Patent Application
20130184164
The present disclosure relates to a DNA chip for genotyping human papillomavirus (HPV), a kit including same and a method for genotyping HPV. More particularly, it relates to a DNA chip (or DNA microarray) on which probes complementarily binding to the nucleic acids of 44 types of HPV, which is the main cause of cervical cancer and the most common cause of sexually transmitted diseases, are spotted, a genotyping kit including same and a genotyping method using same.
Human papillomavirus (HPV) is a virus transmitted to humans through sexual contact and is very important in two aspects.
Firstly, HPV infection is the most common sexually transmitted infection in humans with the highest prevalent rate. In the US, HPV infection is found in 26.8% of women aged between 14 and 59 and it is thought that 80% of women are infected at least once. The infection occurs well particularly in sexually active, fertile women, and the prevalence is estimated to increase. Hence, periodic HPV testing is necessary for adult women and HPV testing is included in testing of sexually transmitted infections (U.S. Department of Health And Human Services, Centers for Disease Control and Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB, Prevention Division of STD Prevention. Sexually Transmitted Disease Surveillance 2008. Division of STD Prevention. 2009: November; Tchernev G. Sexually transmitted papillomavirus infections: epidemiology pathogenesis, clinic, morphology, important differential diagnostic aspects, current diagnostic and treatment options. An Bras Dermatol. 2009; 84(4): 377-89).
Secondly, HPV is clearly proven to cause tumors and cancers in human. HPV, particularly the high-risk type HPV, is the cause of nearly all cases of cervical cancer. HPV infiltrates into the epithelium of human skin or mucous membranes, thereby causing inflammation and hyperproliferation. In most cases, the hyperproliferation is simply skin warts, genital or anal warts, or benign tumors such as condylomata acuminata. However, HPV can cause cancer and, indeed, almost all cervical cancers, most of oral cancers, pharyngeal cancers and laryngeal cancers and a number of anal cancers are caused by HPV. HPV is of great importance in that it can be fatal by causing cancer. Caners and precancerous lesions of the cervix, anus, etc. can be diagnosed early by HPV testing. Indeed, it is shown that HPV testing is superior in prediction sensitivity of cervical cancer than the Papanicolaou test, or Pap smear, which is the standard screening method for diagnosis of cervical cancer. Accordingly, it is approved as the cervical cancer screening test in several countries including the US (Howley P M. Virology. Vol 2, 1996, 2045-2109; Murinoz N et al., N Engl J Med, 2003, 348: 518-27; Parkin M, F. Bray F, J. Ferlay J and P. Pisani P. Global cancer statistics, 2002. C.A. Cancer J. Clin. 2005; National Network of STD/HIV Prevention Training Center. Genital human papillomavirus infection. February 2008). For these reasons, the HPV market is very large and the HPV testing is of great economic value.
Cervical cancer is the second most common cancer in women globally after breast cancer. It is also one of the main causes of cancer-related deaths of women in the developing countries. It is reported that about 440,000 new cases and 270,000 deaths occur each year worldwide. In particular, it is one of the main causes of female death in developing countries. In Korean women, cervical cancer (10.6%) ranks third in incidence following stomach cancer (15.8%) and breast cancer (15.1%). In recent years, human papillomavirus infection has significantly increased in young women of 20s and 30s, accounting for 32% of all sexually transmitted disease patients, and become a severe health concern. According to the 2002 Annual Report of the Korea Central Cancer Registry, Korea shows higher incidence rate with 3,979 cases in 2002 as compared to developed countries. Among the all malignant tumors occurring in women, cervical cancer ranks fifth with 9.1% after breast cancer, stomach cancer, colorectal cancer and thyroid cancer, with the highest incidence in 40s as 29.3%. According to the data from the Korea Central Cancer Registry, cervical cancer ranks 2nd when including carcinoma in situ of the cervix, which is a pre-cancer stage, and ranks 5th when excluding the carcinoma in situ. However, if cervical dysplasia not registered in the cancer statistics is also included, it is still the most important cancer in women. Formerly, about 90% of the cancer of uterine cancer was cervical cancer. But, recently, the incidence of uterine body cancer is increasing and that of cervical cancer is decreasing. Presently, the ratio of cervical cancer to uterine body cancer is about 5:1 (http://www.ncc.re.kr:9000/nciapps/user/basicinfo/each_info.jsp?grpcode=1H00).
Epidemiological studies about the cause of cervical cancer reveal that risk of cervical cancer is higher in women of low level of education or economy or poor hygiene, in women who started sexual intercourse in young ages, in women who have many childbirth experiences, in women who have promiscuous sex partners, and in women who are diagnosed positive in human papillomavirus testing. This suggests that cervical cancer is closely related with sexually transmitted infection and it is widely recognized that human papillomavirus is the major cause of cervical cancer (Jae Won Kim, Ju Won Roh, Moon Hong Kim, Noh Hyun Park, Polymorphisms in E7 Gene of Human Papillomavirus Type 16 Found in Cervical Tissues from Korean Women, J Korean Cancer Assoc. 2000; 32(5) 875-883).
At present, about 120 types of HPV are known based on subtypes or genotypes. Among them, 83 types are known about their base sequence and structure. About 40 types of HPV are the so-called anogenital type or genital HPV infecting the anogenital region, i.e. the skin and mucosa of the vagina, cervix, urethra and penis. While the majority of HPV infections cause no symptoms in most people, some types can cause warts. Others can lead to precancerous lesions such as high grade squamous intraepithelial lesion (HSIL) or cervical intraepithelial neoplasm, and some of them may develop into cancer. HPV types that can lead to precancerous lesions and cancer are called high-risk type HPV and others are called low-risk type HPV. Some researchers classify HPV into high-risk, moderate-risk and low-risk groups. High-risk type HPV includes HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 and 82. And, low-risk type HPV includes HPV type 6, 11, 34, 40, 42, 43, 44, 54, 55, 61, 62, 72 and 81. Probably high-risk type HPVs which are suspected of being high-risk but not identified yet include HPV types 26, 53, 66, 67, 69, 70 and 73. Besides, there are other types that are not clearly identified such as HPV types 7, 10, 27, 30, 32, 57, 83, 84 and 91. Globally, it is reported that 49.9% of cervical cancer patients are infected by HPV type 16, 13.7% by HPV type 18, 7.2% by HPV types 31, 33 and 35, and 8.4% by HPV type 45.
According to the Merck's report, HPV types 16 and 18 are of particular importance. These two types of HPV are reported to cause about 60-70% of cervical cancer, cervical intraepithelial neoplasm (CIN) and HSIL and HPV types 6 and 11 are known to cause about 90% of genital warts. However, there are differences in the epidemiology of HPV types in different races and countries. Indeed, as will be described later, the data from Korea have slight difference from those of other countries. Another report from Korea classifies HPV types 16 and 18 as high-risk HPVs causing cervical cancer, HPV types 31, 33, 35, 45 and 52 as moderate-risk HPVs, and HPV types 6 and 11 as low-risk HPVs and asserts that early screening or diagnosis of cervical cancer is possible through genotyping of HPV (Jae Won Kim, Ju Won Roh, Moon Hong Kim, Noh Hyun Park, Polymorphisms in E7 Gene of Human Papillomavirus Type 16 Found in Cervical Tissues from Korean Women, J Korean Cancer Assoc. 2000; 32(5) 875-883; (http://www.cmcbaoro.or.kr/guide/guide02—02.jsp?dtno=209&dcno=411; Munoz N, Bosch F X, de Sanjose S, Herrero R, Castellsague X, Shah K V, Snijders P J, Meijer C J and International Agency for Research on Cancer Multicenter Cervical Cancer Study Group. Epidemiologic classification of human papillomavirus types associated with cervical cancer. New England Journal of Medicine. 2003; 348: 518-527; Koutsky L A et al., N Engl J Med, 2002, 347: 1645-51; http://www.bosa.co.kr/news_board/view.asp?news_pk=82896).
The HPV genome is about 8-10 kb in size and consists of a double-helical DNA enclosed in a capsid that resembles a golf ball. The genome structure of HPV can be roughly divided into early transcription region E (early gene region), late transcription region L (late gene region) and non-expression region LCR (long control region). The genome structure of HPV greatly affects the outbreak type, risk and prognosis of diseases. Particularly, E6 and E7 genes are integrated into the genome of an infected cell and play an important role in inducing cancer while they remain and are expressed there. The E6 and E7 genes of high-risk types of HPV such as HPV types 16 and 18 react with p53, E6AP, Rb (retinoblastoma, P105RB), P107, P130, etc., which are the most important tumor suppressor genes in human, and inactivate them. As a result, the infected cell is transformed into a cancer cell due to disorder of cell cycle regulation and apoptosis control mechanism. More than 99% of cervical cancer is caused by the high-risk type HPV and HPV gene fragments of E6/E7 are found almost always in the genome of the cancer cell. In contrast, since low-risk types of HPV have low ability to react with the tumor suppressor genes such as p53 or Rb and inactivate them, they normally do not cause cervical cancer. The largest gene of HPV is L1. L1 is present in most HPV types with the base sequence similarly conserved. HPV's capsid protein primarily consists of L1 and L1 has the highest antigenicity.
Once a cervical cell is malignantly transformed by HPV, it advances to so-called carcinoma in situ via precancerous lesion, dysplasia, CIN or squamous intraepithelial lesion (SIL). If the carcinoma in situ invades the basal layer under the cervical epithelium, it becomes carcinoma or invasive carcinoma. In 90% of women infected by HPV, the virus is naturally cleared from the body by the immune system. However, HPV remains in 10% of women who are infected with high-risk type HPV and induces precancerous lesions (Wallin K L, Winklund F, Angstrim T, et al: Type-specific persistence of human papillomavirus DNA before the development of invasive cancer. N Engl J Med 1999; 341: 1633; Bosch F X, Lorincz A, Munoz N, Meijer C J, Shah K V. The causal relation between human papillomavirus and cervical cancer. J Clin Pathol 2002; 55: 244-65). About 8% of the precancerous lesions advance to carcinoma in situ, and about 20% of carcinoma in situ develop into cancer. That is to say, infection of high-risk type HPV maintained 10-20 years or longer, it develops into cervical cancer and the frequency is estimated at about 0.16%. Since such a long time is necessary for the outbreak of cervical cancer and it occurs gradually, it is possible to treat or prevent cervical cancer by early diagnosing precancerous lesions. That is, cancer can be prevented by removing precancerous lesions of the cervix through conservative surgery.
HPV infection is hardly detected by culturing, staining, histological inspection or immunological inspection and can only be accurately diagnosed by genetic testing. There are three kinds of HPV genetic testing. The first is to simply investigate the presence of HPV. A representative example is amplification of the consensus sequence, i.e. invariant nucleotide sequence, of the HPV gene by PCR followed by identification through, for example, electrophoresis. The second is the so-called genotyping analysis of identifying not only the presence of HPV but also its type. The gold standard test is to perform PCR and analyze the genotype by automated nucleotide sequencing of the product. However, since this method requires a lot of cost, time and labor, it is being replaced by the HPV DNA microarray. A plurality of probes specific for HPV types are spotted on a solid support and a PCR product of the sample DNA is placed thereon and hybridized. Then, the result is analyzed using a scanner The third is intermediary of the two test methods. The hybrid capture assay (Digene Corporation, Gaithersburg, Md., USA) is an example. Although it allows to identify whether HPV exists and whether the HPV is high-risk type or low-risk type, accurate genotyping is impossible. In addition, only 13 high-risk type HPVs and 7 low-risk type HPVs can be identified, and other 20 or more HPV types cannot be identified (Kim K H, Yoon M S, Na Y J, Park C S, Oh M R, Moon W C. Development and evaluation of a highly sensitive human papillomavirus genotyping DNA chip. Gynecol Oncol. 2006; 100(1): 38-43; Selva L, Gonzalez-Bosquet E, Rodriguez-Plata M T, Esteva C, Sunol M and Munoz-Almagro C. Detection of human papillomavirus infection in women attending a colposcopy clinic. Diagnostic Microbiology and Infectious Disease. 2009; 64: 416-421).
Another important fact regarding HPV is that prevention of viral infection and cancer is possible through vaccination using the recently developed HPV vaccine. Two types of HPV vaccines are currently available. Gardasil (Merck & Co. Inc., Whitehouse Station, N.J., USA) is a quadrivalent vaccine prepared against HPV types 16, 18, 6 and 11. The other, Cervarix (GlaxoSmithKline Biologicals, Rixensart, Belgium), is a bivalent vaccine designed to prevent infection from HPV types 16 and 18. These vaccines are the most effective for adolescent girls before sexual activity, and the efficacy decreases in women who have been infected by HPV16 or HPV18 before. For this reason, vaccination to adult women is controversial, but, it may be possible unless the HPV infection is by type 16 or 18. Accordingly, it is becoming more and more important to identify not just the HPV infection but the accurate type of HPV (Selva L, Gonzalez-Bosquet E, Rodriguez-Plata M T, Esteva C, Sunol M and Munoz-Almagro C. Detection of human papillomavirus infection in women attending a colposcopy clinic. Diagnostic Microbiology and Infectious Disease. 2009; 64: 416-421; Reynales-Shigematsu L M, Rodrigues E R, Lazcano-Ponce E. Cost-effectiveness analysis of a quadrivalent human papilloma virus vaccine in Mexico. Arch Med Res. 2009 August; 40(6): 503-13).
The Papanicolaou test (Papanicolaou smear or Pap smear) of examining cervical cells has been used as a primary screening test of cervical cancer. However, since the Pap smear is a subjective test, false positive results are not infrequent and, thus, a test method for complementing it has been necessary. Actually, the cytological test based on Pap smear is not so effective for diagnosis of HPV infection, which is the most important cause of cervical cancer, and it is not easy to predict whether an abnormal lesion will be disappear naturally or progress to cancer. Indeed, it is impossible to diagnose non-symptomatic or latent infection through cytomorphological examination under a microscope, particularly to distinguish infection by high-risk type HPV from that by low-risk type HPV. Accordingly, to reduce cervical cancer, a diagnosis method capable of monitoring HPV infection, risk thereof and genotype thereof is required.
As described above, it is necessary to test the presence of HPV and its genotype accurately and quickly, at low cost and in large scale. The DNA microarray (chip) technique is the most suitable in this sense.
HPV diagnosis products used overseas include Hybrid Capture II (Qiagen, Germany; approved by the FDA), Cervista™ HPV HR test (Hologic Women's Health Co.; 14 high-risk types; approved by the FDA), Roche AMPLICOR HPV test (Roche Molecular Systems, USA; CE marking), PapilloCheck HPV screening test kit (Greiner Bio-One GmbH, Germany; 18 high-risk types and 6 low-risk types; CE marking) and Digene HPV genotyping RH test (Qiagen; high-risk types; CE marking).
However, the currently commercialized HPV genotyping DNA chips have the following disadvantages.
Firstly, the number of HPV genotypes that can be tested is limited.
Secondly, although the HPV probes need to be designed based on the base sequence information of the HPV genome of actual clinical samples, most of the HPV DNA chips are designed based on the standard base sequence available from literatures or US GenBank. Since there are numerous variations in the DNA base sequence of the HPV genome, if primers or probes are designed without considering them, PCR or hybridization may not be carried out as desired and error may occur.
Thirdly, since an internal reference gene (control gene) is not used, it is not easy to known whether a negative result is true negative or false negative.
Fourthly, the so-called universal probe capable of testing the presence of all genotypes of HPV is not considered. For this reason, when a negative result is obtained for all the HPV genotypes, it is not easy to determine whether it means that no HPV exists in the sample or other genotypes of HPV may exist.
Fifthly, PCR is the most important step prior to HPV DNA analysis, but the condition is not standardized.
Sixthly, for standardization of the HPV DNA chip and HPV genotyping using same, standard materials for gene cloning are required for each genotype of HPV.
Seventhly, although many HPV DNA diagnosis kits are available, large-scale testing and comparison for investigating how accurate they are as compared to the standard test and how useful they are for screening of cervical cancer and precancerous lesions are insufficient.
The inventors of the present disclosure have studied the presence of anogenital HPVs, types thereof and DNA base sequences thereof for more than 250,000 samples for several years through post-PCR sequencing, DNA microarray testing, and HPV type-specific PCR, and so forth. Based on the result and analysis of the features of commercially available HPV DNA diagnosis kits, they have noticed the problems of the existing art to be solved and invented a new HPV DNA microarray. Details are as follows.
1. Type and Number of Genital and Anal HPVs
According to the literatures, the number of HPV types that can invade the genital and anal regions including the cervix are estimated at about 40 but is not clear. For accurate diagnosis of all the types of genital HPVs, it is prerequisite to test multiple samples for all the types of genital HPVs. However, such data are rare worldwide.
Thus, the inventors of the present disclosure have performed PCR for L1, L2 and E6/E7 genes of HPV for about 16,000 cervical samples from Korean women and analyzed the base sequence of all the PCR products. Based on these data, and referring to the reports from the US and other countries, they have determined the HPV types that should be included in the new HPV DNA chip. The number of the types was 43 and, thus, they have invented a DNA chip capable of analyzing all the 43 types of genital HPVs. This will be described in detail in Example 1.
2. Standard Materials
One of the basic requirements in HPV genotyping is that all standard materials (reference materials) should be prepared for each genotype. This may be HPV itself, the entire genome of HPV, the key genes of HPV or plasmid clones. The kind and number of the standard materials of genital HPVs disclosed in literatures and deposited in GenBank are very restricted.
As described earlier, the inventors have performed PCR for the L1, L2 and E6/E7 genes of HPV for about 15,000 cervical samples from Korean women and analyzed the base sequence of all the PCR products. Based on the result, they have obtained plasmid DNA clones by cloning the L1, L2 and E6/E7 genes for 43 types of genital HPV wholly or partially. They have decided to identify the genotype of the 43 types of HPV by targeting specific regions of the HPV L1 gene and determined plasmid standard materials of HPV L1 gene clones for each type. They were used for the development of a DNA chip and quality control (QC) thereof. This will be described in detail in Example 2.
3. PCR Amplification
For accurate and sensitive analysis is possible using the HPV DNA chip, PCR amplification needs to be performed adequately first. For this, the PCR condition for amplifying the HPV L1 gene to be hybridized on the HPV DNA chip of the present disclosure should be optimized and, most of all, the PCR primers should be designed adequately. Further, it is preferred that the amplification of HPV L1 gene and reference and control genes is achieved in a single tube under the same condition by a single duplex PCR. Since the HPV PCR condition reported in literatures or recommended for the commercially available HPV DNA chips is frequently nested PCR, the amplification process is inconvenient and the risk of contamination is high. Further, some types of HPV are amplified well but others are not and interference often occurs when the reference gene is amplified together.
Thus, through repeated experiments, the inventors have newly established the base sequence of oligonucleotide primers for PCR and the amplification condition based on the base sequence of L1 gene of the 43 types of HPV and standard materials as described earlier. As a result, the amplification of the HPV L1 gene and reference gene could be achieved by a single duplex PCR. This will be described in detail in Example 3.
4. Control Gene
One of the basic requirements in HPV DNA chip analysis is that not only the target gene but also the internal reference or control gene therefor should be investigated as well. This is essential for normalization analysis of the signals from the DNA chip and for distinction from false negative and false positive results. Nonetheless, a number of DNA chip tests are carried out without control genes.
The inventors of the present disclosure have used the human beta-globin gene as a control gene. Further, they have found out that the housekeeping gene beta-actin may be used as another control gene and newly added it in the HPV DNA chip. This will be described in detail in Examples 4-6.
5. Probe Structure
The most important thing in HPV genotyping DNA microarray testing is that hybridization is performed adequately for each genotype of HPV so that it can be identified accurately. The probe is of great importance in this aspect. As described above, the inventors of the present disclosure have performed PCR for L1 gene of HPV for more than 15,000 cervical samples from Korean women and analyzed the base sequence of all the PCR products. Based on the result, they have established plasmid DNA clone standard materials for 43 types of genital HPVs and have determined the basic oligonucleotide structure of the HPV DNA chip. The oligonucleotide is from 18 to 30 base pairs (bp) long. This will be described in detail in Example 5.
6. Final Design and Production of Probe
In general, an oligonucleotide probe is 20-30 by long and has a C6 linker attached thereto. However, the inventors of the present disclosure have empirically found out that a problem may occur during spotting on a glass slide in that case owing to spatial instability.
Thus, the inventors of the present disclosure have designed an oligonucleotide probe having a longer C20 linker This will be described in detail in Example 5. In addition, they have designed a d-shaped probe by introducing a stem part. This will be described in detail in Example 6.
7. Fabrication of DNA Microarray (Chip)
A grid was designed according to the probe and the probe mixed in an adequate buffer was spotted on a glass slide for a microscope. This will be described in detail in Example 7.
8. Reaction on DNA Microarray (Chip)
100 artificial standard samples obtained from various combinations of one, two or three clones for each type of HPV were used as templates for PCR amplification of HPV L1 and beta-actin genes. The PCR products were placed on the chip and hybridization was performed at least 3 times. Then, the optimal condition was established by analyzing with a fluorescence scanner. This will be described in detail in Example 8.
9. Evaluation of Accuracy of DNA Microarray (Chip)
The fabricated new HPV DNA chip of the present disclosure was compared with that of the standard sequencing and HPV-type specific PCR to investigate the accuracy, sensitivity and specificity. Further, it was investigated whether the HPV DNA chip can be used to test the presence of HPV in a clinical sample such as a cervical cell and the genotype thereof. This will be described in detail in Example 9. The existing HPV DNA chips lack such data.
10. Evaluation of Accuracy of Early Diagnosis of Cervical Cancer
The accuracy, sensitivity and specificity of diagnosis of cervical cancer and precancerous lesions of the novel HPV DNA chip fabricated according to the present disclosure were compared with those of the existing Hybrid Capture Assay 2 (HCA-2). In addition, it was investigated whether the HPV DNA chip of the present disclosure can be used to predict cervical cancer or precancerous lesions from a clinical sample such as a cervical cell. This will be described in detail in Example 10. The existing HPV DNA chips lack such data. The HPV DNA chip of the present disclosure was confirmed to be clinically applicable.
The present disclosure is directed to providing a DNA chip for diagnosing HPV capable of accurately and quickly diagnosing infection by 44 types of genital HPV simultaneously.
The present disclosure is also directed to providing an oligonucleotide probe and a PCR primer capable of accurately detecting 44 types of genital HPV with high specificity and sensitivity.
The present disclosure is also directed to providing a kit for genotyping 44 types of genital HPV in which the HPV DNA chip, the PCR primer, a label, etc. are provided “all in one”.
In one general aspect, the present disclosure provides a DNA chip for genotyping human papillomavirus (HPV) from a sample, including a linear oligonucleotide probe having a base sequence selected from SEQ ID NOS 6-109.
In another general aspect, the present disclosure provides a DNA chip for genotyping HPV from a sample, including a d-shaped oligonucleotide probe having a base sequence selected from SEQ ID NOS 110-213.
The DNA chip of the present disclosure is capable of simultaneously genotyping 44 types of HPV including: HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-68a, HPV-68b and HPV-82 as high-risk type HPVs; HPV-26, HPV-53, HPV-66, HPV-67, HPV-69, HPV-70 and HPV-73 as moderate-risk type HPVs; HPV-6, HPV-11, HPV-34, HPV-40, HPV-42, HPV-43, HPV-44, HPV-54, HPV-55, HPV-61, HPV-62, HPV-72 and HPV-81 as low-risk type HPVs; and HPV-90, HPV-10, HPV-27, HPV-30, HPV-32, HPV-57, HPV-83, HPV-84 and HPV-91 as other HPVs.
In an exemplary embodiment of the present disclosure, the oligonucleotide probe having a base sequence selected from SEQ ID NOS 6-97 or SEQ ID NOS 110-201 may bind complementarily to L1 gene region specific for each type of HPV.
In an exemplary embodiment of the present disclosure, the oligonucleotide probe having a base sequence selected from SEQ ID NOS 98-105 or SEQ ID NOS 202-209 may be a universal probe binding complementarily to L1 gene region existing in all types of HPV.
In an exemplary embodiment of the present disclosure, the oligonucleotide probe having a base sequence selected from SEQ ID NOS 106-109 or SEQ ID NOS 210-213 may bind complementarily to beta-actin gene as positive control.
In an exemplary embodiment of the present disclosure, the DNA chip may have 8-24 partitioned wells on which the probe can be spotted.
In an exemplary embodiment of the present disclosure, the concentration of the oligonucleotide probe may be at least 38 pmol.
In an exemplary embodiment of the present disclosure, C6 amine-modified dideoxythymidine may be attached to the oligonucleotide probe as a linker so as to spot the oligonucleotide probe on a superaldehyde-coated support.
In an exemplary embodiment of the present disclosure, the support may be selected from a group consisting of glass slide, paper, nitrocellulose membrane, microplate well, plastic, silicon, DVD and bead.
In an exemplary embodiment of the present disclosure, the sample may be selected from a group consisting of cervical swab, vaginal swab, cervical tissue, penile tissue, urine, anal tissue, rectal tissue, pharyngeal tissue, oral tissue and head and neck tissue.
In an exemplary embodiment of the present disclosure, the sample may be selected from a group consisting of penile cancer cell, urethral cancer cell, anal cancer cell, head and neck cancer cell and precancerous cells thereof
In an exemplary embodiment of the present disclosure, the DNA chip may be used to determine whether HPV vaccine will be administered.
In another general aspect, the present disclosure provides a kit for genotyping HPV, including the DNA chip, a primer for amplifying a target gene by PCR and a label for detecting the amplified DNA.
In an exemplary embodiment of the present disclosure, the primer may be a primer for amplifying human beta-actin gene having a base sequence selected from SEQ ID NOS 1-2 or a primer for amplifying HPV L1 gene having a base sequence selected from SEQ ID NOS 3-5.
In an exemplary embodiment of the present disclosure, the label the may be one or more selected from a group consisting of Cy3, Cy5, Cy5.5, BODIPY, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 660, rhodamine, TAMRA, FAM, FITC, Fluor X, ROX, Texas Red, Orange Green 488X, Orange Green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, BODIPY 630/650, BODIPY 650/665, Calfluor Orange 546, Calfluor Red 610, Quasar 670, biotin, Au, Ag and polystyrene.
In another general aspect, the present disclosure provides a method for genotyping HPV, including:
(a) amplifying a target gene of a sample by single, duplex or nested PCR using a primer having a base sequence selected from SEQ ID NOS 1-5;
(b) labeling an oligonucleotide probe of a DNA chip;
(c) hybridizing the labeled probe with the amplified PCR product; and
(d) detecting the hybridized product.
In an exemplary embodiment of the present disclosure, the labeling in (b) may be performed by labeling the oligonucleotide probe with a label selected from a group consisting of Cy3, Cy5, Cy5.5, BODIPY, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 660, rhodamine, TAMRA, FAM, FITC, Fluor X, ROX, Texas Red, Orange Green 488X, Orange Green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, BODIPY 630/650, BODIPY 650/665, Calfluor Orange 546, Calfluor Red 610, Quasar 670 and biotin.
In an exemplary embodiment of the present disclosure, the labeling in (b) may be performed by labeling a target probe first with an Au nanoparticle and then with silver staining and binding the target probe complementarily to the oligonucleotide probe of the DNA chip.
In an exemplary embodiment of the present disclosure, the labeling in (b) may be performed by labeling a target probe first with an Au nanoparticle and then forming a silver shell and binding the target probe complementarily to the oligonucleotide probe of the DNA chip.
In an exemplary embodiment of the present disclosure, the target probe may have a base sequence selected from SEQ ID NOS 214-215 and C18 linker, A10 and thiol group may be sequentially attached at the 3′-terminal.
In an exemplary embodiment of the present disclosure, the genotyping method may further include analyzing using plasmid vectors in which L1 genes of the 65 types of HPV described in Table 1 are inserted as positive control clones.
In an exemplary embodiment of the present disclosure, the sample may be selected from a group consisting of cervical swab, vaginal swab, cervical tissue, penile tissue, urine, anal tissue, rectal tissue, pharyngeal tissue, oral tissue and head and neck tissue.
In an exemplary embodiment of the present disclosure, the sample may be selected from a group consisting of penile cancer cell, urethral cancer cell, anal cancer cell, head and neck cancer cell and precancerous cells thereof.
The oligonucleotide probe for genotyping HPV, the DNA chip and the diagnosis kit including same and the method for genotyping HPV according to the present disclosure were completed in nine steps as follows.
1. Preparation of Standard and Control Samples
The inventors of the present disclosure performed PCR for L1, L2 and E61E7 genes of HPV for about 16,000 cervical samples from Korean women and analyzed the base sequence of all the PCR products. Based on these data, and referring to the reports from the US and other countries, they determined the HPV types that should be included in a new HPV DNA chip. The number of the types was 43 and, thus, they invented a DNA chip capable of analyzing all the 43 types of genital HPVs.
2. Isolation of DNA
DNA was isolated from the samples obtained in the step 1 using an adequately established method.
3. Duplex PCR
Oligonucleotide primers for amplifying HPV L1 gene and human beta-actin gene were designed and adequate PCR condition was established. PCR was performed in duplex and condition was established for each gene for different primer concentrations. PCR was performed for HPV L1 gene and human beta-actin gene using the DNA isolated in the step 2 as template.
4. Sequencing and Cloning
After the PCR, base sequence of the HPV L1 gene was analyzed by sequencing and a database was made based on the result. The PCR product whose HPV type was identified was cloned into a plasmid vector. Later, the clones were used as standard and control samples during the establishment of reaction condition for the DNA chip of the present disclosure. The clinical DNA samples whose HPV genotype was identified were stored and used for accuracy analysis of the DNA chip of the present disclosure.
5. Probe Design
Based on the sequence database built in the step 4 by genotyping HPV from cervical cells and cancer tissues of Koreans and foreign HPV-related databases, an oligonucleotide probe complementary to L1 gene of all the 43 types of HPV that can infect human cervix and human beta-actin gene and capable of detecting them through hybridization on the DNA chip was designed. Also, a d-shaped oligonucleotide probe having a stem part was designed.
6. Fabrication of DNA Chip
A grid on which the probe designed in the step 5 will be spotted was designed and the probe mixed with an adequate buffer was spotted (or arrayed) on a glass slide for a microscope. The resulting DNA chip was subjected to stabilization and quality control.
7. Establishment of Reaction and Analysis Condition on DNA Chip
HPV L1 and beta-actin genes were amplified by duplex PCR using various combinations of one, two or three clones for each type of HPV obtained in the step 4 as templates. The PCR products were placed on the DNA chip and hybridization was performed several times. Then, the optimal condition was established by analyzing with a fluorescence scanner.
8. Analysis of Clinical Sample on DNA Chip
The DNA of the clinical samples of which the presence and type of HPV were identified in the steps 3 and 4 by PCR and sequencing was subjected again to duplex PCR. The PCR product was placed on the DNA chip fabricated in the step 6 and subjected to hybridization under the condition established in the step 7. After washing, the result was analyzed using a fluorescence scanner. Through this, sensitivity, specificity and reproducibility of the DNA chip of the present disclosure were analyzed and the optimal condition for diagnosis of HPV genotype using the DNA chip of the present disclosure was established again.
9 Analysis of Correlation with Clinical Data Following Analysis of Clinical Sample on DNA Chip
The result of post-PCR DNA chip analysis in the step 8 was compared with clinical data such as those of Pap smear and their correlation was investigated. It was analyzed whether the DNA chip of the present disclosure is useful in predicting cervical cancer or precancerous lesions. As a result, it was confirmed that the DNA chip of the present disclosure is useful not only in genotyping of HPV but also in screening of cervical cancer.
A diagnosis kit using the DNA chip of the present disclosure provides 1) a reagent for extracting DNA from a sample such as cervical swab, paraffin section, etc., 2) a reagent for amplifying HPV L1 and beta-actin genes by PCR, 3) a plasmid DNA clone used as positive control during the amplification of HPV gene, 4) the oligo DNA chip for genotyping HPV and 5) a reaction solution for hybridization using the DNA chip and a washing solution “all in one”.
In accordance with the present disclosure, all the 44 types of HPV invading the genitalia can be detected and coinfection by more than one type of HPV can be diagnosed accurately. The sensitivity and specificity of HPV genotyping is close to 100% and a number of samples can be tested quickly. The present disclosure is very useful in predicting cervical cancer and precancerous lesions.
In particular, the DNA chip for genotyping HPV according to the present disclosure and the kit using same are very useful in large-scale automated diagnosis of infection of samples such as cervical swab, vaginal swab, urine, anal tissue, oral tissue, pharyngeal tissue, etc. by HPV and genotyping thereof. Also, they may be used together with Pap smear or alone to screen cervical cancer and precancerous lesions thereof, reducing cost, labor and time of test. Also, they are useful for customized vaccination since the genotype of HPV can be analyzed accurately.
Accordingly, the present disclosure will contribute greatly to the improvement of health and well-being by reducing HPV-related cancers and deaths caused thereby and is very valuable in medical industry.
Hereinafter, the present disclosure will be described in more detail through examples. But, the present disclosure is not limited by the following examples.
Samples to be used as standard materials were prepared and DNA was extracted therefrom.
As a first sample, human cervical cancer cell of which infection by HPV and type thereof are identified and which have been widely used in HPV genotyping studies was purchased from ATCC (Manassas, Va.20108, USA) and Korea Cell Line Bank (KCLB; Seoul National University Cancer Research Institute, Korea) and used after monolayer culturing. Genomic DNA was isolated therefrom.
A second sample was obtained from the CIN cervical tissue of 100 Korean women who were diagnosed as cervical cancer or carcinoma in situ. Formalin-fixed and paraffin-embedded tissues were cut into 5-10 sections of 10-μm thickness, and attached to a glass slide for a microscope. Then, only the cancer cells were microdissected. Among the 100 cervical cancer lesions, 98 were cervical intraepithelial neoplasm (CIN).
As a third sample, cervical samples were obtained from 15,708 women who visited Hamchun Diagnosis Center (Seoul, Korea) or Korea Gynecologic Cancer Foundation (Seoul, Korea) from 2005 to 2007 and received cervical swab and Pap smear test. Their age was between 16 and 80 years and the average age was 47 years.
DNA was isolated from the samples as follows.
To extract DNA from the cells, cervical swab samples and paraffin section samples, DNA was concentrated and purified using the Labo Pass™ tissue mini kit (CME0112, Cosmo Genetech, Korea). Details are as follows.
A. Isolation of Genomic DNA from Cells
Monolayer cultured cells were isolated and introduced into a 50-mL centrifuge tube. After centrifugation at 3500 rpm for 30 minutes, the supernatant was discarded and pellets were resuspended in 500 μL of PBS and transferred to a 1.5-mL centrifuge tube. After centrifugation again at 12,000 rpm for 2 minutes, the remaining medium was removed by washing and genomic DNA was obtained.
B. Isolation of Genomic DNA from Cervical Swab Sample
1) 1.5 mL of sample solution is transferred to a 1.5-mL centrifuge tube. Cells are settled by centrifuging at 13,500×g for 2 minutes.
2) The supernatant is removed and 500 μL of PBS is added.
3) The cells are mixed well with the solution using a vortex.
4) After centrifugation at 13,500×g for 2 minutes, the supernatant is removed.
5) 200 μL of TL buffer is added.
6) After adding 20 μL of proteinase K, the mixture is mixed well using a vortex.
7) Reaction is performed for 30 minutes in a constant-temperature water bath at 56° C.
8) After the reaction is completed, centrifugation is performed at 6,000×g or higher for about 10 seconds.
9) After adding 400 μL of TB buffer, the mixture is mixed well. Then, centrifugation is performed at 6,000×g or higher for about 10 seconds.
10) The reaction solution is added to a spin column mounted at a collection tube.
11) Centrifugation is performed at 6,000×g for 1 minute.
12) The filtrate that has passed through the column is discarded and a new collection tube is mounted.
13) After adding 700 μL of BW buffer, centrifugation is performed at 6,000×g for 1 minute.
14) The filtrate that has passed through the column is discarded and a new collection tube is mounted.
15) After adding 500 μL of NW buffer, centrifugation is performed at 13,500×g for 3 minutes.
16) The filtrate that has passed through the column is discarded and a new 1.5-mL tube is mounted.
17) After adding 200 μL of AE buffer or purified water, the column is allowed to stand at room temperature for 2 minutes.
18) Centrifugation is performed at 6,000×g for 1 minute.
19) The extracted genomic DNA can be directly used in PCR or may be stored at −20° C. for later use.
20) The extracted genomic DNA may be electrophoresed on 0.8% agarose gel and detected by UV.
C. Isolation of Genomic DNA from Paraffin-Embedded Sample
1) Paraffin-embedded sample is sliced to 20 μm thickness using a microtome or a surgical knife.
2) The sample is transferred to a 1.5-mL tube.
3) After adding 1.2 mL of xylene, the mixture is strongly mixed for 2 minutes using a vortex.
4) After centrifugation at 13,500×g for 5 minutes, the supernatant is removed.
5) After adding 1.2 mL of ethanol, the mixture is strongly mixed for 2 minutes using a vortex
6) After centrifugation at 13,500×g for 5 minutes, the supernatant is removed.
7) The procedure of 3)-5) is repeated to completely remove paraffin.
8) The tube holding the sample is allowed to stand at 37° C. for 15 minutes so that ethanol may be evaporated.
9) 200 μL of TL buffer is added to the sample in the tube.
10) After adding 20 μL of proteinase K, the mixture is mixed well using a vortex. 11) Reaction is performed in a constant-temperature water bath of 56° C. for 30 minutes.
12) After adding 400 μL of TB buffer, the mixture is mixed well. Centrifugation is performed at 6,000×g or higher for about 10 seconds.
13) The reaction solution is added to a spin column mounted at a collection tube.
14) Centrifugation is performed at 6,000×g for 1 minute.
15) The filtrate that has passed through the column is discarded and a new collection tube is mounted.
16) After adding 700 μL of BW buffer, centrifugation is performed at 6,000×g for 1 minute.
17) The filtrate that has passed through the column is discarded and a new collection tube is mounted.
18) After adding 500 μL of NW buffer, centrifugation is performed at 13,500×g for 3 minutes.
19) The filtrate that has passed through the column is discarded and a new 1.5-mL tube is mounted.
20) After adding 200 μL of AE buffer or purified water, the column is allowed stand at room temperature for 2 minutes.
21) Centrifugation is performed at 6,000×g for 1 minute.
22) The extracted genomic DNA can be directly used in PCR or may be stored at −20° C. for later use.
23) The extracted genomic DNA may be electrophoresed on 0.8% agarose gel and detected by UV.
Plasmid DNA clone of HPV L1 gene which would serve as standard material in the following genotyping and analysis was prepared.
First, DNA was extracted from human cervical cancer cell and PCR product of HPV L1 gene was obtained. Second, PCR product of L1 gene of 42 types of HPV was obtained from Korea Food & Drug Administration (KFDA). Third, PCR product of HPV was obtained from cervical cancer tissues of 100 Korean women and cervical swab samples of 15,708 women. After genotyping HPV L1 gene by post-PCR sequencing, the PCR product was cloned to the pGEM-T Easy vector to acquire L1 clones for each HPV genotype. The clones were used as standard and control samples in the establishment of the reaction condition of the DNA chip of the present disclosure. The cloning was performed as follows.
1) The amplified PCR products of L1 gene were isolated using a gel recovery kit (Zymo Research, USA) on agarose gel and the concentration was measured using a spectrophotometer or on agarose gel.
2) pGEM-T Easy vector (Promega, A1360, USA) and 2x rapid ligation buffer that had been stored at −20° C. were melted and mixed slightly by shaking the tube slightly with fingers. After centrifugation at low speed, followed by mixing with insert DNA to be cloned with the following ratio, the mixture was added to a 0.5-mL tube for ligation reaction.
3) After mixing the reaction solution well with a pipette, ligation was performed at room temperature for about an hour. When a large quantity of products were desired, the reaction was performed at 4° C. overnight.
4) Thus ligated sample was transformed with 50 μL of JM109 competent cell (=1×108 cfu/μg DNA) stored at -70° C.
5) 2 μL of the ligated product was added to a 1.5-mL tube and 50 μL of the competent cell was added after thawing in ice bath immediately before the addition. After mixing well, reaction was carried out on ice for 20 minutes.
6) After applying heat shock for 45-50 seconds in a constant-temperature water bath at 42° C., the tube was immediately allowed to stand in ice bath for 2 minutes.
7) After adding 950 μL of SOC medium set to room temperature, the tube was incubated in a shaker at 37° C. for about 1.5 hours.
8) About 100 μL of the culture was applied on LB/ampicillin/IPTG/X-Gal plate. After reversing the plate and incubating in a shaker at 37° C. for about 16-24 hours, colony counting was carried out. Then, only the white colony was selected and cultured in 3 mL of LB/ampicillin broth. Plasmid DNA was miniprepared and it was checked whether the insert DNA was correctly inserted by PCR or using restriction enzymes. For more accurate analysis, all the clones obtained were analyzed using an automated base sequencer. Positive control clones are described in Table 1.
HPV L1 gene and human beta-actin gene as internal control gene were amplified to investigate the genotype of HPV.
For PCR amplification, oligonucleotide primers were selected and designed first. The primers include MY11, GP6-1 and GP6+primers (SEQ ID NOS 1-3) for detecting the HPV L1 gene and ACTB F (forward) and ACTB R (reverse) primers of human beta-actin gene for confirming DNA extraction and. PCR efficiency. The GP6-1, ACTBF and ACTBR primers were designed by the inventors and the other primers were selected from previously known primers. The PCR product of the HPV L1 gene is 185 by in length and that of the beta-actin gene is 102 by long. The base sequence of the PCR primers for each gene is described in Table 2.
Optimal condition for duplex PCR was established and PCR of HPV L1 and human beta-actin genes was performed using the DNA isolated in Example 2 as template. Details are as follows.
A PCR reaction solution for detecting HPV infection was prepared by adding 1 μL (10 pmol) of MY11 primer, 1 μL (8 pmol) of GP6-1 primer, 1 μL (8 pmol) of GP6+ primer, 1 μL (5 pmol) of ACTBF primer and 1 μL (5 pmol) of ACTBR primer to 15 μL of SuperTaq Plus pre-mix (10× buffer 2.5 μL, 10 mM MgCl2 3.75 μL, 10 mM dNTP 0.5 μL, Taq polymerase 0.5 μL) purchased from Super Bio (Seoul, Korea), as described in Table 2. 4 μL (150 ng/μL) of template DNA of the sample was added and the total volume of the reaction solution was adjusted to 30 μL by adding distilled water.
For Duplex PCR, the reaction solution containing each primer was predenatured at 95° C. for 5 minutes and 40 cycles of 95° C. for 30 seconds, 50° C. for 30 seconds and 72° C. for 30 seconds were repeated. Then, extension was carried out at 72° C. for 5 minutes.
The result is shown in
The PCR result for HPV L1 gene for 15,708 cervical clinical samples is given in Table 3. 7,371 samples exhibited positive results. Particularly, HPV-11 or HPV-56 which could not be amplified by the GP6-1 primer could be amplified by the GP6+ primer. Also, non-specific PCR that may occur when the DNA concentration is too low could be overcome through the duplex PCR. Based on this result, the HPV genotype DNA chip of the present disclosure could be designed.
Non-specific chip reaction that may occur in single PCR when the DNA concentration of HPV-negative sample is low could be overcome through the duplex PCR according to the present disclosure. For comparison, the product of single PCR performed using the existing HPV DNA genotyping chip (L1 gene probe & HBB gene probe) for 43 types of HPV and with the product of duplex PCR performed according to the present disclosure were respectively subjected to chip reactions and the chip images were compared after scanning (see
After the PCR in Example 3, automated sequencing analysis of the PCR product was carried out to analyze the base sequence of HPV L1 and a database was built based on the result. In addition, the clinical DNA samples whose HPV genotype was confirmed were stored and used for analysis of accuracy of the DNA chip of the present disclosure. The sequencing reaction was carried out using the ABI 3130XL sequencer and BigDye Terminator v 2.0 according to the known method.
First, 100 paraffin-embedded cervical cancer tissue samples and 50 normal cervical tissue samples were subjected to HPV genotyping using the DNA chip of the present disclosure and by sequencing. As a result, high-risk type HPV was found in 98 out of the 100 cervical cancer tissue samples. In contrast, no high-risk type HPV was found in the normal cervical tissue samples (Table 4).
That is to say, high-risk type HPV was found in 98 out of the 100 cervical cancer tissue samples (98%) as a result of the DNA chip analysis. Among them, 42 samples were HPV-16, 18 samples were HPV-58, 14 samples were HPV-31, 5 samples were HPV-18, 5 samples were HPV-35 and 5 samples were HPV-33. These 7 types accounted for 98%. In contrast to the DNA chip of the present disclosure, only 89 samples (90.8%) could be identified by PCR sequencing. Especially, mixed infection could not be detected with PCR sequencing. This result indicates that the HPV DNA chip of the present disclosure is useful in predicting the pathological condition of the cervix and, particularly, in screening of cervical cancer and carcinoma in situ. Further, it was confirmed again that the mixed HPV infection undetectable with sequencing can be accurately detected.
In order to design oligonucleotide probes to be positioned on the DNA chip, the huge database containing information regarding the base sequence of L1 gene of the 98 types of HPV identified from the benign and malignant cervical samples of Korean women by post-PCR sequencing in Examples 4-5 and the US HPV database were analyzed. Also, intra-variant base sequences present in each gene were analyzed according to HPV genotype and frequency thereof for each human race. As a result, 43 types of genital type HPV invading the cervix were selected and oligonucleotide probes for genotyping them were designed (Table 5).
The oligonucleotide probes were designed as genotype-specific probes capable of specifically binding to the HPV L1 gene DNA of the 43 types of HPV.
Based on (1) HPV database of the US National Center for Biotechnology Information (NCBI), (2) US Los Alamos HPV database and (3) the database of the 45 types of HPV detected from the cervical samples of Korean women in Example 4, genomic DNA base sequences of a total of 79 types of HPV: HPV-1a, -2a, -3, -4, -5, -6b, -7, -8, -9, -10, -11, -12, -13, -15, -16, -16r, -17, -18, -19, -20, -21, -22, -23, -24, -25, -26, -27, -28, -29, -30, -31, -32, -33, -34, -35, -35h, -36, -37, -38, -39, -40, -41, -42, -44, -45, -47, -48, -49, -50, -51, -52, -53, -54, -55, -56, -57, -58, -59, -60, -61, -63, -65, -66, -67, -68a, -68b, -70, -72, -73, -75, -76, -77, -80, -90, -91, MM4(82), MM7(83), MM8(84) and CP8304 were obtained. Based on the obtained DNA sequences, phylogenetic tree was drawn using the computer program DNASTAR (MegAlign™ 5, DNASTAR Inc.) according to the ClustalW method (pairwise alignment and multiple sequence alignment). After screening genotype-specific base sequences for each group, genotype-specific probes were designed using the computer program Primer Premier 5 (Premier Biosoft International Co.).
110 genotype-specific oligonucleotide probes were designed first by setting probe lengths to 20±2 and 18±2 bp. In the HPV DNA chip and diagnosis kit according to the present disclosure, the DNA probes target a total of 43 HPV L1 genes including 14 high-risk type HPV L1 genes, 22 low-risk type HPV L1 genes and 7 moderate-risk type HPV L1 genes. The high-risk type HPVs include HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52 HPV-56, HPV-58, HPV-59, HPV-68, HPV-82, HPV-26, HPV-53, HPV-66, HPV-67, HPV-69, HPV-70 and HPV-73, and the low-risk type HPVs include HPV-6, HPV-11, HPV-34, HPV-40, HPV-42, HPV-43, HPV-44, HPV-54, HPV-55 HPV-61, HPV-62, HPV-72, HPV-81, HPV-90, HPV-10, HPV-27, HPV-30, HPV-32, HPV-57, HPV-83, HPV-84 and HPV-91.
Virtual binding ability of the 110 probes designed above to the 76 different types of HPV was analyzed using the computer program Amplify 1.2 (University of Wisconsin). Probes for HPV-16, HPV-58, HPV-31 and HPV-33 that are common to Koreas and closely related to cervical cancer were designed. Next, probes capable of specifically binding to HPV-18, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52 HPV-56, HPV-58, HPV-59, HPV-68, HPV-82, HPV-26, HPV-53, HPV-66, HPV-67, HPV-69, HPV-70, HPV-73, HPV-6, HPV-11, HPV-34, HPV-40, HPV-42, HPV-43, HPV-44, HPV-54, HPV-55 HPV-61, HPV-62, HPV-72, HPV-81, HPV-90, HPV-10, HPV-27, HPV-30, HPV-32, HPV-57, HPV-83, HPV-84 and HPV91 were selected. The name, SEQ ID NO and type of the linear oligonucleotide probes are summarized in Table 5.
A d-shaped oligonucleotide probe having a stem structure was designed. The d-shaped probe of the present disclosure comprises, in 5′→3′ direction and from left top to right top, (1) a left stem part, (2) a linker part, (3) a right stem part and (4) a right probe part (see
(1) Stem Part
For the d-shaped probe of the present disclosure to be structurally stable, a stem part supporting the probe should be adequately designed. The stem part comprises oligonucleotides having complementary sequences bound to each other. For strong binding, the stem part should comprise C and G bases at least in half and T or A base may be inserted therebetween. The stem part may comprise a naturally occurring telomere. At the end of the chromosome of an eukaryotic organism, a telomere consisting of repetitive base sequences exists. The sequence is TTAGGG, TTTAGGG or T1-3(T/A)G3—for mammals including human and TTGGGG or TTTTGGGG for other organisms (Balagurumoothy P, Brahmachari S K, Mohnaty D, Bansal M and Sasisekharan V. Hairpin and parallel quartet structures for telomeric sequences. Nucleic Acids Research. 1992; 20(15): 4061-4067; Balagurumoothy P and Brahmachari S K. Structure and stability of human telomeric sequence. Journal of Biochemistry. 1994; 269(34): 21858-21869). Accordingly, the stem part of the d-shaped probe of the present disclosure may comprise at least one repeating base selected from the following on one strand.
e.g.)
That is to say, 5-9 oligonucleotides may bind complementarily, and the number of the oligonucleotides can be increased further. In terms of cost and efficiency, the human telomere comprising the nucleotide sequence TTAGGG-AATCCC may be used as the repeating unit. However, the length can be changed variously.
(2) Linker Part
In the present disclosure, amino-modified dideoxythymidine (internal amino modifier CndT; iAmMCnT) with n ranging from 3 to 60 is inserted. In terms of economic efficiency, short iAmMC6T having 6 carbons may be used. At the 5′-terminal of iAmMC6dT, the modified C6 amine linker of the left stem part binds with the aldehyde group coated on the glass slide surface. The base A of the 3′-terminal binds with the base T of the 5′-terminal of the right stem part. The d-shaped probe may be fixed on a chip via binding to the ribose of the iAmMC6dT.
(3) Right Probe Part
The right probe part is designed to be complementary to the target gene to be detected. Any base sequence is possible, but the oligonucleotide sequence and length of the right probe part should be adequately designed. The probe part should be selected such that a secondary structure is not formed. The right probe part may be usually about 15-75 by in length, but the length may be increased to about 150 by or decreased to shorter than 15 by depending on situations. If the sample is a PCR product as in the present disclosure and if it is desired not only to detect HPV infection but also to analyze the accurate type and subtype thereof, the probe length may be about 20 by and it is designed such that the difference in at least three nucleotides at the center portion is discernible.
Grid was designed corresponding to the probes designed in Example 6 and the probes mixed with a suitable buffer were spotted on a glass slide for a microscope. Then, the slide was stabilized with suitable treatment and stored until test after quality control. Details are as follows.
1. Preparation of Grid to be Position on DNA Chip
A grid was prepared so as to determine quickly and easily whether the HPV detected on the chip is high-risk type, moderate-risk type or low-risk type as shown in
In addition to the human beta-actin gene, globin or glyceraldehyde-3-phosphate dehydrogenase gene may be used as standard marker probe.
Each oligonucleotide probe was spotted using an arrayer. The same probes were spotted in duplicate in order that each genotype of HPV is detected at least twice.
2. Preparation of Solution for Spotting Oligonucleotide Probes on Chip and Transfer to Master Plate
Probes synthesized by attaching 5′-C6 amine in Example 6 were purified by high-performance liquid chromatography (HPLC) and dissolved in sterilized triply distilled water to a final concentration of 200 pM. Thus prepared probes were mixed with 4.3 times the volume of a microspotting solution to make the final concentration 38 pM. The resulting mixtures were sequentially transferred to a 384-well master plate.
3. Spotting and Fixation of Probes
Q arrayer2 (Genetixs, UK) or an arrayer comparable thereto was used to transfer the spotting solution containing the probes from the master plate to an aldehyde-coated glass slide and spot each probe in duplicate (double hit). The glass slide may be Luminano Aldehyde LSAL-A, a silicon wafer or a product comparable thereto. Each spot can be 10-200 μm in size. The DNA chip fabricated by spotting the probes onto the glass slide was reacted at room temperature for 15 minutes in a glass jar maintained at 80% humidity and then post-treated according to a known method (Zammatteo, N., L. Jeanmart, S. Hamels, S. Courtois, P. Louette, L. Hevesi, and J. Remacle. 2000. Comparison between different strategies of covalent attachment of DNA to glass surfaces to build DNA microarrays. Anal. Biochem. 280: 143-150.).
4. Washing and Storage of Microarray
A. Preparation of Reagent
1) 10% sodium dodecyl sulfate (SDS; 100 mL): 10 g of SDS (Sigma, L4509-1KG) reagent is weighed into a 500-mL beaker. After adding distilled water (ultrapure water) to make a final volume 100 mL and dissolving, the solution is kept in a sealed container at room temperature.
2) 0.1% SDS (4 L): 10 mL of 10% SDS is added to four respective 1-L containers. After adding distilled water (ultrapure water) to make a final volume 1 L and mixing, the solutions are kept in a sealed container at room temperature.
3) 1 M ethanolamine solution (300 mL): 18.3 mL of 16.6 M ethanolamine solution (Sigma, E0135) is added to a 500-mL container. After adding distilled water (ultrapure water) to make a final volume 300 mL and mixing, the solution is kept in a sealed container at room temperature. Light is blocked since the solution sensitive to light.
4) Blocking solution (425 mL): A blocking solution is prepared immediately before use. 1× PBS (300 mL) is mixed with 100% ethanol (100 mL) and 1 M ethanolamine (25 mL).
5) 1× phosphate buffer: Five PBS buffer tablets (Sigma, P4417) are dissolved by adding 0.9 L of distilled water (ultrapure water). After adjusting pH to 7.4 with 10 N HCl, the final volume is adjusted to 1 L.
6) 25% ethanol solution: 250 mL of 100% ethanol (Merck, 1.00983.2511) is added to 1-L container. After adding distilled water (ultrapure water) to make a final volume 1 L, the solution is kept in a sealed container at room temperature.
B. Washing of Microarray
1) A reactor, a washing container and reagents (0.1% SDS, 1 M ethanolamine, 1× phosphate buffer, 100% ethanol and 25% ethanol) are prepared.
2) 300 mL of 0.1% SDS solution is added to the washing container and the slide is washed for 2 minutes at 150 rpm using a reciprocating shaker. This procedure is repeated twice.
3) The slide is washed for 2 minutes at 150 rpm with triply distilled water using a reciprocating shaker. This procedure is repeated twice.
4) Electrically preheated distilled water is added to a washing container dedicated for distilled water and the chip is kept in the water for 3 minutes.
5) The chip is kept in triply distilled water at room temperature for 1 minute.
6) A blocking solution is prepared immediately before use.
7) The chip is kept in the blocking solution for 30 minutes.
8) 300 mL of 25% ethanol solution is added to a washing container and the slide is washed for 2 minutes at 150 rpm using a reciprocating shaker. This procedure is carried out only once.
9) The slide is washed for 2 minutes at 150 rpm with triply distilled water using a reciprocating shaker. This procedure is repeated twice.
10) After washing is completed, the chip is slowly lifted from the last washing solution (water).
11) Water is removed by centrifuging at 1,000 rpm for 3 minutes (MF-600, Hanil Science). 12) The slide is put in a slide box and stored in a desiccator until use.
The DNA chip of the present disclosure fabricated above was used to perform hybridization as described in Example 8.
100 artificial standard samples obtained from various combinations of one, two or three clones for each type of HPV in Example 5 were used as templates for PCR amplification of HPV L1 and beta-actin genes. The PCR products were placed on the chip prepared in Examples 6-7 and hybridization was performed at least 3 times. Then, the optimal condition was established by analyzing with a fluorescence scanner Details are as follows.
1. Duplex PCR
PCR of HPV L1 and human beta-actin genes was performed as in Example 3. For a reverse primer among the combination of primers, i.e. GP6−1, GP6+ and ACTBR, Cy-5-labeled oligonucleotide was used.
The label may be replaced by Cy3, Cy5, Cy5.5, BODIPY, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 660, rhodamine, TAMRA, FAM, FITC, Fluor X, ROX, Texas Red, Orange Green 488X, Orange Green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, BODIPY 630/650, BODIPY 650/665, Calfluor Orange 546, Calfluor Red 610, Quasar 670, biotin or AuNP (gold nanoparticle having a diameter of 5 nm, 10 nm, 20 nm or 50 nm). Also, silver core shell or silver enhancement may be used. In particular, when AuNP or silver core shell is used as the label, a target probe having a thiol group at 3′-terminal and thus capable of complementarily binding to the PCR template is attached for hybridization with the gold nanoparticle and silver enhancement is carried out or a silver shell is formed on the target probe bound to the gold nanoparticle. After the reaction, reflectivity of the chip is measured using a PD scanner, not a general fluorescence scanner using PMT as a detector, or SEM images are taken for detection.
2. Hybridization Reaction
Hybridization reaction is carried out after placing the HPV PCR products amplified by PCR on a slide substrate on which various HPV oligonucleotide probes are immobilized. A 100-μL 8-well perfusion chamber (Schleicher & Schuell BioScience, Germany) is used as a hybridization chamber. Details are as follows.
1) Fresh 1.5-mL or 200-μL tubes are prepared corresponding to the number of samples.
2) 50 μL of purified water is added to each tube.
3) 15 μL of the duplex PCR products of L1 and ACTB genes are added and mixed well.
4) The tube is allowed to stand on a heat block maintained at 95° C. for 3 minutes.
5) The tube is then allowed to stand on ice for 5 minutes.
6) The reaction tube is centrifuged for 30 seconds.
7) 65 μL of HYB I solution (2 mL of 20×SSC, 6.3 mL of 5× phosphate buffer and 1.7 mL of 90% glycerol, final volume: 10 mL) is added to the tube and mixed well with a pipette.
8) The prepared reaction solution is slowly injected into the injection port on the coverslip attached to the chip surface. It is checked whether foams are observed between the chip and the well cover. If any, the foams are removed by sweeping with a gloved hand.
9) The chip is subjected to hybridization in a reaction bath at 48° C. for 30 minutes.
3. Washing
1) After the hybridization is completed, the well cover is removed from the chip.
2) Previously prepared washing solution 1 is added to a washing container such that the chip is immersed and the chip is washed at room temperature for 2 minutes with a speed of 8 oscillations using a reciprocating shaker. If the number of the chip is one, it may be washed in a 50-mL conical tube holding 40 mL of washing solution by shaking the tube up and down for 2 minutes at a speed of 50 reciprocations per minute. When the washing is carried out manually without using the reciprocating shaker, washing solution is added to a washing container such that the chip is immersed and the washing container is shaken left and right for 2 minutes at a speed of 50 reciprocations per minute.
3) The used washing solution is discarded and fresh washing solution 1 is added. Washing is performed again for 2 minutes.
4) The used washing solution is discarded and fresh washing solution 1 is added. Washing is performed again for 2 minutes.
5) The used washing solution is discarded and fresh washing solution 2 is added. Washing is performed again for 2 minutes.
6) After the washing, a spin dryer or an air compressor may be used to remove the buffer remaining on the chip.
4. Scanning
After the hybridization followed by removal of non-specific signals through washing, the dried slide was scanned with a scanner to analyze chip images. As for the scanner, Genepix 4000B, Easy Scan-1, Affymetrix 428 Array Scanner (Affymetrix, USA), ScanArray Lite (Packard Bioscience, USA) or an instrument comparable thereto may be used.
Duplex PCR was carried out again as described in Example 3 on the DNA of cervical clinical samples of which the presence or absence of HPV and type thereof were identified by post-PCR sequencing in Examples 3-4. The PCR products were placed on the DNA chip fabricated in Examples 6-7 and hybridization was carried out as in Example 8. After washing, analysis was carried out using a fluorescence scanner. Sensitivity, specificity and reproducibility of the DNA chip were analyzed and the optimal condition of the DNA chip of the present disclosure for genotyping of HPV was evaluated again. The results are shown in
That is to say, the 45 probes specific for the HPV types of the DNA chip bound specifically to the DNA of the respective types of HPV without cross-hybridization between the probes. In addition, the samples coinfected by more than one type of HPV could be accurately diagnosed. That is to say, the DNA chip of the present disclosure exhibited 100% sensitivity and 100% specificity for diagnosis of single and mixed infection by HPV. Further, 100% reproducibility was exhibited as the same results were obtained when different testers carried out the diagnosis three or more times with time intervals. The 45 probes synthesized according to the present disclosure could accurately analyze a large number of combinations of HPV types which could not be handled with the existing DNA microarrays.
In particular,
The DNA chip fabricated according to the present disclosure could accurately diagnose the type of HPV from the cervical swab samples. The probe for each HPV type bound specifically to the DNA of specific type of HPV and no cross-hybridization occurred between the probes. In addition, even the samples coinfected by more than one type of HPV, which are difficult to diagnose through direct sequencing and can be diagnosed by many sequencing assays after cloning, could be accurately diagnosed with the DNA chip of the present disclosure. That is to say, the DNA chip of the present disclosure exhibited 100% sensitivity and 100% specificity for diagnosis of single and mixed infection by HPV. Further, 100% reproducibility was exhibited as the same results were obtained when different testers carried out the diagnosis three or more times with time intervals.
The result of analysis using the DNA chip after PCR in Example 9 was compared with clinical data obtained by cervical tissue testing, Pap smear, etc. in order to analyze their correlation and investigate whether the DNA chip of the present disclosure is useful for predicting cervical cancer or precancerous lesions. It was demonstrated that the DNA chip of the present disclosure is useful not only for genotyping of HPV but also for screening of cervical cancer.
Among the 15,708 cervical cell samples from Korean women, HPV infection was identified in 7,371 samples. The prevalence rate was 463.93%. 45 types of HPV were identified. Among the detected HPV types, HPV-16 was the most common, followed by HPV-53, HPV-39, HPV-56, HPV-58, HPV-52, HPV-70, HPV-84, HPV-18, HPV-68 and HPV-35. This result is distinguished from that of Europe where HPV-16 is the most common, followed by HPV-18, HPV-45, HPV-52, HPV-31, HPV-33 and HPV-58 (Murinoz N et al., N Engl J Med, 2003, 348: 518-27).
HPV-53 showed high prevalence rate in Koreans but not in Europeans. Accordingly, it can be seen that HPV-53 is the major cause of cervical cancer in Koreans.
The HPV DNA chip of the present disclosure was used for diagnosis of cervical samples. The purposes of the test were, first, to investigate how accurately the HPV DNA chip can diagnose HPV infection and the genotype of HPV and, second, to evaluate how helpful it is in predicting cancers and important cervical lesions including precancerous lesions. For this, DNA was isolated from cervical swab samples of Korean women who were suspected of cervical HPV infection and lesions and subjected to (1) test with the HPV DNA microarray of the present disclosure, (2) PCR of the HPV L1 gene followed by automated sequencing analysis, and (3) test by Hybrid Capture Assay-II (HCA-II; Digene Corporation) which is an HPV DNA test approved by the USFDA.
The HPV DNA chip of the present disclosure enables detection of all the 43 HPV types invading human cervix, anus, oral cavity, etc., whereas HCA-II tests 12 high-risk type HPVs. Comparison was made while focusing on (1) the sensitivity and specificity of diagnosis of HPV infection, (2) the accuracy of HPV genotype diagnosis, and (3) the accuracy of prediction of cervical cancer and serious lesions including precancerous lesions. The HPV DNA microarray test was carried out as described in Examples 2 and 8 and PCR and base sequencing were performed according to the known method (Kim K H, Yoon M S, Na Y J, Park C S, Oh M R, Moon W C. Development and evaluation of a highly sensitive human papillomavirus genotyping DNA chip. Gynecol Oncol. 2006; 100(1): 38-43). HCA-II test was performed according to the manufacturer's instructions.
The 201 subjects tested were aged between 18 and 81, and the average age was 52.4 years. The result of performing PCR of the HPV L1 gene is summarized in Table 7. HPV infection was identified from 191 subjects out of the 201 subjects. 149 cases were high-risk HPV and 72 cases were mixed infections by more than one HPV type.
The analysis result with the HPV DNA chip of the present disclosure was compared with that of HCA-II (Tables 7-10). The HPV DNA chip of the present disclosure accurately diagnosed all (100%) the 191 cases of positive HPV infection. Among them, 174 cases (91.1%) were accurately genotyped. Although the 149 high-risk cases were accurately identified, rare types of HPV could not be identified with the chip of the present disclosure. Meanwhile, HCA-II failed to detect 40 cases of HPV from the 191 cases of HPV-positive samples and failed to detect 12 cases (8.1%) from among the 149 high-risk HPV infected samples. The HPV DNA chip of the present disclosure could accurately predict all the high-risk type cervical lesions including cervical cancer, cervical intraepithelial neoplasia (CIN) and high-grade squamous intraepithelial lesion (HSIL). In contrast, the HCA-II test failed to detect one of the 8 cases of cervical cancer and one of the 12 cases of HSIL. In addition, the HPV chip of the present disclosure showed better ability of detecting low-grade SIL than HCA-II (92.2% vs. 56.9%, p<0.05).
These results reveal that the HPV DNA chip of the present disclosure exhibits nearly 100% sensitivity in diagnosis of HPV infection and genotyping of HPV, especially high-risk HPV, and is excellent in predicting cervical cancer and precancerous lesions. Further, it is superior to the existing HCA-II test.
HPV can cause cancer not only in the genitalia but also other in organs and tissues. Indeed, a number of oral cancer, pharyngeal cancer, laryngeal cancer and anal cancer are caused by HPV. Accordingly, the HPV DNA chip of the present disclosure was used to analyze HPV infection in cancer and precancerous lesions. For the experiment, 24 tonsil tissue samples and 179 hemorrhoidal tissue samples obtained from Koreans were tested using the chip of the present disclosure.
Among the 24 tonsil tissue samples, 13 were HPV-positive and 19 were HPV-negative. Of the 13 HPV-positive samples, 5 were single infection and 8 were mixed infection. All the 13 HPV-positive samples were infected by high-risk type HPV (HPV-16: 26%, HPV-56: 13%, HPV-33: 13%, HPV-52: 8%).
The 179 hemorrhoidal tissue samples were acquired from Seoul National University Hospital and Asan Medical Center (19 from females, 160 from males aged between 27 and 83; average age: 40 years). Test using the DNA chip of the present disclosure revealed that 63 samples were HPV-positive, 10 from females and 53 from males. Of the 63 HPV-positive samples, 44 were single infection and 19 were mixed infection. Among the 63 HPV-positive samples, 49 were infected by high-risk type HPV (single and mixed infection) and 14 were infected by low-risk type HPV (HPV-16: 21%, HPV-18: 21%, HPV-68: 8%).
Accordingly, it was confirmed that the DNA chip of the present disclosure can be used to diagnose not only the HPV infection causing cervical cancer but also the HPV infection causing anal or laryngeal cancer.
For hybridization in Example 8, the DNA chip was labeled with gold nanoparticles (AuNP; 20 nm in diameter, BBI) or enhanced with silver shell after PCR. That is to say, a target probe having a thiol group at 3′-terminal and thus capable of complementarily binding to the PCR template is attached for hybridization with the gold nanoparticle and silver enhancement is carried out or a silver shell is formed on the target probe bound to the gold nanoparticle. After the reaction, reflectivity of the chip is measured using a PD scanner, not a general fluorescence scanner using PMT as a detector, or SEM images are taken for detection. Details are as follows.
1. Target Probe Design
A target probe for labeling gold nanoparticles is as follows. If the probes spotted on the chip are in forward direction, the PCR template is usually bound in reverse direction. Thus, a sequence capable of complementarily binding to the PCR template bound to the probes on the chip is designed. That is to say, since the terminal of the PCR template binding to the ACTB probe is usually a reverse primer, the target probe is synthesized to have a sequence complementary to the reverse primer. Because the terminal of the target probe should bind with AuNP (20 nm in diameter), an internal C18 linker and 10 adenine residues were inserted following the complementary base sequence and then a 3′-terminal thiol group was added. Thus designed target probe is shown in Table 11. LTP is the target probe for the PCR product of HPV L1 gene and ATP is the target probe for the PCR product of ACTB gene.
2. Attachment of Gold Nanoparticle to PCR Product
The PCR products bound to the oligonucleotide probes spotted on the chip through hybridization are labeled with AuNP by either of the following two methods (
I. Cleavage of Disulfide Group of Thiol-Modified Oligonucleotide
In order to bind gold nanoparticle with the target probe, the thiol group of the target probe should be activated.
1) The oligonucleotide probes described in Table 11 are quick spun and dissolved by mixing well with 1,517 μL of distilled water.
2) 15.4 mg of 0.1 M DTT is dissolved in 1 mL of disulfide cleavage buffer (pH 8.0; 170 nM phosphate buffer, 11.468 g Na2HPO4, 0.509 g NaH2PO4, 500 mL nanopure water).
3) 100 μL of the 0.1 M DTT solution is added to a 1.5-mL tube, mixed well with 100 μL of dissolved oligonucleotide probes (10 nM) and reacted at room temperature for 2 hours.
4) A NAP-5 column (Sephadex G-25 DNA grade, GE Healthcare, Cat. No. 17-0853-02) is prepared by fixing on a stand.
5) The buffer is discarded and the column is washed by filling with DW using a squeeze bottle. This procedure is repeated 3 times for sufficient washing. Then, the column is capped until use.
6) 200 μL of the reacted oligonucleotide probes are loaded in the NAP-5 column. Caution is taken such that bubbles are not formed in the column. After the solution leaves the column (it takes about 1 minute and 25 seconds), 450 μL of distilled water is added. After the solution leaves the column again (it takes about 1 minute and 28 seconds), four drops are collected in each of seven 1.5-mL tubes while adding 950 μL of DW.
II. Determination of Oligonucleotide Probe Concentration
1) Absorbance of 70 μL of the solutions collected in tubes 1, 2 and 5 is measured at 260 nm using a spectrophotometer.
2) The solutions of tubes 1-5 are mixed in tube 2 and absorbance is measured again.
3) Molar concentration is calculated according to the equation C=A/ε.
4) Oligonucleotide probe concentration and AuNP concentration are calculated from the above equation according to the size of AuNP (e.g. 20 nm or 50 nm).
III. Labeling of Target Probe with AuNP
1) Based on the calculation result, 2 mL of AuNP (20 nm) is added to a 15-mL conical tube. After mixing well with 543 μL of oligonucleotide probes, reaction is carried out for 20 minutes in a shaking incubator set to 25° C.
2) After adding 254.356 μL of 100 mM PBS (Na2HPO4 0.562 g+NaH2PO4 0.125 g+H2O 50 mL), the mixture is incubated for 20 minutes.
3) After adding 2.797 μL of 10% SDS, the mixture is incubated for 20 minutes.
4) After adding 140.035 μL of 2 M NaCl, the mixture is incubated for 20 minutes. This procedure is repeated once more.
5) After adding 70.0179 μL of 2 M NaCl, the mixture is incubated for 20 minutes. This procedure is repeated once more and then the mixture is incubated overnight.
6) The solution is dispensed into two 1.5-mL tubes (1.5 mL each) and centrifuged at 10,000 rpm for 20 minutes. The resulting pellets are resuspended by adding 1 mL of 0.01% SDS solution in 0.3 M PBS (10 mM PB, 40 mL+2 M NaCl, 6 mL). After centrifugation at 10,000 rpm for 20 minutes, the pellet resulting pellets are resuspended by adding 1 mL of 0.3 M PBS (NaCl, 8.766 g+Na2HPO4, 0.562 g, NaH2PO4, 0.25 g+DW, 500 mL) twice (2 mL in total).
3. Labeling with Silver Shell (Core Shell) with Gold Nanoparticle as Seed
The silver shell thickness is determined based on the absorbance of the target probe-AuNP measured in the step 2. Then, the total amount of silver (Ag) and the amount of other reagents are determined from the data of Table 12.
1) After sequentially adding the required amounts of DNA-AuNP, 1% PVP, 10−1 M L-SA and 10−3 M AgNO3 and mixing well, the mixture is incubated overnight while shaking at 150 rpm.
2) The solution is dispensed into a 1.5-mL tube and centrifuged at 8,000 rpm for 20 minutes.
3) The supernatant is removed and 1 mL of 0.3 M PBS is added. After mixing well, centrifugation is carried out again at 10,000 rpm for 20 minutes.
4) After removing the supernatant, 0.3 M PBS is added according to the initial volume of AuNP. If the pellets are not resuspended, the mixture is kept in a water bath at 60° C. and then resuspended.
5) Absorbance of the resuspended DNA-AuNP-core shell is measured (λ=260 nm).
4. Hybridization and Washing
1) AuNP-labeled target probe stored at low temperature is suspended in a water bath of 60° C. 100 μL of the target probe is added on the chip and reacted at room temperature for 4 hours.
2) The chip is washed twice with 0.3 M PBS and then dried.
The result of experiments using the probe of the present disclosure is shown in
As described in the foregoing examples, the HPV DNA chip of the present disclosure is useful for detecting the presence of 43 types of HPV invading human genitalia, anus and head and neck and for genotyping thereof. Further, it is more effective for diagnosis of cervical cancer and precancerous lesions than the existing products.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2010-0057676 | Jun 2010 | KR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/KR2010/004164 | 6/25/2010 | WO | 00 | 3/19/2013 |
