US20120276524A1 - Genotyping method - Google Patents

Genotyping method Download PDF

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US20120276524A1
US20120276524A1 US13/510,226 US201013510226A US2012276524A1 US 20120276524 A1 US20120276524 A1 US 20120276524A1 US 201013510226 A US201013510226 A US 201013510226A US 2012276524 A1 US2012276524 A1 US 2012276524A1
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sequence
genotyping
mark
nucleotide
signpost
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Sung Whan An
Myung Sok Oh
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Genomictree Inc
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    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/30—Detection characterised by liberation or release of label
    • C12Q2565/301—Pyrophosphate (PPi)
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • 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.
  • 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.
  • 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.
  • dNTPs deoxynucleotide triphosphates
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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
  • 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,
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ID mark a nucleotide that forms a specific peak according to the dispensation order in this pyrosequencing process
  • ID sequence a sequence comprising the signpost and the endmark, which is a sequence required for forming a single peak by the ID mark
  • ID sequence a sequence comprising the signpost and the endmark, which is a sequence required for forming a single peak by the ID mark.
  • 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 ).
  • nucleotide when one nucleotide is used as an analytical sequence, it can be distinguished by four different peaks on the pyrogram.
  • 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.
  • FIG. 2 there are at most three methods capable of distinguishing a single peak by a single nucleotide
  • 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.
  • 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 ).
  • 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 ).
  • 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.
  • ID mark The single nucleotide separated by the signpost in the analytical sequence.
  • FIG. 4 multiplex genotyping of three types is possible using one ID mark and one signpost.
  • 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).
  • 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.
  • type 1 analytical sequence: AC
  • type 2 analytical sequence: TC
  • type 3 analytical sequence: GC
  • the peak of G appears in dispensation order 3
  • the peak of C appears in dispensation order 4.
  • A, T and G which are the first nucleotides of the analytical sequences are respectively ID marks
  • C which is the second nucleotide of each of the analytical sequences is a signpost.
  • 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.
  • 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
  • the peak of C that is the signpost appears in dispensation sequence 4.
  • 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.
  • 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
  • the peak of C that is the signpost appears in dispensation order 4.
  • 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.
  • the peak of T appears in dispensation order 2
  • the peak of G appears in dispensation order 3
  • the peak of C that is the signpost appears in dispensation order 4.
  • 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.
  • the ID mark can be separated from the next sequence by the signpost and can be present independently of the next sequence.
  • it can advantageously be used in multiplex genotyping.
  • 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.
  • 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 ).
  • 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.
  • 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 ).
  • the signpost functions to separate the single-nucleotide ID mark from the next sequence so as not to be influenced by the next sequence.
  • 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 ).
  • 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.
  • 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.
  • the number N of signposts that can be added is preferably 2-32, and if N is 32, genotyping of 65 types is possible.
  • genotyping of 3 or more types is possible.
  • 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.
  • 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.
  • 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.
  • the sequence consisting of the ID mark, the signpost and the endmark is named “ID sequence”.
  • 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.
  • Nucleotide sequences excluding the ID mark and the endmark are used as the signposts.
  • 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 ).
  • 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
  • an endmark may be any one of A, T and C.
  • 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.
  • the endmarks in the ID sequences may be the same or different.
  • 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.
  • 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 ID sequence has characteristic peaks according to the dispensation order.
  • An ID sequence consists of one ID mark, one or more signposts and at least one endmark.
  • the adjacent nucleotides must differ from each other, and the dispensation order must have the same conditions as described above.
  • the ID sequence may also be designed after determining the dispensation order.
  • three ID marks may be located ahead of signpost 1, and two ID marks may be located between two signposts.
  • the following ID sequence can be made with the dispensation order.
  • the present invention is directed to a genotyping primer comprising a gene-specific sequence for genotyping linked to said ID sequence.
  • 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.
  • 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.
  • the pyrosequencing method is a method in which light emitted from the degradation of ppi (pyrophosphate) generated in a sequencing process
  • 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.
  • 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
  • sequence specific to a virus genotype may be selected among nucleotide sequences shown by SEQ ID NOS: 1 to 15.
  • HPV human papilloma virus
  • HPV human papilloma virus
  • 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.
  • genotyping HPV 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.
  • 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.
  • HPV type 16 and HPV type 18 it is most important to examine persistent infection with carcinogenic HPV infection.
  • 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.
  • primers(GT-HPV 15type primer) containing 15 kinds of ID sequences and sequencing primer sequences
  • a 5′ biotinylated GP6 plus primer were pyrosequenced.
  • 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.
  • a GT-HPV 15 type primer and a 5′ biotinylated GP6 plus primer was amplified by PCR using a GT-HPV 15 type primer and a 5′ biotinylated GP6 plus primer, and the PCR product was pyrosequenced.
  • 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
  • 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.
  • adenocarcinoma of the pancreas 90% of mutations can be found in pancreatic juice and tissue and are known as mutations of codon 12.
  • 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.
  • these mutations are observed in 5-30% of non-small cell lung and are observed mainly in smoking patients.
  • these mutations are found exclusively with EGFR mutations.
  • a method of detecting mutations in codon 12 and codon 13 of the KRAS gene was disclosed.
  • 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.
  • 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 ).
  • sequence specific for a KRAS gene mutation may be selected among nucleotide sequences shown by SEQ ID NOS: 34 to 35.
  • 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,
  • a method of detecting respiratory virus was disclosed.
  • 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.
  • 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.
  • the genes of high-risk HPV (human papilloma virus) types causing cervical cancer were typed.
  • HPV types A GCACATG HPV type 16 T GCACATG HPV type 58 C GCACATG HPV type 18 G A CACATG HPV type 33 G T CACATG HPV type 52 GC G ACATG HPV type 35 GC T ACATG HPV type 45 GCA T CATG HPV type 51 GCA G CATG HPV type 31 GCAC T ATG HPV type 39 GCAC G ATG HPV type 56 GCACA C TG HPV type 59 GCACA G TG HPV type 68 GCACAT A G HPV type 66 GCACAT C G HPV type 82
  • a nucleotide sequence specific to each of 15 HPV types was linked to the 3′ terminal end of each of 15 ID sequences, and a common sequencing primer sequence was linked to the 5′ terminal end, such that 15 types of different ID sequences can be used in pyrosequencing with a single sequencing primer, thereby constructing PCR primers containing the ID sequences (Table 2).
  • HPV viruses The 15 types of HPV viruses were obtained by extracting genomic DNA from Korean female cervicovaginal secretions (Department of Obstetrics & Gynecology, Chungnam National University), identifying the infected genotypes using an HPV DNA chip, and amplifying the L1 gene of the HPV virus by PCR.
  • PCR primers for determining whether the clinical samples were infected with HPV a GP5 plus primer and a GP6 plus primer were used.
  • PCR amplification was performed under the following conditions using forward primers (15 types of GT-HPV primers) consisting of the 15 types of PCR primers shown in Table 2 and a reverse primer consisting of a 5′ biotinylated GP6 plus primer (Bioneer, Korea): 95° C. for 15 min; then 45 cycles each consisting of 95° C. (0.5 min), 45° C. (0.5 min) and 72° C. (0.5 min); then 72° C. (10 min); and then storage at ⁇ 4° C.
  • forward primers 15 types of GT-HPV primers
  • a reverse primer consisting of a 5′ biotinylated GP6 plus primer
  • the PCR products contained a common .
  • a general T7 primer (5′-TAA TAC GAC TCA CTA TAG GG-3′) was used to perform pyrosequencing with the ID sequences, and the pyrograms were analyzed to type HPV ( FIGS. 19 and 20 ).
  • ID marks formed according to the types of HPV in the dispensation order are shown in the upper portion of FIG. 19 .
  • the peaks indicated in red are ID marks, and the peaks indicated in blue are signposts.
  • PCR amplification was performed with the GP5 plus primer and the GP6 plus primer. Then, using a 1:1 mixture of the PCR products for each HPV type as a template, PCR amplification was performed with 15 types of GT-HPV primers and a 5′ biotinylated GP6 plus primer. Then, the PCR products were pyrosequenced using a T7 primer.
  • PCR amplification was performed with 15 types of GT-HPV forward primers and a 5′ biotinylated GP6 plus primer. Then, the PCR products were pyrosequenced using a T7 sequencing primer to obtain pyrograms for the ID sequences.
  • ID sequences for three types of KRAS mutations that is, mutations of codon 12 (GGT>GTT) and codon 13 (GGC>TGC and GGC>GCC), were designed (Table 6).
  • nucleotide sequence specific to each of the three types of KRAS mutations was linked to the 3′ terminal end of each of the ID sequences, and a common sequencing primer sequence to the 5′ terminal end, such that pyrosequencing can be performed using the three different ID sequences with a single sequencing primer, thereby constructing ID sequence-containing PCR primers (Table 7).
  • GT-KRAS ID primers GT-KRAS ID forward primer Sequencing primer ID KRAS mutation- binding site sequence specific sequence Codon12 AACTTGTGGTAGTTGGAGCT GTGCAGT TGGAGCTGT (SEQ ID NO: 33) (GGT > GTT) (SEQ ID NO: 36) Codon13 AACTTGTGGTAGTTGGAGCT GTGCTGT GAGCTGGTT (SEQ ID NO: 34) (GGC > TGC) (SEQ ID NO: 37) Codon13 AACTTGTGGTAGTTGGAGCT CGCACATT AGCTGGTGC (SEQ ID NO: 35) (GGC > GCC) (SEQ ID NO: 38)
  • Templates for mutations corresponding to the ID sequences used as samples were made through gene synthesis (Bioneer, Korea), and the normal KRAS cell line Caco2(ATCC HTB-37) and the mutant cell lines A549(ATCC CCL-185) and HCT116(ATCC CCL-247) were used.
  • Each of the templates was amplified by PCR using the four types of KRAS forward primers and a 5′ biotinylated reverse primer, and the PCR products were pyrosequenced under the following conditions:
  • KRAS mutations were detected using the ID sequences ( FIG. 21 ).
  • New influenza A H1N1
  • seasonal influenza A H1 and H3
  • B viruses prevail in the same season and show similar infection symptoms, but show different responses to antiviral agents. Thus, it is required to accurately identify virus types for treatment.
  • a method of genotyping respiratory virus using the ID sequence was developed.
  • a nucleotide sequence specific to each of the 5 types of respiratory viruses was linked to the 3′ terminal end of each of the ID sequences, and a common sequencing primer sequence was linked to the 5′ end, pyrosequencing for the 5 types of ID sequences can be performed using a single sequencing primer, thereby constructing ID sequence-containing PCR primers (Table 10).
  • GT-RespiVirus ID primers GT-respiratory virus 5 type primer construction
  • Sequencing primer ID Respiratory virus-specific binding sites sequence sequence Influenza A TAATACGACTCACTATAGGG CATA ATATACAACAGGATGGGGGCTGTG virus (SEQ ID NO: 41) (SEQ ID NO: 46) Influenza B TAATACGACTCACTATAGGG G ATA ATCATCATCCCAGGCGACAAAGATG virus ((SEQ ID NO: 42) (SEQ ID NO: 47) RSV B TAATACGACTCACTATAGGG T ATA TGATATGCCTATAACAAATGACCAGAAA (SEQ ID NO: (SEQ ID NO: 43) 48) Rhino TAATACGACTCACTATAGGG A C TA GCCAGAAAGTGGACAAGGTGTGAAGAG virus1 (SEQ ID NO: 44) (SEQ ID NO: 49) Coronavirus TAATACGACTCACTATAGGG AT C A GCAGATTTGCCAGCTTATA
  • cDNAs from virus-infected cells were synthesized using the 5 types of GT-respiratory forward primers and a 5′ biotinylated reverse primer (Table 11), and then amplified by PCR using the GT-RespiVirus ID primers shown in Table 10 and a 5′ biotinylated M13 reverse primer.
  • the PCR products were pyrosequenced to detect virus infection ( FIG. 24 ).
  • the required portions of the virus genes were synthesized, and multiplex PCR was performed using the synthesized virus genes as templates, followed by pyrosequencing for detection of the virus genes.
  • the virus gene templates were mixed at the same ratio, and then amplified by multiplex PCR, followed by pyrosequencing for detection of the viral genes. As a result, multiple infections were normally detected.
  • the ID sequence When pyrosequencing is performed using the ID sequence, a unique and simple pyrogram can be obtained for each genotype.
  • 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.

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CN112852937A (zh) * 2021-03-10 2021-05-28 美格医学检验所(广州)有限公司 一种呼吸道病原微生物检测引物组合、试剂盒及其应用

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