CN109943663A - For recombinase amplification detection method, test strips and its application of 18 genotype of HPV 16 and HPV - Google Patents
For recombinase amplification detection method, test strips and its application of 18 genotype of HPV 16 and HPV Download PDFInfo
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Abstract
The invention discloses a kind of recombinase amplification detection method, test strips and its applications for 18 genotype of HPV 16 and HPV.The sequence of the primer pair for 16 genotype of HPV is respectively as shown in SEQ ID NO.1 and SEQ ID NO.2.For 18 genotype of HPV primer pair sequence respectively as shown in SEQ ID NO.3 and SEQ ID NO.4.The dual lateral flow chromatograph test strip includes support plate, the upper surface of the support plate includes the loading area set gradually, mark zone, drawn area and the suction zones for being combined with the latex beads that Streptavidin is coupled, and is provided in the drawn area respectively in connection with detection line T1, T2 and the nature controlling line C for having FITC antibody, DigiTAb and secondary antibody.Detection method specificity of the invention is good, high sensitivity, specific good, reproducible, easy to operate, strong applicability.
Description
Technical field
The present invention relates to a kind of detection methods of 18 genotype of HPV 16 and HPV, more particularly to are directed to HPV 16 and HPV
The primer pair of 18 genotype, one kind detecting 16 He of HPV based on latex beads label principle and while being based on RPA amplification technique
The dual lateral flow chromatograph test strip and corresponding recombinase amplification detection method of 18 genotype of HPV, belong to biomedical neck
Domain.
Background technique
The early diagnosis and prevention of cancer are always clinical detection field urgent problem to be solved.As Womankind Worldwide group
The cancer that middle incidence and lethality are number four, the detection and prevention of cervical carcinoma are constantly subjected to the concern of whole world people.According to
Estimation, in 2018, the new cases of whole world cervical carcinoma were 570000, death 311000.Existing research shows that people
The main reason for papillomavirus (HPV) infection leads to cervical carcinoma, cervical carcinoma and high risk HPV gene more than 99%
Type is related.It has been demonstrated that the screening method based on HPV can more effectively prevent cervical carcinoma than Pap smear, it is based on
The method of HPV genotype identification is expected to become the primary method that cervical carcinoma early diagnoses and prevents.Studies have shown that high-risk HPV base
Because type has 12 kinds, including HPV 16,18,31,33,35,39,45,51,52,56,58 and 59 types.In the cases of cervical cancer of detection
In, using the recall rate of HPV 16 as highest, followed by HPV 18.Comprehensively consider, develops one kind quickly, reliably, conveniently and simultaneously
The method of screening and the high-risk HPV 16 of detection and 18 genotype of HPV is of great significance.
So far, the developed method for detecting HPV genotype has very much, but only seldom a part is available
In clinical diagnosis.Wherein, insufficient based on immunology and morphologic detection method sensitivity, and cannot distinguish between specific HPV gene
The type of type, therefore it is not able to satisfy the demand of clinical detection and prevention；Two generation hybrid capture technologies can delicately detect 13
Kind HPV genotype, but it is easy to produce cross reaction, so as to cause false positive or the result of testing result inaccuracy.Polymerase
Chain reaction (PCR) technology has benefited from the amplification ability of its shirtsleeve operation and index, it has also become HPV genotype screen and detection
Main means.However hybridization or specific apparatus inspection after being hydrolyzed dependent on the PCR instrument with function of temperature control, electrophoresis, amplified production
The characterization method of survey significantly limits the extensive use of round pcr.Other technologies, such as microarray technology and real time aggregation enzyme chain
React (real-time PCR), it is also desirable to special and expensive equipment, and these equipment may the limited laboratory of resource without
Method obtains, while professional operator being needed to carry out coherent detection work.Therefore, one kind is invented simply, conveniently, sensitively
The high-risk genotype screen of HPV and detection means, for effective development of the cervical carcinoma screening work of base and the deficient area of hardware
It is of great significance.
Summary of the invention
The main object of the present invention provides a kind of drawing for HPV 16 and 18 genotype of HPV aiming at the above status
Object pair, to overcome deficiency in the prior art.
Another main purpose of the invention is to provide recombinase that is a kind of while detecting 18 genotype of HPV 16 and HPV
Amplification detection method.
Another main purpose of the invention is to provide dual side that is a kind of while detecting 18 genotype of HPV 16 and HPV
To stream chromatograph test strip.
Another object of the present invention, which also resides in, to be provided a kind of while detecting the dual lateral of 18 genotype of HPV 16 and HPV
Fluid layer analyses detection method.
For realization aforementioned invention purpose, the technical solution adopted by the present invention includes:
The embodiment of the invention provides a kind of primer pairs for 16 genotype of HPV comprising the first primer and second is drawn
Object, the first primer, the sequence of the second primer are respectively as shown in SEQ ID NO.1 and SEQ ID NO.2, and described first draws
5 ' ends of object are marked through biotin labeling, 5 ' ends of second primer through FITC.
The embodiment of the invention also provides a kind of primer pairs for 18 genotype of HPV comprising third primer and the 4th
Primer, the third primer, the 4th primer sequence respectively as shown in SEQ ID NO.3 and SEQ ID NO.4, and the third
5 ' ends of primer are through biotin labeling, and 5 ' ends of the 4th primer are through digoxigenin labeled.
The embodiment of the invention also provides a kind of dual lateral flow chromatographies for detecting 18 genotype of HPV 16 and HPV simultaneously
Test strips, including support plate, the upper surface of the support plate include the loading area set gradually along direction initialization, are combined with strepto-
Mark zone, drawn area and the suction zones of the latex beads of Avidin coupling, are provided with detection line T1, T2 and matter in the drawn area
Line C is controlled, and the detection line T1, T2 and nature controlling line C are respectively in connection with having FITC antibody, DigiTAb and secondary antibody.
The embodiment of the invention also provides it is aforementioned while detect HPV 16 and 18 genotype of HPV dual lateral fluid layer
Analyse the preparation method of test strips comprising:
Streptavidin and the latex beads of carboxyl modified are coupled, the latex beads of Streptavidin coupling are made；
The latex beads that Streptavidin is coupled are applied to mark zone, by FITC antibody, DigiTAb and secondary antibody point
Detection line T1, T2 and the nature controlling line C of drawn area are not made；And
Loading area, mark zone, drawn area and suction zones are set gradually on the supporting plate along direction initialization, are obtained described
Dual lateral flow chromatograph test strip.
The embodiment of the invention also provides above-mentioned for the primer pair of 16 genotype of HPV, for 18 genotype of HPV
Primer pair or the dual lateral flow chromatograph test strip for detecting 18 genotype of HPV 16 and HPV simultaneously detect 16 He of HPV in preparation
Purposes in the product of 18 genotype of HPV.
Further, using the recombinase amplification detection method packet of 18 genotype of the product testing HPV 16 and HPV
It includes:
Extract the DNA in HPV sample to be detected；
RPA expansion is carried out using the primer pair above-mentioned for 16 genotype of HPV, the primer pair for 18 genotype of HPV
Increase, obtains the double stranded DNA product of difunctionalization；
Dual lateral flow chromatograph test strip above-mentioned is added in obtained double stranded DNA product to detect, according to detection line
Colour developing result realizes the qualitative or quantitative detection of HPV 16 or HPV 18 in HPV sample to be detected.
Compared with prior art, the invention has the advantages that
1) present invention using two component safety pins to the specific function primer pair of 18 genotype of HPV 16 and HPV, according to
In the presence of the high-risk HPV 16 of cervical carcinoma or 18 genotype of HPV, corresponding both ends functionalization is obtained by dual RPA amplification respectively
Double stranded DNA product, using different double stranded DNA products bridge joint act on, Streptavidin be coupled latex beads connect respectively
It is connected in corresponding detection line, to realize the mesh for carrying out the judgement of testing result naked eyes or card reading judgement by latex beads color
's.It is different according to the content of specific high-risk HPV, it is connected to the quantity of the latex beads of the Streptavidin coupling in detection line not
Together, and then keep the depth for detecting line color different, it is established that detect the pass between the depth intensity of line color and specific HPV content
System, and then realize that primary amplification, sample-adding carry out spirit to HPV 16 high-risk in cervical carcinoma clinical sample and 18 genotype of HPV simultaneously
The purpose of quick screening and detection；
2) detection method specificity of the invention is good, high sensitivity (102-103Copy number), specificity is good, and it is reproducible,
Easy to operate, strong applicability can be detected simultaneously by HPV 16DNA and the HPV 18DNA of 500 copies；
3) compared to traditional round pcr, the recombination enzymatic polymerization amplification technique (RPA) that the present invention uses has the excellent of brilliance
Gesture, such as: the amplification condition of isothermal (37-43 DEG C) avoids the use of expensive temperature control instrument, gets rid of the constraint of instrument, more
Suitable for on-site test；The extension for avoiding proliferation time caused by temperature change, highly shortened detection time；It uses
Similar to the primer of PCR, design is simple, low in cost, and allows Multiple detection；Packaging is simple, can exist in dry powder form,
It is on-demand conducive to preservation；It is easy to operate, do not need special technique drill etc.；
4) detection method of the invention does not need special expensive instrument, it is only necessary to common water-bath and corresponding dual side
To stream chromatograph test strip, quick, accurate, the screening simultaneously simultaneously to the high-risk HPV of cervical carcinoma 16 and HPV 18 can be realized, it is suitable
Carry out relevant cervical cancer screening work for base and the deficient area of detection hardware resource.
Detailed description of the invention
Fig. 1 is to carry out screening and detection to actual clinical sample simultaneously using lateral chromatography method in the embodiment of the present invention 1
The schematic diagram of cervical carcinoma high-risk HPV 16 and 18 genotype of HPV.
Specific embodiment
Existing deficiency and defect, this case invention are applied in view of existing cervical carcinoma HPV genotype screen and detection method
People is studied for a long period of time and is largely practiced, and is able to propose technical solution of the present invention, is established a kind of former based on latex beads label
Reason and high sensitivity, the specificity that cervical carcinoma high-risk HPV 16 and 18 genotype of HPV are detected while based on RPA amplification technique
Good and dual lateral flow chromatograph test strip and new detecting method easily to operate, realize it is simpler, sensitive, accurately screening and
Detect the high-risk HPV 16 of cervical carcinoma and 18 genotype of HPV.
The technical solution, its implementation process and principle etc. will be further explained as follows.
More detailed explanation will hereafter be made to technical solution of the present invention.It is understood, however, that in model of the present invention
In enclosing, above-mentioned each technical characteristic of the invention and it is ok between each technical characteristic specifically described in below (e.g. embodiment)
It is combined with each other, to form a new or preferred technical solution.Due to space limitations, I will not repeat them here.
The one aspect of the embodiment of the present invention provides a kind of primer pair for 16 genotype of HPV comprising first draws
Object and the second primer, the first primer, the sequence of the second primer respectively as shown in SEQ ID NO.1 and SEQ ID NO.2, and
5 ' ends of the first primer are marked through biotin labeling, 5 ' ends of second primer through FITC.
Further, the primer for 16 Serotype-dependent of HPV, can be realized to 16 gene of target HPV
Specificity, effectively amplification.
Further, the first primer is biotin functionalized upstream primer, and particular sequence indicates are as follows: 5 '-
BIO-GAACTTTGCaTTATTCGGGATT-3’。
Further, second primer is the downstream primer of FITC functionalization, and particular sequence indicates are as follows: 5 '-FITC-
CTTTGCTTaTTCTTCAGGAAC-3’。
The embodiment of the present invention another aspect provides a kind of primer pairs for 18 genotype of HPV comprising third
Primer and the 4th primer, the third primer, the 4th primer sequence respectively as shown in SEQ ID NO.3 and SEQ ID NO.4,
And 5 ' ends of the third primer, through biotin labeling, 5 ' ends of the 4th primer are through digoxigenin labeled.
Further, the primer for 18 Serotype-dependent of HPV, can be realized to 18 gene of target HPV
Specificity, effectively amplification.
Further, the third primer is biotin functionalized upstream primer, and particular sequence indicates are as follows: 5 '-
BIO-TGTCACGAaGCAACTTAAGAC-3’。
Further, the 4th primer is the downstream primer of digoxin functionalization, and particular sequence indicates are as follows: 5 '-
DIG-CTGGCTTCAaCACTATACAACAA-3’。
The biotin functionalized upstream primer provided by the invention, the downstream of FITC functionalization or digoxin functionalization
Primer is directed to the primer pair of HPV16 and HPV18 for specific designs, convenient for generating both ends functionalization amplified production.
Target HPV genotype is effectively expanded the present invention is based on the combination of above-mentioned upstream primer, downstream primer pair, realization
Meanwhile realizing the both ends of amplified production while marking, it is the lateral flow chromatography screening simultaneously based on amplified production and detection HPV
16 and 18 key Design.
The other side of the embodiment of the present invention additionally provides a kind of while detecting the double of 18 genotype of HPV 16 and HPV
Heavy side is to stream chromatograph test strip, including support plate, the upper surface of the support plate include the loading set gradually along direction initialization
Area, be combined with Streptavidin coupling latex beads mark zone, drawn area and suction zones, be provided with inspection in the drawn area
Survey line T1, T2 and nature controlling line C, and the detection line T1, T2 and nature controlling line C respectively in connection with have FITC antibody, DigiTAb and
Secondary antibody.
In some embodiments, the FITC antibody is selected from the monoclonal antibody of FITC, and concentration is 1.0~3.0mg/mL.
In some embodiments, the latex beads are blue, and partial size is 100~300nm.
Further, the concentration of the Streptavidin is 0.5~2.0mg/mL.
In some embodiments, the DigiTAb is selected from the monoclonal antibody of digoxin, and concentration is 0.3~1.2mg/
mL。
In some embodiments, the secondary antibody is selected from the sheep anti-mouse antibody of Streptavidin, and concentration is 1.0~4.0mg/
mL。
Further, the support plate includes not absorbing water or hydrophobic cardboard or plastic plate, but be not limited only to this.
Further, the material in the loading area includes the glass fibre cotton or polyester film weak to sample adsorption capacity, but not
It is only limitted to this.
Further, the mark zone is the latex beads for being added dropwise and having pre-fixed Streptavidin coupling.
Further, the material of the drawn area includes nitrocellulose filter or pure cellulose film, but is not limited only to this.
Further, FITC antibody, DigiTAb and the two corresponding anti-solution of drawn area suitable concentration make respectively
Detection line T1 and T2 and nature controlling line C.
Further, the suction zones are bibulous filter paper, but are not limited only to this.
The other side of the embodiment of the present invention additionally provides a kind of while detecting the double of 18 genotype of HPV 16 and HPV
Preparation method of the heavy side to stream chromatograph test strip comprising:
Streptavidin and the latex beads of carboxyl modified are coupled, the latex beads of Streptavidin coupling are made；
The latex beads that Streptavidin is coupled are applied to mark zone, by FITC antibody, DigiTAb and secondary antibody point
Detection line T1, T2 and the nature controlling line C of drawn area are not made；And
Loading area, mark zone, drawn area and suction zones are set gradually on the supporting plate along direction initialization, are obtained described
Dual lateral flow chromatograph test strip.
In some preferred embodiments, the preparation method specifically includes: making comprising latex beads, 1- (3- diformazan ammonia
Base propyl) -3- ethyl-carbodiimide hydrochloride and n-hydroxysuccinimide mixed system in room temperature be protected from light activation 20~
Streptavidin is added later and is incubated for, adds BSA solution, obtains the latex beads of Streptavidin coupling by 60min.
Further, carboxyl contained by the latex beads surface, 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide salt
The molar ratio of hydrochlorate and n-hydroxysuccinimide is 1:5:5~1:20:20.
It is described based on latex beads and based on the same of dual RPA amplification among some more specifically case study on implementation
When detection HPV 16 and 18 genotype of HPV dual lateral flow chromatograph test strip preparation method the following steps are included:
1) preparation of the latex beads of Streptavidin coupling: Streptavidin and the latex beads of carboxyl modified are coupled
Prepare the blue latex microballoon of Streptavidin coupling；
2) preparation of mark zone: each mark zone is added dropwise the blue latex microballoon of appropriate Streptavidin coupling, and 25~35
It dries and saves backup in DEG C baking oven；
3) printing of drawn area: the FITC antibody of suitable concentration, DigiTAb and secondary antibody are printed respectively with spray film instrument
It is spare after 25~35 DEG C of oven dryings to the detection line T1 and T2 and nature controlling line C of drawn area；
4) assembling of test strips: loading area, mark zone, drawn area and suction zones are successively assembled on the supporting plate, composition
The dual lateral flow chromatograph test strip of HPV 16 and 18 genotype of HPV are detected while based on latex beads.
Further, the preparation method for the latex beads that Streptavidin is coupled in the step 1) specifically includes:
Activation: 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide salt is added in the latex beads solution toward appropriate concentration
The aqueous solution of hydrochlorate (EDC) and n-hydroxysuccinimide (NHS), makes the molar ratio of latex beads surface carboxyl groups Yu EDC and NHS
For 1:5:5~1:20:20, room temperature is protected from light 20~60min of activation；
Coupling: supernatant is removed in centrifugation (80000~12000g, 4 DEG C, 5~15min), and PB (10mM, pH 7.0) buffering is added
Liquid is resuspended, and suitable Streptavidin is added, and is incubated at room temperature 2h；
Closing: being added the BSA solution that mass volume ratio is 10~20%, is incubated at room temperature 1h；
Be resuspended: supernatant is removed in centrifugation (80000~12000g, 4 DEG C, 5~15min), be added mass volume ratio be 0.5~
1.5% BSA solution is resuspended, 4 DEG C of preservations.
Further, the latex beads are blue, and partial size is 100~300nm.
The other side of the embodiment of the present invention additionally provides the primer pair above-mentioned for 16 genotype of HPV, is directed to
The primer pair of 18 genotype of HPV detects the dual lateral flow chromatograph test strip of 18 genotype of HPV 16 and HPV in system simultaneously
Purposes in standby detection HPV 16 and the product of 18 genotype of HPV.
Further, using the recombinase amplification detection method packet of 18 genotype of the product testing HPV 16 and HPV
It includes:
Extract the DNA in HPV sample to be detected；
RPA expansion is carried out using the primer pair above-mentioned for 16 genotype of HPV, the primer pair for 18 genotype of HPV
Increase, obtains the double stranded DNA product of difunctionalization；
Dual lateral flow chromatograph test strip above-mentioned is added in obtained double stranded DNA product to detect, according to detection line
Colour developing result realizes the qualitative or quantitative detection of HPV 16 or HPV 18 in HPV sample to be detected.
In some embodiments, the recombinase amplification detection method specifically includes: extracting HPV to be detected using paramagnetic particle method
The DNA of sample carries out RPA amplification using the functionalization primer pair for being directed to 18 Serotype-dependent of HPV 16 and HPV respectively, obtains
Obtain the double stranded DNA product of difunctionalization；Amplified production is directly added drop-wise to the loading area of test strips, flows through mark zone and scribing line
Area is connected to latex beads in the corresponding detection line in drawn area using the double stranded DNA product of difunctionalization, passes through naked eyes
The color of latex beads in detection line is directly observed, while realizing the sensitive screening and inspection to 18 genotype of HPV 16 and HPV
It surveys.
In some embodiments, using the recombinase augmentation detection side of 18 genotype of the product testing HPV 16 and HPV
Method specifically includes: making HPV sample dissociation to be detected using cell pyrolysis liquid and Proteinase K Solution, adds the magnetic of carboxyl modified
Pearl and coating buffer, the obtained washed liquid washing of DNA, are resuspended with re-suspension liquid later, obtain the DNA of detection HPV sample.
Further, the magnetic bead that the paramagnetic particle method uses is the magnetic nano-particle of carboxyl modified, the specific scheme is that taking 20
300~700 μ L cell pyrolysis liquids and 10~30 μ L Proteinase K Solutions, which are added, in~100 μ L HPV samples cracks sample completely；Add
Magnetic bead and the absorption of 100~300 μ L coating buffers, separation HPV DNA for entering 5~30 μ g carboxyl modifieds, through 300~1000 μ L cleaning solutions
30~100 μ L re-suspension liquids are added after washing to be resuspended, taking supernatant is mentioned DNA.
Further, the lysate is to contain Tris-HCl buffer, ethylenediamine tetra-acetic acid (EDTA), sodium chloride
(NaCl) and lauryl sodium sulfate (SDS)；Wherein the pH value of Tris-HCl buffer be 7.0~8.0, concentration be 20~
50mmol/L；The concentration of EDTA is 5~20mmol/L, and the concentration of the sodium chloride (NaCl) is 200~1000mmol/L, described
The mass volume ratio of lauryl sodium sulfate and lysate is 0.5~2.5g:100mL, also that is, the mass volume ratio of SDS is 0.5
~2.5%.
Further, final concentration of 0.5~3mg/mL of the Proteinase K Solution.
Further, the coating buffer mainly includes ingredient polyethylene glycol (PEG) and sodium chloride (NaCl), wherein described
The average matter average molecular weight of polyethylene glycol is 400~20000, for example, can be 400,600,800,1000,2000,6000,
10000 or 20000, the mass volume ratio of the polyethylene glycol and coating buffer is 5~30g:100mL, also that is, the mass body of PEG
Product is than being 5~30%, and the concentration of sodium chloride is 0.5~3mol/L in the coating buffer.
Further, the cleaning solution includes the aqueous solution that ethanol content is 55~80vt%.
Further, the main component of the re-suspension liquid includes Tris-HCl buffer and ethylenediamine tetra-acetic acid (EDTA),
Wherein the pH value of Tris-HCl buffer is 7.0~8.0, and concentration is 10~50mmol/L, the ethylenediamine tetra-acetic acid (EDTA)
Concentration be 0.5~10mmol/L.
In conclusion the implementation steps of detection method are as follows: (1) system of the latex beads of Streptavidin coupling
It is standby；(2) embedding of drawn area；(3) production of test strips；(4) extraction of HPV sample DNA and RPA amplification；(5) sample detection.
Present invention uses two pairs to be directed to 18 Serotype-dependent functionalization primer pair of HPV 16 and HPV respectively, according in sample to be tested
When target HPV type and content difference, the product of difunctionalization expanded is different, detection line (T1 and T2) in test strips
Shade is not also identical, to set up the relationship between the depth and corresponding HPV content of detection line color, and then realizes
Simultaneously to the purpose of 18 genotype Sensitive Detection of HPV 16 in cervical carcinoma clinical sample and HPV, opened suitable for basic medical unit
Open up related screening.
Below in conjunction with attached drawing and several preferred embodiments the technical solution of the present invention is further explained explanation, but its
In experiment condition and setup parameter be not construed as the limitation to basic technical scheme of the present invention.And protection scope of the present invention
It is not limited to the following embodiments.
Embodiment 1
Dual lateral flow chromatography detection method packet that is provided in this embodiment while detecting 18 genotype of HPV 16 and HPV
Include following steps:
(1) preparation of the latex beads of Streptavidin coupling
Activation: 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide salt is added in the latex beads solution toward appropriate concentration
The aqueous solution of hydrochlorate (EDC) and n-hydroxysuccinimide (NHS), makes the molar ratio of latex beads surface carboxyl groups Yu EDC and NHS
For 1:5:5, room temperature is protected from light activation 60min；
Coupling: supernatant is removed in centrifugation (8000g, 4 DEG C, 15min), and PB (10mM, pH value 7.0) buffer is added and is resuspended, is added
Suitable Streptavidin is incubated at room temperature 2h；
Closing: being added the BSA solution that mass volume ratio is 10%, is incubated at room temperature 1h；
Be resuspended: supernatant is removed in centrifugation (8000g, 4 DEG C, 15min), and the BSA solution that mass volume ratio is 0.5% is added and is resuspended,
4 DEG C of preservations；
The preparation of mark zone: each mark zone is added dropwise the latex beads of suitable marked by streptavidin, in 25 DEG C of baking ovens
It dries and saves backup.
(2) FITC antibody, DigiTAb and two corresponding anti-solution the embedding of drawn area: are printed on inspection respectively using spray film instrument
Survey line T1 and T2 and nature controlling line C is dried and is saved backup in 25 DEG C of baking ovens.
(3) loading area, mark zone, drawn area and suction zones the production of test strips: are assembled into support plate in order respectively
On.
(4) extraction of HPV sample DNA to be detected and RPA amplification
DNA is extracted using paramagnetic particle method, the carrier extracted using the magnetic nano-particle of carboxyl modified as DNA, specific real
Apply mode are as follows: it is added 20 μ L HPV samples, 300 μ L cell pyrolysis liquids and 10 μ L Proteinase K Solutions in clean centrifuge tube, 60
DEG C be incubated for 15min；Room temperature is cooling, and the magnetic bead and 100 μ L coating buffers of 5 μ g carboxyl modifieds is added, rotates 5min at room temperature；Magnetism is inhaled
It is attached, supernatant is removed, 300 μ L cleaning solutions are added and wash twice；Magnetic absorption removes supernatant, and 30 μ L re-suspension liquids are added and are resuspended, magnetism is inhaled
Attached, taking supernatant is mentioned DNA.
Dual RPA reacts detailed process are as follows: 10 μM of HPV is added in 29.5 μ L lysis hybridization buffers of RPA powdered reagent
16 upstream and downstream primers each 0.8 μ L, 10 μM of each 0.4 μ L of 18 upstream and downstream primer of HPV, 2 μ L of DNA profiling, moisturizing add to 47.5 μ L
Enter after the 2.5 μ L of magnesium acetate solution of 280mM 40 DEG C of reaction 20min immediately.
Wherein, the particular sequence of 16 upstream primer of HPV of use are as follows: 5 '-BIO-GAACTTTGCaTTATTCGGGATT-
3 ', the particular sequence of downstream primer are as follows: 5 '-FITC-CTTTGCTTaTTCTTCAGGAAC-3 '.18 upstream primer of HPV of use
Particular sequence are as follows: 5 '-BIO-TGTCACGAaGCAACTTAAGAC-3 ', the particular sequence of downstream primer are as follows: 5 '-DIG-
CTGGCTTCAaCACTATACAACAA-3’。
(5) sample detection
RPA product in the present embodiment step (4) is directly added drop-wise to the loading area of test strips after sample-loading buffer dilutes,
5min is reacted at room temperature, shoots picture using slr camera, and carry out using intensity of the ImageJ software to detection line T1 and T2
Analysis.Using the screening simultaneously of lateral chromatography method and the high-risk HPV 16 of detection cervical carcinoma and 18 genotype of HPV in the present embodiment
Sensitivity schematic diagram please refers to shown in Fig. 1.
Embodiment 2
Dual lateral flow chromatography detection method packet that is provided in this embodiment while detecting 18 genotype of HPV 16 and HPV
Include following steps:
(1) preparation of the latex beads of Streptavidin coupling
Activation: 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide salt is added in the latex beads solution toward appropriate concentration
The aqueous solution of hydrochlorate (EDC) and n-hydroxysuccinimide (NHS), makes the molar ratio of latex beads surface carboxyl groups Yu EDC and NHS
For 1:10:10, room temperature is protected from light activation 40min；
Coupling: supernatant is removed in centrifugation (10000g, 4 DEG C, 10min), and PB (10mM, pH value 7.0) buffer is added and is resuspended, adds
Enter suitable Streptavidin, is incubated at room temperature 2h；
Closing: being added the BSA solution that mass volume ratio is 15%, is incubated at room temperature 1h；
Be resuspended: supernatant is removed in centrifugation (10000g, 4 DEG C, 10min), and the BSA solution that mass volume ratio is 1% is added and is resuspended, and 4
DEG C save；
The preparation of mark zone: each mark zone is added dropwise the latex beads of suitable marked by streptavidin, in 30 DEG C of baking ovens
It dries and saves backup.
(2) FITC antibody, DigiTAb and two corresponding anti-solution the embedding of drawn area: are printed on inspection respectively using spray film instrument
Survey line T1 and T2 and nature controlling line C is dried and is saved backup in 30 DEG C of baking ovens.
(3) loading area, mark zone, drawn area and suction zones the production of test strips: are assembled into support plate in order respectively
On.
(4) extraction of HPV sample DNA to be detected and RPA amplification
DNA is extracted using paramagnetic particle method, the carrier extracted using the magnetic nano-particle of carboxyl modified as DNA, specific real
Apply mode are as follows: it is added 50 μ L HPV samples, 500 μ L cell pyrolysis liquids and 20 μ L Proteinase K Solutions in clean centrifuge tube, 60
DEG C be incubated for 15min；Room temperature is cooling, and the magnetic bead and 200 μ L coating buffers of 15 μ L carboxyl modifieds is added, rotates 5min at room temperature；It is magnetic
Supernatant is removed in absorption, and 600 μ L cleaning solutions are added and wash twice；Magnetic absorption removes supernatant, and 50 μ L re-suspension liquids are added and are resuspended, magnetism is inhaled
Attached, taking supernatant is mentioned DNA.
Dual RPA reacts detailed process are as follows: 10 μM of HPV is added in 29.5 μ L lysis hybridization buffers of RPA powdered reagent
16 upstream and downstream primers each 0.8 μ L, 10 μM of each 0.4 μ L of 18 upstream and downstream primer of HPV, 2 μ L of DNA profiling, moisturizing add to 47.5 μ L
Enter after the 2.5 μ L of magnesium acetate solution of 280mM 40 DEG C of reaction 20min immediately.
Wherein, the particular sequence of 16 upstream primer of HPV of use are as follows: 5 '-BIO-GAACTTTGCaTTATTCGGGATT-
3 ', the particular sequence of downstream primer are as follows: 5 '-FITC-CTTTGCTTaTTCTTCAGGAAC-3 '.18 upstream primer of HPV of use
Particular sequence are as follows: 5 '-BIO-TGTCACGAaGCAACTTAAGAC-3 ', the particular sequence of downstream primer are as follows: 5 '-DIG-
CTGGCTTCAaCACTATACAACAA-3’。
(5) sample detection
RPA product in the present embodiment step (4) is directly added drop-wise to the loading area of test strips after sample-loading buffer dilutes,
5min is reacted at room temperature, shoots picture using slr camera, and carry out using intensity of the ImageJ software to detection line T1 and T2
Analysis.Using the screening simultaneously of lateral chromatography method and the high-risk HPV 16 of detection cervical carcinoma and 18 genotype of HPV in the present embodiment
Sensitivity schematic diagram please refers to shown in Fig. 1.
Embodiment 3
Dual lateral flow chromatography detection method packet that is provided in this embodiment while detecting 18 genotype of HPV 16 and HPV
Include following steps:
(1) preparation of the latex beads of Streptavidin coupling
Activation: 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide salt is added in the latex beads solution toward appropriate concentration
The aqueous solution of hydrochlorate (EDC) and n-hydroxysuccinimide (NHS), makes the molar ratio of latex beads surface carboxyl groups Yu EDC and NHS
For 1:20:20, room temperature is protected from light activation 20min；
Coupling: supernatant is removed in centrifugation (12000g, 4 DEG C, 5min), and PB (10mM, pH value 7.0) buffer is added and is resuspended, is added
Suitable Streptavidin is incubated at room temperature 2h；
Closing: being added the BSA solution that mass volume ratio is 20%, is incubated at room temperature 1h；
Be resuspended: supernatant is removed in centrifugation (12000g, 4 DEG C, 5min), and the BSA solution that mass volume ratio is 1.5% is added and is resuspended,
4 DEG C of preservations；
The preparation of mark zone: each mark zone is added dropwise the latex beads of suitable marked by streptavidin, in 35 DEG C of baking ovens
It dries and saves backup.
(2) FITC antibody, DigiTAb and two corresponding anti-solution the embedding of drawn area: are printed on inspection respectively using spray film instrument
Survey line T1 and T2 and nature controlling line C is dried and is saved backup in 35 DEG C of baking ovens.
(3) loading area, mark zone, drawn area and suction zones the production of test strips: are assembled into support plate in order respectively
On.
(4) extraction of HPV sample DNA to be detected and RPA amplification
DNA is extracted using paramagnetic particle method, the carrier extracted using the magnetic nano-particle of carboxyl modified as DNA, specific real
Apply mode are as follows: 100 μ L HPV samples, 700 μ L cell pyrolysis liquids and 30 μ L Proteinase K Solutions are added in clean centrifuge tube,
60 DEG C of incubation 15min；Room temperature is cooling, and the magnetic bead and coating buffer of 30 μ g carboxyl modifieds is added, rotates 5min at room temperature；Magnetism is inhaled
It is attached, supernatant is removed, 1000 μ L cleaning solutions are added and wash twice；Magnetic absorption removes supernatant, and 100 μ L re-suspension liquids are added and are resuspended, magnetism is inhaled
Attached, taking supernatant is mentioned DNA.
Dual RPA reacts detailed process are as follows: 10 μM of HPV is added in 29.5 μ L lysis hybridization buffers of RPA powdered reagent
16 upstream and downstream primers each 0.8 μ L, 10 μM of each 0.4 μ L of 18 upstream and downstream primer of HPV, 2 μ L of DNA profiling, moisturizing add to 47.5 μ L
Enter after the 2.5 μ L of magnesium acetate solution of 280mM 40 DEG C of reaction 20min immediately.
Wherein, the particular sequence of 16 upstream primer of HPV of use are as follows: 5 '-BIO-GAACTTTGCaTTATTCGGGATT-
3 ', the particular sequence of downstream primer are as follows: 5 '-FITC-CTTTGCTTaTTCTTCAGGAAC-3 '.18 upstream primer of HPV of use
Particular sequence are as follows: 5 '-BIO-TGTCACGAaGCAACTTAAGAC-3 ', the particular sequence of downstream primer are as follows: 5 '-DIG-
CTGGCTTCAaCACTATACAACAA-3’。
(5) sample detection
RPA product in the present embodiment step (4) is directly added drop-wise to the loading area of test strips after sample-loading buffer dilutes,
5min is reacted at room temperature, shoots picture using slr camera, and carry out using intensity of the ImageJ software to detection line T1 and T2
Analysis.Using the screening simultaneously of lateral chromatography method and the high-risk HPV 16 of detection cervical carcinoma and 18 genotype of HPV in the present embodiment
Sensitivity schematic diagram please refers to shown in Fig. 1.
In conclusion the present invention is deposited according to the high-risk HPV 16 of cervical carcinoma or 18 genotype of HPV by above-mentioned technical proposal
When, the double-stranded DNA amplified production of corresponding both ends difunctionalization is obtained by RPA amplification respectively, double-stranded DNA amplification is utilized to produce
The bridge joint of object acts on, and latex beads is connected in corresponding different detection lines respectively, the content according to different high-risk HPV is different
When, the quantity for being connected to the latex beads in different detection lines is different, and then keeps the depth of different detection line colors different, establishes
The relationship between the depth and different high-risk HPV contents of different detection line colors is played, and then realizes that primary amplification, sample-adding are right simultaneously
The purpose of high-risk HPV 16 and 18 genotype of HPV sensitive screening and detection.The method specificity is good, high sensitivity, repeatability
Good, easy to operate, strong applicability can be detected simultaneously by the cervical carcinoma high-risk HPV 16 and HPV 18DNA of 500 copies.This side
Method does not need special expensive instrument, it is only necessary to which common water-bath and corresponding dual lateral flow chromatograph test strip can be realized
Quick, accurate, the screening simultaneously of HPV 16 and HPV 18 high-risk to cervical carcinoma simultaneously, is suitable for base and detection hardware resource is deficient
Carry out relevant cervical cancer screening work in weary area.
It should be appreciated that the technical concepts and features of above-described embodiment only to illustrate the invention, its object is to allow be familiar with this
The personage of item technology cans understand the content of the present invention and implement it accordingly, and it is not intended to limit the scope of the present invention.It is all
Equivalent change or modification made by Spirit Essence according to the present invention, should be covered by the protection scope of the present invention.
Sequence table
<110>the dark blue medical science and technology limited liability company in Anhui
<120>recombinase amplification detection method, test strips and its application of 18 genotype of HPV 16 and HPV are directed to
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gaactttgca ttattcggga tt 22
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<212> DNA
<213>artificial sequence (artificial sequence)
<400> 2
ctttgcttat tcttcaggaa c 21
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<211> 21
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<213>artificial sequence (artificial sequence)
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tgtcacgaag caacttaaga c 21
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Claims (10)
1. a kind of primer pair for HPV16 genotype, it is characterised in that including the first primer and the second primer, described first draws
Object, the second primer sequence respectively as shown in SEQ ID NO.1 and SEQ ID NO.2, and 5 ' ends of the first primer are through life
5 ' ends of object element label, second primer are marked through FITC.
2. a kind of primer pair for HPV18 genotype, it is characterised in that including third primer and the 4th primer, the third is drawn
Object, the 4th primer sequence respectively as shown in SEQ ID NO.3 and SEQ ID NO.4, and 5 ' ends of the third primer are through life
Object element label, 5 ' ends of the 4th primer are through digoxigenin labeled.
3. a kind of dual lateral flow chromatograph test strip for detecting HPV16 and HPV18 genotype simultaneously, including support plate, feature
Be: the upper surface of the support plate includes the loading area set gradually along direction initialization, is combined with what Streptavidin was coupled
Mark zone, drawn area and the suction zones of latex beads are provided with detection line T1, T2 and nature controlling line C in the drawn area, and described
Detection line T1, T2 and nature controlling line C are respectively in connection with having FITC antibody, DigiTAb and secondary antibody.
4. dual lateral flow chromatograph test strip that is according to claim 3 while detecting HPV16 and HPV18 genotype,
Be characterized in that: the FITC antibody is selected from the monoclonal antibody of FITC, and concentration is 1.0~3.0mg/mL；And/or the latex
Microballoon is blue, and partial size is 100~300nm；And/or the concentration of the Streptavidin is 0.5~2.0mg/mL；And/or
The DigiTAb is selected from the monoclonal antibody of digoxin, and concentration is 0.3~1.2mg/mL；And/or the secondary antibody is selected from chain
The sheep anti-mouse antibody of mould Avidin, concentration are 1.0~4.0mg/mL.
5. dual lateral flow chromatograph test strip that is according to claim 3 while detecting HPV16 and HPV18 genotype,
Be characterized in that: the support plate includes not absorbing water or hydrophobic cardboard or plastic plate；And/or the material in the loading area includes
Glass fibre cotton or polyester film；And/or the material of the drawn area includes nitrocellulose filter or pure cellulose film；And/or
The material of the suction zones includes filter paper.
6. detecting the dual lateral flow chromatographic test paper of HPV16 and HPV18 genotype while described in any one of claim 3-5
The preparation method of item, characterized by comprising:
Streptavidin and the latex beads of carboxyl modified are coupled, the latex beads of Streptavidin coupling are made；
The latex beads that Streptavidin is coupled are applied to mark zone, FITC antibody, DigiTAb and secondary antibody are made respectively
At the detection line T1 of drawn area, T2 and nature controlling line C；And
Loading area, mark zone, drawn area and suction zones are set gradually on the supporting plate along direction initialization, are obtained described dual
Lateral flow chromatograph test strip.
7. preparation method according to claim 6, it is characterised in that specifically include: making comprising latex beads, 1- (3- diformazan
Aminopropyl) -3- ethyl-carbodiimide hydrochloride and n-hydroxysuccinimide mixed system in room temperature be protected from light activation 20~
Streptavidin is added later and is incubated for, adds BSA solution, obtains the latex beads of Streptavidin coupling by 60min；It is preferred that
, carboxyl contained by the latex beads surface, 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and N- hydroxyl amber
The imido molar ratio of amber is 1:5:5~1:20:20.
8. the primer pair described in claim 1 for HPV16 genotype, as claimed in claim 2 for HPV18 genotype
The dual lateral flow that HPV16 and HPV18 genotype is detected while described in any one of primer pair or claim 3-5 chromatographs examination
Purposes of the paper slip in the product of preparation detection HPV16 and HPV18 genotype.
9. purposes according to claim 8, which is characterized in that the application product testing HPV16 and HPV18 genotype
Recombinase amplification detection method includes:
Extract the DNA in HPV sample to be detected；
Using the primer pair described in claim 1 for HPV16 genotype, as claimed in claim 2 it is directed to HPV18 genotype
Primer pair carry out RPA amplification, obtain the double stranded DNA product of difunctionalization；
Dual lateral flow chromatograph test strip described in any one of claim 3-5 is added in obtained double stranded DNA product to examine
It surveys, the qualitative or quantitative detection of HPV16 or HPV18 in HPV sample to be detected is realized according to the colour developing result of detection line.
10. purposes according to claim 9, which is characterized in that apply the product testing HPV16 and HPV18 genotype
Recombinase amplification detection method specifically include: HPV sample dissociation to be detected is made using cell pyrolysis liquid and Proteinase K Solution,
The magnetic bead and coating buffer of carboxyl modified are added, the obtained washed liquid washing of DNA is resuspended with re-suspension liquid later, obtains detection HPV
The DNA of sample；
Preferably, the lysate includes Tris-HCl buffer, ethylenediamine tetra-acetic acid, sodium chloride and lauryl sodium sulfate,
Wherein, the pH value of the Tris-HCl buffer is 7.0~8.0, and concentration is 20~50mmol/L, the ethylenediamine tetra-acetic acid
Concentration is 5~20mmol/L, and the concentration of the sodium chloride is 200~1000mmol/L, the lauryl sodium sulfate and cracking
The mass volume ratio of liquid is 0.5~2.5g:100mL；
Preferably, final concentration of 0.5~3mg/mL of the Proteinase K Solution；
Preferably, the coating buffer includes polyethylene glycol and sodium chloride, wherein the matter average molecular weight of the polyethylene glycol is 400
~20000, the mass volume ratio of the polyethylene glycol and coating buffer is 5~30g:100mL, and sodium chloride is dense in the coating buffer
Degree is 0.5~3mol/L；
Preferably, the cleaning solution includes the aqueous solution that ethanol content is 55~80vt%；
Preferably, the re-suspension liquid includes Tris-HCl buffer and ethylenediamine tetra-acetic acid, wherein the Tris-HCl buffer
PH value be 7.0~8.0, concentration is 10~50mmol/L, and the concentration of the ethylenediamine tetra-acetic acid is 0.5~10mmol/L.
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| CN201910221539.2A CN109943663A (en) | 2019-03-22 | 2019-03-22 | For recombinase amplification detection method, test strips and its application of 18 genotype of HPV 16 and HPV |
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| CN110607354A (en) * | 2019-10-31 | 2019-12-24 | 上海海洋大学 | Rapid detection method of pathogenic vibrio cholerae |
| CN111073997A (en) * | 2018-10-19 | 2020-04-28 | 重庆博艾迈迪森生物科技有限公司 | Kit for early and rapid screening of HPV16-E6 and HPV18-E6 |
| CN111505275A (en) * | 2020-03-20 | 2020-08-07 | 浙江工业大学 | Cas9 nucleic acid isothermal amplification-based immunochromatography multiple gene detection method |
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| CN114292899A (en) * | 2022-01-08 | 2022-04-08 | 安徽医科大学第一附属医院 | Hepatocyte growth factor detection and signal sensitization probe, primer pair and application thereof |
| CN114292899B (en) * | 2022-01-08 | 2024-03-26 | 安徽医科大学第一附属医院 | Hepatocyte growth factor detection and signal sensitization probe, primer pair and application thereof |
| CN115998341A (en) * | 2023-01-18 | 2023-04-25 | 珠海舒桐医疗科技有限公司 | Portable device for menstrual blood sample collection and HPV rapid detection system |
| CN116875440A (en) * | 2023-06-21 | 2023-10-13 | 无锡百泰克生物技术有限公司 | Multiple nucleic acid detector, test paper and method based on chromatography |
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