Detection by PCR of the silkworm infected with Nosema bombycis

家蚕感染家蚕微孢子虫的PCR检测

• 批准号: 06660068
• 负责人: ISHIHARA Ren
• 金额: $ 1.15万
• 依托单位: Nihon University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06660068/
• 关键词: Nosema bombycis; Microsporidia; silkworm; Bombyx mori; diagnosis; PCR

Primers for polymerase chain reaction (PCR) were designed, which were based on the sequence of DNA fragment from Nosema bombycis. One pair of primers successfully amplified DNA extracted from either N.bombycis spores or silkworms (pupae or moths) harboring N.bombycis. The extent of specificity of those primers was tested, using reference stocks of N.bombycis, N.bombycis-like-isolates i.e.one from Spodoptera depravata and the other from S.exigus, Nosema necatrix, and Vairimorpha sp. isolates M_<11> and M_<12>. Of DNA from those microsporidia, DNA from reference stocks of N.bombycis only was amplified, while DNA from all microsporidia used were amplified by the primers which are known to amplify consensus part of small subunit rDNA (SSUrDNA). The sequences of SSUrDNA of the reference stock of N.bombycis and that of S.depravata isolate were determined and compared each other. The length of both rDNA was the same, 1226 bp.Of them, nine bps near c-terminal, ranging from 1201th to 1225th, were different. They appeared to be closely related, but the primers were sensitive enough to discrimiate between them.Investigations are now underway to apply this detection method to the silkworm female moth examination for pebrine.

根据家蚕微孢子虫DNA片段的序列设计了PCR引物。一对引物成功地扩增了从携带N. bombycis的N.bombycis孢子或蚕（蛹或蛾）中提取的DNA。使用来自Spodoptera depravata的N.bombycis、N. bombycis样分离株i.e.one以及来自S.exigus、Nosema necatrix和Vairimorpha sp.分离株M_和M_的另一<11>种参考株，测试了这些引物的特异性程度<12>。在这些微孢子虫的DNA中，仅扩增了来自家蚕微孢子虫参考株的DNA，而使用的所有微孢子虫的DNA均由已知可扩增小亚基rDNA共有部分（SSUrDNA）的引物扩增。对家蚕标准株和腐败链霉菌分离物的SSUrDNA序列进行了测定和比较。两种rDNA的长度相同，均为1226 bp，其中C端有9个bp不同，在1201 ~ 1225位之间。这两种微粒子病的检测方法虽然看起来亲缘关系很近，但引物的灵敏度足以将它们区分开来。

项目成果

Y.Kawakami,T.Inoue,Y.Uchida,Y.Hatakeyama,H.Iwano and R.Ishihara: "Specific amplification of DNA from reference strains of Nosema bombycis(Nosematidae,Microsporidia)" The Journal of Sericultural Science of Japan. 64(in press). (1995)

Y.Kawakami、T.Inoue、Y.Uchida、Y.Hatakeyama、H.Iwano 和 R.Ishihara：“来自家蚕微孢子虫（微孢子虫科、微孢子虫）参考菌株的 DNA 的特异性扩增”日本蚕业科学杂志。

Hidetoshi Iwano and Timothy J.Kurtti: "Identification and isolation of dimorphic spores from Nosema furnacalis (Microspora : Nosematidae)" J.Invertebrate Pathology. Vol.65, No.3. 230-236 (1995)

Hidetoshi Iwano 和 Timothy J.Kurtti：“玉米微孢子虫（微孢子虫：Nosematidae）二形孢子的鉴定和分离”J.无脊椎动物病理学。

Y.Kawakami,T.Inoue,Y.Uchida,Y.Hatakeyama,H.Iwano and R.Ishihara: "Specific amplification of DNA from reference strains of Nosema bombycis (Nosematidae,Microsporidia)" Journal sericultural science of Japan. 64. 165-172 (1995)

Y.Kawakami，T.Inoue，Y.Uchida，Y.Hatakeyama，H.Iwano 和 R.Ishihara：“来自家蚕微孢子虫（微孢子虫科，微孢子虫）参考菌株的 DNA 的特异性扩增”日本蚕业科学杂志。

Yuji Kawakami, Tadashi Inoue, Youko Uchida, Yoshihori Hatakeyama, Hidetoshi Iwano and Ren Ishihara: "Specific amplication of DNA from freference strains of Nosema bombycis" J.Seric.Sci.Jpn.Vol.64, No.2. 165-172 (1995)

Yuji Kawakami、Tadashi Inoue、Youko Uchida、Yoshihori Hatakeyama、Hidetoshi Iwano 和 Ren Ishihara：“来自家蚕微孢子虫参考菌株的 DNA 的特异性扩增”J.Seric.Sci.Jpn.Vol.64，No.2。

Hidetoshi Iwano & Timothy J.Kurtti: "Identification and isolation of dimorphic spores from Nosema furnacalis (Microspora : Nosematidae)" Journal of invertebrate pathology. 65. 230-236 (1995)

岩野秀俊