Amplification Sensitivity And Specificity Of QPCR Influenced By Nucleotide Mismatches Of Primers And Probes

Polymerase chain reaction(PCR)is one of the most powerful diagnosis techniques in molecular biology.The efficacy of PCR is mainly determined by its specificity and efficiency.Amplification efficiency is influenced by a number of factors and the design of primers and probes have a major impact on the PCR amplification.Though there are a set of parameters of common primer design programs,the study of mismatch behavior between primers and templates which may lead to a non-specific amplification or a decrease in amplification efficiency that is still lacking and not systematically investigated and haven't proved to be applicable to clinical applications.Historically,there were no relevant researches on the performance of PCR influenced by probe mismatches.In order to improve the efficiency and reliability of PCR,it is necessary to study the influence of amplification efficiency of the mismatches in primers and the probes.In chapter two of this thesis,we observe single and more mismatches behavior containing different numbers,positions,nucleotide types which were introduced in primers on amplifying the whole nucleic acid of Chlamydia pneumonia.All kinds of 3'-terminal mismatches were included in this experiment and we also considered the impact of DNA Taq polymerases on the PCR detection.After a comparison of the amplification efficiency of seven commercial DNA polymerases used in the market commonly,we chose the Takara and Platinum DNA polymerases which performed well before to study the performance of PCR in the following experiments.Amplification efficiency was determined by real-time PCR methods to calculate their copy numbers.Furthermore,we studied the nucleotide mismatches tolerance of probes to observe its impact on the detection of FERT-qPCR.In chapter three of this thesis,we designed the Babesia-special primers target for 18S rRNA to distinguish Babesia spp.and Theileria spp.based on the results got in this study.The amplified products were confirmed with agar electrophoresis and genomic sequencing.The amplification efficiency of single mismatch on Platinum DNA polymerase decreased much heavily than Takara DNA polymerase which kept the same amplification level with the primers without mismatch(almost 100%).When the nucleotide base C turns into A or T turns into G in 3'-terminal of upstream and downstream primers,the reaction was inhibited obviously or even be stopped.But it is reverse for A turns into C and T turns into C.Single mismatch located 4 bp more away from 3'-terminal has a small inhibitory effect on the reaction(82.3%);The average amplification efficiency of degenerate 2,3 or 4 bases amplified by Platinum DNA polymerase was 59.3%,43%and 33.9%.respectively.We observed identical trends across the two polymerases when mismatches are inserted into central and 5'-terminal of one primer which had an average efficiency by 92.6%and 100%,respectively.The mismatches in 5'-terminal can be eight until the PCR reaction stopped.Five mismatches in one primer in combination with five mismatches in the other primer will not block the amplification completely(more than 40%).We studied influence of mismatches in FAM probe of FRET-qPCR.There was no obviously difference between handled FAM probes and source one except putting five continuous mismatches in the middle of the FAM probe.The Tm value will decrease gradually when the mismatches happened in any position of probe.Mismatches inserted in the central of probe had the most obvious reduction.In the case of 4 nucleotide mismatches,the Tm of the intermediate site decreased by 10? which was more significantly than 3? at which Tm,decreased when the 3'and 5' ends of the probe were mismatched.Babesia spp.and Theileria spp.are both tick-borne protozoan hemoparasites which are clinically difficult to distinguish due to the highly similarity of 18s rRNA sequence.The literature review and preliminary tests of this study show that the most widely recognized PCR method could not specially differentiate them.We designed a PCR system to differentiate Babesia spp.and Theileria spp.according to the results got in this study.The PCR was negative when using Babesia-special primers designed in this study with mismatch G to C in 3' terminal amplified the plasmid inserted part of 18S rRNA of Theileria with two commercial DNA polymerases.Takara DNA polymerase amplified Theileria nucleotides with primers mismatched with T to G in 3' terminal but not for Platinum DNA polymerase.Both of the two polymerases can amplify Theileria while A turns into C.We amplified 40 clinical samples from varied origin using the Babesia-special primers with G to C mismatched showed that the positive rate of Babesia in this study(7.5%)was lower than referenced primers(50%and 30%).The genomic sequencing results of the quoted primers were Theileria spp..In conclusion,the amplification efficiency seemed to be DNA polymerases specific while mismatches introduced in the 3'-terminal primers.Platinum DNA polymerase showed more specific than Takara DNA polymerase while Takara DNA polymerase showed more sensitivity on account of more mismatches can be tolerated.The nucleic acid G turns into C and T turns into G in the 3'-terminal primer will have a pronounced inhibitory effect on amplification.Single mismatch located 4 bp far away from 3'-terminal,mismatches in the central and 5'-terminal of primer will have a moderate effect and the 5'-terminal can endure 8 continuous mismatches.Five mismatches in combination with five mismatches in the other primer will not block the amplification completely.Mismatches inserted into the central of probe have the greater influence than other positions and can tolerate five bases as possible.Study on mismatch of oligonucletides in this experiment demonstrated that the PCR system designed which based on the above conclusions can be used to distinguish Babesia spp.and Theileria spp..This will provide important reference for the differential diagnosis of pathogens with high homology such as Babesia and Theileria.However,the underlying mechanism of mismatch extension is still unknown and need further research.