| Literature DB >> 33927981 |
Tasou Rihne1, Kanwar Pal Singh1, M K Singh1, Akshay Talukdar2.
Abstract
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. © King Abdulaziz City for Science and Technology 2021.Entities:
Keywords: 16S rRNA gene; Candidatus Phytoplasma asteris; DNA extraction; Nested PCR assays; Rosa × hybrida; secA gene
Year: 2021 PMID： 33927981 PMCID： PMC7988127 DOI： 10.1007/s13205-021-02713-y
Source DB: PubMed Journal: 3 Biotech ISSN： 2190-5738 Impact factor: 2.406