Abstract The abaca (Musa textilis Nee) is an important fibre crop in the Philippines. Its fibre yield and quality is severely impacted by two potyviruses, namely, banana bract mosaic virus (BBrMV) and sugarcane mosaic virus (SCMV). A multiplex RT-PCR assay was developed for the simultaneous detection of these two RNA viruses in abaca. Primer sets were designed to amplify fragments of BBrMV coat protein (743 bp), SCMV coat protein (595 bp) and Musa sp. elongation factor 1 (219 bp) as an internal control. The specificity of each primer pair was confirmed by cloning and sequencing of the RT-PCR products. The multiplex RT-PCR assay was optimized by varying primer concentration combinations, dNTP concentration, Mg2+ concentration, Taq polymerase concentration, annealing temperature and extension temperature. The developed multiplex RT-PCR protocol has the same level of sensitivity and specificity when compared with uniplex RT-PCR detection for each virus and internal control. The inclusion of internal control amplification eliminates the risk of obtaining false negative results. The use of the optimized multiplex RT-PCR in simultaneous detection of BBrMV and SCMV from field samples proves that it is reliable, robust and faster than uniplex RT-PCR detection of each virus. This method will be useful for large-scale surveys of these two RNA viruses in abaca and will be vital for epidemiological studies and disease prevention.

中文翻译：

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多重逆转录聚合酶链反应同时检测蕉麻中的香蕉苞叶病毒 (BBrMV) 和甘蔗花叶病毒 (SCMV)

摘要 蕉麻（Musa textilis Nee）是菲律宾重要的纤维作物。其纤维产量和质量受到两种花叶病毒的严重影响，即香蕉苞叶病毒（BBrMV）和甘蔗花叶病毒（SCMV）。为同时检测蕉麻中的这两种 RNA 病毒开发了一种多重 RT-PCR 检测方法。引物组旨在扩增 BBrMV 外壳蛋白 (743 bp)、SCMV 外壳蛋白 (595 bp) 和 Musa sp. 的片段。延伸因子 1 (219 bp) 作为内部对照。每个引物对的特异性通过 RT-PCR 产物的克隆和测序得到证实。通过改变引物浓度组合、dNTP 浓度、Mg2+ 浓度、Taq 聚合酶浓度、退火温度和延伸温度来优化多重 RT-PCR 检测。与针对每种病毒和内部对照的单重 RT-PCR 检测相比，开发的多重 RT-PCR 协​​议具有相同水平的灵敏度和特异性。包含内部控制放大消除了获得假阴性结果的风险。使用优化的多重 RT-PCR 同时检测现场样本中的 BBrMV 和 SCMV 证明，它比对每种病毒的单一 RT-PCR 检测更可靠、稳健且速度更快。这种方法将有助于对蕉麻中这两种 RNA 病毒的大规模调查，对流行病学研究和疾病预防至关重要。使用优化的多重 RT-PCR 同时检测现场样本中的 BBrMV 和 SCMV 证明，它比对每种病毒的单一 RT-PCR 检测更可靠、稳健且速度更快。这种方法将有助于对蕉麻中这两种 RNA 病毒的大规模调查，对流行病学研究和疾病预防至关重要。使用优化的多重 RT-PCR 同时检测现场样本中的 BBrMV 和 SCMV 证明，它比对每种病毒的单一 RT-PCR 检测更可靠、稳健且速度更快。这种方法将有助于对蕉麻中这两种 RNA 病毒的大规模调查，对流行病学研究和疾病预防至关重要。