CN103173565A - Low-risk human papillomavirus (HPV)6/11 detection kit - Google Patents
Low-risk human papillomavirus (HPV)6/11 detection kit Download PDFInfo
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- CN103173565A CN103173565A CN2013100090747A CN201310009074A CN103173565A CN 103173565 A CN103173565 A CN 103173565A CN 2013100090747 A CN2013100090747 A CN 2013100090747A CN 201310009074 A CN201310009074 A CN 201310009074A CN 103173565 A CN103173565 A CN 103173565A
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Abstract
The invention provides a low-risk human papillomavirus (HPV)6/11 detection kit. The kit comprises a nucleic acid releasing agent and PCR (polymerase chain reaction) liquid, wherein the nucleic acid releasing agent comprises 0.01-0.5mM/L of surfactin, 20-300mM/L of potassium chloride, 0.01-2% of sodium dodecyl sulfate and 0.05-1% of ethanol; the PCR liquid comprises a forward primer, a reverse primer and a probe; the primers are used for amplifying targeted polynucleotides; and the probe is used for detecting targeted polynucleotides. The detection results of extracting nucleic acids by using the nucleic acid releasing method in the invention and a boiling method do not have obvious difference. In the invention, when nucleic acids are extracted, the strong protein denaturant is adopted to quickly destroy the protein structures of the pathogen hulls to release the nucleic acids in the pathogens, thus release and extraction of DNA can be completed without heating. The sensitivity of the kit in detecting HPV6/11 can reach 400copies/ml and the quantitative linear range is 400-4.00E+09copies/ml. The kit can be applied to quickly and accurately detecting HPV6/11-DNA in the unknown samples such as wart exfoliative cells and genital secretion and provides a reliable experimental basis for diagnosing HPV6/11 infection.
Description
Technical field
The invention provides a kind of low risk human papillomavirus HPV6/11 detection kit, specifically a kind of HPV6/11-DNA detection kit based on fluorescent PCR.
Background technology
Human papillomavirus (Human papillomavirus, HPV) is the less nonencapsulated double-stranded cyclic DNA virus of molecule amount, and obligate infects the epithelial cell with parasitic human body reproductive organ and other histoorgan.Clinically, virulence size or the large I of carcinogenic risk according to the HPV different subtype is divided into high-risk-type and the large class of low risk two with HPV.Low risk HPV mainly causes exophytic wart class pathology and the low uterine cervix intraepithelial neoplasia of skin of anus and male external genital organs, the large nympha of women, urethral orifice, vagina hypomere, and its virus subtype mainly contains HPV6,11,40,42,43 and 44 types.Pointed condyloma (Genital warts, Condylomata acumlnata, CA) having another name called property wart, anogenital wart, be to be infected the epidermoma sample hyperplasia at caused men and women's sexual organ, perineum and anus position by the HPV of some particular type, multiplely be born between twenty and fifty men and women.This disease is popular in the whole world, is one of modal sexually transmitted disease (STD) of present America and Europe and African country.CA is higher at the sickness rate of China, in recent years increases sharply again, becomes the second largest venereal disease that is only second to gonorrhoea.Have more than 30 kinds relevant to pointed condyloma in nearly hundred kinds of HPV hypotypes, wherein HPV6 type and HPV11 type are again to be identified the Main Subtype that causes pointed condyloma, have the scholar to be referred to as classical hypotype.Therefore, detect fast and accurately HPV6 type and 11 types for its popular etc. having great importance of auxiliary diagnosis, early treatment and the prevention of pointed condyloma.
The laboratory diagnostic method of low risk human papillomavirus mainly contains cytolgical examination, pathological examination, immunohistochemical methods detection, Protocols in Molecular Biology etc. at present.Cytolgical examination is the conventional means of population screening, but drawn materials, smear is made quality, read chip technology affects, accuracy is low, poor repeatability, susceptibility are also limited.Pathological examination is relatively poor to the morphologic features person of not being true to type specificity.Immunohistochemical methods is to come further clear and definite HPV to infect by the capsid antigen that detects HPV, and its gained positive reaction is located clear and definite, and judgement is reliable; But the capsid equipment must be arranged just after HPV DNA replication dna maturation; Negative patient can not be negated diagnosis, and the method susceptibility is lower.In recent years, Protocols in Molecular Biology has manifested its advantage on detecting gradually, thinks that at present round pcr is the best method that detects HPV DNA and somatotype.Fluorescence PCR assay be based on the normal PCR technology and grow up in conjunction with spectroscopic techniques a kind of sensitiveer, more special, more accurate nucleic acid detection technique.Detected result is accurate, and repeatability is high, can dynamically react the forward and backward pathogenic agent dynamic change of patient treatment and reach and clinical relation, and avoid normal PCR to need the problem of aftertreatment in whole process, has reduced pollution.Studies show that in a large number the positive rate that detects HPV DNA with round pcr far above other detection techniques, is to be used for now pointed condyloma and the most frequently used powerful of HPV Infect And Diagnose, and can better be applied to that HPV causes a disease, among the research of mechanism of carcinogenesis.Use round pcr to detect and be mainly concerned with two aspects, the extraction of nucleic acid and the augmentation detection of nucleic acid.
The domestic boiling method that mainly adopts clinically extracts the nucleic acid of human papillomavirus at present, specifically first with secretory product sample concentration, washing, then adds lysate, boil, and high speed centrifugation, getting supernatant is template.The method nucleic acid extraction process is more complicated, sample process length consuming time, and a plurality of steps such as process boiling lysis, high speed centrifugation enrichment DNA when processing sample, there is loss in DNA in sample, especially insufficient to the sample cracking of high density, enrichment is incomplete, can cause a large amount of loss of DNA to cause sample quantitatively on the low side, simultaneously due to the heat step that has adopted water-bath or metal bath, easily cause Aerosol Pollution; In addition, for concentrated this step, the concentrated effect of different manufacturers is different, and what have can see precipitation, and what have can't see; What see precipitation is because virus and albumen have all been concentrated, and is difficult to abundant mixing when causing like this back to add lysate; The operator that can make that can't see precipitation can't determine can or can not blow and beat viral nucleic acid when supernatant is abandoned in suction.
the method that detects clinically HPV6/11-DNA mainly is based on technology and the improvement thereof of real-time fluorescence quantitative PCR at present, Real-Time Fluorescent Quantitative PCR Technique is development in recent years a kind of nucleic acid detection technique rapidly, use a kind of pcr amplification instrument with fluorescence detection device, fluorescence detection device can according to certain property program loop send the exciting light of specific wavelength, collect and detect fluorescent signal, the level of amplification that reflects in real time each circulation of PCR by the dynamic change that detects fluorescent signal, can obtain amplification curve by the software automatic analysis after off-test, according to the intersection point (being the Ct value) of amplification curve and fluorescence threshold line and the shape of amplification curve, can judge the yin and yang attribute result, if quantitative reference material or the standard substance of concentration known are arranged in same reaction, can obtain typical curve by the software automatic analysis, realize thus the definite value (being detection by quantitative) to unknown sample.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 fluorescent energy that the fluorescence report group sends was absorbed by quenching group, presents quenching effect; If the existence of target sequence is arranged in amplification procedure, extension along with target fragment, probe molecule is cut off by the Taq enzymic hydrolysis gradually, fluorescence report group and quenching group dissociate mutually, fluorescence energy transfer effect between having blocked both, the fluorescent signal that the fluorescence report group sends is collected by fluorescence detection device.Along with the carrying out of amplification, fluorescent signal presents linear the enhancing along with the amplification of purpose fragment.After off-test, the software automatic analysis data that can carry by the fluorescent PCR instrument, can obtain the definite value result of yin and yang attribute result and concentration of specimens, therefore, this technology is in the detection and quantitative analysis of target polynucleotide sample, replace gradually traditional PCR method, obtain using very widely.
Existing test kit based on Real-Time Fluorescent Quantitative PCR Technique detection HPV6/11 is applied in clinical detection both at home and abroad at present, but these test kits extract nucleic acid with boiling method mostly, and its detection sensitivity is not high, about 500 ~ 1000copies/ml left and right; In addition, these test kits lack perfect system of quality control mostly, also need further improve and improve technical level, and make this series products more satisfy the needs of clinical Accurate Diagnosis.
Summary of the invention
The invention provides the application of a kind of nucleic acid releasing agent in low risk human papillomavirus HPV6/11 detects, described nucleic acid releasing agent comprises Buddhist Sha graceful (surfactin) 0.01 ~ 0.5mM/L, Repone K 20 ~ 300mM/L, sodium laurylsulfonate 0.01 ~ 2% and ethanol 0.05 ~ 1%.
In nucleic acid releasing agent of the present invention, its solvent can be commonly used for this area, is for example sterilized water or TE damping fluid.The present invention is disclosed in first low risk human papillomavirus HPV6/11 and uses the nucleic acid releasing agent that contains strong protein denaturant in detecting, and simplified to a great extent the step that should virus detects, and detection sensitivity is greatly improved.
The invention provides a kind of low risk human papillomavirus HPV6/11 detection kit, described test kit comprises nucleic acid releasing agent and PCR reaction solution, described nucleic acid releasing agent comprises Buddhist Sha graceful (surfactin) 0.01 ~ 0.5mM/L, Repone K 20 ~ 300mM/L, sodium laurylsulfonate 0.01 ~ 2% and ethanol 0.05 ~ 1%; Comprise upstream primer, the downstream primer for the target polynucleotide amplification in described PCR reaction solution and be used for the probe that target polynucleotide detects.
Use the method for the nucleic acid releasing agent release nucleic acid in test kit of the present invention and the detected result of boiling method extraction nucleic acid there is no notable difference, and adopt strong protein denaturant when extracting nucleic acid in the present invention, rapid damage pathogenic agent coat protein structure, discharge pathogen nucleic acid, need not to heat release and the extraction that to complete DNA; The sensitivity that test kit of the present invention detects HPV6/11 can reach 400copies/ml, and the quantitative linearity scope is 400 ~ 4.00E+09copies/ml.
Concrete, the upstream primer sequence that is used for target polynucleotide is 5 '-CGGCCTAGCGACAGCACAG-3 ', and the downstream primer sequence is 5 '-ACATACGCATCCGTGGCAA-3 ', and probe sequence is 5 '-TGTGCCTCCTCCCAACCCTGTATCC-3 '.Test kit of the present invention has good specificity because adopting above-mentioned primer and/or the probe sequence for detection of target polynucleotide.
In the present invention, also comprise interior mark in preferred described test kit, described interior target sequence is 5 '-CTGCCGTTACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCC CTGGGCAGGTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAA-3 '.Being designated as a segment length who inserts the pUC18T carrier in described in the present invention is the recombinant chou of the DNA artificial sequence synthetic of 103 base pairs, it is plasmid, it prevents as the positive internal reference in the pcr amplification system false negative that causes due to the PCR interfering substance that may exist in sample.Comprise in described test kit in interior target situation, also comprise the upstream primer, downstream primer and the probe that detect for interior mark in described PCR reaction solution; In put on the trip primer sequence be 5 '-CTGCCGTTACTGCCCTGTG-3 ', interior mark downstream primer sequence is 5 '-TTGGTCTCCTTAAACCTGTCTTG-3 ', interior mark probe sequence is 5'-ACCACCAACTTCATCCACGTTCACC-3 '.
Also comprise enzyme mixation in preferred test kit of the present invention, comprise the uracil dna glycosylase (UNG enzyme) of hot resistant DNA polymerase (Taq enzyme) and 0.5 ~ 2U/ μ l in described enzyme mixation, also comprise dUTP simultaneously in described PCR reaction solution.Wherein the function of UNG enzyme is the PCR product that degraded contains dU, utilizes the dUTP in UNG enzyme and PCR reaction solution can play the effect of prevention PCR product pollution, thereby prevents the pattern detection false positive.
In an embodiment, also comprise the quantitative reference material of HPV6/11, HPV6/11 positive control and HPV6/11 negative control in described test kit.
The present invention also provides a kind of HPV6/11 detection kit, described test kit comprises the PCR reaction solution, comprise upstream primer, the downstream primer for the target polynucleotide amplification in described PCR reaction solution and be used for the probe that target polynucleotide detects, and the upstream primer sequence that is used for target polynucleotide is 5 '-CGGCCTAGCGACAGCACAG-3 ', and the downstream primer sequence is 5 '-ACATACGCATCCGTGGCAA-3 '.
The present invention also provides a kind of HPV6/11 detection kit, described test kit comprises the PCR reaction solution, comprise upstream primer, the downstream primer for the target polynucleotide amplification in described PCR reaction solution and be used for the probe that target polynucleotide detects, and being 5 '-TGTGCCTCCTCCCAACCCTGTATCC-3 ' for the probe sequence of target polynucleotide.
The present invention also specifically provides a kind of HPV6/11 detection kit, and described test kit comprises nucleic acid releasing agent, PCR reaction solution, interior mark, enzyme mixation, the quantitative reference material of HPV6/11, HPV6/11 positive control and HPV6/11 negative control; And comprise Buddhist Sha graceful (surfactin) 0.01 ~ 0.5mM/L in described nucleic acid releasing agent, Repone K 20 ~ 300mM/L, sodium laurylsulfonate 0.01 ~ 2%, ethanol 0.05～1% and solvent TE damping fluid; Comprise upstream primer, downstream primer and probe for target polynucleotide amplification and detection in described PCR reaction solution, the upstream primer, downstream primer and the probe that are used for interior mark amplification and detect, 10 * PCR reaction buffer, deoxyribonucleoside triphosphate and/or ribonucleotide triphosphate dNTP; Being designated as a segment length who inserts the pUC18T carrier in described is the recombinant chou of the DNA artificial sequence synthetic of 103 base pairs; Comprise archaeal dna polymerase and uracil dna glycosylase in described enzyme mixation.
HPV6/11 fluorescent quantificationally PCR detecting kit operation provided by the invention fast, method is easy, detection sensitivity is high, sensing range is wide, using this test kit can detect fast and accurately to the HPV6/11-DNA in the unknown sample such as wart surface cast-off cells, genital secretion, provides reliable experimental basis for diagnosis HPV6/11 infects.
Description of drawings
2. be 1. the amplification curve of the positive sample to be tested of low risk human papillomavirus HPV6/11-DNA detected result in embodiment in Fig. 1, be the amplification curve of the negative sample to be tested of HPV6/11-DNA detected result in embodiment in Fig. 1.
Embodiment
Be only below the preferred embodiment of the present invention, protection scope of the present invention is not limited to this, any those skilled in the art in technical scope disclosed by the invention, can be easy to the change carried out or change all be encompassed in protection scope of the present invention within.Therefore, protection scope of the present invention should be as the criterion with the protection domain of claims.
The present embodiment provides a kind of concrete low risk human papillomavirus (HPV6/11) fluorescent quantificationally PCR detecting kit, and it comprises following component:
1. nucleic acid releasing agent: comprise Buddhist Sha graceful (surfactin) 0.1mM/L, Repone K 100mM/L, the ethanol of sodium laurylsulfonate (SDS) 0.1%, 0.1%.
2. mark (positive internal reference) in: for the segment length that inserts the pUC18T carrier is the recombinant chou of the DNA artificial sequence synthetic of 103 base pairs, it is plasmid, concentration is 2.00E+05copies/ml, and the sequence of 103 base pairs is: 5 '-CTGCCGTTACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCC CTGGGCAGGTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAA-3 '.
3. PCR reaction solution: comprise 10 * PCR reaction buffer, 5 μ l, 0.2mmol/L dNTP, the upstream and downstream primer that is used for the target polynucleotide amplification is 0.3 μ mol/L, the probe that is used for the target polynucleotide detection is 0.3 μ mol/L, the upstream and downstream primer that is used for interior tap section amplification is 0.1 μ mol/L, is 0.1 μ mol/L for detection of interior target probe.Wherein, described 10 * PCR reaction buffer is the 200mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride solution that comprises pH7.5,30mmol/L magnesium chloride solution, 500mmol/L Klorvess Liquid, 0.2% Triton solution and 10% formamide soln; Described dNTP comprises dATP, dCTP, dUTP and dGTP; Described is primer and the probe that comes from the conservative region of HPV6 type and HPV11 type nucleic acid for the upstream and downstream primer of target polynucleotide amplification and the probe that detects for target polynucleotide, and its base sequence is respectively: upstream primer: 5 '-CGGCCTAGCGACAGCACAG-3 '; Downstream primer: 5 '-ACATACGCATCCG TGGCAA-3 '; Probe: 5 '-TGTGCCTCCTCCCAACCCTG TATCC-3 '; Describedly be respectively for detection of interior target primer probe sequence: upstream primer: 5 '-CTGCCGTTACTGCCCTGTG-3 '; Downstream primer: 5 '-TTGGTCTCCTTAAACCTGTCTTG-3 '; Probe: 5'-ACCACCAACTTCATCCACGTTCACC-3'.
4. enzyme mixation: comprise the hot resistant DNA polymerase (Taq enzyme) of 5U/ μ l and the uracil dna glycosylase (UNG enzyme) of 1U/ μ l.
5. the quantitative reference material of HPV6/11: derive from the HPV6/11 strong positive plasmid that uses after the HPV6/11 quantitative linearity reference material L1 of enterprise ~ L5 definite value, this HPV6/11 virus quantitatively reference material comprises the gradient reference material that A, B, C, four concentration of D form, and its concentration is respectively 4.00E+07copies/ml(A), 4.00E+06copies/ml(B), 4.00E+05copies/ml(C), 4.00E+04copies/ml(D).
6. HPV6/11 positive control: be the HPV6/11 strong positive sample that clinical hospitals is collected, its concentration is 4.00E+05copies/ml.
7. HPV6/11 negative control: be sterile saline.
The present embodiment provides the operation steps of the described test kit of above-described embodiment 1 for detection of HPV6/11-DNA in the unknown sample such as wart surface cast-off cells, genital secretion:
One, reagent is prepared
Quantity according to sample to be tested, HPV6/11 negative control, HPV6/11 positive control and the quantitative reference material A of HPV6/11 ~ D, get in proportion PCR reaction solution (38 μ l/ person-portion), enzyme mixation (2 μ l/ person-portion) and the interior mark 0.25 μ l/ person-portion of respective amount, fully be mixed into PCR-mix, when for example sample to be tested is 3 person-portion, need altogether the PCR-mix of preparation 9 person-portions (people's umber of above-mentioned four is respectively 3,1,1 and 4); Instantaneous centrifugal rear standby.
Two, sample process
Add nucleic acid releasing agent 2～5 shallow the beating of the dark suction of μ l(suggestion in each PCR reaction tubes, avoid occurring bubble), each pipe adds each 3～5 μ l of sample to be tested, HPV6/11 negative control, HPV6/11 positive control and the quantitative reference material A of HPV6/11 ~ D successively; The interval is more than 10 minutes, and every pipe adds PCR-mix40～45 μ l, the lid upper tube cap.
Three, Fluorescence PCR and interpretation of result (carrying out on the fluorescent quantitative PCR instrument)
1) the PCR reaction tubes is put into the amplification instrument sample cell, by correspondence, sample to be tested title and quantitative reference material concentration are set sequentially.
2) fluorescence detection channel is selected: select FAM passage (Reporter:FAM, Quencher:None) to detect HPV6/11; Select HEX or VIC passage (Reporter:VIC, Quencher:None) to detect interior mark; Reference fluorescence (Passive Reference) is set to none.
3) the quantitative fluorescent PCR reaction conditions sees Table 1:
Table 1
4) interpretation of result
After reaction finished, the automatic saving result of instrument can utilize the software that instrument carries to carry out automatic analysis, and starting value, end value and threshold value that also can the manual regulation baseline be analyzed, and then record sample Ct value and result.The intersection point of amplification curve and threshold line is called Ct value (be cycle threshold, refer to the cycling numerical value that fluorescent signal in the PCR reaction tubes experiences when reaching the threshold value of setting); Instrument software by the typical curve that 4 quantitative reference materials of concentration gradient are drawn, can be tried to achieve the detection by quantitative result of each sample according to each sample Ct value size automatically.For measuring Ct value≤39(Ct value＞0) sample, be reported as the HPV6/11-DNA positive, the amplification curve of sample to be tested is S-type at this moment, as in Fig. 1 1.; For the sample that measure to show without Ct value, simultaneously in mark test positive (Ct value≤39), be reported as the HPV6/11-DNA feminine gender, the sample to be tested amplification curve is straight at this moment, as in Fig. 1 2.; For measuring Ct value〉39 sample, simultaneously in mark test positive (Ct value≤39), be reported as lower than the detection lower limit.If interior mark Ct value〉39 or interior mark show that without the Ct value, the detected result of this sample is invalid, should search and get rid of reason, and this sample is carried out revision test.
Use the specific test of test kit in the present invention to show, itself and common venereal diseases pathogenic agent CT, NG, HSV, UU etc. and the equal no cross reactions of high-risk HPV hypotype such as 16,18,31,52 illustrate that test kit of the present invention has good specificity.
In use the present invention, test kit detects the human mammilla tumor virus L 1 gene type reference material (it contains HPV6 type and HPV11 type reference material) of National Institute for Food and Drugs Control, its yin and yang attribute coincidence rate is 100%, and the detected result of quantitative linearity reference material, sensitivity reference material meets quality standard; In precision test shows batch and batch between good reproducibility, the variation coefficient of its Ct value＜10%, the concentration variation coefficient＜50%.In use the present invention, test kit shows the detection test of clinical sample, and the quantitative linearity scope of this test kit is 400～4.00E+09copies/ml, and detecting lower limit is that sensitivity is 400copies/ml.
Claims (10)
1. the application of nucleic acid releasing agent in low risk human papillomavirus HPV6/11 detects, described nucleic acid releasing agent comprises Buddhist Sha graceful (surfactin) 0.01 ~ 0.5mM/L, Repone K 20 ~ 300mM/L, sodium laurylsulfonate 0.01 ~ 2% and ethanol 0.05 ~ 1%.
2. low risk human papillomavirus HPV6/11 detection kit, described test kit comprises nucleic acid releasing agent and PCR reaction solution, described nucleic acid releasing agent comprises Buddhist Sha graceful (surfactin) 0.01 ~ 0.5mM/L, Repone K 20 ~ 300mM/L, sodium laurylsulfonate 0.01 ~ 2% and ethanol 0.05 ~ 1%; Comprise upstream primer, the downstream primer for the target polynucleotide amplification in described PCR reaction solution and be used for the probe that target polynucleotide detects.
3. detection kit according to claim 2, it is characterized in that, the upstream primer sequence that is used for target polynucleotide is 5 '-CGGCCTAGCGACAGCACAG-3 ', and the downstream primer sequence that is used for target polynucleotide is 5 '-ACATACGCATCCGTGGCAA-3 '.
4. detection kit according to claim 2, is characterized in that, the probe sequence that is used for target polynucleotide is 5 '-TGTGCCTCCTCCCAACCCTGTATCC-3 '.
5. the described detection kit of any one according to claim 2 ~ 4, it is characterized in that, also comprise interior mark in described test kit, described interior target sequence is 5 '-CTGCCGTTACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCC CTGGGCAGGTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAA-3 '.
6. detection kit according to claim 5, is characterized in that, also comprises the upstream primer, downstream primer and the probe that detect for interior mark in described PCR reaction solution; In put on the trip primer sequence be 5 '-CTGCCGTTACTGCCCTGTG-3 ', interior mark downstream primer sequence is 5 '-TTGGTCTCCTTAAACCTGTCTTG-3 ', interior mark probe sequence is 5'-ACCACCAACTTCATCCACGTTCACC-3'.
7. the described detection kit of any one according to claim 2 ~ 4, it is characterized in that, also comprise enzyme mixation in described test kit, the uracil dna glycosylase (UNG enzyme) that comprises hot resistant DNA polymerase (Taq enzyme) and 0.5 ~ 2U/ μ l in described enzyme mixation also comprises dUTP simultaneously in described PCR reaction solution.
8. the described detection kit of any one according to claim 2 ~ 4, is characterized in that, also comprises the quantitative reference material of HPV6/11, HPV6/11 positive control and HPV6/11 negative control in described test kit.
9. HPV6/11 detection kit, described test kit comprises the PCR reaction solution, comprise upstream primer, the downstream primer for the target polynucleotide amplification in described PCR reaction solution and be used for the probe that target polynucleotide detects, and the upstream primer sequence that is used for target polynucleotide is 5 '-CGGCCTAGCGACAGCACAG-3 ', and the downstream primer sequence is 5 '-ACATACGCATCCGTGGCAA-3 '.
10. HPV6/11 detection kit, described test kit comprises the PCR reaction solution, comprise upstream primer, the downstream primer for the target polynucleotide amplification in described PCR reaction solution and be used for the probe that target polynucleotide detects, and being 5 '-TGTGCCTCCTCCCAACCCTGTATCC-3 ' for the probe sequence of target polynucleotide.
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