- 期刊
Using Multiplex RT-PCR Assay for Detection and Differentiation of Three Pepper-Infecting Viruses
使用多重RT-PCR檢測和區分3種感染甜椒病毒
摘要
A multiplex reverse transcription-polymerase chain reaction (multiplex RT-PCR) method was developed to enable the simultaneous detection and differentiation of three tobamoviruses infecting peppers, namely pepper mild mottle virus (PMMoV), tobacco mosaic virus (TMV), and tomato mosaic virus (ToMV). The differentiation was achieved using 3 optimized specific oligonucleotide primer pairs, including 1 universal primer for detecting all tobamoviruses and 3 virus-specific primers as forward in the multiplex RT-PCR. The amplification of these three target viruses was finely tuned by analyzing the PCR primer ratios. The multiplex RT-PCR products generated distinct fragments: 519 base pairs (bp) for PMMoV, 228 bp for TMV, and 177 bp for ToMV. Importantly, this method exhibits a high level of specificity, as no products were amplified from non-target pepper virus RNAs as templates. Additionally, it has been demonstrated that multiplex RT-PCR is a virus-specific, sensitive, and cost-effective method for the multiple detection of pepper-infecting tobamoviruses in the field.
並列摘要
本研究利用多重反轉錄聚合酶鏈鎖反應（multiplex reverse transcription-polymerase chain reaction；multiplex RT-PCR）方法，同時檢測與區分感染甜椒的3種tobamoviruses，分別是辣椒輕斑駁病毒（pepper mild mottle virus；PMMoV）、菸草嵌紋病毒（tobacco mosaic virus；TMV）及番茄嵌紋病毒（tomato mosaic virus；ToMV）。使用1種用於檢測所有tobamoviruses的通用引子與3種病毒專一性引子透過分析PCR引子比率，對這3種目標病毒的擴增進行調整。產生的PCR產物由不同的片段組成：PMMoV為519 base pairs（bp），TMV為228bp及ToMV為177bp。本方法表現出高度的專一性，沒有與其他非目標甜椒病毒擴增產生特定的產物。此外，multiplex RT-PCR已被證明是對檢測甜椒植物中多種tobamoviruses，為一種特異性、靈敏且經濟有效的方法。