CN109609696A - For detecting the nucleic acid reagent, kit, system and method for human papilloma virus - Google Patents

For detecting the nucleic acid reagent, kit, system and method for human papilloma virus Download PDF

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CN109609696A
CN109609696A CN201811641953.0A CN201811641953A CN109609696A CN 109609696 A CN109609696 A CN 109609696A CN 201811641953 A CN201811641953 A CN 201811641953A CN 109609696 A CN109609696 A CN 109609696A
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CN109609696B (en
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王雷
马寅佳
林笑冬
王晓艳
张志强
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Beijing Zhuo Chenghui Biological Polytron Technologies Inc
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Abstract

This disclosure relates to which a kind of nucleic acid reagent, kit, system and method for detecting human papilloma virus is wherein, the nucleic acid reagent includes storage or mutually probe shown in primer and NO.53~77 SEQ ID shown in NO.1~50 SEQ ID of any mixed storage independently of one another respectively.The disclosure establishes nucleic acid reagent, kit, the system and method for detection human papilloma virus by above-described primer and probe, can be realized quick, comprehensive, sensitive, special, automatic testing result and determines.

Description

For detecting the nucleic acid reagent, kit, system and method for human papilloma virus
Technical field
This disclosure relates to field of biotechnology, and in particular, to a kind of nucleic acid reagent for detecting human papilloma virus, Kit, system and method.
Background technique
With popularizing for screening, incidence and the death rate of the cervical carcinoma in developed country or region have been remarkably decreased, some Such as Britain, developed country and Australia year cervical cancer pathogenesis rate from 1991 to 2000 reduce 33%.Since low developed area is sieved Look into do not carry out or it is lack of standardization worldwide therefore cervical carcinoma is the second largest common malignant tumour of women, the death rate is still protected Hold higher level.The cervical carcinoma cause of disease is clear, largely research shows that high-risk human mammilla papillomavirus (Human Papillomavirus, HPV) persistent infection be cervical carcinoma and precancerous lesion main pathogenic.
HPV belongs to papovaviridae (Papovavirdae) Papillomavirus, and HPV, which mainly passes through, directly or indirectly to be connect Touch contaminated articles or the Sex transmitted pathogen mankind.HPV not only has host specificity, and has tissue specificity, can only infect The skin and mucomembranous epithelial cell of people causes a variety of papillomas or wart and mucous membrane genital tract epithelial proliferation of human skin Damage.Continuing HPV infection is the main reason for leading to cervical carcinoma and some genitals tumor-like lesions.
HPV is spherical in shape, nonencapsulated double-stranded DNA virus.Nucleocapsid is in symmetrical 20 face body, is made of 72 capsomeres, capsomere It is made of two kinds of capsid proteins of L1, L2.HPV gene group leader 8000bp encodes 8 main open reading frame (Open Reading Frames, ORF), it is divided into 3 functional areas, i.e. early transcription area (area E), late transcription area (area L) and long control area (Long Control Region, LCR).The area E encodes the early proteins such as E1, E2, E4, E5, E6, E7, participate in virus duplication, The functions such as transcription, translational control and conversion；The area L encodes major capsid protein and secondary capsid protein.L1 accounts for about capsid protein 80%, highly conserved, L2 content is less, makes a variation more.LCR contains needed for replication orgin and the HPV expression of HPV genomic DNA Controlling element, regulate and control virus transcription and duplication.
Existing HPV gene type more than 100 at present, nearly 33% genotype is related with vulneratio genitalis.Infect genital tract Be divided into two kinds: low risk (Low Risk, LR) and high-risk-type (High Risk, HR).Wherein HPV 16,18,31,33, 35,39,45,51,52,56,58,59 and 68 be common HPV high-risk-type infection, has close ties with cervical carcinoma；And 6 He of HPV 11 are HPV low risk.HPV 16 and 18 is most commonly seen in HPV high-risk-type, grinds the cervical carcinoma for making internal disorder or usurp and showing that the whole world has 70% It is related with the infection of HPV 16 and 18.And HPV 6 and 11 is related with most of genitals tumor-like lesion.HPV Genotyping is to facing Bed is significant.Cervical carcinoma can be progressed to by continuing the infection of HPV high-risk-type, some studies have shown that different high-risk HPV gene Type has different carcinogenic risks.According to year U.S.'s gynecatoptron and uterine neck pathology association (American Society for Colposcopy and Cervical Pathology, ASCCP) administration guide about cervical carcinoma screening, HPV 16 and 18 feels The strategy of women's screening of dye is infected different from other high-risk HPVs, 30 years old or more women of the infection of HPV16 and 18, should be direct Row vaginoscopy, and other high-risk-types are positive, repetitive cell and HPV are detected after being spaced 12 months, and detection is positive again Just carry out vaginoscopy.In addition, HPV vaccine, for preventing cervical carcinoma, what oneself listed at present has the four of Merk company production The bivalent vaccine Cervarix of valence vaccine, GlaxoSmithKline PLC company.It is both needed to before vaccine inoculation and after inoculation carry out HPV gene point Type detection.HPV Genotyping also has important value using monitoring for HPV vaccine.
Since HPV cannot be cultivated in traditional cell culture fluid, the direct virology diagnostic techniques of other classics, such as Electron microscope and immunohistochemistry, for the routine testing of HPV, sensibility and specificity is bad.Therefore, all mesh Preceding diagnostic HPV detection reagent (including self-control or commercial reagents) depends on viral nucleic acid detection.HPV nucleic acid inspection Test agent is broadly divided into three classes: (1) the general detection reagent of HR-HPV nucleic acid gene type, which, which only reports, whether there is HR-HPV infection, but specific HPV genotype is not reported, HC2 (Hybrid Capture 2) is wherein most typically representative；(2) While the general detection of HR-HPV nucleic acid gene type, recheck 16/18 genotype of HPV: detection reagent report whether there is HR- HPV infection, while reporting and being infected with the presence or absence of HPV 16/18.Some reagents can be with HR-HPV to the detection infected of HPV 16/18 It carries out simultaneously, whether other reagents are first detected whether there are HR-HPV infection, if detection is positive, deposit to sample reinspection It is infecting, such as Cervista HPV 16/18 (Hologic, Danbury, Ct, USA).(3) HPV Genotyping reagent, the detection Reagent can distinguish the specific genotype of HPV, directly report the result of every kind of genotype infection.Internal reagent producer research and development at present HPV Genotyping reagent belongs to such mostly, and it is also using this kind of that the HPV nucleic acid detection of clinical labororatory Jian exhibition, which has many, Grouping reagents.
HPV infection rate is improved with the beginning of sexual life, and is also mostly mixed infection, more than 12~28.9%, even The HPV of a variety of types can be detected in 85% cases of cervical cancer simultaneously.It is domestic at present clinically to carry out the main of HPV DNA detection Method has real-time fluorescence detection (PCR) method, gene chips, hybrid capture (Hybrid Capture II, HC- Ⅱ).This 3 kinds of methods respectively have superiority and inferiority as the means of clinical cervical carcinoma screening.
Domestic gene chips HPV detection reagent is all mutually to be tied using PCR amplification in vitro with DNA reverse dot blot hybridization at present The DNA chip technology of conjunction.The testing result of gene chips can only carry out qualitative analysis to sample, signal occur with detection site Whether judged, the power of signaling point cannot provide the reference of quantitative aspect.In addition after all gene chips than real-time The more step hybridization colour developings of PCR, so generally operating procedure is still more relatively cumbersome than real-time fluorescence PCR.Due to PCR amplification Afterwards, it is also necessary to uncap and carry out hybrid experiment, increase the risk of laboratory PCR product pollution, it is possible to cause experimental result False positive.
II technology of HC- is actually the integrated use of nucleic acid hybridization, immune response and chemiluminescence, with HPV points Group's (high risk group and low danger group) function, but do not have accurately typing function, this method has been widely used in the screening of cervical carcinoma And follow-up.HC- II has found that the sensitivity of highly squamous intraepithelial lesions (HSIL) is 95%, hence it is evident that it is better than liquid based cytology, but It is specificity is 85%, slightly below liquid based cytology.Since this method is more early in foreign countries' exploitation and listing, what the country developed later Other methods HPV DNA detection reagent, which is substantially all, to be done clinic with the party science of law and compares and verify.Although still can't be HPV Goldstandard is diagnosed, but also the research and development for later country's HPV diagnostic reagent provide an extremely valuable reference frame.But with The extensive use of recent domestic, also gradually expose it is some clinically the shortcomings that and problem, for example to be also easy to produce intersection dirty Dye, cost is higher, high-risk-type and low risk are there are certain cross reaction, and external existing document report exist it is individual High-risk-type omission factor.
Summary of the invention
The nucleic acid reagent for quick, accurate, integrated parting detection human papilloma virus that purpose of this disclosure is to provide one kind, Kit, system and method.
To achieve the goals above, disclosure first aspect: a kind of nucleic acid examination for detecting human papilloma virus is provided Agent, wherein the nucleic acid reagent includes storage or mutually the SEQ ID NO.1-50 institute of any mixed storage independently of one another respectively Probe shown in the primer and SEQ ID NO.53-77 shown.
Optionally, primer shown in the SEQ ID NO.1 relative to 1 μM, draws as shown in SEQ ID NO.2-50 respectively The content of object is respectively 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM and 0.1~0.5 μM, the content of the probe as shown in SEQ ID NO.53-77 is each independently 0.1~0.3 μM respectively.
Optionally, the nucleic acid reagent further includes Quality Control in the positive；
Quality Control contains probe shown in primer shown in SEQ ID NO.51-52 and SEQ ID NO.78 in the positive.
Optionally, the nucleic acid reagent includes A pipe, B pipe, C pipe and D pipe；A pipe contains SEQ ID NO.1-2,5-6,9- Shown in primer shown in 12,19-20,27-28,43-44,51-52 and SEQ ID NO.53,55,57-58,62,66,74,78 Probe；B pipe contains primer and SEQ ID shown in SEQ ID NO.3-4,7-8,13-14,21-22,29-32,45-46,51-52 Probe shown in NO.54,56,59,63,67,68,75,78；C pipe contains SEQ ID NO.7-8,15-16,23-24,29-30, It is visited shown in primer shown in 33-34,39-40,47-48,51-52 and SEQ ID NO.56,60,64,67,69,72,76,78 Needle；D pipe contains primer and SEQ ID shown in SEQ ID NO.5-6,17-18,25-26,35-38,41-42,49-50,51-52 Probe shown in NO.55,61,65,70,71,73,77,78.
Optionally, probe shown in SEQ ID NO.55-56,58-61 has the first fluorescent marker；SEQ ID NO.57, Probe shown in 62-65,67,70 has the second fluorescent marker；It is visited shown in SEQ ID NO.53,54,66,68-69,71-73 Needle set has third fluorescent marker；Probe shown in SEQ ID NO.74-78 has the 4th fluorescent marker；The first fluorescence mark Note, second fluorescent marker, the third fluorescent marker and the 4th fluorescent marker are different, and select each independently It is glimmering from FAM fluorescent marker, JOE fluorescent marker, TAMRA fluorescent marker, CY5 fluorescent marker, ROX fluorescent marker and Quasar670 One of signal.
Optionally, the human papilloma virus include HPV6 type, HPV11 type, HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV42 type, HPV43 type, HPV44 type, HPV45 type, HPV51 type, HPV52 Type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 type, HPV73 type, HPV81 type, HPV82 type and At least one of HPV83 type.
Disclosure second aspect: providing a kind of for detecting the kit of human papilloma virus, which contains this public affairs Open nucleic acid reagent described in first aspect, and optionally, the kit also contain reaction system buffer, archaeal dna polymerase, At least one of magnesium ion, dNTP and water.
The disclosure third aspect: nucleic acid reagent described in disclosure first aspect is provided in preparation for detecting human papilloma Purposes in the kit of virus.
Disclosure fourth aspect: providing a kind of system for detecting human papilloma virus, which includes having the inspection of A pipe Survey PCR instrument, computing device and the output device of device, B pipe detector, C pipe detector and D pipe detector, the A pipe detector, B Pipe detector, C pipe detector and D pipe detector are respectively the nucleic acid reagent storage appearance for being mounted with nucleic acid reagent as described above Device, the PCR instrument include the first fluorescence channel, the second fluorescence channel, third fluorescence channel and the 4th fluorescence channel, described first Fluorescence channel, second fluorescence channel, the third fluorescence channel and the 4th fluorescence channel are different, and respectively solely On the spot for FAM fluorescence channel, JOE fluorescence channel, TAMRA fluorescence channel, CY5 fluorescence channel, ROX fluorescence channel or Quasar670 fluorescence channel；The computing device includes memory and processor, and computer journey is stored in the memory Sequence, the processor is configured to the computer program stored in the memory is executed, to realize following differentiation:
If positive control and negative control are set up, testing result is effective；
If the first fluorescence channel of A pipe has Tm value to be that 66 DEG C of corresponding melting peakss curves are determined as that HPV 16 is positive, if A pipe the It is that 58 DEG C of corresponding melting peakss curves are determined as that HPV 31 is positive that one fluorescence channel, which has Tm value, if the second fluorescence channel of A pipe has Tm Value is that 65 DEG C of corresponding melting peakss curves are determined as that HPV 26 is positive, if it is 55 DEG C corresponding that the second fluorescence channel of A pipe, which has Tm value, Melting peakss curve is determined as that HPV 42 is positive, if it is that 68 DEG C of corresponding melting peakss curves determine that A pipe third fluorescence channel, which has Tm value, It is positive for HPV 51, if it is that 56 DEG C of corresponding melting peakss curves are determined as that HPV 6 is positive that A pipe third fluorescence channel, which has Tm value, if A It is that 63 DEG C of corresponding melting peakss curves are determined as that HPV 73 is positive that the 4th fluorescence channel of pipe, which has Tm value, if the 4th fluorescence channel of A pipe Having Tm value is that 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified；
If the first fluorescence channel of B pipe has Tm value to be that 67 DEG C of corresponding melting peakss curves are determined as that HPV 18 is positive, if B pipe the It is that 54 DEG C of corresponding melting peakss curves are determined as that HPV 33 is positive that one fluorescence channel, which has Tm value, if the second fluorescence channel of B pipe has Tm Value is that 69 DEG C of corresponding melting peakss curves are determined as that HPV 52 is positive, if it is 56 DEG C corresponding that the second fluorescence channel of B pipe, which has Tm value, Melting peakss curve is determined as that HPV 43 is positive, if it is that 63 DEG C of corresponding melting peakss curves determine that B pipe third fluorescence channel, which has Tm value, It is positive for HPV 53, if B pipe third fluorescence channel has Tm value to be determined as that HPV 11 is positive for 51 DEG C of corresponding melting peakss curves, if It is that 60 DEG C of corresponding melting peakss curves are determined as that HPV 81 is positive that the 4th fluorescence channel of B pipe, which has Tm value, if the 4th fluorescence channel of B pipe Having Tm value is that 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified；
If the first fluorescence channel of C pipe has Tm value to be that 67 DEG C of corresponding melting peakss curves are determined as that HPV 18 is positive, if C pipe the It is that 56 DEG C of corresponding melting peakss curves are determined as that HPV 35 is positive that one fluorescence channel, which has Tm value, if the second fluorescence channel of C pipe has Tm Value is that 69 DEG C of corresponding melting peakss curves are determined as that HPV 52 is positive, if it is 54 DEG C corresponding that the second fluorescence channel of C pipe, which has Tm value, Melting peakss curve is determined as that HPV 44 is positive, if it is that 62 DEG C of corresponding melting peakss curves determine that C pipe third fluorescence channel, which has Tm value, It is positive for HPV 56, if C pipe third fluorescence channel has Tm value to be determined as that HPV 66 is positive for 53 DEG C of corresponding melting peakss curves, if It is that 61 DEG C of corresponding melting peakss curves determine that sample is positive for PV 82 that the 4th fluorescence channel of C pipe, which has Tm value, if the 4th fluorescence of C pipe is logical It is that 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified that, which there is Tm value in road,；
If the first fluorescence channel of D pipe has Tm value to be that 66 DEG C of corresponding melting peakss curves are determined as that HPV 16 is positive, if D pipe the It is that 57 DEG C of corresponding melting peakss curves are determined as that HPV 39 is positive that one fluorescence channel, which has Tm value, if the second fluorescence channel of D pipe has Tm Value is that 65 DEG C of corresponding melting peakss curves are determined as that HPV 58 is positive, if it is 55 DEG C corresponding that the second fluorescence channel of D pipe, which has Tm value, Melting peakss curve is determined as that HPV 45 is positive, if it is that 60 DEG C of corresponding melting peakss curves determine that D pipe third fluorescence channel, which has Tm value, It is positive for HPV 59, if D pipe third fluorescence channel has Tm value to be determined as that HPV 68 is positive for 52 DEG C of corresponding melting peakss curves, if It is that 62 DEG C of corresponding melting peakss curves are determined as that HPV 83 is positive that the 4th fluorescence channel of D pipe, which has Tm value, if the 4th fluorescence channel of D pipe Having Tm value is that 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified.
The 5th aspect of the disclosure: a kind of method for detecting human papilloma virus is provided, wherein this method comprises: adopting With nucleic acid reagent as described above, PCR amplification is carried out to the DNA of sample to be tested；The PCR instrument for carrying out the PCR amplification includes the One fluorescence channel, the second fluorescence channel, third fluorescence channel and the 4th fluorescence channel；First fluorescence channel, described second Fluorescence channel, the third fluorescence channel and the 4th fluorescence channel are different, and it is logical to be each independently FAM fluorescence Road, JOE fluorescence channel, TAMRA fluorescence channel, CY5 fluorescence channel, ROX fluorescence channel or Quasar670 fluorescence channel；And Carry out following differentiation:
If positive control and negative control are set up, testing result is effective；
If the first fluorescence channel of A pipe has Tm value to be that 66 DEG C of corresponding melting peakss curves are determined as that HPV 16 is positive, if A pipe the It is that 58 DEG C of corresponding melting peakss curves are determined as that HPV 31 is positive that one fluorescence channel, which has Tm value, if the second fluorescence channel of A pipe has Tm Value is that 65 DEG C of corresponding melting peakss curves are determined as that HPV 26 is positive, if it is 55 DEG C corresponding that the second fluorescence channel of A pipe, which has Tm value, Melting peakss curve is determined as that HPV 42 is positive, if it is that 68 DEG C of corresponding melting peakss curves determine that A pipe third fluorescence channel, which has Tm value, It is positive for HPV 51, if it is that 56 DEG C of corresponding melting peakss curves are determined as that HPV 6 is positive that A pipe third fluorescence channel, which has Tm value, if A It is that 63 DEG C of corresponding melting peakss curves are determined as that HPV 73 is positive that the 4th fluorescence channel of pipe, which has Tm value, if the 4th fluorescence channel of A pipe Having Tm value is that 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified；
If the first fluorescence channel of B pipe has Tm value to be that 67 DEG C of corresponding melting peakss curves are determined as that HPV 18 is positive, if B pipe the It is that 54 DEG C of corresponding melting peakss curves are determined as that HPV 33 is positive that one fluorescence channel, which has Tm value, if the second fluorescence channel of B pipe has Tm Value is that 69 DEG C of corresponding melting peakss curves are determined as that HPV 52 is positive, if it is 56 DEG C corresponding that the second fluorescence channel of B pipe, which has Tm value, Melting peakss curve is determined as that HPV 43 is positive, if it is that 63 DEG C of corresponding melting peakss curves determine that B pipe third fluorescence channel, which has Tm value, It is positive for HPV 53, if B pipe third fluorescence channel has Tm value to be determined as that HPV 11 is positive for 51 DEG C of corresponding melting peakss curves, if It is that 60 DEG C of corresponding melting peakss curves are determined as that HPV 81 is positive that the 4th fluorescence channel of B pipe, which has Tm value, if the 4th fluorescence channel of B pipe Having Tm value is that 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified；
If the first fluorescence channel of C pipe has Tm value to be that 67 DEG C of corresponding melting peakss curves are determined as that HPV 18 is positive, if C pipe the It is that 56 DEG C of corresponding melting peakss curves are determined as that HPV 35 is positive that one fluorescence channel, which has Tm value, if the second fluorescence channel of C pipe has Tm Value is that 69 DEG C of corresponding melting peakss curves are determined as that HPV 52 is positive, if it is 54 DEG C corresponding that the second fluorescence channel of C pipe, which has Tm value, Melting peakss curve is determined as that HPV 44 is positive, if it is that 62 DEG C of corresponding melting peakss curves determine that C pipe third fluorescence channel, which has Tm value, It is positive for HPV 56, if C pipe third fluorescence channel has Tm value to be determined as that HPV 66 is positive for 53 DEG C of corresponding melting peakss curves, if It is that 61 DEG C of corresponding melting peakss curves determine that sample is positive for PV 82 that the 4th fluorescence channel of C pipe, which has Tm value, if the 4th fluorescence of C pipe is logical It is that 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified that, which there is Tm value in road,；
If the first fluorescence channel of D pipe has Tm value to be that 66 DEG C of corresponding melting peakss curves are determined as that HPV 16 is positive, if D pipe the It is that 57 DEG C of corresponding melting peakss curves are determined as that HPV 39 is positive that one fluorescence channel, which has Tm value, if the second fluorescence channel of D pipe has Tm Value is that 65 DEG C of corresponding melting peakss curves are determined as that HPV 58 is positive, if it is 55 DEG C corresponding that the second fluorescence channel of D pipe, which has Tm value, Melting peakss curve is determined as that HPV 45 is positive, if it is that 60 DEG C of corresponding melting peakss curves determine that D pipe third fluorescence channel, which has Tm value, It is positive for HPV 59, if D pipe third fluorescence channel has Tm value to be determined as that HPV 68 is positive for 52 DEG C of corresponding melting peakss curves, if It is that 62 DEG C of corresponding melting peakss curves are determined as that HPV 83 is positive that the 4th fluorescence channel of D pipe, which has Tm value, if the 4th fluorescence channel of D pipe Having Tm value is that 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified.
The beneficial effect of the disclosure is:
The disclosure detects human papilloma virus by ParaDNA and Hybeacon probe technique parting, avoids smear, training It supports and the methods of the RT-PCR detection operating time is long and cumbersome, realize that quick, easy, sensitive, special automatic result is sentenced It is fixed, reach following detection effect:
(1) single tube Multiple detection
The disclosure can disposable parting detection include HPV6 type, HPV11 type, HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV42 type, HPV43 type, HPV44 type, HPV45 type, HPV51 type, HPV52 Type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 type, HPV73 type, HPV81 type, HPV82 type and Human papilloma virus including HPV83 type, testing process is simple, as a result automatic interpretation and reliable, saves time, manpower and examination Agent cost.
(2) easily operation
Clinical sample can directly be detected by ParaDNA system support sampler can be obtained reliably as a result, avoiding Costly and time-consuming sample extraction step, realizes the Emergent detection in addition to Specialty Experiment room.
(3) integrative solution
The disclosure is directed to the demand of the popular hypotype detection of HPV China, provides a set of comprehensive, quick, accurate and operation letter Just integrative solution includes rapidly extracting, fluorescent PCR amplification and the judgement of automation result of nucleic acid.
(4) high sensitivity
The disclosure can be realized the qualitative detection of 25 HPV hypotypes, and the detection of each target gene is sensitive in reaction system Degree can reach 103Copy/mL is suitable with substance real-time fluorescence PCR detection susceptibility.
(5) specificity is good
In the nucleic acid reagent of the disclosure, all primers all pass through BLAST and compare analysis, conservative with height and special Property, can not only will test target mutually distinguishes, and can also, living environment identical bacterium difference close with other kinds It opens, this includes 40 type of human papilloma virus HPV, HPV54 type, HPV61 type, HPV67 type, HPV69 type, HPV70 type, HPV71 type It is read with HPV72 type, herpes simplex virus type II, microspironema pallidum, Ureaplasma urealyticum, mycoplasma hominis, NEISSERIA GONORRHOEAE, white Pearl bacterium, trichomonas vaginalis, chlamydia trachomatis, Acinetobacter bauamnnii and enterococcus faecalis.
Other feature and advantage of the disclosure will the following detailed description will be given in the detailed implementation section.
Specific embodiment
The specific embodiment of the disclosure is described in detail below.It should be understood that described herein specific Embodiment is only used for describing and explaining the disclosure, is not limited to the disclosure.
Disclosure first aspect: it provides a kind of for detecting the nucleic acid reagent of human papilloma virus, wherein the nucleic acid examination Agent includes storage or mutually primer and SEQ ID shown in the SEQ ID NO.1-50 of any mixed storage independently of one another respectively Probe shown in NO.53-77.
The disclosure by ParaDNA and Hybeacon probe technique detect human papilloma virus, avoid smear, culture and The methods of the RT-PCR detection operating time is long and cumbersome, can quickly, accurate, integrally parting detection includes HPV6 Type, HPV11 type, HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV42 type, HPV43 type, HPV44 type, HPV45 type, HPV51 type, HPV52 type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 Human papilloma virus including type, HPV68 type, HPV73 type, HPV81 type, HPV82 type and HPV83 type.
For Hybeacon probe technique to the more demanding of probe, the Tm value of probe is particularly important；In addition, probe and primer Combined effect also has important influence to expanding effect.Above-mentioned primer and probe in the design process, has considered not only difference The primer and probe of target gene is in a reaction system the problem of coamplification, i.e. assessment Tm value, the target Tm that corresponds to probe The difference of value, avoids the occurrence of situations such as hairpin structure and dimer at G/C content, and to guarantee alternative primer and probe section point Above-mentioned a variety of enteroviruses can not be covered comprehensively, and specificity is good and coverage is high.
Further, primer shown in the SEQ ID NO.1 relative to 1 μM, respectively as shown in SEQ ID NO.2-50 The content of primer respectively can for 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM and 0.1~0.5 μM, the content of the probe as shown in SEQ ID NO.53-77 can be each independently respectively 0.1~0.3 μM.
According to the disclosure, to do good quality control, the nucleic acid reagent can also include Quality Control in the positive.Further, Quality Control can contain probe shown in primer shown in SEQ ID NO.51-52 and SEQ ID NO.78 in the positive.At this moment, Primer shown in SEQ ID NO.1 relative to 1 μM, the content of the primer as shown in SEQ ID NO.51-52 respectively may be used respectively Think 0.5~1.0 μM and 0.1~0.5 μM, the content of the probe as shown in SEQ ID NO.78 can be 0.1~0.3 μM.It is logical It crosses and Quality Control in the positive is added, can effectively prompt because false negative caused by the reasons such as operation error, PCR mortifier is examined Survey result.
According to the disclosure, in order to enhance the accuracy of testing result, the nucleic acid reagent can be divided into four pipes, i.e., the described core Acid reagent may include A pipe, B pipe, C pipe and D pipe；A pipe contains SEQ ID NO.1-2,5-6,9-12,19-20,27-28,43- Probe shown in primer shown in 44,51-52 and SEQ ID NO.53,55,57-58,62,66,74,78；B pipe contains SEQ ID Primer shown in NO.3-4,7-8,13-14,21-22,29-32,45-46,51-52 and SEQ ID NO.54,56,59,63,67, Probe shown in 68,75,78；C pipe contains SEQ ID NO.7-8,15-16,23-24,29-30,33-34,39-40,47-48, Probe shown in primer shown in 51-52 and SEQ ID NO.56,60,64,67,69,72,76,78；D pipe contains SEQ ID Primer shown in NO.5-6,17-18,25-26,35-38,41-42,49-50,51-52 and SEQ ID NO.55,61,65,70, Probe shown in 71,73,77,78.
It is possible to further carry out the permutation and combination of fluorescent marker according to the respective Tm value of probe, so that in same system The amplification of different probe identified respectively.For example, as an implementation, shown in SEQ ID NO.55-56,58-61 Probe has the first fluorescent marker；Probe shown in SEQ ID NO.57,62-65,67,70 has the second fluorescent marker；SEQ Probe shown in ID NO.53,54,66,68-69,71-73 has third fluorescent marker；It is visited shown in SEQ ID NO.74-78 Needle set has the 4th fluorescent marker；First fluorescent marker, second fluorescent marker, the third fluorescent marker and described Four fluorescent markers are different, and are each independently selected from FAM fluorescent marker, JOE fluorescent marker, TAMRA fluorescent marker, CY5 One of fluorescent marker, ROX fluorescent marker and Quasar670 fluorescent marker.As a kind of particularly preferred embodiment, Probe shown in SEQ ID NO.55-56,58-61 has FAM fluorescent marker；Shown in SEQ ID NO.57,62-65,67,70 Probe has JOE fluorescent marker；Probe shown in SEQ ID NO.53,54,66,68-69,71-73 has TAMRA fluorescence mark Note；Probe shown in SEQ ID NO.74-78 has CY5 fluorescent marker.In order to enhance dissolution peak effect, above-mentioned target-probe is equal It can be dual labelled probe.In probe FAM be 6- Fluoresceincarboxylic acid, JOE 2, the chloro- 6- Fluoresceincarboxylic acid of 7- dimethyl -4,5 two, TAMRA is 6- carboxyl tetramethylrhodamine, and CY5 is 5H- indoles cyanines.
According to the disclosure, the human papilloma virus may include HPV6 type, HPV11 type, HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV42 type, HPV43 type, HPV44 type, HPV45 type, HPV51 Type, HPV52 type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 type, HPV73 type, HPV81 type, At least one of HPV82 type and HPV83 type.
Disclosure second aspect: providing a kind of for detecting the kit of human papilloma virus, which contains this public affairs Open nucleic acid reagent described in first aspect, and optionally, the kit also contain reaction system buffer, archaeal dna polymerase, At least one of magnesium ion, dNTP and water.
The kit of the disclosure can be realized quick, accurate, sensitive, special, automatic testing result and determine, significantly improve To including HPV6 type, HPV11 type, HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 Type, HPV42 type, HPV43 type, HPV44 type, HPV45 type, HPV51 type, HPV52 type, HPV53 type, HPV56 type, HPV58 type, Human papilloma virus including HPV59 type, HPV66 type, HPV68 type, HPV73 type, HPV81 type, HPV82 type and HPV83 type is same Sensibility, specificity and the simplicity of Shi Jinhang detection.
The disclosure third aspect: nucleic acid reagent described in disclosure first aspect is provided in preparation for detecting human papilloma Purposes in the kit of virus.
Disclosure fourth aspect: providing a kind of system for detecting human papilloma virus, which includes having the inspection of A pipe Survey PCR instrument, computing device and the output device of device, B pipe detector, C pipe detector and D pipe detector, the A pipe detector, B Pipe detector, C pipe detector and D pipe detector are respectively mounted with the above-mentioned nucleic acid reagent including A pipe, B pipe, C pipe and D pipe Nucleic acid reagent tank, the PCR instrument include that the first fluorescence channel, the second fluorescence channel, third fluorescence channel and the 4th are glimmering Optical channel, first fluorescence channel, second fluorescence channel, the third fluorescence channel and the 4th fluorescence channel are each It is not identical, and it is glimmering to be each independently FAM fluorescence channel, JOE fluorescence channel, TAMRA fluorescence channel, CY5 fluorescence channel, ROX Optical channel or Quasar670 fluorescence channel；The computing device includes memory and processor, and meter is stored in the memory Calculation machine program, the processor is configured to the computer program stored in the memory is executed, to realize following differentiation:
If positive control and negative control are set up, testing result is effective；
If the first fluorescence channel of A pipe has Tm value to be that 66 DEG C of corresponding melting peakss curves are determined as that HPV 16 is positive, if A pipe the It is that 58 DEG C of corresponding melting peakss curves are determined as that HPV 31 is positive that one fluorescence channel, which has Tm value, if the second fluorescence channel of A pipe has Tm Value is that 65 DEG C of corresponding melting peakss curves are determined as that HPV 26 is positive, if it is 55 DEG C corresponding that the second fluorescence channel of A pipe, which has Tm value, Melting peakss curve is determined as that HPV 42 is positive, if it is that 68 DEG C of corresponding melting peakss curves determine that A pipe third fluorescence channel, which has Tm value, It is positive for HPV 51, if it is that 56 DEG C of corresponding melting peakss curves are determined as that HPV 6 is positive that A pipe third fluorescence channel, which has Tm value, if A It is that 63 DEG C of corresponding melting peakss curves are determined as that HPV 73 is positive that the 4th fluorescence channel of pipe, which has Tm value, if the 4th fluorescence channel of A pipe Having Tm value is that 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified；
If the first fluorescence channel of B pipe has Tm value to be that 67 DEG C of corresponding melting peakss curves are determined as that HPV 18 is positive, if B pipe the It is that 54 DEG C of corresponding melting peakss curves are determined as that HPV 33 is positive that one fluorescence channel, which has Tm value, if the second fluorescence channel of B pipe has Tm Value is that 69 DEG C of corresponding melting peakss curves are determined as that HPV 52 is positive, if it is 56 DEG C corresponding that the second fluorescence channel of B pipe, which has Tm value, Melting peakss curve is determined as that HPV 43 is positive, if it is that 63 DEG C of corresponding melting peakss curves determine that B pipe third fluorescence channel, which has Tm value, It is positive for HPV 53, if B pipe third fluorescence channel has Tm value to be determined as that HPV 11 is positive for 51 DEG C of corresponding melting peakss curves, if It is that 60 DEG C of corresponding melting peakss curves are determined as that HPV 81 is positive that the 4th fluorescence channel of B pipe, which has Tm value, if the 4th fluorescence channel of B pipe Having Tm value is that 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified；
If the first fluorescence channel of C pipe has Tm value to be that 67 DEG C of corresponding melting peakss curves are determined as that HPV 18 is positive, if C pipe the It is that 56 DEG C of corresponding melting peakss curves are determined as that HPV 35 is positive that one fluorescence channel, which has Tm value, if the second fluorescence channel of C pipe has Tm Value is that 69 DEG C of corresponding melting peakss curves are determined as that HPV 52 is positive, if it is 54 DEG C corresponding that the second fluorescence channel of C pipe, which has Tm value, Melting peakss curve is determined as that HPV 44 is positive, if it is that 62 DEG C of corresponding melting peakss curves determine that C pipe third fluorescence channel, which has Tm value, It is positive for HPV 56, if C pipe third fluorescence channel has Tm value to be determined as that HPV 66 is positive for 53 DEG C of corresponding melting peakss curves, if It is that 61 DEG C of corresponding melting peakss curves determine that sample is positive for PV 82 that the 4th fluorescence channel of C pipe, which has Tm value, if the 4th fluorescence of C pipe is logical It is that 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified that, which there is Tm value in road,；
If the first fluorescence channel of D pipe has Tm value to be that 66 DEG C of corresponding melting peakss curves are determined as that HPV 16 is positive, if D pipe the It is that 57 DEG C of corresponding melting peakss curves are determined as that HPV 39 is positive that one fluorescence channel, which has Tm value, if the second fluorescence channel of D pipe has Tm Value is that 65 DEG C of corresponding melting peakss curves are determined as that HPV 58 is positive, if it is 55 DEG C corresponding that the second fluorescence channel of D pipe, which has Tm value, Melting peakss curve is determined as that HPV 45 is positive, if it is that 60 DEG C of corresponding melting peakss curves determine that D pipe third fluorescence channel, which has Tm value, It is positive for HPV 59, if D pipe third fluorescence channel has Tm value to be determined as that HPV 68 is positive for 52 DEG C of corresponding melting peakss curves, if It is that 62 DEG C of corresponding melting peakss curves are determined as that HPV 83 is positive that the 4th fluorescence channel of D pipe, which has Tm value, if the 4th fluorescence channel of D pipe Having Tm value is that 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified.
The 5th aspect of the disclosure: a kind of method for detecting human papilloma virus is provided, wherein this method comprises: adopting With it is above-mentioned including A pipe, B pipe, C pipe and D pipe nucleic acid reagent, PCR amplification is carried out to the DNA of sample to be tested；Carry out the PCR expansion The PCR instrument of increasing includes the first fluorescence channel, the second fluorescence channel, third fluorescence channel and the 4th fluorescence channel；Described first is glimmering Optical channel, second fluorescence channel, the third fluorescence channel and the 4th fluorescence channel are different, and respectively independent Ground is FAM fluorescence channel, JOE fluorescence channel, TAMRA fluorescence channel, CY5 fluorescence channel, ROX fluorescence channel or Quasar670 Fluorescence channel；And carry out following differentiation:
If positive control and negative control are set up, testing result is effective；
If the first fluorescence channel of A pipe has Tm value to be that 66 DEG C of corresponding melting peakss curves are determined as that HPV 16 is positive, if A pipe the It is that 58 DEG C of corresponding melting peakss curves are determined as that HPV 31 is positive that one fluorescence channel, which has Tm value, if the second fluorescence channel of A pipe has Tm Value is that 65 DEG C of corresponding melting peakss curves are determined as that HPV 26 is positive, if it is 55 DEG C corresponding that the second fluorescence channel of A pipe, which has Tm value, Melting peakss curve is determined as that HPV 42 is positive, if it is that 68 DEG C of corresponding melting peakss curves determine that A pipe third fluorescence channel, which has Tm value, It is positive for HPV 51, if it is that 56 DEG C of corresponding melting peakss curves are determined as that HPV 6 is positive that A pipe third fluorescence channel, which has Tm value, if A It is that 63 DEG C of corresponding melting peakss curves are determined as that HPV 73 is positive that the 4th fluorescence channel of pipe, which has Tm value, if the 4th fluorescence channel of A pipe Having Tm value is that 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified；
If the first fluorescence channel of B pipe has Tm value to be that 67 DEG C of corresponding melting peakss curves are determined as that HPV 18 is positive, if B pipe the It is that 54 DEG C of corresponding melting peakss curves are determined as that HPV 33 is positive that one fluorescence channel, which has Tm value, if the second fluorescence channel of B pipe has Tm Value is that 69 DEG C of corresponding melting peakss curves are determined as that HPV 52 is positive, if it is 56 DEG C corresponding that the second fluorescence channel of B pipe, which has Tm value, Melting peakss curve is determined as that HPV 43 is positive, if it is that 63 DEG C of corresponding melting peakss curves determine that B pipe third fluorescence channel, which has Tm value, It is positive for HPV 53, if B pipe third fluorescence channel has Tm value to be determined as that HPV 11 is positive for 51 DEG C of corresponding melting peakss curves, if It is that 60 DEG C of corresponding melting peakss curves are determined as that HPV 81 is positive that the 4th fluorescence channel of B pipe, which has Tm value, if the 4th fluorescence channel of B pipe Having Tm value is that 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified；
If the first fluorescence channel of C pipe has Tm value to be that 67 DEG C of corresponding melting peakss curves are determined as that HPV 18 is positive, if C pipe the It is that 56 DEG C of corresponding melting peakss curves are determined as that HPV 35 is positive that one fluorescence channel, which has Tm value, if the second fluorescence channel of C pipe has Tm Value is that 69 DEG C of corresponding melting peakss curves are determined as that HPV 52 is positive, if it is 54 DEG C corresponding that the second fluorescence channel of C pipe, which has Tm value, Melting peakss curve is determined as that HPV 44 is positive, if it is that 62 DEG C of corresponding melting peakss curves determine that C pipe third fluorescence channel, which has Tm value, It is positive for HPV 56, if C pipe third fluorescence channel has Tm value to be determined as that HPV 66 is positive for 53 DEG C of corresponding melting peakss curves, if It is that 61 DEG C of corresponding melting peakss curves determine that sample is positive for PV 82 that the 4th fluorescence channel of C pipe, which has Tm value, if the 4th fluorescence of C pipe is logical It is that 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified that, which there is Tm value in road,；
If the first fluorescence channel of D pipe has Tm value to be that 66 DEG C of corresponding melting peakss curves are determined as that HPV 16 is positive, if D pipe the It is that 57 DEG C of corresponding melting peakss curves are determined as that HPV 39 is positive that one fluorescence channel, which has Tm value, if the second fluorescence channel of D pipe has Tm Value is that 65 DEG C of corresponding melting peakss curves are determined as that HPV 58 is positive, if it is 55 DEG C corresponding that the second fluorescence channel of D pipe, which has Tm value, Melting peakss curve is determined as that HPV 45 is positive, if it is that 60 DEG C of corresponding melting peakss curves determine that D pipe third fluorescence channel, which has Tm value, It is positive for HPV 59, if D pipe third fluorescence channel has Tm value to be determined as that HPV 68 is positive for 52 DEG C of corresponding melting peakss curves, if It is that 62 DEG C of corresponding melting peakss curves are determined as that HPV 83 is positive that the 4th fluorescence channel of D pipe, which has Tm value, if the 4th fluorescence channel of D pipe Having Tm value is that 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified.
Wherein, the sample to be tested can be patient's Cervical scrapes sample, and the condition of the PCR amplification can be with are as follows: 98 DEG C, 60s, (98 DEG C, 5s, 58 DEG C, 5s, 72 DEG C, 5s, 49 circulations)；Solubility curve analysis: 98 DEG C, 60s, 35 DEG C, 60s, drop rate is 1.0℃/s；80 DEG C, 5s, liter rate is 0.5 DEG C/s, which collects fluorescence.
Disclosed method can in 80 minutes it is sensitive specifically realize include HPV6 type, HPV11 type, HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV42 type, HPV43 type, HPV44 type, HPV45 Type, HPV51 type, HPV52 type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 type, HPV73 type, The system screening of human papilloma virus including HPV81 type, HPV82 type and HPV83 type, testing process is simple, as a result sentences automatically It reads and reliable, saves time, manpower and reagent cost.
The disclosure is further elaborated by the following examples, but the disclosure is not therefore by any limit System.
Reagent is commercial products in following embodiment, and primer, probe are synthesized in Biosearch (USA) company.
Embodiment
1, primer, probe synthesis
According to probe sequence shown in primer sequence shown in table 1 and table 2, sequent synthesis is carried out.Y represents degeneracy in sequence Base T/C；R represents degeneracy base A/G；W represents degeneracy base A/T；FAM is 6- Fluoresceincarboxylic acid, JOE 2,7- bis- in probe The chloro- 6- Fluoresceincarboxylic acid of methyl -4,5 two, TAMRA are 6- carboxyl tetramethylrhodamine, CY5 is 5H- indoles cyanines.The probe of table 2 Bracket in sequence indicates that the t on the left of bracket has fluorescent marker, the selection of the content representation fluorescent marker in bracket.
Table 1
Table 2
2, sample process
After conventional method acquires patient's Cervical scrapes sample sample, samplers sample swab matched with ParaDNA is used On cervical exfoliated cell, be placed directly within i.e. amplifiable in the reactor of ParaDNA.
3, Hybeacon probe technique detection architecture is constructed
Polymerase Phire Hot Start II DNA Polymerase (article No. F122L), Mg2+, dNTPS is purchased from ThermoFisher company, other biochemical reagents are that import packing or domestic analysis are pure；Fluorescence detector is ParaDNA.
Reaction system is formulated as follows:
Reaction system: 30 μ L of total system is prepared according to following operation.2 × PCR Buffer 15 μ L, magnesium chloride solution 3- 4mM, dNTPS be 1~1.5mM, 0.5~1.0 μM of upstream primer, 0.1~0.5 μM of downstream primer, Hybeacon probe 0.1~ 0.3 μM, 1.2 μ L of polymerase, 5 μ L of template, specific primer and probe content is shown in Table 3, and residue is supplied with water.
Table 3
Kit is divided into A pipe, B pipe, C pipe and 4 reaction tubes of D pipe, and A pipe contains SEQ ID NO.1-2,5-6 in upper table 1, SEQ ID NO.53,55,57-58,62,66 in primer shown in 9-12,19-20,27-28,43-44,51-52 and upper table 2, Probe shown in 74,78；B pipe contains SEQ ID NO.3-4,7-8,13-14,21-22,29-32,45-46,51-52 in upper table 1 Shown in probe shown in SEQ ID NO.54,56,59,63,67,68,75,78 in primer and upper table 2；C pipe is containing in upper table 1 SEQ in primer shown in SEQ ID NO.7-8,15-16,23-24,29-30,33-34,39-40,47-48,51-52 and upper table 2 Probe shown in ID NO.56,60,64,67,69,72,76,78；D pipe contains SEQ ID NO.5-6,17-18,25- in upper table 1 SEQ ID NO.55,61,65,70,71,73,77 in primer shown in 26,35-38,41-42,49-50,51-52 and upper table 2, Probe shown in 78.
Reaction condition: selecting FAM, JOE, CY5 and TAMRA as reporter group, and response procedures are as follows: 98 DEG C, 60s, (98 DEG C, 10s, 58 DEG C, 10s, 30~40 circulations)；Solubility curve analysis: 98 DEG C, 60s, 35 DEG C, 60s, drop rate is 1.0 DEG C/s；80 DEG C, 5s, liter rate is 0.5 DEG C/s, which collects fluorescence.
Reaction result judgement:
If positive control and negative control are set up, testing result is effective；
If it is that 66 DEG C of corresponding melting peakss curves are determined as that HPV 16 is positive that A pipe FAM fluorescence channel, which has Tm value, if A pipe FAM Fluorescence channel has Tm value to be that 58 DEG C of corresponding melting peakss curves are determined as that HPV 31 is positive, if A pipe JOE fluorescence channel has the Tm value to be 65 DEG C of corresponding melting peakss curves are determined as that HPV 26 is positive, if it is 55 DEG C of corresponding melting peakss that A pipe JOE fluorescence channel, which has Tm value, Curve is determined as that HPV 42 is positive, if it is that 68 DEG C of corresponding melting peakss curves are determined as HPV that A pipe TAMRA fluorescence channel, which has Tm value, 51 is positive, if it is that 56 DEG C of corresponding melting peakss curves are determined as that HPV 6 is positive that A pipe TAMRA fluorescence channel, which has Tm value, if A pipe CY5 Fluorescence channel has Tm value to be that 63 DEG C of corresponding melting peakss curves are determined as that HPV 73 is positive, if A pipe CY5 fluorescence channel has the Tm value to be 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified；
If it is that 67 DEG C of corresponding melting peakss curves are determined as that HPV 18 is positive that B pipe FAM fluorescence channel, which has Tm value, if B pipe FAM Fluorescence channel has Tm value to be that 54 DEG C of corresponding melting peakss curves are determined as that HPV 33 is positive, if B pipe JOE fluorescence channel has the Tm value to be 69 DEG C of corresponding melting peakss curves are determined as that HPV 52 is positive, if it is 56 DEG C of corresponding melting peakss that B pipe JOE fluorescence channel, which has Tm value, Curve is determined as that HPV 43 is positive, if it is that 63 DEG C of corresponding melting peakss curves are determined as HPV that B pipe TAMRA fluorescence channel, which has Tm value, 53 is positive, if it is that 51 DEG C of corresponding melting peakss curves are determined as that HPV 11 is positive that B pipe TAMRA fluorescence channel, which has Tm value, if B is managed It is that 60 DEG C of corresponding melting peakss curves are determined as that HPV 81 is positive that CY5 fluorescence channel, which has Tm value, if B pipe CY5 fluorescence channel has Tm Value is that 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified；
If it is that 67 DEG C of corresponding melting peakss curves are determined as that HPV 18 is positive that C pipe FAM fluorescence channel, which has Tm value, if C pipe FAM Fluorescence channel has Tm value to be that 56 DEG C of corresponding melting peakss curves are determined as that HPV 35 is positive, if C pipe JOE fluorescence channel has the Tm value to be 69 DEG C of corresponding melting peakss curves are determined as that HPV 52 is positive, if it is 54 DEG C of corresponding melting peakss that C pipe JOE fluorescence channel, which has Tm value, Curve is determined as that HPV 44 is positive, if it is that 62 DEG C of corresponding melting peakss curves are determined as HPV that C pipe TAMRA fluorescence channel, which has Tm value, 56 is positive, if it is that 53 DEG C of corresponding melting peakss curves are determined as that HPV 66 is positive that C pipe TAMRA fluorescence channel, which has Tm value, if C is managed It is that 61 DEG C of corresponding melting peakss curves are determined as that PV 82 is positive that CY5 fluorescence channel, which has Tm value, if C pipe CY5 fluorescence channel has Tm value It is determined as in the positive that Quality Control is qualified for 52 DEG C of corresponding melting peakss curves；
If it is that 66 DEG C of corresponding melting peakss curves are determined as that HPV 16 is positive that D pipe FAM fluorescence channel, which has Tm value, if D pipe FAM Fluorescence channel has Tm value to be that 57 DEG C of corresponding melting peakss curves are determined as that HPV 39 is positive, if D pipe JOE fluorescence channel has the Tm value to be 65 DEG C of corresponding melting peakss curves are determined as that HPV 58 is positive, if it is 55 DEG C of corresponding melting peakss that D pipe JOE fluorescence channel, which has Tm value, Curve is determined as that HPV 45 is positive, if it is that 60 DEG C of corresponding melting peakss curves are determined as HPV that D pipe TAMRA fluorescence channel, which has Tm value, 59 is positive, if it is that 52 DEG C of corresponding melting peakss curves are determined as that HPV 68 is positive that D pipe TAMRA fluorescence channel, which has Tm value, if D is managed It is that 62 DEG C of corresponding melting peakss curves are determined as that HPV 83 is positive that CY5 fluorescence channel, which has Tm value, if D pipe CY5 fluorescence channel has Tm Value is that 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified.
4, specificity verification
Select 40 type of human papilloma virus HPV, HPV54 type, HPV61 type, HPV67 type, HPV69 type, HPV70 type, HPV71 type and HPV72 type, herpes simplex virus type II, microspironema pallidum, Ureaplasma urealyticum, mycoplasma hominis, stranguria syndrome Neisser The clinical samples such as bacterium, Candida albicans, trichomonas vaginalis, chlamydia trachomatis, Acinetobacter bauamnnii and enterococcus faecalis (above-mentioned sample Derive from national CDC) as specificity assessment sample, after the samplers sample cervical exfoliated cell in system detection, The reaction condition established and optimized using early period is detected on ParaDNA.
As the result is shown under conditions of positive control is set up, dissolution peak of the target to be checked without specificity shows the disclosure Nucleic acid reagent can effective district sorting survey target and non-detection target, there is preferable specificity.
5, minimum detectability is verified
Assessment detection sample: choosing initial concentration is 105Copy/μ L human papilloma virus HPV6 type, HPV11 type, HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV42 type, HPV43 type, HPV44 Type, HPV45 type, HPV51 type, HPV52 type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 type, HPV73 type, HPV81 type, HPV82 type and HPV83 type nucleic acid gradient dilution be 104Copy/μ L, 103Copy/μ L, 102Copy/ μ L, 10 copies/μ L, 100Copy/μ L, the template as minimum detectability assessment.Reaction system and reaction interval as described above Sequence is tested.
The minimum detectability of disclosure kit can reach 10 copies/reaction as the result is shown.
6, coverage is verified
Cervical scrapes sample is selected to use template as coverage assessment.As described above reaction system and response procedures into Row test.
All 250 plants of sample standard deviations can be covered as the result is shown and detected.
7, the storage life test of kit
Strong positive 10 is taken respectively5CFU/mL and weakly positive 103The human papilloma virus HPV6 type of CFU/mL, HPV11 type, HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV42 type, HPV43 type, HPV44 Type, HPV45 type, HPV51 type, HPV52 type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 type, HPV73 type, HPV81 type, HPV82 type and the nucleic acid-templated of HPV83 type were distributed into 10 at the 0th day for assessment detection sample Part freezes in -70 DEG C of refrigerators.The kit that finishes will be set up and be placed in -20 DEG C of preservations, take 0 respectively, 10,15,30,60,90, 120,150,180 and 360 days kits carry out storage life test.
Disclosure kit is stored in -20 DEG C of refrigerators as the result is shown, is the positive in the detection of different storage lives, shows the examination The storage life of agent box is at least 1 year.
Comparative example
1, primer, probe synthesis
According to primer, probe sequence shown in table 4 and table 5, sequent synthesis is carried out.Y represents degeneracy base T/C in sequence；R Represent degeneracy base A/G；W represents degeneracy base A/T；FAM is 6- Fluoresceincarboxylic acid, JOE 2,7- dimethyl -4,5 in probe Two chloro- 6- Fluoresceincarboxylic acids, TAMRA are 6- carboxyl tetramethylrhodamine, CY5 is 5H- indoles cyanines.In the probe sequence of table 5 Bracket indicates that the t on the left of bracket has fluorescent marker, the selection of the content representation fluorescent marker in bracket.
Table 4
Table 5
2, specificity verification
Specificity verification is carried out according to the method for embodiment.The results show that the reaction result of the primer of comparative example, probe is equal For feminine gender.
3, minimum detectability is verified
Minimum detectability verifying is carried out according to the method for embodiment.The comparison of the minimum detectability of embodiment and comparative example is as follows Table 6.
Table 6
Detect target Embodiment Comparative example
HPV6 10 copies/reaction 100 copies/reaction
HPV11 10 copies/reaction 100 copies/reaction
HPV16 10 copies/reaction 100 copies/reaction
HPV18 10 copies/reaction 50 copies/reaction
HPV26 10 copies/reaction 100 copies/reaction
HPV31 10 copies/reaction 100 copies/reaction
HPV33 10 copies/reaction 100 copies/reaction
HPV35 10 copies/reaction 50 copies/reaction
HPV39 10 copies/reaction 100 copies/reaction
HPV42 10 copies/reaction 50 copies/reaction
HPV43 10 copies/reaction 100 copies/reaction
HPV44 10 copies/reaction 50 copies/reaction
HPV45 10 copies/reaction 100 copies/reaction
HPV51 10 copies/reaction 100 copies/reaction
HPV52 10 copies/reaction 50 copies/reaction
HPV53 10 copies/reaction 100 copies/reaction
HPV56 10 copies/reaction 100 copies/reaction
HPV58 10 copies/reaction 50 copies/reaction
HPV59 10 copies/reaction 100 copies/reaction
HPV66 10 copies/reaction 100 copies/reaction
HPV68 10 copies/reaction 100 copies/reaction
HPV73 10 copies/reaction 50 copies/reaction
HPV81 10 copies/reaction 100 copies/reaction
HPV82 10 copies/reaction 100 copies/reaction
HPV83 10 copies/reaction 100 copies/reaction
As shown in Table 6, for the human papilloma virus HPV6 type of trace in sample, HPV11 type, HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV42 type, HPV43 type, HPV44 type, HPV45 type, HPV51 Type, HPV52 type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 type, HPV73 type, HPV81 type, HPV82 type and HPV83 type nucleic acid, disclosure kit have stronger detectability than comparative example.
4, coverage is verified
Coverage verifying is carried out according to the method for embodiment.The coverage of embodiment and comparative example comparison such as the following table 7.
Table 7
As shown in Table 7, the detection coverage of disclosure kit is far longer than the detection coverage of comparative example.
The disclosure can disposably detect to include HPV6 type, HPV11 it can be seen from the comparison of embodiment and comparative example Type, HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV42 type, HPV43 type, HPV44 type, HPV45 type, HPV51 type, HPV52 type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 Human papilloma virus including type, HPV73 type, HPV81 type, HPV82 type and HPV83 type, specificity is high, and minimum detectability is more Low, coverage is wider.
The preferred embodiment of the disclosure is described in detail above, still, during the disclosure is not limited to the above embodiment Detail a variety of simple variants can be carried out to the technical solution of the disclosure in the range of the technology design of the disclosure, this A little simple variants belong to the protection scope of the disclosure.
It is further to note that specific technical features described in the above specific embodiments, in not lance In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the disclosure to it is various can No further explanation will be given for the combination of energy.
In addition, any combination can also be carried out between a variety of different embodiments of the disclosure, as long as it is without prejudice to originally Disclosed thought equally should be considered as disclosure disclosure of that.
Sequence table
<110>biotech inc Beijing Zhuo Cheng Hui Sheng
<120>for detecting the nucleic acid reagent, kit, system and method for human papilloma virus
<130> 12325ABT
<160> 156
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ggacggacaa gattcacaac ctttaaa 27
<210> 2
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gtacacccag acccctcatt tt 22
<210> 3
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
cctttaacac aacattacca aata 24
<210> 4
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tctactatgg cttctaccat aaa 23
<210> 5
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
tgagcaatta aatgacagct cagag 25
<210> 6
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
tgagaacaga tggggcacac aat 23
<210> 7
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gaccttctat gtcacgagca atta 24
<210> 8
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
tgcacaccac ggacacacaa ag 22
<210> 9
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
cgaaattgac ctacgctgct acg 23
<210> 10
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
tggcacacca aggacacgtc ttc 23
<210> 11
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
agcaattacc cgacagctca gat 23
<210> 12
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
gtagaacagt tggggcacac ga 22
<210> 13
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
actgacctay actgctatga gcaa 24
<210> 14
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
tgtgcacags tagggcacac aat 23
<210> 15
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
caactgacct atactgttat gagc 24
<210> 16
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
tgtgaacagc cggggcacac ta 22
<210> 17
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
ttgtatgtca cgagcaatta ggag 24
<210> 18
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
gacactgtgt cgcctgtttg ttta 24
<210> 19
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
cagatgaaga tgaccaagcc aaa 23
<210> 20
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
ttgtctacta tagcttctac acaaaa 26
<210> 21
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
ccagcaagtg aatctacaag ttta 24
<210> 22
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
gtccactgtt gcctctacta taaa 24
<210> 23
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
gacgttacac agccttacca aata 24
<210> 24
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
tctccactat agcctctact agaaa 25
<210> 25
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
gacctgttgt gttacgagca atta 24
<210> 26
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
tgcacaccac ggacacacaa ag 22
<210> 27
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
gctacgagca atttgacagc tcag 24
<210> 28
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
atcgccgttg ctagttgttc gca 23
<210> 29
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
actgacctay actgctatga gcaa 24
<210> 30
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
cagccggggc acacaacttg taa 23
<210> 31
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
acctgcaatg ccatgagcaa ttgaa 25
<210> 32
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
ttatcgcctt gttgcgcaga gg 22
<210> 33
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
acctacartg caatgagcaa ttgg 24
<210> 34
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
tgatgcgcag agtgggcacg tta 23
<210> 35
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
gctatgagca attatgtgac agct 24
<210> 36
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
tgtgcacags tagggcacac aat 23
<210> 37
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
accttgtgtg ctacgagcaa ttac 24
<210> 38
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
gctgcacaca aaggacacac aaa 23
<210> 39
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
acctacartg caatgagcaa ttgg 24
<210> 40
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
tgatgcgcag agtgggcacg tta 23
<210> 41
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 41
ttgtatgtca cgagcaatta ggag 24
<210> 42
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 42
gattactggg tttccgttgc acac 24
<210> 43
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 43
cttacatgtt acgagtcatt ggac 24
<210> 44
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 44
gtttctggaa cagttggggc ac 22
<210> 45
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 45
gtttagttag gctggtggtg ttaa 24
<210> 46
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 46
ctacaacagc ctccaccata aa 22
<210> 47
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 47
gctacgagca atttgacagc tcag 24
<210> 48
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 48
cattgccgat gttagttggt cgca 24
<210> 49
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 49
ggatttggaa gacttacgcc ttattt 26
<210> 50
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 50
ctctcgcacc tttgctgctt t 21
<210> 51
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 51
tttggacctg cgagcg 16
<210> 52
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 52
gagcggctgt ctccacaagt 20
<210> 53
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 53
aacgttcgac tggttgtgca 20
<210> 54
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 54
atgtgacagc aacgtccgac tg 22
<210> 55
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 55
tagatggtcc agctggacaa g 21
<210> 56
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 56
tagatggagt taatcatcaa ca 22
<210> 57
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 57
agatgaagga tgaaacagat aatatg 26
<210> 58
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 58
acagtccagc tggacaagca g 21
<210> 59
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 59
ccagatgagg atgaaggctt gga 23
<210> 60
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 60
attgacggtc cagctggaca a 21
<210> 61
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 61
ggagagtcag aggatgaaat a 21
<210> 62
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 62
aagtctgtta aactcgttgt gc 22
<210> 63
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 63
agtgctctga cagtgacatc a 21
<210> 64
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 64
aaggttcggc tggttgtgca g 21
<210> 65
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 65
atgaagcaga tggagttagt c 21
<210> 66
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 66
ggctggacag gctacgtgtt a 21
<210> 67
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 67
attgtgacat attgtcacag t 21
<210> 68
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 68
atgaggatga agtagaccat 20
<210> 69
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 69
aggatgagga tgaggatgaa gta 23
<210> 70
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 70
attgtaactt gttgttacac t 21
<210> 71
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 71
attgtgtgtg tgtgttgtaa g 21
<210> 72
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 72
atgaggatga aatagaccat ttgc 24
<210> 73
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 73
cagtgtacgt gttgtaagtg taa 23
<210> 74
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 74
agttactgac tgcacgaagt 20
<210> 75
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 75
agtgtgtcct ggctgcgcat 20
<210> 76
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 76
agtagataat atgcgtgacc a 21
<210> 77
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 77
catggaagga gctgtgtatt aa 22
<210> 78
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 78
gttctgacct gaaggctct 19
<210> 79
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 79
cacaaccttt aaaacaacat ttccaaata 29
<210> 80
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 80
tcgtccgcca tcgttgttat 20
<210> 81
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 81
gacatcagac aactacaaga cctttt 26
<210> 82
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 82
caccccgacc cctcatttt 19
<210> 83
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 83
gagcccatta caatattgta acctttt 27
<210> 84
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 84
gaacagatgg ggcacacaat t 21
<210> 85
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 85
gagcaattaa gcgactcaga ggaa 24
<210> 86
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 86
gaaggtcgtc tgctgagctt t 21
<210> 87
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 87
gccagacaag ctggacaaga a 21
<210> 88
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 88
caccaaggac acgtcttcca ttaa 24
<210> 89
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 89
cacatccaat tacaatatcg ttacctttt 29
<210> 90
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 90
ttggggcaca cgattccaaa t 21
<210> 91
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 91
cacttgtaac accacagttc gtttat 26
<210> 92
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 92
gatcggccat tgtagatgat gttta 25
<210> 93
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 93
aaccagacac ctccaattat aat 23
<210> 94
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 94
attccaaatg tgcccattaa taaat 25
<210> 95
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 95
gaacccgacc atgcagttaa t 21
<210> 96
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 96
acacacaaat cctagtgagt ccataaa 27
<210> 97
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 97
gaaacctgca acagatgctt tt 22
<210> 98
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 98
tttgtctact atagcttcta cacaaaa 27
<210> 99
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 99
gacctgctgt tgggcacatt aaa 23
<210> 100
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 100
gtcccactgt tgcctctact ataaa 25
<210> 101
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 101
ctgctgggtt cactggatat att 23
<210> 102
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 102
gttctccact atagcctcta ctagaaa 27
<210> 103
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 103
cgaaccacag cgtcacaaaa tt 22
<210> 104
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 104
caaaggacaa ggtgctcaaa aa 22
<210> 105
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 105
gcaggtgttc aagtgtagta caa 23
<210> 106
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 106
ttcgcacaac acgggcaaa 19
<210> 107
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 107
gcagaacaag ccacaagcaa tta 23
<210> 108
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 108
ccggggcaca caacttgtaa t 21
<210> 109
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 109
gggacgaaca acatccttgt t 21
<210> 110
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 110
tgcccataag catttgttgt aaaat 25
<210> 111
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 111
gtgttaccta atacacgtac cttgtt 26
<210> 112
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 112
agagtgggca cgttactgtt aa 22
<210> 113
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 113
tggcaccacg gttcgttt 18
<210> 114
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 114
catccattgc agatggtgtt tatt 24
<210> 115
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 115
gagccttaca gcagctgttt at 22
<210> 116
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 116
gtaccttccg aatcggccat t 21
<210> 117
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 117
ttacctaatt cacgtacctt gttgtaa 27
<210> 118
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 118
ggtgatgcca ttgcagttat tt 22
<210> 119
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 119
ctgctgttta tggactcact aaattt 26
<210> 120
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 120
tcatcaatgt cattggccat gtt 23
<210> 121
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 121
ccttgccatt gaaagcaaca aa 22
<210> 122
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 122
acctgaatca gccatcttct ttt 23
<210> 123
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 123
cctgaggctg ctgcaacaa 19
<210> 124
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 124
ctacaacagc ctccaccata aa 22
<210> 125
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 125
ggacaggata cgtgttacag aattaaa 27
<210> 126
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 126
cttaggtcgc ccagtaacat tt 22
<210> 127
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 127
cggtacccca ccaacatatt t 21
<210> 128
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 128
gcagttcgtg gtcccactta ata 23
<210> 129
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 129
atggcggtgt ttgcagattt 20
<210> 130
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 130
gattgatagc aacaactgaa tagccaa 27
<210> 131
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 131
actggttgtg cagtgtac 18
<210> 132
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 132
tgtgtcccat ctgcgcac 18
<210> 133
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 133
cggttgtgcg tacaaagcac a 21
<210> 134
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 134
tgttgtgtat gtgttgtaag t 21
<210> 135
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 135
agtatagtgc agctagcag 19
<210> 136
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 136
tcagtgtaag tctacacttc g 21
<210> 137
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 137
agtgaatatt gtgtgcccta c 21
<210> 138
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 138
tgtaaatgtg aggcgacact a 21
<210> 139
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 139
acagtgttcg tgttgtaagt aa 22
<210> 140
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 140
gagtaactgc aatggcggat g 21
<210> 141
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 141
ggtgtgacta ccatggctga taa 23
<210> 142
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 142
atggctgaca atacaggtac ag 22
<210> 143
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 143
attgagctta cagtagagag c 21
<210> 144
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 144
gcgttgtaca gcagatgtta a 21
<210> 145
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 145
ttgtcacagt tgtgatagca ca 22
<210> 146
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 146
tgtaggtgtg agtcgttggt gc 22
<210> 147
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 147
taagtttgtg gtgcagttgg a 21
<210> 148
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 148
agtacaacaa ccgacgtacg a 21
<210> 149
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 149
tgtgtgcagc aaaccagtaa cct 23
<210> 150
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 150
agttggtggt gcagttggac a 21
<210> 151
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 151
atggccaatt gtgaagggct c 21
<210> 152
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 152
agttgcttat gggtacacta g 21
<210> 153
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 153
agtgacatgg cagatgtgga a 21
<210> 154
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 154
agtgttgtac agctcgcagt g 21
<210> 155
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 155
gaggtggatt tggaagactt a 21
<210> 156
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 156
cctgaaggct ctgcgcggac t 21

Claims (10)

1. a kind of for detecting the nucleic acid reagent of human papilloma virus, wherein the nucleic acid reagent includes depositing independently of one another respectively It puts or probe shown in primer and SEQ ID NO.53-77 shown in the SEQ ID NO.1-50 of any mixed storage mutually.
2. nucleic acid reagent according to claim 1, wherein primer shown in the SEQ ID NO.1 relative to 1 μM, respectively The content of the primer as shown in SEQ ID NO.2-50 be respectively 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~ 1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~ 0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~ 1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~ 0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~ 1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~ 0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM, 0.1~0.5 μM, 0.5~ 1.0 μM, 0.1~0.5 μM, 0.5~1.0 μM and 0.1~0.5 μM, the content of the probe as shown in SEQ ID NO.53-77 respectively It is each independently 0.1~0.3 μM.
3. nucleic acid reagent according to claim 1, wherein the nucleic acid reagent further includes Quality Control in the positive；
Quality Control contains probe shown in primer shown in SEQ ID NO.51-52 and SEQ ID NO.78 in the positive.
4. nucleic acid reagent according to claim 3, wherein the nucleic acid reagent includes A pipe, B pipe, C pipe and D pipe；A pipe contains There are primer shown in SEQ ID NO.1-2,5-6,9-12,19-20,27-28,43-44,51-52 and SEQ ID NO.53,55, Probe shown in 57-58,62,66,74,78；B pipe contains SEQ ID NO.3-4,7-8,13-14,21-22,29-32,45-46, Probe shown in primer shown in 51-52 and SEQ ID NO.54,56,59,63,67,68,75,78；C pipe contains SEQ ID Primer shown in NO.7-8,15-16,23-24,29-30,33-34,39-40,47-48,51-52 and SEQ ID NO.56,60, Probe shown in 64,67,69,72,76,78；D pipe contains SEQ ID NO.5-6,17-18,25-26,35-38,41-42,49- Probe shown in primer shown in 50,51-52 and SEQ ID NO.55,61,65,70,71,73,77,78.
5. nucleic acid reagent according to claim 4, wherein probe shown in SEQ ID NO.55-56,58-61 has the One fluorescent marker；Probe shown in SEQ ID NO.57,62-65,67,70 has the second fluorescent marker；SEQ ID NO.53, Probe shown in 54,66,68-69,71-73 has third fluorescent marker；Probe shown in SEQ ID NO.74-78 has the 4th Fluorescent marker；First fluorescent marker, second fluorescent marker, the third fluorescent marker and the 4th fluorescent marker It is different, and be each independently selected from FAM fluorescent marker, JOE fluorescent marker, TAMRA fluorescent marker, CY5 fluorescent marker, One of ROX fluorescent marker and Quasar670 fluorescent marker.
6. nucleic acid reagent described according to claim 1~any one of 5, wherein the human papilloma virus includes HPV6 Type, HPV11 type, HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV42 type, HPV43 type, HPV44 type, HPV45 type, HPV51 type, HPV52 type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 At least one of type, HPV68 type, HPV73 type, HPV81 type, HPV82 type and HPV83 type.
7. a kind of for detecting the kit of human papilloma virus, which contains described in any one of claim 1~6 Nucleic acid reagent, and optionally, the kit also contain reaction system buffer, archaeal dna polymerase, magnesium ion, dNTP and At least one of water.
8. nucleic acid reagent described in any one of claim 1~6 is preparing the kit for detecting human papilloma virus In purposes.
9. a kind of system for detecting human papilloma virus, which includes with A pipe detector, B pipe detector, the inspection of C pipe Survey device and D pipe detector PCR instrument, computing device and output device, the A pipe detector, B pipe detector, C pipe detector and D pipe detector is respectively the nucleic acid reagent tank for being mounted with nucleic acid reagent described in any one of claim 4~6, The PCR instrument includes the first fluorescence channel, the second fluorescence channel, third fluorescence channel and the 4th fluorescence channel, and described first is glimmering Optical channel, second fluorescence channel, the third fluorescence channel and the 4th fluorescence channel are different, and respectively independent Ground is FAM fluorescence channel, JOE fluorescence channel, TAMRA fluorescence channel, CY5 fluorescence channel, ROX fluorescence channel or Quasar670 Fluorescence channel；The computing device includes memory and processor, and computer program, the processing are stored in the memory Device is configured as executing the computer program stored in the memory, to realize following differentiation:
If positive control and negative control are set up, testing result is effective；
If it is that 66 DEG C of corresponding melting peakss curves are determined as that HPV 16 is positive that the first fluorescence channel of A pipe, which has Tm value, if A pipe first is glimmering Optical channel has Tm value to be that 58 DEG C of corresponding melting peakss curves are determined as that HPV 31 is positive, if the second fluorescence channel of A pipe has the Tm value to be 65 DEG C of corresponding melting peakss curves are determined as that HPV 26 is positive, if it is 55 DEG C of corresponding meltings that the second fluorescence channel of A pipe, which has Tm value, Peak curve is determined as that HPV 42 is positive, if it is that 68 DEG C of corresponding melting peakss curves are determined as HPV that A pipe third fluorescence channel, which has Tm value, 51 is positive, if it is that 56 DEG C of corresponding melting peakss curves are determined as that HPV 6 is positive that A pipe third fluorescence channel, which has Tm value, if A pipe the 4th It is that 63 DEG C of corresponding melting peakss curves are determined as that HPV 73 is positive that fluorescence channel, which has Tm value, if the 4th fluorescence channel of A pipe has Tm value It is determined as in the positive that Quality Control is qualified for 52 DEG C of corresponding melting peakss curves；
If it is that 67 DEG C of corresponding melting peakss curves are determined as that HPV 18 is positive that the first fluorescence channel of B pipe, which has Tm value, if B pipe first is glimmering Optical channel has Tm value to be that 54 DEG C of corresponding melting peakss curves are determined as that HPV 33 is positive, if the second fluorescence channel of B pipe has the Tm value to be 69 DEG C of corresponding melting peakss curves are determined as that HPV 52 is positive, if it is 56 DEG C of corresponding meltings that the second fluorescence channel of B pipe, which has Tm value, Peak curve is determined as that HPV 43 is positive, if it is that 63 DEG C of corresponding melting peakss curves are determined as HPV that B pipe third fluorescence channel, which has Tm value, 53 is positive, if it is that 51 DEG C of corresponding melting peakss curves are determined as the HPV11 positive that B pipe third fluorescence channel, which has Tm value, if B pipe the 4th It is that 60 DEG C of corresponding melting peakss curves are determined as the HPV81 positive that fluorescence channel, which has Tm value, if the 4th fluorescence channel of B pipe has the Tm value to be 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified；
If it is that 67 DEG C of corresponding melting peakss curves are determined as that HPV 18 is positive that the first fluorescence channel of C pipe, which has Tm value, if C pipe first is glimmering Optical channel has Tm value to be that 56 DEG C of corresponding melting peakss curves are determined as that HPV 35 is positive, if the second fluorescence channel of C pipe has the Tm value to be 69 DEG C of corresponding melting peakss curves are determined as that HPV 52 is positive, if it is 54 DEG C of corresponding meltings that the second fluorescence channel of C pipe, which has Tm value, Peak curve is determined as that HPV 44 is positive, if it is that 62 DEG C of corresponding melting peakss curves are determined as HPV that C pipe third fluorescence channel, which has Tm value, 56 is positive, if C pipe third fluorescence channel has Tm value to be that 53 DEG C of corresponding melting peakss curves are determined as that HPV 66 is positive, if C pipe the It is that 61 DEG C of corresponding melting peakss curves determine sample for the PV82 positive, if the 4th fluorescence channel of C pipe has Tm that four fluorescence channels, which have Tm value, Value is that 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified；
If it is that 66 DEG C of corresponding melting peakss curves are determined as that HPV 16 is positive that the first fluorescence channel of D pipe, which has Tm value, if D pipe first is glimmering Optical channel has Tm value to be that 57 DEG C of corresponding melting peakss curves are determined as that HPV 39 is positive, if the second fluorescence channel of D pipe has the Tm value to be 65 DEG C of corresponding melting peakss curves are determined as that HPV 58 is positive, if it is 55 DEG C of corresponding meltings that the second fluorescence channel of D pipe, which has Tm value, Peak curve is determined as that HPV 45 is positive, if it is that 60 DEG C of corresponding melting peakss curves are determined as HPV that D pipe third fluorescence channel, which has Tm value, 59 is positive, if D pipe third fluorescence channel has Tm value to be that 52 DEG C of corresponding melting peakss curves are determined as that HPV 68 is positive, if D pipe the It is that 62 DEG C of corresponding melting peakss curves are determined as the HPV83 positive that four fluorescence channels, which have Tm value, if the 4th fluorescence channel of D pipe has Tm value It is determined as in the positive that Quality Control is qualified for 52 DEG C of corresponding melting peakss curves.
10. a kind of method for detecting human papilloma virus, wherein this method comprises: using any in claim 1~6 Nucleic acid reagent described in one carries out PCR amplification to the DNA of sample to be tested；The PCR instrument for carrying out the PCR amplification includes first Fluorescence channel, the second fluorescence channel, third fluorescence channel and the 4th fluorescence channel；It is first fluorescence channel, described second glimmering Optical channel, the third fluorescence channel and the 4th fluorescence channel are different, and be each independently FAM fluorescence channel, JOE fluorescence channel, TAMRA fluorescence channel, CY5 fluorescence channel, ROX fluorescence channel or Quasar670 fluorescence channel；And carry out Following differentiation:
If positive control and negative control are set up, testing result is effective；
If it is that 66 DEG C of corresponding melting peakss curves are determined as that HPV 16 is positive that the first fluorescence channel of A pipe, which has Tm value, if A pipe first is glimmering Optical channel has Tm value to be that 58 DEG C of corresponding melting peakss curves are determined as that HPV 31 is positive, if the second fluorescence channel of A pipe has the Tm value to be 65 DEG C of corresponding melting peakss curves are determined as that HPV 26 is positive, if it is 55 DEG C of corresponding meltings that the second fluorescence channel of A pipe, which has Tm value, Peak curve is determined as that HPV 42 is positive, if it is that 68 DEG C of corresponding melting peakss curves are determined as HPV that A pipe third fluorescence channel, which has Tm value, 51 is positive, if it is that 56 DEG C of corresponding melting peakss curves are determined as that HPV 6 is positive that A pipe third fluorescence channel, which has Tm value, if A pipe the 4th It is that 63 DEG C of corresponding melting peakss curves are determined as that HPV 73 is positive that fluorescence channel, which has Tm value, if the 4th fluorescence channel of A pipe has Tm value It is determined as in the positive that Quality Control is qualified for 52 DEG C of corresponding melting peakss curves；
If it is that 67 DEG C of corresponding melting peakss curves are determined as that HPV 18 is positive that the first fluorescence channel of B pipe, which has Tm value, if B pipe first is glimmering Optical channel has Tm value to be that 54 DEG C of corresponding melting peakss curves are determined as that HPV 33 is positive, if the second fluorescence channel of B pipe has the Tm value to be 69 DEG C of corresponding melting peakss curves are determined as that HPV 52 is positive, if it is 56 DEG C of corresponding meltings that the second fluorescence channel of B pipe, which has Tm value, Peak curve is determined as that HPV 43 is positive, if it is that 63 DEG C of corresponding melting peakss curves are determined as HPV that B pipe third fluorescence channel, which has Tm value, 53 is positive, if B pipe third fluorescence channel has Tm value to be that 51 DEG C of corresponding melting peakss curves are determined as that HPV 11 is positive, if B pipe the It is that 60 DEG C of corresponding melting peakss curves are determined as that HPV 81 is positive that four fluorescence channels, which have Tm value, if the 4th fluorescence channel of B pipe has Tm Value is that 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified；
If it is that 67 DEG C of corresponding melting peakss curves are determined as that HPV 18 is positive that the first fluorescence channel of C pipe, which has Tm value, if C pipe first is glimmering It is that 56 DEG C of corresponding melting peakss curves are determined as the HPV35 positive that optical channel, which has Tm value, if it is 69 that the second fluorescence channel of C pipe, which has Tm value, DEG C corresponding melting peakss curve is determined as that HPV 52 is positive, if it is 54 DEG C of corresponding melting peakss that the second fluorescence channel of C pipe, which has Tm value, Curve is determined as that HPV 44 is positive, if it is that 62 DEG C of corresponding melting peakss curves are determined as HPV that C pipe third fluorescence channel, which has Tm value, 56 is positive, if C pipe third fluorescence channel has Tm value to be that 53 DEG C of corresponding melting peakss curves are determined as that HPV 66 is positive, if C pipe the It is that 61 DEG C of corresponding melting peakss curves determine sample for the HPV82 positive, if the 4th fluorescence channel of C pipe has Tm that four fluorescence channels, which have Tm value, Value is that 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified；
If it is that 66 DEG C of corresponding melting peakss curves are determined as that HPV 16 is positive that the first fluorescence channel of D pipe, which has Tm value, if D pipe first is glimmering Optical channel has Tm value to be that 57 DEG C of corresponding melting peakss curves are determined as that HPV 39 is positive, if the second fluorescence channel of D pipe has the Tm value to be 65 DEG C of corresponding melting peakss curves are determined as that HPV 58 is positive, if it is 55 DEG C of corresponding meltings that the second fluorescence channel of D pipe, which has Tm value, Peak curve is determined as that HPV 45 is positive, if it is that 60 DEG C of corresponding melting peakss curves are determined as HPV that D pipe third fluorescence channel, which has Tm value, 59 is positive, if D pipe third fluorescence channel has Tm value to be that 52 DEG C of corresponding melting peakss curves are determined as that HPV 68 is positive, if D pipe the It is that 62 DEG C of corresponding melting peakss curves are determined as that HPV 83 is positive that four fluorescence channels, which have Tm value, if the 4th fluorescence channel of D pipe has Tm Value is that 52 DEG C of corresponding melting peakss curves are determined as in the positive that Quality Control is qualified.
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