CN106148571A - The reagent set of detection HPV HPV16 and HPV18 - Google Patents
The reagent set of detection HPV HPV16 and HPV18 Download PDFInfo
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- CN106148571A CN106148571A CN201610806214.7A CN201610806214A CN106148571A CN 106148571 A CN106148571 A CN 106148571A CN 201610806214 A CN201610806214 A CN 201610806214A CN 106148571 A CN106148571 A CN 106148571A
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- single stranded
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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
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/708—Specific hybridization probes for papilloma
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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
Abstract
The invention discloses the reagent set of detection HPV HPV16 and HPV18.Reagent set disclosed by the invention is made up of the HPV18 B3 shown in the HPV18 F3 shown in the HPV18 BIP shown in the HPV18 FIP shown in the HPV16 B3 shown in the HPV16 F3 shown in the HPV16 BIP shown in the HPV16 FIP shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8.It is demonstrated experimentally that the reagent set of detection HPV HPV16 and HPV18 disclosed by the invention is specifically high, highly sensitive, accuracy rate is high, can be used for detecting HPV.
Description
Technical field
The present invention relates to biological technical field detects the reagent set of HPV HPV16 and HPV18.
Background technology
HPV (HPV) is a kind of DNA virus, infects human epidermal and mucous membrane tissue, there are about 170 species at present
The HPV of type is identified.Epidemiological study it turned out, and HPV persistent viral infection is the main cause causing cervical carcinoma,
According to the relation of HPV virus and cervical carcinoma, high-risk-type, possible high-risk-type and low risk, wherein, 16 types (HPV16) can be divided into
It is the hypotype that infection rate is the highest in world wide with 18 types (HPV18).
At present, the detection of HPV has two kinds of commercial methods, and a kind of is the hybrid capture II being produced by Qiagen company of Germany
Generation (HC2) detection kit, as the goldstandard of detection additive method, another kind is the LA inspection being produced by Roche company of Switzerland
Survey method, but owing to both approaches is relatively costly, it is difficult to large-scale popularization uses.Additionally, conventional method also has cytology to examine
Look into, PCR (PCR), real-time fluorescence quantitative PCR, Southern Blot method etc., cytolgical examination sensitivity is low,
Being likely to occur false negative, PCR and real-time fluorescence PCR need the complex instrument such as thermal cycler, time-consumingly longer, Southern Blot
Method operation complexity, cost is high.
Content of the invention
The technical problem to be solved is how quickly to detect high-risk human mammilla papillomavirus HPV16 and HPV18,
And improve specific, sensitivity and the reduction testing cost of detection.
For solving above-mentioned technical problem, present invention firstly provides the complete of detection HPV HPV16 and HPV18
Reagent.
The reagent set of detection HPV HPV16 and HPV18 provided by the present invention, by reagent set 1 with become
Set reagent 2 forms；Described reagent set 1 by the single stranded DNA of entitled HPV16-FIP, entitled HPV16-BIP single stranded DNA,
The single stranded DNA composition of the single stranded DNA of entitled HPV16-F3 and entitled HPV16-B3；
Described HPV16-FIP is following a1) to a4) in any one single stranded DNA:
A1) single stranded DNA shown in SEQ ID No.1 in sequence table；
A2) at a1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtains；
A3) and a1) or a2) single stranded DNA that limits has the single stranded DNA of the homogeneity of more than 85%；
A4) under strict conditions with a1) or a2) limit single stranded DNA hybridization single stranded DNA；
Described HPV16-BIP is following b1) to b4) in any one single stranded DNA:
B1) single stranded DNA shown in SEQ ID No.2 in sequence table；
B2) at b1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtains；
B3) and b1) or b2) single stranded DNA that limits has the single stranded DNA of the homogeneity of more than 85%；
B4) under strict conditions with b1) or b2) limit single stranded DNA hybridization single stranded DNA；
Described HPV16-F3 is following c1) to c4) in any one single stranded DNA:
C1) single stranded DNA shown in SEQ ID No.3 in sequence table；
C2) at c1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtains；
C3) and c1) or c2) single stranded DNA that limits has the single stranded DNA of the homogeneity of more than 85%；
C4) under strict conditions with c1) or c2) limit single stranded DNA hybridization single stranded DNA；
Described HPV16-B3 is following d1) to d4) in any one single stranded DNA:
D1) single stranded DNA shown in SEQ ID No.4 in sequence table；
D2) at d1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtains；
D3) and d1) or d2) single stranded DNA that limits has the single stranded DNA of the homogeneity of more than 85%；
D4) under strict conditions with d1) or d2) limit single stranded DNA hybridization single stranded DNA；
Described reagent set 2 is by the single stranded DNA of entitled HPV18-FIP, the single stranded DNA of entitled HPV18-BIP, title
It is the single stranded DNA composition of the single stranded DNA of HPV18-F3 and entitled HPV18-B3；
Described HPV18-FIP is following e1) to e4) in any one single stranded DNA:
E1) single stranded DNA shown in SEQ ID No.5 in sequence table；
E2) at e1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtains；
E3) and e1) or e2) single stranded DNA that limits has the single stranded DNA of the homogeneity of more than 85%；
E4) under strict conditions with e1) or e2) limit single stranded DNA hybridization single stranded DNA；
Described HPV18-BIP is following f1) to f4) in any one single stranded DNA:
F1) single stranded DNA shown in SEQ ID No.6 in sequence table；
F2) at f1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtains；
F3) and f1) or f2) single stranded DNA that limits has the single stranded DNA of the homogeneity of more than 85%；
F4) under strict conditions with f1) or f2) limit single stranded DNA hybridization single stranded DNA；
Described HPV18-F3 is following g1) to g4) in any one single stranded DNA:
G1) single stranded DNA shown in SEQ ID No.7 in sequence table；
G2) at g1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtains；
G3) and g1) or g2) single stranded DNA that limits has the single stranded DNA of the homogeneity of more than 85%；
G4) under strict conditions with g1) or g2) limit single stranded DNA hybridization single stranded DNA；
Described HPV18-B3 is following h1) to h4) in any one single stranded DNA:
H1) single stranded DNA shown in SEQ ID No.8 in sequence table；
H2) at h1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtains；
H3) and h1) or h2) single stranded DNA that limits has the single stranded DNA of the homogeneity of more than 85%；
H4) under strict conditions with h1) or h2) limit single stranded DNA hybridization single stranded DNA.
In the reagent set of above-mentioned detection HPV HPV16 and HPV18, a2) described at a1) 5 ' ends and/or
3 ' ends add the single stranded DNA that one or several nucleotides obtains can for the single stranded DNA shown in SEQ ID No.1 5 ' ends with/
Or 3 ' end add the single stranded DNA that obtains of one to ten nucleotides.B2) described at b1) 5 ' ends and/or 3 ' ends add one or several
The single stranded DNA that individual nucleotides obtains can be for adding one to ten at 5 ' ends of the single stranded DNA shown in SEQ ID No.2 and/or 3 ' ends
The single stranded DNA that individual nucleotides obtains.C2) described at c1) 5 ' ends and/or 3 ' ends add the lists that one or several nucleotides obtains
Chain DNA can be for adding, at 5 ' of the sequence shown in SEQ ID No.3 ends and/or 3 ' ends, the strand that one to ten nucleotides obtains
DNA.D2) described at d1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtains can be at SEQ ID
5 ' ends of the sequence shown in No.4 and/or 3 ' ends add the single stranded DNA that one to ten nucleotides obtains.E2) described at e1) 5 '
The single stranded DNA that end and/or the one or several nucleotides of 3 ' end interpolations obtain can be the 5 ' ends in the sequence shown in SEQ ID No.5
And/or 3 ' end add the single stranded DNA that obtains of one to ten nucleotides.F2) described at f1) 5 ' ends and/or 3 ' ends add one
Or the single stranded DNA that several nucleotides obtains can be for adding one to ten at 5 ' ends of the sequence shown in SEQ ID No.6 and/or 3 ' ends
The single stranded DNA that individual nucleotides obtains.G2) described at g1) 5 ' ends and/or 3 ' ends add the lists that one or several nucleotides obtains
Chain DNA can be for adding, at 5 ' of the sequence shown in SEQ ID No.7 ends and/or 3 ' ends, the strand that one to ten nucleotides obtains
DNA.H2) described at h1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtains can be at SEQ ID
5 ' ends of the sequence shown in No.8 and/or 3 ' ends add the single stranded DNA that one to ten nucleotides obtains.
Term used herein " homogeneity " refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes and this
Bright SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6,
Nucleotide sequence shown in SEQ ID No.7 or SEQ ID No.8 has 85% or higher, or 90% or higher, or 95% or
The nucleotide sequence of higher homogeneity.Homogeneity can with the naked eye or computer software is evaluated.Use computer software, two
Homogeneity between individual or multiple sequence can be represented by percentage (%), and it is same that it can be used to evaluate between correlated series
Property.
In the reagent set of above-mentioned detection HPV HPV16 and HPV18, described stringent condition is at 2 × SSC,
In the solution of 0.1%SDS, hybridize at 68 DEG C and wash film 2 times, each 5min, again in 0.5 × SSC, the solution of 0.1%SDS
In, hybridize at 68 DEG C and wash film 2 times, each 15min；Or, 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS solution in,
Hybridize under the conditions of 65 DEG C and wash film.
Above-mentioned more than 85% homogeneity, can be the homogeneity of the 85%th, 90% or more than 95%.
In the reagent set of above-mentioned detection HPV HPV16 and HPV18, described reagent set 1 is detection human milk
Head tumor virus and the reagent set of HPV16, the reagent set of described detection HPV and HPV18.
In the reagent set of above-mentioned detection HPV HPV16 and HPV18, described reagent set 1 and described complete
2 grams of independent packagings of reagent, each single stranded DNA in described reagent set 1 and described reagent set 2 also all can independent packaging.
In the reagent set of above-mentioned detection HPV HPV16 and HPV18, described described in described reagent set 1
The mol ratio of HPV16-FIP, described HPV16-BIP, described HPV16-F3 and described HPV16-B3 can be 4:4:1:1.Described one-tenth
Described in set reagent 2, the mol ratio of HPV18-FIP, described HPV18-BIP, described HPV18-F3 and described HPV18-B3 can be 4:
4:1:1。
For solving above-mentioned technical problem, present invention also offers following P1) or reagent set P2):
P1) detect the reagent set of HPV HPV16, be described reagent set 1；
P2) detect the reagent set of HPV HPV18, be described reagent set 2.
For solving above-mentioned technical problem, present invention also offers following Q1) or Q2) or Q3) system:
Q1) system (by its named system first) of HPV HPV16 and HPV18 is detected, including described detection
The reagent set of HPV HPV16 and HPV18 and strand displacement type archaeal dna polymerase；
Q2) system (by its named system 1) of HPV HPV16 is detected, including described reagent set 1 and institute
State strand displacement type archaeal dna polymerase；
Q3) system (by its named system 2) of HPV HPV18 is detected, including described reagent set 2 and institute
State strand displacement type archaeal dna polymerase.
In said system, described strand displacement type archaeal dna polymerase can be Bst archaeal dna polymerase.
Described system first can be by the reagent set of described detection HPV HPV16 and HPV18 and described strand displacement
Type archaeal dna polymerase forms.Described system first also can be by the reagent set of described detection HPV HPV16 and HPV18, institute
State strand displacement type archaeal dna polymerase and carry out other reagent required for ring mediated isothermal amplification and/or instrument composition.Carry out ring
Other reagent required for mediated isothermality amplification can be 2 × LAMP Buffer.Described 2 × LAMP Buffer is by solute and solvent
Composition.Described solvent is water, and described solute and concentration thereof are respectively Tris 20mM, KCl 10mM, MgSO4 8mM,(NH4)2SO4
(in four kinds of dNTP, the concentration of every kind of dNTP is for 10mM, Tween 20 0.1%, Betaine 0.8M, dNTPs 1.4mM
1.4mM),MnCl2 0.5mM,Calcein 25μM。
Described system 1 can be made up of described reagent set 1 and described strand displacement type archaeal dna polymerase.Described system 1 also can be by
Described reagent set the 1st, described strand displacement type archaeal dna polymerase and carry out other reagent required for ring mediated isothermal amplification and/
Or instrument composition.Carrying out other reagent required for ring mediated isothermal amplification can be described 2 × LAMP Buffer.
Described system 2 can be made up of described reagent set 2 and described strand displacement type archaeal dna polymerase.Described system 2 also can be by
Described reagent set the 2nd, described strand displacement type archaeal dna polymerase and carry out other reagent required for ring mediated isothermal amplification and/
Or instrument composition.Carrying out other reagent required for ring mediated isothermal amplification can be described 2 × LAMP Buffer.
Said system can be ring mediated isothermal amplification reagent or kit.
For solving above-mentioned technical problem, present invention also offers becoming of described detection HPV HPV16 with HPV18
The preparation method of set reagent.
The preparation method of the reagent set of described detection HPV HPV16 and HPV18 provided by the present invention, bag
Include described HPV16-FIP, described HPV16-BIP, described HPV16-F3, described HPV16-B3, described HPV18-FIP, described
HPV18-BIP, described HPV18-F3 and described HPV18-B3 individually pack.
For solving above-mentioned technical problem, present invention also offers the preparation side of described reagent set 1 or described reagent set 2
Method.
Described reagent set 1 provided by the present invention or the preparation method of described reagent set 2, be following M1) or M2):
M1) include HPV16-FIP described in described reagent set 1, described HPV16-BIP, described HPV16-F3 and institute
State HPV16-B3 individually to pack；
M2) include HPV18-FIP described in described reagent set 2, described HPV18-BIP, described HPV18-F3 and institute
State HPV18-B3 individually to pack.
For solving above-mentioned technical problem, present invention also offers the preparation method of described system.
The preparation method of described system provided by the present invention, including by described detection HPV HPV16 and
Each single stranded DNA in the reagent set of HPV18, described reagent set 1 or described reagent set 2 and each reagent individually wrap
Dress.
For solving above-mentioned technical problem, present invention also offers arbitrary purposes in following X1-X15:
X1, the reagent set of described detection HPV HPV16 and HPV18 are at preparation detection HPV
Application in HPV16 and HPV18 product；
X2, the reagent set of described detection HPV HPV16 and HPV18 produce at preparation detection HPV
Application in product；
X3, the reagent set of described detection HPV HPV16 and HPV18 are suffered from preparation examination HPV
Application in person's product；
X4, described reagent set 1 or described reagent set 2 are at preparation detection HPV HPV16 or HPV18 product
In application；
The application in preparation detection HPV product of X5, described reagent set 1 or described reagent set 2；
X6, described reagent set 1 or described reagent set 2 answering in preparation examination HPV patient product
With；
X7, the reagent set of described detection HPV HPV16 and HPV18 are at detection HPV HPV16
With the application in HPV18；
X8, reagent set the answering in detection HPV of described detection HPV HPV16 and HPV18
With；
X9, described detection HPV HPV16 and HPV18 reagent set in examination HPV patient
Application；
X10, described reagent set 1 or described reagent set 2 answering in detection HPV HPV16 or HPV18
With；
The application in detection HPV of X11, described reagent set 1 or described reagent set 2；
The application in examination HPV patient of X12, described reagent set 1 or described reagent set 2；
The application in detection HPV HPV16 and/or HPV18 of X13, described system；
The application in detection HPV of X14, described system；
The application in examination HPV patient of X15, described system.
In above-mentioned application, described product can be reagent or kit.
For solving above-mentioned technical problem, present invention also offers following H1) or H2) or H3) method:
H1) method of detection or auxiliary detection HPV HPV16, comprising: the genomic DNA with testing sample is
Template, carries out LAMP with described reagent set 1, determines in described testing sample according to the product in LAMP reaction system and is
No containing HPV HPV16 or whether be HPV HPV16；
H2) method of detection or auxiliary detection HPV HPV18, comprising: the genomic DNA with testing sample is
Template, carries out LAMP with described reagent set 2, determines in described testing sample according to the product in LAMP reaction system and is
No containing HPV HPV18 or whether be HPV HPV18；
H3) method of detection or auxiliary detection HPV HPV16 and HPV18, including described H1) and described H2).
Above-mentioned H1) in, carry out with described reagent set 1 described in the reaction system (by its named system 16) of LAMP
The concentration of HPV16-FIP and described HPV16-BIP can be all 40pmol/25 μ L, the concentration of HPV16-F3 and described HPV16-B3
Can be all 10pmol/25 μ L.In described system 16, the concentration of Bst archaeal dna polymerase can be 8U/25 μ L.The described system 16 of 25 μ L
In the amount of each reagent and concentration concretely: described 2 × LAMP Buffer 12.5 μ L, Bst archaeal dna polymerase 8U, described
HPV16-FIP and each 10pmol of each 40pmol of described HPV16-BIP, described HPV16-F3 and described HPV16-B3, testing sample.
Above-mentioned H1) in, determine in described testing sample whether contain human milk according to the product in LAMP reaction system
Head tumor virus HPV16 or be whether that HPV HPV16 can determine for the change according to described LAMP reaction system color
It whether described testing sample contains HPV HPV16 or whether is HPV HPV16, concretely H1a)
And/or H1b):
H1a) in the sunlight, described LAMP reaction system is from the orange yellow that becomes, and described testing sample contains or candidate contains
Have HPV HPV16 or described testing sample for or candidate be HPV HPV16；Described LAMP reaction system
Color is unchanged, and described testing sample does not contains or candidate does not contains HPV HPV16 or described testing sample and is not
Or candidate is not HPV HPV16；
H1b) under uviol lamp, described LAMP reaction system from the colourless fluorescent yellow that becomes, described testing sample contain or
Candidate contain HPV HPV16 or described testing sample for or candidate be HPV HPV16；Described LAMP is anti-
Answering system color unchanged, described testing sample does not contains or candidate does not contains HPV HPV16 or described and treats test sample
Product are not or candidate is not HPV HPV16.
Above-mentioned H2) in, carry out with described reagent set 2 described in the reaction system (by its named system 18) of LAMP
The concentration of HPV18-FIP and described HPV18-BIP can be all 40pmol/25 μ L, the concentration of HPV18-F3 and described HPV18-B3
Can be all 10pmol/25 μ L.In described system 18, the concentration of Bst archaeal dna polymerase can be 8U/25 μ L.The described system 18 of 25 μ L
In the amount of each reagent and concentration concretely: described 2 × LAMP Buffer 12.5 μ L, Bst archaeal dna polymerase 8U, described
HPV18-FIP and each 10pmol of each 40pmol of described HPV18-BIP, described HPV18-F3 and described HPV18-B3, testing sample.
Above-mentioned H2) in, determine in described testing sample whether contain human milk according to the product in LAMP reaction system
Head tumor virus HPV18 or be whether that HPV HPV18 can determine for the change according to described LAMP reaction system color
It whether described testing sample contains HPV HPV18 or whether is HPV HPV18, concretely H2a)
And/or H2b):
H2a) in the sunlight, described LAMP reaction system is from the orange yellow that becomes, and described testing sample contains or candidate contains
Have HPV HPV18 or described testing sample for or candidate be HPV HPV18；Described LAMP reaction system
Color is unchanged, and described testing sample does not contains or candidate does not contains HPV HPV18 or described testing sample and is not
Or candidate is not HPV HPV18；
H2b) under uviol lamp, described LAMP reaction system from the colourless fluorescent yellow that becomes, described testing sample contain or
Candidate contain HPV HPV18 or described testing sample for or candidate be HPV HPV18；Described LAMP is anti-
Answering system color unchanged, described testing sample does not contains or candidate does not contains HPV HPV18 or described and treats test sample
Product are not or candidate is not HPV HPV18.
Above-mentioned H1) neutralize above-mentioned H2) in carry out the temperature of LAMP reaction and can be all 65 DEG C.The time carrying out LAMP reaction is equal
Can be 60min.Concretely constant-temperature incubation 60min at 65 DEG C, 80 DEG C keep 5min.
In the present invention, Bst archaeal dna polymerase concretely NEB Products, article No. is M0275V.
Above, described under uviol lamp concretely under 365nm wavelength.
Compared with prior art, the present invention has a following beneficial technique effect:
1. equipment is simple: this detection method just can be tested by water-bath or heated at constant temperature equipment, eliminates costliness
Heat circulating equipment and the device such as electrophoresis；
2. easy and simple to handle: all reagent of disposable addition before reaction, do not need before detection additionally to add developer, decrease
The possibility of cross pollution, utilize metal ion and indicator shelters the purpose reaching Visual retrieval with release action.
3. high specificity: the method, for each section of DNA to be measured 4 specific primers of design, is drawn relative to two of PCR
For thing, specifically significantly improving, the probability that false positive occurs is substantially reduced.
4. detection efficiency is high: the detection time used by the method is about one hour, and used by conventional PCR amplification, the time is longer, and one
As need more than two hours, shorten the reaction time, improve the efficiency of detection.
The reagent set of detection HPV HPV16 and HPV18 of the present invention, it is only necessary to DNA is carried out to cell sample
Extract and carry out ring mediated isothermal amplification, can quickly detect whether object to be measured infects HPV16 and HPV18 virus, its inspection
Survey result and be better than cytolgical examination, specifically high, highly sensitive (respectively up to 5 × 104Copy HPV16/ μ L and 4 × 103Copy
HPV18/ μ L), accuracy rate is high (up to 100%).Facilitate the limited area use not possessing large-scale instrument and equipment of medical condition, soon
Fast, easy ground carries out HPV Viral diagnosis, and with low cost, has good actual application prospect.
Brief description
Fig. 1 is the LAMP amplification by detection HPV16 virus under daylight.
Fig. 2 is the LAMP amplification of the virus of HPV16 under ultra violet lamp.
Fig. 3 is the LAMP amplification by detection HPV18 virus under daylight.
Fig. 4 is the LAMP amplification of the virus of HPV18 under ultra violet lamp.
Fig. 5 is the specific testing result under fluorescent light of reagent set 1 and reagent set 2.
Fig. 6 is the specific testing result under uviol lamp of reagent set 1 and reagent set 2.
Fig. 7 is to detect HPV16LAMP amplification sensitivity results under daylight.
Fig. 8 is to detect HPV16LAMP amplification sensitivity results under ultra violet lamp.
Fig. 9 is to detect HPV18LAMP amplification sensitivity results under daylight.
Figure 10 is to detect HPV18LAMP amplification sensitivity results under ultra violet lamp.
Figure 11 is clinical sample LAMP augmentation detection result under daylight.
Figure 12 is clinical sample LAMP augmentation detection result under ultra violet lamp.
Detailed description of the invention
Being further described in detail the present invention below in conjunction with detailed description of the invention, the embodiment being given is only for explaining
The bright present invention, rather than in order to limit the scope of the present invention.
Experimental technique in following embodiment, if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all commercially obtain.
Caski cell in following embodiment and Hela cell are Cancer Hospital of Chinese Academy of Medical Sciences product, wherein,
Caski cell contains HPV16 virus, and Hela cell contains HPV18 virus.
The 1st, embodiment detects the preparation of reagent set of HPV HPV16 and HPV18
The reagent set of detection HPV HPV16 and HPV18, is made up of reagent set 1 and reagent set 2；Become
Set reagent 1 is by the single stranded DNA of entitled HPV16-FIP, the single stranded DNA of entitled HPV16-BIP, the list of entitled HPV16-F3
The single stranded DNA composition of chain DNA and entitled HPV16-B3；
HPV16-FIP is the single stranded DNA that nucleotide sequence is SEQ ID No.1 in sequence table；
HPV16-BIP is the single stranded DNA that nucleotide sequence is SEQ ID No.2 in sequence table；
HPV16-F3 is the single stranded DNA that nucleotide sequence is SEQ ID No.3 in sequence table；
HPV16-B3 is the single stranded DNA that nucleotide sequence is SEQ ID No.4 in sequence table；
Reagent set 2 is by the single stranded DNA, entitled of the single stranded DNA of entitled HPV18-FIP, entitled HPV18-BIP
The single stranded DNA composition of the single stranded DNA of HPV18-F3 and entitled HPV18-B3；
HPV18-FIP is the single stranded DNA that nucleotide sequence is SEQ ID No.5 in sequence table；
HPV18-BIP is the single stranded DNA that nucleotide sequence is SEQ ID No.6 in sequence table；
HPV18-F3 is the single stranded DNA that nucleotide sequence is SEQ ID No.7 in sequence table；
HPV18-B3 is the single stranded DNA that nucleotide sequence is SEQ ID No.8 in sequence table.
Each single stranded DNA independent packaging in the reagent set of detection HPV HPV16 and HPV18.
Embodiment the 2nd, detect the reagent set of HPV HPV16 and HPV18 can be used to detect HPV16 and
HPV18
Extract Caski cell and the genomic DNA of Hela cell respectively with TIANamp Micro DNA Kit, obtain
Caski cell genomic dna and Hela cell genomic dna.
1st, the detection of HPV HPV16
The LAMP reaction system of the detection HPV HPV16 of 25 μ L comprises 2 × LAMP Buffer 12.5 μ L,
Bst archaeal dna polymerase 8U (NEB company, M0275V), HPV16-FIP and HPV16-BIP each 40pmol, HPV16-F3 and HPV16-
The each 10pmol of B3, Caski cell genomic dna 1 μ L.Wherein, in 2 × LAMP Buffer each material in this reaction system
Concentration is respectively as follows: Tris (green skies Bioisystech Co., Ltd) 40mM, KCl 20mM, MgSO4(Beijing Chemical Plant) 16mM,
(NH4)2SO4(Beijing Chemical Plant) 20mM, Tween 20 (ladder is uncommon likes (Shanghai) chemical conversion industrial development Co., Ltd) 0.2%,
Betaine (Sigma-Aldrich company) 1.6M, dNTPs (Sangon Biotech (Shanghai) Co., Ltd.) 2.8mM (four
The concentration planting every kind of dNTP in dNTP is 2.8mM), MnCl2(Beijing Chemical Plant) 1mM, Calcein (Beijing Chemical Plant) 50 μ
M。
If the system without Caski cell genomic dna is as negative control.
Constant-temperature incubation 60min, 80 DEG C of holding 5min, the termination reaction at 65 DEG C by above-mentioned system.
Respectively with the naked eye and under ultra violet lamp, observe the change of testing sample color, if in the sunlight reaction system by
Orange become yellow, illustrate that this testing sample contains HPV16, if reaction system is by nothing (under 365nm) under ultra violet lamp
Discoloration is fluorescent yellow, illustrates that this testing sample contains HPV16, if color is unchanged, then this testing sample does not contains HPV16.Knot
Fruit sees that Fig. 1 and Fig. 2, Fig. 1 are LAMP amplification under daylight, and Fig. 2 is LAMP amplification, Fig. 1 and Fig. 2 under ultra violet lamp
Middle left side is negative control, and right side is the testing result of Caski cell.Result shows, in the sunlight negative control reaction
System color is unchanged, and the reaction system of detection Caski cell is become yellow from orange；Negative control reactant under uviol lamp
Being that color is unchanged, the reaction system of detection Caski cell is become fluorescent yellow from colourless.Illustrate, Caski cell contains
HPV16, it is consistent that this contains HPV16 with known Caski cell, shows, the method can be used to detect HPV16.
2nd, the detection of HPV HPV18
The LAMP reaction system of the detection HPV HPV18 of 25 μ L comprises 2 × LAMP Buffer of step 1
12.5 μ L, Bst archaeal dna polymerase 8U, HPV18-FIP and HPV18-BIP each 40pmol, HPV18-F3 and HPV18-B3 is each
10pmol, Hela cell genomic dna 1 μ L.
If the system without Hela cell genomic dna is as negative control.
Constant-temperature incubation 60min, 80 DEG C of holding 5min, the termination reaction at 65 DEG C by above-mentioned system.
Respectively with the naked eye and under ultra violet lamp, observe the change of testing sample color, if in the sunlight reaction system by
Orange become yellow, illustrate that this testing sample contains HPV18, if reaction system is by nothing (under 365nm) under ultra violet lamp
Discoloration is fluorescent yellow, illustrates that this testing sample contains HPV18, if color is unchanged, then this testing sample does not contains HPV18.Knot
Fruit sees Fig. 3 and Fig. 4, and Fig. 3 is for LAMP amplification under daylight, and Fig. 4 is LAMP amplification under ultra violet lamp, Fig. 3 and
In Fig. 4, left side is negative control, and right side is the testing result of Hela cell.Result shows, negative control is anti-in the sunlight
Answering system color unchanged, the reaction system of detection Hela cell is become yellow from orange；Negative control reaction under uviol lamp
System color is unchanged, and the reaction system of detection Hela cell is become fluorescent yellow from colourless.Illustrate, Hela cell contains
HPV18, it is consistent that this contains HPV18 with known Hela cell, shows, the method can be used to detect HPV18.
Result shows, the reagent set of detection HPV HPV16 and HPV18 of the present invention may be used for people's nipple
Tumor virus HPV16 and the experiment of HPV18 ring mediated isothermal amplification, operating process is easy, and result is easy to observe.
The 3rd, embodiment detects reagent set specific of HPV HPV16 and HPV18
Utilizing reagent set 2 to detect the Caski cell genomic dna in embodiment 2, reaction system will be for implementing
Hela cell genomic dna in the LAMP reaction system of the detection HPV HPV18 of 25 μ L of example 2 step 2 is replaced
For Caski cell genomic dna, other all constant systems (system 1) obtaining.If the body without Hela cell genomic dna
System is as negative control.Constant-temperature incubation 60min, 80 DEG C of holding 5min, the termination reaction at 65 DEG C by above-mentioned two system.
Utilize reagent set 2 to detect the Hela cell genomic dna in embodiment 2, reaction system (system 2) with
Condition is with embodiment 2.
Respectively with the naked eye and under ultra violet lamp, observe the change of testing sample color, it was found that in the sunlight and purple
Under outer light irradiation, the color of negative control and system 1 is all unchanged, and system 2 is become yellow from orange in the sunlight, at uviol lamp
Under become fluorescent yellow from colourless.Showing, negative control and system 1 all do not occur LAMP to react, and there occurs that LAMP is anti-in system 2
Should.
Utilizing reagent set 1 to detect the Hela cell genomic dna in embodiment 2, reaction system will be for implementing
Caski cell genomic dna in the LAMP reaction system of the detection HPV HPV16 of 25 μ L of example 2 step 1 is replaced
For Hela cell genomic dna, other all constant systems (system 3) obtaining.If the body without Caski cell genomic dna
System is as negative control.Constant-temperature incubation 60min, 80 DEG C of holding 5min, the termination reaction at 65 DEG C by above-mentioned two system.
Reagent set 1 is utilized to detect the Caski cell genomic dna in embodiment 2, reaction system (system 4)
With condition with embodiment 2.
Difference with the naked eye and observes the change of testing sample color under ultra violet lamp, it was found that it was found that in day
The color of negative control and system 3 is all unchanged under light and under ultra violet lamp, and system 4 is become yellow from orange in the sunlight,
Become fluorescent yellow from colourless under uviol lamp.Showing, negative control and system 3 all do not occur LAMP to react, and occur in system 4
LAMP reaction.
Result is shown in Fig. 5 and Fig. 6, and wherein, 1 expression system 1,2 represents that system 2,3 represents that system 3,4 expression system 4. shows
Reagent set 1 and reagent set 2 have well specific, there is not the phenomenon of cross reaction, can well identify HPV16
With HPV18.
The 4th, embodiment detects the sensitivity of reagent set of HPV HPV16 and HPV18
With TIANamp Micro DNA Kit respectively from 102、103、104、105、106Individual Caski cell extracts DNA, point
Do not obtain 102、103、104、105With 106Individual Caski cell genomic dna, the Caski cellular genome to this 5 variable concentrations
DNA utilizes reagent set 1 and examines according to the LAMP reaction system of the detection HPV HPV16 of embodiment 2 step 1
Survey, if the system without Caski cell genomic dna is as negative control.
Constant-temperature incubation 60min, 80 DEG C of holding 5min, the termination reaction at 65 DEG C by above-mentioned system respectively.
Respectively with the naked eye and under ultra violet lamp, observe the change of testing sample color, result is as shown in Figure 7 and Figure 8, Fig. 7
With Fig. 8 is followed successively by from left to right negative control, 102、103、104、105With 106The reactant of individual Caski cell genomic dna
System, with 102、103、104、105With 106Individual Caski cell genomic dna be template system in the copy number of HPV16 be respectively 5
×104、5×105、5×106、5×107With 5 × 108Copy/μ L, result shows that the sensitivity of reagent set 1 is 102Individual Caski
Cell genomic dna, i.e. 5 × 104Copy HPV16/ μ L.
With TIANamp Micro DNA Kit respectively from 102、103、104、105、106Individual Hela cell extracts DNA, point
Do not obtain 102、103、104、105With 106Individual Hela cell genomic dna, the Hela cellular genome to this 5 variable concentrations
DNA utilizes reagent set 2 and examines according to the LAMP reaction system of the detection HPV HPV18 of embodiment 2 step 2
Survey, if the system without Hela cell genomic dna is as negative control.
Constant-temperature incubation 60min, 80 DEG C of holding 5min, the termination reaction at 65 DEG C by above-mentioned system respectively.
Difference with the naked eye and observes the change of testing sample color under ultra violet lamp, and result as shown in Figure 9 and Figure 10, is schemed
Be followed successively by from left to right in 9 and Figure 10 negative control, 102、103、104、105With 106The reactant of individual Hela cell genomic dna
System, with 102、103、104、105With 106Individual Caski cell genomic dna be template system in the copy number of HPV18 be respectively 4
×103、4×104、4×105、4×106With 4 × 107Copy/μ L, result shows that the sensitivity of reagent set 2 is 102Individual Hela
Cell genomic dna, i.e. 4 × 103Copy HPV18/ μ L.
Embodiment the 5th, actual sample LAMP tests
(1) extraction of actual sample DNA
Extract the genomic DNA of 18 clinical blood samples with TIANamp Micro DNA Kit, obtain testing sample base
Because of group DNA.
(2) ring mediated isothermal amplification
Reagent set 1 that each testing sample genomic DNA is utilized respectively in embodiment 1 and reagent set 2 are according to enforcement
LAMP system in example 2 and condition carry out LAMP amplification.The system without Caski and Hela cell genomic dna that sets respectively is made
For negative control.
(3) sample result is detected
Result is as shown in Figure 11 and Figure 12 and table 1.In Figure 11 and Figure 12, A16-R16 treats test sample for utilizing reagent set 1
The system that product serial number A-R of each sample (in the table 1) detect, A18-R18 is for utilizing reagent set 2 to testing sample (table 1
Serial number A-R of middle each sample) system that detects, the sample number into spectrum of serial number A-R is followed successively by 0285, and 4669,4771,
4776,4655,4724,4786,7825,0286,7819,7835,7838,0278,1578,4589,4617,7805,7831.Inspection
Surveying result and being summarized in following table, in table 1, " 16 " show that testing sample contains HPV16, and " 18 " show that testing sample contains HPV18,
"-" shows that testing sample does not contains HPV16 and HPV18.This result is consistent with the result detecting clinically, uniformity (i.e. accuracy rate)
Reach 100%.
Table the 1st, sample detection result
| Sequence number | Sample number into spectrum | LAMP result |
| A | 0285 | 16 |
| B | 4669 | 16 |
| C | 4771 | 16 |
| D | 4776 | - |
| E | 4655 | 18 |
| F | 4724 | 18 |
| G | 4786 | 18 |
| H | 7825 | 18 |
| I | 0286 | 16,18 |
| J | 7819 | 18 |
| K | 7835 | 18 |
| L | 7838 | 18 |
| M | 0278 | - |
| N | 1578 | - |
| O | 4589 | 18 |
| P | 4617 | 18 |
| Q | 7805 | 18 |
| R | 7831 | 18 |
Claims (10)
1. detect the reagent set of HPV HPV16 and HPV18, be made up of reagent set 1 and reagent set 2；Described
Reagent set 1 is by the single stranded DNA of entitled HPV16-FIP, the single stranded DNA of entitled HPV16-BIP, entitled HPV16-F3
The single stranded DNA composition of single stranded DNA and entitled HPV16-B3；
Described HPV16-FIP is following a1) to a4) in any one single stranded DNA:
A1) single stranded DNA shown in SEQ ID No.1 in sequence table；
A2) at a1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtains；
A3) and a1) or a2) single stranded DNA that limits has the single stranded DNA of the homogeneity of more than 85%；
A4) under strict conditions with a1) or a2) limit single stranded DNA hybridization single stranded DNA；
Described HPV16-BIP is following b1) to b4) in any one single stranded DNA:
B1) single stranded DNA shown in SEQ ID No.2 in sequence table；
B2) at b1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtains；
B3) and b1) or b2) single stranded DNA that limits has the single stranded DNA of the homogeneity of more than 85%；
B4) under strict conditions with b1) or b2) limit single stranded DNA hybridization single stranded DNA；
Described HPV16-F3 is following c1) to c4) in any one single stranded DNA:
C1) single stranded DNA shown in SEQ ID No.3 in sequence table；
C2) at c1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtains；
C3) and c1) or c2) single stranded DNA that limits has the single stranded DNA of the homogeneity of more than 85%；
C4) under strict conditions with c1) or c2) limit single stranded DNA hybridization single stranded DNA；
Described HPV16-B3 is following d1) to d4) in any one single stranded DNA:
D1) single stranded DNA shown in SEQ ID No.4 in sequence table；
D2) at d1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtains；
D3) and d1) or d2) single stranded DNA that limits has the single stranded DNA of the homogeneity of more than 85%；
D4) under strict conditions with d1) or d2) limit single stranded DNA hybridization single stranded DNA；
Described reagent set 2 is by the single stranded DNA, entitled of the single stranded DNA of entitled HPV18-FIP, entitled HPV18-BIP
The single stranded DNA composition of the single stranded DNA of HPV18-F3 and entitled HPV18-B3；
Described HPV18-FIP is following e1) to e4) in any one single stranded DNA:
E1) single stranded DNA shown in SEQ ID No.5 in sequence table；
E2) at e1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtains；
E3) and e1) or e2) single stranded DNA that limits has the single stranded DNA of the homogeneity of more than 85%；
E4) under strict conditions with e1) or e2) limit single stranded DNA hybridization single stranded DNA；
Described HPV18-BIP is following f1) to f4) in any one single stranded DNA:
F1) single stranded DNA shown in SEQ ID No.6 in sequence table；
F2) at f1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtains；
F3) and f1) or f2) single stranded DNA that limits has the single stranded DNA of the homogeneity of more than 85%；
F4) under strict conditions with f1) or f2) limit single stranded DNA hybridization single stranded DNA；
Described HPV18-F3 is following g1) to g4) in any one single stranded DNA:
G1) single stranded DNA shown in SEQ ID No.7 in sequence table；
G2) at g1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtains；
G3) and g1) or g2) single stranded DNA that limits has the single stranded DNA of the homogeneity of more than 85%；
G4) under strict conditions with g1) or g2) limit single stranded DNA hybridization single stranded DNA；
Described HPV18-B3 is following h1) to h4) in any one single stranded DNA:
H1) single stranded DNA shown in SEQ ID No.8 in sequence table；
H2) at h1) 5 ' ends and/or 3 ' ends add the single stranded DNAs that one or several nucleotides obtains；
H3) and h1) or h2) single stranded DNA that limits has the single stranded DNA of the homogeneity of more than 85%；
H4) under strict conditions with h1) or h2) limit single stranded DNA hybridization single stranded DNA.
2. following P1) or P2):
P1) detect the reagent set of HPV HPV16, be reagent set 1 described in claim 1；
P2) detect the reagent set of HPV HPV18, be reagent set 2 described in claim 1.
3. following Q1) or Q2) or Q3):
Q1) system of HPV HPV16 and HPV18 is detected, including detect HPV described in claim 1
The reagent set of HPV16 and HPV18 and strand displacement type archaeal dna polymerase；
Q2) system of HPV HPV16 is detected, including reagent set 1 described in claim 1 and described strand displacement type
Archaeal dna polymerase；
Q3) system of HPV HPV18 is detected, including reagent set 2 described in claim 1 and described strand displacement type
Archaeal dna polymerase.
4. system according to claim 3, it is characterised in that: described system is ring mediated isothermal amplification reagent or reagent
Box.
5. the system according to claim 3 or 4, it is characterised in that: described strand displacement type archaeal dna polymerase is that Bst DNA gathers
Synthase.
6. the preparation method of reagent set described in claim 1, including by HPV16-FIP described in claim 1, described
HPV16-BIP, described HPV16-F3, described HPV16-B3, described HPV18-FIP, described HPV18-BIP, described HPV18-F3
Individually pack with described HPV18-B3.
7. the preparation method of reagent set described in claim 2, is following M1) or M2):
M1) include HPV16-FIP described in claim 1, described HPV16-BIP, described HPV16-F3 and described HPV16-
B3 individually packs；
M2) include HPV18-FIP described in claim 1, described HPV18-BIP, described HPV18-F3 and described HPV18-
B3 individually packs.
8. the preparation method of the arbitrary described system of claim 3-5, including individually pack each single stranded DNA with each reagent.
9. arbitrary purposes in following X1-X15:
Application in preparation detection HPV HPV16 and HPV18 product for the reagent set described in X1, claim 1；
Application in preparation detection HPV product for the reagent set described in X2, claim 1；
Application in preparation examination HPV patient product for the reagent set described in X3, claim 1；
Application in preparation detection HPV HPV16 or HPV18 product for the reagent set described in X4, claim 2；
Application in preparation detection HPV product for the reagent set described in X5, claim 2；
Application in preparation examination HPV patient product for the reagent set described in X6, claim 2；
Application in detection HPV HPV16 and HPV18 for the reagent set described in X7, claim 1；
Application in detection HPV for the reagent set described in X8, claim 1；
Application in examination HPV patient for the reagent set described in X9, claim 1；
Application in detection HPV HPV16 or HPV18 for the reagent set described in X10, claim 2；
Application in detection HPV for the reagent set described in X11, claim 2；
Application in examination HPV patient for the reagent set described in X12, claim 2；
Application in detection HPV HPV16 and/or HPV18 for arbitrary described system in X13, claim 3-5；
Application in detection HPV for arbitrary described system in X14, claim 3-5；
Application in examination HPV patient for arbitrary described system in X15, claim 3-5.
10. following H1) or H2) or H3) method:
H1) method of detection or auxiliary detection HPV HPV16, comprising: with the genomic DNA of testing sample as mould
Plate, carries out LAMP with reagent set 1 described in claim 1, treats according to the product in LAMP reaction system determines
It whether test sample product contain HPV HPV16 or whether is HPV HPV16；
H2) method of detection or auxiliary detection HPV HPV18, comprising: with the genomic DNA of testing sample as mould
Plate, carries out LAMP with reagent set 2 described in claim 1, treats according to the product in LAMP reaction system determines
It whether test sample product contain HPV HPV18 or whether is HPV HPV18；
H3) method of detection or auxiliary detection HPV HPV16 and HPV18, including described H1) and described H2).
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| EP4028560A4 (en) * | 2019-09-09 | 2023-10-25 | Genomtec SA | Primer sets for the detection of human papillomavirus type 16 (hpv16) and human papillomavirus type 18 (hpv18), the method of detecting hpv16 and hpv18 infections, the use of a primer set for the detection of hpv16 and hpv18 infections |
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| EP4028560A4 (en) * | 2019-09-09 | 2023-10-25 | Genomtec SA | Primer sets for the detection of human papillomavirus type 16 (hpv16) and human papillomavirus type 18 (hpv18), the method of detecting hpv16 and hpv18 infections, the use of a primer set for the detection of hpv16 and hpv18 infections |
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