A Multiplex RT-Nested PCR Assay with Newly Designed Primers to Detect and Analyze Human Parainfluenza Viruses Type 1, 2, 3, and 4 from Clinical Specimens

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  • 臨床検体からパラインフルエンザウイルス1，2，3，4 型を検出・遺伝子解析するための新たなMultiplex-RT-Nested PCR 法の検討
  • リンショウケンタイ カラ パラインフルエンザウイルス 1,2,3,4ガタ オ ケンシュツ ・ イデンシ カイセキ スル タメ ノ アラタ ナ Multiplex-RT-Nested PCRホウ ノ ケントウ

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Abstract

<p>The Multiplex-RT-Nested-PCR assay, as previously reported, has been used widely to detect Human parainfluenza virus (HPIV). The assay has high sensitivity to HPIV genes and the capability of typing HPIV. However, the lengths of amplified products by its nested-PCR are too short for constructing phylogenetic trees.<BR> Therefore, we tried to develop a novel Multiplex-RT-Nested-PCR assay that has the ability to amplify the gene products with sufficient length to construct phylogenetic trees as well as high sensitivity for HPIV genes. We designed new primers with reference to base sequences of the HN region and we could amplify various length of PCR products with its nested-PCR, 652 bp in type 1, 950 bp in type 2, 843 bp in type 3, and 552 bp in type 4.<BR> We detected HPIV genes with the novel assay from all 193 clinical specimens, the same as the assay previously reported.<BR> In addition, the novel assay showed a higher percentage of specimens that were positive for HPIV by the 1st-PCR (82.4%), compared with the previously-reported assay (50.3%). The novel assay has no cross reactivity with the other 12 kinds of viruses that cause respiratory illness.<BR> Moreover, we could construct the phylogenetic trees from the sequences with over 500 nt length acquired by the novel assay. By the phylogenetic tree analysis, most of the sequences were classified to existing reported clusters and subtypes.<BR> In conclusion, the newly developed Multiplex-RT-Nested-PCR assay was useful to detect and analyze HPIV genes from clinical specimens.</p>

Journal

  • Kansenshogaku Zasshi

    Kansenshogaku Zasshi 94 (1), 86-96, 2020-01-20

    The Japanese Association for Infectious Diseases

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