Improvement in molecular detection of phytoplasma associated with rose by selection of suitable primers and development of a multiplex PCR assay

被引：0

作者：

Tasou Rihne

Kanwar Pal Namita

M. K. Singh

Akshay Singh

机构：

[1] ICAR-Indian Agricultural Research Institute,Division of Floriculture and Landscaping

[2] ICAR-Indian Agricultural Research Institute,Division of Genetics

来源：

3 Biotech | 2021年 / 11卷

关键词：

× ; DNA extraction; Phytoplasma asteris; Nested PCR assays; gene; gene;

D O I：

暂无

中图分类号：

学科分类号：

摘要：

Sensitive and effective phytoplasma DNA amplification in symptomatic rose cultivars is a long unresolved problem. In the present study, improvement in standardization for PCR assay for phytoplasma detection was established with rose samples by selection of various combinations of nested primer pairs of 16S ribosomal gene and secA gene. CTAB DNA extraction method was slightly modified by adding 2% polyvinyl pyrrolidone and increased the isopropanol volume which yielded better quality DNA. Best amplification results were achieved in nested PCR assay employing P1/P7, R16mF2/R16mR2 and R16F2n/R16R2, P1/P7 and R16mF2/R16mR2, and R16mF2/R16mR2 and fU5/rU3 primer pairs. Besides, a multiplex PCR assay was also developed and optimized for consistent identification of phytoplasma in rose samples by employing primer pairs of 16S rRNA and secA genes together in a single PCR reaction by optimizing annealing temperature at 55 °C.

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