Integrated biotech’s differential detection on syndrome-related pathogens

• Main Authors: Hou Yung-Hua, 侯永華
• Other Authors: Chang Chun-Fan
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/32290124047112230855

碩士 === 中國文化大學 === 生物科技研究所 === 98 === Syndrome-related pathogens (SRP) within specified detection tasks as of challenging detection practice still demands effective strategic platform towards realizing the issue-related differential detection value. With the SRP including virus and bacteria, this thesis proposes a technical platform with strategic solutions towards cracking nutshell issues of whole-set same-time differential detection as to be needed for symptom related viruses group. The specific practical example in the thesis pertains to 9 potyviruses with hard impacts on cut flower production and market value such as Calla lily for preparing cDNA specimens applied in differential detection. Different from conventional PCR detection with unique sequence primers of Nib and CP genes, the minimum-set multiuse-primer amplification (MMA) of 11 primers for performing dual-phase PCR are designed by primer/probe design algorithm (PDA) countermeasures program for implementing the detection platform in order to accomplish whole-set same-time differential detection. During dual phase MMA with minimum-set multiuse-primers, the qPCR analysis is simultaneously performed with specified 3 real-time TaqMan probes among PDA designed unique sequence probes in order for immediate detection on early-phase low-dose severe viral infections. The primer dosage with 200 nM over 400 nM reveals better resolution of Ct linear tendency while plotted at serially diluted templates. With the established standard procedure for qPCR quantization, the assigned reference feature values on the optimal sigmoid kinetic curve with early phase signal recovery may form efficient calibration curve and reveal interference trend. The measurement of initial target quantity maybe enhanced with the interference trend as of target cDNA against high background noise cDNA may thus be feasible for immediate detection on early-phase low-dose severe viral infections. Subsequently with general gel DNA sizing of genotype polymorphism (GTP), the MMA yield at 1/3 amount is separated in gel electrophoresis for low cost differential detection among high-dose mid-to-mild viral infections. The detection sensitivity limit of gel sizing with MMA yield is at 1 fmol level of pure reference DNA templates and at 50 nG total cDNA or lower verified with Actin cDNA detection. Finally with probe array hybridization (PAH), the GTP failed MMA yield from respective cDNA with ZaMV and ZaMMV infection is applied in hybridization for feasible whole set differential detection among low-dose mid-to-mild viral infections. The detection sensitivity limit of PAH with MMA yield of mixed ZaMV/ZaMMV cDNA is at less than 1 fmol level of pure reference DNA templates and at less than 50 nG of mixed ZaMV/ZaMMV cDNA.