CN101130824B - Method and reagent kit for detecting high-risk human papillomavirus - Google Patents
Method and reagent kit for detecting high-risk human papillomavirus Download PDFInfo
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Abstract
The present invention relates to a method for detecting pathogen-high risk human papillomavirus (HR-HPY) of cervical carcinoma and pointed condyloma in clinical sample and its kit. In particular, it relates to a method for diagnosing HR-HPV infection by using real-time quantitative fluorescent PCR technique and its kit.
Description
Affiliated field
The present invention relates to detect method and test kit that human papillomavirus in the clinical sample (HR-HPV) exists, particularly relate to the real-time quantitative fluorescence polymerase chain reaction technique and detect high-risk human mammilla papillomavirus in the clinical sample, think that diagnosing cervical, pointed condyloma provide the clinical detection method of laboratory foundation and finish the employed test kit of this method.
Background of invention
Cervical cancer is one of modal malignant tumour of women.In recent years, along with the change of mode of life, the rejuvenation that more becomes of cervical cancer patient's age of onset, and case load has the trend of continuous rising.Cervical cancer be at present unique a kind of pathogenic factor more clearly tumour, main diseases because of be by high-risk human mammilla papillomavirus (HR-HPV) continue or repeated infection due to [Walboomers JM, Jacobs MV, Manos MM, et al.Humanpapillomavirus is a necessary cause of invasive cervical cancer worldwide.J Pathol, 1999; 189 (1): 50-53].The HPV type of determining has 80 approximately at present, and the position, epithelium place of complying with its infection is divided into skin-type HPV and reproductive tract epithelium HPV, and wherein about 35 types relate to genital tract infection.According to the height of different type HPV and causing danger property of cervical cancer, be divided into low dangerous type HPV and high-risk HPV again, about 20 types are relevant with tumour.Worldwide, the cervical cancer main harm those do not carry out the women of high-risk human mammilla papillomavirus examination.
Studies show that, HPV16,18 types and cervical cancer height correlation, 31,33 is relevant with 35 type moderates.The HPV type is distributed with regional difference, and in Chinese population, HPV16,52,58 types relatively more to be seen.The negative predictive value of HPV reaches 100%, does not promptly have the generation that can get rid of cervical cancer when HPV infects substantially.Cervical cancer early operation result of treatment is splendid, with the strategy that puts prevention first be simply and effective [Nobbenhuis MAE as the main path that reduces the cervical cancer mortality ratio, Walboomers JMM, Helmerhorst TJM, et al.Relation of humanpapillomavirus status to cervical lesions and consequences forcervical-cancer screening:a prospective study.Lancet, 1999; 354:20-25].If can check out in early days and precancerous lesion (can be dormant for many years) that successfully treatment (particularly cancer before intervention treatment) occurs in cervical tissue, just can block the development of precancerous lesion effectively to cervical cancer.
The diagnosis of human papilloma virus infection case mainly relies on laboratory examination.Laboratory examination comprises that morphocytology detects and molecular Biological Detection (order-checking, in situ hybridization, hybrid capture, PCR etc.).Sensitivity of morphocytology detection method and specificity are all lower, are subjected to subjective effect of operators bigger; Order-checking, hybridization in situ technique complexity to having relatively high expectations of operator, are restricted in clinical application; But this method cost height, complicated operation, there is false positive in hybrid capture method by drugs approved by FDA and be applied to clinically, has therefore also limited its large-scale application in cervical cancer screening; Though the qualitative PCR method is highly sensitive and can carry out early detection to virus, because of its complex steps and pollute easily, thereby also limited its clinical use.
The real-time fluorescence quantitative PCR technology is to grow up based on traditional round pcr the mid-90 in last century.Compare with traditional (end point determination) round pcr, the real-time quantitative detection method has not only realized the quantitative analysis of low copy number target polynucleotide, but also has specificity and advantage such as tolerance range is stronger, level of automation is higher and contamination of heavy is littler.The real-time fluorescence PCR technology is to add fluorescence labeling probe in polymerase chain reaction (PCR) system, use a kind of pcr amplification instrument that has electric charge coupling devices (CCD), reflect each round-robin level of amplification of PCR by the dynamic change that detects fluorescent signal in real time.CCD can periodically send the exciting light of specific wavelength according to certain procedure, collect to detect fluorescent signal, and is aggregated into workstation by software analysis and obtains amplification curve.By analysis, can carry out qualitative and quantitative analysis to test sample to amplification curve and cycle threshold (Ct).
The TaqMan round pcr is a kind of (Mackay IM et al.Real-time PCR invirology..Nucleic Acids Res.20025 of real-time fluorescence PCR; 30 (6): 1292-1305; Lie, Y.S., Petropoulos, C.J., Advances in quantitative PCR technology:5 ' nuclease assays.Current Opinion inBiotechnology1998.9,43-48.).Compare with traditional PCR, it has increased the two ends probe of mark fluorescent reporter group and quenching group respectively in reaction system.When probe structure was complete, the energy that the fluorescence report group sends fluorescence was transferred to quenching group, presents quenching effect.If the existence of target sequence is arranged in the amplification procedure, the process middle probe molecule of amplification is hydrolyzed cut-out gradually, and fluorescence report group and quenching group dissociate mutually, have blocked the two FRET (fluorescence resonance energy transfer) effect, and the fluorescence report group sends fluorescent signal.Along with the carrying out of amplification, fluorescent signal presents linear the enhancing along with the segmental amplification of purpose.
In using known real-time fluorescence quantitative PCR technology for detection and quantitative analysis clinical sample in the practice of certain particular target nucleic acid, in order to reduce and avoid the false negative or the false positive of detected result, improve the accuracy of quantitative analysis, critical basic fundamental link is how based on known target polynucleotide sequences Design and prepare suitable primer and oligonucleotide probe.The inventor is being engaged in round pcr particularly on the basis of real-time fluorescence quantitative PCR technical study in the past for many years, this technology is applied to the detection and the quantitative analysis of genome polynucleotide of all known varients of human papillomavirus, has successfully finished the present invention.
Summary of the invention
An object of the present invention is to provide a kind of method of using HR-HPV virus in the real-time fluorescence quantitative PCR technology detection by quantitative sample, this method comprises: (1) provides and comprises sample, nucleic acid amplification system and fluorescence monitoring system are at interior reaction and detection mixture, (2) by the said target polynucleotide of amplified reaction cyclic amplification, (3) make said fluorescence generation group and indirect combination of target polynucleotide that is amplified, (4) determine the fluorescence volume that fluorescence generation group is produced, and determine the existence and the relative quantity (being the copy number of target nucleic acid sequence) thereof of target polynucleotide in conjunction with the quantitative criterion curve; Wherein the nucleic acid amplification system comprise can with the poly-polynucleotide bonded Oligonucleolide primers of target, and the fluorescence real-time monitoring system comprise one can with target polynucleotide specificity bonded oligonucleotide probe; Be characterised in that employed forward and reverse oligonucleotide primer are respectively 5 '-ttg atg aaa atggca atc c-3 ' (SEQ ID NO:1), 5 '-gac aaa aac gga aat cca gt-3 ' (SEQ IDNO:2), 5 '-cat cgg tgt ctg cat ctt c-3 ' (SEQ ID NO:3) and 5 '-cat cgt tttcct tgt cct c-3 ' (SEQ ID NO:4), and employed oligonucleotide probe is 5 '-acc atg tccttt caa aaa aac att tc-3 ' (SEQ ID NO:5) and 5 '-acc acg tcc ttg aga aaa aggatt-3 ' (SEQ ID NO:6).
According to a preferred embodiment of the invention, the wherein said sample clinical sample that is suspicious high-risk HPV the infected.
According to a preferred embodiment of the invention, wherein said high-risk HPV be selected from common in the Chinese population, the related various HPV of development takes place with cervical cancer.
According to a preferred embodiment of the invention, wherein said high-risk HPV is selected from HPV16,18,31,33,35,39,45,52,58,59,67 types.
According to a preferred embodiment of the invention, wherein said target polynucleotide is from high-risk HPV.
According to a preferred embodiment of the invention, wherein said nucleic acid amplification system is a polymerase chain reaction, and the circulation of said amplified reaction is approximately to repeat 40 times polymerase chain reaction circulation.
According to a preferred embodiment of the invention, wherein said nucleic acid amplification system comprise (a) hot resistant DNA polymerase, (b) 2 '-deoxynucleoside triphosphate, (c) can with the forward primer of article one chain combination of double-stranded target polynucleotide and (d) can with the reverse primer of the second chain combination of double-stranded target polynucleotide.
According to a preferred embodiment of the invention, the real-time detection architecture of wherein said fluorescence comprise can combine with the target polynucleotide and two ends respectively bonded the oligonucleotide probe of fluorescence generation group and fluorescent quenching group is arranged.
According to a preferred embodiment of the invention, said method further comprises: required threshold cycle index when (1) determines that fluorescence that the target polynucleotide in the sample is produced reaches fixed threshold more than the baseline after increasing; (2) fixed sample the is hit threshold cycle index of polynucleotide is compared with the threshold cycle index of known quantity target polynucleotide in the standardized solution, to calculate the hit amount of polynucleotide of sample.
Another object of the present invention provides a kind of test kit that uses HPV in the real-time fluorescence quantitative PCR technology detection by quantitative clinical sample, this test kit comprises: (1) is equipped with concentrated solution, DNA extraction liquid, pure water, pcr amplification reaction liquid, diluent, negative control sample, strong positive standard substance and a plurality of reagent bottles that seal or pipe and (2) separation respectively and is concentrated the packing box of these reagent bottles of packing or pipe.
Wherein concentrated solution is made up of PEG8000; The solution that DNA extraction liquid is made up of guanidinium isothiocyanate, sodium hydroxide, SDS.In the PCR reaction tubes is the PCR system, by primer, probe, hot resistant DNA polymerase, dNTPs, H
2O and damping fluid are formed; Diluent is a TE solution.Use the recombinant plasmid that has target gene fragment as the strong positive standard substance in the test kit of the present invention, its concentration is 1 * 10
5Copy/ml uses the recombinant plasmid that has target gene fragment as critical positive criteria product, and its concentration is 1 * 10
2Copy/ml, and use physiological saline as negative control.
According to another preferred embodiment of the present invention, the forward and the reverse primer that wherein are used for target polynucleotide amplification system are respectively 5 '-ttg atg aaa atg gca atc c-3 ' (SEQ ID NO:1), 5 '-gac aaaaac gga aat cca gt-3 ' (SEQ ID NO:2), 5 '-cat cgg tgt ctg cat ctt c-3 ' (SEQ ID NO:3) and 5 '-cat cgt ttt cct tgt cct c-3 ' (SEQ ID NO:4).
According to another preferred embodiment of the present invention, the oligonucleotide probe that wherein is used for amplification of target polynucleotide and monitoring system is 5 '-acc atg tcc ttt caa aaa aac att tc-3 ' (SEQ ID NO:5) and 5 '-acc acg tcc ttg aga aaa agg att-3 ' (SEQ ID NO:6).
The present invention relates to real-time quantitative fluorescence PCR method and test kit, particularly relate to the application in the laboratory diagnosis that common high-risk HPV infects in Chinese population of real-time qualitative, quantitative fluorescence polymerase chain reaction method and test kit thereof.
The invention provides a kind of method that high-risk HPV exists in the real-time fluorescence quantitative PCR technology qualitative and quantitative detection clinical sample of using, this method comprises: (1) provides the sample that comprises the HPV genomic nucleic acids, the nucleic acid amplification system, reaction and detection mixture with the fluorescence monitoring system, (2) by the said target polynucleotide of amplified reaction cyclic amplification, (3) make said fluorescence generation group and indirect combination of target polynucleotide that is amplified, (4) 4) determine the fluorescence volume that fluorescence generation group is produced, and determine the existence and the relative quantity thereof of target polynucleotide in conjunction with the quantitative criterion curve; Wherein the nucleic acid amplification system comprise can with target polynucleotide bonded Oligonucleolide primers, and fluorescent detection system comprise can with target polynucleotide bonded oligonucleotide probe; Be characterised in that employed forward and reverse oligonucleotide primer are respectively 5 '-ttg atg aaa atg gca atcc-3 ' (SEQ ID NO:1) in the said amplified reaction, 5 '-gac aaa aac gga aat cca gt-3 ' (SEQ ID NO:2), 5 '-cat cgg tgt ctg cat ctt c-3 ' (SEQ ID NO:3) and 5 '-cat cgt ttt cct tgtcctc-3 ' (SEQ ID NO:4), and employed oligonucleotide probe is 5 '-acc atg tcc ttt caaaaa aac att tc-3 ' (SEQ ID NO:5) and 5 '-acc acg tcc ttg aga aaa agg att-3 ' (SEQID NO:6).
With finish based on amplified reaction after to carry out the conventional P CR method of single end point determination different, the real-time quantitative polymerase chain reaction method can be monitored the generation of amplified production at any time in the process of amplified reaction, thereby has improved the accuracy and the precision of detection by quantitative greatly.As everyone knows, real-time fluorescence quantitative PCR is to add the specific oligonucleotide probe that primer and 5 ', 3 ' end are marked with fluorescence report group (for example 6-FAM amidite Fluoresceincarboxylic acid 6) and fluorescent quenching group (for example 6-carboxyl tetramethylrhodamin) respectively in pcr amplification simultaneously.If probe does not combine with target complement sequence, its sequence is kept perfectly, and the reporter group fluorescent signal emitted will be absorbed by quenching group, so there is not fluorescent signal to produce; And when pcr amplification reaction carried out, 5 ' of archaeal dna polymerase-3 ' the 5 prime excision enzyme activity fluorescent probe of will degrading cause the fluorescence report group to separate with the fluorescent quenching group, thereby the fluorescence monitoring system can receive and record fluorescent signal.DNA chain of every amplification promptly has a fluorescence molecule to produce, thereby realize that the accumulation of fluorescent signal and PCR product form fully synchronously, monitor whole PCR reaction process in real time, and can carry out quantitative analysis to unknown target polynucleotide (template) by typical curve.
Similar to common real-time quantitative fluorescence PCR technology, comprising equally in the method that the double-stranded target polynucleotide of HPV viral genome exists in the test sample of the present invention that (1) provides comprises (a) sample to be checked, (b) hot resistant DNA polymerase and (c) amplification reaction system of 2-deoxynucleoside triphosphate (dNTP), and comprise (a) can with article one complementary strand bonded forward primer of double-stranded target polynucleotide to be checked, (b) can with the second complementary strand bonded reverse primer of double-stranded target polynucleotide to be checked, and the fluorescent detection system that (c) can be marked with the oligonucleotide probe of fluorescence generation group and fluorescent quenching group respectively with arbitrary the chain combination and two ends of double-stranded target polynucleotide; (2) by the said target polynucleotide of one or many PCR amplification; (3) make after the annealing circumscribed active function that the knot of said fluorescence generation group by probe and target polynucleotide be incorporated in the Taq enzyme down release fluorescence generation group produce fluorescent signal; (4) detect and fluorescence volume that definite fluorescence generation group is produced; (5) analyze the fluorescence volume that the one or many amplification cycles is sent, to detect existing of target poly polynucleotide; Be characterised in that wherein said target polynucleotide is HPV genome polynucleotide, employed forward and reverse oligonucleotide primer be 5 '-ttg atg aaa atg gca atc c-3 ' (SEQ ID NO:1) respectively, 5 '-gac aaa aac gga aat cca gt-3 ' (SEQ ID NO:2), 5 '-cat cgg tgt ctgcat ctt c-3 ' (SEQ ID NO:3) and 5 '-cat cgt ttt cct tgt cct c-3 ' (SEQ ID NO:4), and employed oligonucleotide probe is 5 '-acc atg tcc ttt caa aaa aac atttc-3 ' (SEQ ID NO:5) and 5 '-acc acg tcc ttg aga aaa agg att-3 ' (SEQ ID NO:6).
As previously mentioned, in order to detect the high-risk HPV virus that may exist in the clinical sample, and can accurately and effectively high-risk HPV virus infection and low risk HPV virus, herpes simplex virus type 2, mycoplasma genitalium, cytomegalovirus, ureaplasma urealyticum, chlamydia trachomatis, gonorrhea diplococcus and other common urogenital infections pathogenic agent discriminatings be come, design and preparation realize that the Oligonucleolide primers and the probe of these purposes are very important sport technique segments.For this reason, we carry out homology relatively at the genome nucleotide sequence that uses suitable foranalysis of nucleic acids software to known HPV virus type, and finding out on the basis of homology segment, further use suitable primer-design software (for example Primer Premier5.5) to select and design to have Oligonucleolide primers the 1:5 '-ttg atg aaa atg gca atc c-3 ' (SEQ ID NO:1) that shows nucleotide sequence down, primer 2: 5 '-gac aaa aacgga aat cca gt-3 ' (SEQ ID NO:2), primer 3:5 '-cat cgg tgt ctg cat ctt c-3 ' (SEQ ID NO:3) and primer 4:5 '-cat cgt ttt cct tgt cct c-3 ' (SEQ ID NO:4); And the oligonucleotide probe that shows sequence under having: 5 '-acc atg tcc ttt caa aaa aac att tc-3 ' (SEQ ID NO:5) and 5 '-acc acg tcc ttg aga aaa agg att-3 ' (SEQ ID NO:6).The pairing amplification section of primer is equivalent to the conserved regions of HPV virus E1 gene coded sequence, and length is 107bp-120bp.Designed these primers and probe all have the sequence that is complementary to high-risk HPV viral genome conserved regions, and there is not homology with the nucleotide sequence of other urogenital infections related diseases substances, do not comprise any common endonuclease yet, so the false negative and the false positive that significantly reduce even avoided HPV virus to detect have improved the reliability and the accuracy that detect.
Can use the DNA synthesizer to synthesize required Oligonucleolide primers, with molecular sieve and fast protein liquid chromatography method (FPLC) purifying and carry out ammonia and separate processing.Same synthetic required detection probes, ammonia are separated after the processing respectively at the 6-FAM amidite of its 5 ' end mark as fluorescence generation group (reporter group), and its 3 ' end mark have active connecting arm, as fluorescent quenching or suppress the TAMRA of group.With the fluorescently-labeled probe of FPLC purification by chromatography.Then, can use polyacrylamide gel (20%) electrophoretic method and spectrophotometry physical characterization institute synthetic primer and probe under the sex change condition.
Use conventional DNA extraction method or commercially available DNA extraction test kit, from the cervical exfoliated cell sample that derives from the experimenter, extract DNA and be used for follow-up pcr amplification.
In the reaction system that contains primer 1,2,3 and 4 (each 10pmol), probe 5 and 6 (each 3pmol), dna profiling (pcr amplification target sequence), dNTPs (10mM), hot resistant DNA polymerase (Plantinium archaeal dna polymerase), PCR damping fluid and water, use the automatic fluoroscopic examination thermal cycler of ABI7000 type or other structure types that the dna sequence dna that as above obtains is carried out pcr amplification.Employed reaction conditions is 93 ℃ of pre-sex change after 3 minutes, 93 ℃ 30 seconds, 55 ℃ 45 seconds, carry out 40 circulations altogether.
After reaction is finished, carry out interpretation of result by the automatic analysis system of being furnished with on the device.In this research, if circulation threshold (Ct) value is equal to or greater than 25, the result is promptly negative; If the Ct value is less than 25, the result is then positive.Wherein with preceding 10 fluorescent signals of being produced of circulation of reaction as the fluorescence background signal, the default setting of fluorescence thresholding is 10 times of 1-10 round-robin fluorescent signal standard deviation.In general, the logarithm of the initial copy number of the Ct value of each template and this template is linear.Initial copy number is many more, and the Ct value is more little.Because the stage before the Ct of amplified reaction value, enzyme in the amplification system, primer, probe and dNTPs are all excessive greatly, and the pH value of system and ionic concn etc. are also relatively stable, so the efficient of amplified reaction is a saturated definite value that has nothing to do with the template number at this moment.The PCR system efficiency having only starting template number (copy number of polynucleotide sample) and with sample have nothing to do relevant with the Ct value.
Can utilize the standard substance production standard curve of known initial copy number, wherein the logarithm with the initial copy number of target polynucleotide is an X-coordinate, and is ordinate zou with the Ct value that as above records.After obtaining the Ct value of unknown quantity target polynucleotide, can learn the logarithm of its initial copy number from the typical curve of the data creating that obtains based on parallel laboratory test, the initial copy number that calculates this target polynucleotide then (promptly initially enters the index amplification during phase when amplification cycles reaches the Ct value, the circulation ratio of Ct value is fabulous, and promptly same target polynucleotide is at different time amplification or the same time resulting Ct value constant always that increases in the difference pipe).That is to say, in order to extrapolate the amount (initial copy number) of target polynucleotide based on above-mentioned amplified reaction result, method of the present invention also should further comprise: required threshold cycle index when (1) determines that fluorescence that the target polynucleotide in the sample is produced reaches fixed threshold more than the baseline after increasing; (2) fixed sample the is hit threshold cycle index of polynucleotide is compared with the threshold cycle index of known quantity target polynucleotide in the standardized solution, thereby calculates the hit amount of polynucleotide of sample.
Another object of the present invention provides a kind of test kit that uses high-risk HPV virus in the real-time fluorescence quantitative PCR technology detection by quantitative clinical sample, this test kit comprises: (1) is equipped with concentrated solution, DNA extraction liquid, pure water, the pcr amplification reaction liquid that contains conventional ingredient known in the art, diluent, negative control sample, strong positive standard substance and DNA positive reference substance and a plurality of reagent bottles that seal or pipe and (2) separation respectively and is concentrated the packing box of these reagent bottles of packing or pipe.
According to a preferred embodiment of the invention, wherein concentrated solution is used for concentrated liquid sample to be checked.
According to another preferred embodiment of the present invention, wherein diluent is used to dilute positive quantitative criterion product.
According to another preferred embodiment of the present invention, wherein be used for forward and reverse primer primer difference 5 '-ttg atg aaa atg gca atc c-3 ' (SEQ ID NO:1), 5 '-gac aaaaac gga aat cca gt-3 ' (SEQ ID NO:2), 5 '-cat cgg tgt ctg cat ctt c-3 ' (SEQID NO:3) and 5 '-cat cgt ttt cct tgt cct c-3 ' (SEQ ID NO:4) of target polynucleotide amplification system.
According to another preferred embodiment of the present invention, the oligonucleotide that wherein is used for amplification of target polynucleotide and monitoring system is 5 '-acc atg tcc ttt caa aaa aac att tc-3 ' (SEQ ID NO:5) and 5 '-accacg tcc ttg aga aaa agg att-3 ' (SEQ ID NO:6).
The method of pointing out in the appended working instructions in step or the test kit is used test kit of the present invention as described above.
As previously mentioned, in order to detect the high-risk HPV virus that may exist in the clinical sample, and accurately and effectively high-risk HPV virus infection and low risk HPV virus, herpes simplex virus type 2, mycoplasma genitalium, cytomegalovirus, ureaplasma urealyticum, chlamydia trachomatis, gonorrhea diplococcus and other common urogenital infections pathogenic agent discriminatings are come, the homology that the present invention is based on different type HPV and correlated virus genome nucleotide sequence compares, and has designed the Oligonucleolide primers and the probe of suitable test kit of the present invention especially.The real-time quantitative that uses these primers and probe to finish detects, and has reduced false positive and false negative that high-risk HPV virus polynucleotide detect greatly.Our tolerance range that the present invention detects high-risk HPV viral genome Nucleotide that experiment showed, is almost 100%, and sensitivity reaches 10
2Individual copy.
What be worth special instruction is that in order to ensure the accuracy of high-risk HPV method for detecting virus of the present invention, we also use the recombinant plasmid that carries the HPV viral nucleotide sequences as DNA quantitative measurement standard product.By these standard substance and use test kit of the present invention, carry out parallel detection under the same conditions and make so-called outer marking quantitative typical curve, with the detection tolerance range and the accuracy of further checking test kit of the present invention, and as the additional quality control standard of production test kit of the present invention.
Description of drawings
Fig. 1 shows the typical curve under (Std curve) window.At the template number is 10
2～10
7The reaction system of copy is carried out the TaqMan pcr analysis.When copy number was 100, the Ct value of test sample promptly detected lower limit sensitivity and can arrive 100 copies about 25.
Fig. 2 shows the detection curve of the weak three parts of positive samples of persistent erection.The Ct value of three parts of samples is respectively 25.95,21.45 and 13.47; S-shaped in conjunction with amplification curve, promptly decidable three duplicate samples are all positive.
Fig. 3 shows the detection curve of negative sample.The amplification curve of three samples is more straight broken line, does not have intersection point with the fluoroscopic examination threshold line, perhaps show in the scope of Ct value between 25～30, and amplification curve does not have S shape feature.
Fig. 4 shows the detection curve of samples such as HPV16,18,31,33,52,58 types, and sample is all confirmed as corresponding type through reverse dot blot hybridization and order-checking.Amplification curve is S-shaped, and the decidable test sample is all positive.
Fig. 5 shows the detection curve of positive sample repeated experiment, can find out different curves all in same CT value scope, and this result shows that test kit has good repeatability.
Embodiment
Embodiment 1: high-risk human mammilla papillomavirus detection kit and use thereof
(1) preparation comprises the test kit of following moiety: DNA extraction liquid (500 μ l/ pipe) 1 pipe, PCR reaction tubes (unlabelled pipe) 10 pipes, H
2O (2000 μ l/ pipe) 1 pipe, strong positive standard substance (50 μ l/ pipe) 1 pipe, critical positive criteria product (50ul/ pipe) 1 pipe, negative control (50 μ l/ pipe) 1 pipe.
(2) collection of specimens, transport and preserve: get doubtful human papilloma virus infection patient's cervical exfoliated cell with aseptic cotton swab or Uterine neck bush, and the tissue sample or the wart tissue that are used for biopsy, place freezing preservation in the cryogenic refrigerator (-70 ℃ or-20 ℃).As test, sample needs to transport in environment below 0 ℃ and sent to the laboratory in 24 hours.
(3) detect step and interpretation of result :-
Uterine neck bush, uterine neck swab: add the 1ml stroke-physiological saline solution, fully concussion shakes up, and extracts cotton swab.Draw whole liquid and go in the 1.5ml centrifuge tube, 12,000 centrifugal 5 minutes.Remove supernatant, precipitation directly adds the abundant mixing of 50 μ l DNA extraction liquid (because extracting solution contains water-fast material, so need during sampling elder generation with the abundant mixing of sample injector; Stop up the phenomenon of suction nozzle as occurring because of the suction nozzle mouth back of too carefully can not drawing or take a sample, can with clean free of contamination scissors the suction nozzle mouth be cut one section earlier).After 100 ℃ of heat treated 10 minutes (error is no more than 1 minute), 12, centrifugal 5 minutes of 000rpm gets supernatant liquor 2 μ l and is used for the PCR reaction.
Biopsy, section preparation: the blood of being stained with band with an amount of sterile saline flush away censorship tissue, get about 50mg tissue, adding the 1ml sterile saline grinds to form tissue homogenate and is transferred to .12 in the 1.5ml centrifuge tube with homogenizer, behind centrifugal 5 minutes of the 000rpm, remove supernatant, add the abundant mixing of 50 μ l DNA extraction liquid in throw out, 100 ℃ of constant temperature are handled 10 minutes (error is no more than 1 minute).12,000rpm collects supernatant liquor 2 μ l and is used for the PCR reaction after centrifugal 5 minutes.
With centrifugal 15 seconds of strong positive standard substance 6000rpm, add diluent 450ul, fully be denoted as 10 behind the mixing
5Get 3 new 0.5ml centrifuge tubes then in addition, standard substance are carried out 10 times of gradient dilutions successively (be denoted as 10 respectively
4, 10
3, 10
2) ,-20 ℃ of preservations are standby.
Get sample to be checked (every part of 2ul) then respectively, with the quantitative criterion product and the negative control product of volume, application of sample carries out pcr amplification in the PCR reaction system.The PCR cycling condition is: 93 ℃ of pre-sex change 3 minutes, 93 ℃ 45 seconds, 55 ℃ 60 seconds, carry out 10 circulations, do not detect fluorescent signal in these 10 circulations; 93 ℃ 30 seconds, 55 ℃ 45 seconds, carry out 30 circulations, detect fluorescent signal in the extension stage of reaction.
Reaction finishes the back and preserves the detection data file.Regulate analytical parameters, make the canonical plotting under typical curve (Std curve) window reach best (being correlation values＜-0.95) (referring to Fig. 1).Can be found out respectively that by Fig. 2 and Fig. 3 the fluorescence curve of positive sample and preset threshold line have an intersection point, the fluorescence curve of negative sample then is lower than threshold value.At last calculate the not mensuration numerical value (Qty) of key sample, i.e. the dna content of HPV virus (referring to Fig. 2 and 3) in the sample by the instrument automatic analysing apparatus.
Embodiment 2: the sample of determining the HPV type is detected
Concrete steps are with embodiment 1, and different is that used sample is all confirmed as HPV16,18,31,33,52,58 types through reverse dot blot hybridization and order-checking, and HPV16,18,31,33,52,58 types are that Chinese population infects maximum types.Sample carries out pcr amplification with test kit of the present invention after extracting nucleic acid, is monitored and collect the variation of fluorescent signal in amplification automatically by instrument.Reaction finishes post analysis, uses test kit of the present invention and all can detect other sample of known type (referring to Fig. 4).
Embodiment 3: the reperformance test that clinical samples detects
Concrete steps are with embodiment 1, and different is that used sample is for determining the male sample.Carry out pcr amplification with test kit of the present invention after extracting nucleic acid, each part sample is provided with a plurality of multiple pipes.In amplification, monitor and collect the variation of fluorescent signal automatically by instrument.Reaction finishes post analysis, uses test kit detected result of the present invention and shows the different curve of same sample all in same CT value scope, and this result shows that test kit has good repeatability.(referring to Fig. 5).
Embodiment 4:20 part clinical samples detect and with the comparison of other medicine boxs
20 routine clinical samples are taken from Guangdong People's Hospital, and concrete steps are with embodiment 1, and different is that used sample is used Spain BIOTOOLS B﹠amp simultaneously; M Labs, the BIOPAP QTS of S.A. company test kit detects, and this test kit by the CE of European Union authentication, can detect common high-risk human mammilla papillomavirus.BIOPAP QTS test kit uses the method for fluorescence dye that clinical samples is carried out real-time quantitative and detects.Identify the validity of the said test kit of the present invention in clinical detection by said test kit and BIOPAP QTS test kit among comparison the present invention in the result of clinical samples detection gained.
Table 1: test kit among the present invention and BIOPAP QTS test kit detected result are relatively
| Mark this shop | Test kit detected result among the present invention (with the CT value representation) | BIOPAP QTS test kit detected result (with the CT value representation) |
| 1 | 27.3 | 28.1 |
| 2 | 22.1 | 22.4 |
| 3 | 20.5 | 23.1 |
| 4 | 24.0 | 22.1 |
| 5 | 16.5 | 16.4 |
| 6 | - | - |
| 7 | 24.7 | 24.0 |
| 8 | - | - |
| 9 | 17.4 | 15.6 |
| 10 | 17.8 | 17.0 |
| 11 | 25.1 | 26 |
| 12 | 18.6 | 17.8 |
| 13 | - | - |
| 14 | 14.9 | 16.1 |
| 15 | 22.0 | 21.5 |
| 16 | 21.7 | 21.0 |
| 17 | 23.5 | 24.0 |
| 18 | - | - |
| 19 | 18.4 | 19.8 |
| 20 | - | - |
In the last table '-' expression test kit detect all negative, required threshold cycle index when the fluorescence that the target polynucleotide in the CT value representation sample is produced after increasing reaches fixed threshold more than the baseline.As above table, the result that two kinds of methods obtain compares, and has shown good consistence, and the high credible of test kit of the present invention has been described.Because the present invention uses fluorescent probe, than dye method higher specificity is arranged; U.S. Digene company hybrid capture (hybrid capure II, the HC II) method that test kit of the present invention uses more in the market is in easier application aspect the requiring of instrument and operator, and the sensitivity of detection also is higher than hybrid capture method.
Sequence table
＜110〉Zhongshan University Anda gene limited-liability company
＜120〉method and the test kit of detection high-risk-type papilloma virus
<140>
<141>
<160>6
<210>1
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>1
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>2
<210>3
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>3
<210>4
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>4
<210>5
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, with the probe that increases as detection fluorescent signal in the pcr amplification process.
<400>5
<210>6
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, with the probe that increases as detection fluorescent signal in the pcr amplification process.
<400>6
Claims (1)
1. use the test kit of high-risk human mammilla papillomavirus in the real-time fluorescence quantitative polymerase chain reaction technology detection by quantitative clinical sample, this test kit comprises: a plurality of reagent bottles or pipe that (1) is equipped with concentrated solution, DNA extraction liquid, pure water, amplification reaction solution, diluent, negative control sample, strong positive standard substance respectively and is sealed, (2) packing box of separation and concentrated these reagent bottles of packing or pipe, the strong positive standard substance are that concentration is 1 * 10
5The recombinant plasmid that has target gene fragment of copy/ml, be characterised in that the forward and the reverse primer that wherein are used for target polynucleotide amplification system are respectively forward primer 5 '-ttg atg aaa atg gca atcc-3 ', 5 '-gac aaa aac gga aat cca gt-3 ', with reverse primer 5 '-cat cgg tgtctg cat ctt c-3 ', 5 '-cat cgt ttt cct tgt cct c-3; The oligonucleotide probe that wherein is used for amplification of target polynucleotide and monitoring system is 5 '-acc atg tcc ttt caa aaa aac atttc-3 ' and 5 '-acc acg tcc ttg aga aaa agg att-3 '.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102229930B (en) * | 2011-06-10 | 2012-12-12 | 亚能生物技术（深圳）有限公司 | PCR group, method and kit for detecting human papilloma virus (HPV) |
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| CN102168147A (en) * | 2010-02-26 | 2011-08-31 | 宁波基内生物技术有限公司 | Primer composition, kit and method used to detect human papillomavirus |
| CN102994646A (en) * | 2011-09-19 | 2013-03-27 | 潘振华 | Fluorescent nucleic acid amplification detection method of human papilloma virus |
| CN103409552B (en) * | 2013-02-01 | 2017-04-05 | 港龙生物技术（深圳）有限公司 | The primer sets of the various high risk HPV genotypes of synchronous detecting, probe groups, method and test kit |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5447839A (en) * | 1988-09-09 | 1995-09-05 | Hoffmann-La Roche Inc. | Detection of human papillomavirus by the polymerase chain reaction |
| US5888724A (en) * | 1995-02-17 | 1999-03-30 | The Trustees Of Columbia University In The City Of New York | Detection of high oncogenic-risk papilloma virus in high grade cervical lesions and cancers by a PCR/ELISA assay |
| CN1706966A (en) * | 2004-06-04 | 2005-12-14 | 何以丰 | Typing and quantitative gene detection method of human papillomavirus |
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2006
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5447839A (en) * | 1988-09-09 | 1995-09-05 | Hoffmann-La Roche Inc. | Detection of human papillomavirus by the polymerase chain reaction |
| US5888724A (en) * | 1995-02-17 | 1999-03-30 | The Trustees Of Columbia University In The City Of New York | Detection of high oncogenic-risk papilloma virus in high grade cervical lesions and cancers by a PCR/ELISA assay |
| CN1706966A (en) * | 2004-06-04 | 2005-12-14 | 何以丰 | Typing and quantitative gene detection method of human papillomavirus |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102229930B (en) * | 2011-06-10 | 2012-12-12 | 亚能生物技术（深圳）有限公司 | PCR group, method and kit for detecting human papilloma virus (HPV) |
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