Olmos Antonio, Bertolini Edson, Cambra Mariano
Laboratorio de Virología e Inmunología, Departamento de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), Carretera Moncada-Náquera Km 5, 46113 Moncada, Valencia, Spain.
J Virol Methods. 2007 Jan;139(1):111-5. doi: 10.1016/j.jviromet.2006.09.012. Epub 2006 Nov 7.
A nucleic acid sequence-based amplification method coupled with rapid flow-through hybridisation (NASBA-FH) was developed for diagnosis of Plum pox virus (PPV). The sensitivity level achieved by NASBA-FH was 10 times higher than that obtained by Co-PCR and 1000 times higher than the sensitivity afforded by RT-PCR. In addition, samples from 262 stone-fruit trees collected during winter and spring seasons were analysed. These samples were tested using methods recommended by the European and Mediterranean Plant Protection Organization to detect PPV (DASI-ELISA, RT-PCR and Co-PCR) and by NASBA-FH. Winter PPV diagnostic results by ELISA and NASBA-FH coincided in 90.8%, while ELISA and PCR-based methods coincided in 91.6% and PCR-based methods with NASBA-FH agreed in 95.4%. In spring, diagnostic results were similar with all the molecular techniques, which agreed with ELISA results for 98.8% of the trees. NASBA-FH was able to detect more positive infections in winter, which were later confirmed in spring. These results indicate that NASBA-FH is a suitable molecular method for routine PPV detection in the winter and spring. This user-friendly isothermal RNA amplification coupled with a very fast flow-through hybridisation (15 min) opens up new possibilities for rapid and reliable diagnosis of a variety of pathogens.
开发了一种基于核酸序列扩增方法并结合快速流通杂交技术（NASBA-FH）用于李痘病毒（PPV）的诊断。NASBA-FH达到的灵敏度水平比共聚合酶链反应（Co-PCR）高10倍，比逆转录聚合酶链反应（RT-PCR）的灵敏度高1000倍。此外，对冬春季节采集的262株核果类树木的样本进行了分析。这些样本使用欧洲和地中海植物保护组织推荐的检测PPV的方法（双抗体夹心酶联免疫吸附测定法（DASI-ELISA）、RT-PCR和Co-PCR）以及NASBA-FH进行检测。ELISA和NASBA-FH对冬季PPV的诊断结果一致性为90.8%，而ELISA与基于PCR的方法一致性为91.6%，基于PCR的方法与NASBA-FH的一致性为95.4%。在春季，所有分子技术的诊断结果相似，与ELISA结果对98.8%的树木一致。NASBA-FH能够在冬季检测到更多的阳性感染，这些感染在春季得到了证实。这些结果表明，NASBA-FH是冬春季节常规检测PPV的合适分子方法。这种用户友好的等温RNA扩增技术结合非常快速的流通杂交技术（15分钟）为快速可靠地诊断多种病原体开辟了新的可能性。
