Rotavirus genotype detection method and gene amplification primer set used in same
View Patent ↗The present invention addresses the problem of finding a new gene detection primer capable of more accurately detecting genotypes of rotavirus A and a detection means using the gene detection primer, and provides a rotavirus genotype detection method, a gene amplification primer set used in this method, and a genotype detection kit. In the rotavirus genotype detection method, (A) using a base sequence at positions from 174 to 834 of a VP7 gene segment of a G1 type strain of the rotavirus A as a base sequence counting reference, sense primers each aligned to one to four kinds of base sequence regions selected from (1) the positions from 802 to 834 (corresponding to G3 type), (2) the positions from 747 to 789 (corresponding to equine-like G3 type), (3) the positions from 603 to 636 (corresponding to G9 type), and (4) the positions from 666 to 711 (corresponding to G12 type) are used as basic gene amplification sense primers, and (B) using the above positions from 883 to 1062 as the counting reference, antisense primers each aligned to a region of the base sequence of the counting reference are used as gene amplification common antisense primers.
1 . A method for diagnosing a genotype of a rotavirus infecting a patient comprising:
obtaining a gene from a specimen;
amplifying the gene by applying a PCR method to the gene obtained from the specimen to obtain a gene amplification product; and
determining the genotype of the rotavirus infecting the patient from the gene amplification product;
wherein the amplifying step comprises hybridizing the gene to sense primers (A) and common antisense primers (B),
wherein the sense primers (A) are selected from one or more of the following (A)(1) to (A)(4):
(A)(1) sense primers including the entire sequence of SEQ ID NO: 3;
(A)(2) sense primers including the entire sequence of SEQ ID NO: 4;
(A)(3) sense primers including the entire sequence of SEQ ID NO: 7; and
(A)(4) sense primers including the entire sequence of SEQ ID NO: 8,
and wherein the common antisense primers (B) include the entire sequence of SEQ ID NO: 9.
2 . The method according to claim 1 , wherein sense primers (A) further includes one to four kinds selected from the following (A)(5) to (A)(8):
(A)(5) sense primers including the entire sequence of SEQ ID NO: 1;
(A)(6) sense primers including the entire sequence of SEQ ID NO: 2;
(A)(7) sense primers including the entire sequence of SEQ ID NO: 5; and
(A)(8) sense primers including the entire sequence of SEQ ID NO: 6.
3 . The method according to claim 1 ,
wherein the gene is in the form of an RNA,
wherein between the obtaining step and the amplifying step, an RT-PCR step is used to obtain a template for the amplifying step,
wherein the amplifying step is a second amplification step in a nested-PCR method
wherein, the RT-PCR step comprises hybridizing the RNA with common outer sense primers (O)(1) including the entire sequence of SEQ ID NO: 10, and with common outer sense primers (O)(2) including the entire sequence of SEQ ID NO: 11.