Nucleic acid primer set for detection of drug-resistant strain of hepatitis B virus, assay kit, and method of detecting drug-resistant strain of hepatitis B virus

A method of detecting a drug-resistant or drug-nonresistant strain of hepatitis B virus, comprising:

• amplifying a hepatitis B virus nucleic acid in a sample solution by LAMP with a primer set to yield an amplification product, and
• hybridizing the amplification product with a probe containing a polynucleotide derived from a drug-resistant strain of hepatitis B virus and/or a probe containing a polynucleotide derived from a drug-nonresistant strain of hepatitis B virus, to detect a drug-resistant or drug-nonresistant strain of hepatitis B virus,
• wherein the primer set comprises an FIP primer, an F3 primer, a BIP primer and a B3 primer, and at least one set is selected from the group consisting of:
• a primer set 1 wherein the FIP primer consists of a polynucleotide represented by SEQ ID NO:
• 3, the BIP primer consists of a polynucleotide represented by SEQ ID NO:
• 4, the F3 primer consists of a polynucleotide represented by SEQ ID NO:
• 27, and the B3 primer consists of a polynucleotide represented by SEQ ID NO:
• 28, and
• a primer set 2 wherein the FIP primer consists of a polynucleotide represented by SEQ ID NO:
• 5, the BIP primer consists of a polynucleotide represented by SEQ ID NO:
• 6, the F3 primer consists of a polynucleotide represented by SEQ ID NO:
• 29, and the B3 primer consists of a polynucleotide represented by SEQ ID NO:
• 30,
• which are used for amplification of a nucleotide sequence region containing nucleotide sequences coding for amino acids at positions 181 and 204 in the polymerase region of hepatitis B virus;
• a primer set 3 wherein the FIP primer consists of a polynucleotide represented by SEQ ID NO:
• 7, the BIP primer consists of a polynucleotide represented by SEQ ID NO:
• 8, the F3 primer consists of a polynucleotide represented by SEQ ID NO:
• 31, and the B3 primer consists of a polynucleotide represented by SEQ ID NO:
• 32,
• a primer set 4 wherein the FIP primer consists of a polynucleotide represented by SEQ ID NO:
• 9, the BIP primer consists of a polynucleotide represented by SEQ ID NO:
• 10, the F3 primer consists of a polynucleotide represented by SEQ ID NO:
• 31, and the B3 primer consists of a polynucleotide represented by SEQ ID NO:
• 32,
• a primer set 11 wherein the FIP primer consists of a polynucleotide represented by SEQ ID NO:
• 7, the BIP primer consists of a polynucleotide represented by SEQ ID NO:
• 121, the F3 primer consists of a polynucleotide represented by SEQ ID NO:
• 31, and the B3 primer consists of a polynucleotide represented by SEQ ID NO:
• 123, and
• a primer set 12 wherein the FIP primer consists of a polynucleotide represented by SEQ ID NO:
• 9, the BIP primer consists of a polynucleotide represented by SEQ ID NO:
• 122, the F3 primer consists of a polynucleotide represented by SEQ ID NO:
• 31, and the B3 primer consists of a polynucleotide represented by SEQ ID NO:
• 124,
• which are used for amplification of a nucleotide sequence region containing nucleotide sequences coding for amino acids at positions 204 and 236 in the polymerase region of hepatitis B virus;
• a primer set 5 wherein the FIP primer consists of a polynucleotide represented by SEQ ID NO:
• 15, the BIP primer consists of a polynucleotide represented by SEQ ID NO:
• 16, the F3 primer consists of a polynucleotide represented by SEQ ID NO:
• 33, and the B3 primer consists of a polynucleotide represented by SEQ ID NO:
• 34,
• a primer set 6 wherein the FIP primer consists of a polynucleotide represented by SEQ ID NO:
• 17, the BIP primer consists of a polynucleotide represented by SEQ ID NO:
• 18, the F3 primer consists of a polynucleotide represented by SEQ ID NO:
• 35, and the B3 primer consists of a polynucleotide represented by SEQ ID NO:
• 36,
• a primer set 7 wherein the FIP primer consists of a polynucleotide represented by SEQ ID NO:
• 19, the BIP primer consists of a polynucleotide represented by SEQ ID NO:
• 20, the F3 primer consists of a polynucleotide represented by SEQ ID NO:
• 37, and the B3 primer consists of a polynucleotide represented by SEQ ID NO:
• 38, and
• a primer set 8 wherein the FIP primer consists of a polynucleotide represented by SEQ ID NO:
• 21, the BIP primer consists of a polynucleotide represented by SEQ ID NO:
• 22, the F3 primer consists of a polynucleotide represented by SEQ ID NO:
• 39, and the B3 primer consists of a polynucleotide represented by SEQ ID NO:
• 40,
• which are used for amplification of a nucleotide sequence region containing a nucleotide sequence coding for an amino acid at position 236 in the polymerase region of hepatitis B virus;
• and
• a primer set 9 wherein the FIP primer consists of a polynucleotide represented by SEQ ID NO:
• 23, the BIP primer consists of a polynucleotide represented by SEQ ID NO:
• 24, the F3 primer consists of a polynucleotide represented by SEQ ID NO:
• 41, and the B3 primer consists of a polynucleotide represented by SEQ ID NO:
• 42, and
• a primer set 10 wherein the FIP primer consists of a polynucleotide represented by SEQ ID NO:
• 25, the BIP primer consists of a polynucleotide represented by SEQ ID NO:
• 26, the F3 primer consists of a polynucleotide represented by SEQ ID NO:
• 43, and the B3 primer consists of a polynucleotide represented by SEQ ID NO:
• 44,
• which are used for amplification of a nucleotide sequence region containing a nucleotide sequence coding for an amino acid at position 181 in the polymerase region of hepatitis B virus.

The assay kit for detection of the drug resistance of hepatitis B virus whose genotype is type C, wherein the probe containing a polynucleotide derived from a drug-resistant strain of hepatitis B virus and/or the probe containing a polynucleotide derived from a drug-nonresistant strain of hepatitis B virus comprises at least one probe group which depending on the primer set used, is selected from the group consisting of:

• a probe group 1 comprising at least one probe selected from the group consisting of a probe containing a polynucleotide represented by SEQ ID NO:
• 50 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 51 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 52 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 53 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 54 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 55 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 56 or its complementary strand, and a probe containing a polynucleotide represented by SEQ ID NO:
• 57 or its complementary strand,
• a probe group 2 comprising at least one probe selected from the group consisting of a probe containing a polynucleotide represented by SEQ ID NO:
• 58 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 59 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 60 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 61 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 62 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 63 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 64 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 65 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 66 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 67 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 68 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 69 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 70 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 71 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 72 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 73 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 74 or its complementary strand, and a probe containing a polynucleotide represented by SEQ ID NO:
• 75 or its complementary strand, and
• a probe group 3 comprising at least one probe selected from the group consisting of a probe containing a polynucleotide represented by SEQ ID NO:
• 76 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 77 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 78 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 79 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 80 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 81 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 82 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 83 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 84 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 85 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 86 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 87 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 88 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 89 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 90 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 91 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 132 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 133 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 134 or its complementary strand, a probe containing a polynucleotide represented by SEQ ID NO:
• 135 or its complementary strand, and a probe containing a polynucleotide represented by SEQ ID NO:
• 136 or its complementary strand, and each of the probes is 15 to 45 bases in full length.

The assay kit for detection of the drug resistance of hepatitis B virus whose genotype is type C, wherein the primer set is at least one primer set selected from the group consisting of:

• a primer set 1 wherein the FIP primer consists of a polynucleotide represented by SEQ ID NO:
• 3, the BIP primer consists of a polynucleotide represented by SEQ ID NO:
• 4, the F3 primer consists of a polynucleotide represented by SEQ ID NO:
• 27, and the B3 primer consists of a polynucleotide represented by SEQ ID NO:
• 28,
• a primer set 2 wherein the FIP primer consists of a polynucleotide represented by SEQ ID NO:
• 5, the BIP primer consists of a polynucleotide represented by SEQ ID NO:
• 6, the F3 primer consists of a polynucleotide represented by SEQ ID NO:
• 29, and the B3 primer consists of a polynucleotide represented by SEQ ID NO:
• 30,
• a primer set 3 wherein the FIP primer consists of a polynucleotide represented by SEQ ID NO:
• 7, the BIP primer consists of a polynucleotide represented by SEQ ID NO:
• 8, the F3 primer consists of a polynucleotide represented by SEQ ID NO:
• 31, and the B3 primer consists of a polynucleotide represented by SEQ ID NO:
• 32,
• a primer set 4 wherein the FIP primer consists of a polynucleotide represented by SEQ ID NO:
• 9, the BIP primer consists of a polynucleotide represented by SEQ ID NO:
• 10, the F3 primer consists of a polynucleotide represented by SEQ ID NO:
• 31, and the B3 primer consists of a polynucleotide represented by SEQ ID NO:
• 32,
• a primer set 11 wherein the FIP primer consists of a polynucleotide represented by SEQ ID NO:
• 7, the BIP primer consists of a polynucleotide represented by SEQ ID NO:
• 121, the F3 primer consists of a polynucleotide represented by SEQ ID NO:
• 31, and the B3 primer consists of a polynucleotide represented by SEQ ID NO:
• 123, and
• a primer set 12 wherein the FIP primer consists of a polynucleotide represented by SEQ ID NO:
• 9, the BIP primer consists of a polynucleotide represented by SEQ ID NO:
• 122, the F3 primer consists of a polynucleotide represented by SEQ ID NO:
• 31, and the B3 primer consists of a polynucleotide represented by SEQ ID NO:
• 124, and
• the probe containing a polynucleotide derived from a drug-resistant strain of hepatitis B virus and/or the probe containing a polynucleotide derived from a drug-nonresistant strain of hepatitis B virus is a group consisting of a probe consisting of a polynucleotide represented by SEQ ID NO:
• 93 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 96 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 99 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 105 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 108 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 113 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 117 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 119 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 147 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 149 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 152 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 155 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 157 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 158 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 161 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 164 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 167 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 169 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 171 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 173 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 174 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 176 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 179 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 181 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 183 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 184 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 186 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 188 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 190 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 192 or its complementary strand, a probe consisting of a polynucleotide represented by SEQ ID NO:
• 193 or its complementary strand, and a probe consisting of a polynucleotide represented by SEQ ID NO:
• 195 or its complementary strand.