Study On Real-Time PCR-Based Approaches For Multiple Targets Detection

Real-time PCR has become a powerful tool for quantitative and qualitative nucleic acids analysis,and has been broadly applied in the scientific,medical,and diagnostic communities.However,the number of targets that can be detected in a single real-time PCR is limited to four or five due to the limited number of channels in a real-time PCR apparatus.To address this issue,this dissertation focuses on the development of new real-time PCR strategies for multiple targets detection.It consists of two parts.The first part is the development of a novel multiplexing strategy called multicolor combinational probe coding technology,and the second part is the development of the probe melting curve analysis.In the first part,a new strategy that significantly increases the number of targets identifiable in a single real-time PCR was developed.The strategy,termed multicolor combinational probe coding(MCPC) technology,uses fluorophore combinations in addition to single fluorophores to label probes.The combination rule allows n types of fluorophores to label N= C_n~1+C_n~2 +…+C_n~n= 2~n-1 different probes in a combinational manner.Thus,up to 15 probes can be labeled using 4 different fluorophores,and 15 targets can be detected on a 4-color real-time PCR machine.The feasibility of MCPC technology was tested by identifying multiple targets or detecting multiple mutations.In chapter one of this part,the feasibility of MCPC technology was tested by identifying 8 foodborne pathogens in a single real-time PCR.With MCPC,8 pairs of species-specific tagged primers,1 pair of universal primers,and 8 single-labeled or mix-labeled molecular beacon probes were included in a single reaction tube.As demonstrated by both proof-of-principle experiments and blind test of 118 samples, all 8 foodbrone pathogens targeted were accurately identified and distinguished from other pathogens using MCPC technology.In chapter two,the effect of 5′to 3′nuclease activity of Taq DNA polymerase on displacing probes was studied in order for MCPC technology to detect multiple mutations.Using both kinetic studies and matrix-assisted laser desorption ionization time-of-flight mass spectrometry,we comprehensively examined the 5'-nucIease activity of Taq DNA polymerase on fluorogenic displacing probes of varied structures. We observed that displacing probes with unstable 5'-terminal could be hydrolyzed, and the major cleavage was the removal of the 5'-terminal fluorophore-labeled nucleotide.These observations can serve as guidance for better design of displacing probes with reduced or eliminated background for real-time PCR detection.In chapter three,the concept of MCPC technology was extended to multiplex mutations detection in a single gene,and this was exampled by detecting the five common Chineseβ-thalassemia mutations and an internal control in a single real-time PCR.By coupling MCPC technology and Homo-Tag Assisted Non-Dimer System (HANDS),3 pairs of specific tagged primers,1 universal primer,and 6 single-labeled or mix-labeled displacing probes were included in a single reaction tube.Totally 21 genotypes including wild-type,heterozygous,homozygous and compound heterozygous were covered by this assay.The specificity of the assay was 100%when tested against 129 normal,98 carriers,and 12 patients.Accurate results could be acquired with genomic DNA ranging from 100 ng to 10 pg per reaction.The assay provides a rapid,accurate,and sensitive means for detecting multiple p-thalassemia mutations.In the second part,a new probe melting curve analysis strategy was developed. While multiplex PCR by both probe color and melting temperature(T_m) could greatly expand the power of real-time PCR analysis for multiple targets detection,the existing fluorogenic probes for this aim are inherently limited in combining color multiplexing and T_m multiplexing.Thus,in the first chapter,a variety of fluorogenic probes were evaluated regarding their potentials in melting curve analysis.The result showed that nearly all probes studied could be used for melting curve analysis. Particularly interesting finding was that the self-quenching probes and shared-stem molecular beacons,which were never used for melting curve analysis before,were found to be comparable with the FRET probe.Shared-stem molecular beacons were advantageous in their high signal to noise ratio,narrow melting peak,and more important,in their flexibility of combining color multiplexing and T_m multiplexing.In the second chapter,a new probe melting analysis strategy was established by taking advantage of the shared-stem molecular beacons.The significance of this strategy was that several mutations could be detected by a single probe,thus more mutations could be detected by several differently labeled probes in one reaction.For a proof-of-principle study,more than 15 mutations ofβ-globin gene were detected through the new probe melting curve strategy.The specificity of the multiplex assays was 100%for the detection of 118 clinical samples.Accurate results could be acquired with genomic DNA ranging from 50 ng to 5 pg per reaction.We concluded that the new probe melting curve analysis strategy provided a rapid,accurate, sensitive,low-cost,and high-throughput means for detecting multiple mutations.