- 著者
-
川俣 治
- 出版者
- 新潟医学会
- 雑誌
- 新潟医学会雑誌 = 新潟医学会雑誌 (ISSN:00290440)
- 巻号頁・発行日
- vol.111, no.10, pp.633-646, 1997-10
In order to detect enterovirus and rhinovirus sequences as separately for the virus genera by single test of RT-PCR (reverse transcription-polymerase chain reaction), two sets of the primers were prepared covering the 5'-NTR (nontranslated region) to the VP4 in the virus genome for amplification. The sequences selected for detection included a deletion of ca. 120 b (bases) stretch in the rhinovirus genome so as to get the products separate in the electrophoretic mobilities for each virus groups. The first set of the primers consisted of P3 (5'-GGCCCCTGAATGCGGCTAAT-3') and P5 (3'-GTTCTGGGATCATTTAAGTG-5') and the second was of P4 (5'-ACTTTGGGTGTCCGTGTTTC-3') and P5;the P3 and P4 (for a closer site to the P5 compared to the P3 site) were complementary to the sequences in the 5'-NTR of 95~100% homology and P5 being in the VP4 region complementary to the sequence of 60~90% homology through the two virus groups, each. By considering the relatively low homology in the counterpart site, the P5 primer was employed for initiation of the reverse transcription at a low temperature of 37℃ for promoting the primer annealing to the template RNA. When applied to the prototype strains, the P3-P5 primers gave a single band equivalent to 460 b DNA for the enteroviruses (43/44 serotypes) and that of 340 b DNA for the rhinoviruses (8/8 serotypes), thus enabling to easily discriminate the two virus groups on the separate mobilities of the products. Similarly, the products by the P4-P5 primers were of 340 b and 220 b DNAs for enteroviruses (43/44 serotypes) and rhinoviruses (8/8 serotypes), respectively, again being of easy discrimination of the virus groups. Of these experiments, authenticity of the products was confirmed by base sequence analysis with selected product samples from the experimental groups. The primers prepared were also compared for the efficiencies of virus detection in the clinical specimens with the tissue culture method for virus isolation. Positive ratios resulted were 65/96 by the RT-PCR and 54/96 by the tissue culture method. The primers thus proved effective for rapid and discriminative detection of enteroviruses and rhinoviruses to the laboratory diagnosis.