Genotyping method   

Abstract: The present invention relates to a genotyping method, and more particularly to an ID sequence, which is assigned to each genotype, and a multiplex genotyping method which uses the ID sequence. When pyrosequencing is performed using the ID sequence, a unique and simple pyrogram can be obtained for each genotype. Thus, the use of the ID sequence makes it possible to genotype viral genes, disease genes, bacterial genes and identification genes in a simple and efficient manner. In addition, a genotyping primer of the invention can be used in various genotyping methods which are performed using dispensation orders and sequencing methods.

TECHNICAL FIELD

The present invention relates to a genotyping method, and more particularly to an ID sequence, which is assigned to each genotype, and a multiplex genotyping method which uses the ID sequence.

BACKGROUND ART

Methods which have been developed for detecting infectious organisms include traditional methods of identifying the physical and chemical characteristics of pathogens by cultivation, and methods of detecting the specific genetic characteristics of pathogens. The methods for detecting genetic characteristics include restriction fragment length polymorphism (RFLP) analysis, amplified fragment length polymorphism (AFLP) analysis, pulsed-field gel electrophoresis, arbitrarily-primed polymerase chain reaction (AP-PCR), repetitive sequence-based PCR, ribotyping, and comparative nucleic acid sequencing. These methods are generally too slow, expensive, irreproducible, and technically demanding to be used in most diagnostic settings. All of the above-mentioned methods generally require that a cumbersome gel electrophoretic step be used, that the pathogen be grown in culture, that its genomic DNA be purified, and that the sample not contain more than one type of organism. These limitations also apply to recently developed detection methods which employ high density microarrays (Salazar et al., Nucleic Acids Res. 24:5056-5057, 1996; Troesch et al., J. Clin. Microbiol. 37:49-55, 1999; Lashkari et al., Proc. Natil. Acad. Sci. U.S.A. 94: 13057-13062, 1997). Meanwhile, pyrosequencing is a method of DNA sequencing based on the “sequencing by DNA synthesis” principle, which relies on the detection of pyrophosphate release on nucleotide incorporation, unlike the traditional Sanger sequencing method. In the pyrosequencing method, four deoxynucleotide triphosphates (dNTPs) are sequentially added one by one during polymerization. PPi attached to the dNTPs being polymerized emit light by enzymatic reactions, and the emitted light shows a signal peak according to the reaction order of each of the sequentially added dNTPs, in which the peak shows a pattern which is high or low in proportion to the number of the reacted dNTPs, such that the nucleotide sequence of the pathogen can be determined. In recent years, methods of detecting pathogenic bacteria or viruses in clinical samples based on pyrograms obtained by pyrosequencing of the PCR products of sequences specific to the pathogens have been used (Travasso, C M et al, J. Biosci., 33:73-80, 2008; Gharizadeh, B et al., Molecular and Cellular Probes, 20, 230-238, 2006; Hoffmann, C et al., Nucleic Acid Research, 1-8, 2007).

In the pyrosequencing technique, however, nucleotide sequencing is performed according to the dispensation order of dNTPs, and a nucleotide in a template, which is absent in the dispensation order, does not react, and thus does not form a peak. However, when identical nucleotides in the dispensation order continuously appear, the heights of the peaks are determined according to the intensities of light emitted. Accordingly, when various pathogens exist in the same sample, the peaks of the nucleotides of the various pathogens appear overlapped, thus making it difficult to identify the genotypes through the interpretation of pyrograms. Particularly, as the number of repetitive sequences increases, the peaks of the anterior sequences become relatively lower. Thus, in the case of infection with multiple pathogens, it is difficult to detect a peak according to the degree of infection with each pathogen.

Accordingly, the present inventors have made extensive efforts to enable the genotypes of interest to be identified by unique and simple pyrograms obtained when performing genotyping using pyrosequencing. As a result, the present inventors have found that, when an ID sequence, which has an ID mark, a signpost and an endmark while existing independently of the specific sequence to be typed, is linked with the specific sequence and is used to perform pyrosequencing, a unique and simple pyrogram can be obtained for each genotype, thereby completing the present invention.

It is an object of the present invention to provide an ID sequence which is useful to perform pyrosequencing so as to enable the genotypes of interest to be identified by unique and simple pyrograms.

Another object of the present invention is to provide a genotyping method which uses said ID sequence.

Still another object of the present invention is to provide a method of genotyping HPV using said ID sequence.

Yet another object of the present invention is to provide a method of detecting KRAS gene mutation using said ID sequence.

A further object of the present invention is to provide a method of detecting respiratory virus using said ID sequence.

To achieve the above objects, the present invention provides an ID sequence for genotyping which consists of A(ID−S)n−E, wherein ID is an ID mark which is a single nucleotide selected from among A, T, C and G; S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark; E is an endmark which is a nucleotide different from that of the signpost; and n is a natural number ranging from 1 to 32.

The present invention also provides an ID sequence for genotyping which consists of ID−S, wherein ID is an ID mark which is a nucleotide selected from among A, T, C and G, and S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark.

The present invention also provides a genotyping primer comprising a gene-specific sequence for genotyping linked to said ID sequence.

The present invention also provides a genotyping method which comprises using said genotyping primer.

The present invention also provides a method for genotyping HPV, the method comprising the steps of: (a) designing an ID sequence for genotyping according to the genotype of each HPV virus, the ID sequence consisting of (ID−S)n−E, wherein ID is an ID mark which is a nucleotide selected from among A, T, C and G; S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark; E is an endmark which is a nucleotide different from that of the signpost, and n is a natural number ranging from 1 to 32; (b) constructing a genotyping primer composed of a pyrosequencing primer sequence, the ID sequence, and a sequence specific to a virus genotype corresponding to the ID sequence; (c) amplifying an HPV virus-containing sample by PCR using the genotyping primer; and (d) subjecting the amplified PCR product to pyrosequencing to obtain a sequence for the ID sequence, and distinguishing the genotype of HPV according to the ID sequence.

The present invention also provides a method for detecting KRAS gene mutation, the method comprising the steps of: (a) designing an ID sequence for genotyping according to the gene mutation of each KRAS, the ID sequence consisting of (ID−S)n−E wherein ID is an ID mark which is a nucleotide selected from among A, T, C and G; S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark; E is an endmark which is a nucleotide different from that of the signpost, and n is a natural number ranging from 1 to 32; (b) constructing a detection primer composed of a pyrosequencing primer sequence, the ID sequence, and a sequence specific for a KRAS gene mutation corresponding to the ID sequence; (c) amplifying a KRAS gene-containing sample by PCR using the detection primer; and (d) subjecting the amplified PCR product to pyrosequencing to obtain a pyrogram for the ID sequence, and detecting the KRAS gene mutation according to the ID sequence.

The present invention also provides a method for detecting respiratory virus, the method comprising the steps of: (a) designing an ID sequence for genotyping according to the genotype of each of influenza A virus, influenza B virus, RSV B, rhinovirus, and coronavirus OC43, the ID sequence consisting of (ID−S)n−E wherein ID is an ID mark which is a nucleotide selected from among A, T, C and G; S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark, E is an endmark which is a nucleotide different from that of the signpost, and n is a natural number ranging from 1 to 32; (b) constructing a detection primer composed of a pyrosequencing primer sequence, the ID sequence, and a sequence specific to each respiratory virus gene corresponding to the ID sequence; (c) amplifying a sample, which contains a respiratory virus selected from the group consisting of influenza A virus, influenza B virus, RSV B, rhinovirus, and coronavirus OC43, by PCR using the detection primer; and (d) subjecting the amplified PCR product to pyrosequencing to obtain a pyrogram for the ID sequence, and detecting the respiratory virus according to the ID sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a pyrosequencing process, which is performed according a dispensation order, and the resulting pyrogram.

FIG. 2 shows the change in pyrogram peaks according to analytical sequences.

FIG. 3 shows the change in pyrogram peaks according to analytical sequences.

FIG. 4 shows the change in pyrogram peaks, which results from insertion of a signpost.

FIG. 5 shows pyrograms obtained for a mixture of two different analytical sequences.

FIG. 6 shows pyrograms obtained in the presence or absence of a signpost in a dispensation order.

FIG. 7 shows the changes in pyrogram patterns according to the changes in a sequence posterior to a signpost.

FIG. 8 shows pyrograms obtained in the absence of an endmark.

FIG. 9 shows pyrograms obtained in the absence of an endmark.

FIG. 10 shows the change in the dispensation order according to the change in the order of a signpost.

FIG. 11 shows a method of designing an ID sequence according to a dispensation order.

FIG. 12 shows a method of designing a dispensation order.

FIG. 13 shows pyrograms obtained by ID sequences according to dispensation orders.

FIG. 14 shows a method of designing an ID sequence after determining a dispensation order.

FIG. 15 shows pyrograms obtained by ID sequences according to dispensation orders.

FIG. 16 shows a method of genotyping HPV using an ID sequence of the present invention.

FIG. 17 shows a general system for detecting KRAS mutations.

FIG. 18 shows a method of detecting KRAS mutations using ID sequences of the present invention.

FIG. 19 shows the results obtained by genotyping 15 HPV types using ID sequences of the present invention.

FIG. 20 shows the results obtained by genotyping two or more types of HPV.

FIG. 21 shows the results of detecting KRAS mutations using ID sequences of the present invention.

FIG. 22 shows the results of detecting multiple KRAS mutations using ID sequences of the present invention.

FIG. 23 shows the results of detecting KRAS mutations in colorectal cancer tissue using ID sequences of the present invention.

FIG. 24 shows a method of detecting respiratory virus infection using an ID sequence of the present invention.

FIG. 25 shows the results of detecting single infections of 5 types of respiratory viruses using ID sequences of the present invention.

FIG. 26 shows the results of detecting multiple infections of 5 types of respiratory viruses using ID sequences of the present invention.

Other features and embodiments of the present invention will be more apparent from the following detailed descriptions and the appended claims

In one aspect, the present invention is directed to an ID sequence for genotyping which consists of A(ID−S)n−E, wherein ID is an ID mark which is a nucleotide selected from among A, T, C and G; S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark, E is an endmark which is a nucleotide different from that of the signpost; and n is a natural number ranging from 1 to 32.

In another aspect, the present invention is directed to an ID sequence for genotyping which consists of ID−S, wherein ID is an ID mark which is a nucleotide selected from among A, T, C and G, and S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark.

As used herein, the term “ID sequence” is not a specific sequence conserved in each gene and refers to an artificially constructed nucleotide sequence which can be specifically assigned to each genotype in the genotyping method of the present invention.

As used herein, the term “adjacent ID mark” means an ID mark located ahead of or behind the signpost.

The ID sequence of the present invention is used to perform pyrosequencing such that the pyrogram is distinguished by one nucleotide according to the determined dispensation order using the signpost and the endmark, which allow the pyrogram peak to be formed at a specific location without being influenced by the next sequence.

In this invention, a nucleotide that forms a specific peak according to the dispensation order in this pyrosequencing process is named “ID mark”, and a sequence comprising the signpost and the endmark, which is a sequence required for forming a single peak by the ID mark, is named “ID sequence”. There can be three types of ID marks which are not influenced by a gene sequence located next to the ID mark through the use of one signpost and one endmark, and thus the number of types that can be genotyped is three. In order to perform multiplex genotyping of more than three types, additional signposts and ID marks are required.

In general pyrosequencing, nucleotide sequencing is performed according to the dispensation order (the order of nucleotide addition in DNA synthesis), and if a template has a nucleotide absent in the dispensation order, no reaction will occur, and thus no peak will be formed, but if a sequence identical to the sequence included in the dispensation order is continuously present in the template, the height of the peak is formed according to the intensity of light emitted (FIG. 1).

It is believed that, when one nucleotide is used as an analytical sequence, it can be distinguished by four different peaks on the pyrogram. However, in fact, a sequence next to the analytical sequence is one of A, T, G and C, and thus in at least one case, multiple peaks (if the identical sequences are repeated, one large peak is formed) are necessarily formed. As a result, there are at most three methods capable of distinguishing a single peak by a single nucleotide (FIG. 2).

In addition, an undesired peak is formed due to a sequence following the analytical sequence, and if repeated nucleotides are continuously present following the analysis sequence, polymerization reactions will occur at once to form a single peak. However, because the intensity of light generated in the reactions increases, the height of the peak proportionally increases, and if such repetitive sequences exist, the height of the peak for a single nucleotide relatively decreases (FIG. 3). Because of such problems, there is a limit to multiplex genotyping which uses a single nucleotide. To solve such problems, in the ID sequence of the present invention, a sequence that separates the “analytical sequence” so as not to be influenced by the next sequence and allows additional analysis is named “signpost” (FIG. 4).

In addition, a design of the dispensation order for pyrosequencing varies depending on the signpost sequence.

If one nucleotide in an analytical sequence consisting of two nucleotides is set as a signpost, each of three nucleotides can be located at the site of the remaining one nucleotide (if identical sequences are located, the peaks will overlap, and thus three nucleotides excluding the nucleotide assigned as the signpost can be located at the remaining one nucleotide site), and the nucleotide located ahead of the signpost can be set as shown in FIG. 4 such that the peak can be independently distinguished without being influenced by the nucleotide located behind the analytical sequence.

The single nucleotide separated by the signpost in the analytical sequence is named “ID mark”, and as shown in FIG. 4, multiplex genotyping of three types is possible using one ID mark and one signpost. Herein, the position of the signpost in the dispensation order is after the ID mark (because only the ID mark located ahead of the signpost is not influenced by the sequence located following the analytical sequence).

In one aspect of the present invention, in order to genotype a two-nucleotide analytical sequence consisting of a one-nucleotide ID mark and a signpost, as shown in FIG. 5, analytical sequences of type 1 (analytical sequence: AC), type 2 (analytical sequence: TC) and type 3 (analytical sequence: GC) are synthesized and subjected to pyrosequencing. As a result, for type 1, the peak of A appears in dispensation order 1, and the peak of C appears in dispensation order 4. For type 2, the peak of T appears in dispensation order 2, and the peak of C appears in dispensation order 4. For type 3, the peak of G appears in dispensation order 3, and the peak of C appears in dispensation order 4. Herein, A, T and G which are the first nucleotides of the analytical sequences are respectively ID marks, and C which is the second nucleotide of each of the analytical sequences is a signpost.

To perform multiplex genotyping for the three types of analytical sequences, as shown in FIG. 5(a), a sample consisting of type 1 (analytical sequence: AC) and type 2 (analytical sequence: TC) is pyrosequenced in the dispensation order of A→T→G→C. As a result, the peak of A appears in dispensation sequence 1, the peak of T appears in dispensation sequence 2, no peak appears in dispensation sequence 3, and the peak of C that is the signpost appears in dispensation sequence 4. Herein, the peak of the signpost C is two times higher than the peaks of A and T and present in both the two types, and thus the amount of the reaction is two times larger and the peak intensity is also two times higher than those of A and T.

Similarly, as shown in FIG. 5(b), in the case of a sample consisting of type 1 (analytical sequence: AC) and type 3 (analytical sequence: GC), the peak of A appears in dispensation order 1, the peak of G appears in dispensation order 3, no peak appears in dispensation order 2, and the peak of C that is the signpost appears in dispensation order 4. Herein, the peak of the signpost C is two times higher and present in both the two types, and thus the amount of the reaction is two times larger and the peak intensity is also two times higher.

In addition, as shown in FIG. 5(c), in the case of a sample consisting of type 2 (analytical sequence: TC) and type 3 (analytical sequence: GC), the peak of T appears in dispensation order 2, the peak of G appears in dispensation order 3, no peak appeared in dispensation order 1, and the peak of C that is the signpost appears in dispensation order 4. Herein, the peak of the signpost C is two times higher and is present in both the two types, and thus the amount of the reaction is two times larger and the peak intensity is also two times higher.

Accordingly, the ID mark can be separated from the next sequence by the signpost and can be present independently of the next sequence. Thus, it can advantageously be used in multiplex genotyping.

The results of genotyping in pyrosequencing performed using an analytical sequence consisting of an “ID mark” and a “signpost” are not influenced by whether or not the sequence of the signpost is inserted into the dispensation order. However, if the sequence of the signpost is not inserted into the dispensation order, there will be a problem in that a mechanical error cannot be judged (FIG. 6). For this reason, the sequence of the signpost is preferably inserted into the dispensation order to make it possible to determine whether or not pyrosequencing was normally performed. In addition, because the peak of the ID mark in multiplex genotyping in pyrosequencing isn\'t able to be higher than the peak of the signpost, this can also be used as a reference for judging pyrosequencing error (FIG. 6).

Endmark

The signpost functions to separate the single-nucleotide ID mark from the next sequence so as not to be influenced by the next sequence. However, when the next sequence is identical to the signpost, the height of the peak increases in proportion to the increase in the intensity of light emitted. For this reason, there can occur a phenomenon that the height of the peak of the ID mark changes (FIG. 7). To solve this phenomenon, a nucleotide sequence different from the signpost can be inserted following the signpost in order to prevent the ID mark and the signpost from being influenced by the next sequence. Herein, the inserted sequence is named “endmark”, and the endmark is not inserted in the dispensation order. The endmark functions to prevent the ID mark and the signpost from being influenced by the next sequence and make the peak height constant.

In order words, in the case in which the endmark is absent as shown in FIG. 8, if type 1 (analytical sequence: AC) is followed by CA and if pyrosequencing is performed in the dispensation order of A→T→G→C, the peak of C in dispensation order 3 will be larger than the peak of A in dispensation order 1, because of the overlapping C next to the signpost C. Similarly, if type 3 (analytical sequence: GC) is followed by CC, the peak of G in dispensation order 3 will be much smaller than the excessively large peak of C in dispensation order 4, because C next to the signpost C overlaps three times.

In the case in which the endmark is present as shown in FIG. 9, if type 1 (analytical sequence: AC) is followed by CA and if T as the endmark is inserted therebetween and if pyrosequencing is performed in the dispensation order of A→T→G→C, C will not overlap due to the insertion of T next to the signpost C, and the height of the peak of A in dispensation order 1 and the height of the peak of C in dispensation order 3 will be constant. Similarly, if type 3 (analytical sequence: GC) is followed by CC, C next to the signpost will not overlap due to the insertion of the endmark T next to the signpost C, and the height of the peak of G in dispensation order 3 will be equal to the height of the peak of C in dispensation order 4. In the present invention, the number N of signposts that can be added is preferably 2-32, and if N is 32, genotyping of 65 types is possible.

When a plurality of ID marks and a plurality of signposts are used, genotyping of 3 or more types is possible.

In this case, the adjacent nucleotide sequences should differ from each other in order to obtain pyrograms of single peaks. The ID mark can be located ahead of the signpost or between two signposts. The ID mark located between two signposts may consist of two different nucleotides, because it must have nucleotides different from the signposts located at both sides thereof. In addition, the ID mark located ahead of the signpost may consist of three different nucleotides, because it must have a nucleotide different from the signpost located behind thereof.

Herein, the nucleotide of signpost 1 should not be identical to the nucleotide of signpost 3, and the nucleotide sequence of the most posterior signpost must also not be identical to the base sequence of the endmark.

In the present invention, the sequence consisting of the ID mark, the signpost and the endmark is named “ID sequence”. In the present invention, the ID sequence may also be composed of the ID mark and the signpost. Preferably, it consists of the Id mark, the signpost and the endmark.

Whenever one signpost is added, the ID seqauence of the present invention enables two additional types to be distinguished. Thus, it enables 2N+1 (N=the number of signposts) types to be distinguished by genotyping according to the location of the ID mark.

Nucleotide sequences excluding the ID mark and the endmark are used as the signposts. In order to distinguish the ID marks of the ID sequence in the same dispensation order, the nucleotide sequences of the signposts in the ID sequence must be located in the same order. In other words, only the ID mark should be located ahead of or between the signposts, and the signposts should be arranged in the same order. This is because, when the arrangement of the signposts changes, the dispensation order also changes due to the feature of pyrosequencing (FIG. 10).

Design of ID Sequence

Case of Making ID Sequence After Determining Signposts

In the case in which an ID sequence comprises two signposts, if T and G are used as signpost 1 and signpost 2, respectively, an ID mark which is located ahead of signpost 1 (T) may be any one of A, G and C, an ID mark which is located between signpost 1 and signpost 2 may be A or C, and an endmark may be any one of A, T and C.

Thus, as shown in FIG. 11, there are the following five cases in which the ID sequence consisting of the ID mark, the signpost and the endmark can be produced using one ID mark: three cases (A, G and C) in which the ID mark is located ahead of signpost 1; and two cases (A and C) in which the ID mark is located between signpost 1 and signpost 2.

Herein, because the endmarks are not included in the dispensation order, the endmarks in the ID sequences may be the same or different.

Design of Dispensation Order

In genotyping which is performed using the ID sequence, the ID marks located in the ID sequence sequentially form independent peaks according to the dispensation order. The dispensation order can be designed according to various permutations which can be formed using the signpost as a boundary.

One of dispensation orders which can be formed according tot he ID sequence is shown in the following figure, and the endmark is not included in the dispensation order:

FIG. 12 shows 12 dispensation orders which can be formed according to the ID sequence, and one selected from among the 12 dispensation orders may be used.

The number of dispensation orders that can be formed is 4×6N (N=number of signposts). Thus, if the number of signposts is 2, then 144 dispensation orders may be made.

Accordingly, as shown in FIG. 13, the ID sequence has characteristic peaks according to the dispensation order.

Method of Designing ID Sequence After Determining Dispensation Order

An ID sequence consists of one ID mark, one or more signposts and at least one endmark. In the ID sequence, the adjacent nucleotides must differ from each other, and the dispensation order must have the same conditions as described above. In addition, the ID sequence may also be designed after determining the dispensation order.

In the dispensation order, three ID marks may be located ahead of signpost 1, and two ID marks may be located between two signposts. When this rule is used, the following ID sequence can be made with the dispensation order.

For example, when 9 genotypes are to be separated, 4 signposts are required according to the formula (2N+1). As shown in FIG. 14, if the dispensation order is determined such that A, T, G and C are repeated n times, the signposts will be C, G, T and A, the endmark will be T, and the ID marks will be located between the signposts, thereby constructing an ID sequence. When pyrosequencing is performed according to the dispensation order, the results shown in FIG. 15 can be obtained.

In still another aspect, the present invention is directed to a genotyping primer comprising a gene-specific sequence for genotyping linked to said ID sequence.

In the present invention, the gene-specific sequence for genotyping is preferably a sequence specific to a gene selected from the group consisting of viral genes, disease genes, bacterial genes, and identification genes. The primer preferably additionally contains a sequencing primer sequence at the 5′ terminal end in order to facilitate pyrosequencing.

In yet another aspect, the present invention is directed to a genotyping method which comprises using said genotyping primer.

The genotyping primer comprising the ID sequence of the present invention may be used in various genotyping methods which are performed using dispensation orders and sequencing methods. Preferably, it may be used in pyrosequencing methods and semiconductor sequencing methods, but is not limited thereto.

In the present invention, the pyrosequencing method is a method in which light emitted from the degradation of ppi (pyrophosphate) generated in a sequencing process, and the semiconductor sequencing method is a method in which the change in current by a proton (H+ ion) generated in a sequencing process is analyzed by a chip (Andersona, Erik P. et al., Sens Actuators B Chem.; 129(1): 79, 2008).

The genotyping method of the present invention may comprise the steps of: (a) designing an ID sequence for genotyping according to the genotyping target gene, the ID sequence consisting of (ID−S)n−E, wherein ID is an ID mark which is a nucleotide-selected from among A, T, C and G, S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark, E is an endmark which is a nucleotide different from that of the signpost, and n is a natural number ranging from 1 to 32; (b) amplifying the template of the genotyping target gene by PCR using a genotyping primer comprising a gene-specific sequence for genotyping linked to the designed ID sequence, thereby obtaining a PCR product; and (c) pyrosequencing the PCR product to obtain a pyrogram for the ID sequence.

In a further aspect, the present invention is directed to a method for genotyping HPV, the method comprising the steps of: (a) designing an ID sequence for genotyping according to the genotype of each HPV virus, the ID sequence consisting of (ID−S)n−E, wherein ID is an ID mark which is a nucleotide selected from among A, T, C and G; S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark, E is an endmark which is a nucleotide different from that of the signpost, and n is a natural number ranging from 1 to 32; (b) constructing a genotyping primer composed of a pyrosequencing primer sequence, the ID sequence, and a sequence specific to a virus genotype corresponding to the ID sequence; (c) amplifying an HPV virus-containing sample by PCR using the genotyping primer; and (d) subjecting the amplified PCR product to pyrosequencing to obtain a pyrogram for the ID sequence, and distinguishing the genotype of HPV according to the ID sequence.

In the present invention, the sequence specific to a virus genotype may be selected among nucleotide sequences shown by SEQ ID NOS: 1 to 15.

HPV (human papilloma virus) is one of the most common viruses which infect the human skin or mucous membrane. Today more than about 150 HPV types are known, and about 30 kinds infect the genital tract. About 85% of cancers caused by HPV virus are associated with cervical cancer. Among 30 HPV types that infect the genital tract, 15 types are known as high-risk types that cause cervical cancer. Cervical cancer ranks sixth in cancer incidence among women in Korea, and Pap smears have poor sensitivity and reproducibility, and thus have problems involved in detecting precancerous conditions. In addition, these testing methods incur high cost due to frequent testing. HPV testing methods approved by the FDA to date include HybridCaptureII, but this method can diagnose only HPV infection and cannot determine what type of HPV infection is present.

Because HPV shows different cancer incidences, cancer types and cancer metastatic processes depending on the genotypes, it is important to identify the genotype of HPV, which infected the patient, by genotyping. For example, it was reported that 55% of the incidence of CIN III+ is associated with HPV type 16, 15% with HPV type 18, and the remaining 30% with HPV type 13.

The most important reason for genotyping HPV is that the genotyping makes it possible to monitor genotype-specific HPV infections. A period of persistent infection in older women generally is generally longer than that in younger women, and this is because the older women were highly likely to be infected for a long time. Although a critical period of persistent infection has not yet been clinically determined, it is generally known that an infection period longer than 1 year has increased risk. Although it is also important to identify HPV type 16 and HPV type 18, it is most important to examine persistent infection with carcinogenic HPV infection.

In one example of the present invention, 15 HPV virus types were genotyped using the ID sequence. Each of 15 HPV viral genomes was amplified by PCR using HPV L1 protein specific to 15 HPV virus types, primers(GT-HPV 15type primer) containing 15 kinds of ID sequences and sequencing primer sequences, and a 5′ biotinylated GP6 plus primer, and the PCR products were pyrosequenced. As a result, pyrograms of 15 ID sequences for 15 virus types could be obtained (FIG. 16).

In another example of the present invention, a sample of a mixture of the genome DNA of the CaSki cell line infected with HPV type 16 and the same amount of the genomic DNA of the HeLa cell line infected with HPV type 18 was amplified by PCR using a GT-HPV 15 type primer and a 5′ biotinylated GP6 plus primer, and the PCR product was pyrosequenced. As a result, pyrograms of ID sequences, which were clear without the interference of overlapping peaks and corresponded to HPV type 16 and HPV type 18, could be obtained.

In a still further aspect, the present invention is directed to a method for detecting KRAS gene mutation, the method comprising the steps of: (a) designing an ID sequence for genotyping according to the gene mutation of each KRAS, the ID sequence consisting of (ID−S)n−E wherein ID is an ID mark which is a nucleotide selected from among A, T, C and G; S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark, E is an endmark which is a nucleotide different from that of the signpost, and n is a natural number ranging from 1 to 32; (b) constructing a detection primer composed of a pyrosequencing primer sequence, the ID sequence, and a sequence specific for a KRAS gene mutation corresponding to the ID sequence; (c) amplifying a KRAS gene-containing sample by PCR using the detection primer; and (d) subjecting the amplified PCR product to pyrosequencing to obtain a pyrogram for the ID sequence, and detecting the KRAS gene mutation according to the ID sequence.

The Ras gene was first identified as a retroviral oncogene causing a sarcoma in rats. Since the presence of K-ras in the lymph node of pancreatic cancer patients was identified in 1985, various studies on the K-ras gene have been conducted. The mutation of this oncogene is frequently found in the malignant mutations of the human body. As genes having a structure and function similar to those of this oncogene, H-ras and N-ras are also known as oncogenes. Mutations in codons 12, 13 and 61 of K-ras influence the protein activity to cause excessive activity.

Mutations in the K-ras gene are found in adenocarcinoma of the digestive system. In the case of adenocarcinoma of the pancreas, 90% of mutations can be found in pancreatic juice and tissue and are known as mutations of codon 12. In addition, these mutations are found in 40-45% of colorectal cancer and are known to be associated with a decrease in the response to drugs such as cetuximab or panitumumab, which are used for progressed colon cancer that does not respond to chemotherapy. Furthermore, these mutations are observed in 5-30% of non-small cell lung and are observed mainly in smoking patients. In addition, these mutations are found exclusively with EGFR mutations.

Three mutation types, including a mutation of codon 12 (GGT>GTT) and mutations of codon 13 (GGC>TGC and GGC>GCC), in wild-type KRAS, are difficult to detect by a general pyrosequencing method. Particularly, the mutation of codon (GGT>GTT) has a high frequency of occurrence, and thus is difficult to exclude because of the detection limit (FIG. 17).

In one example of the present invention, a method of detecting mutations in codon 12 and codon 13 of the KRAS gene was disclosed. In the present invention, primers binding specifically to the three types of mutations of codon 12 (GGT>GTT) and codon 13 (GGC>TGC and GGC>GCC) were designed such that the mutations can be detected using ID sequences located ahead of nucleotide sequences specific to the three types of primers. According to the method of the present invention, 12 types of KRAS mutations can be detected by a single PCR process using 3 types of forward primers and 1 type of biotinylated reverse primer (FIG. 18).

In the present invention, the sequence specific for a KRAS gene mutation may be selected among nucleotide sequences shown by SEQ ID NOS: 34 to 35.

In a yet further aspect, the present invention is directed to a method for detecting respiratory virus, the method comprising the steps of: (a) designing an ID sequence for genotyping according to the genotype of each of influenza A virus, influenza B virus, RSV B, rhinovirus, and coronavirus OC43, the ID sequence consisting of (ID−S)n−E wherein ID is an ID mark which is a nucleotide selected from among A, T, C and G; S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark, E is an endmark which is a nucleotide different from that of the signpost, and n is a natural number ranging from 1 to 32; (b) constructing a detection primer composed of a pyrosequencing primer sequence, the ID sequence, and a sequence specific to each respiratory virus gene corresponding to the ID sequence; (c) amplifying a sample, which contains a respiratory virus selected from the group consisting of influenza A virus, influenza B virus, RSV B, rhinovirus, and coronavirus OC43, by PCR using the detection primer; and (d) subjecting the amplified PCR product to pyrosequencing to obtain a pyrogram for the ID sequence, and detecting the respiratory virus according to the ID sequence.

In one example of the present invention, a method of detecting respiratory virus was disclosed. In this method, primers binding specifically to 5 types of respiratory viruses are designed such that the viruses can be detected using ID sequences located ahead of nucleotide sequences specific to the primers. cDNA is synthesized using 5 types of forward primers binding to 5 types of GT-respiratory viruses and 1 type of biotinylated reverse primer, and was amplified by PCR using a GT-RespiVirus ID primer and a 5′-biotinylated M13 reverse primer, and the PCR products were pyrosequenced.

In the present invention, the sequences specific to the respiratory virus genotypes may be nucleotide sequences shown by SEQ ID NO: 41 for influenza A virus, SEQ ID NO: 42 for influenza B virus, SEQ ID NO: 43 for RSV B, SEQ ID NO: 44 for rhinovirus, and SEQ ID NO: 45 for coronavirus OC43.

Hereinafter, the present invention will be described in further detail with reference to examples. It will be obvious to a person having ordinary skill in the art that these examples are illustrative purposes only and are not to be construed to limit the scope of the present invention. That is, the following steps will be described as one illustrative ones and do not limit the scope of the present invention.

Using the ID sequences of the present invention, the genes of high-risk HPV (human papilloma virus) types causing cervical cancer were typed.

ID sequences for 15 high-risk HPV types were designed as shown in Table 1 below.

TABLE 1  ID sequences for 15 HPV types ID sequences HPV types AGCACATG HPV type 16 TGCACATG HPV type 58 CGCACATG HPV type 18 GACACATG HPV type 33 GTCACATG HPV type 52 GCGACATG HPV type 35 GCTACATG HPV type 45 GCATCATG HPV type 51 GCAGCATG HPV type 31

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