CN104805218B - Reaction system and method based on LAMP and molecular beacons detection HPV16 and 18 - Google Patents
Reaction system and method based on LAMP and molecular beacons detection HPV16 and 18 Download PDFInfo
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Abstract
The invention discloses a kind of reaction system and method based on LAMP and molecular beacons detection HPV16 and 18, the reaction system includes outer primer F3, B3 and inner primer FIP, BIP and molecular beacon probe MB 16, MB 18 of detection HPV16 and HPV18.The method of detection HPV16 and 18 of the invention is based on LAMP and molecular beacons technology detection HPV pathogen, the difference that the method sends fluorescence intensity at different temperatures on the basis of traditional LAMP technology using molecular beacon probe is detected to LAMP amplified productions, compared with traditional LAMP technology, the present invention can avoid traditional LAMP technology because the caused false positive results of primer itself hybridization, with high specificity, advantage simple to operate.
Description
Technical field
The invention belongs to technical field of molecular biological detection, more particularly it relates to a kind of be based on LAMP and divide
Reaction system and detection method that sub- beacon is detected to 16 and 18 type HPV pathogen.
Background technology
Cervical carcinoma is the disease for threatening global women's health most serious, and cases of cervical cancer 52.98 ten thousand is newly sent out in the whole world within 2008,
The people of death 25.51 ten thousand, wherein 85% new cases are in developing country.High-risk human mammilla papillomavirus (human
Papillomaviruses, HPV) persistent infection be the main cause for causing cervical carcinoma and its precancerous lesion.Cervical cancer patient
100% carries high-risk HPV infection, and about 97% is HPV positive, low lesion in height lesion (CIN2 or CIN3) patient
(CIN1) positive rate in is also up to 61.4%.Common high-risk HPV has：16、18、31、33、35、39、45、51、52、56、
58th, 59,68,73,82 etc..
Current HPV detection platforms include traditional PCR primer gel electrophoresis, quantitative PCR, the hybridization of reverse film, gene core
Piece, liquid-phase chip etc., but it is confined to possess Grade A hospital clinical laboratory or Center for Disease Control that PCR operates qualification these technologies more
Carry out, expensive, be unfavorable for carrying out extensive examination to crowd.
Loop-mediated isothermal amplification technique (loop-mediated isothermal amplification, LAMP) is
Notomi is characterized in being set for 6 regions of target gene in a kind of novel constant temperature nucleic acid amplification method of exploitation in 2000
Meter 4-6 bar special primers, using a kind of strand displacement archaeal dna polymerase (Bst DNA polymerase) in (60-65 DEG C of isothermy
Left and right) under place 30-60 minute, you can completion nucleic acid amplification reaction.As a kind of simple, quick, low cost, high specificity
Detection technique, is widely applied at aspects such as food security, Micro biological Tests, clinical diagnosises.Number of patent application
201110242077.6 i.e. there is provided a kind of detection method that the common hypotypes of HPV are detected based on LAMP technology.
LAMP technology is applied to clinical greatest problem：It is heavy using observation magnesium pyrophosphate white more than traditional LAMP technology
The method such as shallow lake, HNB dye colours, calcein fluorescence judges positive, but the positive test symbol of the above method can only represent reality
There occurs LAMP courses of reaction during testing, and cannot be distinguished by real being formed by primer and the hybridization of correspondence target dna masterplate
Positive amplification result and between primer, between primer and sample DNA by mistake hybridization occur by mistake expand caused by false positive
As a result, inconvenience is caused to clinical detection.
Molecular beacon (molecular beacon) is that a kind of fluorescence for having loop-stem structure of the inventions such as Tyagi in 1996 is visited
Pin.In the presence of no target sequence, closed in itself during molecular beacon probe low temperature, fluorophor and quenching group it is close to each other and
Do not fluoresce.It is miscellaneous with the double-strand that complementary target sequence forms stabilization during molecular beacon low temperature in the presence of having target sequence
Body is handed over, fluorophor and quenching group is separated, fluorescence is sent, with the rising of temperature, double-stranded hybrid gradually unwinds, and reaches
During fusing point, fluorescence declines rapidly.Fluorescent value is observed by controlling reaction temperature simultaneously, you can judge whether the expansion of target sequence
Increase.In the annealing stage of PCR amplifications, molecular beacon is combined with the target sequence of generation sends fluorescence, is extending the stage, departs from target sequence
Row are expanded without interference, and with the increase of cycle-index, the amount of the molecular beacon combined with template also increases, and final fluorescence is strong
Spend and be directly proportional to the template amount of amplification.The technology has high specificity, and easy to operate, sensitivity is high, particularly it
Real_time quantitative detection can be carried out, even can be used for in-vivo analysis.
In sum, need badly set up one kind can quickly, high specific and sensitivity, while detecting various HPV viruses diseases
The technology of substance.
The content of the invention
Based on this, in order to overcome the defect of above-mentioned prior art, the invention provides a kind of quick, high specific and sensitive
Property, detect the reaction system and detection method of HPV16 and 18 simultaneously based on LAMP and molecular beacon.
In order to realize foregoing invention purpose, this invention takes following technical scheme：
Reaction system based on LAMP and molecular beacons detection HPV16 and 18, the reaction system include detection HPV16 and
The primer sets of HPV18,
The primer sets of the detection HPV16 are：
Outer primer F3 of the nucleotide sequence as shown in SEQ.ID.NO.1；
Outer primer B3 of the nucleotide sequence as shown in SEQ.ID.NO.2；
Inner primer FIP of the nucleotide sequence as shown in SEQ.ID.NO.3；
Inner primer BIP of the nucleotide sequence as shown in SEQ.ID.NO.4；
Molecular beacon probe MB-16；
The primer sets of the detection HPV18 are：
Outer primer F3 of the nucleotide sequence as shown in SEQ.ID.NO.6；
Outer primer B3 of the nucleotide sequence as shown in SEQ.ID.NO.7；
Inner primer FIP of the nucleotide sequence as shown in SEQ.ID.NO.8；
Inner primer BIP of the nucleotide sequence as shown in SEQ.ID.NO.9；
Molecular beacon probe MB-18；
The reaction system includes following components：
Wherein in some embodiments, the reaction system includes following components：
Wherein in some embodiments, the reaction buffer (2X) includes following components：
Wherein in some embodiments, the nucleotide sequence such as SEQ.ID.NO.5 institutes of the molecular beacon probe MB-16
Show, the nucleotide sequence of the molecular beacon probe MB-18 is as shown in SEQ.ID.NO.10.Preferably, the molecular beacon is visited
5 ' ends of the nucleotide sequence of pin MB-16 are connected with fluorescent dye FAM, and 3 ' ends are connected with fluorescent quenching group BHQ1.Preferably,
5 ' ends of the nucleotide sequence of the molecular beacon probe MB-18 are connected with fluorescent dye ROX, and 3 ' ends are connected with fluorescent quenching base
Group BHQ2.
In the present invention, the molecular beacon probe MB-16 and molecular beacon probe MB-18 can also be under other low temperature
Unstressed configuration, such as fluorescence probe for having fluorescence under high temperature, Taqman probes etc..
Present invention also offers the side that HPV16 and 18 are detected using the above-mentioned detection architecture based on LAMP and molecular beacon
Method.
For achieving the above object, this invention takes following technical scheme：
A kind of method based on LAMP and molecular beacons detection HPV16 and 18, comprises the following steps：
(1) DNA of testing sample, is extracted using Axygen companies bacterial genomes DNA extraction kit；
(2), the DNA of testing sample is added in the reaction system described in claim any one of 1-5, at 56-65 DEG C,
Reaction carries out LAMP reactions in 30-90 minutes；
(3), after LAMP reactions, respectively at 4 DEG C, 65 DEG C and 95 DEG C with ultra violet lamp, fluorescence is observed.
Wherein in one embodiment, the reaction condition of the reactions of LAMP described in step (2) is：At 60 DEG C, 60 points are reacted
Clock.
The present invention is to detect 16,18 type human milks with the loop-mediated isothermal amplification technique based on Molecular beacon fluorescence probe technology
Head tumor virus (HPV), fluorescence intensity is sent on the basis of traditional LAMP technology at different temperatures using molecular beacon probe
Difference LAMP amplified productions are detected, compared with traditional LAMP technology, the invention has the advantages that：
(1), the system of detection HPV16 and 18 of the invention is that special dividing is increased on the basis of traditional LAMP detections
Sub- beacon probe as Testing index, the binding site of molecular beacon probe in LAMP amplification regions, but not with LAMP expand
Primer is completely superposed, therefore, after only LAMP amplimers are combined with target dna and amplified reaction occurs really, could expand
Go out substantial amounts of molecular beacon probe binding site to be combined with probe, and mistake hybridization or primer and other non-targeted between primer
DNA causes amplify molecular beacon probe binding site when expanding by mistake when hybridizing by mistake, it is to avoid in traditional LAMP technology
The false positive situation caused because of primer itself hybridization for often occurring；
(2), the method for detection HPV16 and 18 of the invention by design control molecular beacon probe stem, ring portion position
Hybridization temperature, be allowed to meet at a certain temperature, only when LAMP reaction amplify a large amount of target dnas when molecular beacon probe
Can hybridize and send fluorescence with the target dna that obtain of amplification, and LAMP reactions do not occur or expanded not target dna when,
Molecular beacon probe itself stem position complementary series occurs from hybridization without sending fluorescence.So as to traditional LAMP technology it is sensitive,
The specificity of detection is greatly strengthen on the basis of easily；
(3), the method for detection HPV16 and 18 of the invention is simple to operate, for Clinical Institutions at different levels to common HPV infection
Diagnosed.
Specific embodiment
The present invention is described in detail below in conjunction with specific embodiment.
The experimental technique of unreceipted actual conditions in preferred embodiment, generally according to normal condition, such as molecular cloning reality
The condition described in guide (third edition, J. Pehanorm Brookers etc. are write, and Huang Peitang etc. is translated, Science Press, 2002) is tested, or is pressed
Carried out according to the condition proposed by manufacturer.
Raw material used in following examples derives from commercially available.
Embodiment 1 is based on the reaction system and method for LAMP and molecular beacons detection HPV16 and 18
The conserved sequence for choosing HPV16 and HPV18 designs corresponding LAMP primer, including two outer primers (F3, B3) and
Two inner primers (FIP, BIP), and corresponding molecular beacon probe (MB-16, MB-18).Primer synthesis is by raw work bioengineering
(Shanghai) limited company synthesizes, and is purified through HPLC.
The reaction system includes the primer sets of detection HPV16 and HPV18, specifically includes following components：
The reaction system includes following components：
Sequence is as follows respectively：
CATGGAGATACACCTACATTG(SEQ ID NO.1)
GTACGGATGTCTACGTGTG(SEQ ID NO.2)
CTCTGAGCTGTCATTTAATTGCTCAGAATATATGTTAGATTTGCAACCAG(SEQ ID NO.3)
GGACAAGCAGAACCGGACAGCGAAGCGTAGAGTCACAC(SEQ ID NO.4)
CGCGAGACGAAATAGATGGTCCAGTCGCG(SEQ ID NO.5)
ACAAATGTCTGCAGATCCTT(SEQ ID NO.6)
GGTAACAATAGAGCCACTTG(SEQ ID NO.7)
TGCTCTATTCCAAAAATGCCTAGCGGGATTCCATGTTTTTTTGCTT(SEQ ID NO.8)
GGTGACACTGTGCCTCAATCGGGAGAATACAACAGCT(SEQ ID NO.9)
GGCCTTAGAGCAGGTACTATGGGTGACAGGCC(SEQ ID NO.10)
Above-mentioned reaction buffer (2X) includes following components：
In this embodiment, 5 ' ends of the nucleotide sequence of the molecular beacon probe MB-16 are connected with fluorescent dye
FAM, 3 ' ends are connected with fluorescent quenching group BHQ1.5 ' ends of the nucleotide sequence of the molecular beacon probe MB-18 are connected with
Fluorescent dye ROX, 3 ' ends are connected with fluorescent quenching group BHQ2.
A kind of method based on LAMP and molecular beacons detection HPV16 and 18, comprises the following steps：
In this embodiment, the side of HPV16 and 18 is detected using the above-mentioned detection architecture based on LAMP and molecular beacon
Method, comprises the following steps：
(1) DNA of testing sample, is extracted using Axygen companies bacterial genomes DNA extraction kit；
(2), the DNA of testing sample is added in above-mentioned reaction system, at 60 DEG C, it is anti-that reaction carries out LAMP in 60 minutes
Should；
(3), after LAMP reactions, control temperature is bathed respectively at 4 DEG C, 65 DEG C and 95 DEG C with hand-held with PCR instrument or temperature-conditioned metal
Formula ultra violet lamp reaction tube, observes fluorescence；Or 65 DEG C to 90 DEG C solubility curve analyses are carried out on quantitative real time PCR Instrument.
The sensitivity technique of the detection method of the embodiment 1 of embodiment 2
With HPV16, the primers F 3 (SEQ.ID.NO.1 and SEQ.ID.NO.6) of 18 types, B3 (SEQ.ID.NO.2 and
SEQ.ID.NO.7 positive sample DNA) being expanded respectively, amplified fragments being inserted into carrier T and sequence verification, light splitting light is used afterwards
Degree meter quantitative determination OD260/280 values, then the plasmid deionized water or TEBuffer of amplification are diluted to 101-1010Copy
Shellfish/ul is used as standard items.The standard items of various concentrations are carried out into LAMP detections, reaction system and reaction condition are with embodiment 1.
After LAMP reactions, control temperature is bathed with PCR instrument or temperature-conditioned metal purple with hand-held respectively at 4 DEG C, 65 DEG C and 95 DEG C
Outer light irradiation reaction tube, observes fluorescence.Now negative tube is presented unstressed configuration-hypofluorescence-hyperfluorescence and changes as temperature is raised,
And positive pipe is presented hyperfluorescence-hypofluorescence-hyperfluorescence change, yin and yang attribute result is judged with this.
Result shows that the detection sensitivity of HPV16 and 18 types is 100 copies or so.
The specific detection of the detection method of the embodiment 1 of embodiment 3
Choose clinic and determined the DNA sample of HPV infection hypotype with PCR- reverse dot blot hybridization methods, including 16,18,52,
68th, 33 type positive sample, the DNA of testing sample is extracted using Axygen companies bacterial genomes DNA extraction kit, is respectively taken and is treated
The DNA (about 3000 copy human gene group DNA) of 20ng testing samples is surveyed as template, LAMP detections are carried out, reaction system and anti-
Condition is answered with embodiment 1.
Control methods：The DNA sample that clinic has determined HPV infection hypotype with PCR- reverse dot blot hybridization methods is chosen, including
16th, 18,52,68,33 type positive sample, testing sample is extracted using Axygen companies bacterial genomes DNA extraction kit
DNA, the DNA (about 3000 copy human gene group DNA) for respectively taking 20ng testing samples to be measured, as template, carries out LAMP detections, instead
Answer system in addition to being not added with molecular beacon probe (MB-16, MB-18), remaining is same as Example 1, reaction condition is with implementation
Example 1.
After LAMP reactions, control temperature is bathed with PCR instrument or temperature-conditioned metal purple with hand-held respectively at 4 DEG C, 65 DEG C and 95 DEG C
Outer light irradiation reaction tube, observes fluorescence；Now negative tube is presented unstressed configuration-hypofluorescence-hyperfluorescence and changes as temperature is raised,
And positive pipe is presented hyperfluorescence-hypofluorescence-hyperfluorescence change, yin and yang attribute result is judged with this.
Result shows, the no positive knot of the positive DNA samples of negative DNA and non-HPV16 and 18 types of HPV16 and 18 types
Really, the positive DNA of HPV16 and 18 types is illustrated as positive findings；And detected using contrasting detection method, HPV16 and 18
There is the sun of 25.3% positive findings, HPV16 and 18 types in the negative DNA and non-HPV16 of type and the positive DNA samples of 18 types
Property DNA is illustrated as positive findings；Illustrate that the detection method specificity of the embodiment of the present invention 1 is very strong.
The fluorescence solubility curve analysis of the detection method of the embodiment 1 of embodiment 4
After the completion of LAMP reactions, 40 DEG C to 90 DEG C solubility curve analyses are carried out on quantitative real time PCR Instrument, carry out result pair
Than.Solubility curve is analyzed and completed on Qiagen Rotorgene Q, is as a result shown, fluorescence channel is FAM, and fluorescence compensation is 4.
Embodiment described above only expresses several embodiments of the invention, and its description is more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Shield scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (5)
1. a kind of reaction system based on LAMP and molecular beacons detection HPV16 and 18, it is characterised in that the reaction system bag
The primer sets of detection HPV16 and HPV18 are included,
The primer sets of the detection HPV16 are：
Outer primer F3 of the nucleotide sequence as shown in SEQ.ID.NO.1；
Outer primer B3 of the nucleotide sequence as shown in SEQ.ID.NO.2；
Inner primer FIP of the nucleotide sequence as shown in SEQ.ID.NO.3；
Inner primer BIP of the nucleotide sequence as shown in SEQ.ID.NO.4；
Molecular beacon probe MB-16；The nucleotide sequence of the molecular beacon probe MB-16 is as shown in SEQ.ID.NO.5；
The primer sets of the detection HPV18 are：
Outer primer F3 of the nucleotide sequence as shown in SEQ.ID.NO.6；
Outer primer B3 of the nucleotide sequence as shown in SEQ.ID.NO.7；
Inner primer FIP of the nucleotide sequence as shown in SEQ.ID.NO.8；
Inner primer BIP of the nucleotide sequence as shown in SEQ.ID.NO.9；
Molecular beacon probe MB-18；The nucleotide sequence of the molecular beacon probe MB-18 is as shown in SEQ.ID.NO.10；
The reaction system includes following components：
2. the reaction system based on LAMP and molecular beacons detection HPV16 and 18 according to claim 1, its feature exists
In the reaction system includes following components：
3. the reaction system based on LAMP and molecular beacons detection HPV16 and 18 according to claim 1 and 2, its feature
It is that the reaction buffer 2X includes following components：
4. the reaction system based on LAMP and molecular beacons detection HPV16 and 18 according to claim 1, its feature exists
In 5 ' ends of the nucleotide sequence of the molecular beacon probe MB-16 are connected with fluorescent dye FAM, and it is sudden that 3 ' ends are connected with fluorescence
Go out group BHQ1.
5. the reaction system based on LAMP and molecular beacons detection HPV16 and 18 according to claim 1, its feature exists
In 5 ' ends of the nucleotide sequence of the molecular beacon probe MB-18 are connected with fluorescent dye ROX, and it is sudden that 3 ' ends are connected with fluorescence
Go out group BHQ2.
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| CN106148571A (en) * | 2016-09-06 | 2016-11-23 | 清华大学 | The reagent set of detection HPV HPV16 and HPV18 |
| CN107058467A (en) * | 2016-11-11 | 2017-08-18 | 北京晋祺生物科技有限公司 | It is a kind of that gene mutation or SNP method are detected based on isothermal duplication method |
| CN107557492A (en) * | 2017-09-14 | 2018-01-09 | 温州美众医学检验所 | LAMP detections are combined with primer and its reaction system |
| PL240016B1 (en) * | 2019-09-09 | 2022-02-07 | Genomtec Spolka Akcyjna | Set of primers for the detection of human papillomavirus type 16 (HPV16 Human papillomavirus type 16) and human papillomavirus type 18 (HPV18 Human papillomavirus type 18), method of detecting HPV16 and HPV18 infections, the application of a set of primers for the detection of HPV16 and HPV18 infections |
| CN110878381A (en) * | 2019-12-23 | 2020-03-13 | 广西壮族自治区兽医研究所 | Primer composition, kit and method for detecting mycoplasma bovis and infectious bovine rhinotracheitis virus |
| CN110878380B (en) * | 2019-12-23 | 2021-09-07 | 广西壮族自治区兽医研究所 | Primer composition, kit and method for detecting vesicular stomatitis virus Indiana type and new Jersey type |
| CN111270015A (en) * | 2020-03-26 | 2020-06-12 | 广西壮族自治区兽医研究所 | Primer composition, kit and method for detecting CSFV and PRRSV |
| CN111235317A (en) * | 2020-03-26 | 2020-06-05 | 广西壮族自治区兽医研究所 | Primer composition, kit and method for detecting PRRSV (porcine reproductive and respiratory syndrome Virus) and PCV (porcine circovirus Virus) |
| CN114107560A (en) * | 2021-11-03 | 2022-03-01 | 广州朗坤生物科技有限公司 | LAMP primer combination, reaction system and method for detecting 16-type and 18-type HPV |
| CN117327822A (en) * | 2023-11-16 | 2024-01-02 | 河南中检食安生物科技有限公司 | Primer group and kit for detecting hansaibatong bodies |
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| CN103114033A (en) * | 2012-11-08 | 2013-05-22 | 杭州腾越生物科技有限公司 | Array type multiplex microfluidic isothermal amplification chip for HPV (human papillomavirus) detection |
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