CN107988432A - Detect the kit of multiple high-risk human mammilla papillomavirus at the same time in single tube reaction - Google Patents
Detect the kit of multiple high-risk human mammilla papillomavirus at the same time in single tube reaction Download PDFInfo
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Abstract
This case be related to it is a kind of in single tube reaction while detect the kit of multiple high-risk human mammilla papillomavirus, the kit is including at least there is the specific probe SEQ ID NO.1 14 that correspond to this 14 genotype of HPV16,18,31,33,35,39,45,51,52,56,58,59,66 and 68 respectively；And 6 forward primers and 6 reverse primers of comprehensive corresponding 14 genotype.The present invention passes through the improvement to existing HPV nucleic acid detection kit formula so that the technical solution of the application can be used only 6 forward primers and 6 reverse primers and just can realize high sensitivity at the same time in single tube reaction, detect 14 kinds of high-risk human mammilla papillomavirus with high specificity.
Description
Technical field
The invention belongs to in-vitro diagnosis field, and in particular to a kind of to detect multiple high-risk-type human milks at the same time in single tube reaction
The kit of head tumor virus.
Background technology
Human papilloma virus (human papillomavirus, HPV) is a kind of thermophilic epithelial double-stranded DNA spherical virus,
Belong to Papillomaviridae, main infection skin and mucosal tissue.HPV infection can cause wart, benign tumour and cancer.At present
Evidence show HPV infection can cause to include the kinds cancers such as cervical carcinoma, carcinoma of vagina, carcinoma of penis, cancer of anus, oropharyngeal cancer.According to
International human papilloma virus reference center (International Human Papillomavirus Reference Center)
Recent statistics, at present altogether find more than 200 kinds HPV genotype.But not all HPV genotype can induce cancer
Disease.The infection of low risk HPV can induce the mankind's verruca vulgaris being grown on reproductive organs neighbouring skin and mucous membrane, condyloma acuminatum
And benign lesion, the infection of high-risk HPV only few in number such as papilloma being grown on mucous membrane are potentially carcinogenic.Mesh
The example carcinogenic most deep HPV of preceding understanding is women cervical carcinoma.Exactly because also pioneer's sex work in this respect, German section
Scholar Harald zur Hausen obtained Nobel's physiology and Medicine in 2008.
Cervical carcinoma is the malignant tumour occurred at womb hypomere uterine neck, is the 4th malignant tumour occurred frequently in women,
Account for the 4th of the woman cancer death rate.According to the International Cancer Research Center (IARC) of International Health Organization (WHO), 2012
Year, global cervical carcinoma new cases were 52.8 ten thousand, there are about 26.6 ten thousand women and died of cervical carcinoma.The generation of nearly all cervical carcinoma
Caused by being all the persistent infection of high-risk HPV, and need from initial infection to cancer to undergo 5 to 10 years when
Between.Therefore, cervical carcinoma is carried out using the HPV molecular diagnostic techniques using the hereditary material DNA of high-risk HPV as detection object
Examination, can find the etiological cause of the disease in advance, effectively early prevention be carried out to cervical carcinoma, so as to reduce morbidity and mortality.With vinegar
Acid is visually inspected, Pap smear is compared with traditional cervical carcinoma screening means such as liquid based cytology, and the diagnosis of HPV molecules has sensitivity
It is high and do not depend on the advantage of the subjective judgement of pathologist.Therefore, HPV molecules diagnosis has become current developed country's cervical carcinoma
The prefered method of examination.
One challenge of HPV molecular diagnostic techniques maximum is effectively by high-risk-type while detection sensitivity is ensured
HPV is distinguished with low risk HPV, to avoid because mistaken diagnosis and caused by excessively diagnosis and over-treatment and thus to patient produce
Raw unnecessary stress.As it was previously stated, presently found HPV genotype alreadys exceed 200 kinds, and according to WHO/IARC
Deng the achievement in research of authoritative institution, wherein only 14 kinds of genotype are defined as inducing the high-risk-type of cervical carcinoma, i.e.,
HPV16,18,31,33,35,39,45,51,52,56,58,59,66 and 68.Even in addition, in 14 kinds of high-risk HPVs also simultaneously
It is not that each genotype all has identical carciongenic potency.HPV16 and 18 is two genotype for wherein endangering maximum, the two
Cause 55% and 15% cases of cervical cancer respectively.Therefore, it is possible to examine the high-risk HPV molecule of the progress partings of HPV16 and 18
Shunting of the disconnected technology for HPV detection positive patients has original advantage.Further more, not all uterus neck has infected height
The patient of danger type HPV is just bound to suffer from cervical carcinoma, and most of such infection is the immune work(of transient infection, i.e. human body itself
Be able to can effectively it be removed in 6 to 24 months.The determinant that can high-risk HPV be removed by immune system is multi-party
Face, the ability of infection is both removed including patients immune system, also includes the virus load for infecting HPV.Prevalence for many years
Researches show that high-risk HPV infection, there are a clinical threshold value for disease.Virus load is infected less than the high-risk HPV of this threshold value
Often transient infection, can seldom induce cervical carcinoma；And virus load is several higher than the infection-induced cervical carcinoma of this threshold value
Rate will raise significantly.Therefore, the high-risk HPV molecular diagnostic techniques for cervical carcinoma early screening should also be determined with certain
Amount ability is infected with distinguishing the transient infection of low carrying capacity and may finally induce the high carrying capacity of cervical carcinoma.
Mainly there are hybrid capture, PCR- revert dot blot hybridizations, liquid chip method currently for the detection method of HPV nucleic acid
With fluorescence quantitative PCR method etc..The characteristics of for above-mentioned high-risk HPV molecular diagnostic techniques for cervical carcinoma early screening and
The performance indicator of other side, the main advantage and disadvantage of these types of method are summarised in following table：
As can be seen from the above table, consider from methodology angle, can most meet the detection of cervical carcinoma early screening requirement at present
Means are fluorescent quantitative PCR techniques.The sensitivity and specificity of the technology are above hybrid capture and miscellaneous with PCR- reversal points
Friendship method and liquid chip method are suitable.Fluorescence quantitative PCR method can also carry out accurately determining to detection object very in the range of large span
Amount, and realize that the PCR- reversal points of target sequence detection are miscellaneous with the probe hybridization being fixed on film or microballoon using PCR end-products
Friendship method and liquid chip rule cannot quantify.
But in the prior art, HPV nucleic acid detection is carried out, it is necessary to be directed to each using fluorescent quantitative PCR technique
HPV genotype provides a specific probe, a forward primer and a reverse primer, and detection at the same time is more in single tube reaction
During a HPV genotype, some group-specific probe, forward primer and reverse primers will certainly cause more non-specific knots
Close, this have impact on the sensitivity of detection and precision to a certain extent.
The content of the invention
For shortcoming of the prior art, the present invention is intended to provide a kind of high sensitivity, high specific can be
It is common that HPV16,18,31,33,35,39,45,51,52,56,58,59,66 and 68 are detected at the same time in the reaction of one pipe quantitative fluorescent PCR
The kit of 14 kinds of HPV genotype.
To achieve the above object, technical scheme is as follows：
A kind of to detect the kits of multiple high-risk human mammilla papillomavirus at the same time in single tube reaction, it, which is included at least, has point
The specific probe of this 14 genotype of HPV16,18,31,33,35,39,45,51,52,56,58,59,66 and 68 is not corresponded to
SEQ ID NO.1-14；
And comprehensive corresponding 14 genotype and forward primer and reverse primer of the quantity less than 14 pairs.
Preferably, the kit for detecting multiple high-risk human mammilla papillomavirus at the same time in single tube reaction, its
In, the forward primer quantity of comprehensive corresponding 14 genotype is 6, is respectively：SEQ ID NO.15-20.
Preferably, the kit for detecting multiple high-risk human mammilla papillomavirus at the same time in single tube reaction, its
In, the reverse primer quantity of comprehensive corresponding 14 genotype is 6, is respectively：SEQ ID NO.21-26.
Preferably, the kit for detecting multiple high-risk human mammilla papillomavirus at the same time in single tube reaction, its
In, kit further includes the specific probe being useful for being matched as the hemoglobin β gene complementations of Quality Control in human body cell
SEQ ID NO.27。
Preferably, the kit for detecting multiple high-risk human mammilla papillomavirus at the same time in single tube reaction, its
In, kit further includes the forward primer SEQ being useful for being matched as the hemoglobin β gene complementations of Quality Control in human body cell
ID NO.28 and reverse primer SEQ ID NO.29.
The beneficial effects of the invention are as follows：The present invention passes through the improvement to existing HPV nucleic acid detection kit formula so that this
6 forward primers and 6 reverse primers can be used only in the technical solution of application just can realize height at the same time in single tube reaction
Sensitivity, detect 14 kinds of high-risk human mammilla papillomavirus with high specificity.
Each primer in 6 forward primers and 6 reverse primers is not only to act solely on 14 kinds of high-risk HPVs
In one kind, but contribute to multiple HPV genotype at the same time, and 6 forward primers and 6 reverse primers are as an entirety,
Just meet while contribute the demand of 14 kinds of high-risk HPVs of amplification.
Brief description of the drawings
Fig. 1 is the amplification obtained by the concentration gradient standard items of quantitative fluorescent PCR reaction detection difference HPV genotype of the present invention
Curve map (by taking HPV16,18,33 and 51 4 genotype as an example)；The concentration gradient section of HPV16 and 18 is 10~107Copy/
Reaction；The concentration gradient section of HPV33 and 51 is 100~108Copy/reaction；The detection reaction of each concentration gradient includes 3
Technology repeats.
Fig. 2 is testing result comparison diagram of the kit of the present invention to same group of standard items of 3 different batches；Wherein,
P0000002, P0000025 and P0000037 are Mission Number.
Embodiment
The present invention is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art with reference to specification text
Word can be implemented according to this.
Be listed below one embodiment of this case in single tube reaction and meanwhile detect the examination of multiple high-risk human mammilla papillomavirus
Agent box, it includes at least to have corresponds to HPV16,18,31,33,35,39,45,51,52,56,58,59,66 and 68 this 14 respectively
The specific probe SEQ ID NO.1-14 of genotype；
And comprehensive corresponding 14 genotype and forward primer and reverse primer of the quantity less than 14 pairs.
Wherein, the forward primer quantity of comprehensive corresponding 14 genotype is 6, is respectively：SEQ ID NO.15-20.
Wherein, the reverse primer quantity of comprehensive corresponding 14 genotype is 6, is respectively：SEQ ID NO.21-26.
Wherein, kit, which further includes, is useful for mutually recruiting with the hemoglobin β genes (HBB) as Quality Control in human body cell
To specific probe SEQ ID NO.27.
Wherein, kit, which further includes, is useful for and the hemoglobin β gene complementations pairing as Quality Control in human body cell
Forward primer SEQ ID NO.28 and reverse primer SEQ ID NO.29.
Certainly, further preferably include completing the auxiliary agent needed for quantitative fluorescent PCR reaction in kit, such as：PCR buffer solutions, 4
Kind dNTP mixed liquors, thermal starting polymerase etc..
Table 1 shows the sequence of specific primer and probe：
Table 1
In table 1, the fluorophor that HPV16 probes (SEQ ID NO.1) use is JOE；HPV18 probes (SEQ ID
NO.2 the fluorophor) used is CY5；This 10 probe (the SEQ ID of HPV31,33,35,39,51,52,56,59,66 and 68
NO.3,4,5,6,8,9,10,12,13 and the fluorophor 14) used are FAM；HPV45 and 58 (SEQ ID NO.7 and 11) two
The fluorophor that a probe uses is ROX；Fluorophor used in SEQ ID NO.27 is TAMRA.In addition, SEQ ID
NO.3,10,12 and 13 are the patents according to Application No. 201710184261.7《High s/n ratio multiprobe PCR Taqman probes
And its application》The special probe that can significantly reduce quantitative fluorescent PCR background signal of design.Base is quenched in this kind of special probe
Group not oligonucleotide 3 ' end, but in the sequence between some thymidylic acid (T) on, and its 3 ' end then by phosphorus
Acid groups, amino or other closing base closings." t " of small letter represents the T cores with quenching group in above-mentioned 4 probe sequences
Thuja acid, and 3 ' ends are then closed by C3linker.
Table 2 provides including above-mentioned all primer and probes, can detecting all 14 kinds of HPV bases at the same time for a specific embodiment
PCR reaction solution because of type, for single tube multiple fluorescence quantitative PCR.The reaction formula of liquid is shown in Table 2：
Table 2
| Title | React final concentration | Concentration unit |
| Without Mg2+Ion PCR buffer solutions | 1 | X |
| Mg2+ | 4 | mM |
| 4 kinds of dNTP mixed liquors | 0.2 | mM |
| SEQ ID NO.1 | 0.07 | μM |
| SEQ ID NO.2 | 0.05 | μM |
| SEQ ID NO.3 | 0.065 | μM |
| SEQ ID NO.4 | 0.06 | μM |
| SEQ ID NO.5 | 0.05 | μM |
| SEQ ID NO.6 | 0.065 | μM |
| SEQ ID NO.7 | 0.1 | μM |
| SEQ ID NO.8 | 0.07 | μM |
| SEQ ID NO.9 | 0.05 | μM |
| SEQ ID NO.10 | 0.2 | μM |
| SEQ ID NO.11 | 0.1 | μM |
| SEQ ID NO.12 | 0.09 | μM |
| SEQ ID NO.13 | 0.05 | μM |
| SEQ ID NO.14 | 0.05 | μM |
| SEQ ID NO.15 | 0.9 | μM |
| SEQ ID NO.16 | 0.2 | μM |
| SEQ ID NO.17 | 0.2 | μM |
| SEQ ID NO.18 | 0.2 | μM |
| SEQ ID NO.19 | 0.2 | μM |
| SEQ ID NO.20 | 0.2 | μM |
| SEQ ID NO.21 | 0.4 | μM |
| SEQ ID NO.22 | 0.25 | μM |
| SEQ ID NO.23 | 0.25 | μM |
| SEQ ID NO.24 | 0.2 | μM |
| SEQ ID NO.25 | 0.2 | μM |
| SEQ ID NO.26 | 0.2 | μM |
| SEQ ID NO.27 | 0.05 | μM |
| SEQ ID NO.28 | 0.2 | μM |
| SEQ ID NO.29 | 0.2 | μM |
| Thermal starting polymerase (Taq) | 0.12 | U/μL |
In order to verify the technical performance of kit of the present invention, this team is prepared for 14 and includes above-mentioned 14 HPV bases respectively
Mixed because of the plasmid of the DNA target sequence of type, and from human genome DNA to prepare the standard items of different components and various concentrations.
The purpose of human genome DNA is added into standard items in the human DNA certainly existed in clinical sample is simulated.These standards
Human genome DNA origin in product comes from the Jurkat culture cell purifications of T cell leukaemic and obtains.
Embodiment 1：
In order to verify that kit of the present invention can detect different HPV genotype, this case in large span concentration range quantification
It is prepared for 10 times of diluted concentration gradient standard items of different HPV genotype.Quantitative fluorescent PCR is carried out using kit of the present invention
Acquired results are reacted as shown in table 3 and Fig. 1, this explanation is 106In concentration span scope, PCR reactions of the present invention, which can quantify, to be examined
Survey corresponding HPV genotype.
3. kit of table detects 14 kinds of HPV genotype concentration gradient standard items acquired results (Ct values)
Note："/" is represented and not detected.Since HPV16 and 18 is two genotype that carciongenic potency is most strong, harm is maximum, this
Quantitative fluorescent PCR of the present invention reaction is specially directed to the sensitivity design of the two genotype and debugged by case must be higher than other 12 kinds
Genotype.
Embodiment 2：
In order to verify the stability of kit formulation of the present invention, this case is successively using the raw material reagent of 3 sets of different batches
The kit of 3 different batches is prepared for, and same group of standard items are detected with gained kit.Acquired results show 3
The testing result for criticizing kit is very close (table 4 and Fig. 2).
The kit of the present invention of 4. 3 different batches of table detects same group of standard items acquired results and summarizes.
Note：" " represent without amplification curve.
Embodiment 3：
To participate in 93 women of cervical carcinoma screening as detection object, this case completes kit and Kai Jie companies of Germany
(QIAGEN) contrast experiment for the careHPV kit for detecting nucleic acid produced.CareHPV kits can detect identical 14 kinds
HPV genotype, but HPV16 and 18 and other 12 kinds of genotype cannot be distinguished, and negative or positive inspection can only be provided
Survey result.The sampling of cervical exfoliated cell is completed by hospital, every patient adopts two samples.The hospital laboratory is according to careHPV
Kit specification completes the detection to wherein a set of 93 samples.Another set of 93 samples are completed using the technical solution of this case
Detection：The extraction of DNA in sample is completed first；Then DNA after purification is detected using kit of the present invention.The contrast
Experimental result shows that the testing result concordance rate of two kits reaches 95.7% (table 5).For two kit testing results
It is the further studies have shown that kit pair of the present invention of 14 positive and inconsistent two kit testing results cases
Different HPV genotype have preferably specific (table 6)：
1st, careHPV kits are the positive to the testing result of No. 13 patient, and the testing result of kit of the present invention
For feminine gender；Although it can be seen that the specific PCR reactions bands of HPV, sequencing result show that it is in agarose gel electrophoresis
HPV70, a low danger HPV genotype.This is the result shows that the testing result of careHPV kits belongs to by nonspecific reaction
Caused by false positive.
2nd, careHPV kits are the positive to the testing result of the 79th, 84 and No. 103 patient, and kit of the present invention
Testing result is feminine gender；And it can't see the specific PCR reactions bands of HPV in agarose gel electrophoresis.This result also with
The testing result of careHPV kits belongs to the false positive as caused by nonspecific reaction and coincide.
5. kit of the present invention of table and contrast and experiment of the careHPV kits using clinical sample as object.
6. two kit testing results of table are positive and the inconsistent case of testing result further analysis result.
Although embodiment of the present invention is disclosed as above, it is not restricted in specification and embodiment listed
With it can be applied to various suitable the field of the invention completely, can be easily for those skilled in the art
Realize other modification, therefore under the universal limited without departing substantially from claim and equivalency range, it is of the invention and unlimited
In specific details and shown here as the legend with description.
Modification among numbering title sequence two is terminal modified
1 HPV16 probe TTATGTGCTGCCATATCTACTTCAGAAACT 5'-JOE, 3'-BHQ1 of SEQ ID NO. without
2 HPV18 probe CTACACAGTCTCCTGTACCTG 5'-CY5,3'-BHQ2 of SEQ ID NO. without
3 HPV31 probe GTGCTGCAATtGCAAACAGTGATACTAC 5'-FAM, 3'-C3 linker of SEQ ID NO./
iBHQ1dT/
4 HPV33 probe CTTTATGCACACAAGTAACTAGTGAC 5'-FAM, 3'-BHQ1 of SEQ ID NO. without
5 HPV35 probe CTGCTGTGTCTTCTAGTGACAG 5'-FAM, 3'-BHQ1 of SEQ ID NO. without
6 HPV39 probe CTATAGAGTCTTCCATACCTTCTAC 5'-FAM, 3'-BHQ1 of SEQ ID NO. without
7 HPV45 probe CCTGTGCCAAGTACATATGACCC 5'-ROX, 3'-BHQ2 of SEQ ID NO. without
8 HPV51 probe CTGCGGTTTCCCCAAC 5'-FAM, 3'-BHQ1 of SEQ ID NO. without
9 HPV52 probe ACTTTATGTGCTGAGGTTAAAAAGG 5'-FAM, 3'-BHQ1 of SEQ ID NO. without
10 HPV56 probe CTGCTACAGAACAGtTAAGTAAATATG 5'-FAM, 3'-C3 linker of SEQ ID NO./
iBHQ1dT/
11 HPV58 probe TATGCACTGAAGTAACTAAGGAAGG 5'-ROX, 3'-BHQ2 of SEQ ID NO. without
12 HPV59 probe CTACTTCTTCtATTCCTAATGTATACAC 5'-FAM, 3'-C3 linker of SEQ ID NO.
/iBHQ1dT/
13 HPV66 probe CTAAATATGAtGCCCGTGAAATCAATC 5'-FAM, 3'-C3 linker of SEQ ID NO./
iBHQ1dT/
14 HPV68 probe CTGAATCAGCTGTACCAAATATTTATG 5'-FAM, 3'-BHQ1 of SEQ ID NO. without
15 forward primers of SEQ ID NO., 1 TGGTAGATACTACACGCAGTAC is without nothing
16 forward primers of SEQ ID NO., 2 TTGTTTGTTACTGTAGTTGATAC is without nothing
17 forward primers of SEQ ID NO., 3 CAGCTTTTTATTACCTGTGTTG is without nothing
18 forward primers of SEQ ID NO., 4 CAGTTGTTTGTCACAGTTGTGG is without nothing
19 forward primers of SEQ ID NO., 5 CAATTGTTTTTAACAGTTGTAG is without nothing
20 forward primers of SEQ ID NO., 6 CAATTATTTCTTACTGTTGTGG is without nothing
21 reverse primers of SEQ ID NO., 1 GCACAGTTGAAAAATAAACTGTAA is without nothing
22 reverse primers of SEQ ID NO., 2 GCACAATTGAAAAATAAATTGTAAA is without nothing
23 reverse primers of SEQ ID NO., 3 GCATAACTGAAATATAAATTGTAAA is without nothing
24 reverse primers of SEQ ID NO., 4 CATAATTGAAAAATAAATTGCAATTC is without nothing
25 reverse primers of SEQ ID NO., 5 CAAACTGTAGTTCATATTCCTCCAC is without nothing
26 reverse primers of SEQ ID NO., 6 CAACTGAAATATAAATTGCAAATC is without nothing
27 HBB probe GCTCCTGGGAGTAGATTG 5'-TAMRA, 3'-BHQ2 of SEQ ID NO. without
28 HBB forward primers CCAGAAGAGCCAAGGACAGGTACG of SEQ ID NO. are without nothing
29 HBB reverse primers TTTGAGGTTGCTAGTGAACACAG of SEQ ID NO. are without nothing
Claims (5)
1. a kind of kit for detecting multiple high-risk human mammilla papillomavirus at the same time in single tube reaction, it is characterised in that at least
Include and correspond to the special of this 14 genotype of HPV16,18,31,33,35,39,45,51,52,56,58,59,66 and 68 respectively
Property probe SEQ ID NO.1-14；
And comprehensive corresponding 14 genotype and forward primer and reverse primer of the quantity less than 14 pairs.
2. the kit according to claim 1 for detecting multiple high-risk human mammilla papillomavirus at the same time in single tube reaction,
It is characterized in that, the forward primer quantity of comprehensive corresponding 14 genotype is 6, it is respectively：SEQ ID NO.15-20.
3. the kit according to claim 1 for detecting multiple high-risk human mammilla papillomavirus at the same time in single tube reaction,
It is characterized in that, the reverse primer quantity of comprehensive corresponding 14 genotype is 6, it is respectively：SEQ ID NO.21-26.
4. the kit according to claim 1 for detecting multiple high-risk human mammilla papillomavirus at the same time in single tube reaction,
It is characterized in that, kit further includes the spy being useful for being matched as the hemoglobin β gene complementations of Quality Control in human body cell
Specific probes SEQ ID NO.27.
5. the kit according to claim 4 for detecting multiple high-risk human mammilla papillomavirus at the same time in single tube reaction,
It is useful for it is characterized in that, kit further includes with the hemoglobin β gene complementations pairing as Quality Control in human body cell just
To primer SEQ ID NO.28 and reverse primer SEQ ID NO.29.
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