KR19980068858A - PCR primers for simultaneous detection of human papillomavirus types 16 and 18 and diagnostic method for virus infection using the same - Google Patents

Abstract

The present invention relates to a polymerase chain reaction (PCR) primer pair of a specific sequence for simultaneous detection of human papillomavirus (HPV) type 16 and 18, and a method for diagnosing the presence of a vise. The primer pairs of the present invention are specific for primary PCR primer pairs designed and synthesized based on consensus regions of HPV16 and HPV18 to amplify DNA fragments of both HPV16 and HPV18, and HPV16 and HPV18, respectively. It consists of two pairs of primers for the secondary PCR designed and synthesized to amplify DNA fragments of the base sequence while the secondary PCR products are different in size. When the primer pair of the present invention is used for the simultaneous detection of HPV16 and HPV18, four PCRs using four pairs of primers required for conventional nested PCR are not performed, and two PCRs using three pairs of primers are also used. Simultaneous detection is possible, which greatly reduces the procedure and time. Therefore, the first PCR using DNA and primer pair HP1 / HP2 obtained from human serum or tissue, and then the second PCR by adding two pairs of primers HPV161 / HPV162 and HPV181 / HPV182 simultaneously to the first PCR reaction solution. By performing the following procedure, simultaneous infection of HPV16 and HPV18 can be effectively diagnosed.

Description

PCR primers for simultaneous detection of human papillomavirus types 16 and 18 and diagnostic method for virus infection using the same

The present invention relates to a PCR primer (polymerase chain reaction primer) for simultaneous detection of human papillomavirus types 16 and 18 and a method for diagnosing viral infection using the same. More specifically, the present invention can simplify the four pairs of primers and four PCR reactions required until now for the detection of each virus of human papilloma virus type 16 and type 18 with three pairs of primers and two PCR reactions. It relates to a primer of a specific base sequence and a method for diagnosing the infection of the electric virus using the same.

In general, cervical cancer is the most frequent disease among female cancer in Korea, and is reported to be caused by human papillomavirus (hereinafter referred to as 'HPV'). To date, about 70 HPVs have been found, of which HPV16 and 18 are known to be the most important pathogens (Schiffman, M.H. et al., J. Natl. Cancer Inst., 85: 958 (1993)).

To date, for diagnosis of HPV infection, Enzyme linked immunosorbent assay (ELISA) has been reported to test antigens in serum using antibodies to envelope proteins E6 and E7 of HPV16 (Sun Y. et al., J. Clin, Microbiol., 32: 2216 (1994)), animal breeding is required for the production of antibodies, antibody production takes a long time, and it is difficult to secure a large amount of antibodies, so new antibodies should be made whenever needed. Due to various problems such as hassle, the ELISA method is a setting that has not been utilized as a serological test method that can be used normally.

In recent years, polymerase chain reaction (PCR) method, which can rapidly and simply amplify a specific DNA sequence, has been developed and widely used in the field of molecular biology. This genie has also been subjected to PCR in the detection of HPV to solve the problem (Monk BJ et al., Diagn. Mol. Pathol., 3: 283 (1994); Zheng PS et al., Gynecol. Oncol., 53: 179 (1995).

In general, detection of pathogenic microorganisms from clinical samples requires a detection method that provides a high detection sensitivity and reliability, and in order to satisfy the detection using PCR, a second PCR product amplified by the first primer pair is again used. Nested PCR is performed to amplify by primer pairs. Therefore, if the above-described nested PCR is applied to the detection of two viruses of HPV16 and HPV18, two PCRs using two pairs of primers per one virus are required. Therefore, a total of four pairs are used for simultaneous detection of HPV16 and HPV18. Four PCRs using primers should be performed.

Under these technical backgrounds, the present inventors intensively tried to simultaneously detect two viruses, HPV16 and HPV18, which cause cervical cancer. As a result, four procedures using four pairs of primers, which are commonly required for nested PCR, were performed. In order to shorten the time and time, two primers using three pairs of primers were designed and synthesized, which were capable of simultaneously detecting the two viruses of HPV16 and HPV18, and the PCR primer pairs were used as HPV16 and HPV18 standards. As a result of PCR applied to the strain, it was confirmed that HPV16 and HPV18 can be effectively classified and detected, and thus, the present invention was completed.

After all, the first object of the present invention is to provide a pair of PCR primers that can easily detect two viruses of HPV16 and HPV18 at the same time.

It is a second object of the present invention to provide a method for diagnosing infection of HPV16 and HPV18 using an electric primer pair.

1 is a schematic diagram showing the simultaneous detection of HPV16 and HPV18 using the PCR primer of the present invention.

FIG. 2 is a diagram showing the positions of primary and secondary primers for amplifying each DNA fragment characteristic of HPV type and HPV16 and HPV18.

3 is a photograph showing the results of agarose gel electrophoresis of HPV16 and HPV18 specific DNA fragments amplified by primary and secondary PCR.

Hereinafter, the present invention will be described in more detail.

For the simultaneous detection of HPV16 and HPV18 from clinical samples, we design and synthesize primers that can amplify each of the PCR fragments characteristic of HPV16 and HPV18 by simple nested PCR simultaneously, as follows: Both HPV16 and HPV18 In order to amplify DNA fragments of HPV, the sequence of each type of HPV was analyzed, and then the primer pair HP1 / HP2 for primary PCR was designed and synthesized based on the common consensus region of HPV16 and HPV18. do. Subsequently, the following two pairs of primers for secondary PCR, HPV161 / HPV162 and HPV181 / HPV182, are designed and synthesized so as to amplify DNA fragments of nucleotide sequences specific for HPV16 and HPV18, respectively, and have different sizes of secondary PCR products. .

HP1: 5'-CCRYTGCTGYATGYSATAAATGT-3 '

HP2: 5'-CATTTTCGTYCTCGTCATCTGA-3 '

Wherein R is A or G;

Y is C or T; And,

S is G or C.

HPV161: 5'-TCGATGTATGTCTTGTTGCAG-3 '

HPV162: 5'-GGTTACAATATTGTAATGGGC-3 '

HPV181: 5'-CAACATAGCTGGGCACTATAGA-3 '

HPV182: 5'-ACATACACAACATTGTGTGACG-3 '

In order to determine whether the specific DNA fragments of HPV16 and HPV18 are simultaneously amplified by nested PCR using the above-described primer pairs, primer pairs HP1 / HP2 are added to DNA isolated from HPV16 and HPV18 standard strains, and then subjected to the first PCR. The reaction solution was subjected to secondary PCR by adding two pairs of primers HPV161 / HPV162 and HPV181 / HPV182 simultaneously, followed by agarose gel electrophoresis. As a result, it can be seen that the 225 bp DNA fragment, which is the PCR product of HPV16, and the 295 bp DNA fragment, which is the PCR product of HPV18, are simultaneously amplified.

Therefore, when the primer pair of the present invention is used for simultaneous detection of HPV16 and HPV18, two PCRs using three pairs of primers are performed without performing four PCRs using four pairs of primers required for conventional nested PCR. Simultaneous detection is also possible, which greatly shortens the procedure and time.

Finally, the first PCR using DNA and primer pair HP1 / HP2 obtained from human serum or tissue, and then the second PCR by adding two pairs of primers HPV161 / HPV162 and HPV181 / HPV182 simultaneously to the first PCR reaction solution. By performing the following procedure, simultaneous infection of HPV16 and HPV18 can be effectively diagnosed.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 Design of Primers for Simultaneous Detection of HPV16 and HPV18

Primers capable of selectively amplifying a small amount of HPV16 and HPV18 in the sample at the same time were designed by the following procedure: DNA amplification of both HPV16 and HPV18 was performed in the first PCR procedure, where a pair of primers used Was designed based on the common sequence region of HPV16 and HPV18 for simplicity of experiment. Subsequently, selective amplification of HPV16 and HPV18 is performed in the secondary PCR process, wherein each pair of primers is used so that the size of each secondary PCR product is different from each other and amplifies specific sequences capable of distinguishing HPV16 and HPV18. It is designed to. Simultaneous detection of HPV16 and HPV18 using such PCR primers is shown in FIG.

Example 2 Synthesis of Primer for Simultaneous Detection of HPV16 and HPV18

In order to synthesize PCR primers capable of simultaneously detecting HPV16 and HPV18, first, the sequence of each type of HPV having clinical significance reported to date is analyzed, and the common sequence region of HPV16 and HPV18 distinguished from other HPV types. Then, the first PCR primer pair HP1 and HP2 were designed and synthesized as follows. In FIG. 2, (-) in the middle of the sequence represents a deleted base, (*) represents a conserved base, and other positions (-) represent a highly conserved base, box Denotes a portion corresponding to PCR primers used for DNA amplification of HPV16 and HPV18.

HP1: 5'-CCRYTGCTGYATGYSATAAATGT-3 '

HP2: 5'-CATTTTCGTYCTCGTCATCTGA-3 '

Wherein R is A or G;

Y is C or T; And,

S is G or C.

In addition, the secondary PCR primers compare the DNA sequences of HPV16 and HPV18, and primer pairs capable of amplifying base sequences specific to HPV161 / HPV162 and HPV18, which are primer pairs capable of amplifying only HPV16 specific sequences. HPV181 / HPV182 was designed and synthesized.

HPV161: 5'-TCGATGTATGTCTTGTTGCAG-3 '

HPV162: 5'-GGTTACAATATTGTAATGGGC-3 '

HPV181: 5'-CAACATAGCTGGGCACTATAGA-3 '

HPV182: 5'-ACATACACAACATTGTGTGACG-3 '

Example 3: Isolation of DNA from HPV16 and HPV18 Standard Strains

To separate DNA from HPV16 and HPV18 standard strains, each of HPV16 infected CaSki cells (ATCC CRL 1550) and HPV18 infected HeLa cell lines (ATCC CCL 2) were centrifuged at 6,000 rpm for 10 minutes to obtain a precipitate. It was suspended in 100 μl of SE solution (solution consisting of 75 mM NaCl and 25 mM EDTA), and 5 μl of 10% SDS and 1 μl of protease K (20 mg / ml) were added and reacted at 50 ° C. for 2 hours. Then, DNA extraction was performed by extraction with phenol / chloroform and DNA precipitation with alcohol, and suspended in 100 µl of distilled water.

Example 4 Amplification of Specific DNA Fragments of HPV16 and HPV18 by PCR

In order to examine the amplification by PCR of the DNA fragments characteristic of HPV16 and HPV18, 1 μl of the HPV16 / HPV18 DNA-containing solution isolated in Example 3 and the HP1 / HP2 primer pair synthesized in Example 2 were mixed, and at 94 ° C. Pre-denaturation for 5 minutes, followed by 30 times of denaturation (30 seconds at 94 ° C.), annealing (30 seconds at 55 ° C.) and extension (1 minute at 72 ° C.) First PCR was performed.

Subsequently, 1 μl of the completed primary PCR reaction solution and four primers of HPV161 / HPV162 and HPV181 / HPV182 were mixed, premodified at 94 ° C. for 5 minutes, and then denatured (30 seconds at 94 ° C.) and bound (52). A second PCR was performed with 30 seconds) and extension (1 minute at 72 ° C) for a total of 30 seconds.

As a result of the separation of the secondary PCR reaction product thus prepared on an agarose gel, when the HPV16 and HPV18 DNA mixtures were used, the specific PCR product of HPV16 was amplified simultaneously with the 225 bp DNA fragment and the 295 bp DNA fragment, which is particularly the PCR product of HPV18. See lanes 5, 6 and 7 in FIG. 3; When only HPV18 DNA was used, only 295 bp DNA fragment, the specific PCR product of HPV18, was amplified (see lanes 8,9 and 10 in FIG. 3); When only HPV16 DNA was used, it was confirmed that only 226bp DNA fragment, which is a specific PCR product of HPV16, was amplified (Rail 11, 12 and 13 in FIG. 3). In FIG. 3, lane 1 is a DNA size marker, and lanes 2, 3, and 4 are experimental results of a negative control performed by PCR using distilled water.

Therefore, it was found that HPV16 and HPV18 can be detected simultaneously by performing the first PCR using the HP1 / HP2 primer pair and the second PCR using the HPV161 / HPV162 and HPV181 / HPV182 primer pairs.

As described and demonstrated in detail above, when using the primer pair of the present invention for simultaneous detection of HPV16 and HPV18, three pairs of PCR without performing four PCRs using four pairs of primers required for conventional nested PCR Simultaneous detection with two PCRs using primers can greatly reduce the procedure and time. Therefore, the first PCR using DNA and primer pair HP1 / HP2 obtained from human serum or tissue, and then the second PCR by adding two pairs of primers HPV161 / HPV162 and HPV181 / HPV182 simultaneously to the first PCR reaction solution. By performing the following procedure, simultaneous infection of HPV16 and HPV18 can be effectively diagnosed.

Claims (3)

Primary PCR primer pairs of the following base sequences used for simultaneous detection of HPV (human papillomavirus) 16 and HPV18 by nested polymerase chain reaction (NESTED polymerase chain reaction) HP1 / HP2, primer pairs for secondary PCR HPV161 / HPV162 and HPV181 / HPV182: HP1: 5'-CCRYTGCTGYATGYSATAAATGT-3 ' HP2: 5'-CATTTTCGTYCTCGTCATCTGA-3 ' Wherein R is A or G; Y is C or T; And, S is G or C. HPV161: 5'-TCGATGTATGTCTTGTTGCAG-3 ' HPV162: 5'-GGTTACAATATTGTAATGGGC-3 ' HPV181: 5'-CAACATAGCTGGGCACTATAGA-3 ' HPV182: 5'-ACATACACAACATTGTGTGACG-3 ' First PCR (polymerase chain reaction) using DNA and primer pair HP1 / HP2 having the following nucleotide sequence, followed by two pairs HPV161 / HPV162 and the primer having the following nucleotide sequence in the first PCR reaction solution. A method for diagnosing HPV16 and HPV18 simultaneous infection, characterized in that by adding HPV181 / HPV182 simultaneously and performing secondary PCR: HP1: 5'-CCRYTGCTGYATGYSATAAATGT-3 ' HP2: 5'-CATTTTCGTYCTCGTCATCTGA-3 ' Wherein R is A or G; Y is C or T; And, S is G or C. HPV161: 5'-TCGATGTATGTCTTGTTGCAG-3 ' HPV162: 5'-GGTTACAATATTGTAATGGGC-3 ' HPV181: 5'-CAACATAGCTGGGCACTATAGA-3 ' HPV182: 5'-ACATACACAACATTGTGTGACG-3 ' The method of claim 2, A method for diagnosing HPV16 and HPV18 simultaneous infection, wherein the DNA is obtained from human serum or tissue.