Detection of Papaya ringspot virus resistant papaya in food by semi-quantitative and real-time PCR

• Main Authors: Yu-Fan Kao, 高瑜璠
• Other Authors: 方繼
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/31442854180726335507

碩士 === 國立中興大學 === 食品暨應用生物科技學系 === 94 === In recent years, numerous analytical methods have been developed to determine genetically modified organisms (GMOs). These methods can be devided into qualitative and quantitative detections. In this study, a semi-quantitative PCR was developed to detect transgenic papaya resistant to Papaya ringspot virus (PRSV). We expected that semi-quantitative PCR could be instead of qualitative PCR and qualtated and quantitated GM content in food simultaneously to decrease the numbers of samples quantitated by real-time PCR. We chosed two primer pairs, CP-1F/CP-1R(143 bp) used to amplify transgene in papaya designed by our laboratory and Pn-1F/Pn-1R (211 bp) used to amplify the endogenous gene collected from reference and the primers, CP-1F/CP-1R, had good specificity in selecting transgenic papaya. In simplex PCR detection system, the detection limit of the system was 0.1%, and according to the concentration of GM and the ratio of brightness of the correlation coefficient of semi-quantitative standard curve was 0.9755. In duplex PCR system, the two primers didn’t amplify unexpected fragments in one tube, the detection limit of the system was also 0.1% and the correlation coefficient of semi-quantitative standard curve was 0.9824. It showed that duplex PCR system was more accurate and consume fewer time and cost than simplex PCR. The real-time PCR used SYBR Green Ⅰsystem, the correlation coefficients of quantitative standard curve was 0.9995. To compare real-time PCR and semi-quantitative PCR system, we obtained although the accuracy and stability of semi-quantitative PCR system were lower than real-time PCR but it could separate samples from more than 8% or less than 3% . To evaluate the cost of semi-quantitative PCR and real-time PCR, we combined with instrument and procedure of detection and obtained that cost of semi-quantitative PCR was 7 times lower than real-time PCR. Analysis of papaya endogene by simplex PCR with Pn-1F/Pn-1R primers in processed food, we found that DNA would breakdown by processing steps, included dried, low pH, complex manufacture and the sugar content in samples affected the efficiency of PCR detection. Analysis of papaya transgene by duplex PCR with CP-1F/CP-1R and Pn-1F/Pn-1R primers in processed food, we obtained two samples contained coat protein gene of PRSV from seven samples and determined GM content in the two samples.