Establishment of Real-time TaqMan RT-PCR for Quantitation Rotavirus VP7 Gene of Serotype G4

Author: ChenJiJun
Tutor: HanYueWu；ZhouXu
School: Lanzhou University
Course: Biochemistry and Molecular Biology
Keywords: PCR TaqMan Rotavirus G4 VP7 Quantitative reaction Probe technology Genetic methods Primers Amplification efficiency Fluorescence quantitative Standard Real time RNA Children with severe diarrhea Copy number Rotavirus Polymerase Detect Gene
CLC: R440
Type: Master's thesis
Year: 2010
Downloads: 81
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Abstract

Objective To establish a method based on the real-time quantitative RT- PCR detection and quantification of rotavirus G4 VP7 gene TaqMan probe . Method rotavirus (Rotavirus' dissertation">Rotavirus, RV) , severe diarrhea in children under 5 years of age , the most important pathogens , and infection caused deaths accounted for 5% of the number of child deaths , 90% of which occur in developing countries . WHO recommends immunization programs of all countries should include rotavirus vaccine to prevent rotavirus infection . Real - time quantitative PCR (Polymerase chain reaction) can be collected continuously in one or each cycle of the PCR tube, the fluorescent signal of the signal and template copy number dependent . A a perfect quantitative PCR its slope was 3.32 , corresponding to the amplification efficiency of 100% , the intercept between 33-37 cycles and R2 is 1.00 . The purpose of this study is to establish a TaqMan probe ( ? ) Fast, accurate , specific RV fluorescent quantitative reverse transcription-PCR method for the RV G4 VP7 gene detection and quantitation . Rotavirus G4 VP7 gene using RT-PCR (Reverse transcription PCR) the template design primers and probes , probe 5 'end distance upstream primer 3 ' end 8bp, JOE labeled probe 3 'end of the reporting group , 5 ' end-labeled Eclipe quencher . Amplified from rotavirus Type G4 VP7 gene , the gene construct plasmid clone culture . VP7 DNA was amplified from the plasmid and purified as a template, RNA was in vitro transcription . The RNA purification and photometric quantitative , diluted the RNA to 102-107 copies / μl of magnitude as the standard RT-qPCR (Real time quantitative PCR) , optimized PCR system sample G4 virus RNA copy number was determined by the absolute quantification method . RNA standards were obtained as a result of high purity, can be used for the detection and quantification of the Type G4 rotavirus . Create amplification system having a wide dynamic range , the amplification efficiency is greater than 90% , and to construct a standard curve R2 is greater than 0.98 ; compared with the ordinary method of RT-PCR , quantitative PCR, sensitivity is improved by about an order of magnitude , and can be easily self- 100 detection of target gene copies / μl of magnitude sample test reproducibility , specificity and sensitivity . Conclusions based on TaqMan ( ? ) Probe RT-qPCR specific and sensitive detection and quantitation of cell cultures rotavirus G4 VP7 gene .

Establishment of Real-time TaqMan RT-PCR for Quantitation Rotavirus VP7 Gene of Serotype G4

Abstract

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