Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/21/2012
Publication Date: 8/1/2012
Citation: Dey, K.K., Lin, H., Borth, W.B., Melzer, M.J., Hu, J. 2012. A highly sensitive single-tube nested PCR assay for the detection of Pineapple mealybug wilt associated virus-2 (PMWaV-2). Journal of Virological Methods. 183(2):215-218. Interpretive Summary: The pineapple mealy bug wilt associated viruses are a complex of viruses that infect pineapple. In this study, a new diagnostic method was developed for the detection of Pineapple mealybug wilt associated virus-2 (PMWaV-2). In contrast to the conventional nested PCR in which the reactions are carried out in two separate tubes, this new nested PCR detection assay was performed in a single tube. This new detection system is composed of two sets of primer pairs working at different annealing temperatures. The advantages of this single-tube assay are reduced handling time and elimination of cross contamination, while maintaining high sensitivity for reliable detection.
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.