Brucella:
The critical positive quality control product of Brucella is the positive sample of inactivated strain diluent or inactivation, concentration is 1 ×
104copies/mL。
(6) Brucella positive quality control product:
Brucella positive quality control product is the positive sample of inactivated strain diluent or inactivation, concentration is 2.5 ×
106copies/mL。
(7) plasmid standards for quantitation:
Plasmid standards for quantitation is the positive sample of inactivated strain diluent or inactivation, and it is quantitative that concentration is respectively as follows: Brucella
Standard substance I (2.5 × 108Copies/mL), Brucella plasmid standards for quantitation II (2.5 × 107Copies/mL), Brucella is fixed
Amount standard substance III (2.5 × 106Copies/mL), Brucella plasmid standards for quantitation IV (2.5 × 105copies/mL)。
2) PCR reaction
(1) preparation of PCR reaction mixture and sample-adding are carried out in the following proportions: PCR reactant liquor 25 μ L × (n+7: four quantitatively
Standard substance and negative quality-control product, critical positive quality control product, a positive quality control product), often pipe adds brucella primer
Probe mixed liquor 5 μ L；Vibration mixing, every part of 30 μ L divide to PCR pipe, and by the sample to be tested that extracted, (this sample to be tested is fixed
The sample of property test positive) and quality-control product 20 μ L be added in above-mentioned PCR pipe, carry out PCR amplification.
(2) pcr amplification reaction condition is set: 37 DEG C of 2min；95℃3min；94 DEG C of 15s, 60 DEG C of 35s, 10 circulations；94
DEG C 15s, 60 DEG C of 35s fluorescence (phosphor collection), 35 circulations；25℃1min.
2, PCR reaction result interpretation (the detection by quantitative interpretation of result of BK virus)
Using ABI7500 quantitative real time PCR Instrument, be set as FAM fluorescein during fluorescence signal collection, fluorescence signal collection sets
At 60 DEG C
Result judges:
(1) if S type amplification curve does not occur in target gene FAM passage, it is judged to Brucella DNA detection negative.
(2) if S type amplification curve occurs in target gene FAM passage, it is judged to Brucella DNA detection positive, testing result
There is a description below:
1) measured value 0～1000copies/mL sample report be " less than test kit detect lower limit (1000copies/
ML) ", i.e. " < 1000copies/mL ".Should judge in conjunction with other clinical diagnosis index.
2) measured value is 1000～5 × 108Sample between copies/mL, directly reports the numerical value measured；
3) measured value is more than 5 × 108The sample of copies/mL, can be reported as ＞ 5 × 108copies/mL；If need to accurately determine
Amount, can be according to result by Sample Dilution to 5 × 108Below copies/mL repetition measurement, and calculate concentration of specimens according to below equation,
Result is labeled as reporting numerical value.
Concentration × extension rate after numerical value (final sample concentration)=dilution can be reported
Extension rate=(sample size+dilution liquid measure)/sample size
The standard curve utilizing the detection of Brucella plasmid standards for quantitation to be generated calculates the concentration (copies/ of sample to be tested
mL).During detection reaction, through conversion, Brucella plasmid standards for quantitation I (5 × 107Copies/mL), Brucella plasmid standards for quantitation
Ⅱ(5×106Copies/mL), Brucella plasmid standards for quantitation III (5 × 105Copies/mL), Brucella plasmid standards for quantitation IV
(5×104Copies/mL) detecting with sample, if Fig. 2 is plasmid standards for quantitation amplification curve, Fig. 3 is standard substance and treat test sample simultaneously
This amplification curve.According to plasmid standards for quantitation concentration and testing result CT value, system automatically generates a standard curve.According to be measured
The detection CT value of sample, can calculate the concentration of sample target gene.
Utilizing said method that 6 example brucella serum samples are carried out detection by quantitative, the standard generated by computer is bent
Line.Calculate in Fig. 36 samples to be tested (as it is shown on figure 3, except 6 curves of 4 standard substance extra curvatures are sample to be tested curve) from
Left-to-right concentration is respectively, and the concentration calculating 6 positive sample is respectively as follows: 1.2 × 107copies/mL、8.0×105copies/
mL、7.1×104copies/mL、7.8×103copies/mL、1.3×103copies/mL、8.1×102copies/mL。
Result verification: the amplified production that detection is positive is carried out gene sequencing, and sequencing result turns out to be after BLAST comparison
Brucella gene order.
Embodiment 4
The present embodiment 4 provides a kind of test kit detecting BK virus.
The test kit of detection BK virus, including: forward primer: Bcsp-F, downstream primer: Bcsp-R, Taqman fluorescence is visited
Pin Bcsp-FP, purified water；
Described forward primer Bcsp-F comprises the base sequence as shown in SEQ ID NO:1；
Described downstream primer Bcsp-R comprises the base sequence as shown in SEQ ID NO:2；
Described Taqman fluorescence probe Bcsp-FP comprises the base sequence as shown in SEQ ID NO:3.
Preferably, this test kit also includes: internal standard forward primer, internal standard downstream primer, internal standard probe.Internal standard system of quality control
Participate in extracting and amplification.For monitoring reaction system restraining factors that may be present.Internal standard template and target gene are without homology, interior
What mark probe selected is another sense channel not conflicted with target gene probe.
Preferably, this test kit, also include: Brucella feminine gender quality-control product, Brucella positive quality control product, Brucella
Critical positive quality control product and Brucella plasmid standards for quantitation I～IV, described Brucella feminine gender quality-control product is negative serum, described
The critical positive quality control product of Brucella, Brucella positive quality control product are inactivated strain or inactivation specimen, and described Brucella is fixed
Amount standard substance I～IV is inactivated strain or inactivation positive sample, described Brucella plasmid standards for quantitation I～IV, including
Brucella plasmid standards for quantitation I, Brucella plasmid standards for quantitation II, Brucella plasmid standards for quantitation III, Brucella quantitatively mark
Quasi-product IV.
Preferably, the critical positive quality control product of Brucella plasmid standards for quantitation I～IV, Brucella, Brucella positive matter
The construction method of control product comprises the following steps:
(1) extracting inactivated strain nucleic acid or inactivation positive sample nucleic acid, spectrophotometer carries out definite value.
(2) use the normal saline after sterilizing to be diluted by quantitative high concentration bacterial strain, be diluted to 4 gradients 2.5 ×
105～2.5 × 108Copies/mL, respectively as Brucella plasmid standards for quantitation I, Brucella plasmid standards for quantitation of test kit
II, Brucella plasmid standards for quantitation III, Brucella plasmid standards for quantitation IV, it addition, diluted concentration is 2.5 × 106copies/
ML and 1 × 104Copies/mL is respectively as the Brucella positive quality control product of test kit and the critical positive quality control of Brucella
Product.
Preferably, this test kit, including: brucella primed probe mixed liquor, PCR reactant liquor, Brucella quantitatively mark
In quasi-product, Brucella positive quality control product, the critical positive quality control product of Brucella, Brucella feminine gender quality-control product, Brucella
Mark quality-control product.
Preferably, each component composition and manufacture method are:
(1) brucella primed probe mixed liquor:
Brucella primed probe mixed liquor particular make-up composition is: Bcsp-F (50 μMs) 0.6 μ L, Bcsp-R (50 μMs)
0.6 μ L, Bcsp-FP (20 μMs) 0.5 μ L, internal standard forward primer (20 μMs) 0.5 μ L, internal standard downstream primer (20 μMs) 0.5 μ L, internal standard
Probe (10 μMs) 0.375 μ L, purified water 1.925 μ L.Dispensed loading amount is that 120 μ L × 1 manage/24 parts.
(2) PCR reactant liquor:
PCR reactant liquor consists of: 10 × reaction buffer5 μ L, 10mM dNTP Mix 4 μ L, Taq DNA
Polymerase1.2 μ L, UDG enzyme 0.2 μ L, 10mM dUTP1 μ L, purified water 13.6 μ L.Dispensed loading amount is that 600 μ L × 1 manage/24 parts.
(3) Brucella internal standard quality-control product:
Brucella internal standard quality-control product is the recombiant plasmid of external structure, and dispensed loading amount is that 120 μ L × 1 manage/24 parts.
(4) Brucella feminine gender quality-control product:
Brucella feminine gender quality-control product is negative serum, and dispensed loading amount is that 600 μ L × 1 manage/24 parts.
(5) the critical positive quality control product of Brucella:
The critical positive quality control product of Brucella is the positive sample of inactivated strain diluent or inactivation, concentration is 1 ×
104Copies/mL, dispensed loading amount is that 600 μ L × 1 manage/24 parts.
(6) Brucella positive quality control product:
Brucella positive quality control product is the positive sample of inactivated strain diluent or inactivation, concentration is 2.5 ×
106Copies/mL, dispensed loading amount is that 600 μ L × 1 manage/24 parts.
(7) plasmid standards for quantitation:
Plasmid standards for quantitation is the positive sample of inactivated strain diluent or inactivation, and it is quantitative that concentration is respectively as follows: Brucella
Standard substance I (2.5 × 108Copies/mL), Brucella plasmid standards for quantitation II (2.5 × 107Copies/mL), Brucella is fixed
Amount standard substance III (2.5 × 106Copies/mL), Brucella plasmid standards for quantitation IV (2.5 × 105Copies/mL), dispensed loading amount is
600 μ L × 1 manage/24 parts.
Preferably, this test kit consists of, and brucella primed probe mixed liquor, PCR reactant liquor, Brucella quantitatively mark
In quasi-product, Brucella positive quality control product, the critical positive quality control product of Brucella, Brucella feminine gender quality-control product, Brucella
Mark quality-control product, each constituent content is:
The brucellar test kit of above-mentioned detection, is applied to brucellar detection.This detection can be qualitative detection and/
Or detection by quantitative.
Compared with prior art, brucellar detection method of the present invention, test kit, reach following effect:
1. test kit involved in the present invention arranges critical positive quality control product, positive quality control product, plasmid standards for quantitation equal
For inactivated strain or inactivation specimen, participate in nucleic acid extraction and amplification, the truest reflection nucleic acid extraction and amplification.
2. the present invention uses uridnine enzyme (UDG) anti-pollution system, and the heated U-DNA that can optionally degrade, to prevent
The previously pollution of pcr amplification product.
3. the present invention uses internal standard system of quality control, is used for monitoring reaction system restraining factors that may be present.Internal standard template
With target gene without homology, what internal standard probe selected is another sense channel not conflicted with target gene probe.
4. the detection method sensitivity of the present invention is high, and minimum detection can reach 1000copies/mL；Meanwhile, the present invention
The specificity of detection method is fine, other the antibacterial generation cross reaction of getting along well, such as escherichia coli, Salmonella enteritidis, B-mode
Hepatitis virus, ebb virus, human cytomegalic inclusion disease virus etc..
5. the detection method reaction of the present invention quickly, i.e. can get PCR reaction result, and cost for general 1.5 to 2 hours
Low, non-false positive, is suitable for larger scale clinical and carries out.Thus realize to brucellar quickly, the most quantitatively examine
Survey, thus can guarantee that case diagnosis and treatment timely and therapeutic effect monitoring.
Owing to the embodiment of the present application has been described in detail by method part, here to the system related in embodiment
Expansion with method corresponding part describes omits, and repeats no more.Method is referred to for the description of particular content in system implement
The content of example, the most specifically limits.
Described above illustrate and describes some preferred embodiments of the application, but as previously mentioned, it should be understood that the application
Be not limited to form disclosed herein, be not to be taken as the eliminating to other embodiments, and can be used for other combinations various,
Amendment and environment, and can be in application contemplated scope described herein, by above-mentioned teaching or the technology of association area or knowledge
It is modified.And the change that those skilled in the art are carried out and change are without departing from spirit and scope, the most all should be in this Shen
Please be in the protection domain of claims.
Claims (10)
- The most brucellar detection method, comprises the steps:Sample to be tested obtains:Centrifugal column method is used to obtain brucella DNA from serum specimen, it is thus achieved that sample to be tested；Pcr amplification reaction:The brucella DNA of extraction is added in PCR reaction mixture, carries out PCR amplification；Described PCR reaction mixture, including: PCR reactant liquor, brucella primed probe mixed liquor,Described brucella primed probe mixed liquor, including, forward primer Bcsp-F, downstream primer Bcsp-R, Taqman fluorescence Probe Bcsp-FP, purified water；Described forward primer Bcsp-F comprises the base sequence as shown in SEQ ID NO:1；Described downstream primer Bcsp-R comprises the base sequence as shown in SEQ ID NO:2；Described Taqman fluorescence probe Bcsp-FP comprises the base sequence as shown in SEQ ID NO:3, and described Taqman fluorescence is visited Pin BKV-FP5 ' end has fluorescent reporter group, 3 ' ends to have fluorescent quenching group；Result detects:Use quantitative real time PCR Instrument detection reaction result, during fluorescence signal collection, be set as Taqman fluorescence probe BKV-FP 5 ' The fluorescein that end fluorophor is corresponding, collects fluorescence signal at 60 DEG C；To interpretation of result.
- Brucellar detection method the most according to claim 1, it is characterised in that the condition of described pcr amplification reaction For: 37 DEG C of 2min；95℃3min；94 DEG C of 15s, 60 DEG C of 35s, 10 circulations；94 DEG C of 15s, 60 DEG C of 35s fluorescence, 35 circulations；25 ℃1min。
- Brucellar detection method the most according to claim 1, it is characterised in that described BKV primed probe mixed liquor, Also include: internal standard forward primer, internal standard downstream primer, internal standard probe.
- The detection method of BK virus the most according to claim 1, it is characterised in that described pcr amplification reaction, also includes: Be arranged in parallel Brucella feminine gender quality-control product, the critical positive quality control product of Brucella, Brucella positive quality control product and Brucella plasmid standards for quantitation I～IV reaction group, described Brucella feminine gender control product are negative serum, and described Brucella is critical Positive quality control product, the template to be measured of Brucella positive quality control product are inactivated strain or inactivation positive sample, described Brucella The template to be measured of plasmid standards for quantitation I～IV is inactivated strain or inactivation positive sample.
- The most brucellar detection method, it is characterised in that described Taqman fluorescence probe BKV- The fluorescent reporter group 6-CF 5(6)-Carboxyfluorescein of FP, chlordene-6-methyl fluorescein, VIC fluorescent dye, four chloro-6-CF 5(6)-Carboxyfluorescein, Carboxy-X-rhodamine, 6-carboxyl tetramethylrhodamine, Sulforhodamine, 6-carboxyl-4 ', chloro-2 ', the 7 '-dimethoxy of 5-bis-is glimmering At least one in light element succinimide ester, Hua Jing 3, Hua Jing 5 or flower cyanines 5.5；Described fluorescent quenching group is selected from 6-carboxyl Tetramethylrhodamine, 4-(4-dimethylamino-phenylazo) benzoic acid, black hole quencher 1, black hole quencher 2 or black hole cancellation At least one in agent 3.
- Brucellar detection method the most according to claim 1, it is characterised in that described PCR reactant liquor, including UDG The anti-pollution system of enzyme, the described anti-pollution system of UDG enzyme, including: UDG enzyme and dUTP.
- 7. detect brucellar test kit, it is characterised in that include the forward primer Bcsp-F described in claim 1, downstream Primer Bcsp-R, Taqman fluorescence probe Bcsp-FP, purified water；Described forward primer Bcsp-F comprises the base sequence as shown in SEQ ID NO:1；Described downstream primer Bcsp-R comprises the base sequence as shown in SEQ ID NO:2；Described Taqman fluorescence probe Bcsp-FP comprises the base sequence as shown in SEQ ID NO:3.
- The brucellar test kit of detection the most according to claim 7, it is characterised in that also include: internal standard forward primer, Internal standard downstream primer, internal standard probe, Brucella feminine gender quality-control product, Brucella positive quality control product, the critical positive of Brucella Quality-control product and Brucella plasmid standards for quantitation I～IV, described Brucella feminine gender quality-control product is negative serum, described Brucella Critical positive quality control product, Brucella positive quality control product are inactivated strain or inactivation specimen, described Brucella plasmid standards for quantitation I ～IV is inactivated strain or inactivation positive sample, described Brucella plasmid standards for quantitation I～IV, including Brucella quantitative criterion Product I, Brucella plasmid standards for quantitation II, Brucella plasmid standards for quantitation III, Brucella plasmid standards for quantitation IV.
- 9. want the brucellar test kit of detection described in 7 or 8 any one according to right, it is characterised in that described test kit bag Include: brucella primed probe mixed liquor, PCR reactant liquor, Brucella plasmid standards for quantitation, Brucella positive quality control product, The critical positive quality control product of Brucella, Brucella feminine gender quality-control product, Brucella internal standard quality-control product, each constituent content is:
- 10. the brucellar test kit of detection described in any one of claim 7-9, for brucellar detection.
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Application publication date: 20161207 |