Department of Plant and Environmental Protection Sciences, University of Hawaii at Manoa, 3190 Maile Way, Honolulu, HI 96822, USA.
J Virol Methods. 2012 Aug;183(2):215-8. doi: 10.1016/j.jviromet.2012.03.025. Epub 2012 Mar 30.
An assay was developed for the detection of Pineapple mealybug wilt associated virus-2 (PMWaV-2), an important factor in the etiology of mealybug wilt of pineapple. The assay combines reverse transcription of RNA isolated from pineapple with a specific and very sensitive, single, closed-tube nested polymerase chain reaction (PCR) to amplify a segment of the coat protein gene of the PMWaV-2. The outer primers were designed to anneal at higher temperatures than the nested primers to prevent primer competition in consecutive amplification reactions. To reduce potential competition further, the outer primers were used at one-thousandth the concentration of the nested primers. The specificity and sensitivity of this assay are much greater than PCR using only a single primer-pair. A TaqMan(®) probe was also designed for use in quantitative PCR to detect and quantify the PCR amplification products directly in a single-tube assay. The advantages of the single-tube assays using both conventional and quantitative PCR are reduced handling time and prevention of cross contamination compared to regular nested PCR in which the reactions are carried out in two separate tubes.
开发了一种用于检测菠萝绵粉蚧萎蔫相关病毒 2（PMWaV-2）的检测方法，PMWaV-2 是菠萝绵粉蚧萎蔫病病因学中的一个重要因素。该检测方法结合了从菠萝中分离的 RNA 的反转录，以及特异性和非常敏感的单一封闭管嵌套聚合酶链反应（PCR），以扩增 PMWaV-2 外壳蛋白基因的一段。外引物的退火温度设计得比嵌套引物高，以防止连续扩增反应中的引物竞争。为了进一步减少潜在的竞争，外引物的使用浓度是嵌套引物的千分之一。与仅使用单个引物对的 PCR 相比，该检测方法的特异性和灵敏度要高得多。还设计了 TaqMan(®)探针，用于定量 PCR，可在单个管检测中直接检测和定量 PCR 扩增产物。与常规嵌套 PCR 相比，使用这两种常规和定量 PCR 的单管检测具有减少处理时间和防止交叉污染的优势，因为常规嵌套 PCR 反应需要在两个单独的管中进行。
