Esvaran Vijaya Gowri, Mohanasundaram Aarthi, Mahadeva Shruthi, Gupta Tania, Ponnuvel Kangayam M
Genomics Division, Seribiotech Research Laboratory, Carmelaram - Post, Kodathi, Bangalore, 560035 India.
J Parasit Dis. 2019 Mar;43(1):31-38. doi: 10.1007/s12639-018-1053-4. Epub 2018 Nov 26.
Microsporidiosis (Pebrine) caused by the microsporidian parasite is one of the important devastating disease which affect the silk production leading to an unprofitable harvest. Till date ribosomal RNA (rRNA) gene was used as a target for detection of microsporidian species. In this study, we describe conventional and SYBR green based real-time PCR techniques alternatively targeting β-tubulin gene for quantitative detection of microsporidia infecting both the mulberry and non-mulberry silkworms. The modified DNA extraction method followed in our study was found to be easy, economical and could be used for both conventional and real time PCR as template. The real time qPCR revealed the expression of β-tubulin gene in different infected tissues of the silkworm . The sensitivity of the SYBR green based real time PCR was found to be 100 times more than the conventional PCR and PCR was found more sensitive than the microscopic examination. The developed method did not produce any false positive results with the other silkworm pathogens and healthy silkworm. The data suggest that both the developed PCR methods targeting β-tubulin gene could be used effectively in quarantine process at seed centres for early detection of microsporidian infection in silkworms.
由微孢子虫寄生虫引起的微孢子虫病（胚种病）是影响丝绸生产导致收获无利可图的重要毁灭性疾病之一。迄今为止，核糖体RNA（rRNA）基因一直被用作检测微孢子虫物种的靶标。在本研究中，我们描述了传统的和基于SYBR Green的实时PCR技术，它们交替靶向β-微管蛋白基因，用于定量检测感染桑蚕和非桑蚕的微孢子虫。我们研究中采用的改良DNA提取方法简便、经济，可同时用于传统PCR和实时PCR作为模板。实时定量PCR揭示了β-微管蛋白基因在蚕不同感染组织中的表达。基于SYBR Green的实时PCR的灵敏度比传统PCR高100倍，且PCR比显微镜检查更灵敏。所开发的方法对其他蚕病原体和健康蚕未产生任何假阳性结果。数据表明，两种针对β-微管蛋白基因开发的PCR方法均可有效地用于种子中心的检疫过程，以早期检测蚕中的微孢子虫感染。
