Technical Abstract: 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 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. Specificity and sensitivity of this assay are much greater than conventional PCR using only a single primer-pair. A TaqMan® probe also was designed for use in quantitative PCR to detect and quantify 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.