Olmos Antonio, Bertolini Edson, Gil Maite, Cambra Mariano
Departamento de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), Carretera Moncada-Náquera km 5, 46113 Moncada, Valencia, Spain.
J Virol Methods. 2005 Sep;128(1-2):151-5. doi: 10.1016/j.jviromet.2005.05.011.
A TaqMan real-time RT-PCR was developed to detect and quantify RNA-targets from the non-circulative, non-persistently transmitted Plum pox virus (PPV) in individual fresh or aphids captured previously and squashed on paper. Reliable quantitation ranged from 40 up to 4 x 10(8) copies of control transcripts. This technique was applied successfully to plant material and to individual PPV vector (Myzus persicae) and non-vector of PPV (Aphis nerii) aphid species demonstrating acquisition of viral targets by both vector and non-vector aphids. The number of viruliferous aphids detected by real-time RT-PCR and nested RT-PCR in a single closed tube was similar in parallel assays, nevertheless the sensitivity provided by real-time RT-PCR was 100 times higher than nested RT-PCR and 1000 times higher than DASI-ELISA and conventional RT-PCR. The quantities of PPV-RNA targets detected in a single aphid ranged from 40 to more than 2 x 10(3) units. The combined system (immobilization of targets on paper by squash capture and real-time RT-PCR) allows, for the first time, reliable quantitation of PPV targets acquired by individual aphid species and constitute an excellent tool for understanding better PPV epidemiology.
开发了一种TaqMan实时逆转录聚合酶链反应（RT-PCR），用于检测和定量先前捕获并压在纸上的单个新鲜蚜虫或蚜虫中来自非循环、非持久性传播的李痘病毒（PPV）的RNA靶标。可靠定量范围为对照转录本的40至4×10⁸拷贝。该技术已成功应用于植物材料以及单个PPV载体（桃蚜）和PPV非载体（夹竹桃蚜）蚜虫物种，证明载体和非载体蚜虫均能获取病毒靶标。在平行试验中，在单个封闭管中通过实时RT-PCR和巢式RT-PCR检测到的带毒蚜虫数量相似，然而，实时RT-PCR提供的灵敏度比巢式RT-PCR高100倍，比DASI-ELISA和传统RT-PCR高1000倍。在单个蚜虫中检测到的PPV-RNA靶标数量范围为40至超过2×10³单位。该组合系统（通过挤压捕获将靶标固定在纸上并进行实时RT-PCR）首次实现了对单个蚜虫物种获取的PPV靶标的可靠定量，是更好地理解PPV流行病学的优秀工具。
