CN107828920B - Kit for realizing typing of multiple high-risk human papilloma viruses - Google Patents
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Abstract
The scheme relates to a kit for realizing typing of various high-risk human papilloma viruses, which comprises three independently subpackaged reagent tubes; three reagent tubes have specific probes SEQ ID No.1-12 corresponding to HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, respectively; and combining the forward primer SEQ ID NO.13-20 and the reverse primer SEQ ID NO.21-28 corresponding to the 12 genotypes. According to the invention, through improvement of the existing HPV typing kit, the technical scheme of the invention can realize typing of 12 high-risk types of HPV except HPV16 and 18 in a three-tube reaction with high sensitivity and high specificity by only using 8 forward primers and 8 reverse primers.
Description
Technical Field
The invention relates to an HPV typing kit, in particular to a kit for typing multiple high-risk human papilloma viruses, and specifically relates to a kit for typing HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68.
Background
Human Papilloma Virus (HPV) is an epitheliophilic double stranded DNA spherical virus belonging to the family of papillomaviruses that primarily infects skin and mucosal tissues. HPV infection can cause warts, benign tumors, and cancer. There is evidence that HPV infection can lead to a variety of cancers including cervical, vaginal, penile, anal, oropharyngeal cancer. According to the latest statistics of the International Human papilloma virus Reference Center (International Human Papillomavirus Reference Center), more than 200 HPV genotypes are found together at present. However, not all HPV genotypes can induce cancer. Infection with low-risk HPV can induce benign lesions such as common warts, condyloma acuminatum, and papilloma growing on the skin and mucous membranes in the vicinity of the genital organs, and only infection with a few high-risk HPV can cause cancer. The most deeply understood example of HPV carcinogenesis is female cervical cancer. Also because of the pioneering work in this regard, the german scientist Harald zur Hausen acquired a nobel prize for physiology and medicine in 2008.
Cervical cancer is a malignant tumor that occurs in the cervix of the lower uterine segment in women, is the high-grade malignant tumor at the 4 th position in women, and accounts for the 4 th position of cancer mortality in women. According to the international research center for cancer (IARC) of the international health organization (WHO), 52.8 ten thousand new cases of cervical cancer are reported in 2012, and about 26.6 ten thousand women die of cervical cancer. Almost all cervical cancers develop as a result of persistent infection with high-risk HPV types and it takes 5 to 10 years from initial infection to cancer development. Therefore, the early screening of the cervical cancer by using the HPV molecular diagnosis technology taking genetic material DNA of the high-risk HPV as a detection object can discover the etiology in advance and effectively prevent the cervical cancer at early stage, thereby reducing the morbidity and the mortality. Compared with the traditional cervical cancer screening means such as acetic acid visual inspection, pap smear and liquid-based cytology, the HPV molecular diagnosis has the advantages of high sensitivity and independence on the subjective judgment of pathologists. Therefore, HPV molecular diagnosis has become the first choice for cervical cancer screening in developed countries.
One of the biggest challenges of HPV molecular diagnosis technology is to effectively distinguish high-risk HPV from low-risk HPV while ensuring detection sensitivity, so as to avoid over-diagnosis and over-treatment due to misdiagnosis and unnecessary mental stress on patients. As described above, over 200 genotypes of HPV have been found so far, and only 14 genotypes among them are defined as high-risk types that can induce cervical cancer according to the research results of the authorities such as WHO/IARC, i.e., HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68. Furthermore, not every genotype of even 14 high-risk HPV types has the same oncogenic capacity. Currently available epidemiological data show that HPV16 and 18 are the two genotypes with the greatest risk, which lead to 55% and 15% of cervical cancer cases, respectively. Other high-risk types of HPV have led to the remaining approximately 30% of cervical cancer cases, but the difference between the oncogenic abilities of these genotypes has never been clearly defined by the academic community, and particularly in cervical cancer cases there is a clear regional difference in the distribution of different high-risk types of HPV, such as HPV52 and 58 being relatively more prevalent in asia, and HPV33 being relatively more prevalent in europe.
Furthermore, not all patients with high-risk HPV infection of the cervical region must have cervical cancer, and most of these infections are transient infections, i.e., the body's own immune function can effectively clear them within 6 to 24 months. The determinants of whether high risk HPV types are eliminated by the immune system are manifold, including both the ability of the patient's immune system to eliminate infection and the viral load of the infected HPV. Epidemiological studies over the years have shown that there is a clinical threshold for high risk HPV infection. High-risk HPV infections with viral loads below this threshold are often transient infections, rarely inducing cervical cancer; infection with viral loads above this threshold will have a greatly increased chance of inducing cervical cancer. And the determination of this threshold is mainly based on clinical data of HPV16, and is this threshold applicable to other high-risk types? What are the other high-risk types of clinical thresholds, if not applicable? Is the clinical threshold for each high risk type different? To answer these questions, it is necessary to be able to accurately classify these high-risk types, and to be able to quantitatively detect different types.
In addition, since HPV16 and 18 are the two most carcinogenic genotypes, the earliest internationally developed and used prophylactic HPV vaccine was also directed against these two genotypes. Epidemiological data to date have shown that areas where HPV vaccines have been spread and disseminated have seen a dramatic drop in the infection rates of these two genotypes over the last decade, and instead have seen an increase in the infection rates of other high-risk types of HPV. The change of the infection rate inevitably causes the change of the distribution of different high-risk HPV in cervical carcinoma cases in the near future, and then exposes new HPV genotypes with the strongest carcinogenic capacity and the greatest harm.
Therefore, both basic research and clinical applications require molecular biological solutions that can classify other high-risk types of HPV other than HPV16 and 18, in order to further deepen the research on the pathogenesis, epidemiology and etiology of cervical cancer in humans, and to cope with the changes in epidemiology and etiology of cervical cancer caused by the popularization and application of preventive HPV vaccines.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a high-sensitivity and high-specificity kit which can be used for detecting 12 high-risk HPV viruses in a three-tube fluorescent quantitative PCR reaction: kits for typing HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a kit for realizing typing of various high-risk human papilloma viruses comprises three independently-packaged reagent tubes;
wherein, the first reagent tube at least comprises specific probes SEQ ID NO.1, 2, 3 and 5 respectively corresponding to four genotypes of HPV31, 33, 35 and 45; and combining forward primers SEQ ID NO.13, 15, 16 and reverse primers SEQ ID NO.21, 23, 25 corresponding to the four genotypes;
the second reagent tube at least comprises specific probes SEQ ID NO.4, 8, 10 and 12 corresponding to four genotypes of HPV39, 56, 59 and 68 respectively; and combining forward primers SEQ ID NO.13, 16, 19, 20 and reverse primers SEQ ID NO.22, 23, 26, 28 corresponding to the four genotypes;
the third reagent tube at least comprises specific probes SEQ ID NO.6, 7, 9 and 11 corresponding to four genotypes of HPV51, 52, 58 and 66 respectively; and combining the forward primers SEQ ID NO.13, 14, 16, 17, 18 and the reverse primers SEQ ID NO.22, 24, 25, 27 corresponding to the four genotypes.
Preferably, the kit for typing multiple high-risk human papilloma viruses, wherein the first reagent tube further comprises a specific probe SEQ ID No.29 for complementary pairing with a hemoglobin beta gene serving as quality control in human cells.
Preferably, the kit for typing multiple high-risk human papilloma viruses, wherein the first reagent tube further comprises a forward primer SEQ ID NO.30 and a reverse primer SEQ ID NO.31 which are used for complementary pairing with a hemoglobin beta gene serving as quality control in human cells.
Preferably, the kit for typing multiple high-risk human papilloma viruses, wherein the second reagent tube further comprises a specific probe SEQ ID No.29 for complementary pairing with a hemoglobin beta gene serving as quality control in human cells.
Preferably, the kit for typing multiple high-risk human papilloma viruses, wherein the second reagent tube further comprises a forward primer SEQ ID NO.30 and a reverse primer SEQ ID NO.31 which are used for complementary pairing with a hemoglobin beta gene serving as quality control in human cells.
Preferably, the kit for typing multiple high-risk human papilloma viruses, wherein the third reagent tube further comprises a specific probe SEQ ID No.29 for complementary pairing with a hemoglobin beta gene serving as quality control in human cells.
Preferably, the kit for typing multiple high-risk human papilloma viruses, wherein the third reagent tube further comprises a forward primer SEQ ID NO.30 and a reverse primer SEQ ID NO.31 for complementary pairing with a hemoglobin beta gene serving as quality control in human cells.
The invention has the beneficial effects that: according to the invention, through improvement of the existing HPV typing kit, the technical scheme of the invention can realize typing of 12 high-risk types of HPV except HPV16 and 18 in a three-tube reaction with high sensitivity and high specificity by only using 8 forward primers and 8 reverse primers.
Drawings
FIG. 1 is a graph showing the comparison of the results of two different batches of the kit of the present invention for detecting the same set of standards; wherein, the standard template: 12 HPV: mixed solution of 12 high-risk HPV plasmid DNAs with the same concentration; single HPV: a solution containing only the high-risk HPV plasmid DNA of the detection object; the concentration of HPV plasmid DNA of each type in the standard was 1000 copies/reaction.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
The following lists a kit for typing multiple high-risk human papillomaviruses according to an embodiment of the present invention, which includes three independently-packaged reagent tubes;
wherein, the first reagent tube at least comprises specific probes SEQ ID NO.1, 2, 3 and 5 respectively corresponding to four genotypes of HPV31, 33, 35 and 45; and combining forward primers SEQ ID NO.13, 15, 16 and reverse primers SEQ ID NO.21, 23, 25 corresponding to the four genotypes; in the reagent tube, the four HPV genotypes can be distinguished only by 3 forward primers and 3 reverse primers, because each primer in the 3 forward primers or 3 reverse primers not only contributes to one HPV genotype but also contributes to a plurality of genotypes, and the effect formed after the combination is as follows: the 3 forward primers +3 reverse primers as a whole just meet the requirement of simultaneously contributing to the amplification of the four genotypes of HPV31, 33, 35 and 45, namely Ct values measured in the reaction of the 4 genotypes with the same concentration are close, so that a user can more intuitively understand the detection result.
The second reagent tube at least comprises specific probes SEQ ID NO.4, 8, 10 and 12 corresponding to four genotypes of HPV39, 56, 59 and 68 respectively; and combining the forward primers SEQ ID NO.13, 16, 19, 20 and the reverse primers SEQ ID NO.22, 23, 26, 28 corresponding to the four genotypes. The combination of the primer and the probe enables the Ct values of the 4 genotypes with the same concentration to be close in the reaction, so that a user can more intuitively understand the detection result.
The third reagent tube at least comprises specific probes SEQ ID NO.6, 7, 9 and 11 corresponding to four genotypes of HPV51, 52, 58 and 66 respectively; and combining the forward primers SEQ ID NO.13, 14, 16, 17, 18 and the reverse primers SEQ ID NO.22, 24, 25, 27 corresponding to the four genotypes. The reason for using 5 forward primers in the reagent tube is to make Ct values measured in the reaction for the 4 genotypes with the same concentration close, so that the user can more intuitively understand the detection result.
In addition, the combination of the primers and the probes of the 3 reagent tubes also enables Ct values measured in 3 tube reactions of all 12 genotypes with the same concentration to be close, so that a user can understand a detection result more intuitively.
Wherein, the three reagent tubes also preferably comprise a specific probe SEQ ID NO.29 for complementary pairing with a hemoglobin beta gene serving as quality control in human cells.
Wherein, the three reagent tubes also preferably comprise a forward primer SEQ ID NO.30 and a reverse primer SEQ ID NO.31 which are used for complementary pairing with the hemoglobin beta gene serving as the quality control in human cells.
Of course, the three reagent tubes also preferably include the necessary auxiliary agents to complete the fluorescent quantitative PCR reaction, such as: PCR buffer, 4 dNTP mixtures, hot start polymerase, and the like.
Table 1 shows the sequences of specific primers and probes:
TABLE 1
In Table 1, the fluorescent groups used for the HPV31, 56 and 66 probes (SEQ ID NO.1, 8 and 11) are FAM; the fluorescent group used by the three probes HPV33, 39 and 51 (SEQ ID NO.2, 4 and 6) is JOE; the three probes HPV35, 52 and 68 (SEQ ID NO.3, 7 and 12) used a fluorophore CY 5; the fluorophores used for the three probes HPV45, 58 and 59 (SEQ ID NO.5, 9 and 10) were ROX.
The following provides a specific example of PCR reaction solution in three reagent tubes:
a first reagent tube: reaction recipe for fluorescent quantitative PCR reactions typing four genotypes of HPV31, 33, 35 and 45 (table 2.1):
TABLE 2.1
A second reagent tube: reaction recipe for fluorescent quantitative PCR reactions for typing four genotypes of HPV39, 56, 59, and 68 (table 2.2):
TABLE 2.2
| Name (R) | Final concentration of reaction | Concentration unit |
| Free of Mg2+Ionic PCR buffer | 1 | X |
| Mg2+ | 4 | mM |
| 4 kinds of dNTP mixed liquor | 0.2 | mM |
| SEQ ID NO.4 | 0.05 | μM |
| SEQ ID NO.8 | 0.05 | μM |
| SEQ ID NO.10 | 0.01 | μM |
| SEQ ID NO.12 | 0.05 | μM |
| SEQ ID NO.13 | 0.8 | μM |
| SEQ ID NO.16 | 0.2 | μM |
| SEQ ID NO.19 | 0.1 | μM |
| SEQ ID NO.20 | 0.2 | μM |
| SEQ ID NO.22 | 0.8 | μM |
| SEQ ID NO.23 | 0.2 | μM |
| SEQ ID NO.26 | 0.1 | μM |
| SEQ ID NO.28 | 0.2 | μM |
| SEQ ID NO.29 | 0.1 | μM |
| SEQ ID NO.30 | 0.2 | μM |
| SEQ ID NO.31 | 0.2 | μM |
| Hot start polymerase (Taq) | 0.12 | U/μL |
A third reagent tube: reaction recipe for fluorescent quantitative PCR reaction for typing four genotypes of HPV51, 52, 58 and 66 (table 2.3):
TABLE 2.3
| Name (R) | Final concentration of reaction | Concentration unit |
| Free of Mg2+Ionic PCR buffer | 1 | X |
| Mg2+ | 4 | mM |
| 4 kinds of dNTP mixed liquor | 0.2 | mM |
| SEQ ID NO.6 | 0.03 | μM |
| SEQ ID NO.7 | 0.05 | μM |
| SEQ ID NO.9 | 0.05 | μM |
| SEQ ID NO.11 | 0.04 | μM |
| SEQ ID NO.13 | 0.5 | μM |
| SEQ ID NO.14 | 0.3 | μM |
| SEQ ID NO.16 | 0.2 | μM |
| SEQ ID NO.17 | 0.15 | μM |
| SEQ ID NO.18 | 0.2 | μM |
| SEQ ID NO.22 | 0.3 | μM |
| SEQ ID NO.24 | 0.15 | μM |
| SEQ ID NO.25 | 0.2 | μM |
| SEQ ID NO.27 | 0.4 | μM |
| SEQ ID NO.29 | 0.1 | μM |
| SEQ ID NO.30 | 0.2 | μM |
| SEQ ID NO.31 | 0.2 | μM |
| Hot start polymerase (Taq) | 0.12 | U/μL |
In order to verify the technical performance of the kit, 12 plasmids respectively containing the DNA target sequences of the 12HPV genotypes are prepared and mixed with human genome DNA to prepare standard substances with different components and different concentrations. The purpose of adding human genomic DNA to the standards is to mimic human DNA that is necessarily present in clinical samples. The human genomic DNA in these standards was purified from Jurkat cultured cells derived from patients with T cell leukemia.
Example 1:
in order to verify the stability of the formula of the kit, 2 sets of raw material reagents of different batches are sequentially used for preparing the kits of 2 different batches, and the obtained kits are used for detecting the same group of standard substances. The results obtained show that the detection results of the two batches of kits are very similar (FIG. 1).
Example 2:
in order to verify the applicability of the kit to clinical samples, nearly hundred clinical samples are selected. 12 cases were tested as positive using the 12+2 high-risk HPV combined 16/18 genotyping test kit from Kyoto Kappan Biotechnology, Inc. The Kypr kit can directly classify two genotypes of HPV16 and 18 with the highest harm, and other 12 high-risk types are combined in one fluorescence channel for detection due to the limitation of the fluorescence channel which can be used by a fluorescence quantitative PCR instrument. Therefore, if the detection result is positive for the fluorescence channel, it is impossible to determine which one or more of the 12 high-risk types is positive. This is true for 9 of the 12 positive clinical samples described above, and the kit of the present invention successfully typed these 9 clinical samples (table 3). The results show that:
1. for each clinical sample, the type of the high-risk HPV detected by the kit is matched with the fluorescent channel detected by the existing detection kit;
2. the Ct value detected by the kit is similar to the Ct value detected by the existing detection kit;
3. for some high-risk HPV, the clinical samples with positive signals of only one fluorescence channel appear in the detection results of the existing detection kit, the kit successfully detects multiple infections, such as detection of HPV33 and 39 in sample 3 and detection of HPV33 and 56 in sample 4.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
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Claims (7)
1. A kit for realizing typing of various high-risk human papilloma viruses is characterized by comprising three independently subpackaged reagent tubes;
wherein, the first reagent tube comprises specific probes SEQ ID NO.1, 2, 3 and 5 respectively corresponding to four genotypes of HPV31, 33, 35 and 45; and combining forward primers SEQ ID NO.13, 15, 16 and reverse primers SEQ ID NO.21, 23, 25 corresponding to the four genotypes; the two ends of SEQ ID NO.1 are modified into 5 '-FAM, 3' -BHQ 1; the two ends of SEQ ID NO.2 are modified into 5 '-JOE, 3' -BHQ 1; the two ends of SEQ ID NO.3 are modified into 5 '-CY 5, 3' -BHQ 2; the two ends of SEQ ID NO.5 are modified into 5 '-ROX, 3' -BHQ 2;
the second reagent tube comprises specific probes SEQ ID NO.4, 8, 10 and 12 corresponding to four genotypes of HPV39, 56, 59 and 68 respectively; and combining forward primers SEQ ID NO.13, 16, 19, 20 and reverse primers SEQ ID NO.22, 23, 26, 28 corresponding to the four genotypes; the two ends of SEQ ID NO.4 are modified to 5 '-JOE, 3' -BHQ 1; the two ends of SEQ ID NO.8 are modified to 5 '-FAM, 3' -BHQ 1; the two ends of SEQ ID NO.10 are modified to 5 '-ROX, 3' -BHQ 2; the two ends of SEQ ID NO.12 are modified to 5 '-CY 5, 3' -BHQ 2;
the third reagent tube comprises specific probes SEQ ID NO.6, 7, 9 and 11 corresponding to four genotypes of HPV51, 52, 58 and 66 respectively; and combining forward primers SEQ ID NO.13, 14, 16, 17, 18 and reverse primers SEQ ID NO.22, 24, 25, 27 corresponding to the four genotypes; the two ends of SEQ ID NO.6 are modified to 5 '-JOE, 3' -BHQ 1; the two ends of SEQ ID NO.7 are modified to 5 '-CY 5, 3' -BHQ 2; the two ends of SEQ ID NO.9 are modified to 5 '-ROX, 3' -BHQ 2; the two ends of SEQ ID NO.11 are modified to 5 '-FAM, 3' -BHQ 1;
the combination of primers and probes in the three reagent tubes enables Ct values of all 12 genotypes with the same concentration to be similar in the three reagent tube reactions.
2. The kit for realizing typing of multiple high risk human papillomaviruses according to claim 1, wherein the first reagent tube further comprises a specific probe SEQ ID No.29 for complementary pairing with a hemoglobin beta gene as a quality control in human cells, and both ends of the SEQ ID No.29 are modified to be 5 '-TAMRA, 3' -BHQ 2.
3. The kit for typing a plurality of high risk human papillomaviruses according to claim 2, wherein the first reagent tube further comprises a forward primer SEQ ID No.30 and a reverse primer SEQ ID No.31 for complementary pairing with a hemoglobin beta gene as a quality control in human cells.
4. The kit for realizing typing of multiple high risk human papillomaviruses according to claim 1, wherein the second reagent tube further comprises a specific probe SEQ ID No.29 for complementary pairing with a hemoglobin beta gene as a quality control in human cells, and both ends of the SEQ ID No.29 are modified to be 5 '-TAMRA, 3' -BHQ 2.
5. The kit for typing a plurality of high risk human papillomaviruses according to claim 4, wherein the second reagent tube further comprises a forward primer SEQ ID No.30 and a reverse primer SEQ ID No.31 for complementary pairing with a hemoglobin beta gene as a quality control in human cells.
6. The kit for realizing typing of multiple high risk human papillomaviruses according to claim 1, wherein the third reagent tube further comprises a specific probe SEQ ID No.29 for complementary pairing with a hemoglobin beta gene as a quality control in human cells, and both ends of the SEQ ID No.29 are modified to be 5 '-TAMRA, 3' -BHQ 2.
7. The kit for typing a plurality of high risk human papillomaviruses according to claim 6, wherein the third reagent tube further comprises a forward primer SEQ ID No.30 and a reverse primer SEQ ID No.31 for complementary pairing with the hemoglobin beta gene as the quality control in human cells.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104073573A (en) * | 2014-07-15 | 2014-10-01 | 江苏同科医药科技有限公司 | Kit for carrying out genotyping detection on human papilloma viral nucleic acid |
| KR101761701B1 (en) * | 2014-07-29 | 2017-07-26 | 의료법인 제일의료재단 | HPV Specific Probe and DNA Chip for Detecting Genetic Type of HPV Containing Thereof |
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| US6265154B1 (en) * | 1996-10-25 | 2001-07-24 | Abbott Laboratories | Nucleic acid primers and probes for detecting oncogenic human papillomaviruses |
| US9090948B2 (en) * | 2008-09-30 | 2015-07-28 | Abbott Molecular Inc. | Primers and probes for detecting human papillomavirus and human beta globin sequences in test samples |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104073573A (en) * | 2014-07-15 | 2014-10-01 | 江苏同科医药科技有限公司 | Kit for carrying out genotyping detection on human papilloma viral nucleic acid |
| KR101761701B1 (en) * | 2014-07-29 | 2017-07-26 | 의료법인 제일의료재단 | HPV Specific Probe and DNA Chip for Detecting Genetic Type of HPV Containing Thereof |
Non-Patent Citations (2)
| Title |
|---|
| Single-tube multiplex PCR using type-specific E6/E7 primers and capillary electrophoresis genotypes 21 human papillomaviruses in neoplasia;Michael Dictor等;《Infectious Agents and Cancer》;20110117;第6卷(第1期);第1-7页 * |
| 两种人乳头瘤病毒分型检测方法的比较;蒋卫等;《上海交通大学学报（医学版）》;20081231(第8期);第962-966页 * |
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