CN107723384A - A kind of PCR kit for detecting human papilloma virus and preparation method thereof - Google Patents
A kind of PCR kit for detecting human papilloma virus and preparation method thereof Download PDFInfo
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Abstract
The PCR kit of detection human papilloma virus has used the multiple real time fluorescence round pcr containing TaqMan probe.In PCR reactions, a 5 ' ends connection fluorescent reporter dye is introduced, 3 ' ends connect the DNA probe of fluorescence quencher molecule；When in PCR extension amplifications, the Taq enzyme in system cuts off probe molecule so that the reporter dye molecules after being separated with quencher molecule send fluorescence；The beneficial effects of the present invention are：Accuracy in detection is high and can quantify detection, reproducible.The present invention be it is a kind of can in early stage quick, easy, the effective clinical most common 13 kinds of multiple real time fluorescence quantifying PCR detection methods for belonging to high-risk and high-risk hypotype but the uncommon HPV viruse of Chinese of detection.The present invention is external diagnosis reagent, without any harmful components, with person under inspection without directly contacting, securely and reliably.
Description
Technical field
The present invention relates to a kind of PCR kit for detecting human papilloma virus and preparation method thereof, belong to people's mamillary
The detection field of tumor virus hypotype.The present invention relates to application multiple real time fluorescence quantifying PCR technology in four PCR reaction tubes
While the method for quantitatively detecting 13 kinds of human papilloma viruses of rapidly shaping.
Background technology
Human papilloma virus (Human Papilloma Virus, HPV) is a kind of double-stranded DNA virus, belongs to papilloma
Tobamovirus, it is a kind of virus of specific infection skin or mucous membrane stratified epithelium.At present, HPV it has been found that hypotype have it is close
200 kinds, wherein most does not show obvious symptom after infecting the mankind, and there are pathological manifestations a small number of parts, and some can
It can cause mankind's wart, such as be grown in mankind's verruca vulgaris near genitals skin and mucous membrane, condyloma acuminatum.Some are high-risk
Hypotype can cause the tumour of the mankind, cause the generation of cervical carcinoma, carcinoma of vagina, cancer of anus, carcinoma of penis etc..Wherein more than 30 kinds HPV
The propagation of hypotype is contacted by property to cause the lesion of genital area.
HPV infection is the direct cause of disease of cervical carcinoma, and the generation from infection to final cervical carcinoma has for Most patients
One slow incubation period.Therefore, in time periodically to cervical exfoliated cell carry out HPV detection cervical carcinoma early screening,
Auxiliary diagnosis and assess more afterwards etc. is particularly important in link.Cervical carcinoma early symptom unobvious, examination early warning were sent out early stage
Existing cervical carcinoma, it is particularly important to reduce the death rate.HPV diagnosis at present places one's entire reliance upon the detection of HPV DNA in clinical samples.
HPV partings from the binding of clinic have confirmed different HPV genotype and different virus loads have it is different carcinogenic
Property, thus HPV DNA parting quantitatively detect teiology and cancer to cervical carcinoma and female genital tract tumor before forecast there is weight
The meaning wanted.
It is uterine neck to be determined that some hypotypes of HPV for a long time and repeatedly infect HPV in the IARC symposiums of nineteen ninety-five
The main reason for carcinogenesis.At present, the HPV hypotype relevant with uterine neck carcinogenesis is called high-risk hypotype by the mankind, there is HPV16,
18,31,33,35,39,45,51,52,56,58,59,68 etc.；With tumour it is incoherent be referred to as low danger hypotype, the most common are
HPV 6,11 and 44 etc..
Because HPV can not still be cultivated in vitro so far, and without suitable experimental animal, thus it is detected and depended on
Morphological Identification and molecular Biological Detection technology.The main detection of cell pathology in a organized way of HPV detection methods at present, spot
Blotting, fluorescence in situ hybridization, Southern hybrid methods, polymerase chainreaction method (Polymerase Chain
Reaction, PCR) and hybrid capture etc..Cytology method susceptibility and specificity are relatively low, and dot blotting has radioactivity.
The higher method of susceptibility has in situ hybridization and normal PCR method, but ribozyme trace hybridization in situ is complicated, and normal PCR method is special
Property it is very low, false positive rate is higher, and unsuitable larger scale clinical uses.
At present HC-II be one by Food and Drug Administration (FDA) ratify can Clinical practice detection HPV DNA
Technology, but it can not carry out specifically quantifying to HPV, and the problem of crisscrossing produces infection be present.
Multiple real time fluorescence quantifying PCR technology, which can not only detect specific Virus type, can also detect quantity of viruses,
It is a method for having large development prospect.The shortcomings that for existing method and deficiency, we, will be each by continuous effort
Kind of detection method organically combines, and learns from other's strong points to offset one's weaknesses, and develops simple, fast and easy, inexpensive, and sensitiveness, specificity are very high
HPV detection methods are simultaneously applied in the examination of cervical carcinoma.
Real-time fluorescence PCR technology based on fluorescence labeling probe, using the most in current domestic clinical diagnosis
Extensively.In the real-time fluorescence PCR reaction system using sonde method, according to the species of detected pathogen, list is further divided into
Weight or multiple real time fluorescence PCR technologies, one or more pairs of Specific PCR primers and probe are frequently included in reaction system, are passed through
Condition in R＆D process is groped, and probe and primer only combine with corresponding template specificity, and its binding site is at two
Between primer.5 ' ends of probe are marked with reporter group (Reporter, R), such as FAM, VIC, ROX, and 3 ' ends are marked with fluorescence
Quenching group (Quencher, Q), such as BHQ1, BHQ2, TAMRA.When probe is complete, reporter group is launched glimmering
Light energy is quenched group absorptions, and instrument can't detect signal.With PCR progress, Taq archaeal dna polymerases are in chain extension mistake
The probe combined with template is run into journey, its 5 ' → 3 ' exonuclease activity will cut off probe, and reporter group is remote to be quenched
Gone out group, and its energy can not be absorbed, that is, produce fluorescence signal.So often circulated by PCR, fluorescence signal is also and mesh
Fragment it is the same, have the process that sync index increases.Key using each hypotypes of real-time fluorescence PCR technology for detection HPV is
Need to design the PCR primer and probe that can specifically, sensitively detect each hypotype.
There are two class HPV diagnostic kits in the market, one kind is with PCR fluorescence methods and single fluorescent dye production HPV
Diagnostic kit, because probe molecule all in product all uses identical fluorescent dye, this product can not be told
HPV hypotype classifications, it can not also detect the amount of HPV different subtypes.Another kind of product be caught with Za Jiao Pu or gene chips life
Production, this product can tell a variety of HPV hypotypes classifications, but can not detect their amount.In addition genetic chip
The kit of method, complex operation, the testing time is long, cost is high, is not easy to promote.
Patent No. ZL201110087602.1 patent provides the fluorescent PCR examination of detection human papilloma virus hypotype
Agent box, it protects a kind of fluorescent PCR kit for detecting 21 kinds of hypotypes of human papilloma virus, but its detectable nipple
Shape tumor virus is limited, and can not quantify each hypotype HPV viruse load.
The content of the invention
The main object of the present invention is to provide a kind of examination that can accurately detect high-risk hypotype human papilloma virus
Agent box and preparation method thereof, the kit has the advantages of high specificity, high sensitivity, and can quantify and detect.
In order to solve the above problems, the invention provides a kind of PCR kit and its system for detecting human papilloma virus
Preparation Method, the technical scheme that it is used are as follows：
The PCR kit for detecting human papilloma virus is come off born of the same parents' sample using multi-fluorescence probe PCR technology to uterine neck
The specific nucleic acid sequence of HPV (human papilloma virus) genomic DNA expand and detect, so as to judge whether infection pair
The 13 kinds of hypotypes answered, and 13 kinds of Asias are obtained by the linear relationship of the canonical reference gene that World Health Organization confirms and cell to be measured
Carrying capacity of the type in unit cell.This reagents series box it is exclusive employ quadruple fluorescent quantitative PCR technique, be divided into 4 pipes, often
Pipe is divided into tetra- passages of FAM, HEX, ROX, CY5 and distinguishes hypotype.
Detect the PCR kit of human papilloma virus, including following each component：Specific primer, specificity T aqman
Probe, Taq enzyme, PCR reaction solutions, positive reference product, negative reference product, nucleic acid extraction liquid, it is characterised in that：Described PCR is anti-
The pipe of liquid point four is answered, often pipe employs quadruple TaqMan probe real-time fluorescence PCR technology, to 13 kinds of people in cervical exfoliated cell
Papilloma virus gene group DNA specific nucleic acid sequence amplification and parting detection, four described pipe PCR reaction solutions difference
For：101-I is used for the parting of common high-risk HPV (16,18,58, the 52) hypotype of 4 kinds of Chinese and quantitative determination, 101-II are used for
The parting and quantitative determination, 101-III and 101-IV of 3 kind high-risk HPV (39,31, the 35) hypotypes more typical to Chinese are used for
In the parting of the uncommon high-risk HPV hypotypes of 6 kinds of HPV (68,45,59,56,51,33) of Chinese and quantitative determination and conduct
Mankind's normal gene of ginseng.
Preferably, described specificity T aqman probes：
HPV16 probes-CTGACATATCTACTTGAGAAMCTAG；
HPV18 probes-TACTACACAGTYTCGTGTACCTGG；
HPV31 probe-TAGTACTAATA TGTCTATKTGTGCTACAA；
HPV33 probes-TGACTTAATGCGCACAAGTAACTAGTGCCAG；
HPV35 probes-TCGGTGTGTCCTGCTGGGTCTWCTAGTGCC；
HPV39 probes-CCAACTTGACATGATCTTCCTCTATAGAGTGTTCC；
HPV45 probes-TTCTGTGGCTCTACACAACMTCCTGTCC；
HPV51 probes-TTCAACTATTAGTACTGCCACTGCTGC；
HPV52 probes-TCGCCATGACGAAGGTAGTCCTTA；
HPV56 probes-CAAGCCAAAACACCAATGTTGCAGACACAT；
HPV58 probes-TGCCATTATGCACGGAAGTAAMTAATGAAG；
HPV59 probes-TCAACATGTCTGGCATATACYTTAAGAGT；
HPV68 probes-TCCTCAACATGCCTAATATATTCCTTCAA.
Preferably, its corresponding specific primer difference of described specificity T aqman probes is as follows：
- the CATGACGTAGGTATTCCTTAGAGTT-3 ' of HPV16 sense primers 5 '；
- the CTGATGTTGATGCTACACGCAATAC-3 ' of HPV16 anti-sense primers 5 '；
- the TATGTTAGTGTGATAGATTCTAC-3 ' of HPV18 sense primers 5 '；
- the GACAAATAGACTGTAAATCATAGTC-3 ' of HPV18 anti-sense primers 5 '；
- the GAGCAATCAGTTAGTTGTTACCGT-3 ' of HPV31 sense primers 5 '；
- the AAATGCCAATCAAATTCGTCA-3 ' of HPV31 anti-sense primers 5 '；
- the GAGCAATCAGTTATTCGTTACGGT-3 ' of HPV33 sense primers 5 '；
- the GAAATTTAAACTGTTTATCATATTCTCC-3 ' of HPV33 anti-sense primers 5 '；
- the AGCTGATACACCGGTAGTAC-3 ' of HPV35 sense primers 5 '；
- the CACCATGGCTTAACTATTGCTT-3 ' of HPV35 anti-sense primers 5 '；
- the ATTCAATTATTGCTTACTGTTGTGT-3 ' of HPV39 sense primers 5 '；
- the ATAGGTTGCAAATCAATCTCGTGC-3 ' of HPV39 sense primers 5 '；
- the GCGTAACCAGTTGTTTGTTACTGTAG-3 ' of HPV45 sense primers 5 '；
- the AAGTCATATTACTCCACATGACT-3 ' of HPV45 anti-sense primers 5 '；
- the CATGTGTTGATATTACCAGAAGTCC-3 ' of HPV51 sense primers 5 '；
- the ATAAGTTGCATATCATACTCATGC-3 ' of HPV51 anti-sense primers 5 '；
- the TATGTCACAGTTGTAGATACCAC-3 ' of HPV52 sense primers 5 '；
- the GAAATATAAATAGTAAATCATATTC-3 ' of HPV52 anti-sense primers 5 '；
- the CTGCATACTTTAGTGCTGTTGG-3 ' of HPV56 sense primers 5 '；
- the ACACATACTGACAGTTACTTGAC-3 ' of HPV56 anti-sense primers 5 '；
- the TATGTTAGCGTGGTTGATAGCAC-3 ' of HPV58 sense primers 5 '；
- the GAAATACAAACTGAAAGTCATATGC-3 ' of HPV58 anti-sense primers 5 '；
- the AGACTAGTCGCAGCACCTATCT-3 ' of HPV59 sense primers 5 '；
- the GACATATAAACTGCAACTCAAATTC-3 ' of HPV59 anti-sense primers 5 '；
- the ACTATGTTATAGTACACAACGTA-3 ' of HPV68 sense primers 5 '；
- the ACGTTGTCTACTACTGCTGAAGC-3 ' of HPV68 anti-sense primers 5 '.
Preferably, the step of described kit carries out qualitative resolution to 13 kinds of PHV viruses is as follows：
The first step：By cell sample to be measured and kit amplifying nucleic acid extract according to 1:1 ratio is blended in centrifuge tube, is mixed
It is even, after being stored at room temperature 5-10 minutes, 500-1000rpm centrifugation 1-2 minutes, whole supernatants containing DNA are drawn with liquid-transfering gun,
It resulting will be used to real-time fluorescent PCR amplification at once containing DNA solution test, or be placed in -20 DEG C of preservations；
Second step：HPV diagnosticums are taken out, move back ice dissolving completely, the centrifuge tube containing reagent after dissolving mixes in whirlpool to be shaken
2-3 minutes；
3rd step：Drawn with 10-100ul pipettors in the centrifuge tube described in second step a person-portion HPV diagnosticums I,
II, III or IV are put into the union of real-time fluorescence quantitative PCR instrument required standard eight；Then drawn with 0.1-2.5ul pipettors
0.2ul Hot+Taq enzymes are added to eight foregoing unions；The sample of nucleic acid made of 0.1-2.5ul pipettors from the first step is inhaled
2ul is taken to be added in foregoing eight union；The microseism above-mentioned eight unions 1-2 minutes on whirlpool blending instrument, then it is placed on palm centrifugation
1-2 minutes are centrifuged on machine, standing treats machine；
4th step：Repeat step three prepares negative water sample to be tested；Repeat step three prepares standard internal reference sample to be tested.
5th step：The union of sample to be tested eight prepared by the 3rd step and the 4th step is put into the detection sample position of instrument, entered
Row real-time fluorescent PCR amplification；Reporter fluorescence channel selecting FAM, HEX, ROX and Cy5；Setting program is hot 105 DEG C of lid temperature,
Liquid volume added is 20ul；Operation program：
Constant temperature zone：95 DEG C, 4min；Circulation section：95 DEG C, 30s；55 DEG C, 40s.
6th step：According to the automatic read threshold of instrument and reach the Ct values of threshold value and whether standard value multilevel iudge infects 13
Kind PHV virus-virus.
Preferably, described kit carries out quantitative detection to 13 kinds of PHV viruses, and its concrete technical scheme is as follows：
The first step：Standard plasmid gradient dilution：To 13 HPV standard plasmids in HPV kits and internal reference product XB plasmids,
10 gradient dilution is accompanied successively with High Purity water, obtain the plasmid sample of 50ul 5 various concentrations, be put into and indicate various concentrations
5 centrifuge tubes；
Second step：TaqMan probe substance real-time fluorescent PCR amplification is carried out to the plasmid of the same race of 5 various concentrations, obtained
Reach 5 different C of threshold valuetValue；
3rd step：With 5 C obtained by second steptValue and corresponding 5 copies concentration values establish its HPV hypotype
Standards calibration curve, and calculate the slope A of calibration curvex；4th step：In HPV sample TaqMan probe real-time fluorescence PCRs
Its HPV C is obtained in amplification experimenttIt is worth, the slope Ax for the calibration curve for corresponding to plasmid hypotype in this value divided by the 3rd step is
HPV hypotypes load (the copies concentration) Mx；
5th step：4th step more than repeating, obtains mankind's normal gene load (copies concentration) in identical HPV samples
MXB；
6th step：Pass through formula M=Mx/MXB, calculate in the HPV infection sample, in unit mankind's normal gene XB
HPV subtype virus load (copies concentration).
If described 101-IV partings and quantitatively detecting the high-risk hypotypes of all HPV, series of products 101-I, 101-II,
101-III and 101-IV need to be used simultaneously；If parting and the quantitative common and more typical high-risk hypotypes of HPV, in series of products
101-I and 101-II need to be used simultaneously；If parting and the quantitative common high-risk hypotypes of HPV, only need to use 101-I products.
The beneficial effects of the present invention are：Accuracy in detection is high and can quantify detection.The present invention is that one kind can be in morning
Clinical most common 13 kinds of phase quick, easy, effective detection belongs to high-risk and high-risk hypotype but Chinese are uncommon
The multiple real time fluorescence quantifying PCR detection method of HPV viruse.Parting quantitative analysis HPV-16,18,58,52,39,31,35,
68,45,59,56,51,33 are analyzed using sizing is merged, and different subtype detection is not interfere with each other, it was demonstrated that its specificity is preferably；
50 clinical samples of random detection, being contrasted with the result of triumphant general like product detection kit, degree of conformity reaches 99%,
It is and consistent with sequencing result；It is reproducible.The present invention is external diagnosis reagent, without any harmful components, with person under inspection without straight
Contact, securely and reliably.Internal reference product employed in kit are DNA, and negative reference product are empty plasmid, and PCR is anti-
It is that harmless Taq archaeal dna polymerases, salt and water form to answer main component in liquid (PCR Master Mix buffer), the above pair
Safety issue is not present in human body.
Brief description of the drawings
Fig. 1 is the production technological process of the PCR kit of detection human papilloma virus provided by the invention.
Fig. 2 is the HPV diagnosticums -101I of detection human papilloma virus kit RT-PCR curves, in figure 4 from upper
Under
Fig. 3 is the HPV diagnosticums -101II of detection human papilloma virus kit RT-PCR curves, in figure 3 from
Up to lower curve is HPV39 respectively, and 31,35 detect real-time fluorescence RT-PCR curves, HPV39, Ct=15.1；HPV31, Ct=
19.8；HPV35, Ct=23.5.
Fig. 4 is the HPV diagnosticums -101III of detection human papilloma virus kit RT-PCR curves, in figure 4 from
Up to lower curve is HPV68 respectively, and 45,59,56 detect real-time fluorescence RT-PCR curves, HPV68, Ct=17.1, HPV45, Ct
=18.2；HPV59, Ct=18.3；HPV56, Ct=26.5.
Fig. 5 is the HPV diagnosticums -101IV of detection human papilloma virus kit RT-PCR curves, in figure 3 from
Up to lower curve is that XB, HPV51,33 detect real-time fluorescence RT-PCR curve, XB, Ct=15.2 respectively；HPV51, Ct=
17.3；HPV33, Ct=17.4.
Fig. 6 is HPV16 standard curves.
Fig. 7 is HPV18 standard curves.
Fig. 8 is HPV52 standard curves.
Fig. 9 is HPV58 standard curves.
Figure 10 is HPV33 standard curves.
Figure 11 is HPV31 standard curves.
Figure 12 is HPV35 standard curves.
Figure 13 is HPV39 standard curves.
Figure 14 is HPV45 standard curves.
Figure 15 is HPV51 standard curves.
Figure 16 is HPV56 standard curves.
Figure 17 is HPV59 standard curves.
Figure 18 is HPV68 standard curves.
Embodiment
Understand technical scheme provided by the invention in order to clearer, done below in conjunction with the accompanying drawings with specific embodiment into one
The explanation of step.
Detect the PCR kit of human papilloma virus, including following each component：Specific primer, specificity T aqman
Probe, Taq enzyme, PCR reaction solutions, positive reference product；Negative reference product, nucleic acid extraction liquid, it is characterised in that：Described PCR is anti-
The pipe of liquid point four is answered, often pipe employs quadruple TaqMan probe real-time fluorescence PCR technology, to 13 kinds of people in cervical exfoliated cell
Papilloma virus gene group DNA specific nucleic acid sequence amplification and parting detection, four described pipe PCR reaction solutions difference
For：101-I is used for the parting of common high-risk HPV (16,18,58, the 52) hypotype of 4 kinds of Chinese and quantitative determination, 101-II are used for
The parting and quantitative determination, 101-III and 101-IV of 3 kind high-risk HPV (39,31, the 35) hypotypes more typical to Chinese are used for
In the parting of the uncommon high-risk HPV hypotypes of 6 kinds of HPV (68,45,59,56,51,33) of Chinese and quantitative determination and conduct
Mankind's normal gene of ginseng.
Embodiment
【Nucleic acid DNA specimen extraction】
1.1 by cell sample to be measured and kit amplifying nucleic acid extract -101 or -303 according to 1:1 ratio is blended in
1.5ml centrifuge tube.
1.2 liquid transfer gun heads, which are inhaled, plays 6-10 mixing, after being stored at room temperature 5-10 minutes, 500-1000rpm centrifugation 1-2 minutes.
1.3 draw whole supernatants containing DNA with liquid-transfering gun.Resulting it will be used for real-time fluorescence at once containing DNA solution
PCR amplification experiments, or it is placed in -20 DEG C of preservations.
【It is applicable instrument】
ABI Prism 7500,VII；Rich day 9600；The passage above instrument of SLAN fluorescence quantitative PCR detection systems 4 and
The LC 480II instruments of Roche.
【The qualitative resolution of HPV hypotypes】
1. sample preparation to be checked
1.1 take out HPV diagnosticums -101 or the centrifuge tube of HPV diagnosticums -303 from -20 DEG C of or-70 DEG C of refrigerator-freezers, completely
Move back ice dissolving.
Centrifuge tube containing reagent after 1.2 dissolvings mixes vibrations 2-3 minutes in whirlpool.
1.3 use the HPV diagnosticums -101 that 18ul (person-portion) is drawn in centrifuge tube of the 10-100ul pipettors in 1.2
Reagent
I, II, III or IV are put into the union of real-time fluorescence quantitative PCR instrument required standard eight.
1.4 are added to 1.3 eight unions with 0.1-2.5ul pipettors absorption 0.2ul Hot+Taq enzymes.
1.5 are drawn in eight unions that 2ul is added in 1.4 with 0.1-2.5ul pipettors from the sample of nucleic acid of extraction.
1.6 on whirlpool blending instrument eight union 1-2 minutes in microseism 1.5, be then placed on palm centrifuge and centrifuge 1-2
Minute, standing treats machine.
1.7 repetition above 1.3-1.6 prepare negative water sample to be tested.
1.8, which repeat above 1.3-1.6, prepares standard internal reference sample to be tested
2. real-time fluorescent PCR amplification
2.1 according to instrumentation specification, selected on real-time fluorescence PCR instrument reporter fluorescence passage FAM, HEX, ROX and
Cy5。
2.2 according to instrumentation software, and setting program is hot 105 DEG C of lid temperature, liquid volume added 20ul；Operation program：It is permanent
Temperature section：95 DEG C, 4min；Circulation section：95 DEG C, 30s；55 DEG C, 40s.
2.3 according to instrumentation specification, and the union of 1.6,1.7 and 1.8 sample to be tested eight is put into the detection sample of instrument
Position, carry out real-time fluorescent PCR amplification.
【It is applicable instrument】In the automatic read threshold of several instruments and reach the Ct values of threshold value.
The qualitative resolution mass of HPV hypotypes is judged according to lower table analysis.
The qualitative resolution mass standard of the HPV hypotypes of table 2
Note：Ct=36 is the critical value of detection；If Ct>36, can not judged result, illustrate nucleic acid extraction or real-time fluorescence
PCR
Problem be present in detection.Again extract the high nucleic acid of 10 times of concentration and carry out test experience, if Ct values are also greater than critical value
It is judged as feminine gender.
【HPV subtype virus load quantitative detects】
1 standard plasmid gradient dilution
13 HPV standard plasmids (10 in 1.1 pairs of kits of HPV diagnosticums -10110) and internal reference product XB matter copies/ml
Grain (1010Copies/ml), 10 gradient dilution is accompanied successively with High Purity water, obtains the plasmid sample of 50ul 5 various concentrations
This, is put into 5 centrifuge tubes for indicating various concentrations:
a.1010copies/ml；b.109copies/ml；c.108copies/ml；d.107copies/ml；
e.106copies/ml。
It is prepared by 2 samples to be tested
2.1 according to【The qualitative resolution of HPV hypotypes】Middle 1.3-1.4 operations, prepare 5 eight union samples.
2.2 for each HPV hypotypes plasmid and internal reference plasmid, and with 0.1-2.5ul pipettors, centrifuge tube is inhaled from 1.1 respectively
Take 1ul 5
The plasmid of individual various concentrations, it is added separately to 2.1 5 eight unions.
2.3 on whirlpool blending instrument eight union 1-2 minutes in microseism 2.2, be then placed on palm centrifuge and centrifuge 1-2
Minute, standing treats machine.
2.4 repetition above 2.1-2.3 prepare negative water sample to be tested
3. real-time fluorescent PCR amplification
According to【The qualitative resolution of HPV hypotypes】2 are carried out.
4. quantitative result calculates
4.1, for each HPV hypotypes, such as HPV16, reach threshold value 5 are read from real-time fluorescent PCR amplification curve
Various concentrations Ct values.
4.2 make ordinate with the Ct values in 4.1, and the plasmid concentration in 1.1 makees abscissa, establishes standards calibration curve.Such as
Fig. 6 HPV16 standards calibration curves.
The slope A16 of calibration curve is calculated from Fig. 6, other 12 high-risk HPV hypotypes are obtained respectively with same method
Calibration curve slope and internal reference XB plasmid calibration slopes：A16、A18、A31、A33、A35、A39、A45、A51、A52、A56、
A58, A59, A68 and Ar.
4.3 with【The qualitative resolution of HPV hypotypes】3 parts, we obtain the Ct values of HPV sample real-time fluorescent PCR amplifications, this
The slope of the calibration curve of one value divided by its corresponding hypotype plasmid is HPV hypotypes load (copies concentration).
M16=Ct16/A16┈┈┈┈┈┈┈┈┈┈┈┈┈┈┈┈┈(1)
Same method, calculate XB load.
Mr=Ctr/Ar┈┈┈┈┈┈┈┈┈┈┈┈┈┈┈┈(2)
Using the HPV subtype virus load in unit mankind's normal gene XB as quantitative criterion,
Obtained by formula (3) ".
M=M16/Mrr┈┈┈┈┈┈┈┈┈┈┈┈┈┈┈(3)
With above formula 1), 2) and 3) calculate other 12 kinds of HPV subtype virus load in unit human gene XB.
In formula：
Numeral-HPV hypotypes
M-load；
Ct-gained Ct values；
A-slope；
R-internal reference.
By the quality standard of the PCR kit of detection human papilloma virus above as shown in Fig. 2 it is stored in -20 DEG C,
The term of validity is 12 months, and the coincidence rate of its internal reference product false negative must not occur for this kit internal reference product testing result；It is cloudy
The coincidence rate of property reference material:This kit negative reference product testing result must not occur false；The technology quantitative to HPV hypotypes
Quality standard is as follows：
A. lowest detection limit/sensitivity of this kit to plasmid：1x 104copies/ml；
B. positive criteria plasmid concentration detects Ct values in 1x104-1x108copies/ul, corresponding fluorescent PCR：16<Ct<
32。
This kit has used the multiple real time fluorescence round pcr containing TaqMan probe.PCR(Polymerase
Chain Reaction) it is PCR, refer in the case where archaeal dna polymerase is catalyzed, using fundamental chain DNA as template, with specific
Primer is extension starting point, and by being denatured, annealing, the step such as extend, replication in vitro goes out the subchain DNA with the complementation of fundamental chain template DNA
Process.TaqMan probe real-time fluorescence PCR technology is in PCR reactions, introduces a 5 ' ends connection fluorescence report dye
Material, 3 ' ends connect the DNA probe of fluorescence quencher molecule；When in PCR extension amplifications, the Taq enzyme cut-out probe in system divides
Son so that the reporter dye molecules after being separated with quencher molecule send fluorescence；Often pass through a PCR amplification cycles, will collect
To a fluorescence intensity signals, as PCR primer is constantly accumulative, fluorescence signal intensity also equal proportion increase.Therefore, by right
The real-time detection of each circulation products fluorescence signal is qualitative to starting template DNA and quantitative so as to realize in pcr amplification reaction
Analysis.Multiple techniques are more than the two pairs primers of addition in TaqMan probe real-time fluorescence PCR reaction system, more than one
TaqMan probe, while expand and quantify the PCR reactions of multiple nucleic acid fragments.Technical principle based on more than, this kit
Divide four pipes, often pipe employs quadruple TaqMan probe (FAM, HEX, ROX, CY5) real-time fluorescence PCR technology, uterine neck is come off carefully
The specific nucleic acid sequence of 13 kinds of human papilloma viruses (Human Papillomavims, HPV) genomic DNA in born of the same parents expands
Increase and parting detects.
Fig. 2-Figure 14 distinguishes the PCR standards calibration curves of all types of papillomaviruses.
Figure 15-Figure 18 is respectively PCR kit diagnosticum-101I, II, III, the IV for detecting human papilloma virus
RT-PCR curves.
The beneficial effects of the present invention are：Accuracy in detection is high and can quantify detection.The present invention is that one kind can be in morning
Clinical most common 13 kinds of phase quick, easy, effective detection belongs to high-risk and high-risk hypotype but Chinese are uncommon
The multiple real time fluorescence quantifying PCR detection method of HPV viruse.Parting quantitative analysis HPV-16,18,58,52,39,31,35,
68,45,59,56,51,33 are analyzed using sizing is merged, and different subtype detection is not interfere with each other, it was demonstrated that its specificity is preferably；
50 clinical samples of random detection, being contrasted with the result of triumphant general like product detection kit, degree of conformity reaches 99%,
It is and consistent with sequencing result；It is reproducible.The present invention is external diagnosis reagent, without any harmful components, with person under inspection without straight
Contact, securely and reliably.Internal reference product employed in kit are DNA, and negative reference product are empty plasmid, and PCR is anti-
It is that harmless Taq archaeal dna polymerases, salt and water form to answer main component in liquid (PCR Master Mix buffer), the above pair
Safety issue is not present in human body.
One of the foregoing is only a specific embodiment of the invention, but protection scope of the present invention is not limited thereto,
Any those skilled in the art disclosed herein technical scope in, can expect without creative work
Change or replacement, it should all be included within the scope of the present invention.Therefore, protection scope of the present invention should be wanted with right
The protection domain for asking book to be limited is defined.
Claims (8)
1. the PCR kit of human papilloma virus is detected, including following each component：Specific primer, specificity T aqman are visited
Pin, Taq enzyme, PCR reaction solutions, positive reference product, negative reference product, nucleic acid extraction liquid, it is characterised in that：Described PCR reactions
The pipe of liquid point four, often pipe employs quadruple TaqMan probe real-time fluorescence PCR technology, to 13 kinds of people's nipples in cervical exfoliated cell
Simultaneously parting detection, four described pipe PCR reaction solutions are respectively for the specific nucleic acid sequence amplification of shape tumor virus genomic DNA：
101-I is used for parting and the quantitative determination of the common hypotype of high-risk HPV-16,18,58,2 of 4 kinds of Chinese, and 101-II is used for China
The parting of 3 kinds of more typical hypotypes of high-risk HPV-39,31,35 of people and quantitative determination, 101-III and 101-IV are used for Chinese not
The parting of the common high-risk HPV hypotypes in 6 kinds of HPV-68,45,59,56,51,33 and quantitative determination and as internal reference the mankind just
Normal gene.
2. the PCR kit of detection human papilloma virus according to claim 1, it is characterised in that：Comprising following
Specificity T aqman probes：
HPV16 probes-CTGACATATCTACTTGAGAAMCTAG；
HPV18 probes-TACTACACAGTYTCGTGTACCTGG；
HPV31 probe-TAGTACTAATA TGTCTATKTGTGCTACAA；
HPV33 probes-TGACTTAATGCGCACAAGTAACTAGTGCCAG；
HPV35 probes-TCGGTGTGTCCTGCTGGGTCTWCTAGTGCC；
HPV39 probes-CCAACTTGACATGATCTTCCTCTATAGAGTGTTCC；
HPV45 probes-TTCTGTGGCTCTACACAACMTCCTGTCC；
HPV51 probes-TTCAACTATTAGTACTGCCACTGCTGC；
HPV52 probes-TCGCCATGACGAAGGTAGTCCTTA；
HPV56 probes-CAAGCCAAAACACCAATGTTGCAGACACAT；
HPV58 probes-TGCCATTATGCACGGAAGTAAMTAATGAAG；
HPV59 probes-TCAACATGTCTGGCATATACYTTAAGAGT；
HPV68 probes-TCCTCAACATGCCTAATATATTCCTTCAA.
3. the PCR kit of detection human papilloma virus according to claim 2, it is characterised in that：Described is special
Property Taqman probes its corresponding specific primer difference it is as follows：
- the CATGACGTAGGTATTCCTTAGAGTT-3 ' of HPV16 sense primers 5 '；
- the CTGATGTTGATGCTACACGCAATAC-3 ' of HPV16 anti-sense primers 5 '；
- the TATGTTAGTGTGATAGATTCTAC-3 ' of HPV18 sense primers 5 '；
- the GACAAATAGACTGTAAATCATAGTC-3 ' of HPV18 anti-sense primers 5 '；
- the GAGCAATCAGTTAGTTGTTACCGT-3 ' of HPV31 sense primers 5 '；
- the AAATGCCAATCAAATTCGTCA-3 ' of HPV31 anti-sense primers 5 '；
- the GAGCAATCAGTTATTCGTTACGGT-3 ' of HPV33 sense primers 5 '；
- the GAAATTTAAACTGTTTATCATATTCTCC-3 ' of HPV33 anti-sense primers 5 '；
- the AGCTGATACACCGGTAGTAC-3 ' of HPV35 sense primers 5 '；
- the CACCATGGCTTAACTATTGCTT-3 ' of HPV35 anti-sense primers 5 '；
- the ATTCAATTATTGCTTACTGTTGTGT-3 ' of HPV39 sense primers 5 '；
- the ATAGGTTGCAAATCAATCTCGTGC-3 ' of HPV39 sense primers 5 '；
- the GCGTAACCAGTTGTTTGTTACTGTAG-3 ' of HPV45 sense primers 5 '；
- the AAGTCATATTACTCCACATGACT-3 ' of HPV45 anti-sense primers 5 '；
- the CATGTGTTGATATTACCAGAAGTCC-3 ' of HPV51 sense primers 5 '；
- the ATAAGTTGCATATCATACTCATGC-3 ' of HPV51 anti-sense primers 5 '；
- the TATGTCACAGTTGTAGATACCAC-3 ' of HPV52 sense primers 5 '；
- the GAAATATAAATAGTAAATCATATTC-3 ' of HPV52 anti-sense primers 5 '；
- the CTGCATACTTTAGTGCTGTTGG-3 ' of HPV56 sense primers 5 '；
- the ACACATACTGACAGTTACTTGAC-3 ' of HPV56 anti-sense primers 5 '；
- the TATGTTAGCGTGGTTGATAGCAC-3 ' of HPV58 sense primers 5 '；
- the GAAATACAAACTGAAAGTCATATGC-3 ' of HPV58 anti-sense primers 5 '；
- the AGACTAGTCGCAGCACCTATCT-3 ' of HPV59 sense primers 5 '；
- the GACATATAAACTGCAACTCAAATTC-3 ' of HPV59 anti-sense primers 5 '；
- the ACTATGTTATAGTACACAACGTA-3 ' of HPV68 sense primers 5 '；
- the ACGTTGTCTACTACTGCTGAAGC-3 ' of HPV68 anti-sense primers 5 '.
4. the PCR kit of detection human papilloma virus according to claim 1, it is characterised in that：Kit contains
The DNA primer primer of short sequence, the DNA probe agent probe with fluorescer, free Oligo, biology enzyme and chemistry examination
Agent, wherein DNA primer and probe agent are the unique sequences nucleic acid pieces that all cervical carcinoma virus subtype mutant genes design
Section, the technology path of reagent be made by reaction solution and the mixed liquor I containing primer, probe " HPV kits-quadruple-I "；
Made " HPV kits-quadruple-II " by reaction solution and the mixed liquor I I containing primer, probe；Drawn by reaction solution with containing
Thing, the mixed liquor I II of probe make " HPV kits-quadruple-III "；By reaction solution and the mixed liquor containing primer, probe
IV make " HPV kits-quadruple-IV " and technology path composition, some other Taq archaeal dna polymerase and positive plasmid also by
Dilution is divided in kit：
Specific method step is as follows：
1) prepares each composition, prepares reaction solution, optimizes reaction solution.
2) prepares four pipe mixed liquors, per 6-8 different primer and 3-4 kind probes of Guan Zhonghan according to HPV hypotypes.The pipe of optimization four is mixed
Close liquid.
3) is in Quality Control point 1,2,3,4, controls respectively, detects mixed liquor quality in four pipes.
5. the PCR kit of detection human papilloma virus according to claim 1, it is characterised in that：Described reagent
Box carries out qualitative resolution and quantitative detection to 13 kinds of PHV viruses.
6. the PCR kit of detection human papilloma virus according to claim 5, it is characterised in that：Described reagent
The step of box carries out qualitative resolution to 13 kinds of PHV viruses is as follows：
The first step：By cell sample to be measured and kit amplifying nucleic acid extract according to 1:1 ratio is blended in centrifuge tube, is mixed,
After being stored at room temperature 5-10 minutes, 500-1000rpm centrifugation 1-2 minutes, whole supernatants containing DNA are drawn with liquid-transfering gun, by institute
Obtain being used for real-time fluorescent PCR amplification experiment at once containing DNA solution, or be placed in -20 DEG C of preservations；
Second step：HPV diagnosticums are taken out, move back ice dissolving completely, the centrifuge tube containing reagent after dissolving mixes 2-3 points of vibrations in whirlpool
Clock；
3rd step：Drawn with 10-100ul pipettors in the centrifuge tube described in second step a person-portion HPV diagnosticums I, II,
III or IV is put into the union of real-time fluorescence quantitative PCR instrument required standard eight；Then 0.2ul is drawn with 0.1-2.5ul pipettors
Hot+Taq enzymes are added to eight foregoing unions；The sample of nucleic acid made of 0.1-2.5ul pipettors from the first step is drawn 2ul and added
Enter into foregoing eight union；The microseism above-mentioned eight unions 1-2 minutes on whirlpool blending instrument, then it is placed on palm centrifuge and centrifuges
1-2 minutes, standing treat machine；
4th step：Repeat step three prepares negative water sample to be tested；Repeat step three prepares standard internal reference sample to be tested.
5th step：The union of sample to be tested eight prepared by the 3rd step and the 4th step is put into the detection sample position of instrument, carried out real
When fluorescent PCR expand；Reporter fluorescence channel selecting FAM, HEX, ROX and Cy5；Setting program is hot 105 DEG C of lid temperature, liquid volume added
For 20ul；Operation program：
Constant temperature zone：95 DEG C, 4min；Circulation section：95 DEG C, 30s；55 DEG C, 40s.
6th step：According to the automatic read threshold of instrument and reach the Ct values of threshold value and whether standard value multilevel iudge infects 13 kinds
PHV virus-virus.
7. the PCR kit of detection human papilloma virus according to claim 6, it is characterised in that：Described reagent
It is as follows that box carries out the step of quantitative detection to 13 kinds of PHV viruses：
The first step：Standard plasmid gradient dilution：To 13 HPV standard plasmids in HPV kits and internal reference product XB plasmids, with height
Purified water 10 accompanies gradient dilution successively, obtains the plasmid sample of 50ul 5 various concentrations, is put into indicate various concentrations 5
Centrifuge tube；
Second step：TaqMan probe substance real-time fluorescent PCR amplification is carried out to the plasmid of the same race of 5 various concentrations, reached
5 different C of threshold valuetValue；
3rd step：With 5 C obtained by second steptValue and corresponding 5 copies concentration values establish the standard school of its HPV hypotype
Positive curve, and calculate the slope A of calibration curvex；
4th step：Its HPV C is obtained in the experiment of HPV sample TaqMan probes real-time fluorescent PCR amplificationtValue, this value divided by the
The slope Ax that the calibration curve of plasmid hypotype is corresponded in three steps is HPV hypotype load Mx；
5th step：4th step more than repeating, obtains mankind's normal gene load M in identical HPV samplesXB；
6th step：Pass through formula M=Mx/MXB, calculate in the HPV infection sample, the HPV in unit mankind's normal gene XB is sub-
Type quantity of viruses.
8. the PCR kit of detection human papilloma virus according to claim 1, it is characterised in that：Described reagent
Box parting and the high-risk hypotypes of all HPV are quantitatively detected, 101-I, 101-II, 101-III and 101-IV need to be used simultaneously；Parting
The high-risk hypotypes of quantitatively common and more typical HPV, 101-I and 101-II in series of products need to be used simultaneously；Parting and fixed
The high-risk hypotypes of common HPV are measured, only need to use 101-I products.
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| CN111321206A (en) * | 2020-03-06 | 2020-06-23 | 杭州博日科技有限公司 | Detection system of quintuple fluorescent PCR (polymerase chain reaction), application and product thereof |
| CN111455108A (en) * | 2020-04-14 | 2020-07-28 | 天津普瑞赛斯分子诊断技术有限责任公司 | Kit and detection method for detecting 14 high-risk HPV (human papilloma Virus) types |
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| CN111321206A (en) * | 2020-03-06 | 2020-06-23 | 杭州博日科技有限公司 | Detection system of quintuple fluorescent PCR (polymerase chain reaction), application and product thereof |
| CN111321206B (en) * | 2020-03-06 | 2023-09-22 | 杭州博日科技股份有限公司 | Detection system of quintuple fluorescent PCR (polymerase chain reaction), application and product thereof |
| CN111455108A (en) * | 2020-04-14 | 2020-07-28 | 天津普瑞赛斯分子诊断技术有限责任公司 | Kit and detection method for detecting 14 high-risk HPV (human papilloma Virus) types |
| CN111455108B (en) * | 2020-04-14 | 2022-09-13 | 天津普瑞赛斯分子诊断技术有限责任公司 | Kit and detection method for detecting 14 high-risk HPV (human papilloma Virus) types |
| CN111593140A (en) * | 2020-05-21 | 2020-08-28 | 杭州海基生物技术有限公司 | Detection and typing kit for high-risk human papilloma virus |
| CN111593140B (en) * | 2020-05-21 | 2023-04-28 | 杭州海基生物技术有限公司 | High-risk human papilloma virus detection and typing kit |
| CN116286982A (en) * | 2022-09-09 | 2023-06-23 | 广州凯普医药科技有限公司 | HPV genotyping detection positive reference, preparation method and application thereof |
| CN116286982B (en) * | 2022-09-09 | 2024-01-30 | 广州凯普医药科技有限公司 | HPV genotyping detection positive reference, preparation method and application thereof |
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