JP2006280340A - Probe set, classification method and kit for gene type of hbv - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a probe set, classification method and kit for gene type of HBV. <P>SOLUTION: The method for classifying the gene type of HBV comprises a probe set composed of oligonucleotide represented by sequence given by No.17 to No 181, a kit for identifying the gene types of HBV that includes the probe set and the step for amplifying the DNA of HBV and the step where the amplified product is allowed to contact with the above-mentioned probe set for classification. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a probe set, a classification method, and a kit for genotyping HBV.

  Hepatitis B virus (HBV) is a DNA virus having a total length of about 3.2 Kbp. Currently, it is estimated that approximately 350 million people are infected with HBV worldwide. Most of them are people living in Asia and Africa.

  In HBV, seven genotypes are distinguished by more than 8% difference in the entire gene sequence, and they are classified into alphabets A to G. Each of these HBV genotypes has a different geographical distribution. That is, genotype A is the majority in Northwest Europe, North America, and Africa, while genotype B and genotype C are common types in Asia. Genotype D is the most widespread general type in Mediterranean countries. The genotype G is a type limited to East Africa, and the genotype F is a type localized to the new world. Among these, the genotype E added recently has not yet been described about epidemiology.

  Looking at the regional distribution of HBV genotypes in Japan, genotype C accounts for over 90% in Hokkaido, Shikoku, Kyushu, and western Japan, and the ratio of genotype B tends to increase from eastern Japan to the Tohoku region. In Okinawa, on the other hand, it has been reported that genotype B, which exists only in Kyushu, accounts for 60% (Non-patent Document 1).

  Further, when the total base sequences of genotype B widely distributed in neighboring countries in Asia and Japanese genotype B were compared, they were classified into two subtypes (Japanese type: Bj / Asian type: Ba) (Non-patent Document 2). ), It has been revealed that there is a difference in clinical image between them. It has been reported that such subtypes can also be divided into Asia / African type: Aa / Europe / American type: Ae even in genotype A, the most infected person in the world (Non-patent Document 3).

  It is believed that the HBV genotype can affect the signs of disease in the host. In Europe, genotype A is more likely to develop more chronic liver disease than genotype D. In Asia, genotype C has a higher ability to induce disease than genotype B.

  Therefore, development of a method for classifying hepatitis B virus genotypes including two subtypes of type A and type B and a kit therefor has been awaited.

  A method for such HBV genotyping is described in US Pat. However, in this method, the preS1 region of the HBV gene is PCR amplified, the amplified product is brought into contact with a probe specific to each of the genotypes A to G, and the presence or absence of specific hybridization is detected. One well is needed to determine one genotype. Also, it is not possible to detect two subtypes of A type and B type.

  Patent Document 2 describes a method for detecting two subtypes Ba and Bj of HBV genotype B using a restriction length fragment polymorphism method. However, this method requires a lot of instrumental reagents for electrophoresis because it uses a restricted length fragment polymorphism (RFLP) method and requires a long time to obtain a result. Further, in the RFLP method, determination in a state where a plurality of genotype viruses coexist is difficult, and there is a possibility that the genotype is erroneously determined.

Furthermore, despite being a DNA virus, HBV is prone to mutation during its replication process and is rich in diversity. There are hundreds of full-length sequences registered in GenBank, and the sequence to be used as a standard is also known. It is not done. For this reason, it has been extremely difficult to classify HBV genotypes without omission.
JP 2002-355098 A JP 2003-180357 A Orito, E et al .: Geographic distribution of hepatitis B virus (HBV) genotype in patients with chronic HBV infection in Japan. Hepatology 34: 590-594, 2001. Sugachi, F. et al .: Two subtypes of genotype B (Ba and Bj) of heatitis B virus in Japan.Clin Infect Dis 38; 1222-1228, 3004. Sugachi, F et al .: Epipidemiological and sequence differences between two subtypes (Ae and a) of hepatitis B virus genotype AJGen viol 85: 811-820, 2004.

  The present invention provides probe sets, classification methods and kits for HBV genotyping.

  As a result of analyzing the reported gene sequences, paying attention to the core region and S region, and intensive studies, the present inventors have found a clinically useful primer set and completed the present invention. . Accordingly, the present invention relates to the following.

1. The following base sequence pairs:
SEQ ID NO: 17-27 and SEQ ID NO: 28-29,
SEQ ID NOs: 30-42 and SEQ ID NOs: 43-52,
SEQ ID NOs: 53-61 and SEQ ID NOs: 62-67,
SEQ ID NO: 68-75 and SEQ ID NO: 76-78,
SEQ ID NO: 79-88 and SEQ ID NO: 89-96,
SEQ ID NO: 97-103 and SEQ ID NO: 104-105,
SEQ ID NOs: 106-122 and SEQ ID NOs: 123-128,
SEQ ID NOs: 129-141 and SEQ ID NOs: 142-143,
SEQ ID NOs 144-152 and SEQ ID NOs 153-157,
SEQ ID NOs: 158-162 and SEQ ID NOs: 163-164,
SEQ ID NO: 165-171 and SEQ ID NO: 172, SEQ ID NO: 173-179 and SEQ ID NO: 180-183
A set of probes consisting of oligonucleotides indicated by
2. A kit for identifying the genotype of hepatitis B virus, comprising the set of probes according to 1 above.
3. The following process:
A method for classifying the genotype of hepatitis B virus, comprising the steps of amplifying HBV virus DNA and contacting the obtained amplification product with the set of probes described in 1 above.

  According to the present invention, the genotype of HBV is added to genotype B and genotype C which are major Japanese HBV genotypes in addition to genotype B and genotype C, which are major Japanese genotypes. It is classified into type D, and genotype B and genotype A can be further classified into subtypes of genotypes Ba and Bj and genotypes Aa and Ae, respectively. The method of the present invention can also be applied to a mixed type in which a plurality of genotype viruses are mixed.

  The kit of the present invention is designed for classification of HBV genotypes using the invader method.

  The invader method is a method capable of identifying a specific sequence such as a single nucleotide polymorphism (SNP) or gene mutation existing in the genome (Lance Fors et al., 2000, Pharmacogenomics, Volume 1). 219-229). This method is attracting attention because it does not necessarily require DNA amplification such as PCR, which is essential for other SNP typing methods. In the invader method, when the invader oligo causes at least one base intrusion (invasion) in the double strand between the target DNA and the signal probe to form a partial 3-base overlapping structure, the structure-specific 5 It utilizes the fact that the '-endonuclease cleaves the 5' side of the signal probe. Therefore, in order to detect one genotype, a set of at least one invader oligo and a signal probe is required.

  The probes used in the present invention are seven portions of the core region, which are highly conserved as a whole by comparative studies based on known HBV gene sequences and their genotypes, but where differences by each genotype are recognized ( SEQ ID NOs: 2 to 8) and 5 positions in the S region (SEQ ID NOs: 11 to 15) can be selected.

  Then, bases at positions 1978, 1984, 2020, 2056, 2059, 2160 and 2345 in the core region and bases at positions 2887, 2901, 2950, 2980 and 3008 in the S region can be used as the break points by the invader method. .

  For position 1978, a set of signal probes of SEQ ID NOs: 17-27 and invader oligos of SEQ ID NOs: 28 and 29 to recognize the region of SEQ ID NO: 2 can be used.

  For position 1984, a set of signal probes of SEQ ID NOs: 30-42 and invader oligos of SEQ ID NOs: 43-52 to recognize the region of SEQ ID NO: 3 can be used.

  For position 2020, a set of signal probes of SEQ ID NOs: 53 to 61 and invader oligos of SEQ ID NOs: 62 to 67 for recognizing the region of SEQ ID NO: 4 can be used.

  For position 2056, a set of signal probes of SEQ ID NOs: 68-75 and invader oligos of SEQ ID NOs: 76-78 for recognizing the region of SEQ ID NO: 5 (complementary strand) can also be used.

  For position 2059, a set of signal probes of SEQ ID NOs: 79-88 and invader oligos of SEQ ID NOs: 89-96 to recognize the region of SEQ ID NO: 6 can be used.

  For position 2160, a set of signal probes of SEQ ID NOs: 97-103 and invader oligos of SEQ ID NOs: 104 and 105 for recognizing the region of SEQ ID NO: 7 can be used.

  For position 2345, a set of signal probes of SEQ ID NOs: 106-122 and invader oligos of SEQ ID NOs: 123-128 for recognizing the region of SEQ ID NO: 8 can be used.

  For position 2887, a set of signal probes of SEQ ID NOs: 129 to 141 and invader oligos of SEQ ID NOs: 142 and 143 for recognizing the region of SEQ ID NO: 11 can be used.

  For position 2901, a set of signal probes of SEQ ID NOs: 144 to 152 and invader oligos of SEQ ID NOs: 153 to 157 for recognizing the region of SEQ ID NO: 12 can be used.

  For position 2950, a set of signal probes of SEQ ID NOS: 158-162 and invader oligos of SEQ ID NOS: 163 and 164 to recognize the region of SEQ ID NO: 13 can be used.

  For position 2980, a set of signal probes of SEQ ID NO: 165-171 and invader oligo of SEQ ID NO: 172 for recognizing the region of SEQ ID NO: 14 can be used.

  For position 3008, a set of signal probes of SEQ ID NOs: 173 to 179 and invader oligos of SEQ ID NOs: 180 to 183 for recognizing the region of SEQ ID NO: 15 can be used.

Therefore, the present invention
SEQ ID NOs: 17-25 and SEQ ID NOs: 26-27,
SEQ ID NOs: 28-40 and SEQ ID NOs: 41-50,
SEQ ID NO: 51-59 and SEQ ID NO: 60-65,
SEQ ID NOs: 66-73 and SEQ ID NOs: 74-76,
SEQ ID NO: 77-86 and SEQ ID NO: 87-94,
SEQ ID NOs 104-120 and 121-126,
SEQ ID NOs: 127-139 and SEQ ID NOs: 140-141,
SEQ ID NO: 142-150 and SEQ ID NO: 151-155,
SEQ ID NOs: 156-160 and SEQ ID NOs: 161-162,
SEQ ID NO: 163-169 and SEQ ID NO: 170, and SEQ ID NO: 171-177 and SEQ ID NO: 178-181
To a set of probes consisting of the oligonucleotides shown in

  For the purposes of the present invention, it is desirable to first amplify HBV DNA. PCR is generally used as an amplification method, and the following description relates to PCR primers, but is not limited thereto, and any conventionally known DNA amplification technique such as LAMP method can also be used.

  The template HBV gene to be analyzed can be extracted and purified from serum, tissue, etc. from an HBV-infected person. Since HBV is a virus with relatively many mutations, it is desirable to use a site that is highly conserved among HBV genotypes as the primer setting position. In addition, since the PCR amplification efficiency and specificity depend on the primer setting position and primer sequence, it is necessary to set a primer in a portion with good PCR amplification efficiency and high specificity. In addition, during amplification, if there is a large difference in amplification efficiency between genotypes, selection takes place at that point, and there is a possibility that a correct result cannot be obtained by genotyping by hybridization.

  Therefore, in a preferred embodiment of the present invention, as the PCR primer of the present invention, for the region of SEQ ID NO: 2 to 8, SEQ ID NO: 1 (forward primer) and SEQ ID NO: 9 (reverse primer), and SEQ ID NO: 11 For the region -15, SEQ ID NO: 10 (forward primer) and SEQ ID NO: 16 (reverse primer) can be used.

  The present invention also relates to a kit for identifying the genotype of HBV virus comprising the probe set of the present invention.

  Furthermore, the present invention provides a method for classifying the genotype of hepatitis B virus, which comprises the steps of amplifying the DNA of HBV virus and contacting the obtained amplification product with the set of probes according to claim 1. Also related to the method.

  In order to examine the difference in DNA sequence between genotypes, 491 full-length sequences of HBV-DNA were obtained from a database. An alignment of these sequences was made, and bases specific to each genotype that was conserved in each genotype but different from the other types were selected.

  In order to discriminate Aa, Ae, Ba, Bj, C and D which are genotypes detected in Japan, the base region positions 1978, 1984, 2020, 2056, 2059, 2160 and 2345, and the S region Twelve bases at positions 2887, 2901, 2950, 2980, and 3008 were selected, and an invader probe with this portion as the breakpoint was designed.

  Although HBV is a DNA virus, it has many mutations, and even if it is the same genotype, there are very few sequences that perfectly match, so it is difficult to detect all strains with a single invader probe set. . Therefore, it was designed so that all strains classified into each genotype could be detected by combining and reacting 2 to 10 signal probes and 1 to 10 invader oligos.

  In the invader reaction, it is important that the sequence from the cleavage point of the signal probe and the invader oligo to 5 bases does not mismatch with the target DNA. Therefore, for all 5 bases before and after the cleavage point, all sequences found in each genotype are A signal probe and an invader oligo were combined to react.

  An example in which genotype Aa and genotype Ae are determined using the core region position 1984 as a breakpoint will be described below. In 19 cases of genotype Aa, the nucleotide sequence at DNA positions 1984 to 2001 was GGATCTACTHGAHACMSG, whereas in 60 cases of genotype Ae, it was AGATCTYSTRGAYACMG. Therefore, seven types of signal probes for Aa detection (SEQ ID NOs: 30 to 36) and six types for detection of Ae (SEQ ID NOs: 37 to 42) are designed in this portion, and 10 types of invaders are added to DNA sequences 1949 to 1984. Oligos (SEQ ID NOs: 43-52) were designed.

  Probe design software (Invader Creator) manufactured by Third Wave Technologies (TWT) was used for designing a probe for detecting the base sequence.

  For measurement, each invader oligo, signal probe, FRET probe and cleavase enzyme were simultaneously mixed with PCR-amplified DNA using a 384-well plate, reacted at 65 ° C. for 15 to 30 minutes, and then a fluorescent plate reader ( CYTOFLUOR4000 / Applied Biosystems). Judgment criteria are FOZ method (Victor Lyamichev and Bruce Neri, Methods in Molecular Biology, Vol. 212, 2003, pp. 229-240) for determining the presence or absence of a specific reaction based on the ratio of the fluorescence value of the test sample to the fluorescence value of the negative control. ), But the threshold was set to 2.0 in order to make the judgment more strict.

  In the conventional invader method, the reaction temperature is 63 ° C., but in the present invention, the specificity of HBV genotyping can be further improved by setting the reaction temperature to 65 ° C. For example, at position 1978, the FOZ value of the negative sample at 63 ° C. was 1.2 to 1.35, but at 65 ° C., it could be 0.8 to 1.0.

  A comparative study with the DNA probe method (HBV genotype determination kit, Genome Science Laboratories) was performed using 339 clinical isolates.

  As a result of measuring 339 clinical isolates, 10/339 cases (2.95%) were found in which no signal of type A to G was obtained by the DNA probe method. About 10 cases, Ae type 3 examples, Ba type 1 example, Bj type 4 examples, C type 2 examples could be determined. The genotypes of the other 329/339 cases (97.05%) matched.

  When the results of the method of the present invention and the DNA probe method did not match, the HBV-DNA sequence was confirmed by sequence analysis. For the regions of SEQ ID NOs: 2 to 8, SEQ ID NO: 1 (forward primer) and SEQ ID NO: 9 (reverse primer), and for the regions of SEQ ID NOs: 11 to 15, SEQ ID NO: 10 (forward primer) and SEQ ID NO: 16 (Reverse primer) was used for PCR amplification by a known method, and then the HBV-DNA gene sequence was analyzed by direct sequencing or cloning sequencing.

  As a result of measurement by the DNA probe method, 4 cases (1.18%) were found to be a mixed type of B type and C type among 339 cases. In the kit of the present invention, Bj type and It could be determined as a mixed type of C type.

  Probes were also designed for core region positions 1951, 2028 and S region positions 93, 3008. However, at positions 1951, 2028, and 93 other than positions 1984 and 3008, when a clinical isolate whose genotype is known by the sequencing method is measured, the signals of both genotypes Aa and Ae increase, so that specific detection is possible. I couldn't do it. Of the designed probes, 2/5 (40.0%) were useful probes for genotype Aa and Ae determination.

  Invader probes were designed at positions 1605 and 2345 to clearly determine genotypes A and C. In the HBV full-length sequence, only genotype A had a base at position 1605 at T (thymine), and there were 6 inserted bases at position 2345 only in genotype A except for some strains. The clinical isolates from which genotype A signals were obtained were subjected to sequence analysis of the region containing positions 1605 and 2345. As a result, the base at position 1605 was sometimes T (thymine) other than genotype A. As a result, the ratio of probes useful for determining genotypes A and C was 1/2 (50.0%).

  From such a phenomenon, it has been found that it is desirable that the sequences before and after the breakpoint used in the genotyping of HBV be as specific as possible.

  For discrimination between genotypes Bj and Ba, invader probes were designed with the core region positions 1978, 1993, 2020, 2035, 2044, 2056, 2059, 2083, 2089 and 2160 as the breakpoints. There were many differences between the genotypes Bj and Ba in the core region, and a portion having a specific sequence before and after the breakpoint was selected.

  Among these, position 2035 is excellent in reactivity, but in 2 cases out of 52 clinical isolates, the results differed from other cutting points. In addition, the positions 1978, 2020, 2056, 2059 and 2160 were 10 times more sensitive and more reactive than the positions 1993, 2044, 2083 and 2089.

  Therefore, in the kit of the present invention, the region including these five breakpoints is used as a probe for discrimination between Bj and Ba, and the invader reaction does not occur because mutation occurs in any four of the five regions. The probe was selected so that the genotype could be determined at the remaining one location. The proportion of probes useful for determining genotypes A and C was 5/10 (50.0%).

  For discrimination between genotypes B and C, an invader probe was designed with the S region positions 2901, 2950 and 2980 as the breakpoints. The genotypes B and C were determined so that any two of these three regions contained mutations, and even when no invader reaction occurred, the remaining one could be determined.

  The results of the HBV genotype determination by the invader method using the probe set of the present invention and the HBV genotype determination by the DNA probe method using a commercially available HBV genotype determination kit are shown in the following table.

  The present invention can provide clinically useful data by classifying HBV genotypes, and is useful for diagnosis, prevention, treatment and the like of HBV.

It is alignment of HBV-DNA each genotype sequence. Probe design regions (SEQ ID NOs: 2 to 8) are underlined, and invader cleavage points are indicated by numbers representing base positions (core region positions 1978, 1984, 2020, 2056, 2059, 2160 and 2345). It is alignment of HBV-DNA each genotype sequence. Probe design regions (SEQ ID NOs: 2 to 8) are underlined, and invader cleavage points are indicated by numbers representing base positions (core region positions 1978, 1984, 2020, 2056, 2059, 2160 and 2345). It is alignment of HBV-DNA each genotype sequence. Probe design regions (SEQ ID NOs: 2 to 8) are underlined, and invader cleavage points are indicated by numbers representing base positions (core region positions 1978, 1984, 2020, 2056, 2059, 2160 and 2345). It is alignment of HBV-DNA each genotype sequence. Probe design regions (SEQ ID NOs: 2 to 8) are underlined, and invader cleavage points are indicated by numbers representing base positions (core region positions 1978, 1984, 2020, 2056, 2059, 2160 and 2345). It is alignment of HBV-DNA each genotype sequence. Probe design regions (SEQ ID NOs: 2 to 8) are underlined, and invader cleavage points are indicated by numbers representing base positions (core region positions 1978, 1984, 2020, 2056, 2059, 2160 and 2345). It is alignment of HBV-DNA each genotype sequence. Probe design regions (SEQ ID NOs: 2 to 8) are underlined, and invader cleavage points are indicated by numbers representing base positions (core region positions 1978, 1984, 2020, 2056, 2059, 2160 and 2345). It is alignment of HBV-DNA each genotype sequence. Probe design regions (SEQ ID NOS: 11 to 15) are underlined, and invader cleavage points are indicated by numbers representing base positions (S region positions 2887, 2901, 2950, 2980 and 3008). It is alignment of HBV-DNA each genotype sequence. Probe design regions (SEQ ID NOS: 11 to 15) are underlined, and invader cleavage points are indicated by numbers representing base positions (S region positions 2887, 2901, 2950, 2980 and 3008). It is alignment of HBV-DNA each genotype sequence. Probe design regions (SEQ ID NOS: 11 to 15) are underlined, and invader cleavage points are indicated by numbers representing base positions (S region positions 2887, 2901, 2950, 2980 and 3008). It is alignment of HBV-DNA each genotype sequence. Probe design regions (SEQ ID NOS: 11 to 15) are underlined, and invader cleavage points are indicated by numbers representing base positions (S region positions 2887, 2901, 2950, 2980 and 3008). It is alignment of HBV-DNA each genotype sequence. Probe design regions (SEQ ID NOS: 11 to 15) are underlined, and invader cleavage points are indicated by numbers representing base positions (S region positions 2887, 2901, 2950, 2980 and 3008).

Claims (3)

The following base sequence pairs:
SEQ ID NO: 17-27 and SEQ ID NO: 28-29,
SEQ ID NOs: 30-42 and SEQ ID NOs: 43-52,
SEQ ID NOs: 53-61 and SEQ ID NOs: 62-67,
SEQ ID NO: 68-75 and SEQ ID NO: 76-78,
SEQ ID NO: 79-88 and SEQ ID NO: 89-96,
SEQ ID NO: 97-103 and SEQ ID NO: 104-105,
SEQ ID NOs: 106-122 and SEQ ID NOs: 123-128,
SEQ ID NOs: 129-141 and SEQ ID NOs: 142-143,
SEQ ID NOs 144-152 and SEQ ID NOs 153-157,
SEQ ID NOs: 158-162 and SEQ ID NOs: 163-164,
SEQ ID NO: 165-171 and SEQ ID NO: 172, SEQ ID NO: 173-179 and SEQ ID NO: 180-183
A set of probes consisting of oligonucleotides indicated by   A kit for identifying the genotype of hepatitis B virus, comprising the set of probes according to claim 1. The following process:
A method for classifying the genotype of hepatitis B virus comprising the steps of amplifying HBV virus DNA and contacting the resulting amplification product with the set of probes according to claim 1.

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